P0044380 Parchem - Role of Small RNA's in Development

描述(申请人提供)：胚胎干细胞和从体细胞诱导其形成的分子方法是近年来密集研究的主题。这些技术允许科学家绕过使用人类胚胎的伦理争议问题，创造出个性化的多潜能干细胞。这些进展是开始将这些细胞用于治疗目的的令人难以置信的推动的基础。不幸的是，我们产生细胞或组织用于治疗的能力仍然相当有限。Blelloch实验室最近证明，miRNAs对于胚胎干细胞的分化是必要的，并且特定类别的miRNAs通过相反的途径发挥作用，以调节多能性状态的分化。这一发现表明miRNAs是重要的全球分化调节因子。在这一应用中，我假设miR302簇通过调节转录在从多能性到分化胚层的转变中至关重要，而转录是这一至关重要的细胞命运决定的机械基础。为了验证这一假设，我将首先通过表征miR302缺陷小鼠胚胎发育过程中上胚细胞的形成和原肠形成来确定miR302 miRNAs在体内的作用。然后，我将描述缺乏miR302的小鼠胚胎干细胞的分化缺陷。接下来，我将对miR302的分子靶点进行鉴定和功能测试，以确定miR302调节胚层规格的机制。最后，我将剖析miR302基因座中包含不同种子序列的miRNAs的功能，以了解不同靶标共表达的miRNAs在发育中的功能。这些实验将提供关于miRNAs如何调控细胞命运决定以及细胞通过什么机制从多能性转变到初级胚胎生殖层的详细信息。

DESCRIPTION (provided by applicant): Embryonic stem cells and the molecular methods to induce their formation from somatic cells has been the subject of intense study in recent years. These techniques allow scientists to bypass the ethically contentious issue of using human embryos and create personalized pluripotent stem cells. These advances underlie the incredible push to begin using these cells for therapeutic purposes. Unfortunately, our ability to generate cells or tissues for therapeutic use is still quite limited. The Blelloch lab has recently shown tht miRNAs are necessary for differentiation of embryonic stem cells, and that specific classes of miRNAs function through opposing pathways to mediate differentiation out of the pluripotent state. This finding suggests that miRNAs are important global regulators of differentiation. In thi application, I hypothesize that the mir302 cluster of miRNAs is critical in the transition from pluripotency to differentiated germ layers by regulating transcripts that mechanistically underlie this critically important cell-fate decision. To test this hypothesis I will first determine the invivo role of mir302 miRNAs by characterizing epiblast formation and gastrulation during embryonic development of mir302 deficient mice. I will then characterize differentiation defects in mouse embryonic stem cells lacking mir302. Next, I will identify and functionally test the molecular targets of mir302 to identify the mechanism through which mir302 regulates germ layer specification. Finally, I will dissect the function of miRNAs containing different seed sequences within the mir302 locus to understand how co- expressed miRNAs with different targets function in development. These experiments will provide detailed information on how miRNAs regulate cell fate decisions and through what mechanism a cell transitions from pluripotency to the primary embryonic germ layers.