Cellular and molecular mechanisms of aging and regeneration in colonial chordate

描述(由申请人提供)：在老化过程中，生物体表型的几乎每一个方面都会在其功能和形态上发生改变。生物体的复杂性和大量与衰老有关的现象使得很难区分导致衰老的表型和哪些是衰老的影响。在丛生生物体中，像我们一样，最初通过有性繁殖和脊索发育产生的个体可以蜕变为克隆创始人，克隆创始人通过从一小群干细胞发芽每周形成新的个体。个体是短暂的结构，通过大量的凋亡而死亡，连续的芽成熟后每周复制一个完整的新身体。因此，他们的干细胞是组织中唯一的自我更新细胞，也是唯一在该基因的整个生命周期中保留下来的细胞，也是唯一可以保留时间效应的细胞。因此，根据定义，这种生物体中的菌落老化就是干细胞的老化：每周从干细胞再生出其他每一个细胞。在这个群体模式生物中，我们可以清楚地定义，干细胞老化是整个群体衰老的根本原因。最重要的是，我们可以对单个组织或性腺干细胞进行采样，这些干细胞在衰老过程中经历了几十次无性繁殖，并跟踪了干细胞库中发生的针对每个组织和生殖系的一系列变化或克隆选择。在这项建议中，我们概述了研究许氏杆菌组织衰老和再生的细胞和分子机制的研究。具体地说，我们建议描述殖民地脊索动物有性繁殖和无性繁殖的分子途径；识别和表征与强大的再生活动和组织动态平衡相关的分子机制，并测试它们对寿命和与年龄相关的细胞和分子过程的影响；以及调查老年殖民地暴露于年轻殖民地的循环因子(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群体和老年群体中的无性发育途径之间的基因差异表达。这项分析包括比较长寿和短寿的基因类型以及群体内的--证明同时死亡和随机死亡的基因类型。我们还将比较随时间推移来自特定组织和器官的丰富干细胞和干细胞利基的表达谱。利用近交系、自交系或相同基因型的样本的这种方法将允许识别在所测试的基因类型的生命周期内差异表达的基因，这些基因可能会影响衰老和改变组织再生能力。我们将通过原位杂交来鉴定显示差异表达的特定细胞。在这一全面筛选之后，将进行功能实验，通过基因敲除研究来评估基因功能，并在原位改变生物学，因为它们通过克隆之间的血管桥迁移，参与萌芽和生殖细胞形成。我们还将在单细胞水平上确定干细胞和利基细胞的老化特征。 与公共卫生相关：在一种被认为是脊椎动物和无脊椎动物之间的纽带的殖民地生物体--施洛瑟氏杆菌中，个体每周的再生是通过干细胞的迁移、增殖和自我更新来进行的。虽然干细胞在组织和器官中的子代细胞寿命有限，但随着个体群体的衰老，干细胞通过自我更新在有机体几十代人的整个生命中保持。因此，Botryllus模式生物是阐明干细胞老化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.