Regulating retinal cell fate with microRNAs

 描述（由申请人提供）：视网膜疾病或损伤引起的失明和视力受损是具有重大人力和经济成本的健康问题。干细胞研究的最新进展表明，在可预见的未来，人类视网膜修复将是可行的。然而，我们对视网膜细胞生物学和发育的理解仍然不完整，限制了修复策略的合理设计。细胞的复杂性是哺乳动物中枢神经系统（包括视网膜）的关键特征。视网膜包含超过50种不同类型的神经元，基于形态，神经递质和其他分子标记。转录因子和各种信号通路的级联反应与视网膜细胞类型的确定和视网膜细胞多样性的产生有关。最近的研究还表明，在视网膜细胞身份的控制中，microRNA的转录后调控也有牵连。我们已经确定了两个相关的miRNA，改变视网膜发育和促进无长突中间神经元的形成，在其他视网膜细胞类型的代价，异位表达在发育中的小鼠视网膜。我们已经使用Argonaute PAR-CLIP来鉴定新生小鼠视网膜中这些和其他miRNA的内源性靶mRNA。 在这里，我们建议在视网膜中使用CRISPR技术来确定这些miRNAs在无长突细胞形成中的需求。我们还计划分析一个候选靶基因在视网膜发育调控中的作用，我们建议分析mRNA表达的变化，以确定视网膜中受这些miRNA影响的分子和细胞通路。最后，我们建议研究视网膜细胞中表达Ptf 1a转录因子的miRNA调控范围，Ptf 1a转录因子是无长突和水平细胞形成所需的。这些研究将提供深入了解的分子机制，控制发展和细胞的命运决定在哺乳动物视网膜。他们预计将提供信息，将有助于新的战略，以修复视网膜组织。

 DESCRIPTION (provided by applicant): Blindness and impaired vision arising from retinal disease or damage are health problems with substantial human and economic costs. Recent advances in stem cell research suggest that retinal repair in humans will be feasible in the foreseeable future. However our understanding of retinal cell biology and development remains incomplete, limiting rational design of repair strategies. Cellular complexity is a key feature of the mammalian central nervous system, including the retina. The retina contains more than 50 different types of neurons, based on morphology, neurotransmitters, and other molecular markers. Cascades of transcription factors and various signaling pathways have been implicated in retinal cell type determination and the generation of cellular diversity in the retin. Recent studies have also implicated post-transcriptional regulation by microRNAs in the control of retinal cell identity. We have identified two related miRNAs that alter retinal development and promote amacrine interneuron formation, at the expense of other retinal cell types, when ectopically expressed in the developing mouse retina. We have used Argonaute PAR-CLIP to identify endogenous target mRNAs for these and other miRNAs in the neonatal mouse retina. Here we propose to determine the requirements for these miRNAs in amacrine cell formation, using CRISPR technology in retinas. We also plan to analyze a candidate target gene for its role in the regulation of retinal development, and we propose to analyze changes in mRNA expression to identify molecular and cellular pathways in the retina that are affected by these miRNAs. Finally, we propose to investigate the scope of miRNA regulation in retinal cells expressing the Ptf1a transcription factor, which is required for amacrine and horizontal cell formation. These studies will provide insight into the molecular mechanisms that control development and cell fate determination in the mammalian retina. They are expected to provide information that will contribute to new strategies to repair retinal tissue.