TGF-β signaling and Creb5 cooperatively regulate Fgf18 to control pharyngeal muscle development

Article activity feed

1. 

   Version published to 10.7554/elife.80405 on eLife

   Dec 21, 2022
2. 

   eLife

   Nov 14, 2022

   Reviewer #1 (Public Review):

   In this manuscript by Feng et al., the authors investigate the mechanism regulating the development of the levator veli palatini (LVP) in the posterior palate/pharyngeal region. While set up as a model to understand how myogenic progenitors migrate to discrete sites to form individual muscles, it is not clear how applicable the findings are to other subpopulations, though this is not a weakness. The mechanisms driving LVP development are of great interest to a broad group of developmental biologists, as LVP malformation is a common problem even in mild cases of cleft palate. The authors hypothesized that the perimysial population within palatal mesenchyme cells is a niche required for pharyngeal muscle development. Using exquisite analysis of scRNA-seq data from E13.5-E15.5 palatal cells, the authors …

   Reviewer #1 (Public Review):

   In this manuscript by Feng et al., the authors investigate the mechanism regulating the development of the levator veli palatini (LVP) in the posterior palate/pharyngeal region. While set up as a model to understand how myogenic progenitors migrate to discrete sites to form individual muscles, it is not clear how applicable the findings are to other subpopulations, though this is not a weakness. The mechanisms driving LVP development are of great interest to a broad group of developmental biologists, as LVP malformation is a common problem even in mild cases of cleft palate. The authors hypothesized that the perimysial population within palatal mesenchyme cells is a niche required for pharyngeal muscle development. Using exquisite analysis of scRNA-seq data from E13.5-E15.5 palatal cells, the authors illustrate that TGFb signaling is likely involved in perimysial cell development, using gene expression analysis in wild-type palatal sections to show that TGFb signaling precedes the arrival of myogenic cells. Inactivating ALk5 in palatal mesenchyme cells results in failure of LVP formation. The authors continue by identifying a number of transcription factors that presumably function downstream of TGFb signaling that drive LVP development. Among these are Fgf18, in which SMAD sites observed in the upstream region were validated to bind Smad2/3. The authors also identify Creb5 as a potential regulator of Fgf18. Overall, this is a remarkable use of scRNA-seq data, in which findings are supported by subsequent in vivo analysis of gene function using knockout mouse models. These findings will drive further analysis of LVP development and may shed light on the myogenesis of pharyngeal muscle in general.

   Strengths

   1. The treatment of scRNA-seq data using a variety of bioinformatic programs illustrates the utility of this type of data when using sufficient analysis software. The description of the approach is very clear and concise and the controls appear excellent. Further, the use of multiple time points further improves the analysis.

   1. The focus of perimysial cell expression patterns supports the hypothesis of the authors, though as with this type of data, one probably can make a story out of several pathways. The use of RNAscope to carefully examine where TGFb signaling in the posterior pharynx occurs between E12.5 and E16.5 is critical to the setup of this manuscript and is well done. Further aiding the interpretation of these results are cartoons associated with the staining, which illustrate where the staining is occurring, though never over-stating the observed patterns.

   1. Careful histological analysis illustrates the poor myogenic differentiation in the LVP of OSr1-Cre;Alk5fl/fl embryos.

   1. Identifying that TGFb is more important for regulating late perimysial cell development is important in identifying the targets of TGFb signaling.

   1. The use of CellChat to identify sending and receiving cells is well done and further supports the late function of TGFb signaling, in this context working through Fgf18 and Lama4.

   1. The attempt to build a signaling network again using CellChat (Figure 6) is admirable, though there are a few caveats to that approach (see below).

   1. While bead implant studies have been used for 40 years, the approach of culturing a piece of the pharynx and then performing a bead implant to prove that Fgf18 can positively influence myogenic development is admirable.

   Weaknesses

   1. In general, the authors are careful to not suggest that staining is significant unless showing quantification, though, at several points, this is not true.

