Species-specific sensitivity to TGFβ signaling and changes to the Mmp13 promoter underlie avian jaw development and evolution
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Evaluation Summary:
Smith et al. examine jaw development across three different species of birds, chick, quail and duck, all of which have jaws of different shapes and sizes. This study provides interesting new data and insights into jaw development and evolution.
(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. The reviewers remained anonymous to the authors.)
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Abstract
Precise developmental control of jaw length is critical for survival, but underlying molecular mechanisms remain poorly understood. The jaw skeleton arises from neural crest mesenchyme (NCM), and we previously demonstrated that these progenitor cells express more bone-resorbing enzymes including Matrix metalloproteinase 13 ( Mmp13 ) when they generate shorter jaws in quail embryos versus longer jaws in duck. Moreover, if we inhibit bone resorption or Mmp13, we can increase jaw length. In the current study, we uncover mechanisms establishing species-specific levels of Mmp13 and bone resorption. Quail show greater activation of and sensitivity to transforming growth factor beta (TGFβ) signaling than duck; where intracellular mediators like SMADs and targets like Runt-related transcription factor 2 ( Runx2 ) , which bind Mmp13 , become elevated. Inhibiting TGFβ signaling decreases bone resorption, and overexpressing Mmp13 in NCM shortens the duck lower jaw. To elucidate the basis for this differential regulation, we examine the Mmp13 promoter. We discover a SMAD-binding element and single nucleotide polymorphisms (SNPs) near a RUNX2-binding element that distinguish quail from duck. Altering the SMAD site and switching the SNPs abolish TGFβ sensitivity in the quail Mmp13 promoter but make the duck promoter responsive. Thus, differential regulation of TGFβ signaling and Mmp13 promoter structure underlie avian jaw development and evolution.
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Author Response
Reviewer #3 (Public Review):
Smith et al. evaluated a role of bone-resorbing enzyme Mmp13 in craniofacial structures development and the sensitivity of facial tissues to the activation of TGFβ signalling with aim to discover developmental processes driving formation of short beaks in chicken or quail versus long beaks in duck. The main topic can be interesting for broader audience as organ shape regulation during embryogenesis is still not well studied and the manuscript brings new findings to this field.
Authors uncovered that jaw-bone length is governed by neural crest cells-mediated bone resorption and revealed a mechanism contributing to the establishment of specific face shape in selected bird species. Differential gene expression level of TGFβ signaling was found to be associated with shorter beak formation …
Author Response
Reviewer #3 (Public Review):
Smith et al. evaluated a role of bone-resorbing enzyme Mmp13 in craniofacial structures development and the sensitivity of facial tissues to the activation of TGFβ signalling with aim to discover developmental processes driving formation of short beaks in chicken or quail versus long beaks in duck. The main topic can be interesting for broader audience as organ shape regulation during embryogenesis is still not well studied and the manuscript brings new findings to this field.
Authors uncovered that jaw-bone length is governed by neural crest cells-mediated bone resorption and revealed a mechanism contributing to the establishment of specific face shape in selected bird species. Differential gene expression level of TGFβ signaling was found to be associated with shorter beak formation and the activation of the TGFβ pathway was also confirmed by analyses on protein level.
To identify mechanisms that control the differential regulation of Mmp13, authors evaluated the structure and function of the Mmp13 promoter in selected model species and determined several promoter domains, which contained RUNX2 and SMADs binding elements, which are the most probable to regulate MMP13 expression. Design of species-specific reporter constructs with or without these binding elements helped them to uncover differences in promoter activity among species. Higher activity of these binding elements was found for quail and chick with shorter beaks. Two single nucleotide polymorphisms were found directly downstream of the RUNX2 binding element, which again distinguished quail and chick from duck. Series of functional experiments, where these SNPs were switched between species, confirmed their role and direct involvement in the regulation of promoter activity.
The conclusions of this paper are well supported by convincing data.
Strengths: Main question was evaluated at multiple levels to target different aspects of gene regulation of the TGFβ pathway and Mmp13 function and their possible roles in bone resorption, which ultimately underlay the variation of jaw length and drive the species-specific beak morphology in birds. Using of three different models such as chick, quail, and duck embryos enable to associate individual findings with the distinct phenotype of these animals leading to clear outcomes. Careful and complex design of individual experiments enable to target possible role of individual components of MMP13 promoter by functional tests.
Weaknesses: Authors indicate possible evolutionary consequences of their main findings, however there is no discussion about more broad implications from EVO-DEVO perspective or about possible similarities or differences during endochondral skeletogenesis.
We have added a discussion to the Conclusion section on the broader evo-devo implications of our work. While we have also added new details about the fact that the lower jaw does not undergo endochondral ossification, we have not expanded on this point further due to the fact that we have been asked to shorten the manuscript.
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Evaluation Summary:
Smith et al. examine jaw development across three different species of birds, chick, quail and duck, all of which have jaws of different shapes and sizes. This study provides interesting new data and insights into jaw development and evolution.
(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. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
Smith et al. investigated jaw development across three different species of birds. They found that quail have higher TRAP activity and Mmp13 expression than duck in the jaw bones. They have examined the TGFβ signaling activity in chick, quail and duck both in vitro and in vivo and found that quail and chick have higher TGFβ signaling activity than duck and are more sensitive to TGFβ signaling. They also analyzed the function of TGFβ signaling on its downstream target genes in these avian species. Interestingly, they found that two SNPs distinguish chick and quail from duck and that these two SNPs affect the differential species-specific response of the Mmp13 promoter. These data altogether provide an interesting perspective on jaw development and evolution.
