Pou4F2/Brn3B Overexpression Promotes the Genesis of Retinal Ganglion Cell-Like Projection Neurons from Late Progenitors

Abstract

ABSTRACTRetinal ganglion cells (RGCs) are the projection neurons of the retina. In early retinal progenitor cells (RPCs),Atoh7orchestrates the developmental RGC program and regulates the expression of critical downstream targets, includingPou4ffactors. The absence ofPou4f2or more POU4F family genes results in defects in RGC differentiation, aberrant axonal elaboration and ultimately RGC death, confirming the requirement of POU4F factors for RGC development and survival, with a critical role in regulating RGC axon outgrowth and pathfinding. Here, we investigatedin vivowhether ectopicPou4f2expression in late retinal progenitor cells (late RPCs) is sufficient to induce the generation of cells with RGC properties, including projecting axons to the brain. Using a strong ubiquitous promoter to inducePou4f2overexpression in neonates, we observed a change in targeted cell distribution in the retinal tissue, including the presence of cells in the ganglion cell layer and inner plexiform layer with high density of GFP+processes along the retina. Similar results on the induction of neuron processes were obtained when we overexpressedPou4f2inAtoh7knockout mice, suggesting that POU4F2 is sufficient to induce them. Single-cell RNA sequencing (scRNA-seq) analysis shows that several RGC-genes (such asRbpms,Gap-43,Hs6st3, andFoxp2) are upregulated afterPou4f2overexpression. Additionally, gene ontology analysis indicates the induction of genes related to axonogenesis and neuronal differentiation. Imaging throughout the visual pathway revealed high density of axons projecting toward the optic nerve head and extending to brain regions, such as the superior colliculus and geniculate nucleus. Thus,Pou4f2induced neurons with specific RGC characteristics that share similarities with resident RGCs and notably project axons that reach brain targets. In conclusion, these results showed that POU4F2 alone was sufficient to promote critical properties of projection neurons from retinal progenitors outside their developmental window.