Hippo pathway-mediated YAP1/TAZ inhibition is essential for proper pancreatic endocrine specification and differentiation
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Abstract
The Hippo pathway plays a central role in tissue development and homeostasis. However, the function of Hippo in pancreatic endocrine development remains obscure. Here, we generated novel conditional genetically engineered mouse models to examine the roles of Hippo pathway-mediated YAP1/TAZ inhibition in the development stages of endocrine specification and differentiation. While YAP1 protein was localized to the nuclei in bipotent progenitor cells, Neurogenin 3 expressing endocrine progenitors completely lost YAP1 expression. Using genetically engineered mouse models, we found that inactivation of YAP1 requires both an intact Hippo pathway and Neurogenin 3 protein. Gene deletion of Lats1 and 2 kinases ( Lats1&2 ) in endocrine progenitor cells of developing mouse pancreas using Neurog3 Cre blocked endocrine progenitor cell differentiation and specification, resulting in reduced islets size and a disorganized pancreas at birth. Loss of Lats1&2 in Neurogenin 3 expressing cells activated YAP1/TAZ transcriptional activity and recruited macrophages to the developing pancreas. These defects were rescued by deletion of Yap1/Wwtr1 genes, suggesting that tight regulation of YAP1/TAZ by Hippo signaling is crucial for pancreatic endocrine specification. In contrast, deletion of Lats1&2 using β-cell-specific Ins1 CreER resulted in a phenotypically normal pancreas, indicating that Lats1&2 are indispensable for differentiation of endocrine progenitors but not for that of β-cells. Our results demonstrate that loss of YAP1/TAZ expression in the pancreatic endocrine compartment is not a passive consequence of endocrine specification. Rather, Hippo pathway-mediated inhibition of YAP1/TAZ in endocrine progenitors is a prerequisite for endocrine specification and differentiation.
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Reply to the reviewers
Reviewer #1 CROSS-CONSULTATION COMMENTS
All reviewers have similar doubts on quality and quantification of the IHC analyses, so I would agree to give a chance to the authors for a revision, and it is probably doable in between 3 and 6 months (I wrote >6 before).
Response: In our fist submission, our full PDF file did not use the high-resolution figures due to large file size of some images. For re-submission, we uploaded our original, high-resolution figures. We also re-performed several immunostaining experiments and took confocal images to increase the quality of our figures. Please see below for detailed response.
Reviewer #1 (Evidence, …
Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Reply to the reviewers
Reviewer #1 CROSS-CONSULTATION COMMENTS
All reviewers have similar doubts on quality and quantification of the IHC analyses, so I would agree to give a chance to the authors for a revision, and it is probably doable in between 3 and 6 months (I wrote >6 before).
Response: In our fist submission, our full PDF file did not use the high-resolution figures due to large file size of some images. For re-submission, we uploaded our original, high-resolution figures. We also re-performed several immunostaining experiments and took confocal images to increase the quality of our figures. Please see below for detailed response.
Reviewer #1 (Evidence, reproducibility, and clarity (Required)):
Summary:
In this study, Wu et al. confirmed the prominent cross talk of the Hippo pathway in beta-cell development, namely of Ngn3-YAP and beta-cell maturation. Deletion of the Hippo kinases Lats1 and 2 in Ngn3-expressing endocrine progenitor cells activated YAP1/TAZ transcriptional activity and reduced islets size and morphology and induced pancreas inflammation. This is in line with the previously established role of YAP/TAZ as regulator of viral response pathways In contrast, deletion of Lats1&2 later in development had no effect on beta-cell function and morphology. The authors conclude that Hippo pathway-mediated YAP1/TAZ inhibition in endocrine progenitors is a prerequisite for beta-cell maturation.While the effect of Lats1 and 2 in beta-cell development has never been investigated, the outcome of the study is largely confirmative; namely YAP's necessity to switch off whenever an endocrine cell is formed while it also balances inflammation.
The study depends on cell-specific Lats1 and 2-KO mouse models and in-vitro assessments of mechanisms how loss of LATS leads to beta-cell derangement at the time of ngn3 expression and how the inflammatory pathway is activated are missing. Observations are mostly based on stainings of quite low quality and it is unclear how authors performed quantitative evaluations and how many cells from how many mice were counted. Figures show high background, e.g. CD45 in Fig.3 which would make a robust evaluation impossible. Also YAP staining, usually well-expressed in ductal cells, shows low quality and high background. Usually the antibody is well-know for its weak performance in fluorescence and should only be used with chromogenic labels.