   1. The authors identify five putative Smad2 sites upstream of Fgf18, using one of them in a Cut and Run assay whose results suggest enhanced Smad2/3 binding. The problem is that this likely would have worked with the other Smad sites and probably would have worked for any other putative site that one might pick. Proving that a putative site can be bound by its cognate transcription factor is not the same as proving that this occurs in vivo and is sufficient to control the process of LVP development. One would need reporter assays using that TF binding site to better support the points being made by the authors.

   1. In a similar manner, the authors try to define which factors might function with TGFb signaling to regulate myogenic development. Using SCENIC, the authors found a number of genes that might be involved in perimysial fibroblast development. Of these, they illustrate that Creb5 siRNA knockdown decreases Fgf18 expression in cultured palates. The focus on Creb5 was based on it showing, "the most specific expression patterning the late perimysial cells (Figure 6H)....". In fact, Creb5 appears the most broad, appearing to be expressed across the entire LVP, not just in the area where myogenic precursors are found. Thus, any statement or discussion about Creb5 being a direct regulator of Fgf18 should be removed probably needs to be reworded. However, the second problem is that Creb5 knockdown reducing Fgf18 expression does not prove any direct regulation. Both of these are rather circuitous arguments.

   1. While the disorganization of myogenic fibers in the posterior LVP is somewhat obvious, it is not as clear as the authors suggest. This change (which I believe) needs to be better quantified (length, width, area, etc.).

   We thank the reviewer for these “Public Review” comments. For point 1, we have added more quantification for clarification and rephased the wording when quantification was not performed. For point 4, we added measurement to quantify the changes of volume and cross-section area of the LVP in Osr2Cre;Fgf18fl/fl mice (Figure 7M-V).

   Reviewer #2 (Public Review):

   In this study, the authors take advantage of unbiased scRNA-seq datasets of the developing mouse soft palate that they previously reported and performed a new bioinformatic analysis to identify differential signaling pathway activities in the heterogeneous palatal mesenchyme. They found a strong association of TGF-beta signaling pathway activity with the perimysial cells and validated through immunofluorescent detection of pSmad2, which led to their hypothesis that TGF-beta signaling in the perimysial cells might regulate palatal muscle formation. They generated and analyzed Osr2-Cre;Alk5fl/fl mice and showed those mice have cleft soft palate and disruption of the levator veli palataini (LVP) muscle. They then performed a comparative scRNA-seq analysis of the soft palate tissues from E14.5 Osr2-Cre;Alk5fl/fl and control embryos and showed that the Osr2-Cre;Alk5fl/fl embryos exhibited defects in the perimysial cells, in particular reduction in Tbx15+ perimysial fibroblasts that directly associate with the LVP muscle progenitors. The FGF18 is one of the most highly enriched signaling molecules in the perimysial cells and showed that the Osr2-Cre;Alk5fl/fl embryos exhibited reduced Fgf18 expression together with loss of MyoD+ myoblasts in the prospective LVP region. Further data showed that pSmad2 bound in the Fgf18 promoter region in the developing soft palate tissues. In addition, bioinformatic gene regulatory network analysis of the scRNA-seq data identified Creb5 as a potential tissue-specific transcription factor in the perimysial cells and RNAi knockdown assays in palatal mesenchyme culture suggested that Creb5 is required for Fgf18 expression. Further studies identified a subtle deficiency in LVP in Osr2-Cre;Fgf18fl/fl mice and showed that exogenous Fgf18 bead implantation in explants of E14 Osr2-Cre;Alk5fl/fl embryonic head increased the MyoD+ myoblast population in the prospective LVP region. The authors concluded that TGF-beta signaling and Creb5 cooperatively regulate Fgf18 to control pharyngeal muscle development. While the study used multiple complementary approaches and the data presented are solid, important questions need to be addressed to resolve reasonable alternative explanations of the data to the authors' main conclusion.

   We thank the reviewer for the evaluation and suggestions. Responses to each of the suggested revisions are detailed below.

