Abstract
Purpose :
In humans, numerous retinal diseases result in photoreceptor loss and permanent blindness. The ability to replace/regenerate photoreceptors will require detailed knowledge of the mechanisms that govern photoreceptor genesis. In zebrafish, the bHLH transcription factor NeuroD governs photoreceptor genesis through Notch signaling, but information is lacking regarding mechanisms that regulate NeuroD. Members of the miR-17-92 microRNA cluster regulate CNS neurogenesis, and a member of this cluster, miR-18a, is predicted to target neuroD mRNA. The purpose of this study was to determine if miR-18a regulates NeuroD in the retina and if it plays a role in photoreceptor genesis.
Methods :
Quantitative PCR and in situ hybridization were used to analyze miR-18a expression in the embryonic and larval retina. Morpholino oligonucleotides (MO) that target the guide strand of the miR-18a precursor (pre-miR-18a) were used to knock down miR-18a and standard control morpholinos served as controls. Embryos were immersed in 1.5 mM EdU for 20 minutes prior to sacrifice to label proliferating cells. Western blot analysis was performed on embryo heads to measure levels of NeuroD and immunocytochemistry was used to label mature photoreceptors. For use in future experiments, CRISPR/Cas9 gene editing was used to generate mutations in pre-miR-18a at the site targeted by the miR-18a MO.
Results :
At 48 and 72 hpf, corresponding to the start and completion of photoreceptor genesis, miR-18a is expressed throughout the retina. At 48 hpf, relative to controls, knockdown of miR-18a resulted in a 37% increase in NeuroD protein. At 72 hpf, miR-18a knockdown did not alter cell proliferation, but resulted in larger eyes and a 27% increase in the number of mature photoreceptors. These data indicate that miR-18a negatively regulates NeuroD and functions in newly generated photoreceptors to regulate terminal differentiation. Using CRISPR/Cas9 genome editing, three different miR-18a mutant fish lines have been created with insertions or deletions in the guide strand of pre-miR-18a that we predict will prevent formation of mature miR-18a.
Conclusions :
These data indicate that during embryonic development, miR-18a regulates terminal differentiation of photoreceptors and suggest that miR-18a functions through regulation of NeuroD protein levels. We propose that this mechanism regulates the timing and extent of photoreceptor differentiation in the vertebrate retina.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.