Inhibition of the serine protease HtrA1 by SerpinE2 suggests an extracellular proteolytic pathway in the control of neural crest migration
Curation statements for this article:-
Curated by eLife
eLife assessment
This fundamental work substantially advances our understanding of cell migration, especially in that of cranial neural crest. The additional evidence provided to support the conclusion is exceptional, with rigorous biochemical assays for materials used and with intensive genetic interventions. The work will be of broad interest to developmental biologists and cell biologists.
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Abstract
We previously showed that SerpinE2 and the serine protease HtrA1 modulate fibroblast growth factor (FGF) signaling in germ layer specification and head-to-tail development of Xenopus embryos. Here, we present an extracellular proteolytic mechanism involving this serpin-protease system in the developing neural crest (NC). Knockdown of SerpinE2 by injected antisense morpholino oligonucleotides did not affect the specification of NC progenitors but instead inhibited the migration of NC cells, causing defects in dorsal fin, melanocyte, and craniofacial cartilage formation. Similarly, overexpression of the HtrA1 protease impaired NC cell migration and the formation of NC-derived structures. The phenotype of SerpinE2 knockdown was overcome by concomitant downregulation of HtrA1, indicating that SerpinE2 stimulates NC migration by inhibiting endogenous HtrA1 activity. SerpinE2 binds to HtrA1, and the HtrA1 protease triggers degradation of the cell surface proteoglycan Syndecan-4 (Sdc4). Microinjection of Sdc4 mRNA partially rescued NC migration defects induced by both HtrA1 upregulation and SerpinE2 downregulation. These epistatic experiments suggest a proteolytic pathway by a double inhibition mechanism:
SerpinE2 ┤HtrA1 protease ┤Syndecan-4 → NC cell migration.
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Author Response
The following is the authors’ response to the original reviews.
We thank the two reviewers for their very thoughtful suggestions and the editors for writing the eLife assessment. We will submit a revised manuscript that addresses most comments and include a point-by-point response to the reviewers. We will provide evidence that overexpression of the HtrA1 protease and knockdown of its inhibitor SerpinE2 reduce the development of neural crest-derived cartilage elements in the head of Xenopus embryos. This will be done by whole mount in situ hybridization, using a probe for the chondrogenic marker Sox9. We will also provide two time-lapse movies showing (1) collective migration of cranial neural crest cells in culture and (2) failure of these cells to adhere to fibronectin upon SerpinE2 depletion. We will discuss in more …
Author Response
The following is the authors’ response to the original reviews.
We thank the two reviewers for their very thoughtful suggestions and the editors for writing the eLife assessment. We will submit a revised manuscript that addresses most comments and include a point-by-point response to the reviewers. We will provide evidence that overexpression of the HtrA1 protease and knockdown of its inhibitor SerpinE2 reduce the development of neural crest-derived cartilage elements in the head of Xenopus embryos. This will be done by whole mount in situ hybridization, using a probe for the chondrogenic marker Sox9. We will also provide two time-lapse movies showing (1) collective migration of cranial neural crest cells in culture and (2) failure of these cells to adhere to fibronectin upon SerpinE2 depletion. We will discuss in more depth how the SerpinE2-HtrA1 proteolytic pathway and its target, the heparan sulfate proteoglycan Syndecan-4, might regulate FGF signaling and suggest a model, in which serpin secreted by the leader cells and the protease released by the follower cells might establish a chemotactic FGF gradient for the directed migration of the neural crest cohort. The criticism that other factors such as proliferation and cell survival might contribute to the observed craniofacial phenotypes upon misexpression of SerpinE2 and HtrA1, and that it remains unclear to what extent the mechanism reported here is conserved in the trunk neural crest is valid. The reason we focused on the more amenable cranial neural crest in the Xenopus embryo and used a multitude of approaches – structure-function studies, biochemical analyses, in vitro explant assays and epistatic experiments in vivo – was to validate a central finding: that an extracellular proteolytic pathway involving a serpin, a protease and a proteoglycan regulates by a double inhibition mechanism collective cell migration.
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eLife assessment
This fundamental work substantially advances our understanding of cell migration, especially in that of cranial neural crest. The additional evidence provided to support the conclusion is exceptional, with rigorous biochemical assays for materials used and with intensive genetic interventions. The work will be of broad interest to developmental biologists and cell biologists.
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Reviewer #1 (Public Review):
Summary:
A novel serine protease and a inhibitor pair regulate cell migration in the neural crest.
Strengths:
The reproduction of classical cranial neural crest extirpations and their phenocopy by SerpinE2 morpholino are remarkable. Very scholarly written and data of the highest quality.
Weaknesses:
All were improved upon revision.
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Reviewer #2 (Public Review):
Summary:
The authors conducted research on the role of SerpinE2 and HtrA1 in neural crest migration using Xenopus embryos. The data presented in this study was of high quality and supported the authors' conclusions. The discovery of the potential molecular connection between SerpinE2 and HtrA1 in neural crest cell migration in vivo is significant, as understanding this pathway could potentially lead to treatments for aggressive cancers and pregnancy-related disorders.