   Major points:

   1. TGF-beta signaling is known to be crucial for neural crest-derived palatal mesenchyme cell proliferation from E13.5 to E14.5. The Osr2-Cre;Alk5fl/fl mutant embryos exhibited obvious disruption of LVP myogenesis and reduced soft palatal shelf size at E14.5 (Fig3-Sup2A-D and Fig 4H-K). The cellular and molecular defects likely started prior to E14.5. Thus, it is important to examine at earlier stages (E13.5/E14.0) whether the palatal mesenchyme was already defective in cell proliferation/survival and/or perimysial cell marker expression, including Creb5 and Tbx15, to resolve whether the primary defect in the Osr2-Cre;Alk5fl/fl palatal mesenchyme could be a reduction in perimysial progenitor cell proliferation and/or differentiation of the myoblast-associated subset, for which Tbx15 and Fgf18hi act as marker genes rather than direct molecular targets. Furthermore, the apparent loss of Tbx15+ cells coincided with a specific reduction of Fgf18 expression in the myoblast-associated perimysial cells (Fig 4J/K versus Fig 5H-K), which raises the possibility that TGF-beta signaling regulates the differentiation of the Tbx5+ population from the mesenchymal progenitors while the reduction in Fgf18 expression might be a secondary consequence of the cellular defect. The data in Fig 6O showing a lack of significant induction of Fgf18 expression in the palatal mesenchyme culture in both control and Creb5-RNAi cells is also consistent with this alternative explanation.

   We thank the reviewer for the valuable suggestion to identify the primary defects of the perimysial cells. We compared the expression of Creb5, Tbx15 and Fgf18 as well as Smoc2 in E13.5-E14.5 palatal mesenchyme from control and Osr2-Cre;Alk5fl/fl mice (Osr2Cre;Tgfbr1fl/fl mice). We found that expression of Creb5 is prominent from E13.5 to E14.5 and is not affected in Osr2Cre;Tgfbr1fl/fl mice, suggesting that Creb5 may not be a downstream factor but just a “partner” for TGF-β signaling. At E13.5, Tbx15 is not expressed, while Smoc2 is expressed extensively in the palatal mesenchyme but is not affected in the Osr2Cre;Tgfbr1fl/fl mice. In contrast, Fgf18 is expressed as early as E13.5 and this expression was already reduced in the palatal of Osr2Cre;Tgfbr1fl/fl mice relative to controls at this stage, suggesting the changes of Fgf18 expression are primary and precede changes in the perimysial populations. While the proliferation and apoptosis at E13.5 remain unchanged in Osr2Cre;Tgfbr1fl/fl mice, Smoc2 expression in the palate starts to be reduced at E14.0 in Osr2Cre;Tgfbr1fl/fl mice. This suggests that TGF-β signaling is required for the activation of Smoc2 during E13.5-E14.0. In parallel, Tbx15 expression is just starting to be activated in a few cells at E14.0 and this expression increased between E14.0-E14.5 in the control but failed to increase in Osr2Cre;Tgfbr1fl/fl mice. This suggests that TGF-β signaling is also required for the activation of Tbx15 during E14.0-E14.5. Thus, loss of TGF-β signaling leads to differentiation defects of both Smoc2+ and Tbx15+ perimysial cells. For Figure 6O, we performed a time-course experiment of TGF-β induction and found a significant increase of Fgf18 expression after 4 to 18 hours of treatment (instead of 24 hours used in previous experiments), with more obvious changes at 4 hours, confirming the early response of Fgf18 expression to TGF-β induction. These results have been added to Figure 4-figure supplement 2, Figure 5I-L, 5U, Figure 6-figure supplement 2, and Figure 6C.

   1. Since the Osr2-Cre;Fgf18fl/fl mice exhibited much subtler palatal and LVP defects than the Osr2-Cre;Alk5fl/fl mice even though the latter still had a lot of Fgf18-expressing perimysial cells at E14.5, Fgf18 is likely a minor player in the TGF-beta mediated gene regulatory network regulating LVP formation. The major players acting downstream of TGF-beta signaling in the palatal mesenchyme, that control initial LVP progenitor migration to and/or proliferation in the soft palate region, remain to be identified and functionally validated. Whether and how Fgf18 directly regulates the perimysial-myoblast interaction is also not known.

