Dissecting gene regulation of stem cell quiescence in Ciona

项目总结 在发育过程中，异质组织、器官和细胞网络的形成依赖于仔细的 细胞增殖、静止和分化之间的平衡。一旦细胞完全分化，它们的 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 再生或修复。再生疗法的发展将需要利用这种潜在的能力，但 首先，我们需要更深入地了解控制它的过程。因此，理解基因 静止的先祖行为的基础架构是开发和应用新的治疗方法的关键 新技术，如细胞重新编程或基因组编辑。这项建议的目的是 描述控制静止、分化、 海洋无脊椎动物和脊索动物Ciona robusta的增殖。乔纳是我们最接近的无脊椎动物之一 因此，在发育过程中，我们有相似的结构和分子特征。然而，Ciona 经历从幼虫到成虫的戏剧性转变，称为变态，当幼虫的身体计划 退化并被静止的祖细胞取代，后者必须绕过程序性细胞死亡才能重新出现 变形后，并生成成人的身体计划。一个这样的细胞群体是幼虫的神经前体细胞 这种细胞被称为颈部细胞，它们建立在一个离散的干细胞壁龛般的隔间里。信号转导通路 而引导颈部进入、维持和退出静止状态的遗传成分仍未确定。 我建议使用颈部细胞群作为一个模型，以确定调节静止和 可用于未来治疗的再生。这项提议的基本原理是，通过利用 Ciona的易操纵性，这些颈部细胞的可及性，以及它们刻板的细胞行为，我可以密切关注 检查颈部细胞静止、增殖和分化的调控。我将通过追求两个目标来实现这个目标 明确的目标。1)研究了在幼虫阶段控制颈部静止和增殖的方法 细胞外信号和细胞内转录调控的整合。2)研究一种新的机制 转录启动和延缓颈部细胞分化。我将用一种创新的方式追求这些目标 结合细胞谱系特异性、基于CRISPR/Cas9的体细胞基因敲除和荧光的方法 显微镜。拟议工作的预期结果包括确定以前未认识到的功能 对于神经发育中保守但研究很少的基因及其时空调节如何 对精确的静默时间有指导意义。这将为有针对性地调查 一系列人类疾病背后的神经发育过程。

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.