How cells in the embryo coordinate epithelial plasticity with cell fate decision in a fast changing cellular environment is largely unknown. In chick embryos, skeletal muscle formation is initiated by migrating Delta1-expressing neural crest cells that trigger NOTCH signaling and myogenesis in selected epithelial somite progenitor cells, which rapidly translocate into the nascent muscle to differentiate. Here, we uncovered at the heart of this response a signaling module encompassing NOTCH, GSK-3β, SNAI1 and β-catenin. Independent of its transcriptional function, NOTCH profoundly inhibits GSK-3β activity. As a result SNAI1 is stabilized, triggering an epithelial to mesenchymal transition. This allows the recruitment of β-catenin from the membrane, which acts as a transcriptional co-factor to activate myogenesis, independently of WNT ligand. Our results intimately associate the initiation of myogenesis to a change in cell adhesion and may reveal a general principle for coupling cell fate changes to EMT in many developmental and pathological processes.DOI:http://dx.doi.org/10.7554/eLife.14847.001 During early embryogenesis, a succession of extensive tissue rearrangements and cell migration events, intimately associated with rapid cell fate changes, establish the tissues and organs of the future adult. A model where such complex issues are amenable to experimentation is the early formation of skeletal muscles in the chick embryo.Over many days of development, the medial border of the dermomyotome (DML) generates the first skeletal muscle cells that assemble into a primary myotome . This arClassical experiments demonstrated that signaling cues from tissues surrounding the somites act as inducers of muscle formation . It was This raises a paradox: although presumably all cells in the DML are equally exposed to WNTs from axial structures and are fully competent to initiate myogenesis, only a small proportion of DML cells do so at any given time , suggestvia an EMT.We recently introduced NOTCH signaling as a novel player in early myogenesis, by demonstrating that the activation of MYF5 and MYOD in the DML is dependent upon the transient activation of NOTCH signaling, triggered by Delta1-positive neural crest cells migrating from the dorsal neural tube . This fiHere, we discovered that in DML cells, the activation of NOTCH signaling by Delta1-positive neural crest cells strongly decreases GSK-3β activity, independent of NOTCH transcriptional function. This leads to a dramatic stabilization of SNAI1, resulting in the initiation of the EMT program. As a consequence, β-catenin from the cell membrane pool is mobilized, allowing its entry into the nucleus where it activates MYF5 expression in a WNT ligand-independent manner. Therefore, our results suggest that non-canonical functions of NOTCH and β-catenin, associated in an efficient signaling circuitry, explain the coupling of myogenesis with changes in cell adhesion in the DML.The first sign that myogenesis is initiated in the DML is the activation of MYF5 (or MYOD) expression, which serves as a read-out of a cell fate change in this structure . As WNT We found that, within the DML, almost half of the epithelial cells that activated the TOPflash reporter were also positive for the NOTCH reporter . RemarkaDuring this analysis, we made the surprising observation that a large majority of NOTCH reporter-positive cells displayed a high TOPflash fluorescence. On the contrary, most cells that expressed low levels of TOPflash fluorescence were NOTCH-reporter negative . It was The results above raised the possibility that NOTCH and the TCF/β-catenin transcriptional activity may be interconnected during the initial phases of myogenesis. To address this, we first co-electroporated the DML with a constitutively active form of β-catenin  togetherDickkopf (Dkk) genes comprise an evolutionarily conserved family that encode secreted proteins that antagonize WNTs, by inhibiting in the extracellular space the WNT co-receptors Lrp5 and 6 , this led to a near complete absence of TOPflash reporter activity in DML cells , which wTogether with the results presented in In the DML, the initiation of myogenesis is accompanied by an EMT . BecauseTo test whether β-catenin located at the AJ and at the plasma membrane of DML cells is required for the activation of MYF5, we electroporated DML cells with a mutant Y489F β-catenin that cannot be mobilized from the junctional complex . As contWe then tested whether junctional β-catenin is required, downstream of NOTCH, to induce MYF5. We repeated the same experiment as above, this time in combination with NICD. Y489F or WT β-catenin wερε expressed for 10 hr in the DML, after which  NICD expression was induced for 6 hr . In contTogether with the observation that MYF5 expression is associated with high activity of the TOPflash reporter in DML cells  and 4, oSnail (or SNAI) family members. An essential function of SNAI is to repress epithelial gene expression and thereby promote epithelial-mesenchymal transitions (EMT) during vertebrate and invertebrate development and metastatic progression of cancers . Unlike wild-type NICD, which upon electroporation readily enters the nucleus and massively activates the NOTCH reporter , immunosThese results prompted us to test whether CD4-NICD expression would mimic the effects of NICD on SNAI1 and MYF5. Indeed, we observed that the electroporation of CD4-NICD led to i) a massive stabilization of SNAI1 protein comparable to NICD alone  and ii) The findings presented here uncover a unique signaling module, triggered by a signal presented by migrating neural crest cells to selected progenitors in the somites. This signal is transduced into two distinct, but interrelated outcomes i) a cell fate decision in the receiving cells that leads to their entry into the myogenic program and ii) an EMT that allows their translocation into the myotome . While tEMT is intimately associated with essential changes in cell specification in many developmental processes. For instance, the massive and rapid cell rearrangement that takes place during neural crest formation or gastrulation in vertebrates vertebrate are associated with the acquisition of novel features that are characteristic of neural crest cells or of the forming mesoderm and endoderm, respectively . InhibitDrosophila , but alosophila . Our datosophila . It is tWhile it is largely accepted that β-catenin participates in adhesion and signaling functions in a mutually exclusive manner, changes in the transcriptional activity of β-catenin have also been associated with the disassembly of the junctional complex at the plasma membrane in vitro . The resvia the signaling module uncovered here, provokes the release of junctional β-catenin that leads to a high TOPflash response sufficient to trigger myogenesis. The reason for such different cellular responses is only speculative at present. It could be due to varying quantities of β-catenin entering the nucleus, low when WNT is presented by neural crest cells, high when it is (presumably massively) mobilized from the cell membrane. WNT11 and MYF5 would then be morphogen-like responses of DML cells to varying quantities of β-catenin. An alternative hypothesis is that of a qualitative difference in the transcriptional activity of β-catenin when it results from bona fide WNT signaling or when it is mobilized from the cell membrane. Rhee and colleagues have shown that β-catenin phosphorylated at the AJ on Y489 is transported into the nucleus and is transcriptionally active in vitro  expressed by the dorsal neural tube and transported by neural crest cells regulate the expression of WNT11 in the DML through canonical WNT signaling. WNT11 itself plays an essential role in the oriented elongation of early muscle fibers through planar cell polarity signaling . It is tin vitro . It is tFinally, our study also provides a model that reconciles apparently divergent observations on the respective role of NOTCH and WNT signaling in the initial phases of myogenesis. The necessary function of NOTCH in this process  is confiThe somite electroporation technique that was used throughout this study has been described elsewhere ; 2011. BThe following constructs have been previously published:The WNT reporter 12Tf-d2EGFP contains 12 TCF/LEF1 binding sites  upstreamThe CAGGS-H2B–RFP (provided by Dr. S. Tajbakhsh ) containThe CAGGS–EGFP  containsThe constitutively active form of LEF1, CA-LEF1 (provided by Dr. A. Münsterberg) is described in The CAGGS-DN-MAML1–EGFP contains a truncated, dominant-negative form of the human Mastermind (DN-MAML1), fused with EGFP , downstrDKK1 fused to a flag tag downstream of a CMV promoter (pCS2+ DKK1-flag (Addgene #16690) contains a human promoter .U3-DLL1  was madeA Tet-on  inducible system was used to activate NICD expression . The doxCAGGS-cSNAI1 contains the wild type chicken Snail1 gene downstream of the CAAGS promoter upstream of an IRES EGFP .The dominant negative form of SNAI1 consists of the chicken SNAI1 in which the repressor domain was replaced by the VP16 activator domain of the Herpes simplex virus .SNAI1-GFP (Addgene #16225) contains a CMV promoter upstream of a human wild type Snail1 fused to EGFP .CAGGS NICD contains the chicken NICD downstream of the CAGGS promoter .The GSK3 biosensor  contains a CMV promoter upstream of a GFP molecule followed by a polypeptide tail that contains 3 GSK-3β phosphorylation sites, a priming site for MAPK/Erk and a site for the binding of E3 polyubiquitin ligases .HES1–d2EGFP (provided by Dr. R. Kageyama) contains the HES1 