Fibroblast-derived Hgf controls recruitment and expansion of muscle during morphogenesis of the mammalian diaphragm
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Evaluation Summary:
Sefton et al. analyze how fibroblast-derived HGF integrates muscle and nerve development during morphogenesis of the mammalian diaphragm. The new findings are based on in-depth analyses of the development of the diaphragm muscle, and the role of Met and HGF in the process. The work is relevant for the understanding of muscle development, and congenital disease (hernia).
(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 and Reviewer #2 agreed to share their names with the authors.)
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Abstract
The diaphragm is a domed muscle between the thorax and abdomen essential for breathing in mammals. Diaphragm development requires the coordinated development of muscle, connective tissue, and nerve, which are derived from different embryonic sources. Defects in diaphragm development cause the common and often lethal birth defect, congenital diaphragmatic hernias (CDH). HGF/MET signaling is required for diaphragm muscularization, but the source of HGF and the specific functions of this pathway in muscle progenitors and effects on phrenic nerve have not been explicitly tested. Using conditional mutagenesis in mice and pharmacological inhibition of MET, we demonstrate that the pleuroperitoneal folds (PPFs), transient embryonic structures that give rise to the connective tissue in the diaphragm, are the source of HGF critical for diaphragm muscularization. PPF-derived HGF is directly required for recruitment of MET+ muscle progenitors to the diaphragm and indirectly (via its effect on muscle development) required for phrenic nerve primary branching. In addition, HGF is continuously required for maintenance and motility of the pool of progenitors to enable full muscularization. Localization of HGF at the diaphragm’s leading edges directs dorsal and ventral expansion of muscle and regulates its overall size and shape. Surprisingly, large muscleless regions in HGF and Met mutants do not lead to hernias. While these regions are likely more susceptible to CDH, muscle loss is not sufficient to cause CDH.
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Evaluation Summary:
Sefton et al. analyze how fibroblast-derived HGF integrates muscle and nerve development during morphogenesis of the mammalian diaphragm. The new findings are based on in-depth analyses of the development of the diaphragm muscle, and the role of Met and HGF in the process. The work is relevant for the understanding of muscle development, and congenital disease (hernia).
(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 and Reviewer #2 agreed to share their names with the authors.)
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Reviewer #1 (Public Review):
It was previously shown that HGF and Met controls development of the diaphragm muscle. In particular, the signal induces delamination and migration of muscle progenitor cells that colonize the diaphragm. The present manuscript by Sefton and coworkers confirms and extends these observations using (i) conditional mouse lines in which the HGF gene was targeted by Cre/loxP recombination in the pleuroperitoneal folds (Prx1-cre) and at other sites PdgfraCreERT2, and of (ii) Met inhibitors. Overall, the technical quality of the data on diaphragm muscle development is excellent; the conceptual advance over previous work is not exceptional; the evidence for Met/HGF-dependent development of the phrenic nerve is marginal and needs to be strengthened.
The data show that fibroblasts provide HGF signals received by Met in …
Reviewer #1 (Public Review):
It was previously shown that HGF and Met controls development of the diaphragm muscle. In particular, the signal induces delamination and migration of muscle progenitor cells that colonize the diaphragm. The present manuscript by Sefton and coworkers confirms and extends these observations using (i) conditional mouse lines in which the HGF gene was targeted by Cre/loxP recombination in the pleuroperitoneal folds (Prx1-cre) and at other sites PdgfraCreERT2, and of (ii) Met inhibitors. Overall, the technical quality of the data on diaphragm muscle development is excellent; the conceptual advance over previous work is not exceptional; the evidence for Met/HGF-dependent development of the phrenic nerve is marginal and needs to be strengthened.
The data show that fibroblasts provide HGF signals received by Met in muscle progenitor cells that is essential for diaphragm development. The PdgfraCreERT2 line was used to demonstrate that HGF produced by fibroblasts but not by muscle progenitors is essential for diaphragm development. Moreover, development of dorsal and ventral regions of diaphragm muscle requires continuous MET signaling. Thus, HGF is not only required for the delamination of progenitors, but also for proliferation and survival of those muscle progenitors that reached the anlage of the diaphragm.
My major concern is the limited data on the HGF-dependent development of the phrenic nerve (defasciculation). While it is well documented that HGF acts as a trophic factor for motor neurons in culture, its role in development of motor neurons was highly debated due to the fact that some changes observed in Met or HGF mutant mice in vivo are also present in other mutants that lack the muscle groups derived from migrating muscle progenitors. Moreover, careful genetic analyses previously demonstrated indirect mechanisms of Met during motor neuron development, i.e. a non-cell-autonomous function of Met during the recruitment of motor neurons to PEA3-positive motor pools (Helmbacher et al., Neuron 2003).
