Nephronectin-integrin α8 signaling is required for proper migration of periocular neural crest cells during chick corneal development
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Evaluation Summary:
This manuscript examines the role of Nephronectin-Integrin a8 signaling in early stages in the avian corneal development. This is an understudied system with numerous gaps in our comprehension how neural crest derived cells migrate into the "open" space between the corneal epithelium and lens and form the corneal endothelium and stroma. Novel insights are generated on the cellular and molecular mechanisms of this critical process of anterior segment 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 #2 agreed to share their name with the authors.)
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Abstract
During development, cells aggregate at tissue boundaries to form normal tissue architecture of organs. However, how cells are segregated into tissue precursors remains largely unknown. Cornea development is a perfect example of this process whereby neural crest cells aggregate in the periocular region prior to their migration and differentiation into corneal cells. Our recent RNA-seq analysis identified upregulation of nephronectin (Npnt) transcripts during early stages of corneal development where its function has not been investigated. We found that Npnt mRNA and protein are expressed by various ocular tissues, including the migratory periocular neural crest (pNC), which also express the integrin alpha 8 (Itgα8) receptor. Knockdown of either Npnt or Itgα8 attenuated cornea development, whereas overexpression of Npnt resulted in cornea thickening. Moreover, overexpression of Npnt variants lacking RGD-binding sites did not affect corneal thickness. Neither the knockdown nor augmentation of Npnt caused significant changes in cell proliferation, suggesting that Npnt directs pNC migration into the cornea. In vitro analyses showed that Npnt promotes pNC migration from explanted periocular mesenchyme, which requires Itgα8, focal adhesion kinase, and Rho kinase. Combined, these data suggest that Npnt augments cell migration into the presumptive cornea extracellular matrix by functioning as a substrate for Itgα8-positive pNC cells.
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Evaluation Summary:
This manuscript examines the role of Nephronectin-Integrin a8 signaling in early stages in the avian corneal development. This is an understudied system with numerous gaps in our comprehension how neural crest derived cells migrate into the "open" space between the corneal epithelium and lens and form the corneal endothelium and stroma. Novel insights are generated on the cellular and molecular mechanisms of this critical process of anterior segment 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 #2 agreed to share their name with the authors.)
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Reviewer #1 (Public Review)
Authors, for the first time, demonstrated that Nephronectin (Npnt) was abundantly expressed in the migrating periocular neural crest cells during early corneal stromal development in chick. It has been shown that Npnt bound to the integrin a8 (Itga8) for cell migration during kidney development. In this study, authors attempted to test whether Npnt-Itga8 signaling was indispensable for corneal stroma development as well. Interestingly, authors clearly demonstrated that knockdown of Npnt and Itga8 attenuated corneal migration into corneal stroma leading to thinner cornea. In contrast, overexpression of Npnt facilitated cell migration and increase corneal thickness. It is interesting to know that one can control corneal thickness by manipulating Npnt-Itaga8 interaction. In summary, authors concluded that …
Reviewer #1 (Public Review)
Authors, for the first time, demonstrated that Nephronectin (Npnt) was abundantly expressed in the migrating periocular neural crest cells during early corneal stromal development in chick. It has been shown that Npnt bound to the integrin a8 (Itga8) for cell migration during kidney development. In this study, authors attempted to test whether Npnt-Itga8 signaling was indispensable for corneal stroma development as well. Interestingly, authors clearly demonstrated that knockdown of Npnt and Itga8 attenuated corneal migration into corneal stroma leading to thinner cornea. In contrast, overexpression of Npnt facilitated cell migration and increase corneal thickness. It is interesting to know that one can control corneal thickness by manipulating Npnt-Itaga8 interaction. In summary, authors concluded that Npnt-Itga8 signaling was required for proper migration of periocular neural crest cells during chick corneal development.
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Reviewer #2 (Public Review)
Early vertebrate eye development is a complex and fascinating system driven by complex 3D morphogenetic events including cells of the neuroectodermal, ectodermal and neural crest origin. The transparent cornea is the most outer part of the eye and is formed from three different cellular layers, the corneal epithelium formed first from the surface ectoderm, followed by sequential migration of two waves of the neural crest cells/periocular mesenchyme to generate the corneal endothelium followed by corneal stroma. These processes require precise activities of multiple signaling pathways and extracellular matrix proteins (ECMs). Although earlier studies in kidney examined synergistic functions of two proteins, nephronectin (gene name Nptm) and alpha8-integrin (Itga8), in kidney; the findings on their roles in the …
Reviewer #2 (Public Review)
Early vertebrate eye development is a complex and fascinating system driven by complex 3D morphogenetic events including cells of the neuroectodermal, ectodermal and neural crest origin. The transparent cornea is the most outer part of the eye and is formed from three different cellular layers, the corneal epithelium formed first from the surface ectoderm, followed by sequential migration of two waves of the neural crest cells/periocular mesenchyme to generate the corneal endothelium followed by corneal stroma. These processes require precise activities of multiple signaling pathways and extracellular matrix proteins (ECMs). Although earlier studies in kidney examined synergistic functions of two proteins, nephronectin (gene name Nptm) and alpha8-integrin (Itga8), in kidney; the findings on their roles in the cornea are entirely novel and shed very much needed light into the coordination of multiple embryonic processes that occur in parallel in the anterior portion of the developing eye. The data are driven by gene loss- and gain-of-function experiments taking advantages of the chick as experimental model and generation of both wild type and mutated proteins using RCAS virus. Control experiments, biological and technical replicates are at place. Taken together, the data are novel for understudied genes/proteins and their critical roles in the vertebrate anterior segment morphogenesis.
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