The bHLH Transcription Factor NeuroD Governs Photoreceptor Genesis and Regeneration Through Delta-Notch Signaling.

The vertebrate retina is composed of seven cell types, generated in a conserved sequence from a pool of multipotent, mitotic progenitors.1 Several basic helix-loop-helix (bHLH) transcription factors have prominent roles in the retina to regulate cell fate specification,2–5 cell cycle exit, and neuronal differentiation and maturation.6,7 NeuroD is a proneural bHLH transcription factor identified in vivo as a neuronal determination factor,8 and in vitro as a link between cell cycle exit and neuronal differentiation.9 In the mouse retina, NeuroD is required for survival of rods10 and regulates opsin selection among cones.11 In the chick, NeuroD is required for photoreceptor development and survival.12 In medaka fish, NeuroD and Six6 coregulate each other to control amacrine cell and photoreceptor genesis.6 In embryonic zebrafish, neurod is expressed in mitotic photoreceptor progenitors,13,14 and this expression is regulated by the zinc finger protein, Insm1a.15 Within the photoreceptor lineages NeuroD governs cell cycle exit and photoreceptor maturation.7 The Notch pathway mediates cell-to-cell communication through receptor-ligand interactions. Notch receptors are expressed on the cell surface and interact with membrane-bound ligands (e.g., Delta, Jagged), regulating transcription in apposing cells.16,17 In vertebrate retinal development, Notch signaling regulates the balance between neurogenesis and gliogenesis,18–20 maintains progenitors in an undifferentiated, proliferative state,19,21 specifies cell fates, and governs the onset of neurogenesis.22 These events can be regulated in the retina through transcriptional control of Notch signaling molecules. For example, in the chick and mouse, the bHLH transcription factor Ascl1a governs cell cycle exit and differentiation through regulation of the Notch ligand dll,23,24 and in zebrafish the HLH-binding cofactor Id2a regulates the transition from proliferation to differentiation through regulation of Notch ligands and receptors.21 Following retinal damage in zebrafish, Muller glia function as intrinsic stem cells that dedifferentiate, reenter the cell cycle, and produce neurogenic progenitors that regenerate lost neurons.25–33 Recent studies in zebrafish have identified mechanisms that govern the Muller glia response to retinal injury,34–37 but information is lacking around the pathways that govern photoreceptor regeneration from Muller glia–derived progenitor cells. The mechanisms that govern retinal regeneration are expected to largely recapitulate embryonic development,28,29,38 though few molecules have been functionally studied during both events. NeuroD function has not been studied in the adult zebrafish retina, but following photoreceptor ablation neurod is expressed in Muller glia–derived mitotic progenitors,39 suggesting that NeuroD has a role in photoreceptor regeneration. The goal of the current study is to identify the mechanisms that govern photoreceptor genesis from the pool of multipotent progenitors in the embryo and from stem cell-derived progenitors in the adult, by elucidating the pathways through which NeuroD functions during photoreceptor development and regeneration, respectively. In embryos, reciprocal transplant chimeric analysis shows that for cell cycle exit and photoreceptor maturation, NeuroD function is non–cell-autonomous. Knockdown of NeuroD and CRISPR/Cas9 targeted mutation of neurod prevent cell cycle exit and photoreceptor maturation, and increase expression of Notch pathway molecules. Inhibition of Notch signaling rescues deficiencies in cell cycle exit but not photoreceptor maturation. In adults, NeuroD knockdown prevents cell cycle exit among injury-induced progenitors and photoreceptor regeneration, and this, too, is rescued by Notch inhibition. These data demonstrated a conserved function for NeuroD during photoreceptor genesis and regeneration, and identified Notch signaling as a molecular mechanism that links these events.