TLE3 conferred resistance to AR antagonists apalutamide and enzalutamide. Genes differentially expressed upon TLE3 loss share AR as the top transcriptional regulator, and TLE3 loss rescued the expression of a subset of androgen-responsive genes upon enzalutamide treatment. GR expression was strongly upregulated upon AR inhibition in a TLE3-negative background. This was consistent with binding of TLE3 and AR at the GR locus. Furthermore, GR binding was observed proximal to TLE3/AR-shared genes. GR inhibition resensitized TLE3KO cells to enzalutamide. Analyses of patient samples revealed an association between TLE3 and GR levels that reflected our findings in LNCaP cells, of which the clinical relevance is yet to be determined. Together, our findings reveal a mechanistic link between TLE3 and GR-mediated resistance to AR inhibitors in human prostate cancer.Androgen receptor (AR) inhibitors represent the mainstay of prostate cancer treatment. In a genome-wide CRISPR-Cas9 screen using LNCaP prostate cancer cells, loss of co-repressor  Prostate cancer is the second most common cancer and the fifth leading cause of cancer-related death in men worldwide . DeregulAR  was shown to be associated with clinical resistance to enzalutamide (GR enhancer (TLE3) as a modulator of AR inhibitor sensitivity that, upon loss, confers resistance to enzalutamide in prostate cancer cells.Several resistance mechanisms to AR inhibitors have been proposed, including mutations in AR  and exprAR . For exalutamide . Upregullutamide . Using tlutamide . Signifilutamide . GR upreenhancer . How exaenhancer . Using tGR locus affecting AR inhibitor sensitivity in prostate cancer cells.The well-conserved TLE protein family of transcriptional co-repressors is expressed in the nucleus of metazoans and regulate various biological processes including development, cell metabolism, growth and differentiation. At the chromatin, TLE protein family members maintain a silenced chromatin structure . TLE3 isTLE3 as the top hit with all three gRNAs enriched in cells treated with apalutamide compared to untreated cells , as well as short hairpin RNAs (shRNAs) targeting TLE3, conferred resistance to both enzalutamide and apalutamide in LNCaP cells in long-term growth assays (14 days) with drug concentrations up to 8 μM  selectively focusing on AR-responsive genesets. Overall, AR signaling was maintained in the presence of enzalutamide in TLE3KO cells but not in control cells, implying a rescue of AR signaling despite enzalutamide treatment  cells, the expression of TLE3 is induced by enzalutamide   whose expression was found to correlate with TLE3KO and enzalutamide treatment  . We nexts (AREs)  for the  by TLE3 . In Figureatment . Genome-reatment . We founreatment . Furtherreatment . Since TGR was one of the most upregulated genes upon enzalutamide treatment in a TLE3-loss background  . These d). The third patient, having a more profound response , had weak staining for TLE3 and moderate staining for GR at baseline. In the post-treatment staining, TLE3 was low, whereas GR expression had increased , matched tissue samples of metastatic sites were collected before treatment and after progression on enzalutamide treatment for four CRPC patients. These paired biopsies were analyzed by immunohistochemistry for expression of TLE3 and GR, to investigate whether expression of these proteins is altered upon selection pressure by enzalutamide. Two patients had a short PSA response to enzalutamide (<6 months), without radiological response. Tumor tissue of these patients showed moderate to high GR expression at baseline with weak or negative staining for TLE3 . This waFigure 5Dncreased . In thisThe efficacy of the AR antagonists enzalutamide and apalutamide illustrates the importance of persistent signaling through the AR pathway in CRPC . The traTLE3 results in an active-AR-like profile despite anti-hormonal treatment. Our findings are in line with TLE3’s known role as a transcriptional repressor  were previously described to be GR-regulated in a model of AR inhibitor resistance. Together, our results provide novel insights into the regulation of the GR locus in the context of AR inhibition in prostate cancer cells, implicating TLE3 as a regulator of GR-mediated enzalutamide resistance.Pathway reactivation or feedback activation of parallel signaling pathways are commonly described mechanisms found in drug-resistant tumors treated with targeted therapy . In enzaTLE3 conferred resistance to enzalutamide only in LNCaP cells and not in LAPC4 and CWR-R1. The availability of in vitro prostate cancer models is limited, and the heterogeneous nature of resistance mechanisms to antihormonal therapies in prostate cancer may explain why TLE3 loss did not confer resistance to enzalutamide in LAPC4 and CWR-R1 cells. To study broader applicability, we investigated several clinical data-sets. Analysis of RNA expression in two prostate cancer patient cohorts, showed an inverse correlation between TLE3 and GR expression and worse prognosis of prostate cancer patients with low TLE3 expression treated with antihormonal therapy. Additionally, immunohistochemistry on GR and TLE3 of tumor tissue collected from CRPC patients pre- and post-enzalutamide treatment support our findings in LNCaP cells. Although these observations are in agreement with our hypothesis, the clinical implications of our findings are yet to be resolved and need to be determined in larger cohorts. Thus, our results warrant further investigation into the role of TLE3 and enzalutamide resistance in prostate cancer patients.A limitation of our study is the fact that, of the in vitro models we tested, loss of GR expression and drug resistance, complements increasing evidence describing the role of this receptor in bypassing AR blockade in prostate cancer cells.In summary, we have identified TLE3 loss as a novel resistance mechanism to AR-targeted therapeutics in prostate cancer cells. Based on previously reported work and the data in our study, we propose a model in which loss of TLE3 and AR function at the GR enhancer leads to upregulation of GR, which is able to substitute for AR, resulting in enzalutamide resistance . Our datFor an overview of the resources used for this study see 2. All cell lines were STR profiled. Control and TLE3KO cells were created by infecting target cells with lentiviral particles containing LentiCRISPR v2.0 harboring non-targeting or TLE3-targeting gRNAs, which were cloned in using Gibson Assembly (NEB cat#: E2611S) utilizing BsmBI restriction sites. For gRNA and shRNA sequences see The human prostate cancer cell lines were maintained in RPMI  or IMDM (LAPC4). HEK293T cells were cultured in DMEM. Medium was supplemented with 10% FBS and 1% penicillin/streptomycin. Cells were maintained at 37 °C in 5% COLNCaP cells were infected with lentiviral particles containing GeCKO half-library A at low M.O.I. (~0.2) for single viral integration, at a ~ 150 fold coverage, and cultured in the presence of vehicle or 2 μM apalutamide for 6 weeks. Barcodes were recovered and sequenced as described . DESeq2 Colony formation assays were performed as previously described . Used seLive cell proliferation was monitored in real-time using the Incucyte ZOOM (11 days). Cells were seeded in a 384-well plate at 600 cells/well and drugs were added as indicated.R1881, apalutamide, enzalutamide, mifepristone (Medkoo Biosciences) were dissolved in DMSO and stored at −20°C.Typically, LNCaP cells were plated at density of 200,000 cells in 6-well plates and cultured in the presence of enzalutamide for 5 days before harvesting. Samples were prepared and western blot was performed as described previously , using pTLE3KO cells in 10 cm dishes in the presence of 10 μM enzalutamide or vehicle for 5 days, followed by RNA isolation using the ISOLATE II RNA mini kit (Bioline). RNA was then submitted for Illumina sequencing (HiSeq 2500). The differential expression was based on the ratio of normalized read counts . An absolute fold-change threshold of 2 was used. Genes with a coverage <50 in both conditions were excluded from the analyses to prevent spurious results. Data were further analyzed using Enrichr (http://software.broadinstitute.org/gsea). Data was uploaded to GEO (GSE130246).RNA-seq data was generated by seeding 500,000 LNCaP control or  Enrichr  and javaLNCaP cells were plated at density of 200,000 cells in 6-well plates and cultured in the presence of enzalutamide for 5 days before harvesting. Total mRNA isolation, cDNA synthesis and qPCR analysis were performed as described elsewhere . An overThe ChIP-seq data from ChIP-qPCR data was generated according to the protocol described by 0C and TLE3 using clone CL3573 . Bound TLE3 was detected using the OptiView DAB Detection Kit . Glucocorticoid Receptor bound antibody was visualized using Anti-Rabbit HQ  for 12 min at 370C, Anti-HQ HRP  for 12 min at 37°C, followed by ChromoMap DAB Detection Kit . Slides were counterstained with Hematoxylin and Bluing Reagent .Immunohistochemistry of the FFPE tumor samples was performed on a BenchMark Ultra autostainer (TLE3) or Discovery Ultra autostainer (Glucocorticoid Receptor). Briefly, paraffin sections were cut at 3 µm, heated at 75°C for 28 min and deparaffinized in the instrument with EZ prep solution . Heat-induced antigen retrieval was carried out using Cell Conditioning 1  for 64 min at 95°C.Glucocorticoid Receptor clone D6H2L  was detected using 1/600 dilution, 1 hr at 37 In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:Androgen receptor (AR) inhibitors are commonly used to treat prostate cancer, but resistance to these drugs is a problem. Up-regulation of the glucocorticoid receptor (GR) is associated with drug resistance. This paper provides a mechanistic link between the transcriptional co-repressor TLE3 and GR-mediated resistance to AR inhibitors in a prostate cancer cell line. This link involves TLE3 and AR binding to the GR locus as well as GR binding to TLE3/AR regulated genes. An association between TLE3 and GR expression in patient samples suggests the possibility of clinical relevance.Decision letter after peer review:eLife. Your article is being evaluated by three peer reviewers, and the evaluation is being overseen by a guest Reviewing Editor and Kevin Struhl as the Senior Editor.Thank you for sending your article entitled "TLE3 loss confers AR inhibitor resistance by facilitating GR-mediated prostate cancer cell growth" for peer review at Summary:While the reviewers found your work to be of generally high quality, there were concerns about the lack of adequate novel mechanistic insights. There are two main issues. First, there is a worry that the results are observed in a single cell line and that other cell lines behave differently. Thus, the reviewers are interested in additional mechanistic understanding, particularly with respect to this cell-line specificity. Second, given the single cell line issue, the reviewers request some connection of the mechanism shown here with human cancer. For example, is there any evidence for cancer relevance of the pathway in the various databases of human cancer?Reviewer #1:This is a very interesting study by Palit et al. that reports on the loss of the transcription factor co-repressor, TLE3, by CRISPR-library screening that renders LNCaP cells (not any of the other tested CRPC lines) resistant to AR-antagonist treatment with Enzalutamide or Apalutamide. TLE3 appears to be itself negatively regulated by AR. The initial findings were followed by RNA-seq assessment of TLE3 KO cells treated with Enz versus vehicle and led to the identification of GR as one of the top differentially expressed genes. The network analysis indicated that TLE3 KO facilitates reactivation of AR-targeted genes under Enz treatment, suggesting involvement of GR as the most likely mechanism of AR-bypass. Both pharmacological and genetic inhibition of GR was sufficient to reverse the resistant