Cellular and molecular mechanisms of aging and regeneration in colonial chordate

描述（由申请人提供）：实际上，在衰老过程中，生物体表型的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结果变得非常困难。在群集生物中，如猪曲霉（Botryllus schlosseri），最初通过有性繁殖和脊索动物发育而产生的个体，像我们一样，可以蜕变为无性系的创始个体，每周通过一小群干细胞出芽形成新的个体。个体是短暂的结构，通过大量的细胞凋亡而死亡，连续的芽成熟并复制出一个完整的新身体，每周都是如此。因此，他们的干细胞是组织中唯一自我更新的细胞，也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影响的细胞。因此，从定义上讲，这种有机体中菌落的衰老就是干细胞的衰老：每隔一周，干细胞就会再生出另一个细胞。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在衰老过程中经历的数十次无性加倍进行取样，并跟踪每个组织和生殖系的干细胞池中发生的一系列变化或克隆选择。在这篇文章中，我们概述了研究schlosseri组织衰老和再生的细胞和分子机制。具体来说，我们提出表征分子途径强调有性生殖与无性生殖在一个殖民地脊索动物；识别和表征与强大的再生活性和组织稳态相关的分子机制，并测试其对寿命和与年龄相关的细胞和分子过程的影响；并探讨老年菌落暴露于年轻菌落循环因子（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无性发育途径之间基因的差异表达。该分析包括比较长寿命和短寿命基因型以及组内基因型-同时死亡和随机死亡的基因型。我们还将比较来自特定组织和器官的富集干细胞和干细胞壁龛随时间的表达谱。这种方法对近亲杂交、自交系或相同基因型的样本进行处理，将允许鉴定出在被测基因型的生命周期内表达差异的基因，这些基因型可能影响衰老和改变组织再生能力。我们将通过原位杂交鉴定显示差异表达的特定细胞。这一全面筛选之后，将进行功能实验，通过敲低研究来评估基因功能，并在原位改变生物学，因为它们通过菌落之间的血管桥迁移，参与出芽和生殖细胞形成。我们还将在单细胞水平上确定干细胞和生态位细胞的衰老特征。

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.