tested python file, generated dummy data

README.md

@@ -34,6 +34,14 @@ This repository contains the PLOS and eLife datasets, introduced in the EMNLP 20
 
 Each dataset contains full biomedical research articles paired with expert-written lay summaries (i.e., non-technical summaries). PLOS articles are derived from various journals published by [the Public Library of Science (PLOS)](https://plos.org/), whereas eLife articles are derived from the [eLife](https://elifesciences.org/) journal. More details/anlaysis on the content of each dataset are provided in the paper.
 
+Both "elife" and "plos" have 6 features:
+    - "article": the body of the document (including the abstract), sections seperated by "/n".
+    - "section_headings": the title of each section, seperated by "/n".
+    - "keywords": keywords describing the topic of the article, seperated by "/n".
+    - "title" : the title of the article.
+    - "year" : the year the article was published.
+    - "summary": the lay summary of the document.
+
 ### Supported Tasks and Leaderboards
 
 Papers with code - [PLOS](https://paperswithcode.com/sota/lay-summarization-on-plos) and [eLife](https://paperswithcode.com/sota/lay-summarization-on-elife).

dataset_infos.json

@@ -0,0 +1 @@
+{"plos": {"description": "\nThis repository contains the PLOS and eLife datasets, introduced in the EMNLP 2022 paper \"[Making Science Simple: Corpora for the Lay Summarisation of Scientific Literature\n](https://arxiv.org/abs/2210.09932)\". \nEach dataset contains full biomedical research articles paired with expert-written lay summaries (i.e., non-technical summaries). PLOS articles are derived from various journals published by [the Public Library of Science (PLOS)](https://plos.org/), whereas eLife articles are derived from the [eLife](https://elifesciences.org/) journal. More details/anlaysis on the content of each dataset are provided in the paper.\n\nBoth \"elife\" and \"plos\" have 6 features:\n    - \"article\": the body of the document (including the abstract), sections seperated by \"/n\".\n    - \"section_headings\": the title of each section, seperated by \"/n\". \n    - \"keywords\": keywords describing the topic of the article, seperated by \"/n\".\n    - \"title\" : the title of the article.\n    - \"year\" : the year the article was published.\n    - \"summary\": the lay summary of the document.\n", "citation": "\n@misc{Goldsack_2022,\n  doi = {10.48550/ARXIV.2210.09932},\n  url = {https://arxiv.org/abs/2210.09932},\n  author = {Goldsack, Tomas and Zhang, Zhihao and Lin, Chenghua and Scarton, Carolina},\n  title = {Making Science Simple: Corpora for the Lay Summarisation of Scientific Literature},\n  publisher = {arXiv},\n  year = {2022},\n  copyright = {arXiv.org perpetual, non-exclusive license}\n}\n", "homepage": "https://github.com/TGoldsack1/Corpora_for_Lay_Summarisation", "license": "", "features": {"article": {"dtype": "string", "id": null, "_type": "Value"}, "summary": {"dtype": "string", "id": null, "_type": "Value"}, "section_headings": {"dtype": "string", "id": null, "_type": "Value"}, "keywords": {"dtype": "string", "id": null, "_type": "Value"}, "year": {"dtype": "string", "id": null, "_type": "Value"}, "title": {"dtype": "string", "id": null, "_type": "Value"}}, "post_processed": null, "supervised_keys": null, "task_templates": null, "builder_name": "scientific_lay_summarisation", "config_name": "plos", "version": {"version_str": "1.0.0", "description": null, "major": 1, "minor": 0, "patch": 0}, "splits": {"train": {"name": "train", "num_bytes": 1015222548, "num_examples": 24773, "dataset_name": "scientific_lay_summarisation"}, "validation": {"name": "validation", "num_bytes": 56304792, "num_examples": 1376, "dataset_name": "scientific_lay_summarisation"}, "test": {"name": "test", "num_bytes": 56043968, "num_examples": 1376, "dataset_name": "scientific_lay_summarisation"}}, "download_checksums": {"https://drive.google.com/u/1/uc?id=1lZ6PCAtXvmGjRZyp3vQQCEgO_yerH62Q&export=download&confirm=t&uuid=03eb0ca9-2333-4681-ac85-68f0a0e6b5c8": {"num_bytes": 357175122, "checksum": "09b35ac3e96be0263f813d7766a9be7cc19806f722e9d27f5e71271ee6e623a5"}, "https://drive.google.com/u/1/uc?id=1WKW8BAqluOlXrpy1B9mV3j3CtAK3JdnE&export=download&confirm=t&uuid=bbaafa1a-a0be-434f-935f-723033623119": {"num_bytes": 88704247, "checksum": "b0d11e4475f5ffd6a8c1875f7b6b2a6be3c4afc67c4682c2dbf43a1611a8e1a0"}}, "download_size": 445879369, "post_processing_size": null, "dataset_size": 1127571308, "size_in_bytes": 1573450677}, "elife": {"description": "\nThis repository contains the PLOS and eLife datasets, introduced in the EMNLP 2022 paper \"[Making Science Simple: Corpora for the Lay Summarisation of Scientific Literature\n](https://arxiv.org/abs/2210.09932)\". \nEach dataset contains full biomedical research articles paired with expert-written lay summaries (i.e., non-technical summaries). PLOS articles are derived from various journals published by [the Public Library of Science (PLOS)](https://plos.org/), whereas eLife articles are derived from the [eLife](https://elifesciences.org/) journal. More details/anlaysis on the content of each dataset are provided in the paper.\n\nBoth \"elife\" and \"plos\" have 6 features:\n    - \"article\": the body of the document (including the abstract), sections seperated by \"/n\".\n    - \"section_headings\": the title of each section, seperated by \"/n\". \n    - \"keywords\": keywords describing the topic of the article, seperated by \"/n\".\n    - \"title\" : the title of the article.\n    - \"year\" : the year the article was published.\n    - \"summary\": the lay summary of the document.\n", "citation": "\n@misc{Goldsack_2022,\n  doi = {10.48550/ARXIV.2210.09932},\n  url = {https://arxiv.org/abs/2210.09932},\n  author = {Goldsack, Tomas and Zhang, Zhihao and Lin, Chenghua and Scarton, Carolina},\n  title = {Making Science Simple: Corpora for the Lay Summarisation of Scientific Literature},\n  publisher = {arXiv},\n  year = {2022},\n  copyright = {arXiv.org perpetual, non-exclusive license}\n}\n", "homepage": "https://github.com/TGoldsack1/Corpora_for_Lay_Summarisation", "license": "", "features": {"article": {"dtype": "string", "id": null, "_type": "Value"}, "summary": {"dtype": "string", "id": null, "_type": "Value"}, "section_headings": {"dtype": "string", "id": null, "_type": "Value"}, "keywords": {"dtype": "string", "id": null, "_type": "Value"}, "year": {"dtype": "string", "id": null, "_type": "Value"}, "title": {"dtype": "string", "id": null, "_type": "Value"}}, "post_processed": null, "supervised_keys": null, "task_templates": null, "builder_name": "scientific_lay_summarisation", "config_name": "elife", "version": {"version_str": "1.0.0", "description": null, "major": 1, "minor": 0, "patch": 0}, "splits": {"train": {"name": "train", "num_bytes": 260998879, "num_examples": 4346, "dataset_name": "scientific_lay_summarisation"}, "validation": {"name": "validation", "num_bytes": 14257139, "num_examples": 241, "dataset_name": "scientific_lay_summarisation"}, "test": {"name": "test", "num_bytes": 14143535, "num_examples": 241, "dataset_name": "scientific_lay_summarisation"}}, "download_checksums": {"https://drive.google.com/u/1/uc?id=1lZ6PCAtXvmGjRZyp3vQQCEgO_yerH62Q&export=download&confirm=t&uuid=03eb0ca9-2333-4681-ac85-68f0a0e6b5c8": {"num_bytes": 357175122, "checksum": "09b35ac3e96be0263f813d7766a9be7cc19806f722e9d27f5e71271ee6e623a5"}, "https://drive.google.com/u/1/uc?id=1WKW8BAqluOlXrpy1B9mV3j3CtAK3JdnE&export=download&confirm=t&uuid=bbaafa1a-a0be-434f-935f-723033623119": {"num_bytes": 88704247, "checksum": "b0d11e4475f5ffd6a8c1875f7b6b2a6be3c4afc67c4682c2dbf43a1611a8e1a0"}}, "download_size": 445879369, "post_processing_size": null, "dataset_size": 289399553, "size_in_bytes": 735278922}}

dummy/PLOS/test.json

@@ -1 +0,0 @@
-{"id": "journal.ppat.1003207", "year": "2013", "title": "Cooperativity Between CD8+ T Cells, Non-Neutralizing Antibodies, and Alveolar Macrophages Is Important for Heterosubtypic Influenza Virus Immunity", "sections": [["Influenza virus remains a significant threat to global health , and results in 200 , 000 hospitalizations and 3 , 000\u201349 , 000 deaths each year in the United States [1]\u2013[3] .", "The ability of influenza virus to rapidly mutate and/or undergo reassortment , allows the virus to evade protective immunity obtained from previous infections or vaccinations [4] .", "Annual influenza vaccines induce an antibody response specific for the highly variable surface glycoproteins of influenza: neuraminidase ( NA ) and hemagglutinin ( HA ) .", "These seasonal vaccines typically take months to produce and rely on the accurate prediction of the influenza serotypes that will be circulating in the next flu season [5] .", "Thus , if the prediction is not accurate or a pandemic strain emerges , current vaccines offer little protection .", "Much research has therefore , focused on the development of a \u201cuniversal\u201d vaccine that will target the conserved , internal regions of the influenza virus , and confer protection against multiple influenza virus serotypes .", "Significant research in the influenza field has focused on the design of vaccines capable of eliciting influenza virus-specific CD8+ T lymphocytes [6]\u2013[8] .", "Since , CD8+ T cells are able to recognize internal , conserved regions of the influenza virus , these cells may be able provide cross-subtype , or heterosubtypic , protection against the influenza virus [9]\u2013[13] .", "A large body of work supports the potential viability of a CD8+ T-cell based vaccine [14]\u2013[17] .", "In mice , vaccination with internal proteins such as influenza nucleoprotein ( NP ) , leads to higher frequencies of NP-specific CD8+ T cells prior to infection , and lower viral titers after challenge with H1N1 and H3N2 strains of influenza [18]\u2013[22] .", "Furthermore , influenza virus-specific memory T cells are detected in the peripheral blood of healthy adolescents and adults , and there is some evidence for heterosubtypic immunity in humans that has been proposed to be due to T cells [23]\u2013[25] .", "However , several groups have reported that the number of influenza virus-specific memory CD8+ T cells in the lung airways of mice declines over time corresponding with a loss of heterosubtypic protection [26]\u2013[29] .", "While there is some conflicting data over whether heterosubtypic protection wanes , if true this gradual loss of the virus-specific CD8+ T cell population represents a serious concern in the generation of CD8+ T cell based vaccines [30] .", "Interestingly , recent work suggests that non-neutralizing antibodies targeting the internal proteins of influenza , specifically NP , can provide some protection against the influenza virus infection through a mechanism involving Fc receptors [31]\u2013[34] .", "Unlike neutralizing antibodies , which are able to prevent viral entry or exit , non-neutralizing antibodies typically target antigens that reside inside virions and/or infected cells .", "Despite recent progress , it is still not clear what role T cells play in non neutralizing antibody-mediated heterosubtypic protection elicited in an immune competent host .", "Additionally the mechanisms by which non-neutralizing antibodies can provide protection against the influenza virus remain elusive .", "Several groups have also noted the potential role of CD4+ T cells in providing protection against influenza virus [6] , [35] , [36] .", "These reports indicated that CD4+ T cells can form a lung-resident population following influenza virus infection where they can serve a protective role in mediating enhanced viral clearance and survival following lethal challenge through a variety of mechanisms including IFN\u03b3 secretion [35] , [37] .", "Recent studies using human volunteers infected with influenza virus also point to a key role for pre-existing CD4 T cell responses in limiting the severity of influenza virus infection and disease [38] .", "Intriguingly , influenza-specific memory CD4+ T cells have also been reported to synergize with na\u00efve B cells and CD8+ T cells to provide protection against influenza viral infection [36] .", "Whether virus-specific CD8 T cells also exhibit such cooperativity in protective immunity is unclear .", "In this study , we demonstrate that , in most settings , influenza virus-specific CD8+ T cells alone are insufficient to provide optimal protection against influenza virus .", "However , when virus-specific non-neutralizing antibodies are present together with virus-specific CD8+ T cells , complete protection is achieved against a lethal influenza virus challenge .", "Moreover , this cooperative protection is dependent , at least in part , on the presence of alveolar macrophages ( AM ) or other respiratory tract phagocytes , suggesting that non-neutralizing antibodies are able to eliminate influenza virus-infected cells through antibody-dependent cell-mediated cytotoxicity ( ADCC ) and/or phagocytosis .", "We demonstrate a novel mechanism by which antibodies and CD8+ T cells targeted against the conserved regions of the influenza virus act in concert to provide heterosubtypic protection .", "Our results complement recent work on the synergy between memory CD4+ T cells and na\u00efve B and CD8 T cells [36] and suggest that elicitating multiple arms of the adaptive immune response may represent a potent mechanism by which heterosubtypic protection against the influenza virus can be achieved ."], ["It has been reported that CD8+ T cell activity correlates with reduced influenza virus shedding following rechallenge [13] .", "Since , CD8+ T cell epitopes are often located in the internal , conserved regions of the influenza virus , the generation of influenza virus-specific CD8+ T cells may provide protective immunity against heterosubtypic influenza strains .", "Thus , we tested whether influenza virus-specific CD8+ T cells could mediate protective immunity using a recombinant viral approach to identify and track responses .", "We used influenza viruses in which the GP33-41 epitope from lymphocytic choriomeningitis virus ( LCMV ) , was inserted into the NA stalk region of the H3N2 influenza X31 ( X31-GP33 ) and H1N1 influenza PR8 ( PR8-GP33 ) viruses [39] , [40] .", "Influenza viruses expressing the GP33 epitope have been shown to induce a robust GP33 response in mice [39] , [41] .", "Mice were primed with either LCMV Armstrong or X31-GP33 intranasally ( i . n . ) and rechallenged , along with a control group of na\u00efve mice , with PR8-GP33 30 days later .", "The antibodies generated against the surface glycoproteins of the H3N2 X31-GP33 virus do not neutralize the H1N1 PR8-GP33 challenge virus [42]\u2013[44] .", "The GP33-specific CD8+ T cell population elicited from primary viral challenge , however , should be capable of responding to the secondary infection , allowing the role of CD8+ T cells in protection against the influenza virus to be investigated .", "The level of protection conferred upon secondary challenge was determined using three assays .", "Morbidity was assessed by weight loss .", "Pulse oximetry was also used to evaluate lung function .", "Finally , real time quantitative ( qRT-PCR ) was used to detect viral RNA and determine viral load at several time points following infection .", "X31-GP33 primed mice were completely protected from influenza rechallenge by all 3 measures ( Fig . 1A ) .", "These mice experienced almost no impairment of lung function or loss in weight , and had low viral load at all time points measured .", "In contrast , the LCMV Armstrong immunized mice , despite generating a robust GP33-specific CD8+ T cell response , exhibited little if any protection from the PR8-GP33 rechallenge .", "Apart from a slight delay in compromised lung function , the LCMV Armstrong immune mice were indistinguishable from the na\u00efve group and experienced a 25% decline in weight and lung function by day 9 post rechallenge .", "To determine if the difference in protection between the X31-GP33 and LCMV Armstrong immune groups was due to the X31-GP33 immune mice having a larger influenza virus-specific CD8+ T cell response , we quantified the total immune response in these mice 6 days after rechallenge in the lung , bronchoalveolar lavage ( BAL ) , and spleen .", "Responses were analyzed using intracellular staining to evaluate the number of cells in these mice able to produce interferon gamma ( IFN\u03b3 ) in response to stimulation with overlapping peptide pools for the influenza virus proteins HA , NA , non-structural protein 1 ( NS1 ) , NS2 , polymerase acidic ( PA ) , polymerase basic ( PB ) , NP , as well as the LCMV GP33 peptide .", "We found that the LCMV Armstrong immune mice had a similar or slightly larger antiviral CD8+ T cell response directed against the recombinant influenza virus following rechallenge in both the lung and BAL despite lack of protection ( Fig . 1B ) .", "Similar results were obtained using mice immunized intraperitoneally ( i . p . ) with LCMV Armstrong ( Fig . S1A , S1B , S1C ) , though LCMV i . n . immunized mice had slightly enhanced viral control compared to the LCMV i . p . primed mice ( Fig . S2 ) .", "Overall , while the route of priming may have some impact , these results indicate that the magnitude of the virus-specific CD8+ T cell response alone might not be a major determinate of protection against influenza viral challenge and suggest that other factors could be responsible for protection against influenza virus in X31-GP33 immune mice .", "We first sought to evaluate whether the agent used to prime the mice could have an impact on protection in our system .", "The specific priming agent used has been reported to confer differences in protection against influenza virus in several vaccine studies [45] , [46] .", "Thus , mice were primed with LCMV Armstrong , Listeria-GP33 ( LM-GP33 ) , or Vaccinia-GP33 .", "Each group of mice had a similar GP33-specific CD8+ T cell population despite being immunized with different bacterial or viral agents .", "We found that regardless of the priming agent used , all groups experienced severe weight loss , decline in lung function , and high viral load following rechallenge with PR8-GP33 ( data not shown ) .", "Thus , in this setting , the priming agent did not have an obvious direct correlation with whether or not protection was achieved .", "We next examined whether the epitope against which the CD8+ T cells were primed impacted protection .", "Different epitopes have been shown to elicit varying levels of protection to the influenza virus [47] .", "We therefore tested whether priming with another CD8 epitope shared between X31-GP33 and PR8-GP33 , the dominant Db-restricted NP366-374 ( NP366 ) epitope from influenza virus nucleoprotein , could elicit better protection than the GP33 response .", "Mice were primed with recombinant viruses ( or bacteria ) expressing GP33 , NP366 , or a non-influenza determinant ( the LCMV nucleoprotein ) , and challenged 30 days later .", "None of these approaches achieved substantial protection against PR8-GP33 rechallenge , as each group had substantial weight loss , high viral load and reduced lung function ( Fig . 2A ) .", "Thus , at least for the determinants examined , the specific epitope was not a major factor in the lack of protection observed in this model system .", "Next , we used a prime-boost strategy to test whether the more robust immune response induced upon boosting was superior in providing protection compared to non-boosted memory CD8+ T cells .", "Mice were immunized with LM-GP33 and then boosted with LCMV Armstrong 30 days after initial priming .", "These mice were then rechallenged with PR8-GP33 either 8 days or 30 days after the boost .", "We also immunized a group of mice with LCMV Armstrong and rechallenged with PR8-GP33 8 days later .", "The mice rechallenged with influenza virus 8 days after initial priming displayed delayed morbidity with the initiation of weight loss on \u223cday 6 post rechallenge rather than at \u223cday 2\u20133 in na\u00efve mice ( Fig . 2B ) .", "This transient delay in weight loss suggested that GP33-specific effector CD8+ T cell response present at 8 days after acute LCMV infection was capable of providing some initial protection following influenza virus infection .", "This group , however , still lost significant weight by day 9 post rechallenge and had reduced lung function as well as high viral load .", "It is worth noting that the dose of LCMV Armstrong used here has been demonstrated in our lab and others to be cleared by day 8 [48] .", "The prime boost group that was rechallenged 30 days after the \u201cboost\u201d did not experience this transient delay in weight loss and showed kinetics of morbidity similar to the LCMV Armstrong immune groups described above in terms of magnitude of weight loss and decline of lung function suggesting that the greater magnitude of GP33-specific CD8+ T cell response in this group was insufficient to mediate protection .", "In contrast , the prime-boost group that was rechallenged 8 days after the \u201cboost\u201d showed no signs of influenza-related pathology in terms of weight loss and lung function .", "Despite this lack of morbidity these mice still had very high viral loads until day 9 post rechallenge ( Fig . 2B ) .", "While priming ( and the prime-boost regimen ) was able to induce a robust population of GP33-specific response capable of producing IFN\u03b3 and tumor necrosis factor ( TNF\u03b1 ) in response to stimulation ( Fig . S3 ) , this response was still insufficient to mediate viral clearance .", "Thus , although this prime boost group is similar to the X31-GP33 group in terms of weight loss and lung function following rechallenge , viral control was relatively poor .", "The protection from morbidity in the mice challenged 8 days after boosting was not due simply to elevated bystander inflammation as mice subjected to two other prime-boost strategies that lacked CD8+ T cells specific for influenza virus showed rapid weight loss ( Fig . 2C ) .", "Furthermore , the lack of weight loss achieved by the virus-specific prime-boost strategy was recapitulated when LM-GP33 was substituted with VV-GP33 ( Fig . 2C ) , suggesting that the lack of weight loss in these mice is not dependent on the identity of the priming agent , but on the rapid initiation of an influenza virus-specific CD8+ T cell response .", "These results are in line with reports that \u201cboosted\u201d memory CD8+ T cells are better than primary memory CD8+ T cells in controlling some acute infections [49] , [50] .", "However , while pathology was reduced , this immune response was not sufficient to efficiently control viral load .", "We next investigated whether we could achieve enhanced CD8+ T cell-mediated protection using adoptive transfer strategies analogous to adoptive transfer approaches used for influenza virus-specific CD4+ T cells [35] , [36] .", "Ly5 . 1+ mice were immunized with LM-GP33 and 30 days later boosted with LCMV Armstrong .", "On day 8 following the boost , CD8+ T cells were isolated and 0 . 8\u00d7106 , or 1 . 6\u00d7106 GP33+ CD8+ T cells were adoptively transferred to Ly5 . 2+ na\u00efve mice .", "The recipient mice were rechallenged with PR8-GP33 the next day .", "Little to no protection was observed as measured by weight loss and viral load compared to a PBS-treated control group ( Fig . 3A\u2013B ) , despite high numbers of GP33 specific CD8+ T cells present in the lungs of these mice 6 days after rechallenge ( Fig . 3C ) .", "To determine whether the observed lack of protection was due to an insufficient number of in vivo primed adoptively transferred GP33-specific CD8+ T cells , we primed P14 TCR-transgenic CD8+ T cells ( specific for LCMV GP33-41 ) using an in vitro approach that allowed the generation of large numbers of activated GP33-specific CD8+ T cells .", "We then adoptively transferred 2\u00d7106 , 10\u00d7106 or 20\u00d7106 GP33-specific CD8+ T cells into na\u00efve mice and challenged these mice with influenza virus .", "Mice given 20\u00d7106 or 10\u00d7106 GP33-specific CD8+ T cells were almost completely protected from influenza-related morbidity and experienced virtually no decline in weight or lung function ( Fig . 3D ) .", "Additionally , even the group given 2\u00d7106 GP33-specific CD8+ T cells exhibited improved protection with only a 15% decline in weight and a 20% reduction in lung function .", "Despite the greatly reduced morbidity in these mice , the viral loads measured by qRT-PCR for viral RNA were almost indistinguishable from the PBS-treated control group .", "This lack of difference in viral control was confirmed using an assay for infectious virus to ensure that qRT-PCR-based approach was accurately reflecting replicating virus rather than residual viral debris or viral RNA independent of replicating virus ( Fig . S4 ) .", "Thus , similar to the prime-boost group described above ( Fig . 2C ) , the immune response elicited by the adoptively transferred , in-vitro generated effector CD8+ T cells was insufficient to reduce viral load despite the improvement in measures of morbidity .", "Overall , these results indicate that both in vivo and in vitro generated GP33-specific CD8+ T cells alone were insufficient to provide optimal protection against a pathogenic influenza virus challenge .", "While CD8+ T cells in large enough numbers are able to provide some protection as measured by weight loss and lung function , they are unable to significantly reduce viral load .", "Moreover , our results suggest that the mechanism of cross-subtype protection in X31-GP33 immune mice is unlikely to be exclusively CD8+ T cell-dependent .", "To evaluate the mechanism of cross-subtype protection in X31-GP33 primed mice , we next examined the dependence of protection in this setting on T cells ( Fig . 4 ) .", "CD8+ and/or CD4+ T cells were depleted from X31-GP33 immune mice prior to PR8-GP33 rechallenge with depletion being verified as >97% in the lungs .", "In all cases , depletion of CD8+ and/or CD4+ T cells did not increase the severity of weight loss .", "There was a slight but non-significant decrease in oxygen saturation following the depletion of CD8+ T cells , which was amplified when both CD4+ and CD8+ T cells were depleted ( Fig . 4A ) .", "( Note , that CD4+ and CD8+ T cells were depleted simultaneously using anti-Thy1 . 2 , which may also deplete double negative T cells , natural killer ( NK ) cells and innate lymphoid cells ) .", "Interestingly , the depletion of CD8+ T cells resulted in a considerable increase in viral load while CD4+ T cell depletion caused no significant difference in viral load .", "The viral load of the CD8+ T cell depleted group was still lower than that found in na\u00efve mice challenged with PR8-GP33 ( see Fig . 1A ) , although this difference was non-significant .", "Furthermore , the viral load was identical between the group in which only CD8+ T cells were depleted and the one in which both CD8+ and CD4+ T cells were depleted ( Fig . 4A ) .", "This result agrees with previous reports that CD4+ T cells play only a minor role in modulating influenza viral titers , and that depletion of CD4+ T cells does little to alter the course of viral infection [51] , [52] .", "While it is known that memory CD4+ T cells can cooperate with na\u00efve B or CD8+ T cells in the context of influenza infection [36] , it is unknown whether a similar cooperativity occurs with influenza virus-specific CD8+ T cells .", "It is interesting that CD8+ T cells in this setting were needed for control of virus while in the previous experiments ( Fig . 2B , 3D ) CD8+ T cells seemed to be able to control weight loss but not viral load .", "This difference may be due to differences in depletion versus immunization or adoptive transfer approaches or other mechanisms such as changes in immunopathology because of larger numbers of CD8 T cells suppressing other responses ( e . g . CD4 T cells ) .", "X31-GP33 immune mice are largely protected against symptoms of influenza virus infection in the absence of CD8+ and CD4+ T cells , suggesting other possible mechanisms contributing to protection .", "One possibility is that B cells have a role through the action of non-neutralizing antibodies specific for determinants shared between the X31 and PR8 influenza strains .", "To test this notion , we immunized \u00b5MT mice , which lack B cells [53] , with X31-GP33 .", "When challenged 30 days later these mice demonstrated no protection against PR8-GP33 despite a very similar influenza virus-specific CD8 T cell response compared to B6 mice ( Fig . 4B and C ) .", "These data suggested that B cells are essential for heterosubtypic protection .", "One concern is that the immunological response may be altered in \u00b5MT mice due to the total lack of B cells .", "Therefore , we examined MD4 transgenic mice that have normal numbers of B cells , but have a transgenic B cell receptor specific for hen egg lysozyme [54] and are therefore unable to generate an influenza virus-specific antibody response .", "Similar to the \u00b5MT mice , however , MD4 mice immunized with X31-GP33 were also not protected and experienced severe weight loss upon rechallenge with PR8-GP33 ( Fig . 4B ) .", "X31-GP33 immune MD4 mice also had reduced lung function and high viral load ( Fig . 4B ) .", "To test whether the lack of non-neutralizing antibodies might underlie the defect in X31-GP33 immune MD4 mice , we transferred serum collected from X31-GP33 immunized B6 mice ( referred to as X31 serum ) into X31-GP33 primed MD4 mice and rechallenged with PR8-GP33 .", "Protective immunity , as measured by all three parameters was improved ( Fig . 5 ) .", "Collectively , these data suggested that an influenza virus-specific B cell response was essential for X31-GP33 based heterosubtypic protection .", "As cross-neutralizing antibodies are not induced between the X31 and PR8 influenza strains [42]\u2013[44] , non-neutralizing antibodies are likely contributing to protection .", "One possible interpretation of the data presented thus far is that both CD8+ T cells and non-neutralizing antibodies might be necessary for optimal protection .", "To evaluate whether non-neutralizing antibodies in conjugation with influenza virus-specific CD8+ T cells can elicit robust heterosubtypic protection in B6 mice , we transferred serum from X31-GP33 immune mice into LCMV Armstrong immune mice .", "When given X31-GP33 serum , LCMV immune mice containing GP33-specific memory CD8+ T cells displayed significantly reduced weight loss and viral load compared to the LCMV Armstrong immune group that had received PBS or serum from na\u00efve mice ( Fig . 6A ) .", "LCMV immune mice that received X31-GP33 serum also maintained nearly 100% blood oxygen saturation following PR8-GP33 challenge .", "The protection achieved by transfer of X31-GP33 serum to LCMV Armstrong-immune mice in terms of weight loss and lung function was nearly equivalent to that achieved with transfer of serum from PR8-GP33 immune mice containing neutralizing antibodies , although PR8 serum resulted in more effective control of viral replication ( Fig . 6A ) .", "To determine whether the protective factor in the serum was indeed antibodies , we administered serum that had been depleted of IgG and IgA to LCMV Armstrong immune mice and challenged with PR8-GP33 [55] .", "These mice exhibit no evidence of protection indicating that X31-GP33 mediated protection is antibody-dependent ( Fig . 6A ) .", "While it is possible that there may be more total IgG in the transferred X31 serum compared to na\u00efve serum , there was no obvious correlation between the total IgG levels and protection in these experiments ( data not shown ) .", "Our results indicate that optimal heterosubtypic protection against influenza is elicited only when both GP33-specific CD8+ T cells and non-neutralizing antibodies are present .", "While many previous studies have demonstrated that antibodies induced by X31 do not neutralize PR8 and vice versa [42]\u2013[44] , it was possible that a new neutralizing determinant might have been formed due to the insertion of the GP33 sequence into the NA stalk .", "Thus , we transferred X31-GP33 serum into na\u00efve mice one day prior to challenge with PR8-GP33 .", "These mice displayed no signs of protection and experienced severe weight loss and decline in lung function ( Fig . 6B ) .", "The na\u00efve group given PR8 serum was completely protected from challenge with PR8-GP33 due to the presence of neutralizing antibodies ( Fig . 6B ) .", "This result strongly suggested that the antibodies found in X31-GP33 serum were non-neutralizing .", "Additionally the lack of protection found in the na\u00efve group given X31-GP33 serum indicates that both antigen-specific CD8+ T cells and non-neutralizing antibodies were needed for protection .", "To further examine the cooperativity between non-neutralizing antibodies and virus-specific CD8+ T cells in heterosubtypic protection we transferred X31-GP33 serum , na\u00efve serum , or PBS into LCMV Armstrong immune mice .", "We then rechallenged these mice with PR8-WT instead of PR8-GP33 .", "In this setting the LCMV Armstrong primed mice given X31-GP33 serum , as well as the groups given na\u00efve serum or PBS were not protected from PR8-WT rechallenge by any measure ( Fig . 6C ) .", "Thus , the protective immunity in this setting was dependent on both non-neutralizing antibodies and recognition of viral determinants by primed CD8+ T cells .", "To explore whether this cooperativity-based protection could be achieved using natural influenza-virus derived epitopes we immunized mice with VVNP366 to induce an influenza virus-specific T cell response .", "We then waited 30 days and transferred X31 or na\u00efve serum into these mice one day prior to challenge with the H1N1 swine influenza virus strain SW/33 .", "SW/33 is not genetically engineered , but , like PR8-GP33 , is pathogenic in mice .", "Similar to LCMV Armstrong immune mice , VV366 immune mice given X31 serum were protected against viral challenge in terms of both weight and lung function , with these mice also having a trend to lower viral load compared to mice given na\u00efve serum ( Fig . 6D ) .", "This finding strongly indicates that the cooperativity-based protection is not simply an artifact of out recombinant influenza virus system , but rather a likely physiologically relevant mechanism of protection against influenza virus challenge .", "The means by which non-neutralizing antibodies operate in cooperative protection is unclear .", "Among the possible mechanisms are: antibody-dependent cell-mediated cytotoxicity ( ADCC ) , Fc receptor ( FcR ) mediated phagocytosis , and the complement pathway .", "To distinguish between these possibilities we used mice either lacking FcR\u03b3 or interleukin-15 ( IL-15 ) .", "FcR\u03b3 -/- mice are deficient in the gamma chain subunit of the FcgRI , FcgRIII and FceRI receptors resulting in functionally impaired macrophages , neutrophils , mast cells , basophils and Natural Killer ( NK ) cells .", "IL-15 -/- mice , on the other hand , are deficient in NK cells , but not these other cell types allowing the role of NK cell-mediated ADCC in heterosubtypic protection to be tested .", "Wild type , FcR\u03b3 -/- , and IL15 -/- mice were primed with LCMV Armstrong , rested 30 days , and then given either X31-GP33 serum or na\u00efve serum 1 day prior to rechallenge .", "The LCMV immune IL-15-/- mice given X31-GP33 serum were protected upon PR8-GP33 challenge , although the decrease in viral load in these mice was only a trend .", "These results might reflect the moderate defect in CD8 T cell memory in these mice [56]\u2013[58] , although influenza virus-specific T cell memory in the respiratory tract appears independent of IL-15 [59] .", "In contrast , the FcR\u03b3 -/- mice were not protected against infection as measured by any parameters tested regardless of whether the mice were given X31-GP33 or na\u00efve serum ( Fig . 7A ) .", "Together , these results suggested that non-neutralizing antibody-based protection was FcR\u03b3-dependent , but that NK cells were non-essential .", "To further examine cooperative heterosubtypic immunity we first immunized B6 mice with X31-GP33 .", "After 30 days we treated these mice i . n . with clodronate-loaded liposomes to deplete alveolar macrophages ( AM ) ( and possibly other airway-resident phagocytes ) , cobra venom factor to deplete complement , or anti-NK 1 . 1 to deplete NK cells .", "Another group was given empty liposomes as a control .", "Following PR8-GP33 rechallenge , the only group left unprotected was the clodronate treated group in which AM were depleted .", "These mice experienced severe morbidity and high viral load despite having an unimpaired CD8+ T cell response ( Fig . S5A ) .", "All other groups remained healthy and controlled the infection ( Fig . 7B ) .", "This result suggested that heterosubtypic immunity mediated by non-neutralizing antibodies and CD8+ T cells was , at least in part , dependent on cells depleted by clodronate liposomes including AM .", "It is important to note that while we found clodronate liposome treatment to be non-toxic to uninfected mice and to result in \u223c70% depletion of alveolar macrophages in the BAL fluid three days following a single clodronate treatment ( Fig . S5B ) , it is possible that depletion of other airway populations such as dendritic cell or inflammatory macrophages could occur .", "The unimpaired CD8+ response seen in clodronate liposome treated mice however , suggests that any clodronate depletion of dendritic cells in the airway was insufficient to significantly impact presentation of antigen to CD8+ T cells .", "Furthermore , preliminary studies using adoptive transfer of alveolar macrophages obtained from the BAL of na\u00efve mice into LCMV Armstrong immune FcR\u03b3 -/- mice suggested that reintroducing alveolar macrophages could partially rescue weight loss in half the mice when given in conjunction with X31 serum ( Fig . S6 ) .", "While further studies are necessary , these data are consistent with the notion that AM are involved in cooperative heterosubtypic protection .", "To further evaluate the role of AM and NK cells in cooperative heterosubtypic protection , we depleted AM or NK cells in LCMV Armstrong immune mice as described above , and administered X31-GP33 serum to these mice one day prior to rechallenge .", "We found that only the group treated with clodronate liposomes exhibited severe weight loss and a decline in lung function ( Fig . 7C ) .", "The group depleted of NK cells did display a trend towards higher viral loads then the group given only X31-GP33 serum , but this difference was not significant and this group still experienced almost no weight loss or decline in lung function , further suggesting that NK cells ( or perhaps other NK1 . 1+ cells ) only play a minor role in the mechanism of non-neutralizing antibody-based protection .", "Overall , this finding strongly indicates that the mechanism of non-neutralizing antibody-based protection is dependent on cells depleted by clodronate liposomes , including AM , likely through FcR-dependent AM phagocytosis or ADCC of influenza virus-infected cells ."], ["The aim of universal influenza vaccination approaches is to provide long-lasting protection against a wide range of viral serotypes .", "Creating a universal vaccine by inducing CD8+ T cells specific for conserved internal proteins of influenza virus has received considerable attention , but remains an unrealized goal .", "In this study , we demonstrate that influenza virus-specific CD8+ T cells can cooperate with non-neutralizing antibodies to provide efficient cross-subtype influenza virus-specific protection .", "While non-neutralizing antibodies against M2e or other conserved determinants have recently been examined , our data indicate a previously unappreciated role for cooperativity between non-neutralizing antibodies and CD8+ T cell responses in the induction of optimal protection from serologically distinct influenza virus strains .", "This mechanism represents a novel approach by which a universal influenza vaccine could be developed .", "Currently there are several promising universal influenza vaccine candidates in development .", "Among these candidates are broadly neutralizing antibodies , which are able to target the conserved stem region of the influenza virus [60]\u2013[64] .", "These broadly neutralizing antibodies have been found to be cross reactive among different H1 or H3 influenza subtypes and are likely to represent a major advance in generating more effective influenza virus vaccines .", "However , current antibodies specific for the H1 stem are largely effective only against heterologous H1 and H5 viruses , and antibodies against the H3 stem are only effective against H3 viruses [60] .", "Interestingly , neutralizing antibodies were sometimes induced following vaccination with a pandemic H1N1 vaccine , but were of too low magnitude to induce robust heterosubtypic protection [64] .", "Until neutralizing antibodies can be generated against an antigen conserved between many different influenza subtypes , humans will remain vulnerable to the threat of a pandemic from a novel influenza strain such as H7N7 , H9N2 , etc .", "[65] .", "Another promising potential universal influenza vaccine targets the ectodomain of matrix protein 2 ( M2e ) [66] .", "The M2e sequence is conserved across influenza virus subtypes , and humoral anti-M2e immunity has been shown to protect against influenza virus challenge in mice [67] , [68] .", "However , M2e-based protection does not prevent or resolve infection and is of a lower potency that HA-specific antibodies , making an M2e-dependent therapy more likely to act as a safety net in the case of the emergence of pandemic influenza strains rather than a replacement for current vaccines [69] .", "One concern associated with both broadly neutralizing antibodies and M2e based vaccines is that widespread use of these vaccines will introduce immune pressure promoting the evolution of antigenic escape viruses [66] .", "There have already been reports of escape viruses being generated in response to M2e antibodies , with one study finding that virus mutants with antigenic changes in M2e emerged in 65% of virus-infected mice treated with anti-M2e , although some level of protection remained despite these mutations [69] .", "An interesting avenue of future research will be to determine if cooperativity between T cells and non-neutralizing antibody can be used to boost the protection elicited through these vaccination strategies .", "Virus-specific CD8+ T cells do not seem to be generated by HA-stalk or M2e immunization strategies [70] , so a vaccine in which CD8+ T cells can be elicited specific for conserved influenza virus determinants , combined with approaches to generate HA-stalk or M2e targeting antibodies , may offer improved protection .", "Furthermore , the overlapping protection provided by virus-specific CD8+ T cells should help reduce the possibility of an escape virus emerging .", "Recent reports have implicated NP-specific IgG in heterosubtypic immunity to influenza virus [28] , [29] .", "This previous work found that NP protein was detectable in the BAL and nasal washes of influenza virus-infected mice , thereby allowing the NP antigen to interact with NP-specific antibodies and form complexes to stimulate antiviral immune responses .", "These studies also demonstrated that 5 daily antibody injections starting 3 days prior to infection were required to reduce viral load in na\u00efve mice , using a 0 . 25 lethal dose 50% ( LD50 ) influenza rechallenge .", "Our findings extend this work in determining that cooperativity between influenza virus-specific CD8+ T cells and antibodies is important for heterosubtypic protection in immune competent mice .", "NP-specific antibodies are likely to be a primary component of the X31-GP33 serum used in our work .", "The cooperativity we demonstrated may mean that excessively high amounts of non-neutralizing antibodies might not be required if virus-specific CD8+ T cells are also present .", "Since previous influenza virus infections or vaccinations have likely induced anti-NP antibodies and influenza virus-specific memory CD8+ T cells in most adults , it is interesting that more heterologous protection does not seem to exist in humans .", "One reason may be that sufficiently high titers of anti-NP antibodies are not present in adults to mediate cooperative protection .", "Indeed one report indicates that trivalent inactivated influenza virus vaccine ( TIV ) only rarely and modestly boosted existing levels of anti-NP IgG [32] .", "Alternatively , perhaps such cooperative immunity is , in fact , one reason for the relatively low mortality in healthy adults for most strains of influenza virus .", "Our results suggest that it will be interesting to test whether this cooperative immunity might wane with increasing age , a theory supported by several reports [26]\u2013[29] .", "It is possible that the lack of CD8 T cell boosting by the yearly vaccine allows CD8 T cell memory to decline over time even in healthy young adults .", "Future studies will be necessary to test some of these ideas in humans .", "The mechanism by which non-neutralizing antibodies operate in the setting of heterosubtypic immunity remains poorly understood .", "Unlike neutralizing antibodies , non-neutralizing antibodies do not prevent viral entry into host cells and must therefore employ a different means of action to reduce viral load .", "FcRs have been reported to be important mediators of this process , but the specific cell types directly involved in reducing influenza viral load and pathology by this mechanism are unclear .", "Alveolar macrophages have been shown to play a critical role in influenza virus protection , likely through ADCC or phagocytosis [71] , [72] .", "Some reports have also suggested that NK-cells may be involved in influenza virus protection through ADCC [59] , although several recent studies have found this to be unlikely [73] , [74] .", "The complement pathway could also have an important role due to the ability of complement to bind and lyse infected cells or enveloped virus in the presence of antibodies .", "Complement has been demonstrated to be able to neutralize the influenza virus in the presence of natural antibodies [75] , and complement component C3 may be important in T cell priming and migration to the lungs [76] .", "Indeed , C3 deficiency in humans correlates with recurrent infections of the upper and lower respiratory tract [77] .", "In the current studies we found that heterosubtypic protection was dependent , at least in part , on alveolar macrophages .", "Intranasal administration of clodronate liposomes have been shown to selectively deplete alveolar macrophages while leaving the interstitial macrophage population as well as other cell types in the lungs intact [71] , [78]\u2013[80] .", "However , the possibility of off target effects of the clodronate liposome approach cannot be fully excluded .", "The unimpaired CD8+ T cell response found in clodronate-treated mice ( Fig . S3A ) however , suggested that depletion of dendritic cells was unlikely to be responsible for the lack of protection in this setting .", "Thus , alveolar macrophages are likely a major cell type impacted by this treatment and are expected to act to help reduce viral load through recognition of the Fc region of non-neutralizing antibodies .", "This recognition can lead to ADCC and/or antibody-dependent cell-mediated phagocytosis directed against infected cells bound by non-neutralizing antibodies .", "The reduced effectiveness of seasonal influenza vaccines and greater infection-related morbidity and mortality in the elderly is thought to be due to alterations in both the innate and adaptive immune response that occur with age [3] , [81]\u2013[84] .", "Among the alterations reported in the elderly that could influence immunity to infection are changes in macrophages , NK cells , neutrophils , pathogen recognition via Toll-like receptors , innate cell cytokine production [85] , [86] , as well as decreased numbers , proliferation and signaling of B and T cells [87]\u2013[91] .", "Interestingly , there have also been reports of changes in FcRs that occur with age , which could lead to defects in FcR-dependent effector functions [92] .", "While it has been shown that CD8+ T cell and neutralizing antibody-based protection obtained at a young age is still protective many years later [93]\u2013[95] , less is known about non-neutralizing antibody-dependent protection .", "Since this type of protection relies on FcR-dependent effector mechanisms to clear infected cells it will be important to determine if the heterosubtypic protection observed in young mice is also seen in aged groups of animals .", "A major goal of \u201cuniversal\u201d influenza vaccines is to elicit cross-subtype influenza virus protection in both young and aged populations .", "Hence , age-related defects in the immune system are a critical issue that must be addressed in future studies to determine if cooperative protection is an effective strategy in eliciting heterosubtypic influenza protection .", "Collectively , we have shown that influenza virus-specific CD8+ T cells in cooperation with non-neutralizing antibodies are able to provide optimal protection against a lethal influenza virus rechallenge .", "This protection is only exhibited when both influenza virus-specific CD8+ T cells and non-neutralizing antibodies are present .", "Furthermore , non-neutralizing antibodies likely contribute to influenza virus clearance , possibly through a mechanism involving alveolar macrophages .", "It should be pointed out that while we have focused largely on CD8+ T cells , it is possible that cooperative protection will occur for CD4+ T cells and non-neutralizing antibodies .", "Indeed , there is good evidence that CD4+ T cells can contribute to protective immunity to influenza virus [35] , [96] , [97] and cooperate with na\u00efve B and CD8+ T cells [36] .", "It will be important to address this issue in the future .", "This work provides novel insights into cross-subtype influenza virus protection and could have implications for the development of a universal influenza vaccine ."], ["This study was carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health .", "Protocols were approved by the Institutional Animal Care and Use ( IACUC ) committees of the Wistar Institute , ( animal welfare assurance number A3432-01 ) or University of Pennsylvania ( animal welfare assurance number A3079-01 ) .", "The Wistar and University of Pennsylvania Animal Care and Use Programs are fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International ( AAALAC ) .", "C57BL/6 and Ly5 . 1+ mice were purchased from the National Cancer Institute ( Frederick , MD ) or Jackson Laboratories ( Bar Harbor , ME ) .", "Age- and sex matched IL-15-/- mice were obtained from Taconic ( Germantown , NY ) , FcR\u03b3 knockout mice ( FcR\u03b3 KO; strain name B6 . 129P2-Fcer1gtm1RavN12 ) were purchased from Taconic Farms , Inc . ( Hudson , NY ) , and B cell-deficient B6 . 129S2-IghtmICgn/J ( \u00b5MT ) mice and anti-HEL B-cell receptor ( BCR ) -transgenic C57BL/6-TgN ( IghelMD4 ) mice ( referred to as MD4 ) were obtained from the Jackson Laboratory .", "For primary or secondary infections , mice were inoculated using the following pathogens , doses and routes: with LCMV Armstrong ( LCMV Arm; 2\u00d7105 PFU i . p . or 5\u00d7104 PFU i . n ) ; recombinant X31 influenza virus expressing the LCMV GP33 epitope ( X31-GP33; 1 . 6\u00d7105 TCID50 i . n . ) ; vaccinia virus ( VV ) expressing the LCMV GP33 epitope ( VVGP33 ) , VV expressing the influenza virus NP366 epitope ( VVNP366 ) , and VV expressing LCMV Nucleoprotein ( VV-NP ( LCMV ) ) all used at 3\u00d7105 PFU i . n . ; Listeria ( LM ) expressing the GP33 epitope ( LM-GP33; i . v . ) ; Vesicular stomatitis virus expressing the ovalbumin ( OVA ) epitope ( VSV-OVA; 2\u00d7106 PFU i . v . ) ; LCMV Armstrong V35A which lacks the GP33 epitope ( LCMV-V35A; 2\u00d7105 PFU i . p . ) .", "For rechallenge experiments , mice were given either recombinant PR8 influenza virus expressing the LCMV GP33 epitope ( PR8-GP33; 3 LD50 i . n . ) ; wild type PR8 influenza virus ( PR8-WT; 3 LD50 i . n . ) ; wild type swine influenza virus ( SW/33; 3 LD50 i . n ) .", "For both strains of PR8 1LD50\u200a=\u200a\u223c250 TCID50 .", "Prior to i . n . infections , mice were anesthetized by i . p . injection of ketamine hydrochloride and xylazine ( Phoenix Scientific , San Marcos , CA ) in 0 . 2 ml Life Technologies HBSS ( Invitrogen , Carlsbad , CA ) .", "In some experiments , mice were anesthetized with 2 . 5% Avertin ( 0 . 2\u20130 . 35 ml ) i . p . Recombinant influenza virus strains containing the LCMV GP33\u201341 epitope inserted in the neuraminidase stalk region were obtained from Dr . Richard J . Webby ( St . Jude Children's Research Hospital , Memphis , TN ) and have been previously described [35] , [36] .", "These viruses were propagated in eggs , and stored at \u221280\u00b0C .", "The replication and pathogenicity of these recombinant X31 and PR8 strains were not substantially different from their nonrecombinant counterparts ( data not shown ) .", "Viral titers were determined by plaque assay on Vero cell monolayers ( for LCMV and VV ) or on Madin-Darby canine kidney cell monolayers ( for X31-GP33 and PR8- GP33 ) as previously described [98] .", "For all experiments , na\u00efve C57BL/6 mice were also infected with influenza virus to allow comparison of weight loss , viral load , and pulse oximetry between different experiments .", "The concentration of infectious virus in lungs was determined by titration of homogenized tissues in Madin-Darby canine kidney cell ( MDCK ) microcultures as described previously [6] .", "Lung titers are expressed as dilution of lung extract at which 50% of the MDCK cultures revealed virus growth ( TCID50/ml ) .", "For all adoptive transfer experiments , congenic mice differing in Ly5 ( Ly5 . 1 versus Ly5 . 2 ) were used .", "For adoptive transfers CD8+ T cells were purified ( 90% purity ) using magnetic beads ( CD8+ T cell isolation kit , MACS beads; Miltenyi Biotec , Auburn , CA ) .", "The MouseOx Pulse-oximeter ( Starr Life Sciences , Oakmont PA ) was used to measure blood oxygen saturation ( SpO2 ) in PR8-GP33 infected mice during the course of infection .", "A depilatory agent ( Nair , Church & Dwight Co . ) was applied to the neck of anesthetized mice 1 day prior to influenza infection to remove hair and delay future hair growth .", "For readings , the oximeter clip was placed on the neck and percent SpO2 was measured each second over several minutes , data shown is the average of SpO2 readings recorded over 3\u20135 minutes per mouse .", "Alveolar macrophages were isolated and transferred as previously described [71] .", "Briefly AM were isolated from BAL with PBS-EDTA from C57BL/6 mice .", "10 mice were sacrificed as donors for every recipient mouse .", "A 23-gauge cannula was inserted into the trachea , and cells were collected by washing the airway lumen with 3\u00d70 . 5 ml PBS-EDTA .", "The obtained BAL fluid was centrifuged , and cells were washed twice with PBS , counted , and resuspended in PBS .", "4 . 5\u00d7105 cells were then transferred i . n . into recipient mice at a volume of 50 ul/mouse .", "NK1 . 1 cells were depleted in vivo by i . p . injection ( 0 . 2 mg/injection ) of rat mAb PK136 .", "CD8+ T cells were depleted by i . p . injection of rat mAb 53 . 6 , CD4+ T cells were depleted using rat mAb GK1 . 5 and both CD4+ and CD8+ T cells were depleted simultaneously using anti-Thy1 . 2 ( 0 . 2 mg/injection; clone 30H12 , isotype Rat IgG2b obtained from BioXCell ) .", "All antibody treatments were givens days -3 , -1 , 2 , and 5 post PR8-GP33 rechallenge .", "Depletion was confirmed by flow cytometric analysis on day 6 post rechallenge in the lungs .", "All in vivo mouse antibodies were purchased from Bio X Cell ( West Lebanon , NH ) .", "Alveolar macrophages were depleted using the liposome-mediated macrophage depletion technique based on the intracellular delivery of the drug dichloromethylene diphosphonate ( clodronate ) .", "Preparation of clodronate-liposomes and applications of the technique was done as previously described [79] .", "Alveolar macrophages were depleted by i . n . administration of 50 ul of clodronate-liposomes on days -3 , -1 , and 2 post PR8-GP33 rechallenge .", "Viral quantitative real-time RT-PCR was performed essentially as previously described [26] .", "Briefly , total RNA was purified from lungs of PR8-GP33 infected mice using the RNeasy Mini Kit ( Qiagen , Valencia , CA ) .", "Reverse transcriptions were primed with random primers and performed using the High Capacity cDNA Reverse Transcription Kit from Applied Biosystems ( Foster City , CA ) .", "Real-time quantitative PCR ( qRT-PCR ) was performed on cDNA using TaqMan Universal PCR Master Mix ( Applied Biosystems ) and probes and primers specific to the influenza PA protein with all samples analyzed in triplicate .", "Reactions were run on a real-time PCR system ( ABI7500; Applied Biosystems ) .", "Amount of influenza viral RNA per sample was then calculated using known standards .", "The total amount of virus per lung was then calculated using the mass of the lung portion taken for viral RNA determination in relation to the total lung mass .", "The TCID50 of each sample was determined by calculating the volume of virus per lung ( using the viral RNA determination of the PR8-GP33 stock ) and then calculating the total TCID50 in the lungs using the known TCID50 per unit volume of the viral stock .", "The limit of detection was determined by performing qRT-PCR on lung samples from uninfected mice and represented by a dashed line .", "PA sense: CGGTCCAAATTCCTGCTGAT .", "PA antisense: CATTGGGTTCCTTCCATACA .", "PA probe: 6FAMCCAAGTCATGAAGGAGAGGGAATACCGCTTAMRA Lymphocytes were isolated from tissues as previously described [99] .", "Briefly , mice were euthanized and the hepatic vein cut .", "Lungs were perfused by injection of PBS into the hepatic artery or the right heart ventricle .", "Lungs were cut into pieces and incubated in 0 . 2 mg/ml collagenase D ( Roche Diagnostic , Indianapolis , IN ) at 37\u00b0C for 35 min .", "Spleens and lymph nodes were homogenized using a cell strainer .", "In all tissues , red blood cells ( RBCs ) were lysed using ACK lysing buffer ( Quality Biologicals , Gaithersburg , MD ) , and lymphocytes were washed and counted .", "Serum was collected from na\u00efve and day 30+ X31-GP33 , or PR8-GP33 infected mice .", "Serum samples from individual mice were pooled and 1 ml of pooled serum/mouse was injected i . p . into mice on day -1 prior to PR8-GP33 rechallenge .", "In some instances to verify that the antibodies present in the serum were responsible for any protective effects , serum was depleted of IgG and IgA using Protein A and G SpinTrap ( GE Healthcare , Pittsburgh , PA ) according to manufacturer's instructions .", "Effector CD8 T cells were generated in vitro by peptide-stimulation of TCR-transgenic splenocytes ( obtained from a P14 transgenic mouse ) specific for the LCMV glycoprotein peptide ( P14 mice specific for GP33-41 ) .", "Briefly , spleen cells were incubated with 5 \u00b5M GP33 peptide for two hours .", "The peptide was washed off , media replaced and the cells were cultured for 48 hrs in 24-well plate , and maintained afterwards in 75T culture flasks in IL-2 - supplemented media for 5 days .", "The media was changed every 48 hours .", "A daily sample from the culture was examined by flow cytometry for the expression level of the activation markers , CD44 and CD25 .", "On day 5 , the cells were harvested , washed in PBS , counted and resuspended in PBS for adoptive transfer .", "Lymphocytes were stained using standard techniques and analyzed by flow cytometry .", "Virus-specific CD8 T cells were quantified using MHC class I peptide tetramer staining .", "MHC class I peptide tetramers were made and used as described [98] .", "Antibodies to CD8 and CD44 were purchased from eBioscience ( San Diego , CA ) .", "Staining and analysis were performed as previously described [92] .", "Function was investigated by intracellular cytokine staining following antigen stimulation ( IFN\u03b3 , TNF\u03b1 , IL-2 , CD40L ) .", "Briefly , 1\u00d7106 splenocytes were cultured in the absence or presence of the indicated peptide ( 0 . 2 mg/ml ) and brefeldin A for 5 h at 37\u00b0C .", "Influenza virus pooled peptides were used to evaluate the influenza virus-specific CD8+ T cell responses .", "This pool contains 147 overlapping peptides from influenza virus NP and M proteins , and we also included the GP33 peptide in this pool .", "For later experiments the overall influenza-specific CD8+ T cell response was evaluated via intracellular cytokine staining following stimulation with peptides from the influenza proteins HA , NA , NS1 , NS2 , PA , PB , NP , as well as the LCMV epitope GP33 .", "Following staining for surface antigens as described above , cells were stained for intracellular cytokines using the Cytofix/Cytoperm kit ( BD Biosciences ) .", "Samples were collected using an LSRII flow cytometer ( BD Biosciences ) .", "Results represent the mean \u00b1 SEM unless indicated otherwise .", "Statistical significance was determined by paired or unpaired Student's t test .", "Statistical analyses were performed using Prism GraphPad software v5 . 0 .", "( * , p<0 . 05; ** , p<0 . 01; *** , p<0 . 001 ) .", "Neuraminidase-956530; Interferon gamma-15978; Tumor necrosis factor-21926; Hemagglutinin- 956529; Nucleoprotein ( Influenza ) -956531; Nucleoprotein ( LCMV ) -956592; Non-structural protein 1\u2013956533; Non-structural protein 1\u2013956532; Polymerase acidic-956535; Fc receptor-109615; Glycoprotein ( LCMV ) -956590; Matrix protein 2\u2013956528; Interleukin 15\u201316168 .", "All accession ID numbers are recorded from the Entrez Gene database ."]], "headings": ["Introduction", "Results", "Discussion", "Materials and Methods"], "abstract": ["Seasonal epidemics of influenza virus result in \u223c36 , 000 deaths annually in the United States .", "Current vaccines against influenza virus elicit an antibody response specific for the envelope glycoproteins .", "However , high mutation rates result in the emergence of new viral serotypes , which elude neutralization by preexisting antibodies .", "T lymphocytes have been reported to be capable of mediating heterosubtypic protection through recognition of internal , more conserved , influenza virus proteins .", "Here , we demonstrate using a recombinant influenza virus expressing the LCMV GP33-41 epitope that influenza virus-specific CD8+ T cells and virus-specific non-neutralizing antibodies each are relatively ineffective at conferring heterosubtypic protective immunity alone .", "However , when combined virus-specific CD8 T cells and non-neutralizing antibodies cooperatively elicit robust protective immunity .", "This synergistic improvement in protective immunity is dependent , at least in part , on alveolar macrophages and/or other lung phagocytes .", "Overall , our studies suggest that an influenza vaccine capable of eliciting both CD8+ T cells and antibodies specific for highly conserved influenza proteins may be able to provide heterosubtypic protection in humans , and act as the basis for a potential \u201cuniversal\u201d vaccine ."], "summary": ["Influenza virus continues to pose a significant risk to global health and is responsible for thousands of deaths each year in the United States .", "This threat is largely due to the ability of the influenza virus to undergo rapid changes , allowing it to escape from immune responses elicited by previous infections or vaccinations .", "Certain internal determinants of the influenza virus are largely conserved across different viral strains and represent attractive targets for potential \u201cuniversal\u201d influenza vaccines .", "Here , we demonstrated that cross-subtype protection against the influenza virus could be obtained through simultaneous priming of multiple arms of the immune response against conserved elements of the influenza virus .", "These results suggest a novel strategy that could potentially form a primary component of a universal influenza vaccine capable of providing long-lasting protection ."], "keywords": ["medicine", "infectious diseases", "immunology", "biology"]}