mouse promoter followed by a destabilized d2EGFP .The constitutively active form of β-catenin was described in The dominant negative form of Dll1 has been described in A constitutively active form of RBPJ  comprises the CMV promoter driving a VP16 activation domain fused to the mouse RBPJ gene .The dominant-negative GSK-3β (Addgene #29681) is a kinase dead variant of the Xenopus GSK-3β gene fused to an EGFP protein .The SNAI1 siRNA (as well as the Luciferase control) were described and tested in The NOTCH1 siRNA was described in The following plasmids were constructed:HES1-DSRed-Express-DR was created by replacing the d2EGFP from HES1–d2EGFP (provided by Dr. R. Kageyama ) with a The wild-type human β-catenin was amplified from pFG8 (provided by Dr. N. Plachta), then cloned into pCX-Myc .Human β-catenin containing the Y489F (Tyr to Phe ) mutatioThe dominant-negative version of the mouse Wnt1 (obtained from Randy Moon ) was insThe wild-type version of WNT1 (obtained from Randy Moon) was inserted into pCAGGS IRES H2B-GFP  upstream of the IRES element.Danio rerio), the extracellular and transmembrane domain of human CD4 (both obtained from Dr. J Kaslin) followed by the HA tagged NICD previously described, cloned either into the bidirectional vector tetracycline responsive vector or into the pCX-Myc described above.The membrane tagged NICD was constructed using Gibson assembly (NEB) with the signal peptide FGFR2 (from The H2B-BFP has been made by replacing the RFP from the CAGGS-H2B–RFP described above by a TagBFP (Evrogen).The SNAI1-RFP has been made by replacing the GFP from SNAI1-GFP (Addgene #16225) by RFP (Clontech).CAGGS-BFP has been made by cloning the TagBFP (Evrogen) into the pCAGGS vector.Whole-mount antibody staining were performed as described . The folWe have tested the role of β-catenin from the membrane and AJ using a mutant (Y489F) form of β-catenin and a doxycycline inducible (Tet-On system) NICD. The association of β-catenin with proteins partners (α-catenin and cadherin) at the membrane is regulated by its phosphorylation at highly conserved tyrosine residues 142, 489 and 654 . In the Ten hours after electroporation of pCIRX-rtTA and pBI-HA-NICD, doxycycline (300 µl of a 0.75 µg/ml solution) was added onto the embryos. We have shown that the response plasmid is completely silent before doxycycline addition, while it is strongly and rapidly activated 6 hr after doxycycline addition .Dorsal views of somites shown in all figures are projections of stacks of confocal images taken using a 4-channel Leica SP5 confocal microscope (Leica). Confocal stacks of images were visualized and analyzed with the Imaris software suite (Bitplane). Cell counting was performed using the cell counter plugin  within the ImageJ software .Electroporation results in the transfection of a portion of the targeted cell population, which is variable from embryo to embryo. To precisely evaluate the phenotypes obtained after electroporation of cell-autonomously acting cDNA constructs, the number of positive cells was compared to the total number of electroporated cells, recognized by an internal fluorescent reporter construct. On average, more than 700 cells were counted per point and the corresponding quantifications are shown in all figures.To determine the fluorescence intensity of electroporated cells , the surStatistical analyses were performed using the GraphPad Prism software. Mann–Whitney non-parametric two-tail testing was applied to populations to determine the P values indicated in the figures. In each graph, columns correspond to the mean and error bars correspond to the standard deviation. ***p value < 0.001, extremely significant; **p value 0.001 to 0.01, very significant. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Fiona Watt as the Senior Editor.Thank you for submitting your work entitled "Cytoplasmic NOTCH and membranal β-catenin link cell fate choice to EMT during myogenesis" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor, Marianne Bronner, has drafted this decision to help you prepare a revised submission.Summary:This manuscript addresses an interesting developmental biology problem – how cell behavior and fate are coordinated during morphogenesis – using myogenesis of the developing chick somite as a model system. The authors first resolve a previous conundrum in the field to show how Notch and WNT signaling pathways are integrated during myogenesis in the development somite. Interestingly, their results show that Notch triggers release of β-catenin from adherens junctions to the nucleus to activate MYF5, in a WNT ligand independent manner. This in itself is an important result. Although exchange between membrane and nuclear β-catenin pools has been previously postulated, this study directly demonstrates this occurrence in vivo. Second, using a GSK-3β biosensor, they find that Notch ICD inactivates GSK-3β, which in turn stabilizes SNAI1, which promotes EMT and also leads to activation of MYF5 expression. Moreover, this activity of NICD is important in the cytosol and independent of transcription.eLife. However, the presentation of the manuscript, including the title, is confusing and will require rewriting and reorganization in order to present the results and conclusions in a more direct and easier to interpret manner. The following changes should be made.These results are very interesting and highly appropriate for Essential revisions:1) The manuscript clearly shows that both Notch and WNT pathways are required and that the WNT pathway is downstream of Notch. However, there is no need to invoke WNT itself since only β-catenin is required to transcriptionally activate myogenic factors and indeed they conclude this is ligand independent.2) It is clear that SNAI1 is downstream of Notch and that GSK-3 inhibition potentiates SNAI1 function as an EMT inducer. Once SNAI1 is activated and stabilized, DML cells undergo an EMT that is presumably the consequence of cadherin repression and dismantling of adherens junctions. This in turn leads to the release of β-catenin to the cytoplasm, which translocates to the nucleus to activate MYF5. This connection is implied but not stated. They could test this using the Y489F mutant in this experiment to see if NICD still leads to elevated SNAI1-GFP but no activation of MYF5 in the presence of the mutant. Although this experiment is non-essential given the huge amount of data already present in the manuscript, it might tie the stories together a bit better.3) The membrane tethered Y489F mutant is expressed in cells that already have endogenous β-catenin. The authors need to explain better why this construct prevents endogenous β-catenin from translocating to the nucleus.eLife this is may be a question for an independent study.4) It is clear that Notch signaling does not seem to use its canonical RBPJ-mediated transcriptional pathway. The question that remains unsolved is what is the mechanism by which Notch represses GSK-3 activity, but following the principles of 5) In the Discussion, the authors should address how their proposed model reconciles with various reports showing that WNT receptors or membrane bound WNTs  can promote myogenesis.In summary, the relevant players here are not Notch and WNT as stated in the title but rather Notch, SNAI1 and β-catenin. Similarly, alluding to "membrane" β-catenin in the title and in the text is misleading, as it is release from adhesion complexes in the membrane that allows β-catenin to fulfill its role in this process. As such, it is the SNAI1-mediated EMT that releases β-catenin from the membrane. In other words, it is a process mediated by EMT and β-catenin but independent of WNT ligand and Notch-mediated transcription. It is a very good example of how EMT can influence WNT signalling in a WNT-independent manner, as already suggested by Stemmer et al., Oncogene 2008 and discussed in EMT reviews. As already mentioned, all this is compatible with the model in eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Fiona Watt as the Senior Editor.Thank you for submitting your article "Cytoplasmic NOTCH and membrane-derived β-catenin link cell fate choice to epithelial-mesenchymal transition during myogenesis." for consideration by In the present version, the text is difficult to follow and to understand. It is essential that you spell out 'Epithelial-mesenchymal transition' in the title.The clarification about how the Y489F β-catenin works in the presence of the endogenous wild type protein should be added to the manuscript rather than just being in the response letter.It is mandatory to re-write the manuscript to make this study readable for the non-expert. 