Strengths:
Previous research has shown that SerpinE2 and HtrA1 can have both positive and negative effects on cell migration, but their molecular interplay and role in neural crest migration are not well-established. This study is the first to reveal a potential connection between these two proteins in neural crest cell migration in vivo. The …
Reviewer #2 (Public Review):
Summary:
The authors conducted research on the role of SerpinE2 and HtrA1 in neural crest migration using Xenopus embryos. The data presented in this study was of high quality and supported the authors' conclusions. The discovery of the potential molecular connection between SerpinE2 and HtrA1 in neural crest cell migration in vivo is significant, as understanding this pathway could potentially lead to treatments for aggressive cancers and pregnancy-related disorders.
Strengths:
Previous research has shown that SerpinE2 and HtrA1 can have both positive and negative effects on cell migration, but their molecular interplay and role in neural crest migration are not well-established. This study is the first to reveal a potential connection between these two proteins in neural crest cell migration in vivo. The authors found that SerpinE2 promotes neural crest migration by inhibiting HtrA1. Additionally, overexpression of Sdc4 partly alleviates neural crest migration issues caused by SerpinE2 knockdown or HtrA1 overexpression. These findings suggest that the SeprinE2-HtrA1-Sdc4 pathway is crucial for neural crest migration.
Weaknesses:
To further increase the study's credibility, it may be helpful to use techniques like western blotting, qRT-PCR, or in situ hybridization to verify the efficiency of SerpinE2 and HtrA1 knockdown and/or overexpression.
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eLife assessment
This fundamental work substantially advances our understanding of cell migration, especially in that of cranial neural crest. The evidence supporting the conclusion is compelling, with rigorous biochemical assays for materials used and with intensive genetic interventions. The work will be of broad interest to developmental biologists and cell biologists.
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Reviewer #1 (Public Review):
Summary:
A novel serine protease and an inhibitor pair regulate cell migration in the neural crest. This is a very important study that describes a novel pathway controlling neural crest migratory behavior through a pair of protease and inhibitor regulators that act in the extracellular space. Using very high technical standards in Xenopus embryos they show that knockdown of the inhibitor SerpinE2 prevents cell migration and that this is restored by simultaneous knockdown of the serine protease HtrA1.
Strengths:
The reproduction of classical cranial neural crest extirpations and their phenocopy by SerpinE2 morpholino is remarkable. The experiments provided must represent many years of work, and the paper is written in a very scholarly fashion. The data is of the highest quality.
Weaknesses:
The paper is very …
Reviewer #1 (Public Review):
Summary:
A novel serine protease and an inhibitor pair regulate cell migration in the neural crest. This is a very important study that describes a novel pathway controlling neural crest migratory behavior through a pair of protease and inhibitor regulators that act in the extracellular space. Using very high technical standards in Xenopus embryos they show that knockdown of the inhibitor SerpinE2 prevents cell migration and that this is restored by simultaneous knockdown of the serine protease HtrA1.
Strengths:
The reproduction of classical cranial neural crest extirpations and their phenocopy by SerpinE2 morpholino is remarkable. The experiments provided must represent many years of work, and the paper is written in a very scholarly fashion. The data is of the highest quality.
Weaknesses:
The paper is very long and contains many years of experiments, making it at times difficult to read. The paper contains so much data that it would help the readership if the present version were revised in order to make it more digestible.
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Reviewer #2 (Public Review):
Summary:
The authors conducted research on the role of SerpinE2 and HtrA1 in neural crest migration using Xenopus embryos. The data presented in this study was of high quality and supported the authors' conclusions. The discovery of the potential molecular connection between SerpinE2 and HtrA1 in neural crest cell migration in vivo is significant, as understanding this pathway could potentially lead to treatments for aggressive cancers and pregnancy-related disorders.
Strengths:
Previous research has shown that SerpinE2 and HtrA1 can have both positive and negative effects on cell migration, but their molecular interplay and role in neural crest migration are not well-established. This study is the first to reveal a potential connection between these two proteins in neural crest cell migration in vivo. The …
Reviewer #2 (Public Review):
Summary:
The authors conducted research on the role of SerpinE2 and HtrA1 in neural crest migration using Xenopus embryos. The data presented in this study was of high quality and supported the authors' conclusions. The discovery of the potential molecular connection between SerpinE2 and HtrA1 in neural crest cell migration in vivo is significant, as understanding this pathway could potentially lead to treatments for aggressive cancers and pregnancy-related disorders.
Strengths:
Previous research has shown that SerpinE2 and HtrA1 can have both positive and negative effects on cell migration, but their molecular interplay and role in neural crest migration are not well-established. This study is the first to reveal a potential connection between these two proteins in neural crest cell migration in vivo. The authors found that SerpinE2 promotes neural crest migration by inhibiting HtrA1. Additionally, overexpression of Sdc4 partly alleviates neural crest migration issues caused by SerpinE2 knockdown or HtrA1 overexpression. These findings suggest that the SeprinE2-HtrA1-Sdc4 pathway is crucial for neural crest migration.
Weaknesses:
To further increase the study's credibility, the authors could use techniques like western blotting, qRT-PCR, or in situ hybridization to verify the efficiency of SerpinE2 and HtrA1 knockdown and/or overexpression. Furthermore, determining whether the observed craniofacial phenotypes in SerpinE2 and/or HtrA1 mutants were solely due to modified cranial neural crest migration or affected by other factors such as cell proliferation, cell survival, and chondrogenic differentiation could provide more clarity. Lastly, it is unclear whether the SeprinE2-HtrA1-Sdc4 pathway is constant in both cranial and trunk neural crest migration.
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