Cell Cycle Regulation of Cell Fate and Morphogenesis in D. rerio

项目概要/摘要 复杂的形态发生过程是生物正常发育所必需的。这些形态发生 这些过程需要细胞迁移、增殖、侵袭和命运获得的各种组合。的 这些行为的协调必须受到严格的调控，因为这些过程的失调可能导致 发育障碍和疾病状态如癌症。研究复合物的调控 形态发生过程我们转向斑马鱼的发展。斑马鱼中线组织的形态发生 脊索、底板和下索等结构驱动发育中胚胎的轴伸长。 这些组织来源于位于尾芽中的称为中线祖细胞的祖细胞群 细胞（MPC）。MPC经历称为会聚延伸（CE）的形态发生过程，以产生 脊索在CE过程中，相邻的MPC迁移并插入彼此之间形成脊索。 MPC决定采用脊索、底板或下索的命运是基于局部信号线索 例如Wnt和Notch。虽然这些信号通路已被证明可以调节形态发生细胞 越来越多的证据表明，细胞周期也可以调节细胞行为。然而，在这方面， 在尾芽形态发生过程中，细胞周期状态决定细胞行为和细胞命运的机制 仍然不清楚。我将解决这一知识差距，并阐明细胞周期状态和细胞周期之间的关系。 斑马鱼尾芽期脊索发育过程中的细胞命运/形态发生 形态发生作为一个模型。我的实验室和其他实验室发表的数据显示脊索祖细胞处于G1期， 而底板和下索祖细胞可以在细胞周期的所有阶段中发现。而且这些 脊索祖细胞在G1期经历CE，并在加入脊索后重新进入细胞周期，这表明， G1期阻滞促进了这一形态发生过程和脊索命运的获得。在本项目的目标1中，我将 干扰G1期阻滞，特别是在MPC和确定对CE的影响。使用转基因斑马鱼品系， 包括建立细胞周期状态的CDK活性生物传感器和旋转圆盘共聚焦显微镜， 对这些细胞周期紊乱胚胎进行延时成像并定量CE和命运获取。在目标2中，我将 探索负责诱导MPC中G1期阻滞的基因调控网络（GRN），重点是 T-box转录因子Brachyury（tbxta）。已发表的数据表明tbxta是不可缺少的脊索 此外，我们实验室的初步数据显示，tbxta的敲除可以驱动脊索祖细胞 循环并被排除在脊索之外，采用底板或hypochord命运。的组合 CDK活性生物传感器、细胞周期扰动构建体、中线定向细胞移植和旋转 圆盘共聚焦显微镜可以让我彻底而严格地检验我的假设。

PROJECT SUMMARY / ABSTRACT Complex morphogenetic processes are required for proper organismal development. These morphogenetic processes require various combinations of cell migration, proliferation, invasion, and fate acquisition. The coordination of these behaviors must be tightly regulated, as dysregulation of these processes can lead to developmental disorders and disease states such as cancer. To study the regulation of complex morphogenetic process we turn to zebrafish development. In zebrafish, morphogenesis of midline tissue structures such as the notochord, floor plate, and hypochord drive axis elongation of the developing embryo. These tissues are derived from a population of progenitors residing in the tailbud known as midline progenitor cells (MPCs). MPCs undergo a morphogenetic process called convergent extension (CE) to give rise to the notochord. During CE, adjacent MPCs migrate and intercalate between one another to form the notochord. The decision of MPCs to adopt a notochord, floor plate, or hypochord fate is based on local signaling cues such as Wnt and Notch. While these signaling pathways have been shown to regulate morphogenetic cell behaviors, there is growing evidence to suggest that the cell cycle can also modulate cell behaviors. However, the mechanisms by which cell cycle state dictates cell behavior and cell fate during tailbud morphogenesis remain unclear. I will address this gap in knowledge and elucidate the relationship between cell cycle state and cell fate/morphogenesis during development using CE of the zebrafish notochord during tailbud morphogenesis as a model. Published data from my lab and others show notochord progenitor cells are in G1, while floor plate and hypochord progenitors can be found in all phases of the cell cycle. Furthermore, these notochord progenitors undergo CE in G1 and reenter the cell cycle after joining the notochord, suggesting that G1 arrest facilitates this morphogenetic process and notochord fate acquisition. In Aim 1 of this project, I will perturb G1 arrest specifically in the MPCs and determine the effects on CE. Using transgenic zebrafish lines, including a CDK activity biosensor to establish cell cycle state, and spinning disk confocal microscopy, I will time-lapse image these cell cycle perturbed embryos and quantify CE and fate acquisition. In Aim 2, I will explore the gene regulatory network (GRN) responsible for inducing G1 arrest in the MPCs, with a focus on the T-box transcription factor Brachyury (tbxta). Published data show tbxta to be indispensable to notochord formation and moreover, preliminary data from our lab show knockdown of tbxta drives notochord progenitors to cycle and be excluded from the notochord, adopting a floor plate or hypochord fate. The combination of a CDK activity biosensor, cell cycle perturbation constructs, midline directed cell transplantation, and spinning disk confocal microscopy will allow me to test my hypotheses thoroughly and rigorously.