Sefton et al. provide an analysis of a single time point, one histological picture (3G, magnified in 3H) that indicate that in Met+/- animals defasciculation of the phrenic nerve does not occur correctly. This is accompanied by a quantification that barely reaches significance (Fig. 3K). Data shown in Fig. 7 using Met inhibitors show a major change in phrenic nerve branching, which is presumably due to the major change in diaphragm development, as conceded by the authors.
Despite this weakness on the experimental side, the role of HGF/Met in phrenic nerve development is strongly emphasized in abstract /intro/discussion (e.g. line 414: However, PPF-derived HGF is crucial for the defasciculation and primary branching of the nerve, independent of muscle). The data need to be strengthened in order to conclude that HGF coordinates both, diaphragm muscle and phrenic development.
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Reviewer #2 (Public Review):
In this study Sefton et al interrogated the source of HGF in the developing mouse embryo that produces HGF, required for muscularization and also proper innervation of the diaphragm. The authors extended previous results that are over 20 years old by generating cell type specific mutants of Met and Hgf and found that inactivation of Hgf in fibroblasts via PDGFRa-CreERT2 results in muscle-less diaphragms. Similarly, Hgf inactivation in fibroblasts via PDGFRa-CreERT2 mostly abrogated limb muscle formation, formally identifying PDGFRa+ mesenchymal cells as the main source of HGF for generation of muscles in the limb and diaphragm. Similarly, inactivation of Hgf using Prx1-Cre, which targets fibroblasts derived from the pleuroperitoneal folds (PPT) also prevented muscularization of the diaphragm and branching of …
Reviewer #2 (Public Review):
In this study Sefton et al interrogated the source of HGF in the developing mouse embryo that produces HGF, required for muscularization and also proper innervation of the diaphragm. The authors extended previous results that are over 20 years old by generating cell type specific mutants of Met and Hgf and found that inactivation of Hgf in fibroblasts via PDGFRa-CreERT2 results in muscle-less diaphragms. Similarly, Hgf inactivation in fibroblasts via PDGFRa-CreERT2 mostly abrogated limb muscle formation, formally identifying PDGFRa+ mesenchymal cells as the main source of HGF for generation of muscles in the limb and diaphragm. Similarly, inactivation of Hgf using Prx1-Cre, which targets fibroblasts derived from the pleuroperitoneal folds (PPT) also prevented muscularization of the diaphragm and branching of the phrenic nerve. Interestingly, branching of the phrenic nerve was reduced in heterozygous Met mutants with normal diaphragm musculature, indicating that HGF-MET signaling plays a direct role in phrenic nerve branching and that failure of nerve branching in homozygotic Met or Hgf mutants is not solely due to the loss of the musculature. Finally, the authors performed co-cultures between PPFs and myoblasts and found that pharmacological inhibition of MET lowered motility, survival and MyoD expression of myoblasts, leading to the claim that HGF-MET does also play a role in myogenic commitment
The study sheds further light on the source of HGF required for muscularization of the diaphragm and is well executed. However, the gain of knowledge is mainly incremental and deeper molecular insights are missing. The most interesting part of the study is the formal demonstration that HGF is not only required for delamination of muscle progenitor cells from the epithelium of somites but also to maintain migration at later stages. Similar conclusions have been made many years ago based on studies in chicken embryos but the current study clearly goes a step further. The part that claims a role of HGF-MET in myogenic commitment is not that well developed and may need further proof.
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Reviewer #3 (Public Review):
In this MS by Sefton et al., the authors investigate the role of HGF/MET pathway, as well as the cellular source of these molecules, during diaphragm development. In particular, the authors address the function of this pathway on muscle progenitors and phrenic nerve. They further provide evidence for the expression of HGF in pleuroperitoneal folds and for its requirement for muscle progenitor recruitment and maintenance during diaphragm muscle formation. This study is interesting and in general the results support the conclusions. The work could be improved by (1) providing appropriate controls for the role of HGF in the connective tissue and (2) linking the muscleless diaphragms and HGF to the hernia phenotype.
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Author Response:
Reviewer #1:
It was previously shown that HGF and Met controls development of the diaphragm muscle. In particular, the signal induces delamination and migration of muscle progenitor cells that colonize the diaphragm. The present manuscript by Sefton and coworkers confirms and extends these observations using (i) conditional mouse lines in which the HGF gene was targeted by Cre/loxP recombination in the pleuroperitoneal folds (Prx1-cre) and at other sites PdgfraCreERT2, and of (ii) Met inhibitors. Overall, the technical quality of the data on diaphragm muscle development is excellent; the conceptual advance over previous work is not exceptional; the evidence for Met/HGF-dependent development of the phrenic nerve is marginal and needs to be strengthened.
