Department of Biology, University of West Florida, 11000 University Parkway, Pensacola, Florida, 32514.
Ophthalmology and Visual Sciences, University of Michigan, W. K. Kellogg Eye Center, 1000 Wall Street, Ann Arbor, Michigan, 48105.
Dev Neurobiol. 2019 Feb;79(2):202-219. doi: 10.1002/dneu.22666. Epub 2019 Jan 25.
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.
在胚胎视网膜发育过程中，多能祖细胞以严格的时空模式产生六种视网膜神经元。这种模式需要细胞周期退出（即神经发生）和分化以谱系特异性的方式精确调节。在斑马鱼中，bHLH 转录因子 NeuroD 通过 Notch 信号传导来控制光感受器的发生，但也通过目前未知的不同机制来控制光感受器的分化。NeuroD 的调节机制和光感受器发育的时空模式也未知。miR-17-92 微 RNA 簇的成员调节中枢神经系统神经发生，该簇的一个成员 miR-18a 被预测靶向 neuroD mRNA。本研究的目的是确定在发育中的斑马鱼视网膜中，miR-18a 是否调节 NeuroD 及其是否在光感受器发育中发挥作用。定量 RT-PCR 显示，在三个 miR-18 家族成员（miR-18a、b 和 c）中，miR-18a 的表达与 neuroD 的表达最密切相关。形态发生素寡核苷酸和 CRISPR/Cas9 基因编辑用于 miR-18a 的功能丧失（LOF），两者都导致 70 hpf 时幼虫的光感受器更成熟，而不影响细胞增殖。Western blot 显示 miR-18a LOF 增加 NeuroD 蛋白水平，体外双荧光素酶测定显示 miR-18a 直接与 neuroD 的 3'UTR 相互作用。最后，tgif1 突变体中 miR-18a 的表达增加，NeuroD 蛋白减少，成熟的光感受器减少，miR-18a 敲低可挽救光感受器缺陷。总之，这些结果表明，miR-18a 独立于神经发生调节光感受器分化的时间，并表明这是通过 NeuroD 的转录后调节来实现的。
