Stem Cell Multipotency During Quiescence

 描述(由申请人提供)：在对干细胞如何在长时间的细胞静止或不分裂期间保持产生多种细胞类型的能力的理解上存在着根本的差距。由于在静止状态下维持多能性是干细胞功能的一个重要方面，这一差距阻碍了对干细胞生物学的理解。线虫是研究细胞命运调控的有力模型。线虫的静止期可以用达尔幼虫阶段来模拟，达尔幼虫阶段是在发育中期对不利的环境条件做出反应的。在Dauer期间，所有的祖细胞都保持静止，这种静止是由调节哺乳动物干细胞静止的相同途径调节的。这个实验室的长期目标是使用线虫来破译在Dauer过程中促进多能性的机制。FOXO转录因子是跨物种干细胞维持的关键调节因子，也是协调静止和多能性的候选因子。FOXO通过已知的机制控制静止，但人们对FOXO如何调节发育途径以控制多能性知之甚少。这项应用的目的是解开FOXO同源基因DAF-16在Dauer过程中影响发育途径的机制。初步数据表明，FOXO/DAF-16在两种祖细胞类型中调节三种不同的发育途径，以保持Dauer期间的多能性。在这些数据的基础上，提出了三个具体目标。1)分析FOXO/daf-16在调控时间细胞命运通路中的作用。基因和分子实验将为FOXO/daf-16如何调控这一时间命运途径提供第一个机械性的洞察。2)鉴定FOXO/DAF-16靶基因UNK-1在促进多能性中的作用。遗传学实验将确定编码一种保守的锌指蛋白的unk-1与三种发育途径中的基因之间的关系。这一目标的完成将导致FOXO/DAF-16在静息状态下控制多能性的第一个介体的表征。3)确定FOXO/DAF-16下游促进多能性的基因。在FOXO/daf-16突变的达尔幼虫中，哪些基因的表达发生变化尚不清楚。在这一目标中，这些基因将通过下一代测序来识别，并在功能上测试其在细胞命运调控中的作用。这项工作将导致基因组规模的知识，调控FOXO/DAF-16下游的多能性基因。这里提出的工作是创新的，因为使用达氏线虫幼虫建模干细胞将使在体内以单细胞分辨率研究静止的多潜能细胞成为可能，这是对哺乳动物研究的补充。这项拟议的工作意义重大，因为它有望阐明FOXO/DAF-16协调发育途径以实现多能性的紧急特性的机制，促进我们对静止和干细胞命运之间的联系的理解。

 DESCRIPTION (provided by applicant): There is a fundamental gap in the understanding of how stem cells retain the capacity to produce multiple cell types during lengthy periods of cellular quiescence, or non-division. Because the maintenance of multipotency during quiescence is an essential aspect of stem cell function, this gap presents a barrier to the understanding of stem cell biology. C. elegans is a powerful model for studying the regulation of cell fate. Quiescence can be modeled in C. elegans using the dauer larva stage, adopted midway through development in response to adverse environmental conditions. All progenitor cells remain quiescent during dauer, and this quiescence is regulated by the same pathways that regulate quiescence in mammalian stem cells. The long term goal of this lab is to use C. elegans to decipher the mechanisms that promote multipotency during dauer. FOXO transcription factors are key regulators of stem cell maintenance across species, and are candidates for coordinating quiescence and multipotency. FOXO controls quiescence by known mechanisms, but little is known about how FOXO regulates developmental pathways to control multipotency. The objective of this application is to unravel the mechanisms by which the FOXO ortholog, DAF-16 impacts developmental pathways during dauer. Preliminary data establish that FOXO/DAF-16 regulates three different developmental pathways in two progenitor cell types in order to preserve multipotency during dauer. Building on these data, three specific aims are proposed. 1) Analyze the role of FOXO/daf-16 in modulating a temporal cell fate pathway. Genetic and molecular experiments will provide the first mechanistic insight into how FOXO/daf-16 regulates this temporal fate pathway. 2) Characterize the role of the putative FOXO/DAF-16 target gene unk-1 in promoting multipotency. Genetic experiments will establish the relationship between unk-1, encoding a conserved zinc finger protein, and genes within three developmental pathways. Completion of this aim will result in the characterization of the first mediator of FOXO/DAF-16 in controlling multipotency during quiescence. 3) Identify genes downstream of FOXO/DAF-16 that promote multipotency. Genes whose expression changes in FOXO/daf-16 mutant dauer larvae are unknown. In this aim, such genes will be identified by next generation sequencing, and tested functionally for a role in the regulation of cell fate. This work will lead o a genome scale knowledge of genes that regulate multipotency downstream of FOXO/DAF-16. Work proposed here is innovative because modeling stem cells using C. elegans dauer larvae will enable the study of quiescent, multipotent cells in vivo, at single cell resolution, complementing mammalian studies. The proposed work is significant because it is expected to result in the elucidation of mechanisms by which FOXO/DAF-16 coordinates developmental pathways to achieve the emergent property of multipotency, advancing our understanding of the links between quiescence and stem cell fate.