Stem Cell Multipotency During Quiescence

 描述（由申请人提供）：在理解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方面存在根本性的差距。因为在静止期维持多能性是干细胞功能的一个重要方面，这个缺口对理解干细胞生物学构成了障碍。C.线虫是研究细胞命运调控的有力模型。静止可以在C中建模。线虫利用dauer幼虫阶段，通过中途发育，以应对不利的环境条件。所有的祖细胞在dauer过程中保持静止，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。本实验的长期目标是使用C。elegans破译机制，促进多能性在dauer。FOXO转录因子是跨物种干细胞维持的关键调节因子，并且是协调静止和多能性的候选者。FOXO通过已知的机制控制静止，但对FOXO如何调节发育途径以控制多能性知之甚少。本申请的目的是解开FOXO直系同源物，EZ-16影响发育过程中的发育途径的机制。初步数据确定，FOXO/FOX-I6调节两种祖细胞类型中的三种不同发育途径，以在dauer期间保持多能性。在这些数据的基础上，提出了三个具体目标。1)分析FOXO/daf-16在调节颞叶细胞命运途径中的作用。遗传和分子实验将为FOXO/daf-16如何调节这一时间命运途径提供第一个机制性见解。2)表征推定的FOXO/FOS-16靶基因unk-1在促进多能性中的作用。遗传学实验将建立unk-1（编码一种保守的锌指蛋白）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在静止期间控制多能性的第一介体的FOXO/P16的表征。3)确定促进多能性的FOXO/EST-16下游基因。在FOXO/daf-16突变体dauer幼虫中表达变化的基因未知。在这一目标中，这些基因将通过下一代测序来鉴定，并在功能上测试其在细胞命运调控中的作用。这项工作将导致一个基因组规模的知识，调节多能性下游的FOXO/ESTA-16。这里提出的工作是创新的，因为使用C。elegans dauer幼虫将能够以单细胞分辨率在体内研究静止的多能细胞，补充哺乳动物研究。拟议的工作是重要的，因为它有望导致FOXO/FOXO-16协调发育途径以实现多能性的新兴特性的机制的阐明，推进我们对静止和干细胞命运之间联系的理解。

 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.