The Role of MicroRNA-124 in the Development and Differentiation of Photoreceptors
Date of Award
Doctor of Philosophy (PhD)
Photoreceptors are the primary sensory neurons of the retina and are prone to a wide range of neurodegenerative diseases that cause blindness. Elucidating the photoreceptor gene regulatory network is essential to understanding the mechanisms behind photoreceptor differentiation, function and degeneration. Although it had long been thought that most of the regulation of gene expression occurs at the level of transcription, it is now becoming clear that post-transcriptional regulation of gene expression, particularly regulation controlled by microRNAs (miRNAs), plays an important role in cellular proliferation and differentiation. MiR-124 is the most abundantly expressed miRNA in the retina and brain and is predicted to regulate thousands of genes. In my thesis work, I studied the role of miR-124 in the development and differentiation of photoreceptors in the mouse retina. Specifically, I used both knockdown and misexpression approaches to elucidate the function of miR-124. I found that miR-124 knockdown resulted in an increase in the number of cells expressing progenitor cell markers and Müller glial markers at the expensive of the number of cells expressing photoreceptor markers. When miR-124 was misexpressed, more cells expressed photoreceptor markers with a reduction in the number of cells expressing progenitor markers. These results would suggest miR-124 plays a role in photoreceptor differentiation and exit from the cell cycle.
In mouse brain, miR-124 has been shown to regulate adult neurogenesis through its repression of the transcription factor, Sox9. In the retina, Sox9 and the related transcription factor, Sox8, are expressed in all retinal progenitor cells. Expression of these two factors is turned off as photoreceptors and other retinal neurons exit the cell cycle and differentiate. The expression of these factors persist in Müller glia into the adult stage. In this study, I demonstrated that miR-124 targets Sox8 and Sox9 to regulate photoreceptor differentiation. The 3' untranslated regions (UTRs) of both Sox8 and Sox9 contain predicted binding sites for miR-124. I report that mutation of the seed region of these sites in the 3'UTRs of Sox8 and Sox9 resulted in a derepression of the reporter vectors, demonstrating the regulatory properties of these predicted binding sites. In addition, knockdown of Sox8 and Sox9, in the miR-124 knockdown phenotype, evoked a partial restoration of the wild type phenotype. These results show that miR-124 is acting partially through repression of Sox8 and Sox9 in photoreceptor precursors to promote neurogenesis.
Finally, understanding the regulation of miR-124 is essential to understanding its function in the retina. Prior studies have demonstrated that transcription factors, which regulate protein-encoding genes, can also regulate miRNA expression. Here, I report evidence of miR-124 transcriptional regulation by Crx, a transcription factor required for photoreceptor development. Crx ChIP-Seq (from a previous study) and Crx ChIP-qPCR data demonstrate clusters of Crx-bound regions (CBRs) near the miR-124-1 and miR-124-2 loci which suggest that Crx regulates miR-124 at these loci. In addition, I have experimentally demonstrated that many of the CBRs act in a regulatory manner for reporter expression. The regulatory activity shows that these CBRs are functional in the retinal environment. Surprisingly, expression of miR-124 in the Crx -/- mutant strain is not abnormal. The results evaluated together suggest that photoreceptor transcription factor, Crx, may regulate miR-124 expression in the retina. Overall, my thesis work provides a model for the regulatory network in which miR-124 functions. The transcription factor, Crx, regulates expression of miR-124 which in turn represses Sox8 and Sox9 to promote photoreceptor neurogenesis.
Chair and Committee
Joseph C Corbo
David Beebe, Shiming Chen, Kathleen Hall, Vladimir Kefalov, Michael Lovett
Williams, Natecia L., "The Role of MicroRNA-124 in the Development and Differentiation of Photoreceptors" (2013). Arts & Sciences Electronic Theses and Dissertations. 144.