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-{"id": "journal.pgen.0030189", "year": "2007", "title": "The cdx Genes and Retinoic Acid Control the Positioning and Segmentation of the Zebrafish Pronephros", "sections": [["The kidney eliminates metabolic waste in the body using highly specialized structures called nephrons .", "Individual nephrons are composed of a blood filter ( renal corpuscle ) , a tubule that recovers or secretes solutes , and a collecting duct [1] .", "The renal corpuscle contains epithelial cells called podocytes that form the slit-diaphragm filtration barrier and allow collection of substances from the blood [2] .", "In a number of vertebrate species , including some mammals , the renal corpuscle is connected to the tubule by a short stretch of ciliated epithelium called the neck segment that guides filtrate entry into the tubule [3\u20135] .", "The mammalian nephron tubule is subdivided into a series of proximal and distal segments connected to a collecting duct [1 , 6] .", "The polarized epithelial cells in the tubule segments have a unique ultrastructure and express a select cohort of solute transporters [1] .", "Thus , each segment is functionally distinct and performs the transport of particular solutes that are required for proper renal function .", "In higher vertebrates , three kidneys of increasing complexity arise sequentially from the intermediate mesoderm ( IM ) : the pronephros , the mesonephros , and the metanephros [7] .", "The pronephros and mesonephros degenerate in succession , with the metanephros serving as the adult kidney .", "Lower vertebrates , such as fish and amphibians , develop a pronephros during embryonic stages , and then form a mesonephros that will be used throughout their adult life [8\u201310] .", "Each of these kidneys contains the nephron as its basic functional unit [8] .", "To date , much of our knowledge of kidney development has come from gene-targeting studies in the mouse [7 , 11 , 12] .", "These experiments have identified a number of genes that play essential roles in the early stages of metanephros development , but there is a limited understanding of the molecular pathways governing the later stages of kidney ontogeny , when individual nephrons form and become segmented [7] .", "The zebrafish is an ideal genetic and developmental model system for dissecting the molecular mechanisms of nephron formation because of the anatomical simplicity of the pronephros , which contains two nephrons as opposed to the thousands of nephrons in a mammalian metanephros [9] .", "During zebrafish development , bilateral stripes of IM lying on either side of the trunk undergo a mesenchymal-to-epithelial transition to form the pair of pronephric nephrons .", "The anteriormost renal progenitors differentiate into podocytes , which migrate medially and fuse at the midline to form a single renal corpuscle .", "The nephrons also fuse posteriorly at the cloaca to form a shared exitway .", "From a functional standpoint , these pronephric nephrons have been thought to consist of three parts: ( 1 ) the blood-filtering renal corpuscle , ( 2 ) a very short tubule region that transports solutes , and ( 3 ) long pronephric ducts that convey the resulting waste to the cloaca [9] .", "Contrary to this model , recent studies have suggested that the \u2018duct' region possesses regional segmentation , based on the restricted expression boundaries of solute transporter orthologues known to be expressed in the tubule segments of metanephric nephrons .", "For example , a rostral stretch of the pronephric duct expresses the endocytic receptor megalin ( lrp2 ) [13] and the sodium bicarbonate transporter NBC1 ( slc4a4 ) [14] , which are expressed in the proximal tubule in mammals .", "These reports raise the possibility that portions of the pronephros considered to be duct might in fact be tubule , thus suggesting that the organization of the zebrafish pronephros is more complex than previously appreciated .", "However , a complete model of the molecular anatomy of the zebrafish pronephros and whether there is a functional correlation to the segments of the mammalian nephron remain unclear .", "Furthermore , the pathway ( s ) directing segmentation of the pronephros along the embryonic axis are unknown .", "Numerous factors are known to control segmental patterning along the anterior-posterior ( A-P ) axis during vertebrate development and thus provide candidate pathways that might act to establish pronephros segmentation .", "Retinoic acid ( RA ) signaling is vital for directing the A-P regionalization of tissues deriving from all three germ layers , such as the hindbrain , paraxial mesoderm , and gut [15\u201319] .", "Control of RA production via retinaldehyde dehydrogenase ( RALDH ) synthesizing enzymes [20] and the degradation of RA via the CYP26 catabolizing enzymes establishes both the location and timing of RA signaling [21 , 22] .", "In addition to RA , the caudal ( cdx ) transcription factors ( Cdx1 , Cdx2 , and Cdx4 in mammals and cdx1a and cdx4 in zebrafish ) are responsible for determining vertebral identity and directing posterior body formation [23\u201331] .", "cdx genes are known to act as master regulators of the homeobox ( hox ) transcription factors [25] , and in turn , overlapping domains of hox gene expression along the A-P axis are thought to confer segmental identities [32] .", "In mice , loss of Cdx function causes posterior shifts in Hox gene expression that are associated with abnormal vertebral patterning , and posterior truncations due to defects in the extension of the embryo axis [23 , 25\u201327 , 33] .", "Similarly , studies in zebrafish have shown that the loss of cdx4 function or deficiency of both cdx1a and cdx4 causes shifts in hox gene expression domains , a shortened body axis , and altered patterning of the blood , vascular , and neural tissues [24 , 28\u201331] .", "These lines of evidence indicate that the cdx genes play essential roles in controlling cell fates along the embryonic axis; however , the molecular mechanisms underlying these effects have not been elucidated [34] .", "In this study , we undertook a functional genomics approach to identify new markers of the zebrafish pronephros .", "From this analysis , we found that the pronephros is composed of at least eight regions , including two proximal and two distal tubule segments .", "We explored how segmental identity is controlled during nephrogenesis by testing the roles of RA signaling and the cdx genes .", "We found that RA is required to induce proximal segment fates and prevent the expansion of distal segment fates , whereas the cdx genes are necessary for positioning the pronephros along the embryonic axis .", "Embryos deficient in cdx1a and cdx4 displayed a posterior shift in the location of the pronephros and formed proximal but not distal nephron segments .", "The cdx genes were found to control the expression boundaries of raldh2 ( aldh1a2 ) and cyp26a1 , suggesting a model in which the cdx pathway influences where the pronephros forms along the body axis by localizing the source of RA , while subsequent RA signaling acts to direct the segmentation of the pronephros ."], ["To gain insight into the molecular mechanisms that control vertebrate renal development , we undertook a functional genomics approach to identify genes expressed in the kidney .", "We mined two gene collections , one comprising developmentally expressed genes from embryonic zebrafish cDNA libraries [35] and another compiled from an adult zebrafish kidney library [36] .", "Gene expression patterns were analyzed by whole-mount in situ hybridization using wild-type zebrafish embryos between the 5 somite stage and 144 hours post fertilization ( hpf ) .", "We identified a number of genes , including 15 solute transporters , that were expressed within specific subregions of the pronephros .", "In total , eight distinct regions could be visualized , with some genes expressed in more than one region ( Figure 1A ) .", "Representative examples of region-specific genes include wt1b , slc20a1a , trpm7 , slc12a1 , stc1 , slc12a3 , and gata3 , as compared to expression of cdh17 , which is found in all tubule and duct progenitors [37] ( Figure 1A ) .", "We investigated where the mouse or human orthologues of some of these genes are expressed in the mammalian metanephric kidney , and found that many corresponded to segment-specific domains within the nephron .", "For example , Slc9a3 is expressed in podocytes , the proximal convoluted segment ( PCT ) and proximal straight segment ( PST ) ( Figure 1B ) [38] .", "Slc20a1 is expressed throughout the entire nephron epithelium , although stronger expression was observed in proximal tubule segments ( Figure 1B ) .", "Transcripts for Slc13a3 are found in the PST [39] , while Slc12a1 is restricted to the thick ascending limb ( TAL ) and macula densa ( MD ) ( Figure 1B ) [40 , 41] .", "Slc12a3 is expressed in the distal convoluted tubule ( DCT ) ( Figure 1B ) [41] .", "Lastly , GATA-3 expression specifically marks the collecting ducts ( CD ) ( Figure 1B ) [42 , 43] .", "Based on this cross-species gene expression comparison , the following identities were assigned to the zebrafish pronephros segments we observed ( going from proximal to distal ) : podocytes ( pod ) , neck ( N ) , PCT , PST , distal early ( DE ) , corpuscle of Stannius ( CS ) , distal late ( DL ) , and the pronephric duct ( PD ) ( Figure 1A and 1E ) .", "Our division of PCT and PST within the tubule is based on the observation that the slc20a1a-expressing PCT cells undergo morphogenesis from a linear tube into a coiled structure by 5 days post-fertilization ( dpf ) , while the trpm7- and slc13a1-expressing PST segment maintains a linear structure ( Figure 1C ) .", "Expression of trpm7 and slc13a1 is discontinuous within the PST , an observation that has been shown recently to reflect the presence of two cell types in this region: transporting epithelia and multiciliated cells [44 , 45] .", "The renal corpuscle connects to the PCT via a short segment of cells that express the transcription factor rfx2 , and fails to express almost all of our PCT solute transporters ( Figure 1D ) .", "As rfx2 marks ciliated cells and rfx genes are essential regulators of ciliogenesis [46 , 47] , we hypothesize that this region corresponds to the ciliated neck segment found in other fish species as well as mammals [3\u20135] .", "However , a more detailed analysis is needed to confirm this hypothesis .", "In addition to the neck segment , rfx2 expression was also detected in presumptive ciliated cells along the length of the PST and DE segments , as described previously [44 , 45] .", "For the distal tubule , we adopted the DE/DL nomenclature used in Xenopus [48] , although the zebrafish DE appears analogous to the TAL segment in mammals and the DL appears analogous to the mammalian DCT segment according to our gene expression comparison .", "We included the CS as a discrete segment , as it initially arises from the tubular progenitors within the pronephros , but by 48 hpf , it is located just dorsal to the DE/DL boundary [49 , 50] ( unpublished data ) .", "The DL segment expresses slc12a3 and connects to the cloaca via a short segment that expresses gata3 and likely represents the PD .", "Our data are consistent with the notion that the zebrafish pronephric kidney resembles a \u2018stretched-out\u201d' mammalian nephron , and suggests that rather than being composed of mostly nephric duct ( as currently believed ) , it is made up of extensive proximal and distal tubule epithelium ( Figure 1E ) .", "Between 24 and 48 hpf ( the start of blood filtration ) , the pronephros undergoes significant morphogenesis , including the midline migration of podocytes and the growth/extension of the tubules [9] .", "In order to better quantitate these morphological changes , as well as to precisely define the anatomical boundaries of each segment , we mapped the expression domains of segment-specific markers relative to the somites by performing double whole-mount in situ hybridization with myosin heavy chain ( mhc ) at 24 and 48 hpf ( Figures 2 and S1\u2013S4 ) .", "At 24 hpf , podocyte and neck progenitors are arranged in a slight curve at the level of somite 3\u20134 with the anterior boundary of the PCT level with somite 5 ( Figures 2 and S1 ) .", "By 48 hpf , the podocyte progenitors have fused at the midline ( level with somite", "3 ) with the presumptive neck region forming a lateral extension that connects with the PCT also situated at the level of somite 3 ( Figures 2 and S3 ) .", "During this time , the length of the PCT , PST , and DE segments increased , possibly due to cell division within each segment ( Figure 2 and S1\u2013S4 ) .", "This growth may provide the driving force that is responsible for the shift in the anterior boundary of the PCT from somite 5 to somite 3 between 24 and 48 hpf , and for the coiling morphogenesis of the PCT observed between 72 and 144 hpf ( Figure 1C ) .", "However , the DL segment did not increase in length between 24 and 48 hpf , indicating that there is not a uniform expansion in all segments during development .", "During juvenile development ( 2\u20133 wk post-fertilization ) the DL segment is proportionately larger than the other segments , suggesting that its expansion predominates at later stages of development ( unpublished data ) .", "Interestingly , at 24 hpf , we observed an overlap of the DL and PD expression domains at the level of somite 17 ( Figure 2 and Figure S2 ) .", "This overlap may indicate the presence of an additional segment ( such as a discrete CNT equivalent ) , though to date we have not discovered any genes expressed solely in this domain .", "In addition to mapping the morphological changes that occur between 24 and 48 hpf , we also noted segment specific changes in gene expression patterns during this time .", "For example , transcripts for the solute transporters slc13a1 ( inorganic sulphate transporter ) , slc13a3 ( sodium-dicarboxylate carrier ) , and slc22a6 ( organic anion transporter ) were all absent from the PST segment at 24 hpf but were found expressed at 48 hpf ( Figures 2 and S3; and unpublished data ) .", "Similarly , transcripts for trpm7 ( divalent cation-selective ion channel ) and slc41a1 ( Mg 2+ transporter ) were not found in cells of the CS at 24 hpf but could be detected at 48 hpf ( Figure 2 and unpublished data ) .", "The up-regulation of these genes likely reflects the maturation/differentiation of the segment epithelia before the onset of blood filtration , which begins around 40 hpf [51 , 52] .", "However , the glomerulus does not fully mature until 4 dpf , based on the size exclusion of different-sized fluorescent dextrans [53] .", "Further profiling of segment expression patterns at later stages is needed to investigate whether the maturation of the transporting epithelial is also an ongoing process .", "We next sought to characterize the developmental pathways that establish the segmentation pattern of the pronephros .", "Retinoic acid ( RA ) is essential for the development of numerous tissues during embryogenesis [25] .", "In vertebrates , a gradient of RA in the upper trunk is responsible for directing the A-P patterning of the hindbrain into segmental compartments [15 , 54] , and gain or loss of RA also affects vertebral identity [25] .", "Interestingly , RA has been reported to regulate pronephros formation in Xenopus [55] .", "To explore whether RA was needed for pronephros segmentation , we injected wild-type zebrafish embryos with a morpholino to retinaldehyde dehydrogenase 2 ( raldh2 ) , which encodes an enzyme required to produce RA [20] .", "We then examined raldh2 morphants at 48 hpf with our panel of segment-specific markers , in combination with mhc expression to map segment length and location relative to the somites .", "Embryos deficient in raldh2 had fewer podocytes , as evidenced by punctate staining of the podocyte markers wt1b , wt1a , and mafb ( Figure 3A and unpublished data ) .", "Both proximal tubule segments were slightly shortened , based on the reduced expression domains of slc20a1a ( PCT ) and trpm7 ( PST ) ( Figure 3 ) .", "Conversely , the distal tubule was expanded in length , with the clck transporter ( marking both the DE and DL ) expressed in a greater proportion of the cdh17-positive pronephric tubule ( note that the overall length of raldh2 morphants is reduced compared to wild-type ) ( Figure 3 ) .", "Each segment within the distal tubule was moderately expanded , with a lengthened DE shown by expanded slc12a1 expression , enlarged clusters of stc1-expressing cells that comprise the CS , and lengthened DL shown by slc12a3 expression ( Figure 3 ) .", "The expression domain of gata3 ( PD ) was also expanded ( Figure 3 ) .", "In contrast , the cloaca marker aqp3 was unchanged ( Figure 3A ) .", "These data suggest that RA signaling is necessary for podocyte formation and/or survival , as well as for establishing the normal pattern of nephron segmentation .", "Multiple enzymes are capable of synthesizing RA and a recent analysis of the neckless ( nls ) ( now called aldh1a2 ) mutant , which is defective in raldh2 , demonstrated that there are additional sources of raldh-like enzyme activity in the zebrafish embryo [56\u201358] .", "Based on this , we hypothesized that the nephron phenotype in raldh2 morphants represented the effect of reducing RA production , rather than a complete inhibition .", "To more fully block RA signaling , we utilized a competitive , reversible inhibitor of raldh enzymes , 4-diethylaminobenzaldehyde ( DEAB ) [59] , which has been used to effectively prevent de novo RA synthesis in zebrafish embryos [58 , 60] .", "Wild-type embryos were treated with DEAB starting at 60% epiboly ( early gastrula ) until the 15 somite stage , and nephron segmentation was assayed at 48 hpf by double whole-mount in situ hybridization using mhc expression to mark the somites .", "Expression of the podocyte markers wt1b , wt1a , and mafb was absent in DEAB-treated embryos , suggesting that podocyte development was completely blocked ( Figure 3 and unpublished data ) .", "Expression of the PCT and PST markers slc20a1a and trpm7 was also absent , suggesting that these segments had failed to be specified ( Figure 3 ) .", "In contrast , clck expression ( marking the DE and DL segments ) was dramatically expanded , such that it was present throughout the entire tubule territory ( Figure 3 ) .", "These findings suggest that the pronephric tubule adopts a distal tubule identity when RA synthesis is inhibited .", "Within this \u2018distal-only' pronephros , the DE marker slc12a1 was expressed from the anterior limit of the tubule to almost the middle of its length , the DL marker slc12a3 was expressed from the middle of the tubule to near its posterior limit , and the PD marker gata3 was expanded by an additional four somite lengths ( Figure 3 ) .", "A marked expansion of stc1-expressing cells was also detected , with multiple clusters of cells arranged in bilateral stripes , as opposed to the small groups of stc1-expressing cells seen in wild-type embryos ( Figure 3 ) .", "Expression of aqp3 was not altered , suggesting that progenitors of the cloaca are unaffected by the inhibition of RA production over this developmental interval ( Figure 3 ) .", "It is not known if the observed DEAB phenotype represents a full loss of RA signaling , as trace amounts of maternal RA have been detected in the yolk [61] and as zygotic raldh2 transcripts are expressed prior to 60% epiboly [56 , 57] .", "Nevertheless , our results demonstrate that RA , produced by raldh2 and possibly one or more unknown raldh enzymes , plays an essential role in the formation of proximal nephron fates ( podocytes , PCT , PST ) and in suppressing the expansion of distal fates ( DE , CS , DL , PD ) .", "To determine more precisely when RA signaling was needed for the development of proximal segment fates , we performed a DEAB timecourse experiment ( Figure 3 ) .", "Blocking RA signaling from 90% epiboly ( late gastrula ) to the 5 somite stage caused a milder phenotype than the longer 60%- 15-somite exposure that was characterized by a loss of podocytes , reduced lengths of the PCT and PST segments , and small increases in the lengths of the distal segments ( Figure 3 ) .", "A slightly longer DEAB treatment , from 90% epiboly to the 10 somite stage , expanded distal segments further than the raldh2 morphants or DEAB 90% epiboly-5 somite treated embryos ( Figure 3 ) .", "In addition , examination of the slc20a1a and trpm7 expression patterns showed that the PST segment was ablated , while the PCT was only slightly shortened ( Figure 3 ) .", "Loss of PST identity was also confirmed by the absence of slc13a1 and slc22a6 transcripts , which are additional PST markers ( Figure 2 and unpublished data ) .", "These findings suggest that , at least for this DEAB time window , the expansion of distal fates occurs at the expense of the PST segment .", "Finally , we tested whether DEAB treatment during somitogenesis would affect pronephros segmentation .", "DEAB treatment from 5\u201315 somites or 10\u201315 somites had no effect on the segmentation pattern of the pronephros ( unpublished data ) , thus suggesting that an inhibition of RA signaling must be initiated prior to the 5 somite stage in order to affect nephron patterning .", "Taken together , our DEAB timecourse data indicate that RA signaling is required to induce proximal nephron fates and to prevent an expansion of distal fates , thereby establishing the normal pronephric segmentation pattern .", "Interestingly , our data suggest that different segments have different temporal requirements for RA signaling .", "RA is essential between 90% epiboly and the 5 somite stage to induce podocytes , between 90% epiboly and 10 somites to induce PST formation , and between 60% epiboly and 15 somites to form the PCT .", "The PCT segment is the most refractory to RA inhibition and is only lost following the longest DEAB treatment window ( 60% epiboly to 15 somites ) .", "Given these findings , we tested whether increasing the concentration of RA by exogenous treatment would promote proximal nephron fates at the expense of distal fates .", "We exposed wild-type zebrafish embryos to RA during similar developmental intervals used for our DEAB experiments , and then assayed segment marker expression at 24 hpf by double in situ hybridization with mhc to mark the somites .", "Wild-type embryos treated with 1 \u00d7 10\u22127 M RA between 90% epiboly and 5 somites developed a normal number of podocytes , as evidenced by wt1b expression , but displayed expanded proximal tubule domains , shown by expression of slc9a3 ( marking both the PCT and PST ) , slc20a1a ( PCT ) , and trpm7 ( PST ) ( Figure 4 ) .", "Conversely , the clck-expressing distal tubule domain was reduced , due to reductions in the length of the DL ( marked by slc12a3 ) and PD ( marked by gata3 ) ( Figure 4 ) .", "However , the DE segment ( marked by slc12a1 ) was unaffected ( Figure 4 ) .", "The position of the stc1-expressing CS segment cells was shifted posteriorly , and the level of expression was slightly reduced ( Figure 4 ) .", "A longer treatment window , from 60% epiboly\u201315 somites resulted in a more severe \u2018proximalized' phenotype with a longer expanse of proximal tubule and a greater reduction in each distal tubule domain ( Figure 4 ) .", "In these embryos , the position of the stc1-expressing CS population was shifted even more posteriorly , and was located at the distal edge of the yolk sac extension ( Figure 4A ) .", "We next treated wild-type embryos with higher dose of RA ( 1 \u00d7 10\u22126 M ) over this same 60% epiboly\u201315 somites time window , as well as two shorter time periods: 60% epiboly\u20135 somites , and 90% epiboly\u20135 somites .", "Embryos treated for any of these time windows displayed a completely \u2018proximalized' phenotype with the tubule domain being comprised entirely of proximal segment identities ( Figure 4 and unpublished data ) .", "In these embryos , the proximal marker slc9a3 was expressed throughout the cdh17-expressing tubule population ( Figure 4 and unpublished data ) .", "Within this \u2018proximal-only' pronephros , the PCT marker slc20a1a was expressed from the anterior limit of the tubule to somite 13 , and the PST marker trpm7 was expressed from somite 14 to the posterior limit of the tubule , where the trpm7-expressing tubules fused at the prospective site of the cloaca ( Figure 4 and unpublished data ) .", "Expression of all distal segment markers was absent , suggesting that the DE , CS , DL , and PD had failed to be specified ( Figure 4 and unpublished data ) .", "These results show that exogenous RA treatment from gastrulation stages until the 5 somite stage is sufficient to \u2018proximalize' the pronephros , suggesting that this time period is the critical window when RA signaling is required for proximo-distal patterning of pronephric progenitors .", "To further explore the notion that RA alters the patterning of renal progenitors prior to the 5 somite stage , we examined the expression patterns of the Notch ligands deltaC ( dlc ) and jagged2a ( jag2a ) , and the renal transcription factors wt1a , pax2a , pax8 , and evi1 , as these genes are detected in the IM during early somitogenesis and have been implicated in early nephron patterning [44 , 62\u201364] .", "Wild-type embryos were treated with DMSO , DEAB , or 1 \u00d7 10\u22126 M RA from 60% epiboly until the 6 somite stage , and then IM gene expression was assayed by whole-mount double in situ hybridization together with myoD to mark the somites .", "Transcripts for pax2a and pax8 , which label all pronephric progenitors , were found throughout the IM domain in a similar pattern in wild-type , DEAB-treated , and RA-treated embryos ( Figure S5 ) .", "Consistent with our previous results , wt1a expression was absent in DEAB-treated embryos , and wt1a was strongly up-regulated in RA-treated embryos ( Figure S5 ) .", "The expression domains of deltaC and jag2a , normally restricted to a proximal region of the IM adjacent to somites 2\u20135 , were absent in DEAB-treated embryos , and expanded posteriorly in RA-treated embryos ( Figure S5 ) .", "Expression of evi1 , found in the distal portion of the IM starting around somite 6 , was shifted anteriorly to somite 3 following DEAB treatment and reduced to the most posterior group of IM cells following RA treatment ( Figure S5 ) .", "These findings demonstrate that changes in RA dosage during gastrulation to early somitogenesis are associated with gene expression changes in IM progenitors , far in advance of their mesenchymal-to-epithelial transition that creates the pronephric tubules .", "In addition to RA , multiple tissues along the embryonic axis are segmentally patterned by the cdx genes [25 , 34] .", "Loss of cdx gene function leads to an expansion of anterior trunk fates and axial elongation defects that result in a loss/truncation of the posterior trunk and tail [23\u201330 , 34] .", "In zebrafish , cdx4\u2013/\u2013 mutant embryos display expanded wt1a expression at the 15-somite stage , suggesting that the podocyte lineage might be expanded and thus implicating cdx genes in pronephros patterning [24] .", "We therefore examined the formation of the pronephros in cdx4\u2013/\u2013 and cdx1a/4-deficient ( herein referred to as cdx-deficient ) to assess development of the renal corpuscle , tubule , and pronephric duct .", "In addition , we used differential interference contrast ( DIC ) optics to visualize the somite boundaries and determine the size and position of each segment relative to the somites .", "It is important to note that the size of posterior somites in cdx4\u2013/\u2013 embryos , and both the size and number of the somites in cdx-deficient embryos , is greatly reduced toward the posterior , due to the axial elongation defect [24 , 29\u201330] .", "A similar defect has also been observed in mouse Cdx mutants [34] .", "At 24 hpf , wild-type embryos expressed wt1a in presumptive podocytes ( located adjacent to somite 3 , marked by an arrow in Figure 5A ) as well as a population of presumptive mesenchymal cells located next to somites 1\u20133 in a broad lateral domain ( indicated by a bracket and \u2018M' in Figure 5A ) .", "cdx4\u2013/\u2013 embryos had expanded wt1a expression at 24 hpf that ranged from a position anterior to somite 1 to approximately somite 7 , and cdx-deficient embryos showed an even more dramatic posterior expansion that reached somite 12 ( Figure 5A ) .", "As the expansion of wt1a in cdx mutant embryos could indicate increased numbers of podocytes and/or the mesenchymal population , we examined wt1b expression , which specifically marks podocytes [65] .", "Equivalent numbers of wt1b-expressing podocytes were formed in cdx4\u2013/\u2013 mutant and wild-type embryos , but podocytes in cdx4\u2013/\u2013 embryos were located more posteriorly , at the level of somite 5 ( Figure 5A ) .", "cdx-deficient embryos developed a normal number of wt1b-expressing podocytes , though they were arranged in a pair of somewhat irregular linear groupings ( rather than forming bilateral spherical clusters ) , and they were also located more posteriorly , adjacent to somite 7 ( Figure 5A ) .", "We conclude from these observations that the loss of cdx4 and cdx1a/4 function progressively expands wt1a expression without increasing the number of podocytes and leads to podocyte formation at more posterior locations along the embryonic axis .", "To assess tubule formation in cdx mutants , we examined the expression of cdh17 at 24 hpf .", "While cdx4\u2013/\u2013 embryos formed complete tubules that fused at the cloaca , cdx-deficient embryos displayed cdh17 expression that was reduced and discontinuous , with the tubules failing to fuse ( Figure 5A ) .", "Consistent with the posterior shifts in podocyte position , the A-P position of the tubule , marked by cdh17 transcripts , was shifted caudally in both cdx4\u2013/\u2013 and cdx-deficient embryos .", "In wild-type embryos , the tubule spans the length of somites 4\u201318 , but was located from somites 6\u201320 in cdx4\u2013/\u2013 embryos , and from somites 8\u201313 in cdx-deficient embryos ( Figure 5A ) .", "We next characterized the tubule segmentation pattern in cdx mutant embryos by examining the expression patterns of slc20a1a ( PCT ) , trpm7 ( PST ) , slc12a1 ( DE ) , stc1 ( CS ) , slc12a3 ( DL ) , gata3 ( PD ) , and aqp3 ( cloaca ) .", "cdx4\u2013/\u2013 embryos showed a slight expansion of the PCT , which spanned an additional two somites compared to wild-types ( Figure 5A and unpublished data ) .", "The PST , DE , CS , and PD were all shorter than normal in cdx4\u2013/\u2013 embryos , with the PD displaying the most severe reduction in length ( Figure 5A and unpublished data ) .", "Transcripts for aqp3 were not detected in cdx4\u2013/\u2013 embryos , suggesting a defect in cloaca development ( Figure 5A ) .", "These results indicate that loss of cdx4 leads to a slight expansion in PCT fate with corresponding reductions in more distal fates .", "In contrast to cdx4\u2013/\u2013 embryos , cdx-deficient embryos showed discontinuous slc20a1a expression and failed to express trpm7 , slc12a1 , stc1 , slc12a3 , gata3 , or aqp3 ( Figure 5A ) .", "These results suggest that the tubule territory in cdx-deficient embryos acquires a PCT identity , while the remaining nephron segments fail to develop .", "These defects were not the result of delayed development , as at later developmental stages , cdx-deficient embryos continued to possess tubules that only expressed PCT-markers , and the tubules never fused caudally ( unpublished data ) .", "In addition , expression analysis of the podocyte marker mafB in cdx-deficient embryos at 48 hpf revealed that podocytes fail to fuse into a single renal corpuscle .", "Instead , the podocytes formed bilateral corpuscles that were dilated compared to wild-type and cdx4\u2013/\u2013 embryos , presumably due to fluid accumulation ( Figure 5B ) .", "Consistent with this , cdx-deficient embryos had developed glomerular cysts by 72 hpf , as well as severe pericardial edema , indicative of renal failure ( Figure 5C ) .", "In contrast , cdx4\u2013/\u2013 embryos never exhibited glomerular cyst formation or edema , suggesting that although various segment lengths were shortened , these embryos were able to maintain adequate kidney function ( Figure 5C ) .", "In summary , our expression analyses show that cdx deficiency causes a posterior shift in the location of the pronephros along the embryonic axis ( Figure 5D ) .", "While the podocytes and PCT populations formed relatively normally , the PST and distal tubule segments were reduced or absent in cdx4\u2013/\u2013 and cdx-deficient embryos , respectively ( Figure 5D ) .", "Thus the cdx genes , acting either directly or indirectly , are required for the formation of the distal nephron segments and establishing the normal segmentation pattern of the pronephros .", "Given the largely opposite effects of cdx-deficiency and loss of RA signaling on nephron patterning , as well as the recent report that cdx genes control how the hindbrain responds to RA during its patterning [30 , 31] , we wondered if an interplay between these pathways was operative during pronephros segmentation .", "The location and level of RA within tissues is dependent on the expression of raldh-synthesizing enzymes and cyp26-degrading enzymes [15 , 54] .", "We therefore investigated the expression of these genes in cdx-deficient embryos during early somitogenesis , as our previous experiments suggested that the IM is being influenced by RA signaling at this time .", "At the 5 somite stage , expression of raldh2 in the paraxial mesoderm was expanded posteriorly in cdx4\u2013/\u2013 embryos compared to wild-types ( Figure 6A ) .", "An even greater posterior expansion was seen in cdx-deficient embryos , with raldh2 transcripts being detected throughout the entire unsegmented paraxial mesoderm and tailbud region ( Figure 6A ) .", "Expression of the RA-catabolizing enzyme cyp26a1 in the upper trunk region was also expanded posteriorly in cdx4\u2013/\u2013 embryos at the 5 somite stage , and more extensively expanded in cdx-deficient embryos ( Figure 6A ) .", "To visualize how the combined changes in raldh2 and cyp26a1 expression altered the source of RA along the trunk , we generated digital overlays of these expression patterns .", "This analysis suggested that the anterior boundary of RA production ( i . e . , the junction of the cyp26a1 and raldh2 expression domains ) was located more posteriorly in cdx4\u2013/\u2013 mutants compared to wild-types , and that this posterior shift was more pronounced in cdx-deficient embryos ( arrows in Figure 6A ) .", "To better quantitate these posterior shifts , we examined expression of raldh2 and cyp26a1 at the 10 somite stage when the somites could be visualized by staining for myoD transcripts .", "We found that the raldh2 expression boundary in cdx4\u2013/\u2013 and cdx-deficient embryos was shifted posteriorly by 1 and 2 somites , respectively , compared to wild-type embryos ( Figure 6B ) .", "The cyp26a1 domain in wild-type embryos occupied the region of somites 2\u20133 , just rostral and slightly overlapping with raldh2 , as shown by double in situ hybridization ( Figure 6B ) .", "In cdx4\u2013/\u2013 embryos , cyp26a1 transcripts were detected in the region of somites 3\u20135 , whereas in cdx-deficient embryos they extended from somites 3\u20137 ( Figure 6B ) .", "These analyses reveal a striking correlation between the presumptive source of RA at the 10 somite stage and the axial position of the pronephros at 24 hpf in each genotype ( i . e . , the source of RA and the position of the pronephros both start at somites 3 , 5 , and 7 in wild-type , cdx4\u2013/\u2013 , and cdx-deficient embryos , respectively ) .", "The combination of these data , together with the results from our DEAB experiments , suggest a model in which the cdx genes act upstream of raldh2 and cyp26a1 to localize the source of RA along the A-P axis , and that RA , in turn , acts on the IM to induce the proximal segments and prevent an expansion of the distal segment fates .", "If our model is correct , then inhibiting RA synthesis in cdx-deficient embryos should rescue pronephric positioning and the formation of the distal tubule segments .", "To test this , we treated cdx4\u2013/\u2013 and cdx-deficient embryos with DEAB from 90% epiboly to the 5-somite stage and examined pronephros segmentation .", "In support of our model , we found that cdx4\u2013/\u2013 and cdx-deficient embryos exhibited a one-somite anterior shift in the position of the pronephros , as shown by expression of cdh17 ( Figure 7 ) .", "In addition , the development of podocytes was abrogated , and the length of the PCT was reduced in DEAB-treated cdx4\u2013/\u2013 and cdx-deficient embryos ( Figure 7 ) , consistent with our findings in wild-type embryos ( Figure 3 ) .", "We observed that the DE segment was increased in DEAB-treated cdx4\u2013/\u2013 embryos , as shown by an expansion of the slc12a1 expression domain ( Figure 7 ) .", "DEAB treatment also increased the number of CS cells in cdx4\u2013/\u2013 mutants , shown by the expression of stc1 , and increased the DL segment length , evidenced by expansion of the slc12a3 , romk2 , and clck expression domains ( Figure 7 and unpublished data ) .", "In cdx-deficient embryos treated with DEAB , formation of the DE , CS , and DL segments was rescued , shown by expression of slc12a1 , stc1 , and slc12a3 , respectively ( Figure 7 ) .", "A similar rescue of the expression of the DE and DL markers romk2 and clck was also observed ( unpublished data ) .", "These findings demonstrate that cdx gene function is not necessary to specify distal segment identity directly , but instead suggests that the abrogation of distal segment formation in cdx-deficient mutants is related to the level of RA that the renal progenitors are exposed to .", "Taken together with the above results , these finding provide good evidence that the pronephric positioning defect and failure to form the distal tubule identities in cdx-deficient embryos is caused by mis-localization of the RA source along the A-P axis ."], ["Retinoid signaling plays essential roles in the A-P patterning of a number of diverse tissues in the embryo .", "During early development , a source of RA in the upper trunk ( cervical ) region is produced by the action of the RA synthetic enzyme , Raldh2 , which is expressed in the anterior paraxial mesoderm [54] .", "The coordinate expression of RA-catabolizing Cyp26 enzymes in surrounding tissues creates a so-called \u2018sink' for this RA source [54] .", "Collectively , these enzymes are thought to create a gradient of RA activity that diffuses into surrounding tissues [34 , 54] .", "The functions of this RA source have been extensively studied in the developing hindbrain , where the effects of graded RA signaling are thought to create nested expression domains of RA-responsive genes that drive A-P segmentation of the hindbrain into a series of rhombomeres [15 , 54] .", "In addition to regionalizing the overlying neurectoderm , RA produced in the upper trunk paraxial mesoderm has been implicated in the regionalization of the underlying endoderm .", "Studies in zebrafish have shown that RA acts directly on the endoderm to specify hepatopancreatic progenitors that give rise to the liver and pancreas [17 , 18] .", "RA also influences mesodermal cell fate decisions during zebrafish development , including the formation of the pectoral fin field\u2014which arises from the lateral plate mesoderm adjacent to the upper trunk somites [56 , 57 , 66]\u2014and the heart [67] .", "In the latter case , inhibition of RA synthesis leads to an expansion of precardiac mesoderm , resulting in an excessive number of myocardial progenitors [67] .", "These findings indicate that , in addition to acting as an inducer of cell fates such as in the hindbrain and endoderm , RA also plays an important role in restricting certain cell fates .", "Our study now adds the IM as another mesodermal derivative that is patterned by RA .", "Our results show that RA production is essential during gastrulation and early somitogenesis for the induction of proximal nephron fates as well as to restrict the expansion of distal nephron fates .", "Over this period of development , RA is produced by the anterior paraxial mesoderm ( PM ) .", "The IM , which gives rise to the pronephros , is located lateral to the PM ( Figure 8 ) .", "Given the role of RA as a diffusible morphogen in other tissues , we hypothesize that RA diffuses from the PM and establishes a gradient along the IM , with high levels of RA inducing proximal fates and low RA levels being permissive for distal fates ( Figure 8 ) .", "Our time-course experiments with DEAB support this view , with the most severe reduction in proximal fates ( and concomitant expansion in distal segments ) corresponding to the longest treatment window .", "However , further work is needed to determine the nature of the RA gradient , as well as how dynamic fluctuations in retinoid availability [54] affect the dose and length of time that the renal progenitors are exposed to RA .", "A gradient-free model has recently been proposed for RA-dependent hindbrain patterning , based on the finding that sequential expression domains of the cyp26a1 , cyp26b1 , and cyp26c1 genes are essential for rhombomere boundary establishment [68 , 69] .", "It is unclear if a similar mechanism might operate during pronephros segmentation , as cyp26b1 and cyp26c1 do not show a nested pattern of expression in the IM .", "Overall , the effects of RA on the patterning of the IM can be regarded as \u2018anteriorizing .", "' Our finding that exogenous RA treatment induces proximal tubule fates to form throughout the pronephros supports this conclusion .", "Classically , RA is known as a \u2018posteriorizing' factor due to its effects on the central nervous system , where an inhibition of RA signaling causes an expansion of anterior neural fates in the hindbrain [15 , 54] .", "Thus we conclude that RA can actually have both anteriorizing and posteriorizing activities , depending on the tissue in question .", "A unified way to characterize these effects would be to consider the upper trunk RA source as an organizing center , akin to the dorsal organizer in the gastrula , that locally patterns cell types in all three germ layers .", "Previous studies have implicated RA as a regulator of renal development .", "Animal cap experiments in Xenopus showed that RA , together with Activin , is sufficient to induce the formation of pronephric tubules [70] .", "A more recent study in Xenopus reported that overexpressing various RA antagonists results in a complete loss of the pronephros ( glomus , tubules , and duct ) [55] .", "This phenotype is more severe than what we observed and may reflect differences in the efficacy of DEAB to completely block RA production compared with other RA antagonists .", "The pronephric tubules in Xenopus are segmented into proximal and distal segments [48] , similar to the zebrafish pronephros , however a role for RA in Xenopus nephron segmentation has not been reported .", "In mammals , it has long been known that vitamin A deficiency causes severe renal malformations [71] .", "Targeted mutagenesis of the RAR genes in mouse , followed by elegant rescue experiments , established an important role for RA as a dose-dependent inducer of the GDNF receptor Ret [72 , 73] .", "GDNF is an essential regulator of ureteric bud branching morphogenesis , and loss of GDNF signaling results most frequently in renal agenesis [74] .", "Because ureteric bud branching is an essential prerequisite for nephrogenesis , RA serves a key role in stimulating nephron formation .", "At present it is not known whether RA is also involved in the proximodistal patterning of metanephric nephrons .", "Interestingly , Raldh2 transcripts are found in podocyte progenitors , whereas Cyp26a1 is expressed by the tubule anlagen during metanephros development , suggestive of a role for RA in mammalian nephron patterning [75] .", "Transplantation studies in frogs suggest that RA may act directly on pronephric precursors [55] .", "However , the downstream targets of RA in the IM are not known .", "In the hindbrain , the presumptive RA gradient is thought to regulate rhombomere segmentation by activating the expression of the anterior , 3' Hox genes , i . e . , those comprising the 1st through 5th paralog groups [15 , 54 , 76] .", "In zebrafish , transcripts for hoxb1a , hoxb1b , and hoxb5a are found in proximal portions of the IM , thus making them potential candidates for mediating the effects of RA during pronephros segmentation [34] .", "Future studies using single , double , and triple morpholino injections can test the importance of these hox genes for renal development .", "The effects of RA on pronephric segmentation may also be coordinated by the action of non-Hox pathways .", "Our results suggest the intriguing possibility that RA signaling targets may include renal transcription factors as well as members of the Notch signaling pathway .", "The gene evi1 encodes a zinc-finger transcription factor that has been implicated in patterning distal regions of the pronephros in Xenopus , and overexpression of evi1 was found to inhibit proximal segment formation [63] .", "These data are consistent with our results showing that expression of evi1 in the IM was expanded following DEAB treatment , and reduced following exposure to exogenous RA .", "Another renal transcription factor candidate is the odd-skipped related transcription factor 1 ( osr1 ) encoding a zinc-finger repressor .", "Recent studies in Xenopus and zebrafish have shown that osr1 is expressed in the ventral mesoderm during gastrulation and later in an anterior domain of the IM [77] .", "Morpholino-mediated knock-down of osr1 leads to defects in the formation of podocytes and proximal tubule progenitors [77] , consistent with Osr1 participating in a common pathway with RA .", "The Notch pathway may also interact with RA during nephron segmentation .", "Conditional knockout of Notch2 in the mouse metanephros results in a loss of podocytes and proximal tubule fates , whereas distal markers are relatively unaffected [78] .", "In zebrafish , Notch signaling has been shown to regulate the differentiation of multiciliated cells and principle cells in the pronephric tubules [44 , 45] .", "However , a role for Notch signaling in the formation of proximal nephron fates is also suggested by the expression pattern of the Notch ligands deltaC , jag1b , and jag2a , which are restricted to proximal portions of the intermediate mesoderm [62 , 64] .", "Simultaneous knockdown of jag1b/2a results in an abnormally small renal corpuscle and dysmorphic proximal tubules , consistent with a conserved role for Notch signaling in proximal nephron development [79] .", "Our finding that DEAB treatment abrogates deltaC and jag2a expression in the proximal IM , while exogenous RA expands their expression , supports a role for RA acting upstream of the Notch pathway .", "Our study provides evidence that cdx genes control the expression domains of raldh2 and cyp26a1 along the embryonic axis .", "The boundaries of both raldh2 and cyp26a1 are progressively shifted toward the posterior in cdx4 and cdx1a/4-deficient embryos , suggesting that the upper trunk source of RA is posteriorly shifted .", "We hypothesize that this posterior shift in RA production results in a posterior shift in the position of the pronephros ( Figure 8 ) .", "We propose that this effect , combined with the axial elongation defects , leads to reduced or absent distal segment fates .", "The ability to rescue distal segments by treating cdx mutants with a pulse of DEAB is consistent with this model , and also demonstrates that cdx function is not requisite for the induction of distal fates from the intermediate mesoderm .", "Thus additional , as yet unidentified pathways , are responsible for directing distal fates .", "While our data supports the notion that Cdx factors exert their effects by the regulation of RA signaling , it does not rule out the possibility that Cdx factors may also function to repress proximal fates independent of RA signaling .", "Given the mounting evidence that the upper trunk RA source is an important organizing center , we would predict that both the patterning and positioning of numerous organs would be affected in cdx mutants .", "Consistent with this , defects in several mesodermal fates that arise in the anterior trunk region have been observed in cdx-deficient embryos .", "Vascular precursors are progressively expanded when cdx activity is abrogated , and blood precursors are both reduced and shifted posteriorly in cdx mutants [24 , 29] .", "In addition to mesodermal defects , cdx mutants also display patterning defects in the neurectoderm that gives rise to the anterior spinal cord [30 , 31] .", "We hypothesize that many , if not all , of these defects in cdx mutants are caused by the abnormal localization of RA along the A-P axis .", "The loss of cdx gene function in both zebrafish and murine models has been shown to cause global shifts in hox gene expression in the mesoderm and neurectoderm [24 , 25 , 29\u201330 , 34] .", "Given the rostral shifts and expansions of both raldh2 and cyp26a1 expression observed in cdx4 and cdx1a/4-deficient embryos , Hox transcription factors are attractive molecules for regulating raldh2 and cyp26a1 expression .", "Defects in blood formation in cdx4-null zebrafish can be rescued by the overexpression of several hox genes [24] , and the overexpression of hoxa9a also results in a partial rescue of the axis elongation defect in cdx4\u2013/\u2013 embryos [29] .", "Future studies are needed to examine whether hox gene overexpression ( s ) can rescue pronephros positioning and formation of distal segments in cdx mutant embryos .", "In conclusion , our studies have revealed an important link between the cdx genes and localization of RA , and provide evidence that RA signaling is a central determinant of pronephros A-P segmentation .", "Our results establish the zebrafish embryo as a simplified model of vertebrate nephron segmentation that will further our understanding of mammalian nephron segmentation , and provide insights into the causes of kidney birth defects and renal disease in humans ."], ["Zebrafish were maintained and staged as described [80 , 81] .", "T\u00fcbingen strain wild-type embryos were used for all experiments .", "DEAB and all-trans retinoic acid ( Sigma-Aldrich ) were dissolved in 100% dimethyl sulfoxide ( DMSO ) to make a 1 M stock and aliquots were stored at \u221280\u00b0C .", "For DEAB and RA treatments: embryos were incubated in 1 . 6 \u00d7 10\u22125 M DEAB/DMSO in E3 embryo media , 1 \u00d7 10\u22126 M or 1 \u00d7 10\u22127 M RA/DMSO in E3 embryo media , or 1 . 6 \u00d7 10\u22125 M DMSO ( control ) in E3 in the dark over particular developmental intervals , then washed five times with E3 and then fixed at 24 or 48 hpf .", "These experimental treatments were fully penetrant and produced consistent results at the doses and treatment windows that were examined .", "raldh2 morpholino ( CAACTTCACTGGAGGTCATCGCGTC ) was injected into 1-cell wild-type embryos .", "Incrosses of kggtv205 heterozygous adults ( maintained on the T\u00fcbingen strain ) were used to obtain cdx4\u2013/\u2013 embryos and were injected at the 1-cell stage with cdx1a morpholino ( CAGCAGATAGCTCACGGACATTTTC ) as described [29] to obtain cdx-deficient embryos .", "Both raldh2 and cdx1a morpholinos produced fully penetrant effects .", "Embryos were raised to appropriate stages and fixed in 4% paraformaldehyde ( PFA ) /1\u00d7PBST for gene expression analysis .", "For all reported gene expressions , at least 20 embryos were examined .", "Whole-mount in situ hybridization of zebrafish embryos was performed as previously described [24] .", "The expression patterns of cdh17 , clck , cyp26a1 , evi1 , gata3 , mhc , myoD , nbc1 , pax2a , pdzk1 , raldh2 , ret1 , sall1 , sglt1 , slc4a2 , slc20a1a , wt1a , and wt1b were previously reported [14 , 24 , 29 , 37 , 55 , 56 , 61 , 82\u201386] .", "For antisense probe production , we used the following IMAGE clone template plasmids , restriction enzymes for DNA linearization , and RNA enzymes: mafb: 7995399 , pExpress-1 , EcoR1 , T7; rfx2 , pBK-CMV , template was PCR amplified using primers GTGAATTGTAATACGACTCACTATAGGG and TTAACCCTCACTAAAGGGAACAAA , T7; slc9a3: 6996791 , pExpress-1 , EcoRI , T7; slc26a2: 4760214 , pBK-CMV , EcoRI , T7; slc13a1: 6793065 , EcoRV , t7; slc13a3: 4744276 , pCMV-sport6 . 1ccdb , EcoRI , T7; slc22a6: 4744276 , pBK-CMV , SalI , T7; slc12a1: pBK-CMV , EcoRI , T7; slc12a3: 7037010 , pExpress-1 , EcoRI , T7; stc1 was amplified from 24 hpf embryo cDNA using primers ATGCTCCTGAAAAGCGGATTT and TTAAGGACTTCCCACGATGGA and cloned into pGemTEasy , NcoI , Sp6 .", "Gene-specific primers spanning 700\u20131 , 000 bp of the coding sequence were used to amplify DNA fragments from E15 . 5/P0 kidney cDNA pools , and the PCR products of the right size were cloned into the pCRII-Topo vector ( primer sequences available upon request ) .", "DNA templates for riboprobe production were generated by PCR with T7 and Sp6 Ready Made primers ( Integrated DNA Technologies ) from PCRII-TOPO clones or T7 and T3 Ready Made primers from Bmap library clones .", "Digoxigenin-labeled anti-sense riboprobes were synthesized from the PCR product and purified with Micro Bio-spin columns P-30 Tris RNase-free ( Bio-Rad ) .", "Probes were diluted with prehybridization buffer ( 50% formamide , 5\u00d7SSC , pH4 . 5 , 50 \u03bcg/ml yeast tRNA , 1% SDS , 50 \u03bcg/ml heparin ) to 10 \u03bcg/ml and stored at \u221280 \u00b0C .", "Neonatal kidneys were dissected free of surrounding tissues except the ureter and fixed with 4% PFA at 4 \u00b0C for 24 h .", "After PBS washes , they were incubated with 30% sucrose at 4 \u00b0C overnight .", "Kidneys were swirled in five dishes of OCT to remove sucrose and mounted in OCT in a dry ice/ethanol bath .", "The OCT blocks were stored at \u221280 \u00b0C .", "Sections were cut at 20 \u03bcm and air dried .", "Sections were post-fixed with 4% PFA for 10 min , treated with 10 \u03bcg/ml proteinase K for 10 min and post-fixed for 5 min .", "Slides were acetylated ( 1 . 33% Triethanolamine , 0 . 065% HCl , 0 . 375% acetic anhydride ) for 10 min and dehydrated with 70% ethanol and 95% ethanol for 5 min each .", "Slides were air dried then incubated with 500 ng/ml digoxigenin-labeled riboprobes at 68 \u00b0C overnight .", "Hybridized sections were washed with 50% formamide , 1\u00d7SSC , pH4 . 5 for 30 min at 65 \u00b0C , treated with 2 \u03bcg/ml RNase for 15 min at 37 \u00b0C , and washed with 2\u00d7SSC , pH4 . 5 for 30 min , and twice with 0 . 2\u00d7SSC , pH4 . 5 for 30 min at 65 \u00b0C .", "Slides were washed three times at room temperature with 1\u00d7MBST ( 0 . 1 M maleic acid , 0 . 15 M NaCl , 0 . 1% Tween-20 , pH7 . 5 ) for 5 min each , and incubated with blocking solution ( 2% Boehringer Mannheim ( BM ) blocking reagent ) in 1\u00d7MBST , 20% heat-inactivated sheep serum ) for 1 h .", "After incubation with anti-digoxigenin antibody-AP ( Roche , 1:4000 ) at 4 \u00b0C overnight , sections were washed with 1\u00d7MBST at room temperature , 5 min for three times , then with NTMT ( 0 . 1 M NaCl , 0 . 1 M Tris-HCl , pH9 . 5 , 50 mM Mg2Cl , 0 . 1% Tween-20 , 2 mM Levimasole ) for 10 min , and developed with BM purple ( Roche ) .", "Color reactions were stopped with fixatives ( 4% PFA , 0 . 2% glutaraldehyde ) and sections mounted with glycergel mounting media ( DAKO ) .", "Images were captured with a Nikon DXM1200 digital camera attached to a Leitz DMRB microscope ."]], "headings": ["Introduction", "Results", "Discussion", "Materials and Methods"], "abstract": ["Kidney function depends on the nephron , which comprises a blood filter , a tubule that is subdivided into functionally distinct segments , and a collecting duct .", "How these regions arise during development is poorly understood .", "The zebrafish pronephros consists of two linear nephrons that develop from the intermediate mesoderm along the length of the trunk .", "Here we show that , contrary to current dogma , these nephrons possess multiple proximal and distal tubule domains that resemble the organization of the mammalian nephron .", "We examined whether pronephric segmentation is mediated by retinoic acid ( RA ) and the caudal ( cdx ) transcription factors , which are known regulators of segmental identity during development .", "Inhibition of RA signaling resulted in a loss of the proximal segments and an expansion of the distal segments , while exogenous RA treatment induced proximal segment fates at the expense of distal fates .", "Loss of cdx function caused abrogation of distal segments , a posterior shift in the position of the pronephros , and alterations in the expression boundaries of raldh2 and cyp26a1 , which encode enzymes that synthesize and degrade RA , respectively .", "These results suggest that the cdx genes act to localize the activity of RA along the axis , thereby determining where the pronephros forms .", "Consistent with this , the pronephric-positioning defect and the loss of distal tubule fate were rescued in embryos doubly-deficient for cdx and RA .", "These findings reveal a novel link between the RA and cdx pathways and provide a model for how pronephric nephrons are segmented and positioned along the embryonic axis ."], "summary": ["In the kidney , structures known as nephrons are responsible for collecting metabolic waste .", "Nephrons are composed of a blood filter ( glomerulus ) followed by a series of specialized tubule regions , or segments , which recover solutes such as salts , and finally terminate with a collecting duct .", "The genetic mechanisms that establish nephron segmentation in mammals have been a challenge to study because of the kidney's complex organogenesis .", "The zebrafish embryonic kidney ( pronephros ) contains two nephrons , previously thought to consist of a glomerulus , short tubule , and long stretch of duct .", "In this study , we have redefined the anatomy of the zebrafish pronephros and shown that the duct is actually subdivided into distinct tubule segments that are analogous to the proximal and distal segments found in mammalian nephrons .", "Next , we used the zebrafish pronephros to investigate how nephron segmentation occurs .", "We found that retinoic acid ( RA ) induces proximal pronephros segments and represses distal segment fates .", "Further , we found that the caudal ( cdx ) transcription factors direct the anteroposterior location of pronephric progenitors by regulating the site of RA production .", "Taken together , these results reveal that a cdx-RA pathway plays a key role in both establishing where the pronephros forms along the embryonic axis as well as its segmentation pattern ."], "keywords": ["developmental biology", "danio (zebrafish)", "vertebrates", "teleost fishes", "nephrology"]}

