Cellular and gene regulatory mechanisms of whole body regeneration in Botryllus Schlosseri

所有多细胞生物都起源于一小部分多能胚胎干细胞， 扩增并分化为成熟个体的组织和器官，以及来自 蠕虫到人类使用一套高度保守的核心发育途径来完成 胚胎发生的过程。然而，在正常生长和衰老的成年人中，或在受伤后， 分化的细胞和器官必须得到补充或再生。进行再生 通过长寿的，通常是血统受限的干细胞，保持扩增和 在个体的整个生命周期中保持差异化。然而，细胞和分子 大多数组织再生发育的机制还不是很清楚。 更重要的是，尽管胚胎发育途径得到了深度保护，但 不同的生物在损伤后能够再生组织和器官的程度并不是 进化过程中的保守特征：火蜥蜴可以再生被截肢的肢体，但 人类不能。为什么会这样呢？这项资助的重点是研究 基本脊索生物体，集群海鞘动物Botryllus schlosseri，探索这些问题。 使用一个简单的手术程序，我们可以诱导Botryllus再生整个身体， 包括心脏、胃肠道、咽部、血管系统、神经和内分泌系统以及配子， 来自体外血管床的碎片。这个过程被称为全身再生 (WBR)，发生迅速(3-6天)，海鞘是唯一可以再生的脊索动物 整个身体，因此我们可以解剖高度保守的发育途径 在任何组织再生期间重新部署。Botryllus还提供了独特的研究方法 例如，WBR的维管系统是无柄的和透明的，并且易于可视化、标记和 操纵。重要的是，我们最近还开发了一种救援/重建试验，将 使我们能够前瞻性地分离负责的细胞，以及表征和功能 评估WBR背后的发育途径。这项R21拨款旨在发展这一点 新的系统，我们建议：1)利用有限稀释移植试验和细胞 标记移植策略以评估祖细胞是否受到谱系限制或 多能性；以及，2)使用不同阶段的增殖细胞的基因表达谱 再生以确定调节干细胞功能的基因和信号通路 诱导循环干细胞的募集和增殖。然后我们将从功能上测试 使用Small的再生途径和基因调控机制的子集的作用 分子抑制剂和RNAi。我们的目标是在这笔为期两年的拨款结束时，我们将拥有 完成了差异表达基因的大型数据库，完成了初始遗传和 功能表征潜在的再生发育途径，以及 要么已经分离，要么拥有强大的方法来提纯负责和解剖的细胞 它们在再生中的作用。 通过表征负责全身再生的干细胞群体和 识别调控干细胞的基因、信号通路和微环境 在再生生长过程中，这些实验将 极大地促进了Botryllus作为研究干细胞和 再生生物学，并将促进我们对人类干细胞功能和 组织再生。

All multicellular organisms originate from a small set of pluripotent embryonic stem cells that expand and differentiate into tissues and organs of a mature individual, and organisms from worms to humans use a highly conserved set of core developmental pathways to complete the process of embryogenesis. However, in adults during normal growth and aging, or after injury, differentiated cells and organs must be replenished or regenerated. Regeneration is carried out by long-lived, usually lineage-restricted stem cells that retain the capacity to expand and differentiate throughout the lifespan of the individual. However, the cellular and molecular mechanisms underlying regenerative development of most tissues are not well understood. More importantly, despite the deep conservation of embryonic developmental pathways, the degree to which different organisms can regenerate tissues and organs following injury is not a conserved feature throughout evolution: a salamander can regenerate an amputated limb, but a human cannot. Why is this true? This grant is focused on studying the regenerative abilities of a basal chordate organism, the colonial ascidian Botryllus schlosseri, to explore these questions. Using a simple surgical procedure, we can induce Botryllus to regenerate an entire body, including a heart, GI tract, pharynx, vasculature, neural and endocrine system- and gametes, from fragments of an extracorporeal vascular bed. This process, called whole body regeneration (WBR), occurs rapidly (3-6 days), and ascidians are the only chordates that can regenerate entire bodies, thus we can dissect how highly conserved developmental pathways are redeployed during regeneration of any tissue. Botryllus also provides unique ways to study WBR, for example, the vasculature is sessile and transparent, and easy to visualize, label and manipulate. Importantly, we have also recently developed a rescue/reconstitution assay that will allow us to prospectively isolate the cells responsible, as well as characterize and functionally assess developmental pathways underlying WBR. This R21 grant is designed to develop this new system, and we propose to: 1) utilize limiting dilution transplantation assays and cell labeling transplantation strategies to assess whether progenitor cells are lineage restricted or pluripotent; and, 2) use gene expression profiling of proliferating cells at different stages during regeneration to identify genes and signaling pathways that regulate stem cell function and induce recruitment and proliferation of circulatory stem cells. We will then functionally test the role of a subset of regenerative pathways and gene regulatory mechanisms using small molecule inhibitors and RNAi. Our goals are that by the end of this 2-year grant, we will have completed a large database of differentially expressed genes, completed initial genetic and functional characterization the developmental pathways underlying regeneration, as well as either having isolated, or have a robust methodology to purify the cells responsible and dissect their role in regeneration. By characterizing the stem cell population responsible for whole body regeneration and identifying genes, signaling pathways and the microenvironment that regulate stem cell recruitment, proliferation and differentiation during regenerative growth, these experiments will greatly advance Botryllus as a model system for the study of genes conserved in stem cell and regenerative biology, and will advance our understanding of human stem-cell function and tissue regeneration.