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 HB-EGF/Ascl1/Lin28a 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. HB-EGF was significantly up-regulated by 8 h and expression increased through 12 h. By 48 h, up-regulation of Ascl1 and Lin28a 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, Otx2, Nestin, and Vsx2, 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 HB-EGF/Ascl1/Lin28a 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.

该实验室的先前研究已经确定，在成年啮齿动物的眼药水中滴加α7烟碱乙酰胆碱受体（nAChR）激动剂PNU-282987后，穆勒胶质细胞会发生去分化。PNU-282987作用于视网膜色素上皮细胞上的α7nAChR，以刺激Müller来源的祖细胞（MDPC）的产生并最终导致神经发生。本研究旨在检验以下假设：HB-EGF / Ascl1 / Lin28aMüller胶质细胞中的信号通路导致MDPC的起源。在与从PNU-282987处理的视网膜色素上皮（RPE）细胞系收集的上清液接触后，在Müller胶质细胞系（rMC-1）上进行RNA测序。将差异调节基因与已发表的Müller胶质细胞去分化的文献进行了比较，该文献发生在较低的脊椎动物再生和哺乳动物早期发育中。乙肝生长因子在8 h时明显上调了VEGF的表达，在12 h时表达升高。到48小时，Ascl1 和 林28a观察到，两个已知在脱分化斑马鱼Müller胶质细胞中快速诱导的基因。还观察到已知与哺乳动物发育和斑马鱼再生有关的其他基因的上调，以及一些使Müller胶质细胞特性所需的因子下调。使用qRT-PCR验证了RNA-seq结果。使用免疫细胞化学，与MDCP身份相关的标记物的存在，Otx2， 巢蛋白和 VS2发现其在48小时治疗组培养物中表达。这项研究的新颖之处在于，它可以通过刺激与成年啮齿类动物相关的基因来诱导成年啮齿动物的胶质细胞再生。HB-EGF / Ascl1 / Lin28a从PNU-282987处理过的RPE引入条件培养基后，导致MDPC的途径。这项研究使我们进一步了解了成年哺乳动物视网膜中Müller胶质细胞对PNU-282987响应的去分化机理。