1) The manuscript clearly shows that both Notch and WNT pathways are required and that the WNT pathway is downstream of Notch. However, there is no need to invoke WNT itself since only β-catenin is required to transcriptionally activate myogenic factors and indeed they conclude this is ligand independent.2+-dependent pathways). This is the nomenclature we had decided to follow for our manuscript, despite the fact that one of the main messages of our study is that β-catenin can act as a transcription factor independent of WNT ligand. We understand the confusion that may rise from mentioning WNT in the text and we have revised the text throughout to address the point raised by the reviewers.There is a consensus in the WNT field to name the pathway that is dependent on β-catenin-dependent transcription the "WNT/β-catenin-dependent" pathway , to differentiate it from alternate pathways (i.e. the Planar cell Polarity and the Ca2) It is clear that SNAI1 is downstream of Notch and that GSK-3 inhibition potentiates SNAI1 function as an EMT inducer. Once SNAI1 is activated and stabilized, DML cells undergo an EMT that is presumably the consequence of cadherin repression and dismantling of adherens junctions. This in turn leads to the release of β-catenin to the cytoplasm, which translocates to the nucleus to activate MYF5. This connection is implied but not stated. They could test this using the Y489F mutant in this experiment to see if NICD still leads to elevated SNAI1-GFP but no activation of MYF5 in the presence of the mutant. Although this experiment is non-essential given the huge amount of data already present in the manuscript, it might tie the stories together a bit better.Ours and Giulio Cossu's laboratories have shown that the expression of DN forms of TCF or β–catenin lead to an arrest of MYF5 expression . Borello and colleagues have further shown that the transcription of MYF5 in the mouse DML is controlled by 5 TCF/LEF binding sites. Thus, that MYF5 expression in the DML is a direct consequence of the binding of TCF and β–catenin onto its regulatory sequences is known. We have clarified this point in the Introduction (third paragraph).3) The membrane tethered Y489F mutant is expressed in cells that already have endogenous β-catenin. The authors need to explain better why this construct prevents endogenous β-catenin from translocating to the nucleus.We have done this experiment a number of times with each time the same result: Y489F mutant acts (even in the presence of NICD) as a dominant-negative β–catenin molecule. Why this is the case is speculative. We had a long discussion on this very point with Hans Clevers a few weeks ago. His group published in 2009 a study showing that GSK3β is separated into two pools, a minor one (3-5%) that interacts with β–catenin within the APC complex and a major one that acts with its numerous other targets . Coherent with this study, NICD would likely interact with the "free" GSK3β available in the cytoplasm, blocking its activity . In contrast, the GSK3β located within the APC complex could be "immune" to interaction with NICD, being already associated with various proteins within the complex. Thus, when NICD is placed in an Y489F setting, WT β–catenin cannot accumulate enough to drive MYF5 since the APC complex is likely still active.A second hypothesis (based on our observation that MYF5 expression is strictly associated with a high WNT response) is that the amount of β-catenin needed to activate MYF5 cannot be reached (within the time frame of our experiments) by stopping its cytosolic degradation, but instead only after a massive mobilization from the cell membrane.Finally, it is possible that the transcriptional activity of the β-catenin released from the cell membrane is distinct from the one that escapes the APC complex. This is based on very preliminary observation that would need solid confirmation.One or a combination of the hypotheses described above could explain the failure of NICD to rescue MYF5 expression in Y489F conditions. We are pursuing some of those hypotheses at present.4) It is clear that Notch signaling does not seem to use its canonical RBPJ-mediated transcriptional pathway. The question that remains unsolved is what is the mechanism by which Notch represses GSK-3 activity, but following the principles of eLife this is may be a question for an independent study.This is indeed an important point. We have obtained preliminary biochemical data showing that NICD interacts in a complex with GSK3β and Akt in vivo.Those results are encouraging, as they clearly show that NICD and the membrane-tethered NICD (CD4-NICD) co-immunoprecipitate with GSK3β and Akt, but they need to be repeated and improved. While the regulatory role of Akt on the phosphorylation (and therefore the inactivation) of Gsk3β is known, the interaction of NOTCH with Akt was never published. However, while this observation brings us one step further towards an understanding of NOTCH function in the cytosol, it leaves additional questions unanswered: e.g. we have no indication on whether NOTCH and Akt or GSK3 directly or indirectly interact and what is the consequence of this interaction on the activity of NOTCH itself. We provide a figure of those results to the reviewers to satisfy their curiosity, but because they are preliminary and incomplete, we do not wish to include those results in the manuscript. Because the role of NOTCH we uncovered is potentially of importance in a number of cellular contexts, we think these crucial questions deserve to be addressed in an independent study.5) In the Discussion, the authors should address how their proposed model reconciles with various reports showing that WNT receptors or membrane bound WNTs  can promote myogenesis.In fact, we have recently studied how WNT is delivered from neural crest to somites . We showed that WNT does not freely diffuse from migrating neural crest cells, but it is presented to the receiving DML cells by the Heparan Sulfate Proteoglycans family member, Dally-Like-Protein/GPC4. We demonstrated that GPC4 is necessary for the transfer of WNT from neural crest to DML cells. Thus, our results are coherent with those of Farin and colleagues, and in fact it could be that HSPGs are implicated in the cell-cell transfer of WNT they describe in the intestinal crypt. However, we show in the present study that WNT from neural crest does not play a role in myogenesis and we believe integrating the WNT signal from crest into our final model would add an unnecessary layer of complexity to it.In summary, the relevant players here are not Notch and WNT as stated in the title but rather Notch, SNAI1 and β-catenin. Similarly, alluding to "membrane" β-catenin in the title and in the text is misleading, as it is release from adhesion complexes in the membrane that allows β-catenin to fulfill its role in this process. As such, it is the SNAI1-mediated EMT that releases β-catenin from the membrane. In other words, it is a process mediated by EMT and β-catenin but independent of WNT ligand and Notch-mediated transcription. It is a very good example of how EMT can influence WNT signalling in a WNT-independent manner, as already suggested by Stemmer et al., Oncogene 2008 and discussed in EMT reviews. As already mentioned, all this is compatible with the model in derived β-catenin" would better reflect the activity we uncovered in this study. We therefore propose the following title: "Cytoplasmic NOTCH and membrane-derived β-catenin link cell fate choice to EMT during myogenesis".We understand the comment of the reviewers and we think the terms "membrane-In general, the manuscript would benefit from text-editing to make it more concise and to the point. The way it is presently written may make it difficult for the non-expert reader.In an effort to make the text easier to read, we have removed all numbers from the main text. They are now in the figure legends. We believe this considerably improves the flow of the story with the consequence that it is easier to follow.In the present version, the text is difficult to follow and to understand. It is essential that you spell out 'Epithelial-mesenchymal transition' in the title.The clarification about how the Y489F β-catenin works in the presence of the endogenous wild type protein should be added to the manuscript rather than just being in the response letter.It is mandatory to re-write the manuscript to make this study readable for the non-expert.We have gathered all comments made by the reviewers in the first review and have addressed each of them below. As a result, entire paragraphs have been re-written, new experiments added or results presented differently in an effort to make a clearer story.Numbered comments by reviewers1) "there is no need to invoke WNT itself since only β-catenin is required to transcriptionally activate myogenic factors and indeed they conclude this is ligand independent".We agree with this comment: naming WNT could be misleading. To avoid any confusion, we have now changed most references to WNT ligands, as long as the text still made sense after those changes. For instance, we replaced the term "WNT response" by "TOPflash activity" or " TCF/β-catenin transcriptional activity"; we also replaced the term "WNT reporter" by "TOPflash reporter", etc. In this way, we believe that there should be no confusion for the readers as to what they are looking at.2) "They could test this using the Y489F mutant in this experiment to see if NICD still leads to elevated SNAI1-GFP but no activation of MYF5 in the presence of the mutant".We have tested every steps of the signaling module in every possible way. One can indeed think endlessly about new experiments, e.g. blocking GSK3 or NOTCH and rescuing MYF5 with Snail, LEF or β catenin; or as proposed here, blocking β catenin and verifying that all steps upstream are still active. As mentioned by the reviewers, those experiments are non-essential and importantly they do not provide further evidence for the validity of the signaling module we uncovered.3) "The membrane tethered Y489F mutant is expressed in cells that already have endogenous β-catenin. The authors need to explain better why this construct prevents endogenous β-catenin from translocating to the nucleus".We included an explanation  of why this is in the Discussion section, fifth paragraph).4)"The question that remains unsolved is what is the mechanism by which Notch represses GSK-3 activity"We have answered this point in the