The data show that fibroblasts provide HGF signals received by Met …
Author Response:
Reviewer #1:
It was previously shown that HGF and Met controls development of the diaphragm muscle. In particular, the signal induces delamination and migration of muscle progenitor cells that colonize the diaphragm. The present manuscript by Sefton and coworkers confirms and extends these observations using (i) conditional mouse lines in which the HGF gene was targeted by Cre/loxP recombination in the pleuroperitoneal folds (Prx1-cre) and at other sites PdgfraCreERT2, and of (ii) Met inhibitors. Overall, the technical quality of the data on diaphragm muscle development is excellent; the conceptual advance over previous work is not exceptional; the evidence for Met/HGF-dependent development of the phrenic nerve is marginal and needs to be strengthened.
The data show that fibroblasts provide HGF signals received by Met in muscle progenitor cells that is essential for diaphragm development. The PdgfraCreERT2 line was used to demonstrate that HGF produced by fibroblasts but not by muscle progenitors is essential for diaphragm development. Moreover, development of dorsal and ventral regions of diaphragm muscle requires continuous MET signaling. Thus, HGF is not only required for the delamination of progenitors, but also for proliferation and survival of those muscle progenitors that reached the anlage of the diaphragm.
My major concern is the limited data on the HGF-dependent development of the phrenic nerve (defasciculation). While it is well documented that HGF acts as a trophic factor for motor neurons in culture, its role in development of motor neurons was highly debated due to the fact that some changes observed in Met or HGF mutant mice in vivo are also present in other mutants that lack the muscle groups derived from migrating muscle progenitors. Moreover, careful genetic analyses previously demonstrated indirect mechanisms of Met during motor neuron development, i.e. a non-cell-autonomous function of Met during the recruitment of motor neurons to PEA3-positive motor pools (Helmbacher et al., Neuron 2003).
Sefton et al. provide an analysis of a single time point, one histological picture (3G, magnified in 3H) that indicate that in Met+/- animals defasciculation of the phrenic nerve does not occur correctly. This is accompanied by a quantification that barely reaches significance (Fig. 3K). Data shown in Fig. 7 using Met inhibitors show a major change in phrenic nerve branching, which is presumably due to the major change in diaphragm development, as conceded by the authors.
Despite this weakness on the experimental side, the role of HGF/Met in phrenic nerve development is strongly emphasized in abstract /intro/discussion (e.g. line 414: However, PPF-derived HGF is crucial for the defasciculation and primary branching of the nerve, independent of muscle). The data need to be strengthened in order to conclude that HGF coordinates both, diaphragm muscle and phrenic development.
In response to comments from the reviewers, we have more thoroughly investigated the role of Met in the development of the phrenic nerve and include two new sets of genetic experiments. In our first submission, we found a decreased number of phrenic nerve branches at E11.5 in Met Δ/ Δ and Met Δ/+ compared with Met+/+ embryos. In the Met Δ/ Δ embryos, no muscle is present in the diaphragm. Therefore, the greatly reduced branching in these embryos is likely a secondary effect of the requirement of Met in muscle progenitors for diaphragm muscularization. Of particular interest is the reduced branching in the Met Δ/+ embryos. Because the diaphragm is muscularized in these embryos, this suggested that Met may be required intrinsically in the phrenic nerve. One reviewer suggested that the reduced branching in the Met Δ/+ embryos could be due to a developmental delay in the whole embryo. However, we found that Met Δ/ Δ and Met Δ/+ embryos are not overall delayed relative to Met+/+ embryos (as measured by crown rump length or limb length; Figure 3—figure supplement 1). Also, to increase the robustness of these data, we added additional embryos to the analysis. We then extended our analysis of Met Δ/ Δ, Met Δ/+ and Met+/+ embryos to E12.5 (Figure 3—figure supplement 1) to see whether the branching phenotype persisted; we found that while the of Met Δ/ Δ embryos continue to have very few branches, the number of branches in Met Δ/+ embryos recovers and matches that of Met+/+ embryos.
To explicitly test whether Met is required within the phrenic nerve, we used _Olig2Cre/+to conditionally delete Met. This line was chosen for its early expression in motor neurons (Zawadzka et al. 2010). We examined Olig2Cre/+;Met Δ/flox_embryos compared to Olig2Cre/+; Metflox/+ embryos. We chose to include Olig2Cre in our controls because the Olig2Cre is a knock-in/knock-out and Olig2 has important roles in nerve development. However, deletion of Met did not affect the number of branches at E11.5 (Figure 3—figure supplement 2) or E12.5 (data not shown). These data suggest that Met does not intrinsically regulate phrenic nerve branching. This suggests that PPF-derived HGF regulates phrenic nerve branching indirectly via muscle. To test if HGF is sufficient to promote early stages of nerve branching in the absence of muscle, we turned to Pax3SpD/SpD mutants in which a point mutation in Pax3 prevents migration of muscle progenitors into the diaphragm (Figure 3—figure supplement 2). In these embryos, the diaphragm is muscleless, but the PPFs still express HGF. In these diaphragms the number of branches at E11.5 is severely reduced. These data demonstrate that in the absence of muscle the presence of HGF in the PPF fibroblasts is not sufficient to support diaphragm branching.
