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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

基本信息

批准号：
8513373
负责人：
Cortney M Bouldin
金额：
$ 5.39万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
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/8513373
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 Profiling 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

项目摘要

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主要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的增殖是如何调节的。祖细胞是类干细胞，其定义为它们能够保持不分化，直到被提示接受最终命运。在脊椎动物胚胎发生的早期阶段，一群中胚层祖细胞驻留在身体最后端的一个称为尾芽的区域。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，这些体节将成为成体的肌肉和椎骨。中胚层祖细胞的增殖如何被控制以及如何与分化协调尚不清楚。我建议利用斑马鱼系统的特定优势来研究中胚层祖细胞及其后代的增殖调节，包括易于制作转基因系、胚胎的光学透明度以及将转基因细胞移植到非转基因胚胎中的能力。该提案的目的是（1）确定中胚层祖细胞及其后代在活胚胎中是否正在分裂或静止； (2) 检验成纤维细胞生长因子（Fgf）和/或Wnt信号通路调节中胚层祖细胞增殖的假设； (3)确定体节形成过程中的增殖如何影响体节数量、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示器 (Fucci) 的修改版本，我创建了一种新的转基因斑马鱼报告系，并建议使用该新系来识别中胚层祖细胞及其后代何时分裂。我还将确定非分裂细胞在细胞周期中的位置。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型细胞自主通路抑制剂阻断每个通路，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形成，我将使用表达热休克诱导细胞周期检查点调节剂的新斑马鱼转基因系来提高体节形成过程中的增殖率。我将量化这个新转基因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。这些研究旨在利用斑马鱼系统来确定在高度保守的身体形成过程中调节生长控制的机制，并阐明干细胞样祖细胞群中生长控制的整体机制。