phenotype acquired by TLE3 KO. While the phenotypic assessments of TLE3's role in the context of AR and GR inhibition are promising with clear results, the study lacks sufficient mechanistic exploration and evidence for a conclusive story.Major comments:1) Please provide GR protein expression data wherever the transcript levels are analyzed because this is purported to be a critical part of the mechanistic link.2) The CRISPR-screen was performed with treatment and selection of CRISPR-targeted Enz-naïve cells for 6 weeks. However, the emergence and expansion of resistant WT LNCaP cells takes considerably longer. Is TLE3 downregulated in parental cells that become Enz-resistant, when compared to Enz-naïve cells? In other words, is TLE3 depletion naturally required to allow for emergence of Enz resistance?KOTLE3 cells. While the results clearly indicate an enhanced GR induction as a result of TLE3 depletion, no other data presented appears to support TLE3 inhibition as a pre-requisite for GR occupancy.3) To conclude that depletion of TLE3 frees AR binding regions for GR occupancy , the authors would require genome wide ChIP-seq of AR, GR and TLE3 in Enz-treated control vs 4) TLE3 is a known inhibitor of the Wnt pathway whose activation has been reported by several groups to be essential for Enz-resistance. Is it possible that TLE3 loss functions through the Wnt pathway  instead of stimulating GR expression/action? Have the authors tested this alternative hypothesis?5) The TCGA patient dataset is not well described. TCGA data are mainly obtained from local prostate cancer and biochemical recurrence usually refers to recurrence after local therapy , which is usually not relevant to hormone therapy resistance. What is the "anti-hormonal therapy" the authors refer to? How was the expression cut point defined and identified?6) Overall, there appears to be insufficient data to support the model proposed in Figure 5.Reviewer #2:A couple previous studies have indicated an association between TLE3 and AR, but its functional significance for AR function has not been determined. This study found that TLE3 loss enhanced LNCaP cell growth in response to AR antagonist in a CRISPR screen. Further ChIP-seq and transcriptome data support TLE3 coregulation of a subset of AR regulated genes. Finally, in a TLE3 KO background the investigators found that AR inhibition caused an increase in GR, and that GR inhibition could resensitize the cells to ENZ. Together these data support a role for TLE3 in the regulation of a subset of AR regulated genes. However, there are a number of concerns related to functional significance that should be addressed, as indicated below.1) The generality of the findings is unclear as TLE3 depletion only conferred drug resistance in LNCaP, but not in CWR-R1 or LAPC4. It would be of value to explore the basis for this difference a bit more. In particular, does ENZ stimulate TLE3 in these latter cells.2) The significance would also certainly be enhanced by in vivo data confirming that the TLE3 depleted cells are resistant to ENZ.3) AR was the top TF associated with genes that were differentially expressed in TLE3 KO cells in the presence of androgen or with ENZ. One would predict that expression of these genes would increase in the TLE3 KO cells. Figure 2B shows fold change, but it is not clear what is being compared. Moreover, the description is somewhat cryptic (genes that show an interaction with TLE3 KO and ENZ treatment). The effect of TLE3 on AR/TLE3 shared genes, as well as on TLE3 alone genes should be clearly described.4) It is not clear how UGT2B17 fits with the hypothesis that TLE3 is repressing AR regulated genes in response to ENZ .5) It is an attractive hypothesis that AR directly represses TLE3 expression. However, supplemental Figure 2 only shows effects on protein, with no indication of how long the cells were treated. The authors should show a time course of TLE3 mRNA induction and loss in response to ENZ and DHT in order to address whether the effects are likely direct.6) Figure 3 seems to show that TLE3 binding at AR regulated genes does not decrease with R1881 stimulation. This would seem to be inconsistent with the feedback model, and with the marked decrease in TLE3 protein in R1881 treated cells.7) The growth data in Figure 4 is qualitative and only a single plate is shown for each condition. It should be quantified, and a growth curve would help.8) The authors show that GR KO confers sensitivity to ENZ in the TLE3 KO cells, and suggest this reflects GR activation of AR/TLE3 regulated genes. To assess this mechanism, they should address whether GR KO in this context does indeed suppress the expression of AR/TLE3 coregulated genes.9) While GR may contribute to ENZ resistance in TLE3 KO cells, the significance of this finding for TLE3 intact cells is not addressed. Is there increased GR recruitment to AR/TLE3 regulated genes in response to ENZ in wild-type cells?10) In Supplemental Figure 4 it is unclear if the authors are assessing BCR after RP or after ADT. It is probably the former, which would only mean that TLE3 expression is associated with aggressiveness, and would not provide evidence that it is involved with response to ADT.Reviewer #3:The authors identify TLE3 loss as a cause of apalutamide resistance in a genome-wide in vitro CRISPR screen in LNCaP prostate cancer cells. Transcriptomic and ChIP-seq studies support a model whereby TLE3 loss rescues suppression of AR pathway signaling by antiandrogens. Mechanistically this occurs, at least in part, through upregulation of GR based on experiments showing that shRNAs targeting GR restore sensitivity to enzalutamide. This model is supported by ChIP-seq data showing overlapping binding of TLE3 and FOXA1 at various AREs across the genome including a GR enhancer.The data supporting TLE3 as screen hit as well as the proposed mechanism for causing antiandrogen resistance through GR upregulation,is convincing, but the work has two significant shortcomings.1) The apparent context dependence of TLE3 loss for LNCAP cells only (negative results in CWR-R1 and LAPC4) raises concerns about the broader relevance of the TLE3 loss in prostate cancer. The authors could address this in several ways:i) testing of more modelsii) mechanistic insight into why resistance is not seen in CWR-R1 and LAPC4 iii) deeper interrogation of TLE3 status in human datasets, particularly in the castration-resistant setting 2) The prior work on GR in castration resistance diminishes the novelty. This could perhaps be overcome with additional mechanistic insight beyond that reported in the earlier publications. For example, how does TLE3 loss impact the chromatin landscape (particularly repressive histone marks) across the genome and more specifically at the GR locus?Overall I would be supportive of considering a revised manuscript but it would need to have additional data.eLife. Your revised article has been evaluated by Kevin Struhl (Senior Editor) and a guest Reviewing Editor.Thank you for resubmitting your work entitled "TLE3 loss confers AR inhibitor resistance by facilitating GR-mediated human prostate cancer cell growth" for further consideration by The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:Please revise the manuscript as requested by the reviewers.Reviewer #1:Palit, et al., provide a revised manuscript on TLE3 loss and enzalutamide resistance. The strength of the original manuscript is with the enzalutamide resistant phenotype conferred by TLE3 KO but was lacking in mechanistic characterization and there were some concerns about clinical relevance of this finding. In general, the authors have put forth a reasonable effort to address reviewer comments.The additional evaluation on inverse correlation between GR and TLE3 expression from clinical data sets  are not really convincing and requested in vivo experiments by one of the other reviewers on TLE3 KO and resistance are not provided. Together, with a CRISPR KO generated mechanism of resistance that occurs in a single cell line model, if moving forward with this manuscript I would suggest that the authors pull back on the potential clinical relevance and explicitly state this as a major caveat of their story (in the Abstract and Discussion).Reviewer #2:AXIN2.The authors have responded to the points raised in the initial review. There are still concerns that the observations are limited to one cell line and are only in vitro. However, the authors do now show an inverse correlation between TLE3 and GR in public domain data. It should be noted that the previous study from Arora et al. found that GR was not immediately increased by ENZ, but required some substantial time for adaptation. Therefore, overall the data support the hypothesis that loss of TLE3 is a mechanism that contributes to increasing GR in response to ENZ in at least a subset of patients. One minor point is that in response to the question of whether TLE3 loss increased WNT signaling, the authors showed no increase in active b-catenin. However, TLE3 loss would presumably increase TCF activity without an increase in active b-catenin. The more relevant data would be effects on TCF regulated genes such as Reviewer #3:eLife.The authors have responded to my earlier review with additional evidence from clinical datasets that extends the TLE3 work beyond the LNCaP cell line. I believe the revised manuscript is suitable for publication at  Reviewer #1:This is a very interesting study by Palit et al. that reports on the loss of the transcription factor co-repressor, TLE3, by CRISPR-library screening that renders LNCaP cells (not any of the other tested CRPC lines) resistant to AR-antagonist treatment with Enzalutamide or Apalutamide. TLE3 appears to be itself negatively regulated by AR. The initial findings were followed by RNA-seq assessment of TLE3 KO cells treated with Enz versus vehicle and led to the identification of GR as one of the top differentially expressed genes. The network analysis indicated that TLE3 KO facilitates reactivation of AR-targeted genes under Enz treatment, suggesting involvement of GR as the most likely mechanism of AR-bypass. Both pharmacological and genetic inhibition of GR was sufficient to reverse the resistant phenotype acquired by TLE3 KO. While the phenotypic assessments of TLE3's role in the context of AR and GR inhibition are promising with clear results, the study lacks sufficient mechanistic exploration and evidence for a conclusive story.We thank the reviewer for the constructive comments, and are delighted that the reviewer found our manuscript very interesting and promising with clear results. As suggested, we now provide more mechanistic explanation in the revised version, which we hope the reviewer appreciates.Major comments:1) Please provide GR protein expression data wherever the transcript levels are analyzed because this is purported to be a critical part of the mechanistic link.KOTLE3 cells treated with vehicle or enzalutamide for 5 days. This experiment shows clear upregulation of GR on protein level only in KOTLE3 cells when treated with enzalutamide. These findings are in full concordance with our RNA-seq and qPCR-based conclusions that GR levels were increased upon Enzalutamide treatment when TLE3 was lost.This is an excellent point, and we thank the reviewer for this constructive suggestion. As requested, we now added a western blot analysis  showing protein expression levels of GR in control and 2) The CRISPR-screen was performed with treatment and selection of CRISPR-targeted Enz-naïve cells for 6 weeks. However, the emergence and expansion of resistant WT LNCaP cells takes considerably longer. Is TLE3 downregulated in parental cells that become Enz-resistant, when compared to Enz-naïve cells? In other words, is TLE3 depletion naturally required to allow for emergence of Enz resistance?This is an interesting suggestion. We tested this hypothesis by assessing TLE3 protein levels