Altogether our data demonstrate that PPF-derived HGF, via its regulation of muscle, controls the primary branching of phrenic nerve. The Met Δ/+ data demonstrate that Met controls phrenic nerve branching at E11.5 in a dose-dependent manner, but this effect is lost by E12.5. Although we see no obvious defects in muscle of Met Δ/+ diaphragms at later stages, the most parsimonious explanation of the reduced phrenic nerve branching at E11.5 is that this is due to fewer muscle progenitors at this time point.
We thank the reviewers for prompting us to look at the role of HGF/Met in the phrenic nerve more closely. Our revised conclusions are presented in the Results and Discussion. We show that PPF-derived HGF is critical for integrating both muscle and phrenic nerve development, but now demonstrate that HGF’s regulation of phrenic nerve branching is via muscle, which is well-known to express multiple trophic factors required by motor neurons.
In response to the specific point about the Met+/- raised, the images shown in Figure 3G and H are representative whole mount confocal images of Met Δ/+ phrenic nerves. For each genotype, we immunolabeled, confocal imaged, rendered in 3-dimensions the phrenic nerves, and counted (blinded to genotype) the number of branches. We also have added several additional embryos to this analysis. In Figure 7 the branching defects resulting from application of the BMS777607 are similar, as expected, to the severe branching defects seen in the Met Δ/ Δ embryos.
Reviewer #2:
In this study Sefton et al interrogated the source of HGF in the developing mouse embryo that produces HGF, required for muscularization and also proper innervation of the diaphragm. The authors extended previous results that are over 20 years old by generating cell type specific mutants of Met and Hgf and found that inactivation of Hgf in fibroblasts via PDGFRa-CreERT2 results in muscle-less diaphragms. Similarly, Hgf inactivation in fibroblasts via PDGFRa-CreERT2 mostly abrogated limb muscle formation, formally identifying PDGFRa+ mesenchymal cells as the main source of HGF for generation of muscles in the limb and diaphragm. Similarly, inactivation of Hgf using Prx1-Cre, which targets fibroblasts derived from the pleuroperitoneal folds (PPT) also prevented muscularization of the diaphragm and branching of the phrenic nerve. Interestingly, branching of the phrenic nerve was reduced in heterozygous Met mutants with normal diaphragm musculature, indicating that HGF-MET signaling plays a direct role in phrenic nerve branching and that failure of nerve branching in homozygotic Met or Hgf mutants is not solely due to the loss of the musculature. Finally, the authors performed co-cultures between PPFs and myoblasts and found that pharmacological inhibition of MET lowered motility, survival and MyoD expression of myoblasts, leading to the claim that HGF-MET does also play a role in myogenic commitment
The study sheds further light on the source of HGF required for muscularization of the diaphragm and is well executed. However, the gain of knowledge is mainly incremental and deeper molecular insights are missing.
We appreciate this critique and have added data to increase the molecular insight into the role of MET signaling in cell survival in the PPFs (Figure 6—figure supplement 1).
The most interesting part of the study is the formal demonstration that HGF is not only required for delamination of muscle progenitor cells from the epithelium of somites but also to maintain migration at later stages. Similar conclusions have been made many years ago based on studies in chicken embryos but the current study clearly goes a step further.
We agree that this is one of the interesting findings in our study.
The part that claims a role of HGF-MET in myogenic commitment is not that well developed and may need further proof.
We apologize for the misunderstanding here and have altered the text to indicate that we do not propose a role for Met in myogenic commitment, but rather that Met regulates the number of MyoD+ cells by promoting their survival.
Reviewer #3:
In this MS by Sefton et al., the authors investigate the role of HGF/MET pathway, as well as the cellular source of these molecules, during diaphragm development. In particular, the authors address the function of this pathway on muscle progenitors and phrenic nerve. They further provide evidence for the expression of HGF in pleuroperitoneal folds and for its requirement for muscle progenitor recruitment and maintenance during diaphragm muscle formation. This study is interesting and in general the results support the conclusions. The work could be improved by (1) providing appropriate controls for the role of HGF in the connective tissue and (2) linking the muscleless diaphragms and HGF to the hernia phenotype.
We appreciate this review and have added controls for the role of HGF in the connective tissue. Specifically PDGFRaCreER/+; HGF-/fl; RosamTmG/+ embryos have fibroblasts present in muscleless regions. We further link muscleless diaphragms and HGF to the hernia phenotype in our abstract. Absence of muscle is necessary for herniation, but not sufficient.
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