previous rebuttal. We have obtained preliminary data showing that NOTCH co-immunoprecipitates with GSK-3 and Akt. We do not know whether this is a direct or indirect interaction and we are testing at the moment which domain of NOTCH is implicated in this interaction.5)"In the discussion, the authors should address how their proposed model reconciles with various reports showing that WNT receptors or membrane bound WNTs  can promote myogenesis".The paper of Farin et al. does not study myogenesis. If the point that the reviewers want to make is the transport of WNT evoked in that paper, we have recently shown that WNT is not secreted from the neural tube, but rather transported to somites by the migrating neural crest cells. This point is now clearly stated in the first paragraph of the Discussion. But that is it carried rather than secreted does not change the outcome: we show experimentally that the WNT transported by NC does not regulate myogenesis, if we inhibit it (with DN WNT1), or increase it , it is the SNAI1-mediated EMT that releases β-catenin from the membrane".In the summary of the review, reviewers mentioned that The aim of a title is to attract the attention on what is novel in a paper so that potential readers go one step further, at least until the abstract where this is all clearly stated. That an EMT releases β-catenin from the membrane is not novel. Likewise, it is established that β-catenin triggers myogenesis. What is crucial here is that it is the β-catenin derived from the membrane pool, acting as a transcriptional co-factor, and not the cytoplasmic one that does the job. It is our view that this finding has to be in the title as well as the other key finding, the new function of NOTCH in the cytoplasm.On a more fundamental note, we have experimentally demonstrated that every step of this linear module leads to the same outcome, an activation of MYF5. Such that according to one's personal interests or likings, a molecule  or a process  may seem more crucial than another to the mechanism we describe. The fact is, all of them are necessary, and this module works only if all of them are there at the right place and moment. From the comments, it seems that reviewers believe that Snail and EMT are the driving force of myogenesis. In fact, it is easy to understand from the module we describe that the direct activation of MYF5 by CA LEF or CA β-catenin short-circuits the need for Snail or an EMT. We have done those experiments (they are not shown in the paper), we expectedly observe strong MYF5 activation, but indeed with no propensity of cells to translocate into the myotome, thereby dissociating de facto Snail and the EMT from myogenesis. In fine, the only required molecular event in the module we uncovered is the direct activation of MYF5 expression by β-catenin and LEF. The steps upstream are merely means to this end."It is a very good example of how EMT can influence WNT signalling in a WNT-independent manner, as already suggested by Stemmer et al., Oncogene 2008"The paper from Stemmer shows that Snail physically binds to β-catenin and stimulates its transcriptional activity, independent of a transcriptional regulation of cadherin. This is quite different from what we observe in our study and we are not aware that the mode of action of Snail on the transcriptional activity of membrane-derived β-catenin that we uncovered has ever been described."In general, the manuscript would benefit from text-editing to make it more concise and to the point. The way it is presently written may make it difficult for the non-expert reader".In an effort to make this study more understandable, and as suggested by the reviewers and the editor, we have included the responses we had included in the previous rebuttal about the mechanism that may explain how the Y489 mutant form of β-catenin may work in our system .We have also added at various places along the text (Results) and in the Discussion section explanations on the identity of the low TOPflash MYF5-negative DML cells. We have known what these cells are for some time and we extensively published on them. They are cells that respond to WNT carried by neural crest to somites  by activating WNT11 in the DML through canonical WNT signaling . WNT11 itself is a positional cue used by muscle cells to orient in the antero-posterior axis of the embryo through planar cell polarity signaling . Although their presence in the DML alongside the cell population that interests us in the present manuscript makes things a little more complicated, we have not found a way to avoid characterizing them. However, we found that the part of the manuscript that described this population  was rathWe have also added another panel in The Abstract has been modified for more clarity.As explained in the previous rebuttal, we have removed all numbers from the main text. They are now in the figure legends. We have also fixed a few glitches there and there.In conclusion, we are aware that the text is somewhat complex. Its complexity is due to the fact that we took the challenge to dissect an entire pathway, from beginning to end and to place it in its biological context. Every step of the pathway we uncovered was tested in numerous ways. We assayed about 30 distinct constructs and reporters in this study. There are a lot of experiments to describe. We wanted in this study to make a point that the chick system is amazingly powerful to test intricate questions with relative ease and in a short time. We believe we succeeded at this task. Contrasting with this apparent complexity, the end product is a simple signaling module that may have far reaching implications in various models. This is the take home message of the study that we believe most readers will remember.


A large-scale field study in naturally occurring vole populations identified gene expression changes over time and demonstrates how wild mammals exhibit tolerance to chronic parasite infections. Hosts are likely to respond to parasitic infections by a combination of resistance (expulsion of pathogens) and tolerance (active mitigation of pathology). Of these strategies, the basis of tolerance in animal hosts is relatively poorly understood, with especially little known about how tolerance is manifested in natural populations. We monitored a natural population of field voles using longitudinal and cross-sectional sampling modes and taking measurements on body condition, infection, immune gene expression, and survival. Using analyses stratified by life history stage, we demonstrate a pattern of tolerance to macroparasites in mature compared to immature males. In comparison to immature males, mature males resisted infection less and instead increased investment in body condition in response to accumulating burdens, but at the expense of reduced reproductive effort. We identified expression of the transcription factor Gata3 (a mediator of Th2 immunity) as an immunological biomarker of this tolerance response. Time series data for individual animals suggested that macroparasite infections gave rise to increased expression of Gata3, which gave rise to improved body condition and enhanced survival as hosts aged. These findings provide a clear and unexpected insight into tolerance responses (and their life history sequelae) in a natural vertebrate population. The demonstration that such responses  can move from resistance to tolerance through the course of an individual's lifetime emphasises the need to incorporate them into our understanding of the dynamics and risk of infection in the natural environment. Moreover, the identification of Gata3 as a marker of tolerance to macroparasites raises important new questions regarding the role of Th2 immunity and the mechanistic nature of the tolerance response itself. A more manipulative, experimental approach is likely to be valuable in elaborating this further. Hosts do not always resist parasites. And once infection establishes, relatively little is known of how naturally occurring hosts tolerate (mitigate for) adverse effects, or what the life history consequences of this tolerance may be. In this article we demonstrate a pattern of tolerance to parasitic worms and arthropods in wild voles and link this to increased expression of an immunological biomarker. The biomarker, Gata3, is triggered by infection, precedes significant changes in body condition, and impacts on fecundity and survival. These results point to the considerable ecological importance of tolerance in wild vertebrates and to how poorly it is understood, while at the same time giving a new perspective on the natural function of immune response pathways involving Gata3. There is increasing recognition that the ability to tolerate a parasite's presence, where the host accepts infection but actively limits the damage caused, may be a crucial host defence strategy The term “tolerance” is used differently by immunologists and disease ecologists. In moving towards some unification of meaning, we must distinguish between pattern and process. The pattern associated with tolerance, typically the main focus of ecologists, is a relative insensitivity of host health (fitness) to increases in parasite burden The study of tolerance in field systems, then, is an indispensible complement to studies in the laboratory, and there have been calls for the greater integration of data from natural populations on host fitness, immunological variation, and infection pressure Microtus agrestis, using interwoven cross-sectional and longitudinal sampling protocols. This hybrid study design took