Response: ____ To show the co-localization of multiple proteins, immunofluorescence staining is necessary. ____We added and described, in detail, how we performed macrophage quantification in the Methods section.
Reviewer #2 (Evidence, reproducibility, and clarity (Required)):
Summary:
Wu et al. examined the roles of Hippo pathway mediated YAP1/TAZ inhibition in the development stages of endocrine specification and differentiation in vitro and in vivo. This study concluded Hippo pathway-mediated YAP1/TAZ inhibition in endocrine progenitors is a prerequisite for endocrine specification and differentiation. The present study is conducted by solid experiment in some parts, however, there are several major concerns as follows.
Major comments:
Authors concluded that proper Hippo activity was required for the Ngn3 driven differentiation program, further expanding our fundamental understanding of Hippo pathway participation in pancreatic endocrine development. This sentence is too vague. Previous study has already reported that Ngn3 expression and Yap loss occur in parallel within the same cell during development of the endocrine pancreas (Mol Endocrinol Baltim Md. 2015;29: 1594-607. doi:10.1210/me.2014-1375).
Response: ____ As reviewer pointed out, the previous study only reported that Ngn3 expression and YAP1 loss occur in parallel within the same cells, and NGN3 can turn off *Yap1 *transcription in cell culture setting. However, our study showed that NGN3 alone is not sufficient to turn off Yap1 gene in vivo. Using our genetic model, we revealed that the Hippo pathway controling the nuclear localization of YAP1 is essential at the initiation of endocrine differentiation and allows YAP1 to be turned off at the transcriptional level. ____Our rescue model (NLTY mice) confirmed that the endocrine development defects observed in NL pancreas are caused by YAP1/TAZ, suggesting the necessity of controlling YAP1/TAZ by the Hippo pathway during endocrine development. Failure to sequester YAP1/TAZ outside of nuclei by the Hippo pathway in Ngn3-expressing endocrine progenitors halts the development of these cells.
Reviewer #2:
In the present study, removal of YAP1/TAZ rescued the defect in endocrine specification and differentiation in LATS1/2-null pancreas. These results indicate that both of LATS1/2 and YAP/TAZ are not essential for the normal endocrine specification and differentiation. Furthermore, these results suggest that Hippo pathway-mediated YAP1/TAZ inhibition is also unnecessary for proper pancreatic endocrine specification and differentiation. Additionally, these results suggest that just a deletion of YAP/TAZ is sufficient for endocrine specification and differentiation. How is the endocrine specification and differentiation in Ngn3creyap1fl/flTazfl/fl mice?
__Response: ____ Our NL model (deletion of Lats1/2 in Ngn3-expressing cells) showed endocrine development defects, suggesting that Lats1/2 are essential for normal endocrine specification and differentiation. Removal of YAP1/TAZ in Lats1/2 null cells rescued the endocrine defects of NL pancreas. Genetic removal of YAP1/TAZ mimics Hippo pathway-mediated sequestration/degradation of YAP1/TAZ by LATS1/2. Uncontrolled YAP1/TAZ due to genetic removal of Lats1/2 blocked the endocrine specification and differentiation. This further demonstrates the necessity for functional Lats1/2 for inhibition of YAP1/TAZ in Ngn3-expressing cells during endocrine development. Ngn3CreYap1fl/flTazfl/fl mice have no endocrine defects, which is consistent with previous reports that Ngn3 expression and YAP1 loss occur in parallel within the same cells. Thus, we did not present this result. __
Reviewer #2:
In Figure 2B and 3B, YFP expression (an indicator for LATS1/2 deletion) is more detectable in control compared to LATS1/2 null mice, suggesting that the LATS1/2 expression is more decreased in control mice. Is this true?
Response:____ YFP expression shows Cre activity which can be used as an indicator for Lats1/2 deletion. However, the expression level of YFP does not correlate with the expression level of Lats1/2 because YFP is controlled by the Rosa26 gene promoter. We did observe a higher level of YFP expression in endocrine cells compared with non-endocrine cells in NL pancreas. It is possible that the Rosa26 promoter is more active in endocrine cells. However, this is beyond the scope of our research.