   We agree with the reviewer that the phenotype of Osr2-Cre;Fgf18fl/fl mice is much milder than that of Osr2-Cre;Alk5fl/fl mice, as we postulate that Fgf18 is just one of several perimysial-derived signals that may be affected. It will be of great interest to explore the function of other players in future studies. However, we are more inclined toward the possibility that there may be no single “major” player but rather a combination of many signals associated with different aspects of the muscle development. For example, loss of Fgf18 seems to mainly affect the Myf5+ cell proliferation in Osr2-Cre;Fgf18fl/fl mice (Osr2Cre;Fgf18fl/fl mice), as we do not observe any differentiation defect except the reduced muscle size. It is likely that other factors may also play specific functions in specific subpopulations as well. To clarify whether Fgf18 can directly affect the myogenic cell fate, we treated C2C12 mouse myogenic cells with exogenous FGF18 and found that this treatment could indeed significantly increase the proliferation of these cells. We have added these results to Figure 7—figure supplement 2.

   1. While the title and the main conclusion of this manuscript imply a crucial role of Creb5 in the regulation of pharyngeal muscle development, there is no data supporting such a crucial role. Do Creb5-/- mice have specific defects in pharyngeal muscle development?

   We thank the reviewer for this insight. We agree that it is very likely that Creb5 itself may have many roles in the regulation of palatal development or pharyngeal muscle development, given the prominent expression of Creb5 throughout soft palate development and in other myogenic sites of the pharyngeal muscles. Creb5-/- mice (reported as Cre-bpa-/- mice) die immediately after birth; however, the detailed phenotype of this mice was merely described as “data not shown” in a previous publication and defects of craniofacial development in these mice remain unclear (Maekawa et al., 2010). In this study, we focused on the role of Creb5 as a partner of TGF-β signaling, but we plan to generate a Creb5fl/fl mouse model to thoroughly evaluate Creb5’s functions in craniofacial development as an independent study following this work.

   1. Data in Fig 6 are not sufficient to conclude that TGF-beta signaling and Creb5 cooperatively regulate Fgf18. The TGFb1 treatment did not significantly induce Fgf18 expression in either the control or Creb5-RNAi palate mesenchyme cells (Fig 6O). No data regarding how they act cooperatively to regulate Fgf18 expression.

   We appreciate the reviewer for carefully reviewing our data. We re-evaluated the temporal response of Fgf18 expression following TGF- induction and found a significant increase of Fgf18 expression 4 hours post-treatment (instead of 24 hours post-treatment as used in previous experiments). We repeated the Creb5-siRNA treatment experiment using the new experimental condition and replaced the previous Figure 6O with new results showing a significant increase of Fgf18 after TGF-β induction, which was attenuated by Creb5-RNAi treatment, suggesting a requirement of Creb5 for TGF-β-mediated Fgf18 expression. The new result is now included in Figure 6Q.

   Reviewer #3 (Public Review):

   In this study, the authors investigated cell-cell communication between perimysial cells and skeletal muscle progenitors during soft palate development in the mouse. The authors have previously reported on the development of this structure and here they propose that a TGF-β signaling and Creb5 act to regulate Fgf18, and this pathway regulates pharyngeal muscle development through the indicated cell populations. The study is of high quality, very nicely illustrated, and uses multiple approaches including inferences from single cell transcriptomics, validations on sections, and lineage-specific gene activations. In addition, the authors successfully optimized an organ culture system from thick sections to test locally the role of FGF signaling (bead implantation). The results largely confer with the conclusions and provide a valuable example of how subjacent cell populations cooperate to establish an embryonic structure.

   We thank the reviewer for the evaluation and suggestions.

   Read the original source
3. 

   eLife

   Jul 14, 2022

   Evaluation Summary:

   The authors bioinformatically analyze previous scRNA-seq datasets of the developing mouse soft palate to identify differential signaling pathway activities in the heterogeneous palatal mesenchyme. Identifying TGF-beta signaling pathway activity with the perimysial cells, they hypothesize and test whether TGF-beta signaling in the perimysial cells might regulate palatal muscle formation. This paper will be of high interest to developmental biologists interested in the molecular regulation of tissue interactions that occur during mammalian palate morphogenesis.