advantage of the greater range and precision of measurement possible in destructive  sampling, and of the stronger causal inferences possible in capture–recapture  sequences, where driver variables may be observed to precede the responses they trigger We hypothesised, first, that different groups of individuals  will differ in their tolerance—their relationship between health and parasite load—and second, that those individuals that maintain good health in relation to parasite burden  will express more of the physiological pattern associated with tolerance. We monitored immunological gene expression, infection, body condition, and survival in replicated natural populations of field voles, Specifically, adopting the phase space perspective (above), plotting our cross-sectional data comes closest to generating a tolerance curve. However, infection stage cannot be standardized in observations on natural populations. Hence, a less negative, zero, or even positive relationship between health and parasite load will be consistent with tolerance (and suggestive of it), but alternative explanations cannot be excluded. By contrast, our longitudinal data generate sequences of time points on a phase curve for a “typical” individual. A pattern in which health fails to decline (or even increases) following an increase in parasite load will therefore be directly indicative of tolerance. The case for demonstrable tolerance will, of course, be strongest if the results from the cross-sectional and longitudinal studies are consistent with one another. In contrast to tolerance, resistance resulting from acquired responses  and a longitudinal component  and was replicated at two different sites in each of 2 y. Biometric and infection data were recorded at all sampling points. In the cross-sectional component, ligands for Toll-like receptors (TLRs) 2 and 7 were used to stimulate splenocyte cultures, from which we measured the expression of immune genes involved in regulatory  and effector  pathways recruited during antimicrobial pattern recognition responses. In addition, the expression of genes reflecting different T-helper phenotypes was measured in nonspecifically  activated splenocytes. The activity of regulatory T cells was represented by the anti-inflammatory cytokines IL-10 and TGF-β1 and the transcription factor FoxP3. The activity of T-helper cell type 1 (Th1) responses was represented by the pro-inflammatory cytokine IFN-γ and transcription factor Tbet and that of Th2 responses by the transcription factor Gata3. In the longitudinal component, constitutive expression of IFN-γ, IL-10, and Gata3 were measured in peripheral blood.Our sampling, carried out in natural M main) was tested first against similarly reduced immune gene expression variables  or by measures of some influential species . However, the composite measure (PCM main) has the advantage that relationships with host responses will not be diluted when, for example, a low burden for one parasite is accompanied by high burdens for others, such that the response to the low burden is “greater than expected,” driven by the other parasites.Our overall analytical strategy  was to begin with our more detailed cross-sectional data and search first for the patterns of resistance or tolerance described above. We then sought to identify immunological gene expression markers of these patterns and, in turn, to link these to infection and life history variables. Next, we used our independent longitudinal data to corroborate the robustness of the immunological markers, place them within a stronger cause–effect context in relation to the infection and life history variables, and measure their effect on survival. Finally, we returned to the cross-sectional data and used structural equations modelling (SEM) to support the interdependencies between variables indicated by the longitudinal data. In analyses of the cross-sectional data, we primarily represented macroparasite infection using a reduced variable derived as scores from the first principal component of a principal components analysis (PCA) of the most common species that would be expected to be in contact with the host immune system  . In the ariables  and thenM main) tended to accumulate linearly with individual size and age, with no indication that acquired resistance decelerated the acquisition of infection in older animals , but immature males supported less infection as age indicators increased . This pattern is consistent with resistance building up prior to maturity but being absent postmaturity  amongst different life history stages. A particularly robust contrast occurred again between mature males, which showed a strong positive relationship between parasite burden and body condition, and immature males, which showed no such relationship  (Mycobacterium microti) were also in better condition relative to others  weight normalised for individual size – snout-vent length (SVL) and its quadratic term see . Conside = .003) . AmongstTo identify immunological markers for a tolerance strategy and determine their consequences for host fitness, we then focused primarily on the dichotomy between mature and immature males, as the former showed the strongest increased body condition response to macroparasite infection but no epidemiological evidence of resistance. Patterns in females, which may be obscured by immunosuppression during pregnancy, were less clear cut, as discussed briefly below.mit-stim), was consistently and strongly associated with macroparasite burden, host condition, and life history in mature males and also showed a different pattern in immature males. First, Gata3mit-stim correlated with macroparasite burden positively in mature males  in mature males  whether the trend for apparent tolerance rather than resistance in mature versus immature males was linked to variation in immune gene expression. We found that a single parameter, mitogen-stimulated Gata3 expression in cultured splenocytes .Gata3 expression, then, may be a useful biomarker for either resistance or tolerance where a predominant pattern of one or the other can be established within groups, as it can in males. Nonpregnant females showed similar tolerance-like accumulations of macroparasites with age indicators and also increases in condition at higher macroparasite burdens. However, blood) in the following month  in the following month  was linked to the tolerance pattern.In applying SEM to our cross-sectional data  and S15,blood), which decreased with age (using weight as a surrogate), was a significant predictor of survivorship in the longitudinal data .Constitutive Gata3 expression in peripheral blood  animals. Our longitudinal analyses suggest, moreover, that rather than being a mere correlate, elevated Gata3 expression was triggered by macroparasite infection and preceded the life history readjustments. Some corroborative patterns were also seen for another chronic infectious disease, TB, with animals being in better condition if showing overt signs of infection and especially if also expressing high levels of Gata3.These analyses suggest that expression of Gata3 (a master transcription factor involved in the differentiation of Th2 cells) can, in some individuals, be a marker for a physiological programme of tolerance to macroparasite infection, with implications for body condition, fecundity, and survival. Tolerance involved apparent overcompensation in body condition following infection, but accompanying that was downwards readjustment of fecundity, and age-dependent changes in survival, with survival improving in older tolerant (Gata3mit-stim) was inversely associated with macroparasite infection in these animals. This presents a familiar scenario, consistent with the paradigmatic role of acquired Th2 responses in resistance against macroparasites. Mature males, on the other hand, showed a very different and unexpected pattern. They accumulated macroparasites with age (resulting in very high infection levels) and showed a positive association between Gata3mit-stim and infection. Furthermore, body condition increased in heavily infected animals, whereas Gata3mit-stim was also positively associated with body condition  and negatively associated with male fecundity (adjusted testis weight). Our longitudinal time series data for recaptured adult males indicated that peripheral blood Gata3 expression was triggered by macroparasite exposures and preceded host responses that increased somatic maintenance and ultimately survival . This is suggestive of the process, and not just the pattern, of tolerance, as the increase in body condition associated with macroparasite infection and Gata3 expression is indicative of a protective response that does not affect the parasite directly.Our ability to infer tolerance in a complex field situation was facilitated by differing responses, in our cross-sectional study, between immature and mature males. These responses indicated a predominant pattern of resistance to macroparasites in the former and tolerance to macroparasites in the latter. Thus, as immature males aged, their infections reduced, consistent with the development of acquired resistance; also, mitogen-stimulated splenocyte Gata3 expression  are well within the routine scope of ecological researchers focussing on infectious disease. As considered further below, the dual aspect of Gata3 expression  is biologically plausible given the known role of Th2 responses in some pathways leading to resistance to macroparasites mit-stim, body condition, and macroparasite infection in mature and immature