Overexpression of Meis factors in late-stage retinal progenitors yields complex effects on temporal patterning and neurogenesis.

The vertebrate retina serves as a model for studying neurogenesis and cell fate specification, with retinal progenitor cells following a tightly regulated temporal sequence to generate distinct cell types. Meis1 and Meis2 are transcription factors implicated in early retinal development, but their role in late-stage RPCs remains poorly understood. Here, we investigate whether Meis1 and Meis2 overexpression in postnatal mouse RPCs can alter temporal identity and induce early-born cell types. Using electroporation and single-cell RNA sequencing, we find that while these factors modestly upregulate early-stage gene regulatory network components, they do not repress late-stage transcription factors or induce early-born retinal cells. Meis1 overexpression reduces proliferation and inhibits neurogenesis, whereas Meis2 overexpression accelerates neurogenic progression without altering fate commitment. Our findings suggest that overexpression of Meis1 and Meis2 modulate largely non-overlapping aspects of temporal identity and neurogenic competence but are insufficient to fully reprogram late-stage progenitors. These results have implications for regenerative strategies aimed at reprogramming retinal cells for therapeutic purposes. Overall design: Wildtype CD1 murine retinal explants were treated with a pCAGIG- empty control, a pCAGIG-NR2F1, a pCAGIG-NR2F2, or a 50:50 combination of pCAGIG-NR2F1 and pCAGIG-NR2F2 for 2 or 5 days prior to collection and library generation. Retinal explants were lysed into a single cell suspension, GFP+ cells were isolated and run on the 10x Genomics Chromium Single Cell System platform using the Single Cell 3'Reagents Kit v3.1. Libraries were sequenced on Illumina NovaSeq 6000 and processed through the Cell Ranger pipeline using a modified MM10 reference genome with an added loci for GFP transcripts.