in a panel of enzalutamide-resistant and parental cell lines see . InteresKO cells. While the results clearly indicate an enhanced GR induction as a result of TLE3 depletion, no other data presented appears to support TLE3 inhibition as a pre-requisite for GR occupancy.3) To conclude that depletion of TLE3 frees AR binding regions for GR occupancy , the authors would require genome wide ChIP-seq of AR, GR and TLE3 in Enz-treated control vs TLE3We thank the reviewer for bringing up this point and valuable suggestion. We fully agree that investigating GR occupancy would provide important evidence that would further support the model. To address this question, we performed ChIP-qPCR for GR at the loci of several target genes  that were identified in the transcriptomics analyses in Figure 2. We found that GR only occupies the enhancer elements of these genes in the context of TLE3 loss combined with enzalutamide treatment , which is in line with western blot results showing only GR expression in this context . Combined, these experiments support our model that TLE3 inhibition in combination with AR inhibition is a pre-requisite for GR protein upregulation and enhancer occupancy.4) TLE3 is a known inhibitor of the Wnt pathway whose activation has been reported by several groups to be essential for Enz-resistance. Is it possible that TLE3 loss functions through the Wnt pathway  instead of stimulating GR expression/action? Have the authors tested this alternative hypothesis?This is a very interesting hypothesis, and we thank the reviewer for bringing this to our attention. As suggested by the reviewer, we determined the levels of active ß-catenin upon TLE3 loss in combination with vehicle or enzalutamide treatment , indicating that the resistance mediated by TLE3 loss is not the result of increased Wnt activation.5) The TCGA patient dataset is not well described. TCGA data are mainly obtained from local prostate cancer and biochemical recurrence usually refers to recurrence after local therapy , which is usually not relevant to hormone therapy resistance. What is the "anti-hormonal therapy" the authors refer to? How was the expression cut point defined and identified?We apologize for this omission, which is now resolved. Indeed, as the TCGA cohort represents tumors with localized disease, the vast majority of cases did not receive any adjuvant hormone therapy. However, a subset of 65 patients in the entire TCGA cohort did receive hormonal therapy, and the data presented in our manuscript is specifically describing these patients. Unfortunately, the clinical information in the TCGA cohort was insufficiently detailed that no specifics were provided on which type of hormonal therapy was prescribed. Regarding the definition of the expression cut-off in the TCGA analyses, we investigated the 3rd-4th-5th-6th-7th deciles, in which the 3rd decile provided the most-statistically significant difference on biochemical relapse-free survival. This information has now been included in the Materials and methods section.To further strengthen the clinical part of our study, we now include additional analyses from two independent cohorts  showing an inverse correlation between TLE3 and GR mRNA levels. In addition, we now included new data generated from an on-site clinical study, in which biopsies were taken from metastatic lesions before and after enzalutamide exposure . These findings show that for a subset of patient samples we analyzed, TLE3 levels were found decreased upon relapse to Enzalutamide treatment, which was accompanied by an increase of GR in these samples.6) Overall, there appears to be insufficient data to support the model proposed in Figure 5.We thank the reviewer for highlighting this point. We have updated the manuscript with new data ensuring every step in the model is fully experimentally supported, either by us or by previous studies from others. In this revised manuscript, we included several analyses that support our model and have improved this study:– We overlaid the ChIP-seq data derived by Stelloo et al. with data from Shah et al. of the GR enhancer unit and found TLE3 binds at the same locus.KOTLE3 cells treated with enzalutamide. In these conditions, GR binds enhancer elements proximal to the TLE3/AR bound genes most differentially expressed in control cells versus KOTLE3 cells treated with enzalutamide.– We show that GR is expressed on protein level in – We analyzed patient biopsy samples pre- and post-enzalutamide treatment for TLE3 and GR expression and observed low TLE3 and high GR expression in several cases of enzalutamide resistance. Furthermore, we analyzed publicly available RNA-seq data revealing an inverse correlation between TLE3 expression and GR expression in biopsy samples from prostate cancer patients with early-stage disease as well as advanced prostate cancer.eLife. In addition, we feel that our newly added experiments and analyses aided in filling the omissions in the model, and we sincerely hope the reviewer agrees.We hope that by addressing the reviewers comments, and including these additional analyses, the reviewer will find this manuscript suitable for publication Reviewer #2:A couple previous studies have indicated an association between TLE3 and AR, but its functional significance for AR function has not been determined. This study found that TLE3 loss enhanced LNCaP cell growth in response to AR antagonist in a CRISPR screen. Further ChIP-seq and transcriptome data support TLE3 coregulation of a subset of AR regulated genes. Finally, in a TLE3 KO background the investigators found that AR inhibition caused an increase in GR, and that GR inhibition could resensitize the cells to ENZ. Together these data support a role for TLE3 in the regulation of a subset of AR regulated genes. However, there are a number of concerns related to functional significance that should be addressed, as indicated below.1) The generality of the findings is unclear as TLE3 depletion only conferred drug resistance in LNCaP, but not in CWR-R1 or LAPC4. It would be of value to explore the basis for this difference a bit more. In particular, does ENZ stimulate TLE3 in these latter cells.The reviewer raises an excellent point. Indeed, we were also surprised to see that the TLE3-mediated enzalutamide mechanism is not conserved in CWR-R1 and LAPC4 cells. However, as we did find this to be crucial information, we decided to incorporate these results into the manuscript. As suggested by the reviewer, we now added Western blot results for TLE3 expression in LAPC4 and CWR-R1 cells treated with vehicle, R1881 or AR inhibitor . The results show a difference in TLE3 expression levels in LAPC4 cells treated with enzalutamide and R1881 following the same direction as observed in LNCaP cells, though to a lesser extent. This effect was observed to be noticeable but weak in CWR-R1 cells. Potentially, this difference in androgen-regulated TLE3 expression levels could indeed explain the difference regarding the enzalutamide resistance observed in LNCaP and not LAPC4 and CWR-R1.Although additional cell lines showing the same effect of TLE3 loss would be preferred, we do not expect this resistance mechanism to be generally applicable, as there is considerable heterogeneity in resistance mechanisms in prostate cancer patients.Nevertheless, to demonstrate more general applicability of our findings, we tested TLE3 and GR expression by immunohistochemistry in paired biopsies of prostate cancer patients treated with enzalutamide. These analyses revealed low TLE3 expression and high GR expression in several cases of enzalutamide resistance, thus providing evidence for more general applicability of our findings.2) The significance would also certainly be enhanced by in vivo data confirming that the TLE3 depleted cells are resistant to ENZ.We fully agree with the reviewer, that in vivo data would greatly contribute to the significance of our work. To address this, we included two new types of analyses on human prostate tumor datasets. First, we included additional analyses on publicly-available transcriptomics data from two independent prostate cancer cohorts , in which an inverse correlation was observed between GR and TLE3 mRNA levels in vivo, strengthening our cell line-based findings that TLE3 negatively regulated GR transcription. Second, we incorporated data from an on-site clinical trial, in which biopsies from metastatic lesions were taken before enzalutamide treatment and after progression . As expected, heterogeneity is found between patients, as multiple different mechanisms of enzalutamide resistance can occur. For example, one patient (out of 4) showed clear AR amplification arising during enzalutamide treatment. Nevertheless, for the other three patients, we observed low TLE3 and high GR expression associated with resistance. For one of these patients the inverse association between TLE3 and GR became more pronounced upon enzalutamide treatment . These findings are in full concordance with our cell line-based experiments and illustrate that our findings can be recapitalized in vivo.3) AR was the top TF associated with genes that were differentially expressed in TLE3 KO cells in the presence of androgen or with ENZ. One would predict that expression of these genes would increase in the TLE3 KO cells. Figure 2B shows fold change, but it is not clear what is being compared. Moreover, the description is somewhat cryptic (genes that show an interaction with TLE3 KO and ENZ treatment). The effect of TLE3 on AR/TLE3 shared genes, as well as on TLE3 alone genes should be clearly described.KOTLE3 cells treated with enzalutamide). Through this comparison, Figure 2B shows that loss of TLE3 leads to reactivation of AR-driven genes in the presence of enzalutamide, which does not happen in TLE3-proficient cells. We changed the term “interaction” into “correlation” in the manuscript.We thank the reviewer for highlighting this unclarity in the manuscript, which was also raised by reviewer #1. To address this point, we now reformulated the text and figure legend to describe which groups are being compared (control cells versus 4) It is not clear how UGT2B17 fits with the hypothesis that TLE3 is repressing AR regulated genes in response to ENZ .We apologize for the lack of clarity. UGT2B17 is negatively regulated by AR which is illustrated by the fact that expression of this gene goes up upon enzalutamide treatment rather than down. Loss of TLE3 appears to dampen this upregulation, similar to dampening downregulation of genes positively regulated by AR when cells are treated with enzalutamide. In the Results section, this is now more-clearly explained and explicitly mentioned.5) It is an attractive hypothesis that AR directly represses TLE3 expression. However, supplemental Figure 2 only shows effects on protein, with no indication of how long the cells were treated. The authors should show a time course of TLE3 mRNA induction and loss in response to ENZ and DHT in order to address whether the effects are likely direct.The reviewer raises an excellent point. To address this, we analyzed publicly available RNA-seq data  from LNCaP cells treated with R1881 (time course) for TLE3 expression. These data have been added to Figure 2—figure supplement 1H. The results show that TLE3 mRNA levels decrease rapidly, as soon as 4 hours, after stimulation with R1881, providing evidence that AR directly represses TLE3 transcription. These data are in line with the western blot results  and provide evidence that AR directly represses TLE3 transcription.6) Figure 3 seems to show that TLE3 binding at AR regulated genes does not decrease with R1881 stimulation. This would seem to be inconsistent with the feedback model, and with the marked decrease in TLE3 protein in R1881 treated cells.Indeed, as TLE3 is suppressed by AR activation, persistence of TLE3 binding at AR regulated genes after R1881 could potentially be in contrast to the