males. Hence, we can exclude these alternatives.There are alternative possible explanations for some of the cross-sectional associations we observed within mature males. One would be “differential mortality”—that is, high parasite burdens eliciting high Th2 responses, but before our observations are made, animals in poor condition with many macroparasites suffering increased mortality, so that the only animals with high parasite burdens left to observe are those in good condition. A second explanation would be “correlated risk”—that is, increased risk in high condition animals of macroparasite infections, and the Th2 responses they trigger, due, for example, to animals that forage more actively acquiring both more food and more parasites. However, whereas the temporal sequences seen in the longitudinal data directly support the tolerance hypothesis (macroparasites→Gata3→good condition), they support neither differential mortality nor correlated risk, as high Gata3 expression would in neither case precede good condition. Similarly, both differential mortality and correlated risk would predict associations between Gata3 expression and survival that did not change with age, with the former specifically predicting an uncomplicated association of high Gata3 with low survival and poor condition. These predictions, too, are contradicted by our results. Furthermore, differential mortality and correlated risk do not provide explanations for differing cross-sectional associations between Gata3Beyond our own system, these results may also provide some general insight into the nature of tolerance, as both pattern and process. The regulatory component of the immune system has often been seen as a potential driver of tolerance, through its role in suppressing immunopathological effector responses. Indeed, helminth parasites have been suggested to increase tolerance and reduce resistance by the elevation of regulatory responses Mycobacterium infections have been thought to facilitate clinical disease progression In apparent opposition to this, Th2 responses have often been considered to mediate resistance to macroparasites . In the laboratory they may do so via a diverse cascade of effector mechanisms It seems possible that increased body condition and reduced fecundity, in mature males but not immature males, might be adaptive by increasing residual reproductive value during macroparasite infection. This is consistent with a positive influence of Gata3 expression on survival in the oldest males. The contrasting patterns seen between mature and immature males suggest these may face very different cost/benefit scenarios when deploying immune responses against macroparasites. At present we can only speculate about the nature of these pressures, although a number of interesting possibilities might be considered. For example, altered exposure  might raise the immunopathological or energetic costs of resisting infection above thresholds of sustainability. Mature males also, due to their greater prior opportunity to develop robust adaptive immunity to common pathogens, may face fewer consequences from secondarily adopting a more tolerant phenotype. Thus, tolerance processes might have more serious indirect consequences for younger individuals through the inhibition of inflammatory responses against primary microbial infections.These are the first results to indicate a mechanism of tolerance in a naturally occurring mammalian population and to place this within a holistic framework of host fitness: context-dependent cost–benefit outcomes for individual condition, reproductive investment, and survival. This work therefore illustrates how immunological studies in natural populations may not only benefit from the many advances made in the laboratory but might also feed insights back into mainstream immunology. The results support the view that Th2 responses may play a crucial role in the tolerance of infection, and they more generally add to the growing body of evidence arguing for an increased focus on tolerance in our quest to understand host responses to parasitic infection. Further progress is likely to be facilitated by adopting approaches that incorporate experimental manipulations.M. agrestis) in Kielder Forest, Northumberland, United Kingdom, using live-trapping to access individual animals from natural populations. Our study was designed to permit the analysis of individual variation in condition and survival, infection status, and the expression of immune genes  or April (in the 2009–2010 season) and November, during which the capture–recapture study was carried out on the grid and destructive samples were retained from the transects . At eachin vivo expression of a subset  of the genes investigated in the cross-sectional study, in peripheral blood samples.Measurements of gene expression  in the cross-sectional element of the study focussed on cultured splenocytes and are described in detail in Jackson et al (2011) M. microti and carried out PCR diagnostics for Bartonella spp. and Babesia microti using previously established methods  from a PCA of common taxa likely to be in strong contact with the immune system  as the main analytical variable . In the case of macroparasites, we used the first principal component Click here for additional data file.Figure S2Sampling and measurements. Design replicated on two sites in 2008–2009 and two different sites in 2009–2010. Processing steps indicated by solid arrows and measurements by dashed arrows. Gene expression measurements shown in red. For timeline, see (TIF)Click here for additional data file.Figure S3Timeline of sampling.(TIF)Click here for additional data file.Figure S4post hoc testing of individual variables. Arrows indicate steps in the analysis associated with a particular hypothesis test or tests. Blue arrows indicate steps using data from the cross-sectional study . Where a particular individual variable or stratum of hosts was strongly implicated at a given step of the analysis and is focussed upon in the subsequent analysis, this is indicated in red to the right. Yellow arrows indicate steps using longitudinal data . Details of the respective analysis steps are provided in the figures and tables indicated on the left of each arrow (in grey) and the analytical methods employed are indicated on the right (in black) .Flow of main analysis. In order to avoid problems of interpretation due to multiplicity of tests, we carried out our analyses in a stepwise fashion, with each continuation step conditional on significant results in a single or small number of main hypothesis tests in the previous step. For parasite and immune expression data, these main tests initially used reduced variables . Significant main tests involving PC variables were followed by exhaustive (TIF)Click here for additional data file.Figure S5M main  with the abundances of common detritivorous mites (with negative loadings) and was thus also positively linearly associated with SVL and lens weight. There was thus no evidence of ongoing buildups of acquired resistance to the key macroparasitic throughout the lives of individuals, although analyses stratified by life history stage, with different macroparasite on age indicator slopes, indicated that resistance might build up in immature males but then collapse postmaturity . Mature males carry a much heavier macroparasite load than other life history stages.Overall associations between macroparasite abundance and host age indicators and life history stage. (A) Associations between parasite abundance and host size and age indicators, averaging slopes across life history groups. Plotted relationships are predictions (±1 standard error) from LMMs of the form: Parasite variable = Process day+LH+SVL (random component: Year×Sampling Point×Site). A quadratic term for SVL was initially included in models, and equivalent models were also analysed with lens weight terms replacing the SVL terms (with similar results). There were highly significant positive linear associations with the principal component representing key macroparasite species, PCmain see , and witmain see . The com(TIF)Click here for additional data file.Table S1M) was dominated by larger loadings for the more common species. These loadings strongly contrasted laelapid and listrophorid mite abundances with those of small fleas, ticks, and intestinal cestodes. A second PCA was carried out including only common macroparasite species that feed directly on host tissues  or that have an intimate association with host mucosal surfaces and adsorb soluble nutrients utilizable by the host (adult cestodes). This excluded all rare species . It also excluded common species that do not feed directly on host tissues, do not have an intimate association with host mucosal surfaces, and do not adsorb soluble nutrients. This was the case for listrophorid and laelapid mites, which are primarily deritivores, and pinworms (Syphacia), which occur free in the lumen of the colon and feed on bacteria.  