Regulation of Muller Glial Stem Cell Properties: Insights from a Zebrafish Model.

Abstract

In contrast to mammals, zebrafish are capable of regenerating retinal neurons. Essential to regenerative success are the stem cell properties that Müller glia acquire following retinal injury. My dissertation research investigated how Müller glial stem cell properties are regulated. First, we demonstrated that following acute photic lesion in zebrafish, repression of the Transforming Growth Factor beta (TGFβ) signaling pathway is necessary for efficient proliferation of Müller glia-derived retinal progenitor cells and photoreceptor regeneration. Next, we characterized and compared the transcriptional responses of isolated, injury-activated zebrafish Müller glia (as they acquire stem cell properties) to those of isolated, mouse Müller glia in degenerating retinas. This revealed transcriptional changes that are unique to zebrafish Müller glia, and provided candidates to explore as potential therapeutic targets to enhance endogenous Müller glial-dependent retinal regeneration in humans. Lastly, I characterized the mi2004 zebrafish line, in which a transgene insertional event resulted in 1) creation of a recessive, larval lethal mutation and 2) a transgenic reporter for the Müller glial regenerative response. The mutant eye phenotype included defects in retinal structure, cellular localization, proliferation, and Müller glial structure. RNA-sequencing-based, SNP-linkage analysis localized the transgene insertion to a locus on chromosome 9 containing ~40 strongly downregulated gamma-crystallin genes. Attempts to rescue the mutant phenotype by overexpression of this genetic locus have not been successful. Transcriptional analysis of mutant vs. wild-type siblings showed both similarities (increased NF-kappaB signaling, Jak-Stat signaling, arachidonic acid metabolism) and differences (increased TGFβ signaling) with injury-activated Müller glia, which highlights the importance of these signaling pathways in regulating retinal regeneration. In conclusion, this work has enhanced our understanding of Müller glial stem cell properties by demonstrating a novel role for TGFβ signaling regulation in regeneration, by generating a transcriptome database that allows the identification of testable differences in the responses to retinal injury of zebrafish and mouse Müller glia, and by discovering a genetic locus that contains elements that may regulate the regenerative response in Müller glia.