Regulation of retinal amacrine cell generation by miR-216b and Foxn3 [miRNA-seq P0]
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The mammalian retina contains a complex mixture of different types of neurons. We find that the microRNA miR-216b is preferentially expressed in postmitotic retinal amacrine cells in the mouse retina, and expression of miR-216a/b and miR-217 in the retina depend in part on Ptf1a, a transcription factor required for amacrine cell differentiation. Surprisingly, ectopic expression of miR-216b, or the related miR-216a, can direct the formation of additional amacrine cells in the developing retina. In addition, we observe the loss of bipolar neurons in the retina after miR-216b expression. We identify the mRNA for the transcriptional regulator Foxn3 as a retinal target of miR-216b by Argonaute PAR-CLIP and reporter analysis. Inhibition of Foxn3 in the postnatal developing retina by RNAi also increases the formation of amacrine cells and reduces bipolar cell formation, while overexpression of Foxn3 inhibits amacrine cell formation prior to the expression of Ptf1a. Disruption of Foxn3 by CRISPR in embryonic retinal explants also reduces amacrine cell formation. Co-expression of Foxn3 partially reverses the effects of ectopic miR-216b on retinal cell type formation. Our results identify Foxn3 as a novel regulator of interneuron formation in the developing retina and suggest that miR-216b likely regulates expression of Foxn3 and other genes in amacrine cells. Retina microRNA profiles of P0 wild-type mice
哺乳动物视网膜包含由多种不同类型神经元构成的复杂混合群体。我们发现，微小RNA（microRNA）miR-216b在小鼠视网膜的有丝分裂后无长突细胞中优先表达；视网膜内miR-216a/b与miR-217的表达一定程度上依赖于Ptf1a——一种调控无长突细胞分化所必需的转录因子。令人意外的是，异位表达miR-216b或其同源分子miR-216a，可在发育中的视网膜中诱导额外的无长突细胞生成。此外，我们观察到miR-216b表达后，视网膜内双极神经元出现数量丢失。通过Argonaute PAR-CLIP技术与报告基因分析，我们确认转录调控因子Foxn3的mRNA是miR-216b在视网膜中的靶标分子。通过RNA干扰（RNA interference，RNAi）抑制出生后发育视网膜内的Foxn3表达，同样可促进无长突细胞生成并减少双极细胞的形成；而在Ptf1a表达前过表达Foxn3，则会抑制无长突细胞的生成。在胚胎视网膜外植体中通过CRISPR技术敲除Foxn3，同样会减少无长突细胞的生成。共表达Foxn3可部分逆转异位表达miR-216b对视网膜细胞类型形成的影响。本研究结果确认Foxn3是发育视网膜内中间神经元生成的新型调控因子，并提示miR-216b可能在无长突细胞中调控Foxn3及其他基因的表达。P0野生型小鼠的视网膜微小RNA表达谱
