Dissecting gene regulation of stem cell quiescence in Ciona

项目摘要 在发育过程中，异质组织、器官和细胞网络的形成依赖于一个仔细的 平衡细胞增殖、静止和分化。一旦细胞完全分化， 增殖或分化的能力通常会丧失，因此大多数成体细胞通常不能自我增殖。 再生或修复。再生疗法的发展需要利用这种潜在的能力， 首先，我们需要更深入地了解控制它的过程。因此， 静止祖细胞行为的基础结构是开发和应用新兴疗法的关键， 新技术，如细胞重编程或基因组编辑。这项建议的目的是 表征控制静止，分化， 海洋无脊椎动物和脊索动物Ciona robusta的繁殖。玻璃海鞘是最接近我们的无脊椎动物之一 亲戚，因此在发育过程中，我们共享相似的结构和分子特征。然而，Ciona 经历一个戏剧性的转变，从幼虫到成人的形式称为变态，当幼虫的身体计划， 退化并被必须绕过程序性细胞死亡重新出现的静止祖细胞所取代 并生成成年人的身体平面图。一种这样的细胞群是幼虫神经祖细胞 称为颈细胞，它们建立在离散的干细胞龛样隔室中。的信号通路 和遗传成分，直接颈进入，维护，并退出静止仍然没有特点。 我建议使用颈细胞群作为模型，以确定调节静止的独特机制， 可以用于未来治疗的再生。这一建议的理由是，通过利用 玻璃海鞘的易驾驭性，这些颈细胞的可接近性，以及它们刻板的细胞行为，我可以密切地观察到， 检查颈细胞静止、增殖和分化的调节控制。我会通过追踪两个 具体目标。1)探讨白纹伊蚊幼虫期颈部静止和增殖的控制作用 细胞外信号和细胞内转录控制的整合。2)为了研究一种新的机制， 转录引发和Neck细胞分化延迟。我将使用创新的方法来实现这些目标。 结合细胞系特异性、基于CRISPR/Cas9的体细胞基因敲除和荧光的方法 显微镜拟议工作的预期成果包括确定以前未得到承认的职能 保守但研究较少的神经发育基因，以及它们的时空调控如何被 对静止的精确时间有指导意义。这将为有针对性地调查 神经发育过程是一系列人类疾病的基础。

PROJECT SUMMARY During development, the formation of heterogenous tissues, organs, and cellular networks depends on a careful balance between cellular proliferation, quiescence, and differentiation. Once cells have fully differentiated, their ability to proliferate or differentiate are most often lost, and so most adult cells are generally incapable of self- regeneration or repair. The development of regenerative therapies will require harnessing this latent ability, but first we need a deeper understanding of the processes that control it. Thus, understanding the genetic architecture underlying quiescent progenitor behavior is key to developing and applying emerging therapies with new technologies such as cellular reprogramming or genome editing. The objective of this proposal is to characterize the regulation and functions of potentially important genes controlling quiescence, differentiation, and proliferation in marine invertebrate and chordate Ciona robusta. Ciona are among our closest invertebrate relatives, and so during development we share similar structural and molecular features. However, Ciona undergo a dramatic conversion from larval to adult forms called metamorphosis, when the larval body plan degenerates and is replaced by quiescent progenitors which must bypass programmed cell death to reemerge post-metamorphosis and generate the adult body plan. One such cell population are larval neural progenitors called Neck cells which are established in a discrete stem cell niche-like compartment. The signaling pathways and genetic components that direct Neck entry, maintenance, and exit from quiescence remain uncharacterized. I propose using the Neck cell population as a model to identify unique mechanisms regulating quiescence and regeneration that can be harnessed for future therapies. The rationale for this proposal is that, by exploiting the tractability of Ciona, the accessibility of these Neck cells, and their stereotyped cellular behaviors, I can closely examine regulatory control of Neck cell quiescence, proliferation, and differentiation. I will do so by pursuing two specific aims. 1) To investigate the control of Neck quiescence and proliferation during the larval stage by the integration of extracellular cues and intracellular transcriptional control. 2) To investigate a novel mechanism for transcriptional priming and delay of Neck cell differentiation. I will pursue these aims using an innovative approach that combines cell lineage-specific, CRISPR/Cas9-based somatic gene knockouts and fluorescence microscopy. The expected outcomes of the proposed work include identifying previously unrecognized functions for conserved but poorly studied genes in neurodevelopment and how their spatiotemporal regulation can be instructive for the precise timing of quiescence. This will establish a foundation for a targeted investigation of neurodevelopmental processes underlying a wide range of human disorders.