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

灰霉病菌全身再生的细胞和基因调控机制

• 批准号: 9375865
• 负责人: Anthony W De Tomaso
• 金额: $ 18.58万
• 依托单位: UNIVERSITY OF CALIFORNIA SANTA BARBARA
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-01 至 2019-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9375865
• 关键词: Ablation; Adult; Aging; Animal Model; Animals; Biological Assay; Biological Models; Biological Process; Biology; Blood; Blood Vessels; Cell Cycle; Cell Differentiation process; Cell Lineage; Cell physiology; Cells; Chordata; Complex; Databases; Development; Embryo; Embryonic Development; Endocrine system; Evolution; Exhibits; Failure; Fibroblast Growth Factor; Gastrointestinal tract structure; Gene Expression Profiling; Genes; Genetic; Germ Cells; Goals; Grant; Growth; Heart; Human; Individual; Injection of therapeutic agent; Injury; Invertebrates; Label; Ligands; Longevity; Mammals; Messenger RNA; Methodology; Methods; Microsatellite Repeats; Mitomycins; Molecular; Mus; Natural regeneration; Operative Surgical Procedures; Organ; Organism; Outcome; Pathway interactions; Peripheral; Pharyngeal structure; Play; Population; Procedures; Process; Proliferating; RNA Interference; Recruitment Activity; Regulation; Regulator Genes; Role; Salamander; Signal Pathway; Signaling Protein; Site; Small Interfering RNA; Stem cell transplant; Stem cells; Structure; System; Testing; Tissues; Transplantation; Vertebrates; ascidian; base; design; differential expression; embryonic stem cell; experimental study; human stem cells; in vivo; limb amputation; notch protein; novel strategies; progenitor; prospective; receptor; reconstitution; regenerative; relating to nervous system; repaired; response; small molecule inhibitor; stem cell population; tissue regeneration; vascular bed

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.

所有多细胞生物均来自一小组多多能胚胎干细胞 扩展并区分成熟个体的组织和器官，以及从 人类的蠕虫使用一套高度保守的核心发展途径来完成 胚胎发生过程。但是，在正常生长和衰老期间或受伤后的成年人中， 必须补充或再生分化的细胞和器官。进行再生 通过长寿的，通常是谱系限制的干细胞，可保留扩展和 在整个个人的寿命中区分。但是，细胞和分子 大多数组织的再生发展的机制尚不清楚。 更重要的是，尽管对胚胎发育途径进行了深刻的保护，但 受伤后不同生物可以再生组织和器官的程度不是 整个演变中保守的特征：sal可以再生截肢的肢体，但 人不能。为什么这是真的？该赠款的重点是研究 基底脉管有机体，殖民地的海藻botryllus schlosseri，以探索这些问题。 使用简单的外科手术，我们可以诱导botryllus再生整个身体， 包括心脏，胃肠道，咽，脉管系统，神经和内分泌系统和配子， 来自体外血管床的碎片。这个过程称为全身再生 （wbr），迅速发生（3-6天），海鞘是唯一可以再生的弦 整个身体，因此我们可以剖析高度保守的发展途径 在任何组织的再生过程中重新部署。 Botryllus还提供了独特的学习方法 例如，WBR脉管系统是无序且透明的，易于可视化，标签和 操纵。重要的是，我们最近还开发了一种救援/重建测定法 允许我们前瞻性地隔离负责的单元，并在功能上表征和功能 评估WBR的发展途径。 R21赠款旨在开发此事 新系统，我们建议：1）利用限制稀释移植测定和细胞 标记移植策略以评估祖细胞是谱系是否受到限制或 多能； 2）在不同阶段使用增殖细胞的基因表达分析 再生以识别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，我们将在功能上测试 使用小的再生途径和基因调节机制的作用 分子抑制剂和RNAi。我们的目标是，到这项为期两年的赠款结束时，我们将拥有 完成了一个差异表达基因的大数据库，完成了初始遗传和 功能表征开发途径的基础再生以及 具有孤立的方法，或具有鲁棒的方法来净化负责的细胞并进行剖析 它们在再生中的作用。 通过表征负责全身再生和 识别调节干细胞的基因，信号通路和微环境 在再生增长过程中招募，增殖和分化，这些实验将 大大推进博特洛斯作为研究干细胞中保守基因的模型系统和 再生生物学，并将促进我们对人类干细胞功能的理解和 组织再生。