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-{"id": "journal.pgen.1002882", "year": "2012", "title": "Genetics and Regulatory Impact of Alternative Polyadenylation in Human B-Lymphoblastoid Cells", "sections": [["Naturally occurring genetic differences in gene regulation within populations underlie phenotypes of evolutionary and biomedical interest [1]\u2013[3] and can serve as the basis for inference of regulatory networks [4] , [5] .", "A key problem in the field is understanding the molecular mechanisms by which DNA sequence variants give rise to expression change .", "Recent work has emphasized the importance of sequence differences in regions upstream of gene loci that harbor cis-acting determinants of transcription factor binding [6]\u2013[10] and chromatin architecture [11]\u2013[13] .", "Much less is known about the role of 3\u2032-end regulation as a determinant of expression variation between individuals .", "Alternative polyadenylation represents a major regulatory strategy in the human genome , with analysis across tissue types detecting multiple 3\u2032 UTR forms of over half of all human genes [14] .", "Detailed genetic studies have implicated polymorphisms affecting transcript termination in both Mendelian and complex human disease [15]\u2013[20] .", "Genomic analyses have hinted at a broader role for genetic differences in RNA 3\u2032-end processing as a driver of expression variation [6] , [8] , [21] , [22] , but the prevalence and the mechanisms of these changes are incompletely understood .", "Progress in dissecting the genetics of 3\u2032-end processing has been limited in part by fundamental questions about the regulatory information encoded in 3\u2032 UTRs .", "Single-gene studies have made clear that , in addition to its interplay with exonic splicing [23] , [24] , RNA 3\u2032-end processing can dictate the extent of 3\u2032 UTR sequence incorporated into mature transcripts that governs half-life , translation , and localization [25]\u2013[29]; the efficiency of transcription termination itself can also influence steady-state expression level of a given length form [30]\u2013[34] .", "In general , however , identifying the regulatory elements that underlie relationships between 3\u2032 UTR sequence and gene expression remains a primary challenge , and for the majority of human genes , the regulatory impact of alternative polyadenylation is unknown .", "Likewise , the search for molecular players underlying cis-regulation of 3\u2032-end processing at individual gene loci [23] and genome-scale regulation of 3\u2032-end processing in trans [14] , [29] , [35]\u2013[40] is an area of active research .", "A complete understanding of the genetics of alternative polyadenylation will require maps of transcript end site usage and 3\u2032 cis-regulatory elements , and analysis strategies to integrate the data .", "Recently developed short-read sequencing methods for transcript ends [40]\u2013[46] have enabled the possibility of quantitative studies of the regulatory architecture of transcript end forms on a genomic scale .", "In this work , we set out to investigate mechanisms by which alternative polyadenylation impacts gene expression and its variation across genetically distinct human individuals .", "We used 3\u2032-end RNA-seq [42] to maximize the genomic coverage and precision of transcript end positions , and to measure quantitative expression levels of transcript forms .", "The results shed light on the architecture of transcript ends and regulatory elements in human 3\u2032 UTRs and the principles of genetic variation in 3\u2032 length form usage ."], ["To survey the 3\u2032 ends of transcripts in human B-lymphoblastoid cells , we isolated RNA from cell lines derived from six human individuals and subjected each replicate of each sample to 3\u2032-end RNA-seq , which sequences polyadenylated transcript ends on a genomic scale [42] with strong and significant reproducibility ( Figure S1 ) .", "Our analysis pipeline , described in Materials and Methods , filtered out likely products of mispriming from A-rich genomic regions [47] and categorized mapping reads in terms of well-defined peaks or dispersed regions of reads with no peak structure ( Table S1 ) .", "We considered the former to represent the strongest candidates for stable , functional transcript end forms , and focused on these for in-depth analysis , taking the last mapped base of each read as the likely site at which the nascent RNA was cleaved from the processing polymerase and polyadenylated [24] .", "Across such transcript ends mapping to nuclear-encoded loci , the vast majority of reads ( 87% ) originated from 3\u2032 UTRs of coding genes ( Figure 1A and Table S1 ) ; 80% of transcript ends were consistent with the 3\u2032 ends of previously annotated length forms ( Tables S1 and S2 and Figure S2 ) .", "Within 40 base pairs of the inferred cleavage position , most transcript end forms harbored a polyadenylation signal sequence: either the canonical A ( A/U ) UAAA [23] or a close variant , or an A-rich stretch [48]\u2013[50] ( Figure S3A ) .", "Reporter assays confirmed the regulatory importance of A-rich stretches upstream of the inferred cleavage positions in CPSF1 and WDR18 , whose 3\u2032 UTRs lacked canonical polyadenylation signal motifs ( Figure S3C ) .", "We set out to analyze our data set of 3\u2032 transcript ends with respect to alternative polyadenylation , focusing on a maximum of two abundant , distinguishable transcript forms in a given gene .", "We considered three patterns of transcript end usage [30]: class I , indicating genes with a single transcript form terminating in an annotated 3\u2032 UTR; class II , genes with alternative polyadenylation in the same annotated 3\u2032 UTR; and class III , genes in which the two alternative polyadenylation forms differed in their composition of coding sequence ( Figure 1B ) .", "We also observed a small fraction of genes with a single transcript form terminating inside annotated coding exons or introns ( Table S3 ) .", "The breakdown of transcript forms into these classes revealed alternative polyadenylation reaching our threshold of detection in \u223c30% of genes ( Figure 1B ) .", "Across these genes , we observed the expected enrichment of the A ( A/U ) UAAA polyadenylation signal motif in the distal relative to proximal polyadenylation signals ( Figure S3A ) , correlating with the higher expression levels of the long transcript forms [30] , [51] .", "The lower-abundance short transcript forms were more likely to harbor an A-rich stretch or no recognizable polyadenylation signal motif upstream of the inferred cleavage position ( Figure S3B ) , lending credence to the notion that the latter regions represent weak recognition sites for the 3\u2032-end processing machinery [48] .", "We sought to harness our data set of transcript ends to investigate regulatory elements governing translation and transcript half-life , and their relationship to alternative 3\u2032 transcript forms .", "For this purpose , we applied a motif-search strategy to identify putative microRNA binding sites , A/U rich elements ( AREs ) , G/U-rich elements , binding sites for the Pumilio family of proteins , and Alu transposable elements in 3\u2032 UTRs .", "We tabulated rates of sequence variation across human populations and observed marked conservation of most regulatory element motifs relative to the background level of 3\u2032 UTR polymorphism ( Figure 2A ) , reflecting a history of purifying selection on these putatively functional regulatory sequences [52]\u2013[54] .", "We expected that the regulatory logic of alternative polyadenylation would be intimately connected with sequence determinants of transcript half-life or translation in 3\u2032 UTRs .", "For a given gene subject to alternative polyadenylation , we referred to the region of the 3\u2032 UTR upstream of the proximal cleavage site as \u201cshared\u201d among the alternative polyadenylation length forms , and the span of the 3\u2032 UTR in between the proximal and distal cleavage sites as the \u201cdifferential\u201d region ( Figure 2B ) .", "We hypothesized that , if alternative polyadenylation often acted to tune the exposure of cis-regulatory motifs in 3\u2032 UTR sequences , these motifs would preferentially be positioned in differential regions .", "To test this , we tabulated the positions of each type of regulatory element across our set of alternatively polyadenylated genes in class II .", "Significance testing revealed a significant enrichment of genes with motifs in the differential regions of 3\u2032 UTRs relative to those with motifs in shared regions ( Figure 2C ) .", "Analyzing the annotations of genes in these sets , we observed a preponderance of genes with immune-related functions among those with cis-regulatory motifs in the differential regions of 3\u2032 UTRs , while motifs in shared regions of 3\u2032 UTRs were largely detected among genes with housekeeping roles ( Table S5 ) .", "Thus , for genes carrying out immune processes in B-lymphoblastoid cells , the choice between long and short transcript end forms often exposes or eliminates regulatory information in 3\u2032 UTRs , highlighting the importance of 3\u2032-end processing in the control of gene expression levels for specialized cell functions .", "To investigate the genetics of RNA 3\u2032-end processing , we first assessed the contribution of genetic differences , relative to experimental and environmental error , to variation of transcript 3\u2032-end positions across the six genotypes of lymphoblastoid cells in our study .", "For this purpose , we calculated the heritability of length form abundance for each gene , finding 194 coding genes at which the abundances of transcript length forms differed reproducibly ( heritability>0 . 6 ) across human samples ( Table S6 ) .", "To begin to dissect the molecular basis for natural genetic variation in 3\u2032-end usage at these loci , we considered the potential role for DNA sequence differences at polyadenylation signals , the primary determinants of transcript cleavage and polyadenylation .", "We reasoned that although such variants were rare in the human population ( Figure 2A ) , some could underlie differences between human individuals in 3\u2032-end length form usage .", "Consistent with this prediction , genes with highly heritable transcript end positions harbored a single nucleotide polymorphism in polyadenylation signal motif sequence much more often than did the average gene ( 2% of polyadenylation signals at genes with heritable transcript ends , compared to 0 . 3% genome-wide; Fisher's exact p\u200a=\u200a0 . 004; Table S6 ) .", "We hypothesized that naturally occurring genetic variation in RNA 3\u2032-end processing would prove to underlie changes across individuals in steady-state levels of gene expression .", "To test this , we analyzed the genomic relationship between biallelic single-nucleotide polymorphisms in polyadenylation signals and gene expression differences across our set of lymphoblastoid cell lines from distinct human genotypes .", "We used a standard regression test of genetic association to evaluate genotype at each such sequence variant as a predictor of expression of the gene in which it lay .", "The results revealed a significant enrichment of association with expression for variants in polyadenylation signals , relative to the background signal from 3\u2032 UTRs as a whole ( Figure 3A ) .", "We expected that for a given such variant , the allele conferring a closer match to the canonical polyadenylation signal motif would confer more robust transcription termination , and thus more abundant steady-state levels of transcription , than would the allele weakening the match to the canonical motif .", "To quantify this effect , we scored each allele in each variant polyadenylation signal with respect to agreement with the canonical motif , and at each variant position , we calculated the difference in scores between alleles .", "This score difference was a strong predictor of the effect of a given polyadenylation signal variant on steady-state expression levels ( Figure 3B ) , with a departure from the polyadenylation signal motif associated with a drop in expression as predicted .", "To pursue on a molecular basis the impact of variation across humans in polyadenylation signals , we used our 3\u2032-end RNA-seq data to infer the effects of single-nucleotide variants on usage of 3\u2032 transcript forms at individual genes , and we evaluated these predictions in single-gene 3\u2032 UTR reporter assays .", "For each gene , toggling natural variant alleles at one nucleotide position in the polyadenylation signal was sufficient to drive differential usage of short and long 3\u2032 transcript forms ( Figure 4 ) .", "These included variants attenuating usage of 3\u2032 forms of the translation initiation factor EIF2A and the putative DNA methylation enzyme DIP2B , as well as the expected effect of the polymorphic polyadenylation signal on usage of 3\u2032 forms of the inflammation regulator IRF5 [15] .", "In each of the latter genes , the causal variant conferred significant changes in luciferase protein levels as well as usage of transcript forms ( Figure 4A\u2013F ) .", "Our set of confirmed causal variants at polyadenylation signals also included that in the transcription factor NAB1 , which attenuated usage of a minor 3\u2032 transcript form with modest effect on luciferase levels ( Figure 4G , H ) .", "We conclude that sequence differences in polyadenylation signals represent a key mechanism underlying variation between humans in levels of gene expression , with genome-scale trends validated at the single-gene level .", "We next sought to dissect the mechanisms by which natural variation in 3\u2032-end usage impacted gene expression , using as case studies IRF5 and DIP2B , which lie in genomic regions associated with susceptibility to lupus [15] and colorectal cancer [55] , respectively , as well as NAB1 and EIF2A .", "In RNA expression measurements using the 3\u2032 UTR haplotype that produced both long and short transcript forms of a given gene , one form was detected at higher abundance in each case ( Figure 4B , D , F , H ) .", "We hypothesized that these abundance differences between length forms could be in part the result of sequence elements that dictate transcript cleavage , polyadenylation , and termination , and in part the result of regulatory elements that affect transcript half-life .", "To test this , for each gene we first developed expression reporters incorporating only the regions flanking the end positions of each transcript form in turn , which we expected would include the polyadenylation signal and auxiliary sequence motifs underlying 3\u2032-end processing of the respective form while excluding most other 3\u2032 regulatory information .", "Expression measurements confirmed differences in the strength of these 3\u2032-end processing motifs between length forms for EIF2A , IRF5 , and DIP2B , in that reporters incorporating each of the two 3\u2032-end sequences from a given gene exhibited up to 2 . 5-fold differences in expression ( Figure 5A , C , E ) .", "To assess the contribution of 3\u2032 regulatory elements that control transcript half-life , we next measured the decay rate of each transcript length form upon addition of actinomycin D in the context of complete 3\u2032 UTRs .", "Measurements of transcript stability by quantitative PCR and by Northern blot bore out this prediction , with the long transcript form showing reduced half-life relative to the short form for IRF5 [15] and DIP2B ( Figure 5D , F and Figure S4B , C ) , and increased half-life for NAB1 ( Figure 5H and Figure S4D ) .", "Analyzing these results together with the effects of natural variants in polyadenylation signals ( Figure 4 ) indicates that for a given gene , a variant can abrogate production of a transcript form with strong sequence determinants of 3\u2032-end processing , leaving only the less efficiently processed form and giving rise to lower total expression of the gene product , as in IRF5 and EIF2A .", "In addition , a variant abrogating production of a transcript form with longer half-life leaves only the less-stable form and reduces total steady-state levels of the gene product , as in IRF5 and DIP2B .", "As a further investigation of the determinants of abundance of long and short transcript forms for genes subject to natural genetic change in 3\u2032-end processing , we analyzed the role of 3\u2032 regulatory elements in such genes in relation to trans-acting regulatory factors .", "We identified a candidate ARE in the differential region of the 3\u2032 UTR of IRF5 , i . e . between the positions of alternative 3\u2032 ends observed in our 3\u2032-end RNA-seq; a candidate ARE in the differential region of NAB1; and a candidate binding site for the miRNA miR-101 in the differential region of DIP2B ( Figure 6 ) .", "To assess the functional relevance of these motifs , we applied a mutagenesis strategy using 3\u2032 UTR reporter constructs for each gene , as above distinguishing between the 3\u2032 UTR haplotype that produced both long and short transcript forms and the haplotype producing only the long form ( Figure 4D , F , H ) .", "For each inferred cis-regulatory element , we assayed the regulatory response of 3\u2032 UTR reporters to the trans-acting factor predicted to mediate its repressive effect: the ARE-binding proteins TTP and AUF1 for IRF5 and NAB1 , respectively , and a mimic of miR-101 for DIP2B .", "In each case , expression measurements from mutagenized reporter constructs established the respective sequence element as necessary for full repression of the long form of its host UTR ( Figure 6 ) , validating our motif inferences .", "Among experiments that used haplotypes producing both short and long 3\u2032 forms of the respective UTRs , motifs in the differential region were only necessary for repression by trans-acting factors in the case of NAB1 ( Figure 6C ) , for which the population of transcripts arising from this haplotype was dominated by the long form ( Figure 4H ) .", "Taken together , our results illustrate the complexity of regulatory information in 3\u2032 UTRs , as determinants of RNA 3\u2032-end processing and transcript fate each contribute to the final expression level of the host gene .", "We conclude that , for these case studies , integrating predicted regulatory motifs with knowledge of transcript end positions is essential in the effort to relate genotype to gene expression .", "We next aimed to shed light on the biological context in which differences in regulatory responsiveness could manifest between long and short transcript forms .", "For this purpose , we focused on natural variation in alternative polyadenylation at the immune regulator IRF5 .", "In response to the bacterial cell wall component lipopolysaccharide ( LPS ) , immune genes undergo an immediate spike in expression , followed by dampening to a more modest steady-state level mediated by the ARE-binding protein TTP [56]\u2013[58] .", "Motivated by our discovery of a repressive ARE in the differential region of IRF5 ( Figure 6A ) , we hypothesized that the genetically determined production of long and short 3\u2032 mRNA forms of this gene would be associated with different patterns of regulatory behavior after induction .", "To test this , we treated B-lymphoblastoid cell lines from genetically distinct individuals with LPS and , in each culture , measured the recovery of expression levels of IRF5 transcript forms over time .", "The results , shown in Figure 7 , revealed , after an initial overshoot in expression , a difference of up to 2-fold in the time to reach steady-state expression between long and short mRNA forms , with the long form downregulated to steady-state more quickly after induction as predicted , given the presence of the repressive ARE in the latter transcript .", "Variation in IRF5 expression recovery across B-lymphoblastoid lines was associated with genotype at the proximal polyadenylation signal in the IRF5 3\u2032 UTR: haplotypes encoding the long form of IRF5 conferred rapid repression after induction ( Figure 7B ) relative to haplotypes encoding the short form ( Figure 7A ) .", "These findings suggest that genetic variation in 3\u2032-end processing dictates differences across individuals in the regulatory dynamics of IRF5 , further underscoring the power of our approach to identify biologically relevant regulatory effects of 3\u2032-end processing .", "Our molecular confirmation of cis-regulatory elements in 3\u2032 UTRs inferred from sequence search methods ( Figure 6 ) suggested that such inference could provide a mechanistic understanding of gene expression on a genomic scale .", "In particular , we expected that sequence variants between individuals in 3\u2032 regulatory elements would be significant predictors of variation in steady-state expression of the genes in which they lay .", "To test this notion , we first tabulated all single-nucleotide polymorphisms across the cell lines of our data set which overlapped with 3\u2032 regulatory motifs and Alu elements in 3\u2032 UTRs .", "We next classified these motifs according to the impact of alternative polyadenylation on their positions in 3\u2032 UTRs , and we used association tests to assess the strength of each motif variant in each class as a predictor of steady-state expression of its respective gene .", "The results ( Figure 8 ) revealed association with expression across human individuals , for variants in AREs , G/U-rich elements , Pumilio sites , and Alu elements .", "The relationship with expression was striking and significant for variants in regions of 3\u2032 UTRs constitutively incorporated into mature messages ( Figure 8 ) .", "Polymorphic motifs incorporated into low-abundance 3\u2032 length forms showed no evidence of association with expression changes in their respective genes , consistent with the minor contribution of these forms; by the same token , polymorphic motifs in regions incorporated into the predominant 3\u2032 length forms of mRNAs were more strongly associated with expression of their respective genes , though not significantly so ( Figure 8 ) .", "As expected [6] , the relationship between sequence variants and gene expression did not manifest for miRNA sites ( data not shown ) .", "These findings highlight the relevance of 3\u2032 regulatory motifs as predictors of expression variation across human individuals , when integrated with knowledge of transcript length forms from our sequencing strategy ."], ["Alternative polyadenylation is prevalent in the human transcriptome , and in landmark cases , variation across individuals in the use of 3\u2032 length forms of RNAs has been shown to underlie human disease [15]\u2013[20] .", "However , for most human genes , the regulatory importance of changes in transcript ends between individuals is incompletely understood , owing to the challenges of measuring 3\u2032-end usage and identifying functional regulatory elements in 3\u2032 UTRs .", "We have developed a spatially precise , quantitative , high-throughput sequencing approach for 3\u2032 ends , complementing now-classic studies of expressed sequence tags [30] , [36] , [48] , [51] , [59]\u2013[61] .", "We have used the resulting transcript end positions and abundances to pioneer an analysis approach which integrates bioinformatic predictions of 3\u2032 regulatory motifs , genomic analysis , and molecular genetics .", "With this strategy , we have established a regulatory map of transcript ends and functional elements in the 3\u2032 UTRs of lymphoblastoid cells , and we have abstracted genomic principles of alternative polyadenylation and natural genetic variation in this cell type .", "Our mapping of 3\u2032-end length forms and sequence motifs in 3\u2032 UTRs revealed an intuitive logic in which the choice between short and long UTR forms governs the incorporation of regulatory elements into mature messages [36] , [61] .", "We note that the 3\u2032 length forms we report in B-lymphoblastoid cells represent a subset of the total complement of 3\u2032 UTR lengths used across tissues .", "As such , we hypothesize that surveys of tissue types will ultimately reveal transcript forms of many genes , used in particular contexts , that incorporate 3\u2032 regulatory information to different extents .", "The ability to tune the responsiveness to trans-acting input itself distinguishes alternative polyadenylation from other transcriptional and post-transcriptional regulatory mechanisms , providing a compelling model for the particular advantage of 3\u2032-end processing as a regulatory strategy and a rationale for its prevalence in mammalian genomes .", "In comparisons across cell lines from genetically distinct individuals , we analyzed the regulatory importance of genetic changes at 3\u2032 transcript ends .", "We uncovered a key role for polymorphisms in polyadenylation signals as a driver of changes in gene expression , and we detailed the molecular mechanisms at play .", "The polyadenylation variants we study here dictate the production of transcript forms with different determinants of 3\u2032-end processing , different half-lives , and different recognition sites for trans-acting regulators .", "These findings establish a connection between observational studies of transcript 3\u2032 length forms across human populations [21] , [22] and regulatory effects of this variation .", "In the case of IRF5 , we discovered that a naturally occurring genetic change in usage of 3\u2032 RNA forms can serve to tune the kinetics of recovery of expression after induction by lipopolysaccharide .", "Thus , against the backdrop of prior studies of this lupus susceptibility gene [15] , we have uncovered an additional dimension by which variation in RNA 3\u2032-end processing affects regulatory behavior .", "Given these case-study results as a validation of our genome-scale analyses , we speculate that many genetic variants with biologically relevant effects mediated by RNA 3\u2032-end processing remain to be discovered in the human population .", "We have also shown that polymorphisms in 3\u2032 motifs that govern transcript fate can serve as predictors of steady-state levels of the genes in which they lie .", "In light of the ultimate goal of predicting regulatory and phenotypic effects from human genome sequence , our results indicate that analysis strategies using sequence determinants of transcription initiation and splicing alone are likely to provide an incomplete model of expression variation .", "Importantly , however , our work makes clear that binding sites for regulators of mRNA localization , half-life , and translation at 3\u2032 ends are themselves only part of the regulatory landscape .", "Rather , a complete understanding of the genetics of gene expression will integrate the usage of RNA 3\u2032-end processing signals with the effects of 3\u2032 sequence elements that control transcript fate .", "We anticipate that abundances and positions of transcript ends observed in 3\u2032-end RNA-seq will prove to be a key component in the systems-level modeling of regulatory networks and their variation .", "In summary , while the genetic study of RNA 3\u2032-end processing is in its infancy , our work and that of others [15]\u2013[22] establishes that variation at 3\u2032 ends can be a critical determinant of regulatory behaviors .", "However , for the vast majority of human genes , the importance of 3\u2032 regulatory change remains unknown .", "Our single-gene experiments detail the expression effects of 3\u2032 UTR variation in the disease-associated genes IRF5 [15] and DIP2B [55]; the potential for regulatory variants as drivers of human disease will serve as continued motivation for genomic and genetic analyses of expression change ."], ["Two biological replicates of each of the human lymphoblastoid cell lines GM10860 , GM17106 , GM17189 , GM17207 , GM17220 , and GM17253 ( Coriell Institute ) were cultured in RPMI 1640 medium supplemented with 2 mM L-glutamate and 15% fetal bovine serum .", "Cells were incubated at 37\u00b0C under 5% carbon dioxide .", "Total RNA was extracted from \u223c107 cells using TRIzol reagent ( Invitrogen ) , and genomic DNA was removed from RNA using Turbo DNase ( Ambion ) .", "Polyadenylated RNA was selected from 10 \u00b5g of total RNA using the Dynabeads mRNA purification kit ( Invitrogen ) , and was fragmented for 3 minutes at 70\u00b0C using 10\u00d7 Fragmentation Reagent ( Ambion ) .", "After ethanol precipitation , polyadenylated RNA fragments were selected using the Dynabeads mRNA purification kit and reverse transcribed using SuperScript II ( Invitrogen ) and anchored oligo-dT ( Invitrogen ) .", "Double-stranded cDNA was generated using RNase H ( Invitrogen ) and DNA Pol I ( Invitrogen ) , end-repaired using T4 DNA Polymerase ( New England Biolabs ) , Klenow DNA Polymerase ( New England Biolabs ) , and T4 PNK ( New England Biolabs ) , and then adenylated using Klenow 3\u2032 to 5\u2032 exo minus ( New England Biolabs ) .", "Illumina paired-end adapters were ligated to the adenylated cDNA using T4 DNA Ligase ( Enzymatics ) .", "Ligated cDNA was purified on a 2% agarose gel and then amplified by performing 12 cycles of PCR using Phusion HF Polymerase ( New England Biolabs ) .", "Libraries were sequenced using 40 bp paired-end modules on an Illumina 2G Genome Analyzer .", "Consecutive T's from the beginning of all reads were trimmed and classified before mapping: for a given read , if there were more than 20 consecutive T's or if neither mate of a read pair had a stretch of T's , the read was not included in further analysis .", "For mapping , the set of single nucleotide polymorphisms ( SNPs ) segregating in the CEU population was downloaded from HapMap phase II+III , release 27 ( ftp://ftp . ncbi . nlm . nih . gov/hapmap/genotypes ) , and used to modify the human reference genome ( hg18; [62] ) by incorporating the appropriate ambiguous bases at each SNP position .", "All reads were then mapped to this modified genome and to the associated splicing junctions in the Known Genes database of the UCSC genome browser [63] using MOSAIK ( http://bioinformatics . bc . edu/marthlab/Software_Release ) .", "For a given length of trimmed T's , nT , a splicing junction reference was created by concatenating 37 - nT bp from each exon adjacent to the splicing junction to ensure that the reads mapped across the splicing junction .", "If the reads mapped to both the genome and a splicing junction , the mapping with smaller number of mismatches was used .", "Only uniquely mapped reads with two or fewer mismatches in each mate were retained .", "Trimmed T's were then compared to the genome sequence; reads with >2 mismatches to the genome in this poly-T tract were retained for analysis .", "We inferred that a given read was transcribed from the minus strand of the genome if , when it was mapped to the reference genome , the position of its poly-T tract had a lower coordinate position than the mapped position of the other end of the read; we inferred that a read was transcribed from the plus strand of the genome if the mapped position of its poly-T tract had a higher coordinate position than the position of the other end .", "Mapped reads yielded an average coverage of 22 . 6% of UCSC annotated 3\u2032 UTRs with a depth of 97 . 8 reads/bp for the covered bases for each sample .", "The last 100 bp of annotated 3\u2032 UTRs were even more highly represented in libraries , with an average coverage of 43 . 4% and an average depth of 211 . 1 reads/bp .", "Mapped reads from all samples were pooled , sorted according to the polyA positions , defined as the coordinate of the base adjacent to the polyA tail , and then grouped into tag clusters as follows .", "For each strand of each chromosome , the 5\u2032 boundary of a tag cluster was set as the polyA position of the first read , and then reads were sequentially added to this unit until the polyA position of the next read was more than 15 bp away .", "The latter position then became the 5\u2032 boundary of the next tag cluster .", "Most tag clusters spanned less than 24 bp , but if the polyA positions in a cluster spanned more than 40 bp , we applied a peak-finding algorithm as follows .", "For each genome coordinate in the region corresponding to the tag cluster , we defined the read count as the number of reads whose polyA position overlapped the coordinate .", "From these we first identified the genome coordinate ( posM ) with the greatest read count ( MaxHeight ) .", "We then delineated a window 40 bp upstream and 40 bp downstream of this coordinate .", "Within this window , we retained all coordinates with read counts greater than 10% of MaxHeight .", "Of these , we identified the most 5\u2032 and 3\u2032 polyA positions ( posL and posR ) .", "All reads in the tag cluster were then divided into three new candidate tag clusters: reads with positions 5\u2032 to posL , reads with positions including and between posL and posR , and reads with positions 3\u2032 to posR .", "If the distance between posL and posR was longer than 40 bp , the middle candidate tag cluster was eliminated from further analysis .", "If the read counts of all coordinates in a candidate tag cluster were below 10% of MaxHeight , the candidate unit was eliminated .", "If a candidate tag cluster contained coordinates with read counts larger than 10% of MaxHeight , we identified the coordinate with the largest read count within this candidate tag cluster and repeated the peak-finding algorithm .", "For each tag cluster retained for analysis , we defined the polyA position of the unit as the median of the polyA positions of all reads encompassed by the unit .", "After establishing that the stretch of A's in each read represented a mismatch to the genome sequence ( see above ) , we filtered out reads with a potential origin from internal priming from A-rich regions of the genome by removing any tag cluster whose defined polyA position was followed by 10 or more A's in the genome sequence within 20 bp .", "We also filtered out reads with a potential origin as PCR clones or false mapping as follows .", "We expected that for a given set of paired-end reads falling into a tag cluster , the characteristics of read ends originating from biological 3\u2032-end processing should be distinct from the characteristics of read ends originating from fragmentation , reverse transcription , and ligation during RNA-seq library preparation .", "In particular , we reasoned that the polyA positions of a set of reads in a tag cluster with origin as a biologically relevant transcript would be less heterogeneous than the positions of the other mates of the reads .", "If the opposite were true , we considered the tag cluster to be a likely product of false mapping or PCR duplication .", "As such , we retained a tag cluster for analysis only if the precision of polyA positions across the reads of the unit was greater than the precision of the positions of the other mates .", "The precision was calculated by where ni is the number of reads at the ith position and N is the total number of reads in the tag cluster .", "We also filtered out any tag cluster whose total read count across all samples amounted to fewer than 50 reads .", "For tag clusters with read counts between 50 and 100 , we calculated the Pearson correlation coefficient R between each pair of the two biological replicates across the six cell line samples , and eliminated the tag cluster from further analysis if the absolute value of R was less than 0 . 5 .", "For use in searches for regulatory motifs , we harnessed all 3\u2032-end RNA-seq reads in tag clusters from all samples to define a consensus base at each position in 3\u2032 UTRs as follows .", "At every genomic coordinate covered by five or more 3\u2032-end RNA-seq reads , the consensus nucleotide was chosen as that with highest frequency across the sample .", "If the second most abundant base was more than 20% in abundance , it was incorporated into the consensus using an ambiguous base notation ( M\u200a=\u200aA or C , R\u200a=\u200aA or G , W\u200a=\u200aA or T , S\u200a=\u200aC or G , Y\u200a=\u200aC or T , K\u200a=\u200aG or T ) .", "For every tag cluster , the consensus sequence of the region 40 bp upstream from the polyA position was searched for a polyadenylation signal using the known hexamer motifs sorted by their abundance in the human genome from [30] .", "Polyadenylation signals with higher abundance were given higher priority when there was more than one instance in the 40 bp upstream window .", "In the absence of a match to these motifs , the 40 bp upstream window was searched for an A-rich stretch as follows .", "We identified any 9 bp regions containing at least 6 A's and considered each such region a candidate polyadenylation signal; if no such candidate signal were present , we considered the tag cluster not to have an identifiable polyadenylation signal .", "If more than one candidate signal was present , we retained the one with the most A's .", "For each boundary of this region , if the boundary nucleotide was an A , the region was extended to include all consecutive A's .", "If the first base was not an A , it was trimmed until the first base was an A . To find upstream U-rich elements ( USE ) , for a given tag cluster , we identified a candidate USE as the 9-bp window with the highest number of T's in the consensus sequence , within 30 bp upstream of the polyadenylation signal .", "If there were fewer than six T's or if the candidate did not have three consecutive T's , we considered the tag cluster not to have an identifiable USE .", "For each boundary of a given candidate window , if the boundary nucleotide was a T , the window wasextended if there was a T in the adjacent 2 bp and the proportion of T's in the window was above 65%; if the boundary base was not a T , we trimmed the candidate window until a T was reached .", "To find downstream U/G-rich elements ( DSE ) , for a given tag cluster , we identified a candidate DSE as the 9-bp window with the highest number of T's in the consensus sequence , within 40 bp downstream of the poly-A position .", "If there were fewer than five T's or if the window did not contain at least one of the strings TTT , TGTG , GTGT , GTCT , CTGT , TCTG , or TGTC , we considered the tag cluster not to have an identifiable DSE .", "For each boundary of a given candidate window , if the boundary nucleotide was a T , the window was extended if there was a T in the adjacent 2 bp and the proportion of T's in the window was above 50%; if the boundary base was not a T , we trimmed the candidate window until a T was reached .", "Human gene annotations were downloaded from the Known Genes database of the UCSC Genome Browser [63] .", "Among the UCSC transcript annotations that overlapped the start and end positions of a tag cluster , the annotation with the minimum distance between the annotated 3\u2032-end position and the polyA position of the tag cluster was chosen .", "If there were multiple annotations with the same 3\u2032-end positions , we chose the annotation with greatest degree of overlap between the tag cluster consensus sequence and the annotated exons .", "The genomic coordinates of human expressed sequence tags ( ESTs ) with polyA tails were downloaded from the polyA_db2 database [64] .", "The genomic coordinates were converted from hg17 to hg18 using the liftOver tool from the UCSC Genome Browser [65] .", "If the coordinate of polyA_db2 EST was between the start and end position of a tag cluster , we considered the length form corresponding to the unit to be supported by the EST .", "All tag clusters with the same UCSC gene annotation were categorized as associated with the gene , and only the two tag clusters with the highest expression per gene were used for classification of alternative polyadenylation .", "We classified each gene as follows: class I if there was only one tag cluster overlapping with the annotated 3\u2032 UTR of the gene; class II if both tag clusters overlapped with the annotated 3\u2032 UTR of the gene and they were associated with the same UCSC transcript ID; class III if one of the tag clusters overlapped witha coding exon or intron , or if both tag clusters overlapped with 3\u2032 UTRs with different UCSC transcript IDs .", "We calculated the broad-sense heritability in polyA positions using biological replicates of 3\u2032-end RNA-seq across the six human cell lines as follows .", "For a given gene , we considered the polyA position of the most abundant tag cluster in each replicate of each sample as a quantitative trait , and calculated the heritability H2 of this trait from intraclass correlations [66] given by the following equations .", "Here , indices i and j refer to sample and replicate , respectively; is the polyA position for sample i , replicate j; is the average between two replicates for sample i; is the average of all samples and replicates; MSe is the error mean square ( within-individual ) and MSb is the between-individual mean square .", "Heritability of total expression levels for each gene was calculated analogously , using as a quantitative trait the sum of read counts across the gene normalized by the sum of all reads .", "To identify microRNA binding sites in 3\u2032 UTRs , the Perl script from TargetScan Release 5 . 2 [67] was used to predict miRNA binding sites in the 3\u2032 UTR sequences of all expressed genes .", "miRNA sequences and families were downloaded from the TargetScan database .", "Only predicted binding sites with context scores less than \u22120 . 4 were used for analysis .", "To identify AU-rich elements , the class II motif WWWT ( ATTTA ) TTTW was searched in the 3\u2032 UTR sequences of all expressed genes allowing up to one mismatch outside the central pentamer , ATTTA .", "Overlapping motifs were combined .", "To identify GU-rich destabilizing elements , the motif TGTTTGTTTGT was searched in 3\u2032 UTR sequences allowing up to one mismatch .", "To identify Pumilio binding elements , the motif TGTANATA was searched in 3\u2032 UTR sequences .", "Alu transposable element motifs were taken from RepeatMasker ( www . repeatmasker . org ) downloaded from the UCSC Genome Browser .", "We note that each search strategy used human sequence data alone rather than inter- or intra-species conservation to identify motifs .", "SNPs within the human population were downloaded from the 1000 Genomes Project database [68] .", "Genomic coordinates were converted from hg19 to hg18 using the liftOver tool from the UCSC Genome Browser [65] .", "For each regulatory element motif , the SNP rate was calculated as the total number of SNPs within all motif matches divided by the sum of the lengths of all matches .", "For the background model of sequence variation in 3\u2032 UTRs used in Table S4 , we first tabulated all instances of A , C , T , and G across all positions of 3\u2032 UTR sequences in the hg18 human reference genome , where the boundaries of 3\u2032-ends were taken from our compendium of 3\u2032-end RNA-seq data for genes expressed in our samples .", "We refer to these frequencies as P ( A ) , P ( C ) , P ( T ) , and P ( G ) , respectively .", "We then tabulated the SNP rate separately for each of these four sets of base positions from the 1000 Genomes data set , which we refer to as P ( SNP , A ) , P ( SNP , C ) , P ( SNP , T ) , and P ( SNP , G ) , respectively .", "We used these values to calculate the expected density of polymorphisms for the stretch of genome corresponding to a given 3\u2032 motif match as: where is the sequence of bases of length Ns corresponding to the motif match in the human reference genome and P ( SNP|si ) is the probability of a single-nucleotide polymorphism for the nucleotide si at position i , calculated as: For each regulatory motif , P ( SNP|s ) was calculated for every instance of a motif match across all 3\u2032 UTRs in the reference genomefor genes expressed in our samples , and the lower and upper bounds listed in Table S4 were taken as the minimum and maximum values of this distribution , respectively .", "For miRNA binding sites , only the 7-mer seed sequence was used .", "The DEFOG web-based tool ( http://www . mooneygroup . org/defog ) was used for Gene Ontology term enrichment analysis in Table S5 as follows .", "For each type of regulatory element analyzed in Figure 2C , we tabulated a list of class II genes with motifs in the shared region only , and combined these lists across elements .", "For each Gene Ontology term , we then evaluated the significance of the representation of genes annotated in the term in this list , relative to a background set of class II genes harboring at least one motif , using DEFOG with default parameters .", "Separately , we tabulated an analogous combined list of class II genes with motifs in the differential region only and repeated the DEFOG analysis .", "To find polymorphisms in 3\u2032 UTRs across the six individuals of our sample , we generated mRNA-seq libraries [69] from one biological replicate of each cell line sample , and sequenced using 36 bp paired-end modules on an Illumina 2G Genome Analyzer , resulting in 21 to 24 million reads per sample .", "The sequenced reads were mapped to the human genome ( hg18 ) using MOSAIK ( http://bioinformatics . bc . edu/marthlab/Software_Release ) .", "Mapped reads ( 10 to 14 million per sample ) yielded an average coverage of 48% of UCSC annotated exons with an average depth of 27 reads/base for the covered bases .", "Mapped reads were used to call SNPs using GigaBayes ( http://bioinformatics . bc . edu/marthlab/Software_Release ) with options \u2013ploidy diploid \u2013O 3 \u2013indel \u2013CAL 10 .", "Only SNPs with a quality score higher than 0 . 99 were retained for analysis .", "This set of SNPs was used in association tests with total gene expression levels as follows .", "Given tag cluster definitions for each gene from analysis of 3\u2032-end RNA-seq libraries ( see above ) , we calculated a normalized expression level for each tag cluster in each sample as the ratio between the number of 3\u2032-end RNA-seq reads mapping within the tag cluster boundaries in the sample and the total number of mapped 3\u2032-end RNA-seq reads in the sample .", "We then defined the expression level of a given gene in a given sample as the sum of all normalized expression levels across all tag clusters in that gene .", "To get a final estimate of gene expression level for use in association tests in Figure 3 and Figure 8 , we summed expression values across the two replicate samples from each cell line .", "For each SNP in the 3\u2032 UTR of a gene , we identified the major allele across all cell lines and scored each diploid genotype in terms of the number of major alleles ( values ranging from 0 to 2 ) .", "Given the complete matrix of gene expression levels and genotypes across all six cell lines , we calculated the Pearson correlation coefficient R between allele counts and the started logarithm of the gene expression level .", "For Figure 3A and Figure 8 , we used the absolute value of R as the association statistic , and for Figure 3B , we used the signed value of R . In cases of alternative polyadenylation , SNPs in polyadenylation signals upstream of a maximum of two major length forms were considered for association tests .", "To analyze the effect of genetic variation in polyadenylation signal strength on expression in Figure 3B , each allele of each polyadenylation signal was assigned a score: 1 if the signal was AATAAA or ATTAAA , 0 . 5 if the signal was a match to the \u201cvariant\u201d polyadenylation motifs in [30] , and 0 otherwise .", "The polyadenylation signal strength difference was calculated by subtracting the strength of the minor allele from the major allele .", "In Figure 3A , we analyzed SNPs in 3\u2032 UTRs for 4214 genes and SNPs in polyadenylation signals for 33 genes .", "In Figure 8 , we analyzed regulatory element SNPs in 62 , 16 , 7 , and 20 genes respectively in 3\u2032 UTRs of class I genes , shared regions of 3\u2032 UTRs of class II genes , differential regions of 3\u2032 UTRs of class II genes whose long forms were more abundant , and differential regions of 3\u2032 UTRs of class II genes whose short forms were more abundant .", "A psiCheck-2 vector ( Promega ) was modified to generate a 3\u2032 UTR reporter vector , pOKY001 ( Figure S5 and Table S8 ) , in which we removed the SV40 late polyA signal from the firefly luciferase gene and replaced it with a tag for ligation independent cloning ( LIC ) [70] of 3\u2032 UTR sequences of interest .", "Genomic DNA was isolated from lymphoblastoid cell lines using the MasterPure DNA Purification Kit ( Epicentre ) , and 3\u2032 UTR sequences were amplified from genomic DNA using Phusion HF Polymerase ( New England Biolabs ) with PCR primers with LIC tags .", "PCR products were cloned into pOKY001 using LIC , and plasmids were purified using Plasmid Midi Kit ( Qiagen ) .", "Site-directed mutagenesis of plasmids was performed using QuikChange XL Site-Directed Mutagenesis Kit ( Agilent ) according to the manufacturer's instructions .", "All primers used for PCR and mutagenesis are listed in Table S7 .", "Cloned sequences of all reporter vectors were checked by capillary sequencing .", "The list of reporter vectors is provided in Table S8 .", "HEK293T cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and 1% non-essential amino acids in six-well culture plates .", "For each of two independent transfections for each construct , at 90% confluency , 1 \u00b5g of plasmid DNA was transfected using Lipofectamine 2000 ( Invitrogen ) according to the manufacturer's instructions .", "After incubating at 37\u00b0C under 5% carbon dioxide for 24 h , the cells were washed with Dulbecco's Phosphate-Buffered Saline ( DPBS ) , detached from plates by adding 300 \u00b5L of trypsin-EDTA and incubated at 37 degrees for 5 minutes .", "After adding 900 \u00b5L of media , the detached cells were split into three 2 mL tubes , and pelleted by spinning at 1000\u00d7 g for 3 minutes .", "The cell pellets were washed with DPBS and then flash-frozen in liquid nitrogen and stored at \u221280\u00b0C .", "Given one cell pellet from each of two reporter transfections ( see above ) , cells from each pellet were lysed and used for two technical replicates of a dual luciferase assay using the Dual Luciferase Reporter Assay System ( Promega ) .", "Luminescence from the activities of firefly and Renilla luciferases were measured sequentially in a Turner BioSystems Veritas Luminometer .", "The ratio of luminescence measurements between Firefly and Renilla luciferases was used for all analyses .", "To measure expression of short and long forms for a given gene with alternative polyadenylation , we considered quantitative PCR assays that would interrogate regions just upstream of the inferred cleavage sites of the short and long forms ( which we refer to below as SU and LU , respectively ) .", "We reasoned that expression measurements of SU would reflect abundance of both the short and long transcript forms , whereas LU would reflect abundance of the long form only .", "For a given transcript in a given experiment , the absolute mRNA level ( calculated from quantitative PCR reactions on a biological sample and standards , as described below ) from the primer set amplifying LU was used as the expression level for the long form; the expression level of the short form was calculated by subtracting the absolute mRNA level of LU from the absolute mRNA level of SU for all genes except IRF5 .", "Primer sequences for IRF5 were taken from [15] , where the primer set for SU hybridized to the polyA tail and only amplified the short form .", "Thus , for IRF5 , the mRNA count from SU was used as the expression level for the short form .", "All other primers were designed using Primer3 Plus [71] , checked for potential hairpins and primer dimers using BeaconDesigner Web Edition ( PREMIER Biosoft International ) .", "Primers were synthesized by Elim Biopharmaceuticals .", "For each transcript in each experiment , measurements were normalized by absolute mRNA counts of Renilla luciferase or GAPDH genes .", "All primer sequences are in Table S7 .", "Expression measurements on cells transfected with 3\u2032 UTR reporters were performed as follows .", "Given one cell pellet from each of two transfections ( see above ) , total RNA was isolated from each pellet separately using TRIzol ( Invitrogen ) .", "For each sample , genomic DNA was removed from 10 \u00b5g of total RNA using TurboDNase ( Ambion ) .", "Single-stranded cDNA was synthesized from 2 \u00b5g of DNased RNA using oligo ( dT ) ( Invitrogen ) and SuperScript III reverse transcriptase ( Invitrogen ) , and then RNA was removed using RNase H ( Invitrogen ) .", "For each of two technical replicates for each primer set and sample , the cDNA was amplified using the DyNamo HS SYBR Green QPCR Kit ( Thermo Scientific ) for 40 cycles on a Strategene MX3000P qPCR instrument .", "We calculated absolute numbers of mRNA molecules amplified from each primer set in each experiment by comparing the number of amplification cycles taken to reach a given threshold against a standard curve constructed using samples of a synthetic template ( gBlocks Gene Fragments , Integrated DNA Technologies ) of known absolute numbers of molecules , in serial dilutions extending from 107 to 101 molecules .", "Reporters incorporating 3\u2032 processing signals were constructed as follows .", "For each length form of each gene , we aimed to clone a region centered on the cleavage site as inferred from the transcript end position observed in 3\u2032-end RNA-seq , bounded by the 40 bp upstream and 40 bp downstream in the genomic sequences and flanked by LIC tags ( region sequences for EIF2A , IRF5 , DIP2B , and NAB1 were taken from the haplotype giving rise to production of both long and short forms for cell lines GM17220 , GM10860 , GM10860 , and GM17220 respectively ) .", "This 115-bp construct was produced by PCR stitching two opposite-strand DNA oligos of 60 bp and 78 bp ( Integrated DNA Technologies ) with 23 bp overlap using Phusion HF Polymerase ( New England Biolabs ) for 3 cycles .", "PCR products were cloned into pOKY001 and purified as described above .", "Transfection and dual luciferase assay were performed as above .", "For each transcript half-life measurement , cells transfected with a luciferase reporter harboring the complete 3\u2032 UTR from the haplotype giving rise to production of both long and short forms of a given gene were incubated at 37\u00b0C under 5% carbon dioxide for \u223c24 h .", "A stock solution of actinomycin D dissolved in DMSO was added to achieve a final actinomycin D concentration in tissue culture media of 10 \u00b5g/mL .", "After incubation times as indicated in Figure 5 and Figure S4 , cells were harvested for quantitative PCR measurements as above or Northern blotting as described below .", "To generate expression vectors for ARE-binding proteins , the tristetraprolin ( gene name ZFP36 ) coding sequence was amplified from the vector pGFP-TTP and the AUF1 p37 coding sequence from pCDEF-His-AUF1-p37 ( both kind gifts from B . Glaunsinger ) .", "Each gene was cloned into the pcDNA3 mammalian expression vector ( Invitrogen ) .", "Syn-has-miR-101 , a miRNA mimic of has-miR-101 , was purchased from Qiagen ( MSY0000099 ) .", "For dose-response assays , expression vectors or miRNA mimic were mixed with the corresponding luciferase reporter vectors ( Figure 6 ) and then transfected as above .", "Lipopolysaccharide from E . coli K12 ( Invivogen ) was added to each of the human B-lymphoblastoid cell lines GM10860 , GM17106 , and , GM17207 to a final concentration of 5 \u00b5g/mL in 6-well tissue culture plates , and incubated at 37\u00b0C under 5% carbon dioxide .", "The cells were harvested after 0 , 1 , 2 , 4 , 6 , 8 , 12 , and 16 hours by centrifuging at 1000\u00d7 g for 3 minutes , washing with DPBS , and then flash-freezing in liquid nitrogen .", "RNA was isolated using Trizol ( Invitrogen ) .", "The DIG Northern Starter Kit ( Roche ) was used for Northern blotting .", "A 400 bp region of firefly luciferase gene was amplified from luciferase reporter vector pOKY001 , and cloned into pcDNA3 vector ( Invitrogen ) .", "A digoxigenin ( DIG ) labeled RNA probe for firefly luciferase was created by in vitro transcription with SP6 RNA polymerase after linearizing the plasmid with BamHI .", "Total RNA isolated using Trizol ( Invitrogen ) was cleaned using RNeasy columns ( Qiagen ) with on-column DNase-digestion .", "Between 1 and 10 \u00b5g of total RNA was loaded and run on 1% denaturing agarose gels .", "The gels were stained with SYBR Gold ( Invitrogen ) and then the separated RNA was blotted onto nylon membranes ( Roche ) and UV cross-linked .", "Both the gels and the membranes were imaged on Blue Light Transilluminator ( Invitrogen ) to check the transfer of SYBR-stained RNA .", "28S ribosomal RNA bands on the membranes were used as the loading control in Figure S4 .", "Each membrane was subjected to prehybridization , probe hybridization , low and high stringency washing , and detection procedures recommended by the manufacturer ( Roche ) .", "Anti-digoxigenin-AP ( Roche ) and CDP-Star ( Roche ) were used for chemiluminescent detection of the DIG-labeled RNA probe .", "To maximize length resolution for EIF2A , total RNA was incubated at 37\u00b0C for 30 min with RNaseH ( Invitrogen ) and an oligo antisense to a 20 bp region upstream of the binding site for the Northern probe in the firefly gene .", "For DIP2B , the denaturing agarose gel was treated with 0 . 05% NaOH and washed with water before blotting onto the nylon membrane .", "The expression data reported in this paper have been deposited in the Gene Expression Omnibus ( GEO ) ( http://www . ncbi . nlm . nih . gov/geo ) database ( series accession number GSE33154 ) ."]], "headings": ["Introduction", "Results", "Discussion", "Materials and Methods"], "abstract": ["Gene expression varies widely between individuals of a population , and regulatory change can underlie phenotypes of evolutionary and biomedical relevance .", "A key question in the field is how DNA sequence variants impact gene expression , with most mechanistic studies to date focused on the effects of genetic change on regulatory regions upstream of protein-coding sequence .", "By contrast , the role of RNA 3\u2032-end processing in regulatory variation remains largely unknown , owing in part to the challenge of identifying functional elements in 3\u2032 untranslated regions .", "In this work , we conducted a genomic survey of transcript ends in lymphoblastoid cells from genetically distinct human individuals .", "Our analysis mapped the cis-regulatory architecture of 3\u2032 gene ends , finding that transcript end positions did not fall randomly in untranslated regions , but rather preferentially flanked the locations of 3\u2032 regulatory elements , including miRNA sites .", "The usage of these transcript length forms and motifs varied across human individuals , and polymorphisms in polyadenylation signals and other 3\u2032 motifs were significant predictors of expression levels of the genes in which they lay .", "Independent single-gene experiments confirmed the effects of polyadenylation variants on steady-state expression of their respective genes , and validated the regulatory function of 3\u2032 cis-regulatory sequence elements that mediated expression of these distinct RNA length forms .", "Focusing on the immune regulator IRF5 , we established the effect of natural variation in RNA 3\u2032-end processing on regulatory response to antigen stimulation .", "Our results underscore the importance of two mechanisms at play in the genetics of 3\u2032-end variation: the usage of distinct 3\u2032-end processing signals and the effects of 3\u2032 sequence elements that determine transcript fate .", "Our findings suggest that the strategy of integrating observed 3\u2032-end positions with inferred 3\u2032 regulatory motifs will prove to be a critical tool in continued efforts to interpret human genome variation ."], "summary": ["Messenger RNAs carry the instructions necessary to synthesize proteins that do work for the cell .", "Extending beyond the protein-coding sequence of a given mRNA is an additional stretch of sequence , harboring signals that govern how much protein is made and how long the mRNA remains in the cell before it is broken down .", "The incorporation of this end region into mature mRNA is itself subject to change; for the vast majority of human genes , how and why cells use different mRNA ends remains largely unknown .", "In this work , we surveyed mRNA ends from \u223c10 , 000 genes in immune cells from genetically distinct human individuals .", "We found that mRNA end positions were not randomly distributed , but rather preferentially flanked the locations of regulatory signals that govern mRNA fate .", "The usage of these mRNA length forms and regulatory elements varied across individuals and could be dissected molecularly .", "Our results uncover key mechanisms and regulatory effects of transcript end processing , particularly as these are perturbed by genetic differences between humans ."], "keywords": ["genetics", "biology", "genomics", "genetics and genomics"]}

