Reprogramming lineage-traced Müller glia for robust proliferation and neurogenesis in adult mammalian retinas

The therapeutic potential of adeno-associated virus (AAV)-mediated one-step glia-to-neuron conversion has been challenged following rigorous lineage-tracing analyses. In zebrafish, Müller glia (MG) serve as retinal stem cells to replenish lost neurons after injury. In contrast, mammalian MG do not spontaneously re-enter the cell cycle, and limited neurogenesis occurs in response to neurotoxic injury. Here, we demonstrate that activation of FGF2/MAPK signaling, following AAV-mediated gene transfer of Ascl1, effectively stimulates lineage-traced MG to undergo robust cell-cycle re-entry in adult mice, independent of neurotoxic injury. With the addition of retinoic acid, this approach further reprograms a significant proportion of proliferative MG-derived progenitor-like cells into regenerative states, driving enhanced in vivo neurogenesis. Using multiplex techniques, we reveal distinct phases of cell fate transitions during in vivo MG-derived neurogenesis. This approach provides a two-step strategy for inducing MG proliferation and subsequent MG-derived neurogenesis, which may represent a potent avenue toward retinal regeneration.