model. However, we would like to highlight the aspect of protein stability and turnover as a variable. In Figure 3, the ChIP-seq was performed using cells that were exposed to R1881 for 4 hours to facilitate AR translocation to the nucleus and chromatin binding. At this timepoint, TLE3 protein levels are not yet affected .Therefore, we believe these findings do not contradict the model. We updated the Discussion section, and explicitly refer to the data from Stelloo et al. to further explain this aspect.7) The growth data in Figure 4 is qualitative and only a single plate is shown for each condition. It should be quantified, and a growth curve would help.KOTLE3 cells treated with enzalutamide.We agree that quantitative data for the growth assays would be superior over the mere qualitative data as provided in the original paper. To address this point, we now quantified the data for the growth assays shown for this figure . The conclusion remains identical to the original observations, and statistical testing confirmed that there is significant difference in growth between control and 8) The authors show that GR KO confers sensitivity to ENZ in the TLE3 KO cells, and suggest this reflects GR activation of AR/TLE3 regulated genes. To assess this mechanism, they should address whether GR KO in this context does indeed suppress the expression of AR/TLE3 coregulated genes.In the original manuscript, we performed shRNA targeting of TLE3. Using the same model system for RT-QPCR analyses on AR/TLE3 regulated genes, we were unable to consistently detect impact of GR knockdown on expression of these genes, suggesting that the remaining low levels of GR sufficed to preserve the phenotype on a transcriptomics level. In order to address the reviewer question, we reverted to the original observation from the Sawyers lab , in which these very same genes as we study in our paper   were tested for expression after targeting GR. Thus, for several of the most differentially expressed TLE3/AR target genes we identified  GR was shown to be functionally and critically involved in the regulation of these genes, as was originally shown by the Charles Sawyers lab. We highlighted this in the Results and Discussion sections.9) While GR may contribute to ENZ resistance in TLE3 KO cells, the significance of this finding for TLE3 intact cells is not addressed. Is there increased GR recruitment to AR/TLE3 regulated genes in response to ENZ in wild-type cells?KOTLE3 cells treated with enzalutamide, but not in untreated- or enzalutamide-treated WT cells.While we agree it would be interesting to investigate GR occupancy in this context, GR appears to be only upregulated when both TLE3 and AR are perturbed in our model as shown in Figure 2. Newly added western blot results  show no detectable GR protein in WT cells treated with enzalutamide, making GR chromatin occupancy unlikely in this context. We updated the Discussion section to highlight this point. Our newly added GR ChIP-qPCR data  confirms this, as GR occupancy is only observed proximal to AR/TLE3 genes in 10) In Supplemental Figure 4 it is unclear if the authors are assessing BCR after RP or after ADT. It is probably the former, which would only mean that TLE3 expression is associated with aggressiveness, and would not provide evidence that it is involved with response to ADT.We apologize for the unclarity. Indeed, as the TCGA cohort represents tumor with localized disease, the vast majority of cases did not receive any adjuvant hormone therapy. However, a subset of 65 patients in the entire TCGA cohort did receive hormonal therapy, and the data presented in our manuscript is specifically describing these patients. As such, the BCR as described in Figure 5C is after hormonal therapy. Unfortunately, the clinical information in the TCGA cohort was insufficiently detailed in the sense that no specifics were provided on which type of hormonal therapy was prescribed.We further strengthened the connection between TLE3 and Enzalutamide resistance in two ways, as we mention in reply to point 2 of this reviewer: 1) correction of TLE3 and GR mRNA levels in 2 prostate cancer cohorts. 2) Decrease of TLE3 levels and increased GR levels in patient samples relapsing after enzalutamide in an on-site performed clinical trial. We believe these new additions further strengthen the clinical implications of our work, and we sincerely hope the reviewer agrees.Reviewer #3:The authors identify TLE3 loss as a cause of apalutamide resistance in a genome-wide in vitro CRISPR screen in LNCaP prostate cancer cells. Transcriptomic and ChIP-seq studies support a model whereby TLE3 loss rescues suppression of AR pathway signaling by antiandrogens. Mechanistically this occurs, at least in part, through upregulation of GR based on experiments showing that shRNAs targeting GR restore sensitivity to enzalutamide. This model is supported by ChIP-seq data showing overlapping binding of TLE3 and FOXA1 at various AREs across the genome including a GR enhancer.The data supporting TLE3 as screen hit as well as the proposed mechanism for causing antiandrogen resistance through GR upregulation,is convincing, but the work has two significant shortcomings.We thank the reviewer for the constructive comments and valuable suggestions. As further elaborated below, we did our utmost best to address the two issues what were raised by this reviewer.1) The apparent context dependence of TLE3 loss for LNCAP cells only (negative results in CWR-R1 and LAPC4) raises concerns about the broader relevance of the TLE3 loss in prostate cancer. The authors could address this in several ways:i) testing of more modelsii) mechanistic insight into why resistance is not seen in CWR-R1 and LAPC4 iii) deeper interrogation of TLE3 status in human datasets, particularly in the castration-resistant setting We thank the reviewer for highlighting this critical point. Indeed, the impact of TLE3 loss in prostate cancer cells was quite context dependent, and the observations in LNCaP cells could not be recapitulated in CWR-R1 and LAPC4 cells. However, as we did find this to be crucial information, we decided to incorporate these results into the manuscript. To provide more insights into this discrepancy, we now added Western blot results for TLE3 expression in LAPC4 and CWR-R1 cells treated with vehicle, R1881 or AR inhibitor . The results show a difference in TLE3 expression levels in LAPC4 cells treated with enzalutamide and R1881 following the same direction as observed in LNCaP cells, though to a lesser extent. This effect is far less prominent in CWR-R1 cells. Potentially, this difference in androgen-regulated TLE3 expression levels could indeed explain the difference regarding the enzalutamide resistance observed in LNCaP and not LAPC4 and CWR-R1To address the reviewer’s concern regarding human datasets, we significantly increased the amount of data describing TLE3 in human datasets. We now include additional analyses from two independent prostate cancer patient cohorts , showing an inverse correlation between TLE3 and GR mRNA levels. In addition, we generated important additional data from an on-site clinical trial, in which biopsies were taken from metastatic lesions before and after enzalutamide exposure . These analyses revealed low TLE3 expression and high GR expression in several cases of enzalutamide resistance.With these new data, we sincerely hope the reviewer finds this issue sufficiently addressed.2) The prior work on GR in castration resistance diminishes the novelty. This could perhaps be overcome with additional mechanistic insight beyond that reported in the earlier publications. For example, how does TLE3 loss impact the chromatin landscape (particularly repressive histone marks) across the genome and more specifically at the GR locus?Indeed, prior work from the Sawyers group showed that GR can mediate Enzalutamide resistance. However, this previous work did not explain how GR upregulation was mediated. Our results show that TLE3 acts as a transcriptional repressor that plays a critical role in inhibiting the expression of GR in prostate cancer cells. Upon TLE3 loss and AR inhibition, this suppression is alleviated, resulting in an increase of GR levels, both on the transcript  and protein levels , giving rise to Enzalutamide resistance. With this, we feel that our findings do provide substantial novelty, in explaining how GR upregulation in Enzalutamide resistance can occur: through a loss of the transcriptional repressive effects of TLE3. We have now updated the Discussion section to communicate this message more clearly.To address the question how loss of TLE3 affects the chromatin at the GR locus, we performed H3K27ac ChIP-qPCR for the GR enhancer occupied by TLE3. TLE3 is known to facilitate a repressed chromatin structure by recruiting HDACs. Loss of TLE3 was shown to positively regulate H3K27 acetylation in breast cancer cells, affecting chromatin structure at TLE3/ERα binding sites resulting in derepression of these loci/genes .KOTLE3 cells when compared to TLE3-intact cells . These data are in line with the expression data for RND3 in Figure 2, and newly added ChIP-qPCR data showing binding of GR proximal to RND3 and several other target genes despite enzalutamide treatment .In our model, loss of TLE3 resulted in upregulation of H3K27ac when compared to WT cells . We also looked beyond the GR enhancer by analyzing the RND3 locus as this gene showed upregulation upon TLE3 loss. Also here we found that H3K27 acetylation was increased significantly in Overall I would be supportive of considering a revised manuscript but it would need to have additional data.We thank the reviewer for the constructive remarks and hope that these new findings on the transcriptional regulation of the GR locus, along with the new clinical datasets that strengthen the GR/TLE3 connection, sufficiently address the points raised by this reviewer and would render our manuscript acceptable for publication.To summarize, our most important new data includes:– Addition of TLE3 and GR expression analysis in prostate cancer patient samples.– Analysis of publicly available RNA-seq data analyzing TLE3 and GR expression in prostate cancer cohortsKOTLE3 cells treated with enzalutamide and TLE3 expression in LAPC4 and CWR-R1 cells in various conditions– GR protein expression by western blot in control and – Analysis of time course of TLE3 expression in publicly available RNA-seq data  from LNCaP cells treated with R1881KOTLE3 cells, as well as ChIP-qPCR-analysis for GR at several other TLE3/AR target genes.