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-{"id": "elife-37443-v3", "year": "2018", "title": "Cerebellar implementation of movement sequences through feedback", "sections": [["Most movements are comprised of sequences .", "From the complex routines that gymnasts perform to intricate dance numbers to simply reaching for an object , our movements are comprised of sequences of movements that are learned through practice .", "The cerebellum has been long implicated in learning and execution of accurate movements ( Doyon et al . , 1997; Shin and Ivry , 2003; Leh\u00e9ricy et al . , 2005; Krupa et al . , 1993; Lisberger , 1994; Diener and Dichgans , 1992 ) .", "Movement sequences as well as multi-joint movements are particularly sensitive to cerebellar dysfunction ( Shin and Ivry , 2003; Diener and Dichgans , 1992; Doyon et al . , 2002 ) .", "For example , one of the hallmark deficits of cerebellar pathology is dysdiadochokinesia ( Diener and Dichgans , 1992 ) \u2013 an inability to perform a rapid alternating sequence of movements .", "Patients with cerebellar lesions display severe deficits ( Doyon et al . , 1997 ) in sequence learning or are unable to learn sequences at all ( Shin and Ivry , 2003 ) , even with modest impairments in learning of single-component , directly cued movements ( Spencer and Ivry , 2009 ) .", "Studies of sequence learning in nonhuman primates ( Desmurget and Turner , 2010; R\u00fcnger et al . , 2013 ) demonstrated that with repeated training components of movement in the sequence start to be initiated predictively , before the arrival of sensory cue .", "Since one of the fundamental properties of cerebellar learning is the ability to learn a predictive response ( Marr , 1969; Bastian , 2006; Shadmehr et al . , 2010; Therrien and Bastian , 2015 ) , these results also indirectly imply a strong cerebellar contribution to sequence learning .", "Although results of these studies suggest cerebellar involvement in the learning and execution of movement sequences , most of what we know about cerebellar mechanisms of learning comes from studies utilizing single-component movements .", "These include adaptation of the vestibule-ocular reflex , adaptation of saccadic and smooth pursuit eye movements and conditioning of eyelid responses .", "In principle , the cerebellar mechanisms underlying movement sequences could be different from , or at least somewhat different from those mediating single-component movements .", "If so , these mechanisms are largely unknown .", "In contrast , we tested the hypothesis that a simple elaboration of cerebellar mechanisms that mediate learning single-component movements is sufficient to explain cerebellar learning and implementation of movement sequences .", "Three possible ( but not mutually exclusive ) ways of implementing cerebellar learning of movement sequences are illustrated in Figure 1 .", "With the first possibility a specific external cue is associated with a specific movement component in the sequence ( Figure 1A ) .", "The second option is a variant of the first one .", "Here only single external cue is present , but it persists in time through the whole sequence , so that different movement components are elicited by signals associated with different times during the cue ( Figure 1B ) .", "Finally , Figure 1C illustrates a possibility where feedback signals from one movement component are used to learn the next component .", "The design of most experiments does not permit distinguishing between these possibilities .", "For example , a number of studies ( Choi and Moore , 2003; Moore and Choi , 1997; Freeman et al . , 2003; Halverson et al . , 2015; Jirenhed et al . , 2017 ) used eyelid conditioning to train subjects to respond with a sequence of two eyelid responses .", "In these experiments , however , the external cue either explicitly extended through the whole sequence , or involved an auditory signal that can elicit persistent activity in working-memory areas such as mPFC ( Siegel et al . , 2012 ) , precluding the possibility of determining which type of sequence learning applies ( Figure 1B and Figure 1C are both possible ) .", "We utilized the practical and conceptual advantages of eyelid conditioning to test explicitly the sufficiency of feedback signal ( s ) ( FS ) for sequence learning ( Figure 1C ) .", "We demonstrate directly that the cerebellum can learn to chain together a sequence of inter-connected movement components by using FS from one component to serve as a cue for the next component in the sequence ( Figure 1C ) .", "In a standard eyelid conditioning experiment , activation of mossy fiber inputs by a sensory stimulus is the cue the cerebellum uses to learn a predictive conditioned eyelid response ( CR ) ( Figure 1C , first movement ) .", "A sensory stimulus can be replaced by direct electrical stimulation of mossy fibers , which is an equally effective cue to support learning ( Steinmetz et al . , 1985 ) , but has the advantage of restricting inputs only to the cerebellum .", "We employed electrical stimulation of mossy fibers as a cue to learn a single-component response .", "We then arranged the timing of stimuli in subsequent training so that the second component in the sequence could not be supported by the mossy fiber stimulation eliciting the first component and thus , only option shown in Figure 1C would be possible .", "We demonstrate robust learning of later components , including sequences of movements produced by the same muscle and sequences with different muscle groups used for the different components .", "A variety of control experiments showed how the presence and timing of the later components of responses were coupled to the first component and not to the mossy fiber stimulation cue that initiated the first component .", "In vivo recordings from cerebellar cortex showed that Purkinje cell activity relates as strongly to the latter components of sequences as it has been shown to relate to single-component eyelid responses .", "Together , these results show how the well-characterized cerebellar mechanisms of learning single movement components can be extended to learning of movement sequences and provide a general framework for how the cerebellum can use feedback signals to learn to chain together appropriately timed responses to produce a movement sequence ."], ["To test the hypothesis that the cerebellum can use feedback signals from a movement as the cue for the next movement in a sequence , we started by training rabbits using electrical stimulation of mossy fibers as a cue ( to which we refer as the conditioned stimulus or CS ) .", "All subjects ( New Zealand albino rabbits ) were initially trained by pairing a mossy fiber stimulation CS with a reinforcing unconditioned stimulus ( US , electrical stimulation of the skin near the eye ) .", "The inter-stimulus interval ( ISI ) between CS onset and US onset was 500 ms . As has been previously demonstrated , this training yields robust and well-timed conditioned eyelid responses in response to the mossy fiber CS ( Steinmetz et al . , 1985; Kalmbach et al . , 2011 ) .", "Example conditioned eyelid responses are shown in Figure 2A .", "Use of mossy fiber stimulation as the CS ensured that the CS was restricted to the cerebellum and did not propagate to areas that could provide a delayed secondary input to the cerebellum ( Siegel and Mauk , 2013; Halverson et al . , 2010 ) , which can be the case for auditory stimuli commonly used in eyelid conditioning .", "Moreover , the control over CS duration allowed control over the time gap between CS offset and the US .", "Once subjects reached robust responding to the mossy fiber CS training was switched to protocols teaching a sequence of CRs .", "The design of an ipsilateral sequence training protocol is illustrated in Figure 2B .", "Training trials involved presentation of the same mossy fiber stimulation CS ( 500 ms long ) used during initial training , but now , depending on the amplitude of the CR , the US was presented at one of two different times .", "If the CR amplitude was below the target amplitude ( 3 mm , half of full closure ) the US was presented as normal at CS offset ( designated US1 ) .", "When the CR amplitude was at or above the target value at the scheduled US1 time , the US was instead presented 600 ms after CS offset ( designated as US2 ) .", "The purpose of the US1 trials was to ensure continued robust responding to the mossy fiber CS , whereas the purpose of the US2 trials was to train a second component of the movement .", "With this procedure we could then test whether the expression of the first CR could serve as a signal for the cerebellum to learn the second CR .", "A 600 ms gap between the offset of the mossy fiber stimulation CS and the presentation of US2 was used based on previous findings that a temporal gap larger than 400 ms between the offset of a mossy fiber stimulation CS and the onset of a US does not support cerebellar learning of eyelid CRs ( Kalmbach et al . , 2010a ) .", "Given these factors , the ability to learn a second CR elicited near the time of US2 would suggest that the feedback information about the first CR is sufficient for the cerebellum to use as a new \u2018CS\u2019 to learn the subsequent CR .", "All subjects ( N\u00a0=\u00a08 ) successfully acquired a sequence of ipsilateral CRs ( Figure 2C\u2013F ) with the timing of the second peak appropriate for the time at which US2 was presented ( Figure 2\u2014figure supplement 1A ) .", "The probability of the second CR , defined as the fraction of trials with first CR where second CR was present too , and second CR amplitude grew monotonically over several sessions of training ( Figure 2C and D respectively , blue lines , one-way ANOVA , F ( 9 , 63 ) = 6 . 44 , p<10\u22126; F ( 9 , 63 ) = 7 . 16 , p<10\u22127 for second CR probability and amplitude respectively ) , and eventually reached asymptotic value , paralleling typical acquisition curves in eyelid conditioning .", "Cerebellar learning is associative .", "As a consequence , presentation of trials with omitted US diminishes CRs in a process called extinction .", "As a test of whether the second CRs are associatively learned , we tested whether they can be selectively extinguished like normal CRs .", "Over three extinction sessions ( Figure 2C , D , G , H ) , the US1 was still presented according to the rule described above , but US2 was omitted .", "If second CRs are learned associatively , the absence of reinforcing US2 should lead to their gradual extinction .", "As expected , the probability and amplitude of the second CRs ( blue lines ) monotonically decreased ( One-way ANOVA , F ( 23 , 120 ) = 4 . 96 , p<10\u22129; F ( 23 , 120 ) = 4 . 92 , p<10\u22129 for second CR probability and amplitude respectively ) during extinction sessions , without any effect on the probability and amplitude of the first CRs ( black lines ) ( One-way ANOVA , F ( 23 , 120 ) = 1 . 45 , p=0 . 10 , F ( 23 , 120 ) = 1 . 35 , p=0 . 15 for first CR probability and amplitude ) .", "Similar to extinction in conventional eyelid conditioning protocols , spontaneous recovery ( Weidemann and Kehoe , 2004; Thanellou and Green , 2011; Ohyama et al . , 2010 ) of the second CRs was present at the beginning of the second and third extinction sessions ( paired Wilcoxon signed rank test , p=0 . 023 for second CR probability , p=0 . 032 for second CR amplitude ) .", "Since the number of trials with first CR amplitude larger than the 3 mm target was variable between sessions and animals , the same data plotted as a function of total trial number is shown in Figure 2\u2014figure supplement 1C , D .", "Plotted this way , the acquisition and extinction rates appear similar to what was observed in conventional eyelid conditioning training protocols .", "These observations are consistent with the notion that the second components in the sequence are acquired through standard cerebellar-dependent associative learning .", "The primary hypothesis we sought to test was that the cerebellum uses a feedback signal ( FS ) of some sort about the first CR as a separate \u2018CS\u2019 to learn the second CR ( Figure 2I ) .", "Such a FS should occur anytime there is a CR , whether the CR is elicited by the mossy fiber stimulation CS or not .", "This suggests the prediction for ipsilateral sequence of CRs , illustrated in Figure 2I .", "When a well-trained subject is presented with CS-alone trials , the mossy fiber stimulation CS will elicit the first CR , then a FSL from the first CR will elicit the second CR .", "Because the second CR is produced by the same muscles of left eyelid and is driven by likely the same neurons in cerebellar nuclei , then if the second CR is large enough , its FS should be similar to the FSL from the first CR .", "Since the cerebellum already learned to associate FSL with upcoming US2 , it should produce a predictive CR with appropriate timing .", "Thus , we would expect to see a third ( and perhaps fourth and fifth , etc . ) CRs on CS-alone trials , though subjects were never explicitly trained to produce them .", "This is indeed what we observed ( Figure 2G , J ) .", "Third and following CRs appeared in late acquisition sessions once robust second CR performance was established and disappeared with the extinction of the second CRs ( Figure 2J , H ) .", "On 36% of CS-alone trials where there was a second CR , there was also a third CR .", "The relative time between peaks of second and third CRs was similar to the time between peaks of first and second CRs ( Figure 2\u2014figure supplement 1B ) .", "Though third CRs were never explicitly reinforced , they remained through late acquisition sessions and subsequent control experiments , as long as second CRs also remained .", "This observation is consistent with the idea that both second and third CR share the same \u2018CS\u2019 as FSL and reinforcement of second CRs on a portion of trials was sufficient to maintain both second and third CRs .", "Because we recorded eyelid position for 2500 ms on each trial , we were only able to observe up to four CRs with ipsilateral sequence training .", "The existence of third CRs is inconsistent with the idea that the original mossy fiber stimulation CS is driving the expression of the second ( and later ) CRs .", "Complex movements typically involve more than one muscle group and often bilateral coordination ( Castiello et al . , 1993; Kelso et al . , 1979 ) .", "We therefore asked whether this training protocol can support a sequence where a left eyelid CR would be followed by a right eyelid CR ( Figure 3A ) .", "As before , subjects were initially trained with mossy fiber stimulation CS to produce left eyelid CRs at ISI 500 ms . After successful acquisition , subjects were switched to a contralateral CRs sequence protocol .", "Here again , if the left eyelid CR amplitude was lower than the target ( 3 mm , half-sized CR ) , USL was presented to the left eye to maintain robust responding of the left eyelid CRs .", "If however , the amplitude of the left CR was higher than the target , USL was omitted and USR was presented to the right eye .", "During initial acquisition , the interval between CS offset and USR was typically 400 ms ( N\u00a0=\u00a04 ) , but for some subjects was 300 ms ( N\u00a0=\u00a01 ) or 500 ms ( N\u00a0=\u00a01 ) .", "We chose to use a shorter duration of the gap interval compared to ipsilateral sequence training , because the pilot data showed that most subjects were unable to learn a contralateral sequence with 600 ms gap interval from na\u00efve right eyelid state .", "In this situation , however , there is less concern about the ability of the mossy fiber stimulation CS to drive the right eyelid CR , since the mossy fiber stimulation CS was delivered through electrodes implanted in the left middle cerebellar peduncle .", "The only way that CS could propagate to the right cerebellar hemisphere was by antidromic activation of neurons in pontine nucleus that have bilateral axons projections .", "Since the number of such neurons is extremely low ( Serapide et al . , 2002; Tan and Gerrits , 1992; Kratochwil et al . , 2017 ) , it is unlikely that mossy fiber stimulation CS could support acquisition of right eyelid CRs .", "Several control experiments described later confirm this notion .", "All subjects ( N\u00a0=\u00a06 ) successfully acquired a contralateral sequence of CRs where a left eyelid response was followed by a right eyelid response ( Figure 3 B\u2013E , F ( 9 , 46 ) = 14 . 28 , p<10\u22129; F ( 9 , 46 ) = 9 . 05 , p<10\u22126 for right CR probability and amplitude respectively ) .", "Right eyelid CRs were adaptively timed , peaking near the time of USR delivery and thus corresponding to the gap interval used during training ( Figure 3\u2014figure supplement 1A , B ) .", "Unlike the ipsilateral training protocol , here there were no third CRs produced by either left or right eyelid .", "Assuming that all but the first ( left ) CRs are driven by FS , and assuming that FSL from left eyelid CRs and FSR from the right eyelid CRs are different signals , this is the expected result .", "In this protocol the FSL are paired with a right eyelid USR and the FSR are never paired with a US .", "Thus there should only be a left eyelid CR driven by the mossy fiber stimulation CS and a right eyelid CR driven by FSL from left eyelid CR .", "Through 86 sessions with contralateral sequence protocol from 6 subjects , responses following right eyelid CR were not observed for either the left or right eyelid ( Data not shown ) .", "As with the ipsilateral sequence protocol , we verified the associative nature of right eyelid CRs by performing three extinction sessions of right eyelid CRs ( red lines in Figure 3B , C , F , Figure 3\u2014figure supplement 1C , D , One-way ANOVA , F ( 23 , 96 ) = 4 . 04 , p<10\u22127; F ( 23 , 96 ) = 6 . 89 , p<10\u221212 for right CR probability and amplitude respectively ) .", "Extinction of right eyelid CRs did not influence the performance of preceding left eyelid CRs ( One-way ANOVA , F ( 23 , 96 ) = 0 . 96 , p=0 . 52; F ( 23 , 96 ) = 0 . 82 , p=0 . 70 for left eyelid CR probability and amplitude respectively ) .", "Spontaneous recovery of right eyelid CRs at the beginning of second and third extinction sessions was present here as well ( paired Wilcoxon signed rank test , p=0 . 055 for right CR probability , p=0 . 020 for right CR amplitude ) .", "Following extinction sessions , subjects were switched to contralateral training protocol with a longer interval between CS offset and USR ( 500 ms , transition not shown ) .", "In the analysis of contralateral sequence data , data produced on sessions with different gap intervals are indicated with different colors and corresponding legend , if applicable .", "The hypothesis that the cerebellum generates the later CRs using FS from the first CR gives rise to several testable predictions .", "We designed two control experiments and performed additional analyses on training session data as a formulation of these predictions to test relatively directly whether the second CR in a sequence ( second left eyelid CR in ipsilateral protocol and right eyelid CR in contralateral protocol ) is driven by FS from the first CR and not by the mossy fiber stimulation CS itself .", "The design of the first control experiment is based on a straightforward prediction \u2013 the absence or a decline in the first CRs should also lead to an absent or diminished second CRs .", "We applied an extinction protocol designed to promote extinction of the first CRs and tested the effects on the second CRs .", "The setup of this experiment is shown in Figure 4A , D .", "Regardless of first CR amplitude , on these sessions the second CR was always reinforced with US2 ( or USR in the contralateral sequence ) , while US1 ( USL ) was never delivered .", "A decline of the second CRs despite the presence of reinforcing US2 ( or USR ) , would indicate that expression of the second CRs requires the expression of the first CR in the sequence .", "Figure 4B , E shows CRs amplitudes is a sequence as a function of block number .", "Since extinction typically took longer for subjects trained with the ipsilateral sequence , most required two extinction sessions .", "The same type of plot for CRs probability is shown in Figure 4\u2014figure supplement 1A , B .", "For subjects trained in\u00a0either the ipsilateral or contralateral sequences , as the amplitude and probability of left eyelid CR decreased , so did the amplitude of the second left eyelid CR in ipsilateral sequence ( r\u00a0=\u00a00 . 86 , p<10\u221213 ) ; or right eyelid CRs in contralateral sequence ( r\u00a0=\u00a00 . 80 , p<10\u221212 ) .", "To quantify this effect further we evaluated the probability of the second CRs conditioned on first CR amplitude .", "For that , we first plotted second CR amplitude versus first CR amplitude ( Figure 4\u2014figure supplement 1C , D , each dot represents a single trial from this control experiment ) .", "We then used that data to calculate the probability of the second CR conditioned on first CR amplitude ( Figure 4C , F ) .", "For both ipsilateral and contralateral sequence , the probability of observing second CRs decayed with decrease in first CR amplitude ( Chi-square analysis , \u03c72 ( 6 , 836 ) =60 . 90 , p<10\u221211 , \u03c72 ( 6 , 572 ) =92 . 7 , p<10\u221218 , for ipsilateral and contralateral sequence protocols respectively ) .", "On trials without the first left eyelid CR , the probability of second CRs was negligible ( probability of second left eyelid CR\u00a0=\u00a00 . 026\u00a0\u00b1\u00a00 . 009 for ipsilateral sequence protocol , probability of right eyelid CR\u00a0=\u00a00 . 023\u00a0\u00b1\u00a00 . 01 for contralateral sequence protocol ) .", "These data show that the presence of a first CR is required for the expression of a second CR .", "If a FS is the cue that drives the second CR , then it should not be necessary for the first CR to be elicited by the original training CS .", "Any CR produced by that eyelid , however it was elicited , should serve as an effective source of a FS to produce a second CR .", "The schematic of the second control experiment that we used to test this prediction is shown in Figure 5A , E .", "We started by training subjects to produce left eyelid CRs ( ISI 500 ms ) with two different types of CS: CS1 and CS2 .", "Electrical stimulation of mossy fibers was always used as CS1 , CS2 was either a 500 ms mossy fibers stimulation delivered through a separate electrode ( N\u00a0=\u00a05 , 2\u00a0subjects for ipsilateral sequence and 3 subjects for contralateral sequence ) or a 500 ms 1 kHz tone ( N\u00a0=\u00a05 , 3 subjects for ipsilateral sequence and 2 subjects for contralateral sequence ) .", "At the end of this pre-training each subject elicited robust CRs to the presentation of either CS1 or on separate trials to CS2 .", "Then , during sequence training as described above , only CS1 was used , CS2 was never used for sequence training , neither for training left eyelid CRs at ISIs other than 500 ms nor for right eyelid CR training .", "After successful acquisition of ipsi- or contralateral sequence of CRs , subjects were then presented with occasional CS2-alone probe trials .", "This input should elicit left eyelid CRs , since this is how subjects were trained .", "However , the essential test is whether the second CRs in the sequence are also present on CS2-alone trials .", "Because the US2 ( USR ) was never presented during the CS2 trials , the presence of second CRs would indicate that the cerebellum did not use CS1 to learn the second CR in the sequence , but rather used FS from the first CR .", "Again , this is indeed what we observed .", "Example eyelid responses on CS2-alone trials are shown in Figure 5B , F for subjects trained in ipsilateral or contralateral sequence respectively .", "On most CS2-alone trials with the first left eyelid CR present we also observed the rest of CRs in the sequence the subject was trained to with CS1 .", "The summary across all sessions with CS2 test trials is shown in Figure 5C , G .", "Here each dot shows the probability within a session of the second CR in a sequence , the color indicates a group of trials during which the second CR probability was calculated , based either on CS type ( CS1 or CS2 ) or amplitude of the first left eyelid CR .", "On trials where the amplitude of first left eyelid CR was larger than 3 mm , the probability of observing other responses in a sequence was the same on trials with either CS1 or CS2 ( Tukey\u2019s post hoc test , p=0 . 73 , blue versus brown bars for ipsilateral sequence; p=0 . 51; red versus brown bars for contralateral sequence ) .", "Importantly , on CS2 trials without the first response ( violet bars ) , there were no other CRs in the sequence ( ipsilateral sequence: one-way ANOVA comparing brown versus violet bars: F ( 3 , 61 ) = 187 , p<10\u221228; Tukey\u2019s post hoc test p<10\u22129; contralateral sequence: one-way ANOVA comparing brown versus violet bars: F ( 3 , 62 ) = 159 , p<10\u221229; Tukey\u2019s post hoc test , p<10\u22129 ) .", "In addition , we repeated CS2-alone test sessions with subjects either not trained with ( N\u00a0=\u00a01 and N\u00a0=\u00a03 for ipsi- and contralateral sequence respectively ) or extinguished from ( N\u00a0=\u00a04 and N\u00a0=\u00a02 for ipsi- and contralateral sequence respectively ) producing a sequence of CRs .", "In this case , while first left eyelid CRs amplitudes were larger than 3 mm , CS2 alone trials did not elicit other CRs in the sequence ( ipsilateral sequence: dark blue bars , second CR probability\u00a0=\u00a00 . 006\u00a0\u00b1\u00a00 . 008 , comparing violet versus dark blue bars with Tukey\u2019s post hoc test results in p=0 . 99; contralateral sequence: dark blue bars , right CR probability\u00a0=\u00a00 . 041\u00a0\u00b1\u00a00 . 028 , comparing violet versus dark blue bars with Tukey\u2019s post hoc test results in p=0 . 84 ) .", "In addition , for subjects trained to produce a sequence of CRs , we observed a significant correlation across sessions between probabilities of second CRs in the sequence on CS1 and CS2 trials ( Figure 5D , H , r\u00a0=\u00a00 . 48 , p=0 . 026 for ipsilateral sequence , r\u00a0=\u00a00 . 6 , p=0 . 007 for contralateral sequence ) .", "Thus , on sessions with a more robust sequence of CRs performed on CS1 trials , subjects also showed larger probability of producing a sequence of CRs on CS2-alone trials .", "Together , these experiments demonstrate that while the first CR in the sequence is driven by the mossy fiber stimulation CS , CRs that follow are associated with FS from the previous CR .", "Two additional predictions can be tested through additional analyses of the normal sequence-training sessions .", "Parallel to the logic of the first control experiment described above , we should expect that on trials without a first CR there should be no second CR .", "To test this prediction we used eyelid responses on CS-alone trials from sessions where the overall probability of second CRs was greater than 40% .", "Panels A and B in Figure 6 show two example trials from subjects trained respectively in ipsilateral and contralateral sequence of CRs .", "Data from all subjects on all CS-alone trials are shown in Figure 6C , D .", "The amplitude of the second CR plotted versus amplitude of the first CR for subjects trained in ipsilateral sequence of CRs is shown in Figure 6C .", "Each dot represents data from a single CS-alone trial .", "Similarly , Figure 6D shows right eyelid CR amplitudes versus left eyelid CR amplitudes for subjects trained to produce contralateral sequence of CRs .", "Data corresponding to the example trials are indicated by grey dots .", "In both plots , horizontal and vertical solid black lines represent nonCR cutoffs ( CR amplitudes\u00a0<0 . 3 mm ) , diagonal is shown by a dotted black line .", "While there were trials with first CR , but no second CR ( dots below horizontal line on the bottom ) , there were no trials without a first CR , but with the second CR ( to the left of vertical line ) .", "Figure 6E and F show the same data plotted as a probability of the second CR ( or right eyelid CR ) , conditioned on the first CR amplitude .", "For both protocols , the probability of the second CR started to decrease on trials with the first CR amplitude smaller than 3 mm , which was used during sequence training ( Chi-square analysis , \u03c72 ( 6 , 571 ) =70 . 13 , p<10\u221213 , \u03c72 ( 6 , 578 ) =92 . 4 , p<10\u221220 , for ipsilateral and contralateral sequence protocols respectively ) .", "Most importantly , on trials without a first CR , the probability of second CRs was negligible ( probability of second left eyelid CR\u00a0=\u00a00 . 037\u00a0\u00b1\u00a00 . 047 for ipsilateral sequence protocol , probability of right eyelid CR\u00a0=\u00a00 for contralateral sequence protocol ) .", "There is a natural trial to trial variability in the timing of the CRs .", "If a FS from the first CR is a signal for the second CR , then it follows that the timing of the first and second CRs should co-vary on a trial by trial basis .", "For example , on trials with relatively early first CRs the following CRs should also happen earlier than average; on trials with late first CRs the following CRs should also happen later .", "Example trials with earlier ( on top ) and later ( on bottom ) first CR onset times are shown in Figure 7A and B from ipsilateral and contralateral sequence respectively .", "One can notice even from example trials that the whole sequence of CRs is shifted with the timing of the first CR .", "We investigated the degree of co-variation between timing of CRs in sequence using a variety of CR timing measures , spanning from CR onset time to CR peak time .", "These timing measures are indicated by orange dots on eyelid position profile in Figure 7A .", "The timing measures were defined for every trial when the amplitude of both responses in a sequence was larger than 2 mm ( except for analysis of third and later CRs in ipsilateral sequence , only CS-alone trials were used there ) .", "Data from two of pairs of timing measures are shown for each protocol in Figure 7C\u2013F .", "Here each dot represents a single trial , with color indicating the pair of CRs for ipsilateral sequence or the gap interval for contralateral sequence .", "We applied two types of analyses to investigate the temporal dependence of CRs in the sequence .", "First , for every pairwise combination of CR timing measures we calculated a Pearson correlation coefficient .", "We found that the majority of combinations of measures showed a significant trial-to-trial co-variation ( Figure 7\u2014figure supplement 1 ) , for both ipsilateral and contralateral sequence of CRs .", "Second , for every trial of each CR timing measures we calculated an inter-CR time interval; correspondingly color-coded distributions of inter-CR time intervals are shown on the right of each panel in Figure 7C\u2013F .", "We than randomly permuted the timing of the first CR and calculated a new shuffled distributions of inter-CR intervals shown in grey .", "If the timing of the second CR is independent of the timing of the first CR , the shuffled distributions should be identical to the true distributions .", "We found however a significant difference in distributions for timing measure pairs shown in Figure 7 ( Kolmogorov-Smirnov test , see Figure 7\u2014source data 1 ) .", "These results show that the timing of the second CR in sequence is influenced by the timing of the first CR and support the hypothesis that a FS from the first response drives the learning and expression of the second response .", "The cerebellar cortex has been shown previously to be necessary for acquisition and expression of well-timed eyelid CRs ( Garcia and Mauk , 1998; Kalmbach et al . , 2010b ) .", "Purkinje cells ( PCs ) , the principal neurons and sole output of the cerebellar cortex , have been a target of several studies .", "Because PCs are inhibitory neurons , decreases in their activity result in increased cerebellar output that drives eyelid CR ( Heiney et al . , 2014 ) .", "For single-component eyelid CRs , the timing and magnitude of the decrease in PCs activity have been shown to encode the kinematic features of CRs ( Halverson et al . , 2015; ten Brinke et al . , 2015 ) .", "Two studies have also shown that under training conditions that produce two CRs , eyelid PCs also showed two correspondingly timed decreases in activity ( Halverson et al . , 2015; Jirenhed et al . , 2017 ) .", "Unlike the present study , in these previous studies both CRs were elicited by the external CS .", "Thus , to begin to understand the cerebellar mechanisms of movement sequences involving feedback signals , we investigated PC activity during the ipsilateral sequence protocol .", "If the second CRs are mediated by the usual cerebellar mechanisms , simply driven by a FS rather than CS , then: ( 1 ) the same PCs should control kinematics of both first and second CRs , since both responses are produced by the same muscle , and ( 2 ) the relationship between PC activity and both responses timing and kinematics should be similar .", "We tested these predictions with in vivo recordings and analysis described below .", "Tetrode microdrives were chronically implanted in three subjects targeting the region of cerebellar cortex previously shown to be necessary for acquisition and expression of well-timed eyelid CRs ( Halverson et al . , 2015; Garcia and Mauk , 1998; Kalmbach et al . , 2010b ) .", "These subjects were trained to produce the ipsilateral CRs sequence , as before employing electrical stimulation of mossy fibers as the CS .", "We recorded 156 well-isolated single units during ipsilateral CRs sequence training sessions .", "Out of those , 42 were classified as PCs based on the presence of both simple and complex spikes and out of those 16 were classified as eyelid PCs ( Halverson et al . , 2015 ) based on the US-evoked complex spikes .", "All subsequent analyses involved only these eyelid PCs .", "Data from an example recording session are shown in Figure 8A and B , with eyelid CRs at the bottom and the raster plot showing eyelid PC simple spikes with corresponding PSTH at the top .", "Panel A shows trials with first CR amplitude smaller than 3 mm , resulting in delivery of US1 .", "Simple spike activity of eyelid PCs developed a decrease during first CR expression , consistent with previous studies .", "Panel B shows eyelid PC activity and behavioral responses on trials with first CR amplitude larger than 3 mm , resulting in omission of US1 and delivery of US2 .", "Even in this single example it is readily apparent that the same PC develops a decrease in activity corresponding in time to each of CRs in ipsilateral sequence .", "Previous work has shown that for a single-component CR , the timing of behavioral CRs and the timing of decreases in eyelid PCs activity co-vary ( Halverson et al . , 2015 ) .", "We therefore investigated if the same relationship holds for both first and second CRs in ipsilateral sequence protocol .", "First , as a replication of previous findings , we studied the timing of eyelid PCs responses as a function of first CRs timing .", "For that , we separated trials into three groups based on first CRs onset times ( non-CRs , early CRs and late CRs ) and calculated corresponding average firing rates of eyelid PCs .", "Results related to the first CR timing are shown in Figure 8C .", "The timing of decrease in eyelid PCs firing rate corresponded to the timing of first CRs , similar and consistent with published results ( Halverson et al . , 2015; ten Brinke et al . , 2015 ) .", "Namely , on non-CR trials ( black lines ) eyelid PCs firing rate barely deviated from baseline activity .", "On trials with early first CRs ( red lines ) decreases in eyelid PCs firing rate happened robustly earlier than on late first CR trials ( blue lines ) .", "For results shown here and below , the absence of overlap in 95% confidence intervals between eyelid PCs activity corresponding to different groups was used as evidence of reliable separation .", "Next , we investigated whether the same relationship holds for the timing of second CRs in the sequence and eyelid PC activity .", "Results demonstrating that the timing of the second decrease in eyelid PCs activity also matches the timing of second CRs are shown in Figure 8D .", "Here we similarly separated trials into three groups , now based on the presence and timing of the second CRs .", "Now on trials without the second CRs ( black lines ) , PCs firing rate returned to the baseline level after the decrease corresponding to the first CR .", "On trials with second CRs present ( red and blue lines ) PCs activity demonstrated double decrease corresponding to the first CR and second CRs in the sequence .", "Moreover , the timing of the second decrease in PCs activity followed the timing of second CRs ( Figure 8D , early versus late second CRs shown by red and blue lines correspondingly ) .", "Previous work ( Halverson et al . , 2015 ) demonstrated that for single-component CRs , the decrease in eyelid PCs activity precedes the onset of CR , consistent with the causal relationship between eyelid PCs responses and CRs expression .", "To examine whether changes in eyelid PCs activity precede the expression of first and second CRs in the ipsilateral sequence , we aligned eyelid PCs activity to the onset of first and second CRs ( Figure 8E and F ) .", "Figure 8E shows first CR time profile ( top ) and corresponding PCs activity ( bottom ) aligned to the first CR onset time ( black vertical dotted line ) .", "Purkinje cells activity was calculated on first CR trials ( blue line ) and non-CR trials ( black line ) .", "Non-CR trials were aligned by CR onset by randomly sampling from the distribution of first CR onset times .", "Similar to published results during conventional eyelid conditioning sessions ( Halverson et al . , 2015 ) , PCs activity on CR trials demonstrated a robust decrease in activity prior to CR onset .", "We next performed a similar analysis for the second CRs , with results shown in Figure 8F .", "Eyelid PCs activity reliably separated prior to the second CR onset on trials with second CRs ( blue line ) comparing to non-second-CRs trials ( black line ) .", "The amount of decrease in PCs activity from baseline at the moment of CR onset was similar for first and second CRs ( 0 . 766\u00a0\u00b1\u00a00 . 015 at the time of first CR onset; 0 . 796\u00a0\u00b1\u00a00 . 034 at the time of second CR onset; p=0 . 45 , Wilcoxon rank sum test ) .", "The above results demonstrate that individual eyelid PCs show decreases in activity that precede the onset and match the presence and timing of both first and second eyelid CRs in an ipsilateral sequence .", "We next examined whether this similarity holds for the relationship between eyelid CRs amplitude and the amount of PCs decrease , as has been demonstrated for the single-component CRs ( Halverson et al . , 2015; ten Brinke et al . , 2015 ) .", "First , as additional replication of previous findings , we separated trials into three groups based on the first CR amplitude and calculated average eyelid PCs responses ( Figure 8F ) .", "As expected , the amount of decrease in eyelid PCs activity scaled with first CR amplitude .", "We next investigated if this relationship holds for the second CRs .", "For that we performed a similar analysis by separating trials based on the second CR amplitude ( Figure 8G ) .", "Eyelid PCs activity showed no change on trials without the second CR ( black line ) and correspondingly scaled amount of decrease on trials with smaller and larger second CRs ( red and blue lines respectively ) .", "Overall , recordings from eyelid PCs demonstrate that individual PCs learn both first and second components in a sequence and the way PC responses relate to these components is not distinguishable .", "This suggests that the same cerebellar mechanisms are involved in generation of both CRs in the sequence .", "These data are consistent with the general notion that FS can be used by the cerebellum to learn following responses in the sequence through processes similar to learning the first response using the CS ."], ["While we have demonstrated the sufficiency of FS from an eyelid CR to be used by the cerebellum as a new \u2018CS\u2019 , the nature and source of this FS remains an open question .", "Several sources of feedback are possible .", "The most direct candidate feedback route would originate from the deep cerebellar nucleus neurons ( DCN ) , which provide the output of the cerebellum .", "Increases in DCN neuron activity are known to drive the expression of eyelid CRs ( Halverson et al . , 2010; McCormick and Thompson , 1984 ) .", "DCN neurons are also known to have axon collaterals that return to the cerebellar cortex ( Houck and Person , 2015; Ankri et al . , 2015; Gao et al . , 2016 ) and form mossy-fiber like synaptic connections .", "Thus the information about the ongoing expression of a CR can be passed back to granule cells in the cerebellar cortex via a monosynaptic excitation by axon collaterals of glutamatergic DCN neurons ( Houck and Person , 2015; Gao et al . , 2016 ) or via a reduction of inhibition from Golgi cells that receive inhibitory projections ( Ankri et al . , 2015 ) from GABA/glycinergic DCN neurons .", "More indirect routes to convey DCN activity back to cerebellar cortex also exist , for example via thalamic ( Halverson et al . , 2010 ) or red nucleus feedback ( Cartford et al . , 1997 ) to pontine nuclei neurons .", "It is also possible that the FS may not originate directly from cerebellar output activity , but rather from proprioceptive information driven by the movement itself .", "All of these possible routes can contribute to a FS , none are mutually exclusive , and their relative contributions will likely depend on temporal constrains and/or laterality of movement sequence .", "For example , nucleo-cortical feedback routes have been shown ( Houck and Person , 2015 ) to have more dominant ipsilateral projections to areas of cerebellar cortex .", "This can be a possible explanation for the need to use shorter time-intervals for subjects trained in contralateral sequence comparing to ipsilateral .", "While we know that the feedback information is supplied to the cerebellar cortex ( Giovannucci et al . , 2017 ) , further studies are needed to investigate relative contributions of possible feedback pathways .", "Apart from the cerebellum , the basal ganglia is another prominent brain system implicated in learning and execution of movement sequences ( Doyon et al . , 1997; Leh\u00e9ricy et al . , 2005; Seidler et al . , 2005 ) .", "However the relative involvement of the cerebellum and basal ganglia in learning and production of motor sequences might differ .", "While several imaging ( Leh\u00e9ricy et al . , 2005; Doyon et al . , 2002; Seidler et al . , 2005 ) and recording ( Jog et al . , 1999 ) studies have reported changes in basal ganglia activity during sequence learning , studies involving lesions or neurodegenerative diseases were generally less conclusive .", "Studies in Parkinson\u2019s patients showed only a partial ( Doyon et al . , 1997; Shin and Ivry , 2003; Jackson et al . , 1995 ) impairment of sequence learning compared to control groups and patients with focal basal ganglia lesions did not display deficits ( Shin et al . , 2005 ) .", "Moreover , pharmacological lesion of globus pallidus internus , the primary basal ganglia output region , resulted in decrease of movement velocity and acceleration , but did not impair the production of learned sequences of movements ( Desmurget and Turner , 2010 ) .", "While we addressed in our work the cerebellar involvement in learning a movement sequence , the results do not preclude significant basal ganglia contributions to learning and expression of movement sequences .", "In our experiments we trained subjects to produce a sequence of discrete responses .", "The results of our study however should not rely on this aspect of the responses , which we utilized for the clarity of analysis .", "Natural complex multi-joint movements , such as locomotion or reaching movements , can be broken down into a sequence of several movement components .", "In these cases our results imply that the feedback about the each portion of the movement can be used by the cerebellum to learn a correct continuation of the movement .", "This general framework can also be used to describe how the cerebellum can learn to guide cortical activity to produce a desired sequence of states .", "The cerebellum and cerebral cortex areas , such as premotor and primary motor cortices , are known to form a closed loop system via cortico-ponto-cerebellar projections ( Kelly and Strick , 2003; Glickstein et al . , 1985; Evarts and Thach , 1969 ) , where the cerebellum sends information back to the cerebral cortex via the thalamus .", "Electrical ( Penfield and Boldrey , 1937; Graziano et al . , 2002 ) or optogenetic ( Harrison et al . , 2012 ) stimulation of motor cortex area is known to produce complex multi-joint movements .", "A common interpretation of these results is a notion of motor program ( Mink , 1996; Summers and Anson , 2009 ) , stored either entirely within a motor cortex or at least partially within downstream areas .", "Our study provides new evidence towards an interpretation that motor programs could be stored in part within the cerebellum .", "This way , the initial command from motor cortex serves as a trigger to initiate the movement , while the later feedback signals from cerebellar output and movement itself are used by the cerebellum to learn a proper output that guides motor cortex activity along the correct trajectory in state space , which in turn results in a correct movement trajectory .", "While speculative at this point , such a framework is fully compatible with results of the present study ."], ["In all experiments subjects were 14 male New Zealand albino rabbits weighing 2 . 5\u20133 . 5 kg at experiment onset .", "Treatment of rabbits and surgical procedures were in accordance with National Institutes of Health guidelines and an institutionally approved animal welfare protocol .", "All subjects were maintained on a 12 hr light/dark cycle .", "One week before the start of experiment , subjects were removed from the home cage and anesthetized with a cocktail of acepromazine ( 1 . 5 mg/kg ) and ketamine ( 45 mg/kg ) .", "After onset of anesthesia , the subjects were placed in a stereotaxic frame , intubated , and maintained on isoflurane ( 1~2% mixed in oxygen ) for the remainder of the surgery .", "Under sterile conditions the skull was exposed with a midline incision ( ~5 cm ) , and four holes were drilled for anchor screws .", "Some anchor screws also functioned as ground screws for subjects with mossy fiber stimulation implants or a microdrive ground for subjects with microdrive implant .", "The rabbit\u2019s head was then positioned with lambda 1 . 5 mm ventral to bregma .", "For subjects prepared only for behavioral experiments involving electrical stimulation of mossy fibers , a craniotomy was drilled out at 5 . 5 mm lateral and 3 mm anterior from lambda , ipsilateral to the trained eye .", "Skull fragments were carefully removed from the craniotomies , the dura matter was carefully opened under visual guidance .", "One or two laterally spaced ( by 1 mm ) tungsten stimulating electrodes ( A-M Systems , Carlsborg , WA; tip exposed to obtain impedance of 100\u2013200 k\u03a9 ) were implanted in the middle cerebellar peduncle ( 16 mm ventral to lambda ) .", "For subjects prepared for microdrive implantation in the cerebellar cortex , a craniotomy was also drilled out at 5 . 9 mm posterior and 6 . 0 mm lateral to lambda .", "Skull fragments were carefully removed from the craniotomies , the dura matter was carefully opened under visual guidance .", "A custom-made microdrive ( 16 tetrodes and 2 references ) fitted with an electronic interface board ( EIB-36-16TT , Neuralynx ) was implanted in the left anterior lobe of the cerebellar cortex at a 40\u00b0 angle posterior to vertical and 17 . 8 mm ventral to lambda .", "This region of the anterior lobe has been shown to be involved in acquisition and expression of well-timed conditioned eyelid responses ( Halverson et al . , 2015; Kalmbach et al . , 2010a; Garcia et al . , 1999 ) .", "The primary target of tetrode recordings were PCs with evoked complex spikes from US , referred throughout the manuscript as eyelid PCs .", "The bundle cannula of the microdrive was lowered to the surface of the brain .", "The craniotomies were sealed with low viscosity silicon ( Kwik-Sil; World Precision Instruments ) .", "The head bolt to mount eyelid detector , anchor/ground screws , stimulation electrodes and microdrive were secured with dental acrylic ( Bosworth Fastray , Pink; The Harry J . Bosworth Company ) , and the skin was sutured where the skull and muscle were exposed .", "Finally , two stainless steel loops terminating in gold pins were inserted into the anterior and posterior periorbital region of the left eye ( and optionally right eye for subjects trained in contralateral sequence of CRs ) for delivery of the stimulation US .", "Subjects were given postoperative analgesics and antibiotics for 2 days after surgery and were allowed to recover for a week before experiments began .", "The subjects were trained in custom-designed , well-ventilated , and sound attenuating chambers measuring 90\u00a0\u00d7\u00a060\u00a0\u00d7\u00a060 cm ( length , width , height ) .", "Each rabbit was placed in a plastic restrainer with their ears stretched over a foam pad and taped down to limit head movement .", "To measure eyelid position , an infrared emitter/detector system was attached directly to the head stage of each rabbit to record movements of the left external eyelid .", "These detectors provide a linear readout of eyelid position ( \u00b10 . 1 mm ) at 1 kHz sampling rate by measuring the amount of infrared light reflected back to the detector , which increases as the eye closes ( Ryan et al . , 2006 ) .", "At the start of each daily session , the gain of the eyelid position detector was calibrated by delivering a test US to elicit full eyelid closure ( defined as 6 . 0 mm , typical for an adult rabbit ) .", "A trial would not start until the rabbit\u2019s eyelid was sufficiently open .", "Stimulus presentation was controlled by custom-designed software for all experiments .", "Each conditioning chamber was equipped with a speaker that was connected to a stereo equalizer and receiver which were connected to a computer that generated the tone .", "For subjects trained using tone as the CS , the CS was set as a 1 kHz , 500 ms , 75 dB sinusoidal tone with a rise and fall time of 5 ms to avoid audible clicks from the speaker .", "For subjects trained with electrical stimulation of mossy fibers , the CS was a constant frequency pulse train of cathodal current pulses ( 100 Hz , 500 ms , 0 . 1 ms pulse width , 100\u2013150 \u03bcA ) , generated by a stimulus isolator ( model 2300 , A-M Systems , Carlsborg , WA ) and passed through the electrode ( s ) implanted in the middle cerebellar peduncle .", "The current pulses were controlled by custom written software ( available from authors upon request ) and delivered through an isolated Pulse Stimulator ( model 2100 , A-M Systems , Carlsborg , WA ) .", "Electrical leads from a separate stimulator ( model 2100 ) were attached to the periorbital electrodes to deliver pulses of electrical stimulation to the left eyelid as the US .", "The US was a 50 ms train of constant current pulses ( 50 Hz , 0 . 7\u20131 ms pulse width , 1\u20133 mA ) delivered through the periorbital electrodes .", "US intensity was adjusted for each rabbit to produce a full eyelid closure without any pain reactions .", "All types of trials in all sessions were separated by a mean inter-trial interval of 30\u00a0\u00b1\u00a010 s .", "For initial training , subjects were given daily eyelid conditioning sessions comprised of 12 blocks of 9 trials each .", "All subjects were initially trained at an inter-stimulus interval ( ISI ) of 500 ms to produce left eye CRs .", "Each block consisted of 1 CS-alone trial and 8 paired trials .", "After reaching a robust CR performance , subjects were switched to the sequence training protocols ."]], "headings": ["Introduction", "Results", "Discussion", "Materials\u00a0and\u00a0methods"], "abstract": ["Most movements are not unitary , but are comprised of sequences .", "Although patients with cerebellar pathology display severe deficits in the execution and learning of sequences ( Doyon et al . , 1997; Shin and Ivry , 2003 ) , most of our understanding of cerebellar mechanisms has come from analyses of single component movements .", "Eyelid conditioning is a cerebellar-mediated behavior that provides the ability to control and restrict inputs to the cerebellum through stimulation of mossy fibers .", "We utilized this advantage to test directly how the cerebellum can learn a sequence of inter-connected movement components in rabbits .", "We show that the feedback signals from one component are sufficient to serve as a cue for the next component in the sequence .", "In vivo recordings from Purkinje cells demonstrated that all components of the sequence were encoded similarly by cerebellar cortex .", "These results provide a simple yet general framework for how the cerebellum can use simple associate learning processes to chain together a sequence of appropriately timed responses ."], "summary": ["Imagine a gymnastics competition in which participants take turns to cartwheel and somersault across the floor .", "The routines on display comprise sequences of precisely timed movements learned through practice .", "This is also true for many of the actions we perform every day , such as reaching for a cup of coffee .", "A region of the brain called the cerebellum helps us learn sequences of movements .", "But how does it do this ?", "To find out , Khilkevich et al . came up with a new version of an old experiment .", "Rabbits were first trained to blink their eye in response to a specific external cue .", "This type of learning , called associative learning , has been shown before in the cerebellum .", "But Khilkevich et al . wondered whether the cerebellum could also use internal feedback signals from the eyeblink as a cue to learn the next movement ?", "If so , this might explain how the cerebellum can chain movements together in a sequence .", "As predicted , Khilkevich et al . found that rabbits could learn to blink their eye in response to an initial signal , and then blink again in response to the first blink .", "Control experiments confirmed that the second eyeblink was coupled to the first , and not to the original cue .", "Moreover , on many trials the rabbits showed a third and even fourth eyeblink .", "This is because feedback signals from the first , second or third blink were the same .", "Thus , the feedback signals from the first blink triggered the second blink , feedback from the second triggered the third , and so forth .", "Rabbits could also learn to use a blink of the left eye as a cue for a blink of the right eye .", "Similar patterns of neuronal activity accompanied each blink , suggesting that the same mechanism generated them all .", "The cerebellum can thus use feedback from one movement as a cue to learn the proper timing of the next movement in a sequence .", "A key question is whether this mechanism of sequence learning extends beyond movement .", "The cerebellum has extensive connections to the brain\u2019s outer layer , the cortex , including many areas involved in cognition .", "Future experiments should test whether the cerebellum might help guide sequences of cortical activity during cognitive tasks ."], "keywords": ["neuroscience"]}

