Late retinal progenitor microRNA depletion impairs bipolar cell generation

Abstract

Purpose : There is increasing evidence that epigenetic factors such as microRNAs (miRNAs) are required for proper retinal development and overall retinal health. While numerous factors have been identified that are essential for the formation and differentiation of the various retinal cells including Muller glia (MG), the role of miRNAs in postnatal retinogenesis is still poorly understood.

Methods : We used the Ascl-CreER:tdTomato^stopfl/fl reporter mouse (referred to as wildtype) and created a Dicer1 conditional knock mouse to specifically deplete mature miRNAs in late retinal progenitor cells (RPCs), i.e. the Ascl-CreER:Dicer^fl/fl:tdTomato^stopfl/fl mouse (further referred to as cKO_RPC). The CreER was activated at postnatal day (P) 1-3. Optical coherence tomography (OCT) and electroretinograms (ERGs) in combination with immunofluorescent labeling were used to investigate the structural integrity, function, and cellular composition of retinas of wildtype and cKO_RPC mice at postnatal day P56.

Results : Cellular analysis of the adult Ascl1:tdTomato mouse (wildtype) revealed that the progenies from late RPCs (P1-3) differentiated into bipolar cells (BCs), MG as well as a subset of amacrine cells (ACs). This reporter signal declined with age but was still found in a few immature MG in the peripheral retina until P13. In the P56 cKO_RPC, we found a thinning of the outer and inner nuclear layers suggesting neuronal loss. The area of inner segment/RPE was also reduced but no functional impairment of the retina was found. Histological analysis showed that the retinas of cKO_RPC mice had a smaller BC-progeny population (~20% less), and an increased AC-progeny population (doubled cell number), but an unaffected MG-progeny population, compared to age-matched wildtype littermates. Moreover, a new, immature (solely Sox9+) population (~17%) was present in the cKO_RPC retinas. We identified Elavl3 which encodes for the AC marker HuC, as a target of miR-25, a highly expressed miRNA in late RPCs. The reduction of miR-25 probably caused the shift to AC identity.

Conclusions : Our data suggests that the P1-3 RPC-miRNAs are essential for proper BC generation. Furthermore, RPC-miRNAs seem not to be required for MG generation, but probably for their maturation. The increase in the AC population seems to be due to the RPC-miRNA miR-25 which appears to negatively regulate AC differentiation via Elavl3.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.