Regulation of stem cell fate by FOXO and RNA binding proteins

对于如何调节发育途径以维持发育的理解存在根本性差距 干细胞在长时间静止或不分裂期间具有多能性。 FOXO 家族 转录因子是干细胞维持和静止的关键调节因子。然而， FOXO 蛋白影响发育途径以控制细胞命运的机制尚不清楚 明白了。该应用程序利用线虫系统的力量来解决 单一 FOXO 直向同源物 daf-16 调节保守发育途径的机制 在静止期间保持干细胞的多能性。为了模拟干细胞静止，我们将使用 静止的多尔幼虫阶段，在发育中途采用，以应对不利的环境 条件。这种方法是创新的，因为线虫系统使我们能够研究静止、 单细胞分辨率的体内多能细胞，补充了哺乳动物研究。长期目标是 该实验室旨在破译在 dauer 期间促进多能性的机制。表皮缝细胞， 干细胞模型是多能的，并且在细胞分裂时经历自我更新的特征模式 每个幼虫阶段直到成年分化。在 dauer 期间，接缝细胞处于静止和活跃状态 机制维持多能性。初步数据表明，在 dauer 期间，FOXO/daf-16 区块 通过正向调节编码 RNA 结合蛋白的三个基因的表达来调节成体细胞的命运 （RBP）。这些 RBP 的直系同源物调节干细胞和祖细胞的增殖和功能 苍蝇和哺乳动物。本申请的目的是揭示 FOXO/daf-16 的机制 在静止期，通过 RBP 调节成体细胞的命运。三个具体目标是 提议实现这一目标。 1)确定FOXO/daf-16与RBPs之间的亲缘关系。 功能丧失和功能获得实验将建立 FOXO/daf-16（一种 控制细胞命运的新机制。 2) 确定阻断成体细胞命运的直接 RBP 靶标 静止。 RBP 的直接 mRNA 靶点将通过 iCLIP 进行识别。功能测试将确定 哪些靶标参与缝细胞命运的调节。这些实验将共同阐明 FOXO/daf-16 调节的 RBP 与成体细胞命运之间的联系。 3）剖析转录 静止期间成体细胞命运标记的调节。初步数据表明，在 dauer 期间， FOXO/daf-16 和三个 RBP 阻断成人特异性基因转录报告基因的表达， 广泛用于标记成体细胞的命运。候选转录的启动子解剖和功能测试 这些因素将破译关键成体细胞命运标记的静止特异性调节。拟议的工作 很重要，因为它将阐明发育途径的调节如何与 多能细胞静止的调节。

A fundamental gap exists in the understanding of how developmental pathways are regulated to maintain stem cell multipotency during extended periods of quiescence, or non-division. The FOXO family of transcription factors are key regulators of stem cell maintenance and quiescence. However, the mechanisms by which FOXO proteins impact developmental pathways to control cell fate are poorly understood. This application capitalizes on the power of the C. elegans system to address the mechanisms by which the single FOXO ortholog, daf-16, regulates conserved developmental pathways to preserve stem cell multipotency during quiescence. To model stem cell quiescence, we will use the quiescent dauer larva stage, adopted midway through development in response to adverse environmental conditions. This approach is innovative because the C. elegans system allows us to study quiescent, multipotent cells in vivo at single cell resolution, complementing mammalian studies. The long-term goal of this lab is to decipher the mechanisms that promote multipotency during dauer. Epidermal seam cells, the stem cell model, are multipotent and undergo a characteristic pattern of self-renewing cell divisions at each larval stage until differentiating at adulthood. During dauer, seam cells are quiescent and active mechanisms maintain multipotency. Preliminary data establish that during dauer, FOXO/daf-16 blocks adult cell fate by positively regulating the expression of three genes that encode RNA-binding proteins (RBPs). The orthologs of these RBPs regulate the proliferation and function of stem and progenitor cells in flies and mammals. The objective of this application is to unravel the mechanisms by which FOXO/daf-16 acts via RBPs to regulate adult cell fate during the quiescent dauer stage. Three specific aims are proposed to meet this objective. 1) Determine the genetic relationship between FOXO/daf-16 and RBPs. Loss-of-function and gain-of-function experiments will establish the regulation of RBPs by FOXO/daf-16, a novel mechanism to control cell fate. 2) Identify direct RBP targets that block adult cell fate during quiescence. Direct mRNA targets of RBPs will be identified by iCLIP. Functional testing will determine which targets are involved in the regulation of seam cell fate. Together these experiments will elucidate the connection between FOXO/daf-16-regulated RBPs and adult cell fate. 3) Dissect the transcriptional regulation of an adult cell fate marker during quiescence. Preliminary data establish that during dauer, FOXO/daf-16 and the three RBPs block expression of a transcriptional reporter of an adult-specific gene, widely used to mark adult cell fate. Promoter dissection and functional testing of candidate transcription factors will decipher the quiescence-specific regulation of a key adult cell fate marker. The proposed work is significant because it will illuminate how the regulation of developmental pathways is coordinated with the regulation of quiescence in multipotent cells.