The MicroRNA, miR‐18a, Regulates NeuroD and Photoreceptor Differentiation in the Retina of Zebrafish

Abstract

During embryonic retinal development, six types of retinal neurons are generated from 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, in the developing zebrafish retina, miR‐18a regulates NeuroD 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 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 3′ UTR 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.

Disruption ofmiR-18aalters proliferation, photoreceptor replacement kinetics, inflammatory signaling and microglia/macrophage numbers during retinal regeneration in zebrafish

“…In miR-18a mi5012 retinas compared with WT, more cells continue to proliferate at 7 and 10 dpi, when most photoreceptor progenitors have normally exited the cell cycle, indicating that miR-18a regulates proliferation of MG-derived progenitors. This differs from findings in the developing embryonic retina in which miR-18a regulates the timing of photoreceptor differentiation but does not regulate cell proliferation [30]. The function of miR-18a to limit progenitor proliferation also differs from the general function of miR-18a in cancer, in which it typically promotes the proliferation of tumor cells [47,48] including glioblastoma cells [49].…”

“…Several miRNAs have been identified as important regulators of inflammation and could be key neuroinflammatory regulators in the injured retina [26,27], but studies linking miRNAs with retinal inflammation are lacking. The miRNA miR-18a was recently identified as an important regulator of photoceptor differentiation in the developing embryonic retina [30], and miR-18a is predicted to interact with mRNAs of more than 25 inflammation-related molecules (http://www.targetscan.org/fish_62), suggesting that miR-18a could regulate neuroinflammation in the retina, but the roles of miR-18a in injury-induced inflammation and photoreceptor regeneration had not been previously investigated.…”

“…The effect of miR-18a on the cell cycle during photoreceptor regeneration is strikingly different from embryonic development, in which miR-18a regulates photoreceptor differentiation but not cell proliferation [30]. This indicates that in the injured retina, miR-18a regulates pathways that are specific to the post-injury response.…”

“…MicroRNAs are small, 18-25 nucleotides long, non-coding RNAs that generally function by binding to the 3' untranslated region of mRNA and inhibit translation and/or promote mRNA degradation [28,29]. The miRNA, miR-18a, was recently found to regulate photoreceptor differentiation in larval zebrafish by suppressing levels of the transcription factor, NeuroD [30]. The role of miR-18a during photoreceptor regeneration in zebrafish is currently unknown.…”

The microRNA miR-18a Links Proliferation and Inflammation During Photoreceptor Regeneration in the Injured Zebrafish Retina

“…MicroRNAs are small, 18-25 nucleotides long, non-coding RNAs that generally function by binding to the 3' untranslated region of mRNA and inhibit translation and/or promote mRNA degradation [28,29]. The miRNA, miR-18a, was recently found to regulate photoreceptor differentiation in larval zebra sh by suppressing levels of the transcription factor, NeuroD [30]. The role of miR-18a during photoreceptor regeneration in zebra sh is currently unknown.…”