Reviewer #2:
Please show the expression of YFP, NGN3, and YAP/TAZ in figure 6A to confirm their expression status.
Response:____ In Figure 6, we intend to show that loss of YAP1/TAZ can rescue NL mice defects at P1 where Ngn3 expression is low. The NLTY pancreas showed much more endocrine cells compared to NL pancreas, suggesting that Lats1&2 functions to control YAP1/TAZ. Genetic ablation of YAP1/TAZ in Ngn3-expressing endocrine cells equals sequestering of YAP1/TAZ outside of nuclei by LATS1/2. Thus, we did not show expression of YFP, NGN3, and YAP1/TAZ in Figure 6A. Instead, we reperformed immunostaining for Figure 5B and took high-magnification confocal images to show expression pattern of NGN3 and YAP1 in endocrine progenitors.
Reviewer #2:
Please present the sequential changes of LATS1/2, YAP/TAZ, NGN3 expressions in the control mice and LATS1/2 null mice at least E12.5, E16.5, and P1, to make ii easy to understand them for general readers.
Response:____ At E12.5, the Hippo pathway plays important roles in pancreas development. See reference 11. Ngn3 peaks at E15.5 and only expresses in endocrine progenitors. We have published a review paper (ref. 7) “Wu Y, Aegerter P, Nipper M, Ramjit L, Liu J, Wang P. Hippo Signaling Pathway in Pancreas Development. Front Cell Dev Biol. 2021;9: 663906. doi:10.3389/fcell.2021.663906” as a detailed background for this manuscript, including pancreas development, up-to-date publication on Hippo pathway in pancreas development, and a model for YAP1 function in endocrine development. Thus, we did not include this background information in this manuscript.
Reviewer #2:
Minor comments: Please describe the affiliation of authors (number 3 and 4).
Response:____ It has been included in the manuscript: 3Department of Molecular Medicine, 4Department of Population Health Sciences.
Reviewer #2:
In table 1, the details of secondary antibodies were not described.
Response:____ We have added tables to show all primary and secondary antibodies used in the paper.
Reviewer #2 (Significance (Required)): Previous studies have already reported that Ngn3 expression and Yap loss occur in parallel manner during development of the endocrine pancreas. The primary aim of the present study is whether the YAP loss is mediated by LATS1/2 in Ngn3 positive cells.
Response:____ The primary aim of our study is to understand if the Hippo pathway plays an important function in endocrine pancreas development.
Reviewer #2:
Furthermore, although authors concluded that Hippo pathway (LATS1/2)-mediated YAP1/TAZ inhibition is essential for proper pancreatic endocrine specification and differentiation, the null condition both of LATS1/2 and YAP/TAZ in Ngn3 positive cells provided the normal endocrine specification and differentiation (Figure 6). These findings do not support the conclusion.
Response:____ The defects in our NL (Lats1/2 null) pancreas in endocrine development strongly suggest the essential role of Hippo pathway in endocrine development. The null conditions of both Lats1/2 and Yap1/Taz (NLTY mice) mimic the effects of LATS1/2-mediated control of YAP1/TAZ in endocrine progenitors via genetic ablation rather than through the canonical Hippo kinase cascade. This further demonstrates the importance of proper Hippo signaling during endocrine development to inhibit YAP1/TAZ via the Hippo kinase cascade in Ngn3-expressing endocrine progenitors.
Reviewer #3 (Evidence, reproducibility, and clarity (Required)):
Summary: In this study, Wu et al. use murine Cre-lox model systems to demonstrate that the core Hippo pathway components, Lats1/2, promote pancreatic endocrine specification and differentiation through Yap1/Taz. The roles of the Hippo pathway in mammals are complicated and context-dependent. Prior studies have implicated the core kinase cascade of the Hippo pathway, containing MST1/2, LATS1/2 and YAP1/TAZ, in pancreatic cell lineage differentiation and morphogenesis. However, the necessity of the Hippo pathway in the development of the endocrine pancreas in vivo remains unsettled. This study by Wu et al. demonstrates that the Hippo pathway is essential for endocrine progenitor specification and differentiation but not for pancreatic beta cell function in mice. Their results are in line with prior studies, but some major issues ensue largely due to the lack of data quantification to substantiate the authors' claims, the use of ambiguous/imprecise terminologies, and making unsubstantiated claims.