This second PCA produced a very dominant first component (PCM main) with large loadings of similar magnitude and the same sign for small fleas, ticks, and adult cestodes, indicating a pattern of general positive covariation among these. In subsequent analyses, PCM main was used as the primary index of macroparasite infection pressure , but PCM was also analyzed secondarily (with similar results).Multivariate reduction of macroparasite data . PCA was used to generate single composite variables  representing the main axis of covariation amongst sets of macroparasite variables. Scores from these PC1 were then used in subsequent analyses to represent “grouped” macroparasite variation. An initial PCA included all of the macroparasite variables. PC1 (designated PC(DOC)Click here for additional data file.Table S2tlr-stim and PC2tlr-stim ; PC1tlr-index and PC2tlr-index ; PC1mit-stim and PC2mit-stim ; and PC1mit-index and PC2mit-index .Multivariate reduction of immune expression data . PCA was used to reduce sets of immune expression variables into smaller sets of nonredundant composite variables  reflecting major axes of covariation among the starting variables. Scores from these PCs were then used in subsequent analyses to represent grouped immune responses . Immune expression data were considered in two different ways: (1) expression under stimulatory conditions relative to a calibrator sample  and (2) expression indexed to a corresponding unstimulated control culture. Each of these sets of variables was reduced separately, with distinct analyses being carried out within each set for data from TLR-stimulated and mitogen-stimulated cultures. This gave four separate PCAs, from each of which we considered the two largest components (PC1 and PC2) in further analyses. The components were designated: PC1(DOC)Click here for additional data file.Table S32+LH.SVL+LH.SVL2+Parasite variable (random term = Year×Sampling Point×Site). Both of the macroparasite principal component variables (PCM and PCM main), several of the individual macroparasite variables, and presence/absence of overt TB all showed significant positive associations with body condition. The only negative association was for Listrophoridae (fur mites), which may possibly have been due to an association between poor condition and compromised grooming. Significant positive association in the main hypothesis test is highlighted in orange; significant associations in post hoc tests are highlighted in yellow (positive associations) or grey (negative associations).Association of body condition with parasitic infections across all host stages . Body condition was represented in LMMs by body weight (the response) adjusted for covariates SVL and its quadratic term, initially averaging slopes for these across life history stages. Models considered all of the sampled stages and were of the form: Body weight = LH+Process group+SVL+SVL(DOC)Click here for additional data file.Table S42+LH.SVL+LH.SVL2+Parasite variable (random term = Year×Sampling Point×Site). Both of the macroparasite principal component variables (PCM and PCM main), two of the individual macroparasite variables, and presence/absence of overt TB all showed significant  positive associations with liver condition. The only significant negative association was for ear mites, which may possibly have been due to an association between poor condition and compromised grooming. Significant positive association in main hypothesis test is highlighted in orange; significant  associations in post hoc tests are highlighted in yellow (positive associations) or grey (negative associations).Association of liver condition with parasitic infections across all host stages . Liver condition was represented in LMMs by liver weight (the response) adjusted for covariates SVL and its quadratic term, initially averaging slopes for these across life history stages. Models considered all stages sampled and were of the form: Liver = LH+Process group+SVL+SVL(DOC)Click here for additional data file.Table S510 spleen weight = LH+Process group+SVL+SVL2+LH.SVL+LH.SVL2+Parasite variable (random term = Year×Sampling Point×Site). Both of the macroparasite principal component variables (PCM and PCM main) and several of the individual macroparasite variables showed significant  positive associations with spleen condition. The only significant negative association for a macroparasite was for Listrophoridae (fur mites), which may possibly have been due to an association between poor condition and compromised grooming. The relative development of the spleen is a complex indicator of individual condition, potentially reflecting a combination of generalised individual condition, standing investment in immune defences, and ongoing host responses. The strong positive association of B. microti with spleen condition (but not with body or liver condition) is likely to be due to a host response, given that splenomegaly often develops in Babesia infections . Furthermore, the strong negative association of Bartonella spp. with spleen condition  is likely due to confounding from B. microti. Bartonella in our data and in previous studies shows a strong negative interaction with B. microti (and the significant result for Bartonella spp. disappears if B. microti is added as an explanatory term to the LMM). Significant positive association in the main hypothesis test is highlighted in orange; significant  associations in post hoc tests are highlighted in yellow (positive associations) or grey (negative associations).Association of spleen condition with parasitic infections across all host stages . Spleen condition was represented in LMMs by spleen weight (the response) adjusted for covariates SVL and its quadratic term, initially averaging slopes for these across life history stages. Models considered all stages sampled and were of the form: Log(DOC)Click here for additional data file.Table S610 testis weight = Process group+SVL+SVL2+Parasite variable (random term = Year×Sampling Point×Site). Both of the macroparasite principal component variables (PCM and PCM main) and several individual macroparasite variables showed significant  positive associations with testis condition. The only significant negative association was for Listrophoridae (fur mites), which may possibly be due to an association between poor condition and compromised grooming. Significant positive association in the main hypothesis test is highlighted in orange; significant  associations in post hoc tests are highlighted in yellow (positive associations) or grey (negative associations).Association of testis condition with parasitic infections in adult males . Testis condition was represented in LMMs by testis weight (the response) adjusted for covariates SVL and its quadratic term. Models considered only mature males  and were of the form: Log(DOC)Click here for additional data file.Table S7M main) across life history stages , all life history stages were initially analysed in a single LMM of the form described in M main as the parasite variable and, additionally, a Life History Stage (LH)×PCM main interaction. This interaction was highly significant , indicating divergent reaction norms. When females were excluded from the analysis, due to the complex and sometimes ambiguous transitions between different reproductive states, there was still highly significant heterogeneity of slopes between mating and nonmating males  . Significant positive associations are highlighted.Association between body condition and grouped macroparasite infection in different life history stages : differing epidemiological reaction norms (ERNs). To assess the heterogeneity of adjusted body condition slopes on grouped macroparasite infection (PC(DOC)Click here for additional data file.Table S8M main as a further covariate. Models included adult males only and were of the form: Body weight = Process group+SVL+SVL2+PCM main+Immunological variable (random term = Year×Sampling Point×Site). The table shows the results of the main hypothesis tests  that were initially carried out on the eight immunological principal component (PC) variables, followed by post hoc testing of individual variables. PC variables that would be significant at p<.01 after multiplicity adjustment  are highlighted in orange. Individual variables from post hoc testing that would be significant at p<.01 after a multiplicity adjustment are also highlighted in orange. PC and individual variables that were only significant  without multiplicity adjustment are highlighted in yellow (nonsignificant results for post hoc testing not shown). PC2mit-stim (which featured a large loading for Gata3mit-stim) and Gata3mit-stim itself were the only two significant variables following multiplicity adjustment (for the whole table of tests carried out). Gata3mit-index was also relatively highly significant at an individual level. When Gata3mit-stim was considered in a model of the above form additionally including immature males and different slopes on Gata3mit-stim and PCM main for mature and immature males, there were very significantly different stage-specific Gata3mit-stim slopes, with mature males showing a positive slope and immature males no association .Association between body condition and immunological gene expression in adult males . Body condition was represented in LMMs by body weight (the response) adjusted for covariates SVL and its quadratic term. Body condition was also adjusted for the association with macroparasites see  by the i(DOC)Click here for additional data file.Table S9mit-stim) in adult males . Organ condition was represented in LMMs by organ weight (the response) adjusted for covariates SVL and its quadratic term. Organ condition was also adjusted for the association with macroparasites . Significant positive associations are highlighted in yellow and significant negative associations in grey.Associations between organ condition and mitogen-stimulated Gata3 expression Click here for additional data file.Table S10mit-stim), overt TB lesions, and body condition . Post hoc analyses of body condition focussing on TB found a highly significant interaction