Cellular and molecular mechanisms of aging and regeneration in colonial chordate

描述（由申请人提供）：在衰老过程中，生物体表型的几乎每个方面都经历了功能和形态的改变。生物体的复杂性和大量与衰老相关的现象使得区分导致衰老的表型和衰老的影响变得非常困难。在菌落生物中，如Botryllus schlosseri，最初通过有性生殖和脊索发育产生的个体，就像我们一样，可以变形为克隆创始人，每周通过一小群干细胞的萌芽形成新个体。个体是短暂的结构，通过大量的细胞凋亡死亡，连续的芽成熟，每周复制一个全新的身体。因此，它们的干细胞是组织中唯一能自我更新的细胞，是唯一能在基因型的整个生命周期中保留下来的细胞，也是唯一能保留时间效应的细胞。因此，根据定义，这种生物体中殖民地的老化是干细胞的老化：每隔一周，每隔一周，干细胞都会再生。在这个群体模式生物中，我们可以清楚地定义干细胞衰老是整个群体衰老的根本原因。最重要的是，我们可以在群体老化过程中经历的数十次无性繁殖殖民地中，对单个组织或性腺干细胞进行采样，并跟踪每个组织和生殖系干细胞库中发生的系列变化或克隆选择。在这个建议中，我们概述了研究，以调查的细胞和分子机制的老化和再生的组织B. schlosseri。具体来说，我们建议的分子途径，强调性与无性生殖在殖民脊索动物的特点，以确定和表征的分子机制与强大的再生活动和组织的稳态，并测试其对寿命的影响，和年龄相关的细胞和分子过程;并研究将老集落暴露于来自年轻集落的循环因子（通过血管吻合）对其分子表达谱和再生潜力的影响。我们将使用Illumina高通量mRNA测序平台来比较年轻菌落与老年菌落中无性发育途径之间的基因差异表达。该分析包括比较长寿和短命的基因型和组内基因型，表现出同时与随机死亡。我们还将比较富集的干细胞和干细胞龛随着时间的推移从定义的组织和器官的表达谱。使用相同基因型的近交系、自交系或样品的这种方法将允许鉴定在测试基因型的寿命期间差异表达的基因，所述基因可能影响衰老并改变组织再生能力。我们将通过原位杂交鉴定显示差异表达的特定细胞。这一全面的筛选之后将进行功能实验，通过敲除研究评估基因功能，并在它们穿过菌落之间的血管桥迁移以参与出芽和生殖细胞形成时改变原位生物学。我们还将在单细胞水平上确定干细胞和小生境细胞中的衰老特征。 公共卫生相关性：在一个殖民生物体，Botryllus schlosseri，被认为是脊椎动物和无脊椎动物之间的联系，通过干细胞的迁移，增殖和自我更新发生个体的每周再生。虽然它们在组织和器官中的子细胞具有有限的寿命，但通过自我更新，干细胞随着个体的殖民地年龄的增长而在整个生物体的几十代中保持。因此，葡萄球菌模式生物是阐明干细胞衰老在衰老和再生过程中的作用的有力工具。

DESCRIPTION (provided by applicant): Virtually every aspect of an organism's phenotype undergoes modification in its functionality and morphology during aging. The complexity of organisms and the vast amount of aging related phenomena make it very difficult to distinguish between phenotypes that cause aging and which are the effect of it. In colonial organisms, like Botryllus schlosseri, individuals originally derived, like us, by sexual reproduction and chordate development can metamorphose to clonal founders that undergo weekly formation of new individuals by budding from a small group of stem cells. Individuals are transient structures which die through massive apoptosis and successive buds mature to replicate an entire new body, every week. As a result, their stem cells, which are the only self renewing cells in a tissue, are the only cells which remain through the entire life of the genotype and are the only cells that can retain the effects of time. Therefore, aging of the colony in this organism is, by definition, aging of the stem cells: every other cell is regenerated from them on a weekly basis. In this colonial model organism, we can clearly define that stem cell aging is the root cause of senescence of the entire colony. Most importantly, we can sample single tissue or gonad stem cells over the dozens of asexual doublings the colony undergoes while aging, and follow the serial changes or clonal selections that occur in the stem cell pools for each tissue, and for the germline. In this proposal, we outline studies to investigate the cellular and molecular mechanisms of aging and regeneration in tissues of B.schlosseri. Specifically, we propose to characterize the molecular pathways which underline sexual versus asexual reproduction in a colonial chordate; to identify and characterize the molecular mechanisms associated with robust regeneration activity and tissue homeostasis and to test their effects on longevity, and age-related cellular and molecular processes; and to investigate the effect of exposure of old colonies to circulating factors from young colonies (through vascular anastomosis), on their molecular expression profile and regeneration potential. We will use the Illumina high throughput, mRNA sequencing platform to compare differential expression of genes between: asexual developmental pathways in young colonies versus old. This analysis includes comparing long and short lived genotypes and within groups-genotypes that demonstrate simultaneous versus random death. We will also compare expression profiles of enriched stem cells and stem cell niches from defined tissues and organs over time. This approach with inbred, self crossed lines or samples of the same genotype will allow identification of genes which are expressed differentially over the life span of the tested genotypes that are likely to affect aging and alter tissue regeneration capacities. We will identify the specific cells that show the differential expression by in situ hybridization. This comprehensive screen will be followed by functional experiments that evaluate gene function by knockdown studies, and altered biology in situ as they migrate across vascular bridges between colonies to participate in budding and germ cell formation. We will also determine, at the single cell level the aging signatures in both stem cells and niche cells. PUBLIC HEALTH RELEVANCE: In a colonial organism, Botryllus schlosseri, believed to be the link between vertebrates and invertebrates, weekly regeneration of individuals occurs via migration, proliferation, and self renewal by stem cells. While their daughter cells in tissues and organs have a limited lifespan, by self-renewal stem cells remain throughout the entire life of dozens of generations of the organism as the colony of individuals ages. Thus the Botryllus model organism is a powerful tool to elucidate the role of stem cell aging on aging and regeneration processes.