Major comments:
Immunofluorescence was used to evaluate the expression, co-localization, and subcellular localization of proteins of interest (Figures 2~7). However, except for the estimation of macrophage densities (4D and 6C), none of the other immunofluorescence experiments are accompanied by quantifications and statistical analyses to substantiate the authors' claims. In addition, the red channel in several figure panels (eg. 2A and 4A) was suboptimal making interpretations very difficult. Quantification of immunofluorescence data can be done by manual counting or automated counting using software such as ImageJ or QuPath, followed by statistical analyses to provide objective evidence.
Response:____ We did not upload high resolution images for Bioxriv publication. We now uploaded the high resolution images. We also repeated a few immunostaining experiments and have taken confocal images to increase clarity.
Reviewer #3:
The endocrine component constitutes only a portion of the pancreas. The authors use whole pancreases to compare the expression levels of cell type-specific or Yap1 target genes between the knockout and control mice through qPCR. While there may be technical feasibility reasons limiting the direct assessments of gene expression in the endocrine progenitor cells, the caveats of the experiments (eg. inferring cell type-specific gene expression changes from whole organ) should be highlighted along with any inconsistent results. For example, among the three ductal genes tested in Figure 1C, only Krt19 has significantly higher mRNA expression in NL vs control, but the possible reasons for such discrepancy between ductal markers were not discussed. The authors used immunofluorescence, which is only semi-quantitative, to determine that Yap1 protein abundance is increased in Lats1&2 knockout cells. qPCR should also be performed for Yap1 in addition to Yap1 target genes to augment their claim of Yap1 expression being increased. More details on how the relative mRNA expression was computed is also necessary for readers to accurately interpret the results.
Response:____ As reviewer pointed out that there are technical feasibility reasons limiting the direct assessments of gene expression in the endocrine progenitor cells. We can only quantify gene expression level through RT-qPCR. We have discussed the high level of Krt19 and unchanged level of Sox9 in Figure 3B and 3C when we performed immunostaining. The results from the two experiments are consistent. We observed KRT19 positive staining but no SOX9 staining in Lats1/2 null Ngn3-expressing cells. This result is consistent with RT-qPCR result. We have postulated in the Discussion section that Krt19 may be directly controlled by YAP1.
__Furthermore,* Lats1/2* controls the protein level and localization of YAP1/TAZ, thus immunostaining can show the cellular localization. __
In addition, we added the n’s used to perform RT-qPCR experiments to our manuscript and figures. We also added details to provide clarity on calculations made for relative mRNA expression from RT-qPCR experiments in the Methods section.
Reviewer #3:
The increased immunofluorescent detection of Yap1 in the NL pancreases at E16.5 is interesting but warrants further investigation. Is the increase restricted to endocrine progenitor cells or all endocrine compartments? George et al. (Mol Endocrinol. 2015) used RNA in situ hybridization and found that Yap1 mRNA expression is undetectable in the endocrine pancreas at E16.5, but here the authors observe increased Yap1 protein detected by immunofluorescence in the pancreases of animals with Lats1&2 knockout at E16.5. Although the authors speculate on a possible mechanism that may explain the discrepancies, it is important to evaluate whether the knockout really results in reactivation of Yap1 transcription and whether Yap1 auto-regulates on the transcriptional level.
__Response:____ The increase of YAP1 staining is restricted to endocrine progenitor cells and blocks the endocrine differentiation. The endocrine cells in NL pancreas are escapers of Lats1/2 deletion and have no YAP1 expression, which is consistent with George et al. findings. The model we proposed is that in Ngn3-expressing cells, Lats1/2 are required to keep YAP1/TAZ out of nuclei so that Ngn3 can repress YAP1 expression. Loss of Lats1/2 led to high nuclear YAP1 which may block Ngn3’s ability to suppress YAP1. ChIP-seq or ChIP-PCR on YAP1 promoter will answer the question whether YAP1 auto-regulates on the transcriptional level. However, the small number of cells in mouse pancreas limits us to perform this experiment. __
Reviewer #3:
The numbers of animals used are unclear except for those depicted by the barplots. There is also no mention of the number of cells or fields analyzed for immunofluorescence experiments, which is essential for any quantitative comparisons and claims. The n's should be added throughout.
