RT Journal Article SR Electronic T1 The microRNA, miR-18a, regulates NeuroD and photoreceptor differentiation in the retina of the zebrafish JF bioRxiv FD Cold Spring Harbor Laboratory SP 440263 DO 10.1101/440263 A1 Taylor, Scott M. A1 Giuffre, Emily A1 Moseley, Patience A1 Hitchcock, Peter F. YR 2018 UL http://biorxiv.org/content/early/2018/10/10/440263.abstract AB During embryonic retinal development, six types of retinal neurons are generated from a pool of multipotent progenitors in a strict spatiotemporal pattern. This pattern requires cell cycle exit (i.e. neurogenesis) and differentiation to be precisely regulated in a lineage-specific manner. In zebrafish, the bHLH transcription factor NeuroD governs photoreceptor genesis through Notch signaling but also governs photoreceptor differentiation though distinct mechanisms that are currently unknown. Also unknown are the mechanisms that regulate NeuroD and the spatiotemporal pattern of photoreceptor development. 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 development. Quantitative RT-PCR showed that, of the three miR-18 family members (miR-18a, b and c), miR-18a expression most closely parallels neuroD expression. Morpholino oligonucleotides and CRISPR/Cas9 gene editing were used for miR-18a loss-of-function (LOF) and both approaches resulted in larvae with more mature photoreceptors at 70 hpf without affecting cell proliferation. Western blot showed that miR-18a LOF increases NeuroD protein levels and in vitro dual luciferase assay showed that miR-18a directly interacts with the 32UTR of neuroD. Finally, tgif1 mutants have increased miR-18a expression, less NeuroD protein and fewer mature photoreceptors, and the photoreceptor deficiency is rescued by miR-18a knockdown. Together these results show that, independent of neurogenesis, miR-18a regulates the timing of photoreceptor differentiation and indicate that this occurs through post-transcriptional regulation of NeuroD.This work was supported by grants from the National Institutes of Health (NEI)-R01EY07060 (PFH), T32EY013934 (SMT), P30EY07003 (PFH) and an unrestricted grant from the Research to Prevent Blindness, New York. The authors declare no competing financial interests. The authors thank Laura Kakuk-Atkins and Dilip Pawar for technical assistance. Fish lines and reagents provided by ZIRC were supported by NIH-NCRR Grant P40 RR01.