Specification and maintenance of quiescent progenitor cells set aside for the biphasic life cycle of an invertebrate chordate

项目概要/摘要 该提案的目的是描述指定，维持， 保护预图案化但静止和未分化的祖细胞的离散隔室 （被称为“脖子”）在被囊玻璃海鞘。像玻璃海鞘这样的被囊类动物是最接近 与脊椎动物有关，具有独特的双相生命周期，在短暂的， 活动的幼虫和无柄的成虫。在变态过程中，大量的细胞程序性死亡 消除大多数分化的幼虫细胞，同时保留未分化的成体祖细胞 形成了大多数的成年细胞类型和结构。尽管这种异时性， 分化和完全重塑后变质成人，幼虫和成人的身体 在胚胎发育期间，平面是连续的并且同时形成图案。 这种特殊的排列方式为研究离散的干细胞隔室 可以被放在一边，并保护以后的发展潜力，尽管分化和细胞 死亡围绕着他们。中心假设是，定型的规格，维护， 颈部静止祖细胞的存活受以下精确的发育调节控制： 编码多种生化途径限速成分的基因。所依据的理由 拟议的研究是，通过利用玻璃海鞘的基因组和细胞简单性及其 从一个能动的幼虫身体计划到一个无柄的成年人，我们可以从一个激进的，但自然定型的过渡， 在更大的空间和时间细节上理解组织重塑和细胞周转。 中心假设将通过追求三个具体目标进行测试：1）我们将测试Pax 2/5/8是否 是建立颈作为成人祖细胞的离散隔室所必需的。2)我们将 测试FGF和Hedgehog信号在维持颈细胞作为静止细胞群中的作用。 未分化细胞3)我们将测试在凋亡波期间Neck细胞的存活是否 发生在变态需要抗凋亡作用的一氧化氮信号和一个独特的 钒蛋白是一类与钒结合的金属蛋白，钒是一种重金属， 被囊细胞，但没有已知的生物学功能。这些目标将通过创新的 将细胞系特异性CRISPR/Cas9为基础的体细胞基因敲除与下一个- 代测序，体内荧光显微镜和体外生物化学测定。的 拟议工作的预期成果将是：1）揭示保守的生物学机制， 可能与人类共享，和/或2）识别高度不同的蛋白质和途径， 尽管如此，它仍然可以被用于治疗人类疾病和损伤的新的治疗工具。

PROJECT SUMMARY/ABSTRACT The objective of this proposal is to characterize the molecular mechanisms that specify, maintain, and protect a discrete compartment of pre-patterned but quiescent and undifferentiated progenitor cells (termed the “Neck”) in the tunicate Ciona. Tunicates like Ciona are the invertebrates most closely related to vertebrates, and possess a unique biphasic life cycle alternating between a short-lived, motile larva and a sessile adult. During metamorphosis, a mass wave of programmed cell death eliminates most differentiated larval cells while sparing set-aside undifferentiated adult progenitor cells that go on to form the majority of adult cell types and structures. Despite this heterochrony in differentiation and the complete remodeling of the post-metamorphic adult, the larval and adult body plans are contiguous and simultaneously patterned during embryonic development. This peculiar arrangement offers a unique opportunity to study how discrete stem cell compartments can be set aside and protected for later developmental potential in spite of the differentiation and cell death around them. The central hypothesis is that the stereotyped specification, maintenance, and survival of quiescent progenitor cells of the Neck are controlled by precise developmental regulation of genes encoding rate-limiting components of diverse biochemical pathways. The rationale underlying the proposed research is that, by exploiting the genomic and cellular simplicity of Ciona and their radical yet naturally stereotyped transition from a motile larval body plan to a sessile adult one, we can understand tissue remodeling and cellular turnover in much greater spatial and temporal detail. The central hypothesis will be tested by pursuing three specific aims: 1) We will test whether Pax2/5/8 is required for establishing the Neck as a discrete compartment of adult progenitor cells. 2) We will test the roles of FGF and Hedgehog signaling in maintaining Neck cells as a quiescent population of undifferentiated cells. 3) We will test whether the survival of Neck cells during the wave of apoptosis that occurs in metamorphosis requires the anti-apoptotic effects of Nitric oxide signaling and a unique class of metalloproteins that bind vanadium, a heavy metal that has been observed to accumulate in tunicate cells but without a known biological function. These aims will be pursued using an innovative approach that combines cell lineage-specific CRISPR/Cas9-based somatic gene knockouts with next- generation sequencing, in vivo fluorescence microscopy and in vitro biochemical assays. The expected outcomes of the proposed work would be to 1) reveal conserved biological mechanisms that might be shared with humans, and/or 2) identify highly divergent proteins and pathways that could nonetheless be harnessed for novel therapeutic tools to treat human disease and injury.