Response:____ We added n’s to all figures and in our manuscript.
Reviewer #3:
The authors repeatedly refer to the dataset from Cebola et al. (Nat Cell Biol 2015), which was generated from human embryonic pancreatic progenitor cells, to speculate that TEAD1 binds to the promoter regions of YAP1, CDH1 and KRT19 and therefore may promote YAP1 autoregulation or expression of CDH1 and KRT19, explaining some of their immunofluorescence observations. However, they fail to acknowledge potential differences that may ensue due to the different species examined. Co-staining of Yap1 and Cdh1/Krt19 would indicate whether co-expression of Yap1 and Cdh1/Krt19 is indeed evident in the context of their study and provide further evidence to support their speculations.
__Response:____ Reviewer is correct that the dataset of TEAD1 ChIP-seq from Cebola et al. was generated from human embryonic pancreatic progenitor cells. These data are in line with our mouse experimental results where cells with high YAP1, due to loss of Lats1&2, continue to have high YAP1, CDH1 and KRT19. We do not have evidence to point out the potential differences between mouse and human. __
Reviewer #3:
Several observations are over-interpreted or over-stated, and should be qualified as preliminary or speculative with proper wording. For example, P14: "differentiation...was blocked by lack of expression of ISL1 and NKX2.2". Although the authors observe low Isl1 and Nkx2.2 staining in NL vs control pancreases, no experiments were done to substantiate the claim that the reduction in ISL1 and NKX2.2 directly block the differentiation in this context.
Response:____ We did not claim that the reduction in ISL1 and NKX2.2 directly block the differentiation in NL pancreas. ISL1 and NKX2.2 are markers for endocrine differentiation. The lack of ISL1 and NKX2.2 expression indicates that endocrine differentiation has been blocked.
Reviewer #3:
P15: "...KRT19 expression is not controlled by SOX9, but instead by YAP1". The authors observe that Krt19 proteins are increased in Lats1&2-null Ngn3+ cells, whereas Sox9 proteins were unchanged. However, they do not provide evidence that Yap1 controls Sox9 expression.
__Response:____ No Sox9 expression was observed in Ngn3-expressing cells in both Control and NL pancreases (Figure 3B and 3C) while we observed YAP1 nuclei staining in NGN3-positive cells (Figure 5B), suggesting that YAP1 does not control Sox9 expression.
__
Reviewer #3:
Minor comments:
It is mentioned that deletion of Lats1&2 in Ngn3+ cells results in fewer acinar cells and smaller islets, evident by reduction in Ins+ or Gcg+ cells, whereas such genetic ablation in pancreatic beta cells does not result in any phenotype. Did the deletion of Lats1&2 in Ngn3+ cells similarly lead to reduction in other endocrine cell types?
__Response:____ We showed that there was no positive staining for ISL1 and NKX2.2 in progeny of Lats1&2 null Ngn3-expressing cells, suggesting the block of endocrine differentiation including all endocrine cells. The INS+ and GCG+ cells in NL pancreas are the escapers in which Lats1&2 were not deleted. Other endocrine cells should be affected too. We observed smaller sized NL mice with low blood glucose levels. We have postulated that brain expression of Ngn3Cre may contribute to these phenotypes. __
Reviewer #3:
The authors use the word "expression" without specification to refer to both mRNA expression and marker fluorescence levels throughout the text. This is inaccurate and potentially confusing. More specific terminologies should instead be used to avoid ambiguity.
__Response:____ We have added mRNA in the appropriate places where we discuss results from RT-qPCR experiments. All other places are protein expression results by immunostaining. We followed the general guideline for formatting gene and protein name throughout the manuscript: mouse gene symbols are italicized, with only the first letter in upper-case; protein symbols are not italicized, and all letters are in upper-case. __
Reviewer #3:
Figure S1D is mis-referred to as S1E in the text. Figure 7G is missing. Table S1 is mis-referred to as S Table 2 in the text. Typo on page 19: "Controlcontrol" Figure S6B is mis-referred to as Figure S6A in the text.
