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.
Highlights
In the developing retina, six types of neurons are generated from a pool of multipotent, mitotic progenitors in a sequence that is highly conserved among vertebrates (Bassett & Wallace, 2012; Centanin & Wittbrodt, 2014; Wallace, 2011)
Post-transcriptional regulation can occur through small ~22 nucleotide singlestranded RNA molecules called microRNAs that bind to target mRNA through complementary base paring and regulate protein expression by blocking translation and/or causing mRNA degradation (Huntzinger & Izaurralde, 2011)
Mature miRNAs typically function by binding via a specific “seed” sequence comprising ~6-8 nucleotides near the 5’ end of the miRNA, to a complementary sequence in the 3’ untranslated region (UTR) of the target mRNA (Bartel, 2004; Broughton, Lovci, Huang, Yeo, & Pasquinelli, 2016)
Summary
Six types of neurons are generated from a pool of multipotent, mitotic progenitors in a sequence that is highly conserved among vertebrates (Bassett & Wallace, 2012; Centanin & Wittbrodt, 2014; Wallace, 2011). NeuroD is required for photoreceptor progenitors to exit the cell cycle and differentiate, and if NeuroD levels are regulated by miR-18 miRNAs, knockdown of these molecules is expected to affect the rate of photoreceptor development. To determine if miR-18 miRNAs regulate photoreceptor development, morpholino oligonucleotides targeted to the mature sequences of miR-18a, mir-18b or miR-18c were injected into embryos at the single cell stage.
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