Regulating retinal cell fate with microRNAs

 描述(申请人提供)：由视网膜疾病或损害引起的失明和视力受损是健康问题，会造成巨大的人力和经济成本。干细胞研究的最新进展表明，在可预见的未来，人类的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了解仍然不完整，限制了修复策略的合理设计。细胞的复杂性是包括视网膜在内的哺乳动物中枢神经系统的一个关键特征。根据形态、神经递质和其他分子标记，视网膜包含50多种不同类型的神经元。转录因子和各种信号通路的级联参与了视网膜细胞类型的确定和视黄素细胞多样性的产生。最近的研究还表明，microRNAs在控制视网膜细胞特性方面具有转录后调控作用。我们已经确定了两个相关的miRNAs，它们在发育中的小鼠视网膜中异位表达时，会改变视网膜发育并促进无长突间神经元的形成，而牺牲了其他视网膜细胞类型。我们已经使用ArgAerte PAR-CLIP来识别这些和其他miRNAs在新生小鼠视网膜中的内源性靶向mRNAs。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，并建议分析mRNA表达的变化，以确定视网膜中受这些miRNAs影响的分子和细胞通路。最后，我们建议研究视网膜细胞表达Ptf1a转录因子的miRNA调节范围，Ptf1a转录因子是无长突细胞和水平细胞形成所必需的。这些研究将为控制哺乳动物视网膜发育和细胞命运的分子机制提供洞察力。预计他们将提供有助于修复视网膜组织的新策略的信息。

 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.