Response:____ We have made all appropriate changes to the manuscript to correct these mistakes.
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Referee #3
Evidence, reproducibility and clarity
Summary
In this study, Wu et al. use murine Cre-lox model systems to demonstrate that the core Hippo pathway components, Lats1/2, promote pancreatic endocrine specification and differentiation through Yap1/Taz. The roles of the Hippo pathway in mammals are complicated and context-dependent. Prior studies have implicated the core kinase cascade of the Hippo pathway, containing MST1/2, LATS1/2 and YAP1/TAZ, in pancreatic cell lineage differentiation and morphogenesis. However, the necessity of the Hippo pathway in the development of the endocrine pancreas in vivo remains unsettled. This study by Wu et al. demonstrates that the Hippo …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #3
Evidence, reproducibility and clarity
Summary
In this study, Wu et al. use murine Cre-lox model systems to demonstrate that the core Hippo pathway components, Lats1/2, promote pancreatic endocrine specification and differentiation through Yap1/Taz. The roles of the Hippo pathway in mammals are complicated and context-dependent. Prior studies have implicated the core kinase cascade of the Hippo pathway, containing MST1/2, LATS1/2 and YAP1/TAZ, in pancreatic cell lineage differentiation and morphogenesis. However, the necessity of the Hippo pathway in the development of the endocrine pancreas in vivo remains unsettled. This study by Wu et al. demonstrates that the Hippo pathway is essential for endocrine progenitor specification and differentiation but not for pancreatic beta cell function in mice. Their results are in line with prior studies, but some major issues ensue largely due to the lack of data quantification to substantiate the authors' claims, the use of ambiguous/imprecise terminologies, and making unsubstantiated claims.
Major comments:
- Immunofluorescence was used to evaluate the expression, co-localization, and subcellular localization of proteins of interest (Figures 2~7). However, except for the estimation of macrophage densities (4D and 6C), none of the other immunofluorescence experiments are accompanied by quantifications and statistical analyses to substantiate the authors' claims. In addition, the red channel in several figure panels (eg. 2A and 4A) was suboptimal making interpretations very difficult. Quantification of immunofluorescence data can be done by manual counting or automated counting using software such as ImageJ or QuPath, followed by statistical analyses to provide objective evidence.
- The endocrine component constitutes only a portion of the pancreas. The authors use whole pancreases to compare the expression levels of cell type-specific or Yap1 target genes between the knockout and control mice through qPCR. While there may be technical feasibility reasons limiting the direct assessments of gene expression in the endocrine progenitor cells, the caveats of the experiments (eg. inferring cell type-specific gene expression changes from whole organ) should be highlighted along with any inconsistent results. For example, among the three ductal genes tested in Figure 1C, only Krt19 has significantly higher mRNA expression in NL vs control, but the possible reasons for such discrepancy between ductal markers were not discussed. The authors used immunofluorescence, which is only semi-quantitative, to determine that Yap1 protein abundance is increased in Lats1&2 knockout cells. qPCR should also be performed for Yap1 in addition to Yap1 target genes to augment their claim of Yap1 expression being increased. More details on how the relative mRNA expression was computed is also necessary for readers to accurately interpret the results.
- The increased immunofluorescent detection of Yap1 in the NL pancreases at E16.5 is interesting but warrants further investigation. Is the increase restricted to endocrine progenitor cells or all endocrine compartments? George et al. (Mol Endocrinol. 2015) used RNA in situ hybridization and found that Yap1 mRNA expression is undetectable in the endocrine pancreas at E16.5, but here the authors observe increased Yap1 protein detected by immunofluorescence in the pancreases of animals with Lats1&2 knockout at E16.5. Although the authors speculate on a possible mechanism that may explain the discrepancies, it is important to evaluate whether the knockout really results in reactivation of Yap1 transcription and whether Yap1 auto-regulates on the transcriptional level.
- The numbers of animals used are unclear except for those depicted by the barplots. There is also no mention of the number of cells or fields analyzed for immunofluorescence experiments, which is essential for any quantitative comparisons and claims. The n's should be added throughout.
