Regulation of Mesodermal Progenitors in Transgenic Zebrafish

描述(申请人提供)：脊椎动物早期发育的一个标志是胚体从前部(A)到后部(P)的渐进生长，这依赖于位于胚胎最后端的多能干状祖细胞群体，该区域称为尾芽。这种祖细胞群体逐渐释放生长在体节(主要是肌肉)中的中胚层细胞，以及形成脊髓的神经细胞，直到建立完整的A-P轴。为了身体的正常形成，尾芽的释放速度和细胞的增殖都必须仔细控制，以便沿着整个A-P轴产生正确的细胞比例。这些过程是如何被监管和整合的，人们知之甚少。这项提议的第一个目的是研究细胞离开尾芽进入体节的机制。根据最近对一个新的转基因品系的初步结果，将通过检验Wnt功能的两个假说来阐明Wnt信号在调节这一过程中的作用。此外，分析tbx16/spaDetail启动子中的一个独特的小元件，它在细胞决定离开祖细胞群体时被激活，将提供对调节随着身体伸长而承诺中胚层命运的初始步骤的分子机制的关键洞察。识别控制细胞分配的机制将是理解脊椎动物胚胎如何准确地调节尾芽细胞释放的重要一步。第二个目标将确定为什么细胞增殖在原肠胚后的胚胎中受到严格调控，以至于祖细胞处于静止状态，只有在它们开始分化时才分裂。这一目标将验证这样的假设，即使用我们最近培育的一种新的转基因株，这种调控对于允许中胚祖细胞及其衍生物的正常信号和/或形态发生是必不可少的。确定脊椎动物胚胎为什么严格控制增殖对于了解胚胎如何调节竞争需求至关重要，以增加细胞数量，同时维持建立胚胎身体计划所需的复杂信号和形态发生过程。研究，特别是在哺乳动物中，表明后祖细胞是一个类似干细胞的群体，它贡献了不同类型的细胞。由于斑马鱼能够产生表达时间受控的信号和细胞增殖调节因子的转基因株系，以及能够轻松地下调基因功能，因此为了解脊椎动物干细胞在体内是如何调控的提供了一个很好的模型系统。由于干细胞在治疗许多疾病方面有很大的希望，这里描述的研究将提供关于控制干细胞维持和组织形成的信号网络和调节因子的有价值的信息。

DESCRIPTION (provided by applicant): A hallmark of early vertebrate development is the progressive growth of the embryonic body from the anterior (A) to the posterior (P), which relies on a multipotent stem-like progenitor population located at the most posterior end of the embryo, in a region called the tailbud. This progenitor population gradually releases mesodermal cells that populate the somites (primarily muscle), as well as neural cells that form the spinal cord, until the complete A-P axis has been established. For the body to form normally, both the rate of release from the tailbud and cell proliferation must be carefully controlled so tht the correct proportion of cells is produced along the entire A-P axis. How these processes are regulated and integrated is poorly understood. The first aim of this proposal will examine the mechanisms by which cells leave the tailbud and enter the somites. Based on recent preliminary results with a new transgenic line, the role of Wnt signaling in regulating this process will be elucidated by testing two hypotheses for Wnt function. In addition, analysis of a small unique element within the tbx16/spadetail promoter, which is activated just as cells make the decision to leave the progenitor population, will provide key insight into the molecular mechanism that regulates the initial step in the commitment to the mesodermal fate as the body elongates. Identifying the mechanisms that control cell allocation will be a major step forward in understanding how the vertebrate embryo precisely regulates the release of cells from the tailbud. The second aim will determine why cell proliferation is tightly regulated in the post-gastrula embryo such that the progenitors are quiescent, and only divide when they first begin to differentiate. This aim will test the hypotheses that this regulation is essential to allow normal signaling and/or morphogenesis of the mesodermal progenitors and their derivatives using a novel transgenic line we have recently produced. Determining why the vertebrate embryo strictly controls proliferation is essential for understanding how the embryo regulates the competing needs to increase cell number yet maintain the complex signaling and morphogenetic processes necessary to establish the embryonic body plan. Studies, particularly in mammals, show that the posterior progenitors are a stem-cell like population, which contribute to a variety of cell types. With the ability to produce transgenic lines expressing temporally controlled regulators of signaling and cell proliferation, as well as the ability to easily knock down gene function, zebrafish provides an excellent model system for understanding how vertebrate stem cells are regulated in vivo. As stem cells have great promise for the treatment of many diseases, the studies described here will provide valuable information about the signaling networks and regulatory factors that control stem cell maintenance and tissue formation.