microRNAs in Retinal Development and Müller glia Reprogramming

Abstract

"Retinal diseases lead to visual impairments with loss of neurons and often cause blindness. Since the mammalian retina has no regenerative capability, one of attractive strategies is cell replacement, achieved by reprogramming endogenous mature cells. There are attractive molecules that regulate gene expression and are elementary during retinal development (retinogenesis), and also can induce reprogramming in mouse Müller glia (MG). MG, predominant glia in the retina, are known to have regenerative capacity naturally in fish, unlike mammalian. Recent studies demonstrated that microRNAs (miRNAs) play an important role in the embryonic phase of retinogenesis, and the set of miRNAs can induce mouse MG reprogramming into neuronal-like cells that express neuronal markers and have neuronal morphologies. The miRNAs were, however, poorly understood in the postnatal processes of retinogenesis. Furthermore, the questions include which cell types can be generated and, most importantly whether these neuronal cells are mature and functional were not answered yet. Therefore, the aims of this study were to 1) analyze the effects of loss of miRNAs in the late retinal progenitor cells (RPCs) during postnatal retinal development, to 2) evaluate the reprogramming efficiency of the RPC-miRNA miR-25 with regard to the generation of functional neurons. Aim 2 included the establishment of an improved cell culture protocol together with calcium imaging in order to investigate the functionality and maturation state of these newly generated neuronal-like cells. Aims one and two included the establishment of luciferase reporter assays to identify a possible underlying mechanism. Loss of miRNAs in late RPCs is caused by a genetic knock-out of the enzyme Dicer, required for the formation of mature, functional miRNA and Ascl1CreER:tdTomato strain (RPC reporter mouse). The tissue was examined at different timepoints and evaluated histologically. For MG reprogramming, RPC-miRNA miR-25 was overexpressed using artificially made miRNAs called mimics, in mouse primary MG. Cells were monitored and evaluated over the time course of three weeks. miRNA target gene analyses were performed to identify potential underlying mechanisms. Our data showed that loss of miRNAs in late RPC reduced populations of bipolar cells, rod photoreceptors, MG and increased an amacrine cell population. We found that miR-25 directly target amacrine cell mRNAs, Elavl3, a gene that encodes for HuC, a protein expressed in amacrine cells. Using the RPC-miRNA miR-25 to reprogram young primary MG led to a conversion of the cells to more immature cells. We show for the first time that one miRNA alone, namely miR-25, was sufficient to successfully reprogram primary MG into progenitor cells that subsequently differentiate into functional neurons. This cell conversion might be partly due to the inhibition of the neuronal repressor Rest, a direct target of miR-25. Furthermore, we found that miRNA molecules were taken up efficiently by all glia, however, not all glia were reprogrammed. This suggests that the competence state of the glia might play a role in reprogramming. Taken together, late RPC-miRNAs are essential for the proper development of late-born cells and miRNA loss leads to reduced bipolar cells, photoreceptors, and MG populations but an increase in amacrine cells. The reduced bipolar cell number might be partly due to a competent shift of the progenitors towards amacrine cell generation. Furthermore, the RPC-miRNA miR-25 alone is sufficient to successfully reprogram primary MG into progenitor cells that subsequently differentiate into functional neurons. The neuronal repressor Rest might be one of the primary key players in this process. Since miRNA molecules were taken up efficiently, remained at least 4 weeks after transfection, and no toxicity was detected, miR-25 might be a promising tool for in vivo MG reprogramming. "