Nephronectin-Integrin α8 signaling is required for proper migration of periocular neural crest cells during chick corneal development

  1. Justin Ma
  2. Lian Bi
  3. James Spurlin
  4. Peter Lwigale  Is a corresponding author
  1. Rice University, United States

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 (Itga8) receptor. Knockdown of either Npnt or Itga8 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 or 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 Itga8, focal adhesion kinase (FAK) and Rho kinase (ROCK). Combined, these data suggest that Npnt augments cell migration into the presumptive cornea ECM by functioning as a substrate for Itgα8-positive pNC cells.

Data availability

All data generated or analyzed during this study are included in the manuscript and supporting file; Source Data files have been provided for Figures 2-7.

Article and author information

Author details

  1. Justin Ma

    Department of Biosciences, Rice University, Houston, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Lian Bi

    Department of Biosciences, Rice University, Houston, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. James Spurlin

    Department of Biosciences, Rice University, Houston, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Peter Lwigale

    Department of Biosciences, Rice University, Houston, United States
    For correspondence
    lwigale@rice.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1799-4905

Funding

National Eye Institute (EY031381)

  • Peter Lwigale

National Eye Institute (EY022158)

  • Peter Lwigale

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Animal experimentation: This study was conducted in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Fertilized chick embryos incubated between 1 to 17 days were handled according to the approved institutional animal care and use committee (IACUC) protocol (#IACUC-20-190) of Rice University.

Reviewing Editor

  1. Carole LaBonne, Northwestern University, United States

Publication history

  1. Received: September 29, 2021
  2. Preprint posted: October 13, 2021 (view preprint)
  3. Accepted: March 2, 2022
  4. Accepted Manuscript published: March 3, 2022 (version 1)
  5. Version of Record published: March 11, 2022 (version 2)

Copyright

© 2022, Ma et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 843
    Page views
  • 96
    Downloads
  • 1
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Justin Ma
  2. Lian Bi
  3. James Spurlin
  4. Peter Lwigale
(2022)
Nephronectin-Integrin α8 signaling is required for proper migration of periocular neural crest cells during chick corneal development
eLife 11:e74307.
https://doi.org/10.7554/eLife.74307

Further reading

    1. Developmental Biology
    2. Neuroscience
    Emily L Heckman, Chris Q Doe
    Research Advance Updated

    The organization of neural circuits determines nervous system function. Variability can arise during neural circuit development (e.g. neurite morphology, axon/dendrite position). To ensure robust nervous system function, mechanisms must exist to accommodate variation in neurite positioning during circuit formation. Previously, we developed a model system in the Drosophila ventral nerve cord to conditionally induce positional variability of a proprioceptive sensory axon terminal, and used this model to show that when we altered the presynaptic position of the sensory neuron, its major postsynaptic interneuron partner modified its dendritic arbor to match the presynaptic contact, resulting in functional synaptic input (Sales et al., 2019). Here, we investigate the cellular mechanisms by which the interneuron dendrites detect and match variation in presynaptic partner location and input strength. We manipulate the presynaptic sensory neuron by (a) ablation; (b) silencing or activation; or (c) altering its location in the neuropil. From these experiments we conclude that there are two opposing mechanisms used to establish functional connectivity in the face of presynaptic variability: presynaptic contact stimulates dendrite outgrowth locally, whereas presynaptic activity inhibits postsynaptic dendrite outgrowth globally. These mechanisms are only active during an early larval critical period for structural plasticity. Collectively, our data provide new insights into dendrite development, identifying mechanisms that allow dendrites to flexibly respond to developmental variability in presynaptic location and input strength.

    1. Developmental Biology
    Hidenobu Miyazawa, Marteinn T Snaebjornsson ... Alexander Aulehla
    Research Article

    How cellular metabolic state impacts cellular programs is a fundamental, unresolved question. Here we investigated how glycolytic flux impacts embryonic development, using presomitic mesoderm (PSM) patterning as the experimental model. First, we identified fructose 1,6-bisphosphate (FBP) as an in vivo sentinel metabolite that mirrors glycolytic flux within PSM cells of post-implantation mouse embryos. We found that medium-supplementation with FBP, but not with other glycolytic metabolites, such as fructose 6-phosphate and 3-phosphoglycerate, impaired mesoderm segmentation. To genetically manipulate glycolytic flux and FBP levels, we generated a mouse model enabling the conditional overexpression of dominant active, cytoplasmic PFKFB3 (cytoPFKFB3). Overexpression of cytoPFKFB3 indeed led to increased glycolytic flux/FBP levels and caused an impairment of mesoderm segmentation, paralleled by the downregulation of Wnt-signaling, reminiscent of the effects seen upon FBP-supplementation. To probe for mechanisms underlying glycolytic flux-signaling, we performed subcellular proteome analysis and revealed that cytoPFKFB3 overexpression altered subcellular localization of certain proteins, including glycolytic enzymes, in PSM cells. Specifically, we revealed that FBP supplementation caused depletion of Pfkl and Aldoa from the nuclear-soluble fraction. Combined, we propose that FBP functions as a flux-signaling metabolite connecting glycolysis and PSM patterning, potentially through modulating subcellular protein localization.