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-{"id": "elife-35500-v1", "year": "2018", "title": "National and regional seasonal dynamics of all-cause and cause-specific mortality in the USA from 1980 to 2016", "sections": [["It is well-established that death rates vary throughout the year , and in temperate climates there tend to be more deaths in winter than in summer ( Campbell , 2017; Fowler et al . , 2015; Healy , 2003; McKee , 1989 ) .", "It has therefore been hypothesized that a warmer world may lower winter mortality in temperate climates ( Langford and Bentham , 1995; Martens , 1998 ) .", "In a large country like the USA , which possesses distinct climate regions , the seasonality of mortality may vary geographically , due to geographical variations in mortality , localized weather patterns , and regional differences in adaptation measures such as heating , air conditioning and healthcare ( Davis et al . , 2004; Braga et al . , 2001; Kalkstein , 2013; Medina-Ram\u00f3n and Schwartz , 2007 ) .", "The presence and extent of seasonal variation in mortality may also itself change over time ( Bobb et al . , 2014; Carson et al . , 2006; Seretakis et al . , 1997; Sheridan et al . , 2009 ) .", "A thorough understanding of the long-term dynamics of seasonality of mortality , and its geographical and demographic patterns , is needed to identify at-risk groups , plan responses at the present time as well as under changing climate conditions .", "Although mortality seasonality is well-established , there is limited information on how seasonality , including the timing of minimum and maximum mortality , varies by local climate and how these features have changed over time , especially in relation to age group , sex and medical cause of death ( Rau , 2004; Rau et al . , 2018 ) .", "In this paper , we comprehensively characterize the spatial and temporal patterns of all-cause and cause-specific mortality seasonality in the USA by sex and age group , through the application of wavelet analytical techniques , to over three decades of national mortality data .", "Wavelets have been used to study the dynamics of weather phenomena ( Moy et al . , 2002 ) and infectious diseases ( Grenfell et al . , 2001 ) .", "We also used centre of gravity analysis and circular statistics methods to understand the timing of maximum and minimum mortality .", "In addition , we identify how the percentage difference between death rates in maximum and minimum mortality months has changed over time ."], ["The strengths of our study are its innovative methods of characterizing seasonality of mortality dynamically over space and time , by age group and cause of death; using wavelet and centre of gravity analyses; using ERA-Interim data output to compare the association between seasonality of death rates and regional temperature .", "A limitation of our study is that we did not investigate seasonality of mortality by socioeconomic characteristics which may help with understanding its determinants and planning responses ."], ["We used wavelet and centre of gravity analyses , which allowed systematically identifying and characterizing seasonality of total and cause-specific mortality in the USA , and examining how seasonality has changed over time .", "We identified distinct seasonal patterns in relation to age and sex , including higher all-cause summer mortality in young men ( Feinstein , 2002; Rau et al . , 2018 ) .", "Importantly , we also showed that all-cause and cause-specific mortality seasonality is largely similar in terms of both timing and magnitude across diverse climatic regions with substantially different summer and winter temperatures .", "Insights of this kind would not have been possible analysing data averaged over time or nationally , or fixed to pre-specified frequencies .", "Prior studies have noted seasonality of mortality for all-cause mortality and for specific causes of death in the USA ( Feinstein , 2002; Kalkstein , 2013; Rau , 2004; Rau et al . , 2018; Rosenwaike , 1966; Seretakis et al . , 1997 ) .", "Few of these studies have done consistent national and subnational analyses , and none has done so over time , for a comprehensive set of age groups and causes of death , and in relation to regional temperature differences .", "Our results on strong seasonality of cardiorespiratory diseases deaths and weak seasonality of cancer deaths , restricted to older ages , are broadly consistent with these studies ( Feinstein , 2002; Rau et al . , 2018; Rosenwaike , 1966; Seretakis et al . , 1997 ) , which had limited analysis on how seasonality changes over time and geography ( Feinstein , 2002; Rau et al . , 2018; Rosenwaike , 1966 ) .", "Similarly , our results on seasonality of injury deaths are supported by a few prior studies ( Feinstein , 2002; Rau et al . , 2018; Rosenwaike , 1966 ) , but our subnational analysis over three decades revealed variations in when injury deaths peaked and in how seasonal differences in these deaths have changed over time in relation to age group which had not been reported before .", "A study of 36 cities in the USA , aggregated across age groups and over time , also found that excess mortality was not associated with seasonal temperature range ( Kinney et al . , 2015 ) .", "In contrast , a European study found that the difference between winter and summer mortality was lower in colder Nordic countries than in warmer southern European nations ( Healy , 2003; McKee , 1989 ) ( the study\u2019s measure of temperature was mean annual temperature which differed from the temperature difference between maximum and minimum mortality used in our analysis although the two measures are correlated ) .", "The absence of variation in the magnitude of mortality seasonality indicates that different regions in the USA are similarly adapted to temperature seasonality , whereas Nordic countries may have better environmental ( e . g . housing insulation and heating ) and health system measures to counter the effects of cold winters than those in southern Europe .", "If the observed absence of association between the magnitude of mortality seasonality and seasonal temperature difference across the climate regions also persists over time , the changes in temperature as a result of global climate change are unlikely to affect the winter-summer mortality difference .", "The cause-specific analysis showed that the substantial decline in seasonal mortality differences in adolescents and young adults was related to the diminishing seasonality of ( unintentional ) injuries , especially from road traffic crashes , which are more likely to occur in the summer months ( Liu et al . , 2005 ) and are more common in men .", "The weakening of seasonality in boys under five years of age was related to two phenomena: first , the seasonality of death from cardiorespiratory diseases declined , and second , the proportion of deaths from perinatal conditions , which exhibit limited seasonality ( Figure 9\u2014figure supplement 2 and Figure 10\u2014figure supplement 3 ) , increased ( MacDorman and Gregory , 2015 ) .", "In contrast to young and middle ages , mortality in older ages , where death rates are highest , maintained persistent seasonality over a period of three decades ( we note that although the percent seasonal difference in mortality has remained largely unchanged in these ages , the absolute difference in death rates between the peak and minimum months has declined because total mortality has a declining long-term trend ) .", "This finding demonstrates the need for environmental and health service interventions targeted towards this group irrespective of geography and local climate .", "Examples of such interventions include enhancing the availability of both environmental and medical protective factors , such as better insulation of homes , winter heating provision and flu vaccinations , for the vulnerable older population ( Katiyo et al . , 2017 ) .", "Social interventions , including regular visits to the isolated elderly during peak mortality periods to ensure that they are optimally prepared for adverse conditions , and responsive and high-quality emergency care , are also important to protect this vulnerable group ( Healy , 2003; Lerchl , 1998; Katiyo et al . , 2017 ) .", "Emergent new technologies , such as always-connected hands-free communications devices with the outside world , in-house cameras , and personal sensors also provide an opportunity to enhance care for the older , more vulnerable groups in the population , especially in winter when the elderly have fewer social interactions ( Morris , 2013 ) .", "Such interventions are important today , and will remain so as the population ages and climate change increases the within- and between-season weather variability ."], ["We used data on all 85 , 854 , 176 deaths in the USA from 1980 to 2016 from the National Center for Health Statistics ( NCHS ) .", "Age , sex , state of residence , month of death , and underlying cause of death were available for each record .", "The underlying cause of death was coded according to the international classification of diseases ( ICD ) system ( 9th revision of ICD from 1980 to 1998 and 10th revision of ICD thereafter ) .", "Yearly population counts were available from NCHS for 1990 to 2016 and from the US Census Bureau prior to 1990 ( Ingram et al . , 2003 ) .", "We calculated monthly population counts through linear interpolation , assigning each yearly count to July .", "We also subdivided the national data geographically into nine climate regions used by the National Oceanic and Atmospheric Administration ( Figure 18 and Table 2 ) ( Karl and Koss , 1984 ) .", "On average , the Southeast and South are the hottest climate regions with average annual temperatures of 18 . 4\u00b0C and 18\u00b0C respectively; the South also possesses the highest average maximum monthly temperature ( 27 . 9\u00b0C in July ) .", "The lowest variation in temperature throughout the year is that of the Southeast ( an average range of 17 . 5\u00b0C ) .", "The three coldest climate regions are West North Central , East North Central and the Northwest ( 7 . 6\u00b0C , 8 . 0\u00b0C , 8 . 2\u00b0C respectively ) .", "Mirroring the characteristics of the hottest climate regions , the largest variation in temperature throughout the year is that of the coldest region , West North Central ( an average range of 30 . 5\u00b0C ) , which also has the lowest average minimum monthly temperature ( \u22126 . 5\u00b0C in January ) .", "The other climate regions , Northeast , Southwest , and Central , possess similar average temperatures ( 10\u00b0C to 14\u00b0C ) and variation within the year of ( 23\u00b0C to 26\u00b0C ) , with the Northeast being the most populous region in the United States ( with 19 . 8% total population in 2016 ) .", "Data were divided by sex and age in the following 10 age groups: 0\u20134 , 5\u201314 , 15-24 , 25\u201334 , 35\u201344 , 45\u201354 , 55\u201364 , 65\u201374 , 75\u201384 , 85+\u00a0years .", "We calculated monthly death rates for each age and sex group , both nationally and for sub-national climate regions .", "Death rate calculations accounted for varying length of months , by multiplying each month\u2019s death count by a factor that would make it equivalent to a 31 day month .", "For analysis of seasonality by cause of death , we mapped each ICD-9 and ICD-10 codes to four main disease categories ( Table 1 ) and to a number of subcategories which are presented in the Supplementary Note .", "Cardiorespiratory diseases and cancers accounted for 56 . 4% and 21 . 2% of all deaths in the USA , respectively , in 1980 , and 40 . 3% and 22 . 4% , respectively , in 2016 .", "Deaths from cardiorespiratory diseases have been associated with cold and warm temperatures ( Basu , 2009; Basu and Samet , 2002; Bennett et al . , 2014; Braga et al . , 2002; Gasparrini et al . , 2015 ) .", "Injuries , which accounted for 8% of all deaths in the USA in 1980 and 7 . 3% in 2016 , may have seasonality that is distinct from so-called natural causes .", "We did not further divide other causes because the number of deaths could become too small to allow stable estimates when divided by age group , sex and climate region .", "We obtained data on temperature from ERA-Interim , which combines predictions from a physical model with ground-based and satellite measurements ( Dee et al . , 2011 ) .", "We used gridded four-times-daily estimates at a resolution of 80 km to generate monthly population-weighted temperature by climate region throughout the analysis period .", "We used wavelet analysis to investigate seasonality for each age-sex group .", "Wavelet analysis uncovers the presence , and frequency , of repeated maxima and minima in each age-sex-specific death rate time series ( Hubbard , 1998; Torrence and Compo , 1998 ) .", "In brief , a Morlet wavelet , described in detail elsewhere ( Cazelles et al . , 2008 ) , is equivalent to using a moving window on the death rate time series and analysing periodicity in each window using a short-form Fourier transform , hence generating a dynamic spectral analysis , which allows measuring dynamic seasonal patterns , in which the periodicity of death rates may disappear , emerge , or change over time .", "In addition to coefficients that measure the frequency of periodicity , wavelet analysis estimates the probability of whether the data are different from the null situation of random fluctuations that can be represented with white ( an independent random process ) or red ( autoregressive of order one process ) noise .", "For each age-sex group , we calculated the p-values of the presence of 12 month seasonality for the comparison of wavelet power spectra of the entire study period ( 1980\u20132016 ) with 100 simulations against a white noise spectrum , which represents random fluctuations .", "We used the R package WaveletComp ( version 1 . 0 ) for the wavelet analysis .", "Before analysis , we de-trended death rates using a polynomial regression , and rescaled each death rate time series so as to range between 1 and \u22121 .", "To identify the months of maximum and minimum death rates , we calculated the centre of gravity and the negative centre of gravity of monthly death rates .", "Centre of gravity was calculated as a weighted average of months of deaths , with each month weighted by its death rate; negative centre of gravity was also calculated as a weighted average of months of deaths , but with each month was weighted by the difference between its death rate and the year\u2019s maximum death rate .", "In taking the weighted average , we allowed December ( month 12 ) to neighbour January ( month 1 ) , representing each month by an angle subtended from 12 equally-spaced points around a unit circle .", "Using a technique called circular statistics , a mean ( \u03b8- ) of the angles ( \u03b81 , \u03b82 , \u03b83\u2026 , \u03b8n , ) representing the deaths ( with n the total number of deaths in an age-sex group for a particular cause of death ) is found using the relation below:\u03b8-=arg\u2211j=1nexp\u2061 ( i\u03b8j ) , where arg denotes the complex number argument and \u03b8j denotes the month of death in angular form for a particular death j .", "The outcome of this calculation is then converted back into a month value ( Fisher , 1995 ) .", "Along with each circular mean , a 95% confidence interval ( CI ) was calculated by using 1000 bootstrap samples .", "The R package CircStats ( version 0 . 2 . 4 ) was used for this analysis .", "For each age-sex group and cause of death , and for each year , we calculated the percent difference in death rates between the maximum and minimum mortality months .", "We fitted a linear regression to the time series of seasonal differences from 1980 to 2016 , and used the fitted trend line to estimate how much the percentage difference in death rates between the maximum and minimum mortality months had changed from 1980 to 2016 .", "We weighted seasonal difference by the inverse of the square of its standard error , which was calculated using a Poisson model to take population size of each age-sex group through time into account .", "This method gives us a p-value for the change in seasonal difference per year , which we used to calculate the seasonal difference at the start ( 1980 ) and end ( 2016 ) of the period of study .", "Our method of analysing seasonal differences avoids assuming that any specific month or group of months represent highest and lowest number of deaths for a particular cause of death , which is the approach taken by the traditional measure of Excess Winter Deaths .", "It also allows the maximum and minimum mortality months to vary by age group , sex and cause of death ."]], "headings": ["Introduction", "Results", "Discussion", "Materials and methods"], "abstract": ["In temperate climates , winter deaths exceed summer ones .", "However , there is limited information on the timing and the relative magnitudes of maximum and minimum mortality , by local climate , age group , sex and medical cause of death .", "We used geo-coded mortality data and wavelets to analyse the seasonality of mortality by age group and sex from 1980 to 2016 in the USA and its subnational climatic regions .", "Death rates in men and women \u2265 45 years peaked in December to February and were lowest in June to August , driven by cardiorespiratory diseases and injuries .", "In these ages , percent difference in death rates between peak and minimum months did not vary across climate regions , nor changed from 1980 to 2016 .", "Under five years , seasonality of all-cause mortality largely disappeared after the 1990s .", "In adolescents and young adults , especially in males , death rates peaked in June/July and were lowest in December/January , driven by injury deaths ."], "summary": ["In the USA , more deaths happen in the winter than the summer .", "But when deaths occur varies greatly by sex , age , cause of death , and possibly region .", "Seasonal differences in death rates can change over time due to changes in factors that cause disease or affect treatment .", "Analyzing the seasonality of deaths can help scientists determine whether interventions to minimize deaths during a certain time of year are needed , or whether existing ones are effective .", "Scrutinizing seasonal patterns in death over time can also help scientists determine whether large-scale weather or climate changes are affecting the seasonality of death .", "Now , Parks et al . show that there are age and sex differences in which times of year most deaths occur .", "Parks et al . analyzed data on US deaths between 1980 and 2016 .", "While overall deaths in a year were highest in winter and lowest in summer , a greater number of young men died during summer \u2013 mainly due to injuries \u2013 than during winter .", "Seasonal differences in deaths among young children have largely disappeared and seasonal differences in the deaths of older children and young adults have become smaller .", "Deaths among women and men aged 45 or older peaked between December and February \u2013 largely caused by respiratory and heart diseases , or injuries .", "Deaths in this older age group were lowest during the summer months .", "Death patterns in older people changed little over time .", "No regional differences were found in seasonal death patterns , despite large climate variation across the USA .", "The analysis by Parks et al . suggests public health and medical interventions have been successful in reducing seasonal deaths among many groups .", "But more needs to be done to address seasonal differences in deaths among older adults .", "For example , by boosting flu vaccination rates , providing warnings about severe weather and better insulation for homes .", "Using technology like hands-free communication devices or home visits to help keep vulnerable elderly people connected during the winter months may also help ."], "keywords": ["epidemiology and global health"]}