- The authors repeatedly refer to the dataset from Cebola et al. (Nat Cell Biol 2015), which was generated from human embryonic pancreatic progenitor cells, to speculate that TEAD1 binds to the promoter regions of YAP1, CDH1 and KRT19 and therefore may promote YAP1 autoregulation or expression of CDH1 and KRT19, explaining some of their immunofluorescence observations. However, they fail to acknowledge potential differences that may ensue due to the different species examined. Co-staining of Yap1 and Cdh1/Krt19 would indicate whether co-expression of Yap1 and Cdh1/Krt19 is indeed evident in the context of their study and provide further evidence to support their speculations.
- Several observations are over-interpreted or over-stated, and should be qualified as preliminary or speculative with proper wording. For example, a. P14: "differentiation...was blocked by lack of expression of ISL1 and NKX2.2". Although the authors observe low Isl1 and Nkx2.2 staining in NL vs control pancreases, no experiments were done to substantiate the claim that the reduction in ISL1 and NKX2.2 directly block the differentiation in this context. b. P15: "...KRT19 expression is not controlled by SOX9, but instead by YAP1". The authors observe that Krt19 proteins are increased in Lats1&2-null Ngn3+ cells, whereas Sox9 proteins were unchanged. However, they do not provide evidence that Yap1 controls Sox9 expression.
Minor comments:
- It is mentioned that deletion of Lats1&2 in Ngn3+ cells results in fewer acinar cells and smaller islets, evident by reduction in Ins+ or Gcg+ cells, whereas such genetic ablation in pancreatic beta cells does not result in any phenotype. Did the deletion of Lats1&2 in Ngn3+ cells similarly lead to reduction in other endocrine cell types?
- The authors use the word "expression" without specification to refer to both mRNA expression and marker fluorescence levels throughout the text. This is inaccurate and potentially confusing. More specific terminologies should instead be used to avoid ambiguity.
- Figure S1D is mis-referred to as S1E in the text.
- Figure 7G is missing.
- Table S1 is mis-referred to as S Table 2 in the text.
- Typo on page 19: "Controlcontrol"
- Figure S6B is mis-referred to as Figure S6A in the text.
Significance
The exact roles of the Hippo pathway in pancreas organogenesis are far from being delineated. The current study proposes a new model wherein the Hippo pathway kinase cascade acts to sequester and inhibit Yap1 in the cytosol for Ngn3 to drive endocrine specification, including inhibition of Yap1 expression. This work is potentially valuable to further our understanding of the physiological roles of the Hippo pathway in the context of endocrine pancreas development.
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Referee #2
Evidence, reproducibility and clarity
Summary:
Wu et al. examined the roles of Hippo pathway mediated YAP1/TAZ inhibition in the development stages of endocrine specification and differentiation in vitro and in vivo. This study concluded Hippo pathway-mediated YAP1/TAZ inhibition in endocrine progenitors is a prerequisite for endocrine specification and differentiation. The present study is conducted by solid experiment in some parts, however, there are several major concerns as follows.
Major comments:
- Authors concluded that proper Hippo activity was required for the Ngn3 driven differentiation program, further expanding our fundamental understanding of Hippo pathway …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #2
Evidence, reproducibility and clarity
Summary:
Wu et al. examined the roles of Hippo pathway mediated YAP1/TAZ inhibition in the development stages of endocrine specification and differentiation in vitro and in vivo. This study concluded Hippo pathway-mediated YAP1/TAZ inhibition in endocrine progenitors is a prerequisite for endocrine specification and differentiation. The present study is conducted by solid experiment in some parts, however, there are several major concerns as follows.
Major comments:
- Authors concluded that proper Hippo activity was required for the Ngn3 driven differentiation program, further expanding our fundamental understanding of Hippo pathway participation in pancreatic endocrine development. This sentence is too vague. Previous study has already reported that Ngn3 expression and Yap loss occur in parallel within the same cell during development of the endocrine pancreas (Mol Endocrinol Baltim Md. 2015;29: 1594-607. doi:10.1210/me.2014-1375).
