Abstract
Purpose:
In humans, photoreceptor death causes permanent visual impairment, whereas, in zebrafish, photoreceptor death stimulates a regenerative response that restores photoreceptors and visual function. Identifying the mechanisms that govern photoreceptor regeneration in zebrafish can provide critical information for developing regenerative therapies that restore photoreceptors in humans. In embryonic zebrafish, the bHLH transcription factor NeuroD governs photoreceptor genesis through Delta-Notch signaling (Taylor et al., under review), but the role of NeuroD during photoreceptor regeneration is unknown. The purpose of this study was to test the role of NeuroD in adult photoreceptor regeneration and identify mechanisms through which NeuroD functions.
Methods:
Following light-induced photoreceptor ablation, NeuroD was knocked down by electroporating ATG-targeted morpholinos into the retina. Standard control morpholinos were used as controls. 5-Ethynyl-2’-Deoxyuridine (EdU) was injected IP at 3 days post lesion (dpl), and 5-Bromo-2’-Deoxyuridine (BrdU) was administered by immersion at 5-6 dpl to label dividing cells and determine the percentage of progenitors remaining in the cell cycle at 6 dpl. In-situ hybridization was used to label regenerated rods and cones at 6 and 7 dpl and to evaluate the expression of Notch pathway molecules. Finally, following NeuroD knockdown, the gamma secretase inhibitor DAPT was used to inhibit Notch signaling.
Results:
NeuroD knockdown resulted in an increased proportion of EdU+ cells co-labeled with BrdU at 6 dpl, indicating that NeuroD is required for injury-induced progenitors to exit the cell cycle. Correlated with this, there were significantly fewer regenerated photoreceptors in experimental retinas compared to controls, indicating that NeuroD is required for photoreceptor regeneration. NeuroD knockdown caused up-regulation of notch1a, deltaA and deltaD, as well as the Notch targets her4 and ascl1a, suggesting that NeuroD transcriptionally represses Notch signaling. Following NeuroD knockdown, inhibition of Notch signaling with the Gamma secretase inhibitor DAPT rescued the defects in cell cycle exit and photoreceptor regeneration.
Conclusions:
These data are interpreted to show that following photoreceptor ablation, NeuroD governs progenitor cell cycle exit and photoreceptor regeneration by negatively regulating Delta-Notch signaling.