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-{"id": "elife-15477-v3", "year": "2016", "title": "Increasing Notch signaling antagonizes PRC2-mediated silencing to promote reprograming of germ cells into neurons", "sections": [["Cell-fate decisions are controlled , on the one hand , by intercellular signaling and , on the other hand , by intrinsic mechanisms such as epigenetic chromatin modifications .", "The Notch signaling pathway is a highly conserved and widespread signaling mechanism ( Artavanis-Tsakonas et al . , 1999; Greenwald and Kovall , 2013 ) , which has been implicated in key cell-fate decisions such as the decision between proliferation and differentiation ( Liu et al . , 2010 ) .", "Notch signaling has also been implicated in cellular reprograming .", "Upon inhibition of Notch signaling , the oncogenic genes KLF4 and cMyc become dispensable for the generation of induced pluripotent stem cells ( iPSCs ) from mouse and human keratinocytes ( Ichida et al . , 2014 ) .", "In this setting , Notch inhibits reprograming .", "Conversely , Notch signaling promotes transdifferentiation of pancreatic acinar cells to ductal cells ( Sawey et al . , 2007 ) , or the conversion of hepatocytes into biliary cells in liver primary malignancy intrahepatic cholangiocarcinoma ( ICC ) ( Sekiya and Suzuki , 2012 ) .", "Notch signaling can also affect reprograming in normal development .", "In C . elegans , signaling through the GLP-1 and LIN-12 Notch receptors impedes reprograming during embryogenesis and , during larval development , signaling through LIN-12 is required for the conversion of a rectal epithelial cell into a motorneuron ( Jarriault et al . , 2008; Djabrayan et al . , 2012 ) .", "The role of epigenetic regulators in cell-fate decisions has been studied mostly in pluripotent cells cultured outside of their normal tissue environment ( Meshorer and Misteli , 2006; Spivakov and Fisher , 2007; Lessard and Crabtree , 2010; Orkin and Hochedlinger , 2011 ) .", "Therefore , the\u00a0epigenetic regulation of stem cell identity in intact tissues remains poorly understood .", "Additionally , the impact of external cues , for example signaling from a stem cell niche to the recipient cell\u2019s chromatin remains equally unresolved .", "By contrast , C . elegans has been used as a model to study reprograming in an intact organism ( Horner et al . , 1998; Fukushige et al . , 1998; Zhu et al . , 1998; Fukushige and Krause , 2005; Ciosk et al . , 2006; Jarriault et al . , 2008; Yuzyuk et al . , 2009; Riddle et al . , 2013 ) .", "In this model , germ cells can be directly reprogrammed into neurons by depleting specific chromatin modifiers such as LIN-53 ( Rbbp4/7 ) or components of PRC2 , and by concomitant overexpression of the transcription factor CHE-1 , which induces glutamatergic neuronal fate in a process which we refer to as Germ cell Conversion ( GeCo ) ( Tursun et al . , 2011; Patel et al . , 2012 ) .", "Here , we identify the Notch signaling pathway as a critical player in this reprograming model .", "This was unanticipated , since signaling through the Notch receptor GLP-1 ( henceforth GLP-1Notch ) from the somatic gonadal niche is known to maintain germline stem cell/progenitor fate ( Kimble and Crittenden , 2007 ) .", "To understand this novel , reprograming-promoting role of GLP-1Notch , we combined genetics with tissue-specific expression profiling .", "We identified genes regulated by GLP-1Notch , including genes recently shown to maintain the germline stem/progenitor cells ( Kershner et al . , 2014 ) .", "Additionally , and unexpectedly , we found that many genes activated by GLP-1Notch signaling were also repressed by the cell fate-stabilizing chromatin regulator PRC2 .", "We show that GLP-1Notch and PRC2 have an antagonistic effect on germ cell-fate decisions and demonstrate co-regulation of their common target , utx-1 .", "Importantly , UTX-1 is a histone demethylase known to erase the gene-silencing methylation of histone H3 dependent on PRC2 ( Maures et al . , 2011; Jin et al . , 2011; Vandamme et al . , 2012 ) .", "Thus , we propose that the GLP-1Notch\u2013dependent induction of UTX-1 facilitates reprograming by alleviating PRC2-mediated repression of alternative cell fates ."], ["Germ cells can be converted into neuronal cells in intact C . elegans upon overexpression of the neuronal transcription factor CHE-1 , simply by depleting the chromatin modifier LIN-53 ( Tursun et al . , 2011; Patel et al . , 2012 ) .", "This GeCo phenotype can be followed in living animals by monitoring a reporter GFP expressed from the gcy-5 promoter , which otherwise is induced in glutamatergic ASE neurons ( Altun-Gultekin et al . , 2001 ) .", "In contrast to the spontaneous teratomatous differentiation of meiotic germ cells , observed in the absence of specific RNA-binding proteins ( Ciosk et al . , 2006; Biedermann et al . , 2009; Tocchini et al . , 2014 ) , GeCo is preferentially observed in the pre-meiotic , proliferating germ cells ( Tursun et al . , 2011; Patel et al . , 2012 ) .", "Consistently , removing the proliferating germ cells , by inhibiting GLP-1Notch signaling , prevents GeCo ( Tursun et al . , 2011 ) .", "However , because the proliferating germ cells were eliminated , these experiments did not address a possible direct effect of GLP-1Notch signaling on GeCo .", "We began addressing this issue by examining the gonads of animals carrying the gain-of-function glp-1 allele ( ar202 ) ( Pepper et al . , 2003 ) .", "These gonads are filled with proliferating germ cells and , upon depleting LIN-53 and overexpressing CHE-1 , we observed that significantly more germ cells converted to ASE neurons ( Figure 1A , Figure 1\u2014source data 1 ) .", "We refer to this enhanced GeCo as 'GeCo+' .", "Detailed quantification revealed that the GeCo+ gonads contained more than twice the number of converted cells ( Figure 1\u2014figure supplement 1A , Figure 1\u2014source data 1 ) .", "The nuclei of these converted cells were reminiscent of neuronal nuclei and the cells displayed axo-dendritic projection ( Figure 1\u2014figure supplement 1B ) , as previously described ( Tursun et al . , 2011; Patel et al . , 2012 ) .", "To confirm that the GeCo enhancement depends on the canonical Notch signaling pathway , rather than an independent function of the GLP-1Notch\u00a0receptor , we RNAi-depleted the transcriptional effector of GLP-1Notch signaling , LAG-1 ( Christensen et al . , 1996 ) .", "We exposed animals only after hatching to lag-1 RNAi in order to avoid sterility , which is caused when animals are subjected to lag-1 RNAi earlier ( Supplemental file 1 ) .", "RNAi-mediated knock-down of lag-1 strongly inhibited GeCo ( Figure 1B , Figure 1\u2014source data 1 ) .", "Importantly , under these experimental conditions , we did not observe any obvious reduction of germ cell numbers ( Figure 1C , Figure 1\u2014source data 1 ) , suggesting a proliferation-independent effect of GLP-1Notch signaling on cell-fate conversion .", "To investigate this further , we tested GeCo efficiency on germ cells proliferating independently of GLP-1Notch signaling .", "We took advantage of mutants in which , in the absence of two meiosis/differentiation-promoting factors GLD-1 and GLD-2 , germ cells proliferate independently of GLP-1Notch ( Kadyk and Kimble , 1998 ) .", "Specifically , we examined GeCo in the loss-of-function gld-1 ( q497 ) gld-2 ( q485 ) mutants , which carried either wild-type glp-1 or the loss-of-function glp-1 ( q175 ) allele ( Austin and Kimble , 1987 ) .", "Both mutant combinations have previously been described to have tumorous germlines and impaired meiotic entry ( Kadyk and Kimble , 1998; Hansen et al . , 2004 ) .", "In contrast to efficient GeCo observed in the gld-1 ( q497 ) gld-2 ( q485 ) gonads , GeCo was strongly diminished in the gld-1 ( q497 ) gld-2 ( q485 ) ; glp-1 ( q175 ) gonads , despite the ongoing germ cell proliferation ( Figure 1D , Figure 1\u2014source data 1 ) .", "Counting the number of germ cells in these gonads revealed only a slight difference ( a 15% increase in the numbers in the double vs . triple mutant gonads ) , suggesting that the strong enhancement of GeCo by GLP-1Notch signaling cannot be explained by the\u00a0increased number of germ cells ( Figure 1\u2014figure supplement 2 , Figure 1\u2014source data 1 ) .", "Since it has been proposed that dividing cells have a higher propensity for cellular reprograming ( Egli et al . , 2008; Hanna et al . , 2009 ) , we also tested whether blocking the cell cycle would affect the observed GeCo enhancement in glp-1 ( gf ) gonads .", "As previously described ( Fox et al . , 2011; Patel et al . , 2012 ) , we used hydroxyurea ( HU ) treatment to block the cell cycle in the S phase .", "Blocking the cell cycle by HU did not diminish the GeCo+ phenotype ( Figure 1\u2014figure supplement 3 , Figure 1\u2014source data 1 ) .", "Combined , these results suggest that GLP-1Notch enhances GeCo independently from its role in promoting germ cell proliferation . 10 . 7554/eLife . 15477 . 003Figure 1 . GLP-1Notch signaling promotes reprograming of germ cells .", "( A ) GLP-1Notch enhances germ cell conversion ( GeCo ) into neuronal-like cells .", "Left: Fluorescent ( top ) and combined fluorescent/differential interference contrast ( DIC ) micrographs ( bottom ) of adult animals .", "All animals ectopically expressed the pro-neuronal transcription factor CHE-1 from a heat-shock promoter .", "glp-1 ( ar202 ) is a temperature-sensitive gain-of-function allele of the Notch receptor .", "Animals were subjected to either mock ( control ) or lin-53 RNAi .", "Reprogrammed cells expressed a GFP reporter driven from the neuronal gcy-5 promoter ( here an in other figures nGFP ) and are outlined here and elsewhere in yellow .", "Any signal outside the outlined region comes from somatic tissues .", "GeCo+ indicates animals that displayed a strongly enhanced GeCo phenotype .", "Scale bars = 10 \u03bcm .", "The cartoons depicting the GeCo and GeCo+ phenotypes are on the top right .", "The gonads are shaded in grey and GFP-positive converted germ cells are green .", "Fractions of animals displaying GeCo and GeCo+ are indicated below .", "At least 250 animals were quantified per condition .", "P-values were calculated using Student's t-test: p1<0 , 0001; p2=0 , 0006 .", "Error bars represent SEM .", "( B ) The transcriptional effector of the GLP-1Notch signaling pathway , LAG-1 , is required for the GLP-1Notch\u2013mediated enhancement of GeCo .", "Left: Fluorescent micrographs of adults expressing CHE-1\u2013induced nGFP as explained above .", "GeCo is diminished upon the depletion of LAG-1 .", "White dashed lines outline the animal body .", "Scale bars = 10 \u03bcm .", "Right: The corresponding quantifications .", "At least 400 animals were quantified per condition .", "P-values were calculated using Student's t-test: p1<0 , 0001; p2=0 , 0018 .", "Error bars represent SEM .", "( C ) GLP-1Notch signaling enhances GeCo independently from germ cell proliferation .", "Shown are DAPI-stained gonads of glp-1 ( ar202 ) animals , expressing CHE-1\u2013induced nGFP , treated with either mock or lin-53 RNAi .", "Germ cells were counted from the DTC ( yellow asterisk ) to the turn of the gonad arm ( dashed yellow line ) .", "15 gonad arms per condition were counted .", "Scale bars = 10 \u03bcm .", "Quantifications are on the right .", "While greatly inhibiting GeCo , lag-1 RNAi did not change the number of germ cells .", "P-values were calculated using Student's t-test: p1=0 , 89 .", "Error bars represent SEM .", "( D ) GLP-1Notch enhances GeCo independently from proliferation .", "Left: Fluorescent micrographs of adults ( with indicated genotypes ) , expressing CHE-1\u2013induced nGFP .", "The first panel on the left shows a control , heterozygous ( wild-type ) gld-1 gld-2/++; glp-1/+ animal .", "The other panels show the homozygous gld-1 ( q497 ) gld-2 ( q485 ) mutants , carrying either a loss-of-function ( q175 , center ) or a wild-type ( right ) allele of glp-1 .", "Despite proliferating , germ cells in the gld-1 gld-2; glp-1 gonads have lost the ability to undergo GeCo .", "Scale bars = 10 \u03bcm .", "Right: the corresponding quantifications .", "At least 250 animals were quantified per condition .", "P-values were calculated using Student's t-test: p1=0 , 0478; p2=0 , 0201 .", "Error bars represent SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 15477 . 00310 . 7554/eLife . 15477 . 004Figure 1\u2014source data 1 . Quantification of GeCo in glp-1 ( gf ) and lag-1 RNAi\u00a0animals .", "( A ) Quantification of GeCo+ phenotype upon RNAi against lin-53 in glp-1 ( ar202 ) mutants .", "( B ) Quantification of GeCo dependency on LAG-1 .", "( C ) Quantification of germ cells in glp-1 ( ar202 ) gonads with our without lag-1 RNAi treatment .", "( D ) Quantification of GeCo in different genetic backgrounds with highly proliferative germlines upon RNAi against lin-53 .", "Figure 1\u2014figure supplement 1A source data: Quantification of gfp-positive germ cells .", "Figure 1\u2014figure supplement 2 source data: Quantification of germ cells in different genetic backgrounds with highly proliferative germlines .", "Figure 1\u2014figure supplement 3A source data: Quantification of GeCo after cell cycle block with HU treatment .", "More details can be found in the corresponding figure legends . DOI: http://dx . doi . org/10 . 7554/eLife . 15477 . 00410 . 7554/eLife . 15477 . 005Figure 1\u2014figure supplement 1 . glp-1 ( gf ) gonads contain more than twice the number of converted cells which display neuronal characteristics .", "( A ) For the quantification of gcy-5::gfp-positive cells per gonadal arm only the GeCo category of wt vs . glp-1 ( gf ) was used because GeCo+ animals already show an extensive area of the gonad filled with gcy-5::gfp-positive cells with usually >100 cells/gonad making reliable counting impossible .", "Notably , animals with a seemingly similar extend of GeCo in wt vs . glp-1 ( gf ) show a clear increase of gcy-5::gfp-positive cells per gonadal arm from approx .", "10 in wt to > 30 in glp-1 ( gf ) .", "n ( wt ) = 75 gonaldal arms , n ( glp-1 ( ar202 ) ) = 221 gonadal arms .", "The background of the loss of function allele glp-1 ( q175 ) leads to a significant decrease in GeCo as shown previously ( Tursun et al . , 2011 ) .", "( B ) A magnified view of gcy-5::gfp-positive ( nGFP ) cells , in a GeCo+ gonad from a glp-1 ( gf ) animal .", "The converted cells show axo-dendritic projections ( white arrow heads ) .", "The inset in the corresponding DIC image , magnified in the right-bottom corner , shows the nuclear morphology of a converted germ cell , which has lost the germ cell-specific \u2018fried-egg\u2019-like shape and instead shows nuclear speckles characteristic of a neuronal cell .", "Scale bar = 1 \u03bcm . DOI: http://dx . doi . org/10 . 7554/eLife . 15477 . 00510 . 7554/eLife . 15477 . 006Figure 1\u2014figure supplement 2 . Germ cell numbers are similar between gld-1 gld-2 double and gld-1 gld-2; glp-1 triple mutants . DAPI-stained gonads of gld-1 ( q497 ) gld-2 ( q485 ) or gld-1 ( q497 ) gld-2 ( q485 ) ; glp-1 ( q175 ) mutants , carrying the hsp::che-1 and gcy-5::gfp transgenes .", "The gonads were imaged by fluorescent microscopy using Z-stack acquisitions .", "Germ cells from the DTCs ( yellow asterisks ) to the turn of the gonad arm ( dashed lines ) were counted .", "Below:15 gonad arms per condition of L4 animals were counted .", "The numbers of germ cells differ only slightly ( 15% ) in the double mutant vs . triple mutants background .", "Scale bars = 10 \u03bcm .", "Error bars represent SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 15477 . 00610 . 7554/eLife . 15477 . 007Figure 1\u2014figure supplement 3 . Blocking the cell cycle with hydroxyurea does not inhibit GeCo+ .", "( A ) We used hydroxyurea ( HU ) treatment for 5 hr to chemically block the cell cycle , which makes germ cells arrest in the S phase of the cell cycle .", "This arrest does not diminish the GeCo+ phenotype in glp-1 ( gf ) gonads upon lin-53 RNAi and che-1 ( oe ) .", "At least 150 animals were quantified per condition .", "P-values were calculated using Student's t-test: p1=0 , 1409; p2=0 , 4583 .", "Error bars represent SEM .", "The right panel shows examples of GeCo+ displaying animals based on gcy-5::gfp ( nGFP ) for HU-untreated ( -HU ) and HU-treated ( +HU ) animals .", "( B ) The gonads were stained for EdU incorporation .", "Dashed lines outline gonads .", "Asterisks indicate distal tips of gonads .", "Scale bars = 10 \u03bcm .", "( C ) The gonads of glp-1 ( ar202 ) gf animals , which were treated with HU for 12 hr and stained with DAPI and H3Ser10ph ( pH3 ) antibody .", "The pH3-positive cells indicate condensed chromosomes of dividing cells .", "After 12 hr of HU treatment , the gonads contained , as expected , enlargement nuclei ( arrowheads ) ( Gartner et al . , 2004; Fox et al . , 2011 ) .", "The loss of pH3-positive cells indicates a cell cycle arrest .", "Asterisks indicate distal tips of gonads , dashed lines outline gonad .", "Scale bars = 10 \u03bcm . DOI: http://dx . doi . org/10 . 7554/eLife . 15477 . 007 To understand the effects of GLP-1Notch on GeCo , we set out to identify genes regulated by GLP-1Notch signaling in germ cells .", "To conduct the\u00a0analysis in morphologically similar tissue , we again took advantage of the gld-1 gld-2 double mutants that , combined with either loss-of-function or gain-of-function glp-1 alleles , have morphologically similar gonads , filled with proliferating , undifferentiated germ cells ( Figure 2\u2014figure supplement 1 ) ( Kadyk and Kimble , 1998; Hansen et al . , 2004 ) .", "We combined gld-1 ( q497 ) gld-2 ( q485 ) mutations with either the temperature-sensitive loss-of-function ( lf ) glp-1 allele ( e2144 ) , or the temperature-sensitive gain-of-function ( gf ) glp-1 allele ( ar202 ) ( Priess et al . , 1987; Pepper et al . , 2003 ) .", "Because GLD-1 and GLD-2 regulate gene expression at the posttranscriptional level only , we expected that transcriptionally regulated GLP-1Notch targets could be identified in this background .", "To analyze gene expression , gonads were dissected from animals grown at the restrictive temperature in two independent experiments , and transcripts were analyzed with tiling arrays ( GEO accession number GSE49395 ) .", "We identified around 100 transcripts that were differentially expressed between the gld-1 gld-2; glp-1 ( lf ) ( Notch OFF ) and gld-1 gld-2; glp-1 ( gf ) ( Notch ON ) gonads ( Figure 2A and Figure 2\u2014figure supplement 2 , Figure 2\u2014source data 1 ) .", "These changes were confirmed by quantitative RT-PCR ( RT-qPCR ) on selected transcripts ( Figure 2\u2014figure supplement2A , Figure 2\u2014source data 1 ) .", "Most differentially expressed transcripts were upregulated in the \u2018Notch ON\u2019 gonads , indicating a predominantly activating role of GLP-1Notch in germ cells .", "For simplicity , we will refer to the transcripts upregulated at least two fold in the Notch ON gonads as \u2018Notch\u2013activated\u2019 .", "Some Notch-activated genes , such as sel-8/Mastermind , lst-1 , and epn-1/Epsin , have been implicated in Notch signaling in other cell types ( Doyle et al . , 2000; Yoo , 2004; Tian et al . , 2004; Singh et al . , 2011; Kershner et al . , 2014 ) .", "However , it remains possible that , rather than being direct GLP-1Notch targets , some of the Notch-activated genes were upregulated as an indirect consequence of increased GLP-1Notch signaling . 10 . 7554/eLife . 15477 . 008Figure 2 . GLP-1Notch and PRC2 regulate common targets and are functionally connected .", "( A ) Notch-activated genes are biased for the sex chromosome linkage .", "Left: Changes in transcript abundance in the \u2018Notch ON\u2019 versus \u2018OFF\u2019 dissected gonads ( genotypes explained in Figure 2\u2014figure supplement 1A\u2013B ) were analyzed by microarrays .", "Transcripts upregulated at least 2-fold in the \u2018Notch ON\u2019 gonads are marked in red , those downregulated at least 2-fold in blue .", "Selected transcripts verified by RT-qPCR in Figure 2\u2014figure supplement 2A are additionally circled in black .", "Right: 5426 genes can be considered expressed in the gonad , based on the bimodal distribution of expression values .", "Only 3% of those expressed genes are X-linked .", "In contrast , nearly half ( 46% ) of the expressed and Notch-activated transcripts are X-linked ( see Figure 2\u2014figure supplement 2B for numbers ) .", "( B ) GLP-1Notch and PRC2 interact genetically .", "Left: DAPI-stained gonads from animals of the indicated genotypes .", "The mes-2 ( bn11 ) M+Z- single mutant gonads have wild-type appearance at 20\u00b0C .", "The glp-1 ( ar202 ) gain-of-function mutants have an almost wild-type appearance at this temperature , except for an extended proliferative zone in the gonad , referred to as 'distal tumor' .", "At the same temperature , mes-2 ( bn11 ) M+Z-; glp-1 ( ar202 ) double mutants developed germline tumors in 32/32 of the examined gonads .", "The insets show close-ups from the indicated gonadal regions: the distal-most regions contain undifferentiated , proliferative germ cells in all mutants ( a , c , e ) .", "However , while the single mutants contain oocytes with characteristically condensed chromosomes in the proximal gonads ( b ,", "d ) , the proximal gonads of the double mutants harbor proliferative germ cells ( f ) .", "Scale bar = 30 \u03bcm .", "Right: quantification of the phenotypes .", "'Distal tumor' indicates the presence of an elongated distal proliferative zone ( approximately \u00bd of the distal gonad arm ) .", "'Extended' tumor indicates an extended distal tumor , few oocytes , and frequently also a proximal tumor .", "'Fully tumorous' indicates the absence of all differentiated cell types except for sperm produced during larval development .", "( C ) GLP-1Notch and PRC2 target the same genes on the X chromosomes .", "The plots correlate changes in gene expression in M+Z- mes-2 mutants with changes in gene expression changes in M+Z- mes-6 mutants .", "Results are shown separately for X-linked ( left ) and autosomal ( right ) transcripts .", "Notch-activated genes ( red in Figure 2A ) are marked in red .", "Lightly shaded areas indicate transcripts that are at least 2-fold upregulated .", "The overlap between transcripts upregulated by GLP-1Notch and transcripts upregulated by the loss of CePRC2 is highly significant , particularly for the X-linked genes .", "The significance of the correlation was measured by hypergeometric distribution; X-linked Notch-activated vs . mes-2 derepressed: p=1 . 31e-31; X-linked Notch-activated vs . mes-6 de-repressed: p=7 . 41e-25; autosomal Notch-activated vs . mes-2 derepressed: p=1 . 47e-22; autosomal Notch-activated vs . mes-6 de-repressed: p=1 . 8e-12 . DOI: http://dx . doi . org/10 . 7554/eLife . 15477 . 00810 . 7554/eLife . 15477 . 009Figure 2\u2014source data 1 . Microarray results .", "( A ) Microarray results of differentially expressed genes in Notch ON/OFF gonads .", "Figure 2\u2014figure supplement 3 source data: Quantification of germlines with wild-type morphology versus germlines with tumors upon germline-autonomous RNAi against genetic interactors of PRC2 .", "More details can be found in the corresponding figure legends . DOI: http://dx . doi . org/10 . 7554/eLife . 15477 . 00910 . 7554/eLife . 15477 . 010Figure 2\u2014figure supplement 1 . Examining transcriptional effects of GLP-1Notch signaling .", "( A ) GLP-1Notch signaling counteracts the meiosis-promoting activity of several RNA binding proteins , of which GLD-1 and -2 are indicated .", "( B ) Mutants that were used in this study: while the loss-of-function ( lf ) alleles of glp-1 cause the loss of germ cells , gain-of-function ( gf ) alleles result in a constitutive proliferation of germ cells .", "However , in the absence of the meiosis-promoting GLD-1 and GLD-2 proteins , germ cells continue to proliferate in the absence of GLP-1Notch activity .", "( C ) Three representative gonads of Notch ON: gld-2 ( q497 ) gld-1 ( q485 ) ; glp-1 ( ar202 ) and Notch OFF: gld-2 ( a497 ) gld-1 ( q485 ) ; glp-1 ( e2144 ) animals are shown after dissection and DAPI staining .", "The central planes of the gonads were imaged .", "Nuclei were counted from those images using the CellCounter plugin with ImageJ .", "For each genetic background , germ cells in the entire gonad of 10 dissected gonads were counted .", "The quantification below revealed that the numbers of germ cells in both backgrounds are not changed .", "Error bars represent SD .", "Scale bar = 10 \u03bcm . DOI: http://dx . doi . org/10 . 7554/eLife . 15477 . 01010 . 7554/eLife . 15477 . 011Figure 2\u2014figure supplement 2 . Analysis of Notch-activated genes .", "( A ) Changes in the abundance of several transcripts ( marked black in Figure 2A ) were verified independently from the genomic quantification by RT-qPCR .", "Colors correspond to fold-changes detected by microarrays ( green ) , by RT-qPCR on genetically identical gonads ( black ) , or by RT-qPCR on gonads with a different loss-of-function glp-1 allele , q175 ( white ) .", "*This experiment was performed only once .", "The error bars represent SEM .", "( B ) Although the number of genes on the arrays is almost equally distributed between the different chromosomes , the expression of X-linked genes is largely silenced in the germline .", "The table displays the numbers and fractions of genes on the different chromosomes , and the distributions of the Notch-activated genes .", "The cutoff between 'not expressed' and 'expressed' genes was set according to the bimodal distribution of expression values in the Notch ON and OFF arrays . DOI: http://dx . doi . org/10 . 7554/eLife . 15477 . 01110 . 7554/eLife . 15477 . 012Figure 2\u2014figure supplement 3 . The PRC2 component MES-6 and most enhancers/suppressors of glp-1 ( ar202 ) induced tumors appear to interact genetically with GLP-1Notch signaling in a germline-autonomous manner . Each bar indicates the relative proportion of germlines with wild-type morphology ( grey ) , germlines that contain eggs but also a proximal or distal tumor ( red ) , and germlines that are fully tumorous ( black ) .", "'n' indicates the number of DAPI-stained gonads scored for each column .", "Empty vector 1\u20133 represent three independent replicates of the empty vector control and demonstrate the robustness of the experiment .", "We observed that , for an unknown reason , the rrf-1 ( pk1417 ) ; glp-1 ( ar202 ) double mutants were less likely to produce tumors at the semi-permissive temperature of 20\u00b0C .", "Nonetheless , the double mutant strain reacts to enhancers , suppressors , and depletion of PRC2 components in a similar manner as the glp-1 ( ar202 ) single mutant strain , with the exception of mbk-1 and B0416 . 5 RNAi .", "Depletion of mes-2 and mes-3 by RNAi was ineffective , since it did not enhance the tumourous phenotype in either of the two strains . DOI: http://dx . doi . org/10 . 7554/eLife . 15477 . 01210 . 7554/eLife . 15477 . 013Figure 2\u2014figure supplement 4 . Global levels of H3K27me3 are unaffected by neither loss-of-function nor gain-of-function mutations in glp-1 . Stainings of wild-type , gain-of-function glp-1 ( ar202 ) , Notch ON ( gld-2 ( q497 ) gld-1 ( q485 ) ; glp-1 ( ar202 ) ) and Notch OFF ( gld-2 ( a497 ) gld-1 ( q485 ) ; glp-1 ( q175 ) ) gonads with antibodies against H3K27me3 performed on whole worms ( left panel , scale bars = 10 \u03bcm ) or dissected gonads ( right panel , scale bars = 1 \u03bcm ) .", "The H3K27me3 levels do not differ globally between the different mutant backgrounds .", "Asterisks indicate distal tips of gonads , dashed lines outline gonad . DOI: http://dx . doi . org/10 . 7554/eLife . 15477 . 013 To demonstrate that Notch-activated genes are functionally relevant for germ cell proliferation , we performed RNAi knockdown of Notch-activated genes ( n\u00a0=\u00a064 ) on animals carrying the gain-of-function glp-1 ( ar202 ) allele , and screened for enhancement or suppression of the tumorous gonad phenotype ( Supplementary file 1; for detailed experimental procedure see Materials and Methods ) .", "Knocking down some of the Notch-activated genes suppressed the tumorous phenotype , which agrees with predominantly proliferation-promoting role of GLP-1Notch .", "Interestingly , knocking down a smaller\u00a0subset of the Notch-activated genes enhanced the tumor ( Supplementary file 1 ) , suggesting that some of the Notch-activated genes may counteract proliferation .", "While some of these genes may function autonomously in the germline , others could affect the germline indirectly from the soma .", "To test this , we RNAi-depleted selected candidates in the rrf-1 ( pk1417 ) mutant background , which is permissive for RNAi in the germline but deficient in RNAi in many ( but not all ) somatic tissues ( Kumsta and Hansen , 2012 ) .", "While depleting most candidates in the rrf-1 background had similar effects on the germline as in the wild type ( suggesting germline-autonomous function ) , in some cases the effects were abolished , suggesting that these genes function in the soma ( Figure 2\u2014figure supplement 3 , Figure 2\u2014source data 1 ) .", "Strikingly , we noticed that Notch-activated genes were enriched on the X-chromosome , the C . elegans sex chromosome .", "45% of the Notch-activated genes were X-linked , which is four-fold more than expected by chance ( p=2 . 99e-14; Figure 2A and Figure 2\u2014figure supplement 2B , Figure 2\u2014source data 1 ) .", "When analyzing only genes with higher than baseline germline expression values , the disproportional X-linkage of Notch-activated genes was even more striking ( fifteen times more than expected by chance ( p=2 , 19e-38; Figure 2A and Figure 2\u2014figure supplement 2B , Figure 2\u2014source data 1 ) .", "In the C . elegans germline , X-linked genes are largely silenced by the C . elegans PRC2 ( Fong et al . , 2002 ) .", "Thus , the X chromosome bias among Notch-activated genes suggested a possible antagonistic relationship between GLP-1Notch and PRC2 .", "The C . elegans PRC2 consists of MES-2 , -3 , and -6 ( Bender et al . , 2004 ) and levels of the corresponding transcripts were essentially not altered by GLP-1Notch signaling ( mes-2: absolute fold change ( fc ) -1 . 3747; mes-3: fc 1 . 003; mes-6: fc 1 . 037 ) .", "To test for a functional relationship between GLP-1Notch and PRC2 , we examined genetic interactions between GLP-1 and PRC2 mutants .", "At 20\u00b0C , both the temperature-sensitive gain-of-function glp-1 ( ar202 ) and the homozygous loss-of-function mes-2 ( bn11 ) mutants , derived from heterozygous mothers providing maternal MES-2 ( mes-2\u2009M+Z- mutants ) , were viable and produced gonads with nearly wild-type appearance .", "The double mes-2\u2009M+Z-; glp-1 ( ar202 ) mutants , however , displayed distal and proximal tumors at the same temperature ( Figure 2B; 32/32 examined gonads ) .", "PRC2 and GLP-1Notch thus interact functionally , and they appear to do so in a germ cell autonomous manner ( Figure 2\u2014figure supplement 3 , Figure 2\u2014source data 1 ) .", "Given the striking enrichment of Notch-activated genes on the X chromosome , and the genetic interaction between PRC2 and GLP-1Notch , we speculated that GLP-1Notch and PRC2 act on similar genes .", "To determine whether Notch-activated genes are also PRC2-repressed , we first determined putative PRC2 targets by expression analyses on isolated wild-type , M+Z- mes-2 or mes-6 mutant gonads ( GEO accession number GSE49395 ) .", "Consistent with the joint function of MES-2 and MES-6 in the PRC2 complex , a very similar set of genes was upregulated upon the loss of either protein ( Figure 2C; Figure 2\u2014source data 1; henceforth \u2018PRC2 repressed\u2019 genes; also see ( Gaydos et al . , 2012 ) .", "Indeed , those PRC2-repressed genes overlapped strongly with Notch-activated genes , particularly those linked to the X chromosome .", "Nearly all of the X-linked Notch-activated genes were also derepressed upon the loss of PRC2 ( Figure 2C ) .", "This is consistent with the observed genetic interaction and suggests that increased GLP-1Notch signaling can induce expression of specific PRC2-repressed genes .", "This activation of the PRC2-repressed genes is not due to a global loss of the repressive tri-methylation of Histone H3 at lysine residue 27 ( H3K27me3 ) , since , upon examining gonads of different GLP-1Notch mutants , we observed no global loss of H3K27me3 in the germline ( Figure 2\u2014figure supplement 4 ) .", "Germ cell conversion to neurons can be triggered not only by LIN-53 depletion but also by the depletion of PRC2 ( Patel et al . , 2012 ) .", "Potentially , the depletion of LIN-53 could facilitate reprograming by inhibiting PRC2 , since the depletion of LIN-53 results in a global loss of H3K27me3 in the germline ( Patel et al . , 2012 ) .", "Considering the antagonistic relation between GLP-1Notch and PRC2 on cell proliferation and gene regulation , we wondered whether GeCo triggered by the depletion of PRC2 would be sensitive to Notch signaling .", "Indeed , GeCo was strongly enhanced in PRC2-depleted ( mes-2 , mes-3 or mes-6 RNAi ) animals , when they also carried the gain-of-function glp-1 ( ar202 ) allele ( Figure 3A , Figure 3\u2014source data 1 ) .", "Moreover , similar to the LIN-53\u2013depleted gld-1 ( q497 ) gld-2 ( q485 ) ; glp-1 ( q175 ) gonads ( Figure 1D ) , the loss of GLP-1 effectively prevented GeCo in PRC2\u2013depleted gld-1 ( q497 ) gld-2 ( q485 ) ; glp-1 ( q175 ) gonads ( Figure 3B , Figure 3\u2014source data 1 ) .", "Together , these results suggest that GLP-1Notch stimulates GeCo in PRC2-compromised gonads . 10 . 7554/eLife . 15477 . 014Figure 3 . GLP-1Notch enhances reprograming upon the depletion of PRC2 . ( A ) Notch and PRC2 genetically interact in GeCo .", "Left: Fluorescent micrographs of glp-1 ( ar202 ) gain-of-function mutants expressing CHE-1\u2013induced neuronal GFP .", "The animals were subjected to control RNAi or RNAi against PRC2 components ( MES-2 , 3 , and 6 ) , as indicated .", "Increased GLP-1Notch signaling enhanced the GeCo+ phenotype upon PRC2 depletion .", "Control RNAi ( mock ) for each genetic background did not result in any GeCo ( images not shown \u2013 see quantification ) .", "Right: The corresponding quantifications .", "P-values were calculated using Student's t-test: p1=0 , 0006; p2<0 , 0001; p3=0 , 0536; p4=0 , 0001; p5=0 , 4035; p6=0 , 0003 .", "At least 200 animals were scored per condition .", "Error bars represent SEM .", "( B ) GLP-1Notch is required for GeCo in PRC2-depleted animals independently from proliferation .", "Left: Fluorescent micrographs of adults expressing CHE-1\u2013induced nGFP , with the genotypes indicated above the panels .", "The animals were subjected to RNAi as indicated on the left .", "The first column shows heterozygous , the other two homozygous animals carrying the loss of function alleles gld-1 ( q497 ) , gld-2 ( q485 ) and , in the central panels , glp-1 ( q175 ) .", "The animals were subjected to control RNAi or RNAi against PRC2 components ( MES-2 , 3 , and 6 ) .", "In the absence of GLP-1Notch , depletion of PRC2 components did not induce GeCo .", "Scale bars = 10 \u03bcm .", "Right: The corresponding quantifications .", "P-values were calculated using Student's t-test: p1<0 , 0456; p2=0 , 0337; p3=0 , 0070; p4=0 , 0637; p5=0 , 0080; p6=0 , 1259 .", "At least 70 animals were scored per condition .", "Error bars represent SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 15477 . 01410 . 7554/eLife . 15477 . 015Figure 3\u2014source data 1 . Quantification of GeCo upon PRC2 depletion .", "( A ) Quantification of GeCo+ phenotype upon RNAi against PRC2 subunits in glp-1 ( ar202 ) mutants .", "( B ) Quantification of GeCo in different genetic backgrounds with highly proliferative germlines upon RNAi against PRC2 subunits .", "More details can be found in the corresponding figure legends . DOI: http://dx . doi . org/10 . 7554/eLife . 15477 . 015 To determine how GLP-1Notch might counteract PRC2 , we depleted candidate Notch-activated genes ( Supplementary file 1 ) , and examined GeCo efficiency ( Figure 4A , Figure 4\u2014source data 1 ) .", "The strongest suppression of the GeCo+ and GeCo phenotype was observed upon the depletion of utx-1 , which also suppressed mes-3 RNAi-mediated GeCo+ in glp-1 ( ar202 ) gonads ( Figure 4\u2014figure supplement 1 ) .", "Depletion of two other candidates , the uncharacterized C07G1 . 6 and the aldolase ortholog aldo-1 , also suppressed GeCo+ , albeit less efficiently ( Figure 4A ) .", "Because UTX-1 was suggested to effect gonadal development by functioning in the somatic gonad ( Vandamme et al . , 2012 ) , we re-examined GeCo efficiency upon utx-1 RNAi in the rrf-1 ( pk1417 ) background .", "Importantly , in rrf-1 mutants , RNAi is impaired in the somatic gonad , including the distal tip cell ( DTC ) , which constitutes the germline stem cell niche ( Kumsta and Hansen , 2012 ) .", "Because the suppression of GeCo+ upon utx-1 RNAi was observed also in the rrf-1 background , UTX-1 does not seem to enhance GeCo by functioning in the somatic gonad ( Figure 4\u2014figure supplement 1A ) .", "Furthermore , different genetic backgrounds and RNAi agains utx-1\u00a0do not affect the expression levels of\u00a0CHE-1 in the germline ( Figure 4\u2014figure supplement 1B ) . 10 . 7554/eLife . 15477 . 016Figure 4 . The H3K27 demethylase UTX-1 is required for GeCo enhancement .", "( A ) UTX-1 is critical for GeCo enhancement .", "Candidate Notch-activated genes , selected from Supplementary file 2 with available RNAi clones , were assayed for a role in GeCo in glp-1 ( ar202 ) animals , expressing CHE-1\u2013induced nGFP and treated with lin-53 RNAi .", "While the additional depletion of utx-1 had the strongest impact on GeCo+ and GeCo , the depletion of C07G1 . 6 and aldo-1 had a weaker effect .", "Representative fluorescence micrographs are below the quantification chart .", "White dashed line outline the animal body , yellow lines outline gonadal areas with GeCo .", "P-values for GeCo+ were calculated using Student's t-test: p1=0 , 000013; p2=0 , 026; p3=0 , 021; p4>0 , 1 .", "At least 250 animals were scored per condition .", "Error bars represent SEM .", "nGFP = gcy-5::gfp .", "Scale bars = 10 \u03bcm .", "( B ) As in A , but RNAi was performed against jmjd-1 . 2 ( H3K9/27me2 demethylase ) ; jmjd-3 . 1 , jmjd-3 . 2 , and jmjd-3 . 3 , ( H3K27me2/3 demethylases ) ; and jmjd-2 ( H3K9/36 demethylase ) .", "Only RNAi against jmjd-1 . 2 suppresses GeCo+ , though to a lesser degree compared to utx-1 RNAi .", "Representative fluorescence micrographs are below the quantification chart .", "P-values for GeCo+ were calculated using Student's t-test: p1=0 , 0042; p2=0 , 035; p3>0 , 2 .", "At least 190 animals were scored per condition .", "Error bars represent SEM .", "Scale bars = 10\u00a0\u03bcm . DOI: http://dx . doi . org/10 . 7554/eLife . 15477 . 01610 . 7554/eLife . 15477 . 017Figure 4\u2014source data 1 . Quantification of GeCo upon double RNAi against lin-53 and Notch-activated genes .", "( A ) Quantification of GeCo+ upon RNAi against lin-53 and Notch-activated genes .", "( B ) Quantification of GeCo+ upon RNAi against lin-53 and Histone demethylases .", "Figure 4\u2014figure supplement 1A source data: Quantification of GeCo+ upon RNAi against lin-53 and utx-1 with and without rrf-1 ( pk1417 ) background .", "More details can be found in the corresponding figure legends . DOI: http://dx . doi . org/10 . 