Regulation of proliferation in vertebrate embryonic mesodermal progenitors

脊椎动物胚胎中胚层祖细胞增殖的调节

• 批准号: 8326850
• 负责人: Cortney M Bouldin
• 金额: $ 5.22万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-29 至 2014-08-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8326850
• 关键词: Adult; Affect; Bypass; Cancer Biology; Cell Cycle; Cell Cycle Checkpoint; Cell Cycle Progression; Cell Transplants; Cell division; Cell physiology; Cells; Cues; Dermis; Embryo; Embryonic Development; Fibroblast Growth Factor; Gene Expression; Growth; Heat-Shock Response; Interphase Cell; Knowledge; Left; Length; Life; Mesoderm; Mesoderm Cell; Morphogenesis; Muscle; Optics; Pathway interactions; Phase; Population; Process; Regulation; Reporter; Research; Research Design; Signal Pathway; Signal Transduction; Somites; Staging; Stem cells; System; Tail; Techniques; Testing; Transgenic Organisms; Transplantation; Ubiquitin; Undifferentiated; Zebrafish; bone; daughter cell; exhaust; exhaustion; extracellular; gastrulation; in vivo; inhibitor/antagonist; insight; muscle form; neoplastic cell; overexpression; progenitor; somitogenesis; spine bone structure; stem; stem cell biology; stem cell population; tumor

DESCRIPTION (provided by applicant): A key difference between a stem cell and a tumor cell is the ability to regulate proliferation. In most cases, however, it is not known how proliferation is regulated in stem cell populations in vivo. Progenitor cells are stem-like cells defined by their ability to remain undifferentiated until cued to accept a terminal fate. During the early stages of vertebrate embryogenesis, a population of mesodermal progenitor cells resides at the most posterior end of the body in a region called the tailbud. Cells progressively leave the progenitor population of the tailbud and differentiate, producing blocks of mesoderm called somites that will become the muscle and vertebrae of the adult body. How proliferation in the mesodermal progenitors is controlled and coordinated with differentiation is poorly understood. I propose to investigate regulation of proliferation in the mesodermal progenitors and their progeny by using the specific advantages of the zebrafish system including ease of making transgenic lines, optical clarity of the embryos, and the ability to transplant transgenic cells into nontransgenic embryos. This proposal is aimed (1) to determine if the mesodermal progenitors and their descendants are dividing or quiescent in living embryos; (2) to test the hypothesis that Fibroblast growth factor (Fgf) and/or Wnt signaling pathways regulate proliferation in mesodermal progenitors; and (3) to determine how proliferation during somite formation affects somite number, somite size, and the differentiation and morphogenesis of mesodermal cells. Using a modified version of a recently developed technique, Fluorescent Ubiquitin Cell Cycle Indicators (Fucci), I have created a new transgenic zebrafish reporter line, and propose to use the new line to identify when the mesodermal progenitors and their progeny divide. I will also determine where in the cell cycle non-dividing cells are held. To identify if the Fgf or Wnt signaling pathways regulate the rate of proliferation in the mesodermal progenitors, I will block each pathway using a heat shock-inducible, cell autonomous pathway inhibitor followed by analysis of proliferation in transplanted cells using Fucci. Finally, to determine how proliferation affects somite formation, I will increase the rate of proliferation during somite formation with a new zebrafish transgenic line expressing a heat-shock inducible, cell cycle checkpoint regulator. I will quantify the number and size of somites formed in this new transgenic line, and analyze cell fates in transplanted cells by gene expression analysis. These studies are designed to take advantage of the zebrafish system to identify mechanisms regulating growth control throughout the highly conserved process of body formation and elucidate global mechanisms of growth control in a stem cell-like progenitor population.

描述（由申请人提供）：干细胞和肿瘤细胞之间的关键区别是调节增殖的能力。然而，在大多数情况下，尚不清楚体内干细胞群中增殖是如何调节的。祖细胞是干细胞样细胞，其定义是它们保持未分化直到接受终末命运的能力。在脊椎动物胚胎发生的早期阶段，一群中胚层祖细胞位于身体的最后端称为尾芽的区域。细胞逐渐离开尾芽的祖细胞群并分化，产生称为体节的中胚层块，这些体节将成为成年身体的肌肉和椎骨。中胚层祖细胞的增殖是如何控制和协调分化的知之甚少。我建议调查中胚层祖细胞及其后代的增殖调节，通过使用斑马鱼系统的具体优势，包括易于制作转基因系，胚胎的光学清晰度，以及将转基因细胞移植到非转基因胚胎的能力。该提议旨在（1）确定中胚层祖细胞及其后代在活胚胎中是分裂还是静止;（2）检验成纤维细胞生长因子（Fgf）和/或Wnt信号传导途径调节中胚层祖细胞增殖的假设;和（3）确定体节形成期间的增殖如何影响体节数目、体节大小以及中胚层细胞的分化和形态发生。使用最近开发的技术，荧光泛素细胞周期指标（Fucci）的修改版本，我已经创建了一个新的转基因斑马鱼报告线，并建议使用新的线来确定中胚层祖细胞和他们的后代分裂时。我还将确定在细胞周期中非分裂细胞的位置。为了确定Fgf或Wnt信号通路是否调节中胚层祖细胞的增殖速率，我将使用热休克诱导的细胞自主通路抑制剂阻断每种通路，然后使用Fucci分析移植细胞的增殖。最后，为了确定增殖如何影响体节的形成，我将增加一个新的斑马鱼转基因系表达热休克诱导，细胞周期检查点调节器的体节形成过程中的增殖率。我将量化在这个新的转基因株系中形成的体节的数量和大小，并通过基因表达分析来分析移植细胞中的细胞命运。这些研究的目的是利用斑马鱼系统，以确定整个高度保守的身体形成过程中的生长控制调节机制，并阐明干细胞样祖细胞群体中生长控制的全球机制。