– ChIP-qPCR analysis for the TLE3-mediated H3K27 acetylation at the GR locus in WT and Reviewer #1:Palit, et al., provide a revised manuscript on TLE3 loss and enzalutamide resistance. The strength of the original manuscript is with the enzalutamide resistant phenotype conferred by TLE3 KO but was lacking in mechanistic characterization and there were some concerns about clinical relevance of this finding. In general, the authors have put forth a reasonable effort to address reviewer comments.The additional evaluation on inverse correlation between GR and TLE3 expression from clinical data sets  are not really convincing and requested in vivo experiments by one of the other reviewers on TLE3 KO and resistance are not provided. Together, with a CRISPR KO generated mechanism of resistance that occurs in a single cell line model, if moving forward with this manuscript I would suggest that the authors pull back on the potential clinical relevance and explicitly state this as a major caveat of their story (in the Abstract and Discussion).We are delighted the reviewer appreciates the changes made to the original manuscript and we are thankful for the constructive comments helping us to further improve the manuscript. We have made changes to the Abstract and Discussion as requested. In the Abstract, we now mention that TLE3 and GR expression in clinical samples reflect our findings in LNCaP cells, but that clinical relevance is yet to be determined. This is also stated in the Discussion, where we  mention the limitations of the in vitro data. We state that our findings warrant further investigation into the clinical relevance of this resistance mechanism in patients.eLife.We hope the reviewer appreciates the changes made to the revised manuscript and considers the manuscript suitable for publication at Reviewer #2:The authors have responded to the points raised in the initial review. There are still concerns that the observations are limited to one cell line and are only in vitro. However, the authors do now show an inverse correlation between TLE3 and GR in public domain data. It should be noted that the previous study from Arora et al. found that GR was not immediately increased by ENZ, but required some substantial time for adaptation. Therefore, overall the data support the hypothesis that loss of TLE3 is a mechanism that contributes to increasing GR in response to ENZ in at least a subset of patients. One minor point is that in response to the question of whether TLE3 loss increased WNT signaling, the authors showed no increase in active b-catenin. However, TLE3 loss would presumably increase TCF activity without an increase in active b-catenin. The more relevant data would be effects on TCF regulated genes such as AXIN2.We are pleased that the reviewer appreciates the changes made to the manuscript in response to the comments made in the initial review. We thank reviewer#2 for the constructive comments and we now mention the adaptation time of the LREX model used in the study by Arora et al., 2013 in the Discussion.AXIN2 in KOTLE3 cells treated with vehicle or enzalutamide for 5 days , and found no significant changes relative to control cells, indicating Wnt signaling is not altered in this context. We hope that our manuscript is now suitable for publication at eLife.To address the point regarding Wnt signaling through TCF activity mentioned by the reviewer, we now added qPCR data for the TCF target gene Reviewer #3:The authors have responded to my earlier review with additional evidence from clinical datasets that extends the TLE3 work beyond the LNCaP cell line. I believe the revised manuscript is suitable for publication at eLife.eLife.We thank the reviewer for the valuable suggestions and constructive comments and are pleased the reviewer appreciates the changes made to the article and believes that it is suitable for publication at


To investigate the impact of disease duration on the ocular surface during the course of type 2 diabetes mellitus compared with nondiabetic controls.  One hundred twenty diabetic patients were divided into three groups according to disease duration: less than 5 years, 5–10 years, and over 10 years. All eyes were imaged using a corneal topographer (Oculus Keratograph 5M). Tear film measurements and meibography were also recorded. Meibomian gland changes were scored from 0 to 6 (meiboscore). P=0.0056  and  P=0.010, resp.). Tear meniscus height (TMH) was significantly lower in the over 10 years diabetic group compared with the control group (P=0.0016) and the 5 years group (P=0.0061). We also found that more patients in the over 10 years diabetic group showed bulbar and limbal hyperemia compared with the control group . The meiboscore in the over 10 years diabetic group was significantly higher compared with the other three groups (P < 0.05). Bulbar hyperemia showed a significant negative correlation with NIKBUT-1st in the over 10 years diabetic group (r=−0.35  and  P < 0.05).  The noninvasive breakup time first (NIKBUT-1st) and noninvasive breakup time average (NIKBUT-avg) were significantly shorter in the over 10 years diabetic group compared with the control group ( Ocular surface damage in long-term type 2 diabetes is more severe than that in patients with shorter disease duration. Type 2 diabetes mellitus is one of the largest public health problems worldwide, especially in developing nations such as China , due to However, according to a clinical study, up to 73.6% of type 2 diabetic patients suffer from corneal complications, such as punctate keratopathy, endothelial dystrophy, and recurrent erosions . During In contrast to aqueous-deficient dry eye, which is usually caused by a lack of tear production, evaporative dry eye is due to lid-related and ocular surface-related causes such as meibomian gland dysfunction (MGD) and is more frequent . It is aCorneal changes usually depend on the type, duration, and compensation of diabetes mellitus. In this study, we aimed to analyze the tear film stability and morphological changes in meibomian glands using keratography in diabetic patients compared with nondiabetic controls and to better understand the impact of disease duration on the ocular surface during the course of type 2 diabetes mellitus.The study was approved by the Investigational Review Board of Shanghai Ninth People's Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China. All subjects enrolled were informed of the aims of this study.One hundred twenty patients diagnosed with type 2 diabetes mellitus met the inclusion criteria and were enrolled in the study at the Shanghai Ninth People's Hospital between April 2016 and January 2017. Data were obtained from the right eye of each subject unless this eye was excluded, in which case data were collected from the left eye. The inclusion criteria were as follows: at least 40 years of age and willingness to participate in the study. Patients were excluded if they used topical medications; wore contact lenses; had undergone ocular surgery in the past year or had evidence of other ocular surface diseases; had active ocular infection, inflammation, or systemic disease; or were taking medications that would alter the ocular surface. All diabetic patients had a blood glucose level within the normal range. Diabetic patients were divided into the following three groups according to duration of diabetes since the first diagnosis: less than 5 years, 5–10 years, or more than 10 years. Forty subjects were recruited as nondiabetic controls. The exclusion criteria were similar to those of the control group. Fasting blood glucose was measured to rule out diabetes even in those without a history of diabetes. All patients underwent noninvasive ocular surface examinations in the following order: tear meniscus height (TMH), noninvasive tear film breakup time, bulbar and limbal hyperemia, and grading of meibomian gland loss with the Oculus Keratograph 5M . All patients were examined by the same physician (He FL).All diabetic patients were from the Department of Endocrinology in our hospital. These patients had been followed up over a long period, and disease duration was confirmed by their professional endocrinologists. The data in this article are reliable and were obtained from medical files. The groups were matched by age.Oculus Keratograph 5M can provide automated measurements of tear film dynamics and meibographic images using infrared light without topical anesthetic, fluorescein staining, cobalt blue light, or manual timing. Measurements of noninvasive breakup time (NIKBUT) obtained with the advanced corneal topographer provide a simple, noninvasive screening test for dry eyes with acceptable sensitivity, specificity, and repeatability . MeibogrTMH was measured twice in each eye using infrared images obtained from the keratograph. The lower tear film meniscus images were captured 5 s after blinking, and the values were graded perpendicular to the lower eyelid margin at the central point.NIKBUT was measured twice in each eye using the Oculus noninvasive Keratograph tear breakup time (NIKBUT) tool. The participants were instructed to blink twice before screening and to keep their eyes open to the best of their ability when recording. The NIKBUT was then determined by Keratograph 5M, which automatically generated two measures for NIKBUT: NIKBUT first (NIKBUT-1st) and NIKBUT average (NIKBUT-avg). NIKBUT-1st represents the time point at which the tear film starts to break up. NIKBUT-avg represents the average time for the overall tear film to break up.Increased conjunctival redness is often one of the first signs indicating abnormal strain or pathological changes in the eye. Patients were required to open their eyes as wide as possible and focus on a point inside the camera while a keratograph image was captured. The images were then analyzed by the R-SCAN tool following the evaluation protocol. The software analyzed the image automatically and assigned a red eye index (accurate to 0.1 unit).The upper and lower eyelids were ectropionized, and their respective infrared images were captured. Meibomian gland loss was graded as described by Arita et al. . The meiP values less than 0.05 were considered statistically significant. Data are presented as mean ± SD.Pearson's chi-squared test was applied to analyze sex and age differences between the groups. The Shapiro–Wilk test or Kruskal–Wallis test was performed accordingly. Normally distributed continuous parameters were analyzed between the groups by one-way ANOVA and Welch ANOVA tests. The Spearman correlation test was used to calculate correlations. All analyses were performed using the statistical software package GraphPad Prism (version 6.00 for Mac). All From April 2016 to January 2017, 120 eyes of 120 type 2 diabetic patients and 40 eyes of 40 nondiabetic patients  were included in this study. In the diabetic group, the duration of diabetes ranged from 1 to 25 years: in 44 eyes of 44 patients , the duration was less than 5 years; in 40 eyes of 40 patients , the duration was 5–10 years; and the duration was more than10 years in 36 eyes of 36 patients . Medians and ranges of clinical parameters in the control group and diabetic groups are shown in Figures P values for pairwise comparisons between the groups are presented in Tables The results of clinical parameters in each group and P < 0.01) and the 5 years group . Similarly, NIKBUT-avg was significantly shorter in the over 10 years diabetic group compared with the control group . The association between NIKBUT-1st and time from diagnosis was calculated and indicated a slightly negative Spearman correlation  and the 5 years group . The association between TMH and time from diagnosis was also calculated and indicated a slightly negative Spearman correlation .The bulbar and limbal redness scan found that more patients in the over 10 years diabetic group showed bulbar and limbal hyperemia compared with the control group , which indicated that the over 10 years diabetic patients suffered a loss of meibomian glands.The over 10 years diabetic group showed more meibomian gland changes including dropouts, shortening, distortion, and dilation. The meiboscore in this group was significantly higher compared with the other three groups , which is shown in In addition to higher bulbar hyperemia and shorter NIKBUT-1st in the over 10 years diabetic group compared with the nondiabetic group, we also found that bulbar hyperemia had a significant negative correlation with NIKBUT-1st in the over 10 years diabetic group , and bulbar hyperemia had a significant negative correlation with NIKBUT-1st in the over 10 years diabetic group (r=−0.35  and  P < 0.05). Conjunctival inflammation is a hallmark of dry eyes [The bulbar conjunctiva and anterior episclera are nourished by vessels from the anterior and long posterior ciliary arteries , and thedry eyes , 21. We dry eyes . In addiWe compared meibographic findings in the 4 groups, and a significant difference in the meiboscore was found between the control patients and the over 10 years diabetic patients. This was consistent with previous findings , 24 wherThe meibomian gland synthesizes and produces lipids and proteins which form the outermost layer of the tear film. These lipids decrease evaporation and promote stability of the tear film. The International Workshop on Meibomian Gland Dysfunction suggested that MGD is the most prevalent cause of evaporative dry eye and may play a role in aqueous-deficient dry eye . The meiIn addition, Oculus Keratograph 5M is a corneal topographer with additional noninvasive imaging tools for the assessment of tear film kinetics and meibography. It includes the ability to capture clear images of the meibomian gland architecture using infrared light, eliminating the need for instillation of sodium fluorescein into the tear film or the use of white light that exacerbates photophobia in patients with ocular surface disturbance. However, there is no consistent method for meibographic analysis in clinical practice. Our results provide an objective grading method to illustrate complementary data of glandular density.In summary, our data suggest that long-term type 2 diabetes predisposes to various changes on the ocular surface, which should be noted at an early stage and treated appropriately in order to prevent more severe eye complications. Therefore, close attention should be paid to the ocular surface, especially in long-term diabetics. Further studies are needed to expand the sample size and include fluctuations in blood sugar as a key factor in studying the ocular surface.