   (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

   Read the original source
4. 

   eLife

   Jul 14, 2022

   Reviewer #1 (Public Review):

   In this manuscript by Feng et al., the authors investigate the mechanism regulating the development of the levator veli palatini (LVP) in the posterior palate/pharyngeal region. While set up as a model to understand how myogenic progenitors migrate to discrete sites to form individual muscles, it is not clear how applicable the findings are to other subpopulations, though this is not a weakness. The mechanisms driving LVP development are of great interest to a broad group of developmental biologists, as LVP malformation is a common problem even in mild cases of cleft palate. The authors hypothesized that the perimysial population within palatal mesenchyme cells is a niche required for pharyngeal muscle development. Using exquisite analysis of scRNA-seq data from E13.5-E15.5 palatal cells, the authors …

   Reviewer #1 (Public Review):

   In this manuscript by Feng et al., the authors investigate the mechanism regulating the development of the levator veli palatini (LVP) in the posterior palate/pharyngeal region. While set up as a model to understand how myogenic progenitors migrate to discrete sites to form individual muscles, it is not clear how applicable the findings are to other subpopulations, though this is not a weakness. The mechanisms driving LVP development are of great interest to a broad group of developmental biologists, as LVP malformation is a common problem even in mild cases of cleft palate. The authors hypothesized that the perimysial population within palatal mesenchyme cells is a niche required for pharyngeal muscle development. Using exquisite analysis of scRNA-seq data from E13.5-E15.5 palatal cells, the authors illustrate that TGFb signaling is likely involved in perimysial cell development, using gene expression analysis in wild-type palatal sections to show that TGFb signaling precedes the arrival of myogenic cells. Inactivating ALk5 in palatal mesenchyme cells results in failure of LVP formation. The authors continue by identifying a number of transcription factors that presumably function downstream of TGFb signaling that drive LVP development. Among these are Fgf18, in which SMAD sites observed in the upstream region were validated to bind Smad2/3. The authors also identify Creb5 as a potential regulator of Fgf18. Overall, this is a remarkable use of scRNA-seq data, in which findings are supported by subsequent in vivo analysis of gene function using knockout mouse models. These findings will drive further analysis of LVP development and may shed light on the myogenesis of pharyngeal muscle in general.

   Strengths
   1. The treatment of scRNA-seq data using a variety of bioinformatic programs illustrates the utility of this type of data when using sufficient analysis software. The description of the approach is very clear and concise and the controls appear excellent. Further, the use of multiple time points further improves the analysis.
   2. The focus of perimysial cell expression patterns supports the hypothesis of the authors, though as with this type of data, one probably can make a story out of several pathways.
   2. The use of RNAscope to carefully examine where TGFb signaling in the posterior pharynx occurs between E12.5 and E16.5 is critical to the setup of this manuscript and is well done. Further aiding the interpretation of these results are cartoons associated with the staining, which illustrate where the staining is occurring, though never over-stating the observed patterns.
   3. Careful histological analysis illustrates the poor myogenic differentiation in the LVP of OSr1-Cre;Alk5fl/fl embryos.
   4. Identifying that TGFb is more important for regulating late perimysial cell development is important in identifying the targets of TGFb signaling.
   5. The use of CellChat to identify sending and receiving cells is well done and further supports the late function of TGFb signaling, in this context working through Fgf18 and Lama4.
   6. The attempt to build a signaling network again using CellChat (Figure 6) is admirable, though there are a few caveats to that approach (see below).
   7. While bead implant studies have been used for 40 years, the approach of culturing a piece of the pharynx and then performing a bead implant to prove that Fgf18 can positively influence myogenic development is admirable.