between overt TB lesions and Gata3 expression in mitogen-stimulated splencocytes (Gata3mit-stim) across all stages. This analysis is based on the following LMM: Body weight = Log10 Gata3mit-stim+LH+Process group+SVL+SVL2+LH.SVL+LH.SVL2+Overt TB+Overt TB×Log10Gata3mit-stim (random term = Year×Sampling Point×Site). There was only a marginally significant overt TB×Gata3mit-stim interaction when the analysis was restricted to adult males (p = .08).Gata3 expression (Gata3(DOC)Click here for additional data file.Table S11mit-stim) in adult males . The association of Gata3mit-stim with grouped parasitic infection was analyzed in LMMs of the form: Parasite variable = Process group+SVL+Log10 Gata3mit-stim (random term = Year×Sampling Point×Site). In mature males there were significant positive associations with both of the macroparasite principal component variables (PCM main and PCM) (see table). When immature males were added to the above model for PCM main, with different age-specific slopes, there was significant variation in the Gata3mit-stim slopes, with a positive slope for mature males and a negative slope for immature males . A similar interaction also occurred in the case of PCM (p = .031). Significant positive association in main hypothesis test is highlighted in orange; significant association in post hoc test is highlighted in yellow.Associations between parasitic infections and mitogen-stimulated Gata3 expression (Gata3(DOC)Click here for additional data file.Table S12blood) in peripheral blood in adult males . (A) Time lagged associations. Association of Gata3blood with infection variables earlier in time (t-1 = 1 mo earlier). When analyzed alone, ticks and small fleas were represented by continuous log-transformed abundance variables. When ticks and small fleas were analyzed together, this was as a binary presence/absence factor . Table shows significant explanatory terms from models of the form: Gata3blood = Weight+Parasite variable . Results for weight are for the model with a factor for ticks and or small fleas. Significant positive associations highlighted in yellow and significant negative associations in grey. Earlier infection tended to be positively associated with Gata3blood expression. (B) Instantaneous association. Association of Gata3blood with infection at the same time point. When analyzed alone, ticks and small fleas were represented by continuous log-transformed abundance variables. When ticks and small fleas were analyzed together this was as a binary presence/absence factor . Table shows significant explanatory terms from models of the form: Log10 Gata3blood = Weight+Parasite variable . Results for weight are for the model with a factor for ticks and or small fleas. Significant  negative associations highlighted in grey. Contemporaneous infection tended to be negatively associated with Gata3blood expression, perhaps due to some protective (resistance) effect of this response.Instantaneous and time-lagged associations between exposure to blood-feeding ectoparasites and Gata3 expression (Gata3(DOC)Click here for additional data file.Table S13blood) in peripheral blood in adult males . Association of month-on-month weight gain (adjusted for starting weight) with Gata3blood expression 1 mo earlier. Table shows significant explanatory terms from LMMs of the form: Final weight = Starting weight+Gata3blood . Gata3blood was positively associated with adjusted weight gain in the following month. There was no association between Gata3blood and weight gain in the preceding month . The effects of individual parasite variables were examined in these base models (taking each variable in turn), but were nonsignificant. Macroparasite variables examined included the abundance of fleas, ticks, laelapid mites, listrophorid mites, and lice and also an overall ectoparasite index; microparasite variables (presence/absence) included B. microti, Bartonella spp., and overt TB. Ectoparasite index was an additive score based on the sum of standardized abundances for the different ectoparasites. Significant positive associations are highlighted in yellow.Time-lagged association between month-on-month weight gain and Gata3 expression (Gata3(DOC)Click here for additional data file.Table S14p<.05) or near-significant (.05<p<.08) parameters; and optimal model fit  and AIC statistics. The table shows the two best supported models with six or seven significant (or near-significant) parameters. Parameter estimates for model 2 are shown in M main, see 10 Gata3 expression in mitogen-stimulated splenocytes; C, body condition; T, testis condition. Prior to the SEM analysis, all of the variables were adjusted, in general linear models, for spatiotemporal sampling point (sampling time and site nested within year) and for host linear dimensions (SVL+SVL2) if SVL did not contribute to the variable already.SEM of key variables from cross-sectional study. In the SEM analysis of the cross-sectional data, we searched for models with: the causality indicated by the longitudinal analyses in (DOC)Click here for additional data file.Table S15M main, see 10 Gata3 expression in mitogen-stimulated splenocytes; C, body condition; T, testis condition. Prior to the SEM analysis, all of the variables were adjusted, in general linear models, for spatiotemporal sampling point (sampling time and site nested within year) and for host linear dimensions (SVL+SVL2) if SVL did not contribute to the variable already. A causal effect of macroparasites (P) upon Gata3 expression (G) and of G upon body condition (C) was independently suggested by analyses of the longitudinal dataset ; P, infection with key influential macroparasites (PC(DOC)Click here for additional data file.Table S16blood) and age-specific primary session return rate . Return rate of mature males (at the next primary trapping session) (A) was initially analyzed with respect to Gata3 expression in peripheral blood (Gata3blood) using a GLMM with binomial errors . The base model was of the form: Return∼Weight+Gata3blood+Peripheral row . The peripheral row factor accounted for animals captured in the outer trap row of the grid, which might putatively have lower recapture probabilities (as their ranges are more likely to have been centred outside the grid). Other models (not shown) examined the effects of individual parasite variables in this base model (taking each in turn). Macroparasite variables included the abundance of fleas, ticks, laelapid mites, listrophorid mites, and lice and also an overall ectoparasite index; microparasite variables (presence/absence) included B. microti, Bartonella spp., and overt TB. Ectoparasite index was an additive score based on the sum of standardized abundances for the different ectoparasites. These were only included in the final model if significant. A similar set of models was then investigated (post hoc) for all stages (B), additionally including life history stage as a factor. The main effect for Gata3blood was not significant in adult males but was significantly negative in the analysis across all stages . Significant Weight×Gata3blood interactions occurred in both analyses . Although return rate might act as a reasonable surrogate for survival .Gata3 expression in peripheral blood (Gata3rved see ), furtherved see . Signifi(DOC)Click here for additional data file.Table S17blood) was analyzed in CJS models in a subset of males with >1 capture and no or few missing values for Gata3blood (n = 107).  Time-specific body weight covariates were highly significant  when added to an optimal base model for survival and recapture that included seasonal (monthly) variation in survival (φ) and variation across years in recapture probability (p). Gata3blood and Gata3blood×Body Weight covariates were then added to the base model already containing body weight covariates. The best resulting models contained covariates for Gata3blood and Gata3blood×Body Weight only in the last 2 mo  or last month of the season . In these months, survival tended to increase in larger animals expressing a greater amount of Gata3blood. The following parameter values were estimated for a model in which overall p and φ for the first time interval (φApril→May) were constrained to be 1. This was because p was generally estimated to be very high  and because φApril→May was limited by the nature of the data, which only included animals with multiple recaptures. Real function parameters for survival were: φMay→June = 0.49±0.07, φJune→July = 0.76±0.06, φJuly→August = 0.87±0.05, φAugust→September = 0.73±0.05, φSeptember→October = 0.55±0.08. The logit link function parameter for the September Gata3blood×Body Weight covariate was: βSeptember = 0.3±0.11.Gata3 expression and survival: CJS analysis with time-specific individual covariates . The effect of Gata3 expression in peripheral blood (Gata3(DOC)Click here for additional data file.Table S18Real-time PCR primers used to measure the expression of immunological genes in peripheral blood.(DOC)Click here for additional data file.Table S19Bartonella spp. and B. microti may have had unequal sensitivities in 2008 and 2009 due to the use of different DNA extraction methods. Prevalence data are thus not directly comparable across years.Infection statistics for parasites at Kielder broken down by Locality×Year of Study. A, abundance±standard error. P, prevalence, followed by 95% CI. *Abundance data for Listrophoridae relate to a semiquantitative abundance index. †PCR diagnosis of (DOC)Click here for additional data file.Methods S1Supplementary Methods.(DOC)Click here for additional data file.