Gallus gallus, supported by Wnt1-Cre lineage analysis in Mus musculus. To test the intriguing possibility that neural crest cells contribute to heart repair, we examined Danio rerio adult heart regeneration in the neural crest transgenic line, Tg(−4.9sox10:eGFP). Whereas the adult heart has few sox10+ cells in the apex, sox10 and other neural crest regulatory network genes are upregulated in the regenerating myocardium after resection. The results suggest that neural crest cells contribute to many cardiovascular structures including cardiomyocytes across vertebrates and to the regenerating heart of teleost fish. Thus, understanding molecular mechanisms that control the normal development of the neural crest into cardiomyocytes and reactivation of the neural crest program upon regeneration may open potential therapeutic approaches to repair heart damage in amniotes.Cardiac neural crest cells contribute to important portions of the cardiovascular system including the aorticopulmonary septum and cardiac ganglion. Using replication incompetent avian retroviruses for precise high-resolution lineage analysis, we uncover a previously undescribed neural crest contribution to cardiomyocytes of the ventricles in  Before birth, unspecialized stem cells go through a process called differentiation to form the many types of cells found in the adult. Neural crest cells are a group of these stem cells found in all animals with backbones (i.e. vertebrates) including humans. These cells migrate extensively during development to form many different parts of the body. Due to their contributions to diverse organs and tissues, neural crest cells are very important for healthy development.The heart ventricle is one of the tissues to which neural crest cells contribute during embryonic development in fish and amphibians. However, it was unclear whether this is also the case for birds or mammals or whether neural crest cells have any roles in the regeneration of the adult heart after injury in fish and amphibians.To address these questions, Tang, Martik et al. used cell biology techniques to track neural crest cells in living animals. The experiments revealed that neural crest cells contribute to heart tissue in developing birds and mammals and help repair the heart in adult zebrafish. Further results showed that the contribution of neural crest cells to the heart is controlled by the same genes during both the growth of the embryonic heart and the repair of the adult heart.These results provide new insights into the repair and healing of damaged heart muscle in fish. They also show that similar processes could exist in mammals, including humans, suggesting that activating neural crest cells in the heart could treat damage caused by heart attacks and related conditions. The neural crest is an important stem cell population characterized by its multipotency, migratory behavior, and broad ability to differentiate into derivatives as diverse as elements of the cardiovascular system, craniofacial skeleton, and peripheral nervous system. However, not all neural crest cells are alike, with distinct populations existing along the body axis. One of the most unique neural crest populations is the ‘cardiac neural crest’ that contributes to the outflow septum and smooth muscle of the outflow tract of the heart. Ablation studies in chick embryos show that removal of the cardiac crest results in a broad range of defects, including persistent truncus arteriosus, abnormal myocardium function, and misalignment of the arch arteries . These dWnt1-Cre driven β-galactosidase in mammals  RIA  that indelibly and precisely marks neural crest progenitors for long term lineage analysis at single cell resolution and without the need for tissue grafting. To this end, the post-otic neural tube of the hindbrain adjacent to somites 1–3 was injected at Hamburger and Hamilton (HH) stage 9–10 with high-titer (1 × 10/mL) RIA , which dpromoter . At thisVirally infected embryos were then allowed to develop for 1–9 days post injection, cryo-sectioned, and analyzed using confocal microscopy. One day after injection, whole mount imaging revealed RIA-labeled cells migrating in a stream along pharyngeal arch 3 , that suImportantly, by E3 and onward, virally labeled neural crest cells were observed in the myocardium of both the outflow tract and the ventricles, where they expressed the myocardial markers, Troponin T and Myosin Heavy Chain , consistfl/flWnt1-Cre;ZsGreen transgenic mice in which neural crest cells were labeled with cytoplasmic GFP  without ectopic activation of canonical Wnt/β-catenin pathway , that labels all embryonic migratory neural crest lineages to address whether neural crest-derived cardiomyocytes reactivated their developmental program upon injury   ; n = 6. on (dpa) ; n = 6. ve cells , suggestsox10 and other bona fide neural crest markers such as tfap2a, were upregulated endogenously, we performed in situ hybridization on paraffin sections of regenerating and uninjured ventricles. The results reveal upregulation of expression of sox10 and tfap2a transcripts after injury, whereas they were mostly absent from uninjured ventricles , which permanently labels all sox10-derived lineages with mCherry  is a double transgenic line for the sox10:GAL4-UAS-Cre transgene and the ubi:Switch reporter in which the sox10 promoter drives expression of Cre recombinase. Upon activation of sox10 expression in neural crest cells, eGFP is excised and so cells of the sox10 lineage are permanently labeled with mCherry  into single cell suspensions and performed FAC-sorting of sox10:mRFP+ cells (sox10+ cells compared to sox10- cells of the same injured ventricles (sox10+ cells to: 1) our recently published chick developmental cardiac neural crest gene regulatory program, 2) known zebrafish neural crest genes, and 3) core neural crest gene regulatory network genes expressed at all axial levels  may represent a key population that proliferates and differentiates into new cardiomyocytes after injury.sox10 and other neural crest gene regulatory network genes. The finding that sox10-derived cells are primarily in the proximal trabecular myocardium of the zebrafish heart suggests that these cells must be migrating into to the wound site after injury. Of course, we cannot rule out the possibility that the cells that reactivate sox10 and the neural crest program may come from another adult lineage. But in the adult, their molecular signature strongly correlates with that of embryonic neural crest cells  corrects possible ectopic expression problematic in the original Wnt1-Cre;ZsGreen line  of the trabeculated myocardium in the proximal part of both ventricles. Interestingly, this percentage is similar to what has been reported in zebrafish . In amniIn summary, the present results show, for the first time, the common ability of cardiac neural crest cells across diverse vertebrates to contribute to heart muscle. Moreover, these cells appear to be critical for cardiac regeneration in zebrafish. If the results extrapolate to other species, the mechanisms that control the normal development of the neural crest into cardiomyocytes may be harnessed to stimulate these cells to proliferate and regenerate new cardiomyocytes, thus offering potential therapeutic approaches to repair heart damage in mammals including humans.CVCL_0570, Certificate of Analysis with negative mycoplasma testing at the ATCC website) were transfected with RIA-H2B-YFP plasmid (RRID:Addgene_96893) and ENV-A plasmid in 15 cm culture dishes. Cell culture medium was collected 24 hr post-transfection, and twice per day for four days, then centrifuged at 26,000 rpm for 1.5 hr. The supernatant was dried with aspiration, and the pellet was dissolved in 20–30 μl of DMEM to a final titer of 1 × 107 ifu/mL. Viral aliquots were stored in −80°C until the time of injection.Using a standard transfection protocol, chick DF1 cells  to generate the working solution, which was mixed with 0.3 μl of 2% food dye  as indicator. The lumen of the neural tube adjacent to the middle of the otic vesicle to the level of somite three was injected with 0.2 μl of working in HH8-10 chicken embryos. Embryos were sealed with surgical tap and incubated at 37°C for 1–9 days, harvested at HH14 (n = 5), HH18 (n = 5), HH21 (n = 4), HH25 (n = 4), HH28 (n = 12), HH32 (n = 4) and E10 (n = 4). At the time of harvesting, chick embryos were dissected, fixed in 4%PFA in PBS for 30 mins at 4°C, then embedded in gelatin and sectioned (Microm HM550 cryostat).Viral stock was diluted 1:2 with Ringer’s solution , 21dpa (n = 3) and sham operated (n = 3) hearts in an area of 2 × 105 μm2 at the apex. One section per heart at the middle of the apex was quantified and presented in To quantify RIA-labeled cells in chick embryos, three consecutive sections of the same axial level were imaged per embryo. The number of YFP-positive cells was averaged to account for variability due to sampling. n = 4–6 embryos were analyzed at each stage as biological replicates. The results are presented as presence or absence of virally labeled cardiac neural crest derivatives at different anatomical locations in Tg(−4.9sox10:eGFP) or Tg(sox10:mRFP) line, according to published protocols (Adult zebrafish heart resection was conducted with the rotocols . ResecteAB_528495); 1:10 Myosin Heavy Chain ALD58, DSHB (RRID:AB_528361); 1:10 Myosin Heavy Chain F59, DSHB (RRID:AB_528373); 1:500 Mouse anti-smooth muscle actin, Sigma-Cat# A5228-200uG; 1:500 Mouse anti phospho-histone H3, Abcam-ab14955; 1:500 rabbit anti caspase-3,R and D Systems # AF835; 1:500 goat anti GFP, Abcam Cat#ab6673, all in blocking reagent 1xPBS with: 5% vol/vol normal donkey serum, 0.3% vol/vol Triton-X100). Subsequently, sections were washed for 3 times with 1xPBS, incubated with secondary antibody for 40 mins at room temperature and counterstained with DAPI. Secondary antibodies include: Goat anti-mouse IgG2a Alexa-568 (RRID:AB_2535773), Goat anti-mouse IgG1 Alexa-568(RRID:AB_2535766), Goat anti-rabbit IgG Alexa-568(RRID:AB_2534121), Donkey anti-goat IgG Alexa-488(RRID:AB_2534102); 1:1000, Molecular Probes. Zeiss AxioImager.M2 with Apotome.2 and Zeiss LSM 800 confocal microscope were utilized for imaging. Images were cropped, rotated, and intensity was linearly adjusted for visualization.After cryosectioning, slides were incubated in 1xPBS at 42°C to remove gelatin. 