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

斑马鱼细胞命运和形态发生的细胞周期调控

基本信息

批准号：
10463258
负责人：
Samantha Stettnisch
金额：
$ 3.65万
依托单位：
STATE UNIVERSITY NEW YORK STONY BROOK
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-05-19 至 2025-05-18
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10463258
关键词：
Address Adopted Affect Anterior Behavior Biosensor Brachyury protein CDK2 gene CDK4 gene CDKN1C gene Cell Cycle Cell Cycle Arrest Cell Cycle Inhibition Cell Cycle Regulation Cell Transplantation Cells Complex Confocal Microscopy Cues Cyclin-Dependent Kinase Inhibitor Cyclin-Dependent Kinases Data Development Disease Disseminated Malignant Neoplasm Embryo Floor G1 Arrest Gene Expression Genes Goals Knowledge Lead Malignant Neoplasms Mediating Modeling Morphogenesis Movement Organism Outcome Phase Play Population Process Publishing Regulation Research Role Signal Pathway Signal Transduction Structure Testing Time Tissues To specify Transcriptional Regulation Transgenic Organisms Up-Regulation Work Zebrafish base cancer cell cell behavior cell fate specification cell motility cellular imaging convergent extension developmental disease gene regulatory network in vivo in vivo Model insight knock-down migration mutant new therapeutic target notch protein notochord notochord development novel novel therapeutics overexpression prevent progenitor single-cell RNA sequencing stem cells transcription factor zebrafish development

项目摘要

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.

项目概要/摘要适当的有机体发育需要复杂的形态发生过程。这些形态发生过程需要细胞迁移、增殖、侵袭和命运获得的各种组合。这这些行为的协调必须受到严格监管，因为这些过程的失调可能会导致发育障碍和癌症等疾病状态。研究复杂的调控我们转向斑马鱼的形态发生过程。在斑马鱼中，中线组织的形态发生脊索、底板和下脊索等结构驱动发育中胚胎的轴伸长。这些组织源自尾芽中的一群祖细胞，称为中线祖细胞细胞（MPC）。 MPC 经历称为收敛延伸 (CE) 的形态发生过程，以产生脊索。在 CE 期间，相邻的 MPC 相互迁移并插入以形成脊索。 MPC 决定采用脊索、底板或次弦命运是基于本地信号线索例如Wnt和Notch。虽然这些信号通路已被证明可以调节细胞形态发生行为，越来越多的证据表明细胞周期也可以调节细胞行为。然而，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不清楚。我将解决这一知识空白并阐明细胞周期状态和细胞周期之间的关系使用斑马鱼尾芽期间脊索的 CE 进行发育过程中的细胞命运/形态发生形态发生作为模型。我的实验室和其他实验室公布的数据显示脊索祖细胞处于 G1 期，而底板和下索祖细胞则存在于细胞周期的所有阶段。此外，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 停滞促进了这种形态发生过程和脊索命运的获得。在这个项目的目标 1 中，我将干扰 MPC 中的 G1 逮捕并确定对 CE 的影响。使用转基因斑马鱼品系，包括用于建立细胞周期状态的 CDK 活性生物传感器和转盘共聚焦显微镜，我将对这些细胞周期扰动的胚胎进行延时成像并量化 CE 和命运获取。在目标 2 中，我将探索负责诱导 MPC 中 G1 期停滞的基因调控网络 (GRN)，重点关注 T 盒转录因子 Brachyury (tbxta)。已发表的数据显示 tbxta 对于脊索来说是不可或缺的此外，我们实验室的初步数据显示 tbxta 驱动脊索祖细胞的敲低循环并被排除在脊索之外，采用底板或下弦命运。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焦显微镜将使我能够彻底而严格地检验我的假设。