7554/eLife . 15477 . 01710 . 7554/eLife . 15477 . 018Figure 4\u2014figure supplement 1 . UTX-1 is required for the GeCo+ enhancement upon the depletion of PRC2 . ( A ) Adult glp-1 ( ar202 ) animals treated with lin-53 or mes-3 RNAi were additionally subjected to either control or utx-1 RNAi .", "Depletion of utx-1 strongly suppressed the GeCo+ phenotype .", "Suppression upon utx-1 co-depletion with lin-53 is also detectable in the rrf-1 ( pk1417 ) background which is permissive for RNAi in the germline but not in the somatic gonad and the DTC .", "Right: quantification: n = 715 were scored for lin-53; utx-1 RNAi; n = 500 for mes-3; utx-1 RNAi and n = 270 were scored for lin-53; utx-1 RNAi in rrf-1 ( pk1417 ) .", "P-values were calculated using Student's t-test: p1=0 , 0588; p2=0 , 0042; p3=0 , 2454; p4=0 , 01713; p5=0 , 40479; p6=0 , 00271 .", "Error bars represent SEM .", "( B ) anti-HA antibody staining for the 3xHA-tagged CHE-1 protein , which is being induced after heat-shock treatment in the different genetic backgrounds: glp-1 ( ar202 ) ; otIs305 ( hspprom::che-1::3xHA ) ntIs1 ( gcy-5prom::gfp ) treated with our without RNAi against lin-53 and utx-1 .", "As additional controls the strain otIs305 ( hspprom::che-1::3xHA ) ntIs1 ( gcy-5prom::gfp ) with or without lin-53 RNAi and heat shock treatment was used .", "No obvious changes in the induction of CHE-1::3xHA in the germlines of the different genetic backgrounds can be detected .", "Scale bars = 1 \u03bcm . DOI: http://dx . doi . org/10 . 7554/eLife . 15477 . 018 Importantly , utx-1 encodes a conserved H3K27me3 demethylase , an enzyme erasing the repressive mark deposited by PRC2 ( Agger et al . , 2007; Jin et al . , 2011; Maures et al . , 2011 ) , potentially explaining how its depletion impairs GeCo efficiency .", "However , a number of other H3K27me3 demethylases exist in C . elegans , which prompted us to test whether depletion of these demethylases might have an effect on GeCo in the glp-1 ( ar202 ) gonads .", "We RNAi-depleted jmjd-1 . 2 , encoding a H3K9/27me2 demethylase ( Kleine-Kohlbrecher et al . , 2010 ) , jmjd-3 . 1 , jmjd-3 . 2 , and jmjd-3 . 3 , which were reported to demethylate H3K27me2/3 ( Agger et al . , 2007; Kleine-Kohlbrecher et al . , 2010; Zuryn et al . , 2014 ) , and , as a control , jmjd-2 , encoding a H3K9/36 demethylase ( Whetstine et al . , 2006; Greer et al . , 2014 ) ( Figure 4B , Figure 4\u2014source data 1 ) .", "Only the depletion of JMJD-1 . 2 suppressed GeCo+ significantly , though to a lesser degree than the depletion of UTX-1 ( Figure 4B , Figure 4\u2014source data 1 ) .", "The suppression of GeCo by the depletion of UTX-1 or JMJD-1 . 2 stresses the importance of counteracting PRC2 in reprograming .", "However , only the expression of utx-1 is activated by the GLP-1Notch signaling , suggesting that it is the activity of UTX-1 which is key for the reprograming dependent on GLP-1Notch signaling .", "The inhibition of GeCo enhancement upon utx-1 RNAi in the rrf-1 background suggested that UTX-1 functions in the germline .", "To test the potential expression of utx-1 in the germline , we constructed a strain expressing single copy-integrated , FLAG and GFP-tagged , functional UTX-1 ( expressed from the endogenous utx-1 promoter under the control of endogenous utx-1 3'UTR ) .", "We found that this protein was indeed expressed in the germline ( Figure 5\u2014figure supplement 1 ) .", "To examine the potential transcriptional regulation of utx-1 expression by GLP-1Notch and PRC2 , we also created a strain expressing GFP-tagged histone H2B from the utx-1 promoter ( putx-1 reporter ) , under the control of an unregulated ( tbb-2 ) 3\u2019UTR .", "This construct was also single-copy integrated into a defined genomic location .", "With both the UTX-1 fusion protein and the putx-1 reporter , we expected expression patterns similar to that of other reported GLP-1Notch target genes ( Lamont et al . , 2004; Lee et al . , 2006; Kershner et al . , 2014 ) .", "Among these , lst-1 and sygl-1 account for the role of GLP-1Notch in stem cell maintenance , and both genes are expressed in the distal-most stem cells/progenitors ( Kershner et al . , 2014 ) .", "By contrast , both the UTX-1 fusion protein and the putx-1 reporter were little or not expressed in the distal-most , proliferative part of the germline ( Figure 5A\u2013C; Figure 5\u2014figure supplement 1 ) .", "Concomitantly with progression through meiosis , their expression increased toward the proximal end of the gonad ( Figure 5A\u2013C ) .", "When examining the existing mRNA hybridization patterns of Notch-activated and PRC2-repressed genes ( 33 genes ) , we noticed that half of these ( 18 , all X-linked ) are similarly expressed in the medial and/or proximal , but not the distal-most , gonads ( Supplementary file 2 ) , arguing against direct transcriptional activation of these genes by GLP-1Notch in the wild type .", "Nevertheless , in agreement with the expression analyses , we observed increased expression of the putx-1 reporter in PRC2-depleted gonads; this increase occurred throughout the gonad , including the distal-most region ( Figure 5A ) .", "In situ hybridization for the endogenous utx-1 transcript also showed increased expression throughout the gonad in the absence of PRC2 ( Figure 5\u2014figure supplement 2 ) .", "Moreover , expression of the putx-1 reporter was higher upon increased GLP-1Notch activity in glp-1 ( ar202 ) mutants , including the distal-most region where , in the wild\u00a0type , utx-1 is little or not expressed ( Figure 5B ) .", "Importantly , we found that the activation of the utx-1 promoter by Notch signaling was depended on the putative LAG-1/CSL binding sites within the promoter ( Yoo et al . , 2004 ) , as deleting those sites reduced reporter expression by approximately one-fourth ( Figure 5C ) .", "The interaction between LAG-1 and utx-1 was also tested by chromatin immunoprecipitation ( ChIP ) , performed on a strain expressing FLAG-tagged LAG-1 in either wild-type or glp-1 ( ar202 ) background ( Figure 5D and Figure 5\u2014figure supplement 3 ) ; the previously identified GLP-1Notch targets , lst-1 and sygl-1 ( Kershner et al . , 2014 ) , served as positive controls for the ChIP .", "Expectedly , we observed the enhanced binding of LAG-1 to the utx-1 promoter , indicating that utx-1 is a transcriptional target of GLP-1Notch signaling .", "Summarizing , based on the observations in mutant backgrounds , PRC2 and GLP-1Notch signaling have antagonistic effects on utx-1 transcription .", "However , in wild type , the endogenous levels of GLP-1Notch signaling are apparently insufficient to overcome PRC2-mediated repression of utx-1 in the distal-most part of the gonad . 10 . 7554/eLife . 15477 . 019Figure 5 . UTX-1 is regulated by GLP-1Notch and PRC2 . ( A\u2013C ) Expression of utx-1 is regulated by PRC2 and GLP-1Notch .", "Top: dissected gonads expressing a GFP reporter , driven from the utx-1 promoter ( putx-1::GFP , fused to histone 2B for nuclear localization to facilitate quantification ) , subjected to the indicated RNAi ( A ) , crossed into the indicated genetic background ( B ) or carrying the indicated mutations in the reporter gene ( C ) .", "a , b , and c indicate gonadal regions containing germ cells in mitosis", "( a ) , and leptotene/zygotene", "( b ) or pachytene", "( c ) stages of meiosis .", "Below: the corresponding GFP quantifications .", "The diagrams show GFP intensities relative to the control ( indicated by green arrows ) in regions a-c .", "Results are represented as average changes in the GFP intensity ( relative to mock RNAi-ed or untreated animals ) .", "The error bars represent SEM .", "The numbers of analyzed gonads were as follows: n = 44 for wild-type reporter; n = 36 for glp-1 ( ar202 ) ; n = 55 for wild-type reporter on control RNAi; n = 48 for mes-2 ( RNAi ) ; n = 15 for mes-3 ( RNAi ) ; n = 29 for mes-6 ( RNAi ) , and n = 20 for the LAG-1 binding sites deleted reporter .", "( A ) The putx-1::GFP reporter is repressed by PRC2 .", "In all mes-depleted gonads , the reporter was de-repressed in proliferating cells", "( a ) as well as in more proximal gonadal regions ( b-c ) .", "( B ) The putx-1::GFP reporter is upregulated by GLP-1Notch .", "In the gain-of-function glp-1 ( ar202 ) mutant , the reporter was strongly derepressed in the proliferating cells in the distal-most gonad", "( a ) .", "Its expression was also increased in the more proximal regions ( b-c ) , which , in this mutant background , contain proliferating cells instead of meiotic cells .", "( C ) putx-1::GFP expression depends on the predicted LAG-1/CSL binding sites in the promoter .", "Upon deletion of putative LAG-1/CSL binding sites , the reporter expression was abolished .", "The changes in GFP intensities were highly significant ( p-values were measured by independent t-tests ) p1=4 . 85\u201313 , p2=1 . 38\u201320 , p3=1 . 18\u20137 .", "( D ) LAG-1 binds the utx-1 promoter .", "Lysates of animals expressing FLAG-tagged LAG-1 ( strain wgIs591; lag-1::TY1::EGFP::3xFLAG ) , either in wild-type or glp-1 ( ar202 ) background , were subjected to ChIP-qPCR analysis of the indicated genes .", "Negative controls and additional tested genes are shown in Figure 5\u2014figure supplement 3 .", "The qPCR amplicons were tested in at least three independent experiments .", "The results are shown as fold enrichment in anti-FLAG IP compared to IP with unspecific antibody .", "The 3\u2019UTR of lst-1 serves as a negative control .", "Interestingly , LAG-1 binding in the glp-1 ( ar202 ) gain-of-function background is stronger to the utx-1 promoter than to the reported GLP-1Notch targets lst-1 and sygl-1 .", "The asterisk indicates\u00a0a p-value < 0 . 05 ( Students t-test ) .", "Error bars represent SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 15477 . 01910 . 7554/eLife . 15477 . 020Figure 5\u2014figure supplement 1 . The functional utx-1 transgene is expressed in the same pattern as a GFP reporter coupled to the utx-1 promoter . Shown is an adult with an outlined gonad .", "The full-length GFP-tagged functional UTX-1 ( rrrSi189 ) is repressed in the distal , proliferative part .", "Nuclei entering meiosis and developing oocytes express the fusion protein progressively stronger .", "This expression pattern is identical with the pattern seen in the utx-1 promoter reporter strains ( rrrSi185 , rrrSi281 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 15477 . 02010 . 7554/eLife . 15477 . 021Figure 5\u2014figure supplement 2 . The endogenous utx-1 mRNA is upregulated in the absence of the PRC2-component MES-6 . Shown are representative in-situ hybridisations against endogenous utx-1 mRNA in dissected gonads .", "In M+Z- mes-6 ( bn66 ) mutants , utx-1 is upregulated throughout the gonads compared to the control wild-type gonads .", "'AS' and 'S' indicate antisense and sense probes . DOI: http://dx . doi . org/10 . 7554/eLife . 15477 . 02110 . 7554/eLife . 15477 . 022Figure 5\u2014figure supplement 3 . Testing LAG-1 binding to additional genes by ChIP . Worm lysates ( corresponding to 4 mg protein ) of animals , with or without lag-1::TY1::EGFP::3xFLAG ( wgIs591 ) transgene , were used for ChIP .", "Samples were incubated with 50 \u00b5l of FLAG ( \u2018specific\u2019 FLAG antibody ) or HA antibodies ( \u2018unspecific\u2019 HA antibody ) coupled to \u00b5MACS microbeads ( Milteny ) .", "As negative control , lysate N2 or glp-1 ( ar202 ) worm lysates , which do not express the recombinant target protein , were used .", "Both negative control lysates did not show any differences during the ChIP experiment when tested with either specific antibody ( anti-FLAG coupled to \u00b5MACS beads ) or unspecific antibody ( anti-HA coupled to \u00b5MACS beads ) .", "Lysates of worms expressing the recombinant target protein in N2 or glp-1 ( ar202 ) background were incubated with specific ( anti-FLAG ) and unspecific ( anti-HA ) antibodies coupled to \u00b5MACS beads .", "The qPCR amplicons were tested in a minimum of three independent ChIP-qPCR experiments .", "Quantification results are shown as fold enrichment of anti-FLAG \u00b5MACS\u2122 beads using wgIs591 lysate over anti-FLAG \u00b5MACS beads using lysate without wgIs591 ( no lag-1::TY1::EGFP::3xFLAG ) .", "Primer for qPCRs ( sequence details above ) were designed using Primer3Plus ( Untergasser et al . , 2007 ) .", "The FLAG-beads using lysates with lag-1::TY1::EGFP::3xFLAG show specific enrichment for tested target genes thereby validating the specificity of the ChIP .", "The asterisks indicates p-values < 0 . 05 ( Students t-test ) .", "Error bars represent SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 15477 . 022"], ["In the C . elegans germline , GLP-1Notch signaling is essential for maintaining a pool of undifferentiated stem cells/progenitors .", "Here , we report an unexpected role of GLP-1Notch signaling in promoting cell fate reprograming .", "To understand this phenomenon , we identified genes activated upon increased GLP-1Notch signaling .", "While the identified genes include the physiological GLP-1Notch targets , sygl-1 and lst-1 , many other genes , including utx-1 , appear to be only weakly or not expressed in the distal-most region of the wild-type gonad , where both sygl-1 and lst-1 are induced by GLP-1Notch ( Kershner et al . , 2014 ) .", "Thus , the wild-type levels of GLP-1Notch signaling are either insufficient to induce expression of many potential target genes ( see below ) , or their expression is controlled by additional mechanisms , perhaps similarly to lip-1 mRNA , which , while induced by GLP-1Notch , is post-transcriptionally degraded in the distal-most gonad ( Hajnal and Berset , 2002;\u00a0Lee et al . , 2006 ) .", "In addition to its major proliferation-promoting function , GLP-1Notch has been suggested to have a lesser role in promoting germ cell differentiation ( Hansen et al . , 2004 ) .", "Some of the identified Notch-activated genes appear to promote germ cell differentiation , potentially explaining the proposed differentiation-promoting function of GLP-1Notch .", "However , whether these genes are activated by GLP-1Notch and promote differentiation under physiological conditions remains to be determined .", "Many of the Notch-activated genes are repressed by PRC2 , suggesting that the expression of these genes is determined by the crosstalk between the extrinsic intercellular signaling pathway , Notch , and the intrinsic chromatin modifier PRC2 .", "Indeed , at least in the case of utx-1 , PRC2 appears to prevent its inappropriate expression in the distal-most gonad , which , nevertheless , can be overcome upon increased GLP-1Notch signaling .", "Our findings suggest that GLP-1Notch antagonizes PRC2 , at least in part , by stimulating expression of the H3K27 demethylase UTX-1 , which is essential for the enhancement of cellular reprograming .", "Previously , it was suggested that UTX-1 influences the germline by functioning in the somatic gonad ( Vandamme et al . , 2012 ) .", "However , by using the rrf-1 background , which displays defective RNAi in the somatic gonad , including the\u00a0DTC ( Kumsta and Hansen , 2012 ) , we found that the reprograming-promoting role of UTX-1 is unlikely to depend on its function in the somatic gonad .", "Although we cannot fully exclude the possibility that the reprograming-facilitating role of UTX-1 depends on its expression in another somatic tissue ( such as the intestine , in which RNAi remains functional in the rrf-1 mutant; Kumsta and Hansen , 2012 ) , the germline expression of UTX-1 reported here suggests a germline-autonomous function .", "Consistent with this hypothesis , manipulating either PRC2 or GLP-1Notch affected the germline expression of utx-1 .", "Although additional factors , such as the uncharacterized C07G1 . 6 and the ortholog of the human aldolase A ( Kuwabara and O'Neil , 2001; Shaye and Greenwald , 2011 ) might contribute to reprograming , they appear to be less critical .", "In addition to UTX-1 , another H3K27/H3K9-demethylating enzyme , JMJD-1 . 2 ( Kleine-Kohlbrecher et al . , 2010 ) , is required for enhanced reprograming .", "Similar to UTX-1 , JMJD-1 . 2 is likely to be directly involved in regulating chromatin accessibility , since its depletion results in increased levels of the repressive H3K9me2 and H3K27me2 modifications ( Kleine-Kohlbrecher et al . , 2010 ) .", "The reprograming-promoting role of JMJD-1 . 2 might indicate that , besides further demethylation of H3K27me2 , perhaps also the removal of H3K9me2 facilitates GeCo .", "Future studies will shed light on this interesting question .", "UTX-1 orthologs in other species contribute to tissue-specific chromatin signatures , for example during myogenesis or in cardiac development ( Seenundun et al . , 2010; Lee et al . , 2012 ) , and have been implicated in germ-cell and somatic reprograming ( Mansour et al . , 2012 ) .", "Together with our data , these findings underscore the importance of UTX-1 in cellular reprograming .", "Here , we suggest that one way the activity of UTX-1 may be stimulated to promote reprograming is through its Notch signaling-dependent transcriptional activation .", "Interestingly , an antagonistic relationship between Notch and PRC2 has also\u00a0been observed in T-cell leukemia ( Ntziachristos et al . , 2012 ) .", "A fascinating possibility is that a regulatory principle described here could help explain the etiology of this and perhaps other human diseases linked to a pathological increase in Notch signaling ."], ["Standard procedures were used to maintain animals .", "Worms ( RRID:WB_DL226 ) were grown at 25\u00b0C unless stated otherwise .", "All heat-shock and temperature-sensitive strains were kept at 15\u00b0C .", "The C . elegans lines used in this study are listed and described in detail in the Supplementary file 3A .", "The enhancer-suppressor screen on Notch targets was performed by feeding the animals with bacteria containing RNAi clones from the Ahringer and Vidal RNAi libraries .", "The clones not present in either of these libraries were cloned using primers as described in detail in the Supplementary file 3B .", "Experiments were performed at 15\u00b0C , 20\u00b0C or 25\u00b0C using bleached embryos or overnight-synchronized L1 animals as stated in Supplementary file 1 .", "The percentage of adult animals with the germline tumor phenotype was scored .", "To test germ-cell autonomy , RNAi clones that induced significant suppression or enhancement in either setting were re-tested in a strain carrying the glp-1 ( ar202 ) ( RRID:WB_GC833 ) allele and , additionally the rrf-1 ( pk1417 ) ( RRID:WB_NL2098 ) mutation , which impairs RNAi primarily in the soma ( Sijen et al . , 2001 ) .", "For this test , gravid adults were picked to RNAi plates and allowed to lay eggs overnight at the semi-permissive temperature of 20\u00b0C .", "Progeny was scored for enhancement or suppression of the germline tumorous phenotype at the young adult stage by DAPI staining of dissected gonads and scoring gonads as either wild-type , as containing a proximal or distal tumor but still some eggs , or as completely tumorous .", "Reprograming experiments were carried out as F1-RNAi .", "Worms were put on RNAi plates and the following F1 generation was screened for phenotypes .", "Used RNAi clones are described in the Supplementary file 3B .", "The genotype of the worms used for germ cell reprograming assays is BAT28 ( otIs305 ntIs1 , RRID:WB_OH9846 , details in Supplemental Materials and methods ) .", "Animals were synchronized by bleaching and eggs were put on NGM-agar containing E . coli OP50 ( RRID:WB_OP50 ) as a\u00a0food source to grow at 15\u00b0C until worms reached the L4-stage .", "At this stage 15\u201320 worms were put per well of a 6-well plate , containing bacteria expressing dsRNA or carrying an empty RNAi vector , and grown at 15\u00b0C until most of the F1 progeny reached the L3/L4 stage .", "The plates were heat-shocked at 37\u00b0C for 30\u2009min followed by an overnight incubation at 25\u00b0C .", "Next day ( ~16\u2009hrs post heat-shock ) the plates were screened for progeny showing ectopic GFP induction in the germline .", "To induce the Glp\u00a0phenotype in glp-1 ( ar202 ) , the animals were shifted to room temperature 9\u2009hrs before the heat-shock .", "For double RNAi , bacteria were grown as saturated culture .", "The OD600 was measured to ensure that the bacteria were mixed in an appropriate 1:1 ratio and subsequently seeded on RNAi 6-well plates .", "The RNAi screen was performed as described above .", "The p-values were calculated using Student's t-test .", "Hydroxyurea ( HU ) treatment was carried out as previously described ( Fox et al . , 2011; Patel et al . , 2012 ) .", "HU was added to seeded RNAi-plates at a final concentration of 250 \u00b5M .", "L4 worms ( strain BAT32 , details in Supplementary file 3A ) grown on RNAi-plates were transferred to HU plates and incubated at room temperature for 5\u2009hrs prior to heat-shock in order to induce CHE-1 expression .", "To test HU efficiency , control animals were treated with HU for 12\u2009hrs with subsequent staining for DAPI and H3Ser10ph ( pH3 ) antibody ( Abcam #ab5176 ) .", "After overnight incubation , the worms were assessed for GFP induction in the germline as described above .", "To assess the efficiency of the HU treatment , the E . coli strain MG1693 ( E . coli stock center ) , with incorporated 5-ethynyl-20-deoxyuridine ( EdU ) , was used to feed L3/L4 worms .", "EdU in combination with DAPI staining was performed similar to the procedure described previously ( Ito and McGhee , 1987 ) and according to the manufacturer's instructions ( Invitrogen , Europe ) of the EdU labeling kit .", "The Click-iT EdU reaction buffer ( Invitrogen , Europe ) was mixed with Alexa Fluor azide ( \u2018click\u2019 reaction ) dye to detect cells that were replicating DNA .", "Staining was performed by freeze cracking worms after fixation with 2% formaldehyde .", "Parental animals were raised at 15\u00b0C and shifted to 25\u00b0C for egg laying .", "Offspring was dissected after the L4-adult transition .", "Fifty gonads per analyzed genotype in triplicates were dissected in M9 containing levamisole .", "RNA was isolated using the PicoPure RNA Isolation Kit ( Applied Biosystems ) according to the manufacturer\u2019s recommendations .", "Total RNA ( 200 ng for the Notch ON/OFF experiments or 100 ng for the mes/wild-type experiments ) was amplified using the Affymetrix GeneChip WT Amplified Double Stranded cDNA Synthesis Kit according to the manufacturer\u2019s instructions .", "The Affymetrix GeneChip WT Double-Stranded cDNA Terminal Labeling Kit was used for the fragmentation and labeling of 7 . 5\u2009\u03bcg cDNA .", "The labeled material was loaded onto Affymetrix GeneChip C . elegans Tiling 1 . 0R Arrays and hybridized at 65\u00baC for 16\u2009hrs .", "The arrays were washed in an Affymetrix Fluidics station with the protocol FS450_0001 and scanned in an Affymetrix GeneChip Scanner 3000 with autoloader using Affymetrix GCC Scan Control v . 3 . 0 . 0 . 1214 software .", "All tiling arrays were processed in R ( 32 , 33 ) using Bioconductor ( 34 ) and the packages tilingArray and preprocessCore .", "The arrays were RMA background corrected and log2 transformed on the oligo level using the following command: expr <- log2 ( rma . background . correct ( exprs ( readCel2eSet ( filenames , rotated = TRUE ) ) ) ) .", "We mapped the oligos from the tiling array ( bpmap file from www . affymetrix . com ) to the C . elegans genome assembly ce6 ( www . genome . ucsc . edu ) using bowtie ( Langmead et al . , 2009 ) , allowing no error and unique mapping position .", "Expressions for individual transcripts were calculated by intersecting the genomic positions of the oligos with transcript annotation ( WormBase WS190 ) and averaging the intensity of the respective oligos .", "For the mes-4/wt experiment , no quantile normalization was performed as the distributions for the mes-4 mutant and the wt differed substantially .", "In the case of the Notch ON/OFF dataset quantile normalization was performed on the level of transcripts .", "The p-values were calculated in R with the hypergeometric distribution function 'phyper' .", "RNA was isolated as described above .", "cDNA was synthesized with oligo dT primer using the ImProm II Reverse Transcription System from Promega according to the manufacturer\u2019s instructions .", "cDNA was used for qPCR with the Absolute QPCR SYBR Green ROX Mix ( AbGene ) on an ABI PRISM 7700 system ( Applied Biosystems ) .", "qPCR reactions were performed as described previously ( Biedermann et al . , 2009 ) .", "At least one primer in each pair is specific for an exon-exon junction .", "Sequences of the used primers are described in detail in the Supplementary file 3C .", "Mouse RNA was added before RNA isolation and reverse transcription , allowing for normalization to cyt-c and thereby correcting for variations in RNA isolation and reverse transcription .", "Standard curves for quantification were generated from a serial dilution of input cDNA for each primer pair .", "The amount of target present in each replicate was derived from the standard curve; an average was calculated for each of the triplicates .", "To compare total mRNA levels , the qPCR results were normalized to mouse cyt-c and to the C . elegans tubulin gene tbb-2 , and fold enrichments were calculated .", "RNA in situ hybridization was performed and analyzed as previously described ( Biedermann et al . , 2009 ) .", "Description of the primer pairs to generate probes from cDNA can be found in the Supplementary file 3C .", "Images were captured with a Zeiss AxioImager Z1 microscope , equipped with a Zeiss AxioCam MRc camera .", "Images were taken in linear mode of the Axiovision software ( Zeiss ) and processed with Adobe Photoshop CS4 in an identical manner .", "ChIP was performed as described ( Askjaer et al . , 2014 ) .", "In brief , worms ( strains OP591 , RRID:WB_OP591 , and BAT890 ) were washed with M9 buffer and frozen as \u2018worm popcorn\u2019 in liquid nitrogen prior to pulverization with a mortar and pestle .", "The powder was dissolved in 10 volumes of 1 , 1% formaldehyde in PBS + 1\u00a0mM PMSF and incubated 10\u2009mins with gentle rocking at room temperature with subsequent quenching for 5\u2009mins at room temperature by adding 2 , 5 molar glycine ( final concentration 125\u2009mM ) .", "After centrifugation with 4 . 000\u2009g at 4\u00b0C the pellet was washed with ice-cold PBS+1\u00a0mM PMSF and resuspended in 50\u2009mM FA buffer ( 50\u2009mM HEPES/KOH pH 7 , 5; 1\u00a0mM EDTA; 1% Triton-X 100; 0 , 1% sodium deoxycholate; 150\u2009mM NaCl ) + 1% sarkosyl + protease-inhibitors .", "Samples were sonicated twice using the Biorupter ( 15 times 30 s ON , 30 s OFF ) on high settings at 4\u00b0C followed by spinning at 13 . 000\u2009g , 15\u2009min , 4\u00b0C .", "The supernatant corresponding to 4 mg of protein measured by Bradford assay was used for ChIPs .", "Samples were incubated with 50 \u00b5l of FLAG or HA antibodies coupled to \u00b5MACS microbeads ( Milteny ) ( all blocked with 5% milk in FA-buffer ) .", "After incubating 1\u2009hr at 4\u00b0C , the beads where washed in \u00b5MACS matrix columns in a magnetic rack with FA buffer containing 1\u00a0M and 0 . 5\u2009M NaCl and subsequent wash with TE and TEL buffer ( 0 , 25\u2009M LiCl; 1% Sodium deoxycholate; 1\u2009mM EDTA; 10\u2009mM Tris pH 8 , 0 ) .", "Bound material was eluted with 65\u00b0C pre-warmed 125 \u00b5l ChIP elution buffer ( 1% SDS , 250\u2009mM NaCl , 10\u2009mM Tris pH 8 . 0 , 1\u2009mM EDTA ) and fixation was reversed using 2 \u00b5l of 10 mg/ml Proteinase K at at 50\u00b0C for 1\u2009hr followed by 65\u00b0C incubation overnight .", "DNA was purified using the Quiaquick PCR purification kit in a final volume of 40 \u00b5l and 1 \u00b5l was used for qPCR .", "Negative controls were used to assess the quality of the ChIP and fold enrichment of the target genes: lysates ( N2 worms ) which do not express the recombinant target protein with specific antibody ( anti-FLAG coupled to \u00b5MACS beads ) and lysates of worms expressing the recombinant target protein with unspecific antibody ( anti-HA coupled to \u00b5MACS beads ) controls , respectively .", "Primer for qPCRs was designed using Primer3Plus ( Untergasser et al . , 2007 ) with the following settings: amplified region min . 100 bp \u2013 max .", "200 bp; GC content: 50\u201360%; min . primer length: 18 nt \u2013 max .", "length 24 nt; melting temperature: min . 58\u00b0C \u2013 max .", "63\u00b0C; max .", "; 3' self complementary allowance set to 1; max .", "allowed length of a mononucleotide repeat ( max . poly-x ) : 3 .", "Sequences of the used primers are listed in in the Supplementary file 3C .", "The qPCRs were run on CFX96 Touch Real-Time PCR Detection System from BioRad using the Maxima SYBR Green/ROX qPCR Master Mix ( 2X ) .", "The data analysis was performed by calculating the \u0394\u0394Ct-values .", "Differences were assessed using Student's t-test .", "Worms were transferred to a slide and fixed by adding 10\u201320 \u00b5l 95% ethanol and letting evaporate the ethanol .", "The ethanol fixation was repeated 2 more times before adding the DAPI solution in microscopy mounting media ( vectashield from Vector or similar ) .", "The samples were sealed with a coverslip and nail polish before microscopy .", "Fluorescent micrographs were recorded with Zeiss AxioImager Z1 microscope and the SensicamII camera ( PCO ) and the micromanager software was used to capture Z-stack images with 0 . 5 \u00b5m slice steps .", "Images subject to direct comparison were taken at identical exposure times .", "Counting of germ cells within the range from the DTC to the turn of gonadal arms of glp-1 ( ar202 ) ; hsp::che-1; gcy-5::gfp animals treated with either mock or lin-53 RNAi was performed using the Z-stacks .", "Micromanager was used to control the Z-stack levels and the ImageJ plugin for cell counting for scoring the number of germ cells .", "Germ cell counts in gonads of Notch ON: gld-2 ( q497 ) gld-1 ( q485 ) ; glp-1 ( ar202 ) and Notch OFF: gld-2 ( a497 ) gld-1 ( q485 ) ; glp-1 ( e2144 ) ( Figure 1\u2014figure supplement 2 ) and germline tumor phenotype in glp-1 ( ar202 ) and glp-1 ( ar202 ) ; rrf-1 ( pk1417 ) were scored after dissection , formaldehyde fixation and DAPI staining .", "For Notch ON and Notch OFF mutants , the central plane of the gonads was imaged and germ cells in the entire dissected gonad were counted using the CellCounter plugin with ImageJ .", "For each of the two strains , germ cells in the entire gonad of 15 dissected gonads were counted .", "Antibody stainings of intact worms were performed using a freeze-crack procedure as described ( Duerr , 2006 ) .", "In brief , after washing , worms were resuspended in 0 . 025% glutaraldehyde , and frozen between two frost-resistant glass slides on dry ice .", "Separating the glass slides while frozen creates additional cracks in the cuticle .", "Acetone/methanol or 4% paraformaldehyde in 0 . 1\u00a0M phosphate buffer for 1\u2009hr on ice fixation was used .", "Worms were washed off the slides in PBS , blocked with 0 . 2% gelatin + 0 . 25% Triton in PBS , and stained .", "Primary antibodies were diluted in PBS with 0 . 1% gelatin and 0 . 25% Triton and fixed worms were incubated 4\u2009hrs - overnight at 4\u00b0C .", "After PBS washes secondary antibody was applied for 3\u2009hrs .", "After PBS washes worms were mounted with DAPI-containing mounting medium ( Dianova , #CR-38448 ) on glass slides .", "The primary antibodies used were anti-H3K27me3 ( 1:400; gift from Dr . Hiroshi Kimura ) ; anti-HA ( 1:100 , Roche #12CA5; acetone fixation ) ; anti-H3Ser10ph ( 1:400 , Abcam , #ab14955; acetone fixation ) .", "Secondary antibodies were Alexa Fluor dyes applied at 1:1000 dilution .", "Stainings for H3K27me3 on dissected gonads were performed using anti-H3K27me3 from Millipore ( catalogue number 07\u2013449 , Lot 1959680; courtesy of Jan Padeken , Gasser laboratory ) on dissected gonads .", "The\u00a0adult animals were dissected in M9 containing levamisole , fixed with 2% paraformaldehyde in PBS on poly-lysine coated slides , snap-frozen on dry ice , freeze-cracked , incubated for 5 mins in ice-cold DMF at \u221220\u00b0 , washed for 5\u2009mins in PBS 0 . 1% Tween-20 at room temperature , blocked for 20\u2009mins in PBS 0 . 1% Tween-20 + 5% BSA and incubated with the primary antibody over night at 4\u00b0C .", "Secondary antibodies ( Alexa 488 , goat anti rabbit , 1:500 ) were applied for 2\u00a0hrs at room temperature .", "Slides were then washed three times for 5\u2009mins in PBS 0 . 1% Tween-20 at room temperature and mounted with Vectashield mounting medium containing DAPI .", "The transcriptional reporter gene putx-1::gfp-h2b ( rrrSi185 ) was constructed from the 1302 bp putative promoter region of the gene utx-1 ( human UTX ( Ubiquitously transcribed TPR on X ) homolog - 1 ) fused to sequences encoding for GFP-H2B and the ubiquitously expressed tbb-2 3\u2019UTR using the Gateway Reporter Cloning System ( Merritt et al . , 2008 ) .", "The reporter gene putx-1::gfp-h2b ( rrrSi185 and rrrSi281 ) was constructed with the following primers: putx-attB4: GGGGACAACTTTGTATAGAAAAGTTGGGATTTTATCTTCATCGGACCTG putx-attB1 : GGGGACTGCTTTTTTGTACAAACTTGTGGCGGTGTGAGAAGCGATAC The full-length functional transgene putx-1::flag-gfp-utx-1::utx-1 3\u2019UTR ( rrrSi189 ) was constructed with the following primers: utx-1+3UTR+attB2 L ggggacagctttcttgtacaaagtggACGACGAATCAGAACCTCTGCCGGAGGAGCGTCATgtaag utx-1+3UTR+attB3 R ggggacaactttgtataataaagttgaatgcggatactgccttctc The functional UTX-1 transgene putx-1::flag-gfp-tev::utx-1::utx-1 3\u2019utr ( rrrSi189 ) contains the same promoter as the transcriptional reporter , the full-length utx-1 genomic sequence as well as the endogenous 3\u2019UTR , and was equipped with N-terminal GFP , FLAG , and TEV tags .", "Transgenic animals were produced\u00a0as\u00a0single-copy integrants\u00a0using the MosSCI direct insertion protocol ( Fr\u00f8kjaer-Jensen et al . , 2008 ) .", "The rrrSi189 transgene is functional , as it rescues the utx-1 mutant ( ok3553 allele ) .", "For GFP quantification , gonads were dissected from live animals in M9 buffer containing levamisole and mounted to glass microscopy slides ( Fr\u00f8kjaer-Jensen et al . , 2008 ) .", "Fluorescent micrographs were recorded with Zeiss AxioImager Z1 microscope and a Zeiss Axioncam MRm REV 2 CCD camera was used to capture images .", "Fluorescence intensities were quantified using ImageJ .", "GFP intensities were normalized to the picture background and corrected with the average autofluorescences measured in wild type ( N2 ) gonads at the corresponding temperatures .", "Images subject to direct comparison were taken at identical exposure times and were processed with Adobe Photoshop CS4 in an identical manner .", "The numbers of analyzed gonads were as follows: n = 44 for wild-type reporter; n = 36 for glp-1 ( gf ts ) ; n = 55 for wild-type reporter on control RNAi; n = 48 for mes-2 ( RNAi ) ; n = 15 for mes-3 ( RNAi ) ; n = 29 for mes-6 ( RNAi ) , and n = 20 for LAG-1 binding sites deleted reporter .", "Alleles used were glp-1 ( ar202 ) and mes-2 ( bn11 ) .", "Worms were grown at the semi-permissive temperature of 20\u00b0C and gonads were dissected and DAPI-stained shortly after the L4-young adult transition .", "The experiment was performed twice .", "In a first round , a low number of gonads were examined to identify whether the double mutant had a phenotype and to define phenotypic categories to score .", "Based on the observation of a clear and penetrant phenotype , gonads were scored in a second round according to the categories defined in the first round ."]], "headings": ["Introduction", "Results", "Discussion", "Materials and methods"], "abstract": ["Cell-fate reprograming is at the heart of development , yet very little is known about the molecular mechanisms promoting or inhibiting reprograming in intact organisms .", "In the C . elegans germline , reprograming germ cells into somatic cells requires chromatin perturbation .", "Here , we describe that such reprograming is facilitated by GLP-1/Notch signaling pathway .", "This is surprising , since this pathway is best known for maintaining undifferentiated germline stem cells/progenitors .", "Through a combination of genetics , tissue-specific transcriptome analysis , and functional studies of candidate genes , we uncovered a possible explanation for this unexpected role of GLP-1/Notch .", "We propose that GLP-1/Notch promotes reprograming by activating specific genes , silenced by the Polycomb repressive complex 2 ( PRC2 ) , and identify the conserved histone demethylase UTX-1 as a crucial GLP-1/Notch target facilitating reprograming .", "These findings have wide implications , ranging from development to diseases associated with abnormal Notch signaling ."], "summary": ["The DNA in genes encodes the basic information needed to build an organism or control its day-to-day operations .", "Most cells in an organism contain the same genetic information , but different types of cell use the information differently .", "For example , many of the genes that are active in a muscle cell are different from those that are active in a skin cell .", "These different patterns of gene activation largely determine a cell\u2019s identity and are brought about by DNA-binding proteins or chemical modifications to the DNA ( which are both forms of so-called epigenetic regulation ) .", "Nevertheless , cells occasionally change their identities \u2013 a phenomenon that is referred to as reprograming .", "This process allows tissues to be regenerated after wounding , but , due to technical difficulties , reprograming has been often studied in isolated cells grown in a dish .", "Seelk , Adrian-Kalchhauser et al . set out to understand how being surrounded by intact tissue influences reprograming .", "The experiments made use of C . elegans worms , because disturbing how this worm\u2019s DNA is packaged can trigger its cells to undergo reprograming .", "Seelk , Adrian-Kalchhauser et al . show that a signaling pathway that is found in many different animals enhances this kind of reprograming in C . elegans .", "On the one hand , these findings help in understanding how epigenetic regulation can be altered by a specific tissue environment .", "On the other hand , the findings also suggest that abnormal signaling can result in altered epigenetic control of gene expression and lead to cells changing their identity .", "Indeed , increased signaling is linked to a major epigenetic mechanism seen in specific blood tumors , suggesting that the regulatory principles uncovered using this simple worm model could eventually provide insights into a human disease .", "A future challenge will be to determine precisely how the studied signaling pathway interacts with the epigenetic regulator that controls reprograming .", "Understanding this interaction in molecular detail could help to devise strategies for controlling reprograming .", "These strategies could in turn lead to treatments for people with conditions that cause specific cells types to be lost , such as Alzheimer\u2019s disease or injuries ."], "keywords": ["developmental biology"]}