0.3% vol/vol Triton-X100in 1xPBS was used to permeabilize the tissue. Sections were incubated with primary antibody underneath a parafilm layer at 4°C overnight . After several washes with MABT, chromogenic color was developed using NBT/BCIP precipitation (Roche).sox10-mRFP-positive and sox10-mRFP-negative cells were collected by FAC-sorting on a BD Biosciences FACSAriaFusion Cell Sorter. cDNA from mRFP-positive and negative cells was prepared using SMART-seq Ultra Low Input RNA Kit V4 (Takara) according to the manufacturer’s protocol. Sequencing libraries were built according to Illumina Standard Protocols and sequenced using an Illumina HiSeq2500 sequencer at the Millard and Muriel Jacobs Genetics and Genomics Laboratory . 50 million, 50 bp, single-ended reads from two biological replicates were mapped to the zebrafish genome (GRCz10) using Bowtie2 (For each replicate (n = 2), regenerating ventricles (n = 12) were isolated at 21 days post injury and dissociated into a single cell suspension using a pestle-A tissue homogenizer followed by incubation in Accumax  at 30°C.  Bowtie2 . Transcr Bowtie2 . Protein Bowtie2 . Heatmap 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 Didier Stainier as the Senior Editor and Reviewing Editor, and three reviewers. The reviewers have opted to remain anonymous.Thank you for submitting your article "Cardiac neural crest contributes to cardiomyocytes in amniotes and heart regeneration in zebrafish" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.As noted by all three reviewers, the manuscript addresses an important issue, that of neural crest contribution to the myocardium, and provides interesting and significant new findings. While some experiments were suggested that would strengthen the paper, the consensus from the discussions was that they were not essential.However, as detailed in reviewer #2's comments, the interpretation of some of the zebrafish data needs to be adjusted.I have pasted below the full reviews as all the comments should be taken into account when revising the manuscript.Reviewer #1:The current manuscript reports interesting findings on the role of the cardiac neural crest cells to the development of the heart in amniotes. Using a series of elegant lineage tracing analysis in chicken and mouse embryos Bronner and colleagues describe a previously unknown contribution of the cranial neural crest cells to cardiomyocytes of the outflow tract and ventricle in vertebrates. The analyses are well performed and provide interesting data. Remarkably, the authors also report in a model with heart regenerative capacities, the adult zebrafish, a reactivation of embryonic programs related to the neural crest development upon a cardiac injury . Then they speculate that the cranial neural crest-derived cardiomyocytes of the ventricles could contribute to the regenerative process in the injured adult heart. While these speculations are certainly relevant, the manuscript would benefit from further analysis to support this proposal.Essential revisions:Mef2 and Tbx5 in the neural crest-derived cardiomyocytes in response to injury.- Since both proliferation and trans-differentiation are prominent processes during heart regeneration, it is relevant to assess whether there is also reactivation of a proliferation gene signature (beyond gata4) and early cardiac transcriptional regulators such as Nkx2.5, - Can the authors comment on why have they decided to do the transcriptomic analysis at 21 dpi instead of 7 dpi, which is the early onset of reactivation of the developmental neural crest program?- In order to rule out an epicardial origin ), it is also relevant to determine whether or not the neural crest-derived cardiomyocytes express bona fide epicardial markers such as Tbx18 or Tcf21.- Given that the neonatal mammalian heart is also capable of regeneration after various injury models , assessing the reactivation of neural crest gene regulatory programs in mice would further support the most exciting conclusions of this study.Reviewer #2:In this manuscript, Tang, Martik, and colleagues examine the contributions of the cardiac neural crest to the myocardium. This is an important and controversial topic with substantial relevance to the causes of congenital heart disease and to the evolution of neural crest derivatives, as well as potential relevance to mechanisms underlying tissue regeneration. It is therefore valuable that the authors provide new lineage analysis data sets that document the contribution of some cardiac neural crest cells to the myocardium in both chick and mouse. This work is carefully performed and interpreted and provides new information of interest to the field. In addition, the authors demonstrate that sox10, as well as other genes associated with the migratory neural crest, are upregulated in the myocardium of the regenerating zebrafish heart, following injury. These new gene expression data add to our understanding of how the adult zebrafish heart responds to injury, and the authors are interested in drawing a connection between these observations and the contributions of cardiac neural crest-derived cells to regeneration. These data are also valuable to those seeking to understand the regulation of cardiac regeneration, but would benefit from some interpretative adjustments. Specifically:1) The induction of expression of sox10 and other neural crest-associated genes is very interesting, but it is inappropriate to interpret this as evidence that cardiac neural crest cells contribute to the regenerative capacity of the zebrafish heart without evidence that the cells activating expression of these genes are indeed neural crest-derived. The injury stimulus may induce expression of sox10 et al. in cardiomyocytes that were not originally neural crest-derived, and a different type of lineage tracing experiment would be required to determine whether neural crest-derived cardiomyocytes indeed respond to injury differently than lateral mesoderm-derived cardiomyocytes do. Alternatively, the authors could amend their interpretation – these changes in gene expression are certainly interesting whether or not the responding cells are necessarily neural crest-derived. 2) Along the same lines, the data shown here associate the induction of sox10 expression  with regeneration but do not directly demonstrate the significance of these genes to the regenerative response. Demonstrating their significance would surely require substantial additional experimentation; here, the authors should simply take caution to avoid implying that these particular genes are associated with regulation of regeneration.3) Could the authors comment on the relationship of the differential gene expression that they document in Figure 4H with other published datasets that examine the transcriptomic changes triggered by injury? Is it the case that the same neural crest-associated genes are differentially expressed in those datasets ?Reviewer #3:This is an interesting paper showing additional contributions of the neural crest to parts of the heart across vertebrates. Moreover, the authors present evidence that during regeneration of the zebrafish heart, neural crest-derived heart cells contribute to the regenerate and redeploy a neural crest gene expression programme. This suggests previously unappreciated heterogeneity in the cells contributing to regeneration.Overall, the findings are important, but the manuscript could be substantially improved by more explanations and quantifications of data.1) The manuscript has two parts and it is not entirely clear how these are connected. In the first part, the authors use lineage tracing to find new and known populations of crest cells that contribute to heart development. These cells seem to be rare in the apex "…"no neural crest-derived cardiomyocytes were observed in the apex of the heart"…"). However, in the second part of the manuscript they remove the apex for regeneration purposes. How are the two parts of the manuscript connected?2) The authors say that they have found novel crest-derived cell populations in the heart. At the same time, they state that previous research was controversial, and tools were inadequate due to high background. It is unclear whether previous papers already described these populations and this is here confirmed with more robust tools, or whether these populations are entirely new. Moreover, it would be good to at least discuss in more detail how they know that their tools are superior in their hands (e.g. "the improved Wnt1 line").3) Many results rely on scoring for the presence or absence of cells. How were tissues scored? More quantifications of the relative contribution to the tissue in question should be shown for lineage tracing and zf heart regeneration, such that the relative importance of the crest contribution can be appreciated.4) Figure 4—figure supplement 1: Were there also cells that expressed sox10 mRNA de novo? The prediction would be that only lineage-traced cells re-express sox10 – otherwise non-neural crest derived cells contribute to the population of sox10+ cells in regeneration. Can this be clearly observed in the combination of colorimetric ISH and transgenic fluorescence?5) More information is needed on how the "the cardiac neural crest migratory gene regulatory module" was identified in the expression profile of regenerated heart cells in zf. e.g. did they use GO analysis? They highlight 20 out of 656 genes – what are the other genes? Why is 21 days post-injury the best time point? Reviewer #1:[…] Remarkably, the authors also report in a model with heart regenerative capacities, the adult zebrafish, a reactivation of embryonic programs related to the neural crest development upon a cardiac injury . Then they speculate that the cranial neural crest-derived cardiomyocytes of the ventricles could contribute to the regenerative process in the injured adult heart. While these speculations are certainly relevant, the manuscript would benefit from further analysis to support this proposal.Essential revisions:- Since both proliferation and trans-differentiation are prominent processes during heart regeneration, it is relevant to assess whether there is also reactivation of a proliferation gene signature (beyond gata4) and early cardiac transcriptional regulators such as Nkx2.5, Mef2 and Tbx5 in the neural crest-derived cardiomyocytes in response to injury.Thank you for this suggestion. We now include information about genes associated with cell proliferation as well as early cardiac transcription factors, including Nkx2.5, Gata4 and Tbx5 in our dataset. From our RNA-seq data, we do not see enrichment of these factors compared to injured, sox10-negative cells. While not enriched, these genes are expressed at low levels, perhaps because of the time point examined.To address the reviewer’s comment, we have included a heatmap of normalized counts in our supplemental data  to show enrichment of known regeneration genes in our 21dpa dataset. As suggested by the reviewer, the RNAseq data reveal expression of cell cycle and proliferation genes, consistent with an important role for proliferation of sox10+ cells. We now include data on enrichment of cell cycle genes in Figure 4—figure supplement 2.- Can the authors comment on why have they decided to do the transcriptomic analysis at 21 dpi instead of 7 dpi, which is the early onset of reactivation of the developmental neural crest program?We selected the 21 day time point for practical reasons, since we noted a significant number of Sox10-expressing cells in the heart at this time after regeneration as opposed to fewer cells at 7 days. We validated gene expression by in situ hybridization at both time points but focused the RNA-seq to the later time point. Moreover, at 21 days, we see the full progression of neural crest gene regulatory states from induction to migration that may have been missed at an earlier time point. However, the reviewer raises an excellent point and we will try to profile more time points for future experiments.