AUTHOR=Stanchfield Megan L. , Webster Sarah E. , Webster Mark K. , Linn Cindy L. 

TITLE=Involvement of HB-EGF/Ascl1/Lin28a Genes in Dedifferentiation of Adult Mammalian Müller Glia

JOURNAL=Frontiers in Molecular Biosciences

VOLUME=7

YEAR=2020

URL=https://www.frontiersin.org/journals/molecular-biosciences/articles/10.3389/fmolb.2020.00200

DOI=10.3389/fmolb.2020.00200

ISSN=2296-889X

ABSTRACT=<p>Previous studies from this lab have determined that dedifferentiation of Müller glia occurs after eye drop application of an α7 nicotinic acetylcholine receptor (nAChR) agonist, PNU-282987, to the adult rodent eye. PNU-282987 acts on α7 nAChRs on retinal pigment epithelial cells to stimulate production of Müller-derived progenitor cells (MDPCs) and ultimately lead to neurogenesis. This current study was designed to test the hypothesis that the activation of genes involved in the <italic>HB-EGF/Ascl1/Lin28a</italic> signaling pathway in Müller glia leads to the genesis of MDPCs. RNA-seq was performed on a Müller glial cell line (rMC-1) following contact with supernatant collected from a retinal pigment epithelial (RPE) cell line treated with PNU-282987. Differentially regulated genes were compared with published literature of Müller glia dedifferentiation that occurs in lower vertebrate regeneration and early mammalian development. <italic>HB-EGF</italic> was significantly up-regulated by 8 h and expression increased through 12 h. By 48 h, up-regulation of <italic>Ascl1</italic> and <italic>Lin28a</italic> was observed, two genes known to be rapidly induced in dedifferentiating zebrafish Müller glia. Up-regulation of other genes known to be involved in mammalian development and zebrafish regeneration were also observed, as well as down-regulation of some factors necessary for Müller glia cell identity. RNA-seq results were verified using qRT-PCR. Using immunocytochemistry, the presence of markers associated with MDCP identity, <italic>Otx2</italic>, <italic>Nestin</italic>, and <italic>Vsx2</italic>, were found to be expressed in the 48 h treatment group cultures. This study is novel in its demonstration that Müller glia in adult rodents can be induced into regenerative activity by stimulating genes involved in the <italic>HB-EGF/Ascl1/Lin28a</italic> pathway that leads to MDPCs after introducing conditioned media from PNU-282987 treated RPE. This study furthers our understanding of the mechanism by which Müller glia dedifferentiate in response to PNU-282987 in the adult mammalian retina.</p>