scientific_lay_summarization.py → scientific_lay_summarisation.py

@@ -100,7 +100,7 @@ class ScientificLaySummarisation(datasets.GeneratorBasedBuilder):
     def _split_generators(self, dl_manager):
         """Returns SplitGenerators."""
         dl_paths = dl_manager.download_and_extract(_URLS)
-        path = os.path.join(dl_paths[self.config.name], self.config.name + "-dataset")
+        path = dl_paths[self.config.name]
         return [
             datasets.SplitGenerator(
                 name=datasets.Split.TRAIN,
@@ -119,8 +119,8 @@ class ScientificLaySummarisation(datasets.GeneratorBasedBuilder):
     def _generate_examples(self, path=None):
         """Yields examples."""
         with open(path, encoding="utf-8") as f:
-            f = json.loads(f)
-            for line in f:
+            f = json.loads(f.read())
+            for d in f:
                 # Possible keys are:
                 # "id": str,                      # unique identifier
                 # "year": int,                    # year of publication
@@ -131,14 +131,13 @@ class ScientificLaySummarisation(datasets.GeneratorBasedBuilder):
                 # "summary": List[str],           # lay summary, in sentences
                 # "keywords": List[str]           # keywords/topic of article
 
-                d = json.loads(line)
-
                 sections = [" ".join(s).strip() for s in d["sections"]]
                 abstract = " ".join(d['abstract']).strip()
+                full_doc = [abstract] + sections
                 summary = " ".join(d["summary"]).strip()
 
                 yield d["id"], {
-                    _DOCUMENT: "\n".join([[abstract] + sections]),
+                    _DOCUMENT: "\n".join(full_doc),
                     _SUMMARY: summary,
                     "section_headings": "\n".join(["Abstract"] + d["headings"]),
                     "keywords": "\n".join(d["keywords"]),