- In the present study, removal of YAP1/TAZ rescued the defect in endocrine specification and differentiation in LATS1/2-null pancreas. These results indicate that both of LATS1/2 and YAP/TAZ are not essential for the normal endocrine specification and differentiation. Furthermore, these results suggest that Hippo pathway-mediated YAP1/TAZ inhibition is also unnecessary for proper pancreatic endocrine specification and differentiation. Additionally, these results suggest that just a deletion of YAP/TAZ is sufficient for endocrine specification and differentiation. How is the endocrine specification and differentiation in Ngn3creyap1fl/flTazfl/fl mice?
- In Figure 2B and 3B, YFP expression (an indicator for LATS1/2 deletion) is more detectable in control compared to LATS1/2 null mice, suggesting that the LATS1/2 expression is more decreased in control mice. Is this true?
- Please show the expression of YFP, NGN3, and YAP/TAZ in figure 6A to confirm their expression status.
- Please present the sequential changes of LATS1/2, YAP/TAZ, NGN3 expressions in the control mice and LATS1/2 null mice at least E12.5, E16.5, and P1, to make ii easy to understand them for general readers.
Minor comments:
Please describe the affiliation of authors (number 3 and 4).
In table 1, the details of secondary antibodies were not described.
Significance
Previous studies have already reported that Ngn3 expression and Yap loss occur in parallel manner during development of the endocrine pancreas. The primary aim of the present study is whether the YAP loss is mediated by LATS1/2 in Ngn3 positive cells.
Furthermore, although authors concluded that Hippo pathway (LATS1/2)-mediated YAP1/TAZ inhibition is essential for proper pancreatic endocrine specification and differentiation, the null condition both of LATS1/2 and YAP/TAZ in Ngn3 positive cells provided the normal endocrine specification and differentiation (Figure 6). These findings do not support the conclusion.
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Referee #1
Evidence, reproducibility and clarity
In this study, Wu et al. confirmed the prominent cross talk of the Hippo pathway in beta-cell development, namely of Ngn3-YAP and beta-cell maturation. Deletion of the Hippo kinases Lats1 and 2 in Ngn3-expressing endocrine progenitor cells activated YAP1/TAZ transcriptional activity and reduced islets size and morphology and induced pancreas inflammation. This is in line with the previously established role of YAP/TAZ as regulator of viral response pathways In contrast, deletion of Lats1&2 later in development had no effect on beta-cell function and morphology. The authors conclude that Hippo pathway-mediated YAP1/TAZ inhibition in …
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Referee #1
Evidence, reproducibility and clarity
In this study, Wu et al. confirmed the prominent cross talk of the Hippo pathway in beta-cell development, namely of Ngn3-YAP and beta-cell maturation. Deletion of the Hippo kinases Lats1 and 2 in Ngn3-expressing endocrine progenitor cells activated YAP1/TAZ transcriptional activity and reduced islets size and morphology and induced pancreas inflammation. This is in line with the previously established role of YAP/TAZ as regulator of viral response pathways In contrast, deletion of Lats1&2 later in development had no effect on beta-cell function and morphology. The authors conclude that Hippo pathway-mediated YAP1/TAZ inhibition in endocrine progenitors is a prerequisite for beta-cell maturation.
While the effect of Lats1 and 2 in beta-cell development has never been investigated, the outcome of the study is largely confirmative; namely YAP's necessity to switch off whenever an endocrine cell is formed while it also balances inflammation. The study depends on cell-specific Lats1 and 2-KO mouse models and in-vitro assessments of mechanisms how loss of LATS leads to beta-cell derangement at the time of ngn3 expression and how the inflammatory pathway is activated are missing. Observations are mostly based on stainings of quite low quality and it is unclear how authors performed quantitative evaluations and how many cells from how many mice were counted. Figures show high background, e.g. CD45 in Fig.3 which would make a robust evaluation impossible. Also YAP staining, usually well-expressed in ductal cells, shows low quality and high background. Usually the antibody is well-know for its weak performance in fluorescence and should only be used with chromogenic labels.
Referees cross-commenting
All reviewers have similar doubts on quality and quantification of the IHC analyses, so I would agree to give a chance to the authors for a revision, and it is probably doable in between 3 and 6 months (I wrote >6 before).
Significance
Largely confirmative. Single model approach with relatively low quality of the IHC analyses.
My expertise: inflammation, beta-cell, islets, Hippo pathway
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