   Weaknesses
   1. In general, the authors are careful to not suggest that staining is significant unless showing quantification, though, at several points, this is not true.
   2. The authors identify five putative Smad2 sites upstream of Fgf18, using one of them in a Cut and Run assay whose results suggest enhanced Smad2/3 binding. The problem is that this likely would have worked with the other Smad sites and probably would have worked for any other putative site that one might pick. Proving that a putative site can be bound by its cognate transcription factor is not the same as proving that this occurs in vivo and is sufficient to control the process of LVP development. One would need reporter assays using that TF binding site to better support the points being made by the authors.
   3. In a similar manner, the authors try to define which factors might function with TGFb signaling to regulate myogenic development. Using SCENIC, the authors found a number of genes that might be involved in perimysial fibroblast development. Of these, they illustrate that Creb5 siRNA knockdown decreases Fgf18 expression in cultured palates. The focus on Creb5 was based on it showing, "the most specific expression patterning the late perimysial cells (Figure 6H)....". In fact, Creb5 appears the most broad, appearing to be expressed across the entire LVP, not just in the area where myogenic precursors are found. Thus, "most specific" probably needs to be reworded. However, the second problem is that Creb5 knockdown reducing Fgf18 expression does not prove any direct regulation. Both of these are rather circuitous arguments.
   4. While the disorganization of myogenic fibers in the posterior LVP is somewhat obvious, it is not as clear as the authors suggest. This change (which I believe) needs to be better quantified (length, width, area, etc.).

   Read the original source
5. 

   eLife

   Jul 14, 2022

   Reviewer #2 (Public Review):

   In this study, the authors take advantage of unbiased scRNA-seq datasets of the developing mouse soft palate that they previously reported and performed a new bioinformatic analysis to identify differential signaling pathway activities in the heterogeneous palatal mesenchyme. They found a strong association of TGF-beta signaling pathway activity with the perimysial cells and validated through immunofluorescent detection of pSmad2, which led to their hypothesis that TGF-beta signaling in the perimysial cells might regulate palatal muscle formation. They generated and analyzed Osr2-Cre;Alk5fl/fl mice and showed those mice have cleft soft palate and disruption of the levator veli palataini (LVP) muscle. They then performed a comparative scRNA-seq analysis of the soft palate tissues from E14.5 Osr2-Cre;Alk5fl/fl …

   Reviewer #2 (Public Review):

   In this study, the authors take advantage of unbiased scRNA-seq datasets of the developing mouse soft palate that they previously reported and performed a new bioinformatic analysis to identify differential signaling pathway activities in the heterogeneous palatal mesenchyme. They found a strong association of TGF-beta signaling pathway activity with the perimysial cells and validated through immunofluorescent detection of pSmad2, which led to their hypothesis that TGF-beta signaling in the perimysial cells might regulate palatal muscle formation. They generated and analyzed Osr2-Cre;Alk5fl/fl mice and showed those mice have cleft soft palate and disruption of the levator veli palataini (LVP) muscle. They then performed a comparative scRNA-seq analysis of the soft palate tissues from E14.5 Osr2-Cre;Alk5fl/fl and control embryos and showed that the Osr2-Cre;Alk5fl/fl embryos exhibited defects in the perimysial cells, in particular reduction in Tbx15+ perimysial fibroblasts that directly associate with the LVP muscle progenitors. The FGF18 is one of the most highly enriched signaling molecules in the perimysial cells and showed that the Osr2-Cre;Alk5fl/fl embryos exhibited reduced Fgf18 expression together with loss of MyoD+ myoblasts in the prospective LVP region. Further data showed that pSmad2 bound in the Fgf18 promoter region in the developing soft palate tissues. In addition, bioinformatic gene regulatory network analysis of the scRNA-seq data identified Creb5 as a potential tissue-specific transcription factor in the perimysial cells and RNAi knockdown assays in palatal mesenchyme culture suggested that Creb5 is required for Fgf18 expression. Further studies identified a subtle deficiency in LVP in Osr2-Cre;Fgf18fl/fl mice and showed that exogenous Fgf18 bead implantation in explants of E14 Osr2-Cre;Alk5fl/fl embryonic head increased the MyoD+ myoblast population in the prospective LVP region. The authors concluded that TGF-beta signaling and Creb5 cooperatively regulate Fgf18 to control pharyngeal muscle development. While the study used multiple complementary approaches and the data presented are solid, important questions need to be addressed to resolve reasonable alternative explanations of the data to the authors' main conclusion.

