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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.

In a prior study, a S163R mutation in the complement-1q tumor necrosis factor-related protein 5 (CTRP5/ C1QTNF5) was reported to be associated with early-onset long anterior zonules (LAZ) and late-onset retinal degeneration (L-ORD). The ocular tissues involved in the phenotype are the retinal pigment epithelium (RPE) in the posterior segment and ciliary epithelium (CE) and lens in the anterior segment. The purpose of this study was to characterize the spatial and temporal expression of the mouse Ctrp5 gene, determine tissue and subcellular localization, and study the effect of the S163R mutation.The expression of the Ctrp5 gene in the mouse was studied by quantitative (q)RT-PCR and in situ hybridization. CTRP5 protein expression and distribution were studied by Western blot analysis, immunohistochemistry, and immunoelectron microscopy. Cellular location of wild-type and mutant CTRP5 in MDCK and COS-7 cells was determined by immunofluorescence and immunoblot analysis.A significant level of Ctrp5 expression was detected in the adult mouse in the ciliary body (CB) and RPE, and expression started at a very early stage of embryogenesis. Immunohistochemical analysis showed CTRP5 protein in the apical processes of the RPE and forming a hexagonal lattice associated with the RPE lateral membranes. In the ciliary body, CTRP5 was localized to the apical aspects of the CE, the region between the bilayered ciliary epithelial cells. The membrane association of CTRP5 in the RPE and CE was further confirmed by immunoelectron microscopy. Furthermore, cultured cells were used to show that the CTRP5 is a secretory protein and that its secretion is impaired by the S163R mutation.CTRP5, a secretory and membrane-associated protein, is localized to the lateral and apical membranes of the RPE and CB. Impaired secretion of the mutant protein may underlie the pathophysiology of L-ORD and LAZ.

Purpose To report a novel mouse model of achromatopsia with a cpfl3 mutation found in the ALS/LtJ strain. Methods The effects of a cpfl3 mutation were documented using fundus photography, electroretinography (ERG), and histopathology. Genetic analysis was performed using linkage studies and PCR gene identification. Results Homozygous cpfl3 mice had poor cone-mediated responses on ERG at 3 weeks that became undetectable by 9 months. Rod-mediated waveforms were initially normal, but declined with age. Microscopy of the retinas revealed progressive vacuolization of the photoreceptor outer segments. Immunocytochemistry with cone-specific markers showed progressive loss of labeling for alpha-transducin, but the cone outer segments in the oldest mice examined remained intact and positive with peanut agglutinin (PNA). The cpfl3 mapped to mouse chromosome 3 at the same location as human GNAT2, known to cause achromatopsia. Sequence analysis revealed a missense mutation due to a single base pair substitution in exon 6 in cpfl3. Conclusions The Gnat2(cpfl3) mutation leads to cone dysfunction and the progressive loss of cone alpha-transducin immunolabeling. Despite a poor cone ERG signal and loss of cone alpha-transducin label, the cones survive at 14 weeks as demonstrated by PNA staining. This mouse model of achromatopsia will be useful in the study of the development, pathophysiology, and treatment of achromatopsia and other cone degenerations. The gene symbol for the cpfl3 mutation has been changed to Gnat2(cpfl3).