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

项目摘要/摘要 这项提议的目标是描述分子机制的特征，这些分子机制规定、维持和 保护预先构图但静止和未分化的祖细胞的离散隔间 (称为“颈”)在被囊状乳突中。像Ciona这样的被囊动物是最接近无脊椎动物的 与脊椎动物有亲缘关系，并具有独特的双相生命周期，在短暂的， 活动的幼虫和一只固着的成虫。在变态过程中，程序性细胞死亡的大量浪潮 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 这些细胞继续形成大多数成体细胞类型和结构。尽管存在这种异步性， 变态后成虫、幼虫和成体的分化和完全重塑 计划是连续的，并在胚胎发育期间同时形成模式。 这种特殊的排列提供了一个独特的机会来研究离散的干细胞隔间 可以被搁置并为以后的发育潜力而保护，尽管分化和细胞 死亡围绕着他们。中心假设是刻板的规范、维护和 颈部静止的祖细胞的存活受精确的发育调节控制 编码不同生化途径的限速成分的基因。背后的基本原理 拟议的研究是，通过利用Ciona和它们的基因组和细胞的简单性 从活动的幼体计划到固执的成体计划的激进但自然刻板的转变，我们可以 在更大的空间和时间细节上了解组织重塑和细胞周转。 核心假设将通过追求三个具体目标来检验：1)我们将检验Pax2/5/8 是建立颈部作为一个独立的成体祖细胞隔间所必需的。2)我们会 检测成纤维细胞生长因子和Hedgehog信号在维持颈部细胞静止种群中的作用 未分化的细胞。3)我们将测试颈部细胞在凋亡浪潮中的存活情况 发生在变态中需要一氧化氮信号的抗凋亡作用和一种独特的 一类与钒结合的金属蛋白，钒是一种重金属，观察到在 被囊状细胞，但没有已知的生物功能。这些目标将通过创新的 结合细胞谱系特异性CRISPR/Cas9的体细胞基因敲除和NEXT- 世代测序、体内荧光显微镜和体外生化分析。这个 拟议工作的预期结果将是1)揭示保守的生物机制， 可能与人类共享，和/或2)识别高度差异的蛋白质和通路，这些蛋白质和通路可以 尽管如此，仍可被利用为治疗人类疾病和伤害的新型治疗工具。

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.