Regulation of Mesodermal Progenitors in Transgenic Zebrafish

描述（由申请人提供）：早期脊椎动物发育的一个标志是胚胎体从前部(A)到后部(P)的渐进式生长，这依赖于位于胚胎最后端，称为尾芽的区域的多能茎样祖群体。这个祖细胞群逐渐释放出填充体（主要是肌肉）的中胚层细胞，以及形成脊髓的神经细胞，直到完整的A-P轴建立。为了使身体正常形成，必须仔细控制从尾芽释放的速度和细胞增殖的速度，以便沿着整个A-P轴产生正确比例的细胞。这些过程是如何调节和整合的，人们知之甚少。这个提议的第一个目的是研究细胞离开尾芽进入体的机制。基于最近一个新的转基因品系的初步结果，我们将通过验证Wnt功能的两个假设来阐明Wnt信号在调节这一过程中的作用。此外，对tbx16/spadetail启动子中一个小而独特的元件的分析将提供关键的见解，该元件在细胞决定离开祖细胞群时被激活，从而调节身体延长时向中胚层命运承诺的初始步骤。确定控制细胞分配的机制将是理解脊椎动物胚胎如何精确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胚胎中受到严格的调控，使得祖细胞处于静止状态，只有在它们开始分化时才会分裂。这一目标将测试的假设，这种调节是必不可少的，以允许正常的信号和/或形态发生的中胚层祖细胞及其衍生物使用新的转基因系我们最近生产。确定脊椎动物胚胎严格控制增殖的原因对于理解胚胎如何调节增加细胞数量的竞争需求，同时维持建立胚胎体计划所必需的复杂信号传导和形态发生过程至关重要。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，它有助于多种细胞类型。斑马鱼有能力产生表达暂时受控的信号和细胞增殖调节因子的转基因系，以及容易敲除基因功能的能力，为了解脊椎动物干细胞如何在体内受到调节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具有很大的前景，本文所述的研究将提供有关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.