   Major points:
   1. TGF-beta signaling is known to be crucial for neural crest-derived palatal mesenchyme cell proliferation from E13.5 to E14.5. The Osr2-Cre;Alk5fl/fl mutant embryos exhibited obvious disruption of LVP myogenesis and reduced soft palatal shelf size at E14.5 (Fig3-Sup2A-D and Fig 4H-K). The cellular and molecular defects likely started prior to E14.5. Thus, it is important to examine at earlier stages (E13.5/E14.0) whether the palatal mesenchyme was already defective in cell proliferation/survival and/or perimysial cell marker expression, including Creb5 and Tbx15, to resolve whether the primary defect in the Osr2-Cre;Alk5fl/fl palatal mesenchyme could be a reduction in perimysial progenitor cell proliferation and/or differentiation of the myoblast-associated subset, for which Tbx15 and Fgf18hi act as marker genes rather than direct molecular targets. Furthermore, the apparent loss of Tbx15+ cells coincided with a specific reduction of Fgf18 expression in the myoblast-associated perimysial cells (Fig 4J/K versus Fig 5H-K), which raises the possibility that TGF-beta signaling regulates the differentiation of the Tbx5+ population from the mesenchymal progenitors while the reduction in Fgf18 expression might be a secondary consequence of the cellular defect. The data in Fig 6O showing a lack of significant induction of Fgf18 expression in the palatal mesenchyme culture in both control and Creb5-RNAi cells is also consistent with this alternative explanation.
   2. Since the Osr2-Cre;Fgf18fl/fl mice exhibited much subtler palatal and LVP defects than the Osr2-Cre;Alk5fl/fl mice even though the latter still had a lot of Fgf18-expressing perimysial cells at E14.5, Fgf18 is likely a minor player in the TGF-beta mediated gene regulatory network regulating LVP formation. The major players acting downstream of TGF-beta signaling in the palatal mesenchyme, that control initial LVP progenitor migration to and/or proliferation in the soft palate region, remain to be identified and functionally validated. Whether and how Fgf18 directly regulates the perimysial-myoblast interaction is also not known.
   3. While the title and the main conclusion of this manuscript imply a crucial role of Creb5 in the regulation of pharyngeal muscle development, there is no data supporting such a crucial role. Do Creb5-/- mice have specific defects in pharyngeal muscle development?
   4. Data in Fig 6 are not sufficient to conclude that TGF-beta signaling and Creb5 cooperatively regulate Fgf18. The TGFb1 treatment did not significantly induce Fgf18 expression in either the control or Creb5-RNAi palate mesenchyme cells (Fig 6O). No data regarding how they act cooperatively to regulate Fgf18 expression.

   Read the original source
6. 

   eLife

   Jul 14, 2022

   Reviewer #3 (Public Review)

   In this study, the authors investigated cell-cell communication between perimysial cells and skeletal muscle progenitors during soft palate development in the mouse. The authors have previously reported on the development of this structure and here they propose that a TGF-β signaling and Creb5 act to regulate Fgf18, and this pathway regulates pharyngeal muscle development through the indicated cell populations. The study is of high quality, very nicely illustrated, and uses multiple approaches including inferences from single cell transcriptomics, validations on sections, and lineage-specific gene activations. In addition, the authors successfully optimized an organ culture system from thick sections to test locally the role of FGF signaling (bead implantation). The results largely confer with the conclusions …

   Reviewer #3 (Public Review)

   In this study, the authors investigated cell-cell communication between perimysial cells and skeletal muscle progenitors during soft palate development in the mouse. The authors have previously reported on the development of this structure and here they propose that a TGF-β signaling and Creb5 act to regulate Fgf18, and this pathway regulates pharyngeal muscle development through the indicated cell populations. The study is of high quality, very nicely illustrated, and uses multiple approaches including inferences from single cell transcriptomics, validations on sections, and lineage-specific gene activations. In addition, the authors successfully optimized an organ culture system from thick sections to test locally the role of FGF signaling (bead implantation). The results largely confer with the conclusions and provide a valuable example of how subjacent cell populations cooperate to establish an embryonic structure.

   Read the original source
7. 

   Version published to 10.1101/2022.05.25.493396v1 on bioRxiv

   May 26, 2022