- In order to rule out an epicardial origin ), it is also relevant to determine whether or not the neural crest-derived cardiomyocytes express bona fide epicardial markers such as Tbx18 or Tcf21.Thank you for this suggestion. To address this possibility, we have looked through our datasets for early cardiac transcription factors. The results show epicardial markers such as Tbx18 or Tcf21 are not enriched in our Sox10+ cells compared to Sox10- cells in the regenerating heart.- Given that the neonatal mammalian heart is also capable of regeneration after various injury models , assessing the reactivation of neural crest gene regulatory programs in mice would further support the most exciting conclusions of this study.We agree with the reviewer that it would be logical to extend this to neonatal mice. As this will require learning a new injury model, we would prefer to pursue this in a future publication, but have added this as a discussion point to be addressed in the future.Reviewer #2:[…] These new gene expression data add to our understanding of how the adult zebrafish heart responds to injury, and the authors are interested in drawing a connection between these observations and the contributions of cardiac neural crest-derived cells to regeneration. These data are also valuable to those seeking to understand the regulation of cardiac regeneration, but would benefit from some interpretative adjustments. Specifically:1) The induction of expression of sox10 and other neural crest-associated genes is very interesting, but it is inappropriate to interpret this as evidence that cardiac neural crest cells contribute to the regenerative capacity of the zebrafish heart without evidence that the cells activating expression of these genes are indeed neural crest-derived. The injury stimulus may induce expression of sox10 et al. in cardiomyocytes that were not originally neural crest-derived, and a different type of lineage tracing experiment would be required to determine whether neural crest-derived cardiomyocytes indeed respond to injury differently than lateral mesoderm-derived cardiomyocytes do. Alternatively, the authors could amend their interpretation – these changes in gene expression are certainly interesting whether or not the responding cells are necessarily neural crest-derived. In the revised manuscript, we raise the caveat that we cannot rule out the possibility that these cells are reactivating Sox10 and the neural crest program but may come from another lineage.In support of a neural crest origin, Abdul-Wajid, 2018 have also shown that ablation of the embryonic neural crest yields few or no sox10+ cells in the adult heart. This suggests there are no post-embryonic neural crest additions to the heart. This is now discussed.On a personal note, I find it very unlikely that the Sox10+ cells would come from another lineage. To my knowledge, there are no non-neural crest-derived Sox10-expressing cells in the periphery. There are a few Sox10+ non-neural crest-derived cells in the CNS, but they are confined to the brain. Therefore, I strongly suspect that these are indeed neural crest-derived. That said, I agree that this would need to be more rigorously validated and therefore we are more circumspect in the conclusions drawn in the manuscript.2) Along the same lines, the data shown here associate the induction of sox10 expression  with regeneration but do not directly demonstrate the significance of these genes to the regenerative response. Demonstrating their significance would surely require substantial additional experimentation; here, the authors should simply take caution to avoid implying that these particular genes are associated with regulation of regeneration.We agree that a much more detailed analysis would be necessary to directly demonstrate mechanism. We have amended the manuscript to make this point and be more circumspect.3) Could the authors comment on the relationship of the differential gene expression that they document in Figure 4H with other published datasets that examine the transcriptomic changes triggered by injury? Is it the case that the same neural crest-associated genes are differentially expressed in those datasets ?sox10+ enriched genes on RegenDbase.org , we find many of our enriched genes are present at 21dpa, but not statistically significant when compared to the 0dpa vs 21dpa dataset.By investigating the expression of our Reviewer #3:This is an interesting paper showing additional contributions of the neural crest to parts of the heart across vertebrates. Moreover, the authors present evidence that during regeneration of the zebrafish heart, neural crest-derived heart cells contribute to the regenerate and redeploy a neural crest gene expression programme. This suggests previously unappreciated heterogeneity in the cells contributing to regeneration.Overall, the findings are important, but the manuscript could be substantially improved by more explanations and quantifications of data.1) The manuscript has two parts and it is not entirely clear how these are connected. In the first part, the authors use lineage tracing to find new and known populations of crest cells that contribute to heart development. These cells seem to be rare in the apex "…"no neural crest-derived cardiomyocytes were observed in the apex of the heart"…"). However, in the second part of the manuscript they remove the apex for regeneration purposes. How are the two parts of the manuscript connected?Thank you for pointing out this issue. We have tried to integrate the two parts of the manuscript better. Basically, given that neural crest cells are a well-known stem cell population, the finding that the they can contribute to cardiomyocytes in amniotes similar to what had been previously shown in anamniotes made us wonder if they might contribute to heart regeneration. Zebrafish seemed like the most appropriate model for this due to their extensive capacity for heart regeneration.sox10 is downregulated in the heart as development proceeds . We confirm this by showing minimal sox10 expression in control adult hearts.To improve the connection between the two parts of the paper, we now more thoroughly reference and discuss several previously published papers that use the sox10 promoter for labeling cardiac neural crest cells . These studies show that in zebrafish, the neural crest contribution to the ventricles is primarily to the trabecular myocardium, similar to what we have found in chick and mouse. The study by Cavanaugh and colleagues also shows that sox10-derived cells are primarily in the proximal trabecular myocardium of the zebrafish heart suggests that these cells must be migrating into to the wound site after injury. We now discuss this interesting possibility more clearly.The finding that 2) The authors say that they have found novel crest-derived cell populations in the heart. At the same time, they state that previous research was controversial, and tools were inadequate due to high background. It is unclear whether previous papers already described these populations and this is here confirmed with more robust tools, or whether these populations are entirely new. Moreover, it would be good to at least discuss in more detail how they know that their tools are superior in their hands (e.g. "the improved Wnt1 line").Thank you for pointing out these issues. We have added further discussion of why the contribution to cardiomyocytes may have been missed in birds and previous hints from the literature of a neural crest contribution to the mammalian heart. In birds, this population was totally missed. There is no mention of a contribution of neural crest cells to cardiomyocytes in any of the quail/chick chimera papers. In mice, the lineage tracing was done with P0 mice which is not a neural crest specific marker, but rather labels Schwann cells and notochord cells, making these results inconclusive at the time. In fact, Fukada and colleagues recently published a paper  describing the differences between P0 and Wnt1 lineage labeling in mice and described Wnt1 as the “gold standard” for neural crest labeling.Wnt1-Cre2+: R26mTmG) is because the original line (Wnt1-Cre:ZsGreen), used in most publications, is known to have ectopic expression in some locations due to Wnt1 activation caused by an in-frame ATG located upstream of Wnt1 start codon, thus complicating the results. The Wnt1-Cre2+ corrects this issue and is therefore a more reliable reporter. We think that previous authors missed the neural crest contribution due to the myocardium in Wnt1 mice because the early experiments done with Wnt1-cre lineage tracing used colorometric lacZ as a readout. As there is high background from endogenous enzyme activity in the heart, it is likely that any labeling observed in this location was ignored as high background. Thus, with our tools, including retroviral lineage tracing in chick and fluorescent Wnt1-cre2+ mouse lines, we both confirmed all other previously described contributions of cardiac neural crest to the heart  as well as discovering a novel contribution to the myocardium. We have clarified this point in the paper and thank the reviewer for point out this confusion.In our study, we use two different Wnt1 lines to examine the contribution of neural crest cells to the murine heart. The reason for using the “improved” Wnt1 line (3) Many results rely on scoring for the presence or absence of cells. How were tissues scored? More quantifications of the relative contribution to the tissue in question should be shown for lineage tracing and zf heart regeneration, such that the relative importance of the crest contribution can be appreciated.We thank the reviewer for the suggestion. To address this, we quantitated the numbers of labeled neural crest cells we observe in various cardiac locations. As we do not label the entire neural crest population in chick, it’s most accurate to compare the% of labeled cells in each location (explained Supplementary file 1 legend). For the mouse, we label the entire neural crest population and we find that 16.8% of the labeled neural crest cells are in the proximal ventricles . Supplementary file 1A presents the quantified virally labelled cells in the chick embryo and E15.5 mouse and include the average percentage of neural crest contribution in the ventricular myocardium in mouse. We clarify that tissue were scored for numbers of GFP+ cells and clarify how counting was done in sections through chick, mouse and fish tissue in the methods. We have added approximate numbers of GFP+ cells to Supplementary file 1C in the zebrafish regeneration experiments as well.4) Figure 4—figure supplement 1: Were there also cells that expressed sox10 mRNA de novo? The prediction would be that only lineage-traced cells re-express sox10 – otherwise non-neural crest derived cells contribute to the population of sox10+ cells in regeneration. Can this be clearly observed in the combination of colorimetric ISH and transgenic fluorescence?sox10 during regeneration. However, not all mCherry+ cells express sox10, presumably because they lose expression as they differentiate into cardiomyocytes. We now include an updated figure that is clearer.Thanks for raising this. We now clarify that only cells that lineage trace with the sox10 marker reexpress 5) More information is needed on how the "the cardiac neural crest migratory gene regulatory module" was identified in the expression profile of regenerated heart cells in zf. e.g. did they use GO analysis? They highlight 20 out of 656 genes – what are the other genes? Why is 21 days post-injury the best time point?sox10+ dataset, focusing on transcription factors associated with modules of the neural crest gene regulatory network . We have also highlighted known zebrafish neural crest genes on our volcano plot . This gene list was acquired by a literature search as well as ZFIN. We now clarify this in the text.Thank you for raising this issue as we now realize that we were unclear. For comparison, we use data from a recently published paper in which we profiled migratory cardiac neural crest genes in chick . We then looked for similar gene signatures in our zebrafish