Strengths: The authors used three different avian …
Reviewer #1 (Public Review):
Smith et al. investigated jaw development across three different species of birds. They found that quail have higher TRAP activity and Mmp13 expression than duck in the jaw bones. They have examined the TGFβ signaling activity in chick, quail and duck both in vitro and in vivo and found that quail and chick have higher TGFβ signaling activity than duck and are more sensitive to TGFβ signaling. They also analyzed the function of TGFβ signaling on its downstream target genes in these avian species. Interestingly, they found that two SNPs distinguish chick and quail from duck and that these two SNPs affect the differential species-specific response of the Mmp13 promoter. These data altogether provide an interesting perspective on jaw development and evolution.
Strengths: The authors used three different avian species to study jaw development and the mechanism uncovered in this work provides important clues about jaw evolution. The authors have also used different approaches to evaluate the TGFβ-Runx2-Mmp13 axis in jaw development in these three avian species. It has potential impact on both developmental biology and evolutionary biology.
Weaknesses: There should be more in vivo analysis to validate their findings. In addition, more evidence should be provided at the cellular level to support their conclusions.
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Reviewer #2 (Public Review):
In this manuscript, the authors propose a novel mechanism by which jaw length might be regulated in vertebrates. The jaw skeleton is derived from neural crest mesenchyme. They demonstrate that in developing quail embryos, the lower jaw, which is significantly smaller compared to duck, actively upregulates TGFβ signaling pathway and in turn display a high MMP13 activity and this subsequently leads to increased bone remodeling. They further correlate this process to the smaller mandible size in quail embryos. The main point of the manuscript is therefore that active bone remodeling can regulate organ size and may contribute to size/ shape variation of mandible across species, however in its current form, this point does not come across so clearly. The manuscript provides further comparative data highlighting …
Reviewer #2 (Public Review):
In this manuscript, the authors propose a novel mechanism by which jaw length might be regulated in vertebrates. The jaw skeleton is derived from neural crest mesenchyme. They demonstrate that in developing quail embryos, the lower jaw, which is significantly smaller compared to duck, actively upregulates TGFβ signaling pathway and in turn display a high MMP13 activity and this subsequently leads to increased bone remodeling. They further correlate this process to the smaller mandible size in quail embryos. The main point of the manuscript is therefore that active bone remodeling can regulate organ size and may contribute to size/ shape variation of mandible across species, however in its current form, this point does not come across so clearly. The manuscript provides further comparative data highlighting promoter elements in species-specific regulation of Mmp13 expression.
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Reviewer #3 (Public Review):
Smith et al. evaluated a role of bone-resorbing enzyme Mmp13 in craniofacial structures development and the sensitivity of facial tissues to the activation of TGFβ signalling with aim to discover developmental processes driving formation of short beaks in chicken or quail versus long beaks in duck. The main topic can be interesting for broader audience as organ shape regulation during embryogenesis is still not well studied and the manuscript brings new findings to this field.
Authors uncovered that jaw-bone length is governed by neural crest cells-mediated bone resorption and revealed a mechanism contributing to the establishment of specific face shape in selected bird species.
Differential gene expression level of TGFβ signaling was found to be associated with shorter beak formation and the activation of …Reviewer #3 (Public Review):
Smith et al. evaluated a role of bone-resorbing enzyme Mmp13 in craniofacial structures development and the sensitivity of facial tissues to the activation of TGFβ signalling with aim to discover developmental processes driving formation of short beaks in chicken or quail versus long beaks in duck. The main topic can be interesting for broader audience as organ shape regulation during embryogenesis is still not well studied and the manuscript brings new findings to this field.
Authors uncovered that jaw-bone length is governed by neural crest cells-mediated bone resorption and revealed a mechanism contributing to the establishment of specific face shape in selected bird species.
Differential gene expression level of TGFβ signaling was found to be associated with shorter beak formation and the activation of the TGFβ pathway was also confirmed by analyses on protein level.
To identify mechanisms that control the differential regulation of Mmp13, authors evaluated the structure and function of the Mmp13 promoter in selected model species and determined several promoter domains, which contained RUNX2 and SMADs binding elements, which are the most probable to regulate MMP13 expression. Design of species-specific reporter constructs with or without these binding elements helped them to uncover differences in promoter activity among species. Higher activity of these binding elements was found for quail and chick with shorter beaks. Two single nucleotide polymorphisms were found directly downstream of the RUNX2 binding element, which again distinguished quail and chick from duck. Series of functional experiments, where these SNPs were switched between species, confirmed their role and direct involvement in the regulation of promoter activity.The conclusions of this paper are well supported by convincing data.
Strengths:
Main question was evaluated at multiple levels to target different aspects of gene regulation of the TGFβ pathway and Mmp13 function and their possible roles in bone resorption, which ultimately underlay the variation of jaw length and drive the species-specific beak morphology in birds.
Using of three different models such as chick, quail, and duck embryos enable to associate individual findings with the distinct phenotype of these animals leading to clear outcomes.
Careful and complex design of individual experiments enable to target possible role of individual components of MMP13 promoter by functional tests.Weaknesses:
Authors indicate possible evolutionary consequences of their main findings, however there is no discussion about more broad implications from EVO-DEVO perspective or about possible similarities or differences during endochondral skeletogenesis. -
