Control and Implementation of a Morphogenetic Program

形态发生程序的控制和实施

• 批准号: 10592885
• 负责人: David H. A. Fitch
• 金额: $ 1.17万
• 依托单位: NEW YORK UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10592885
• 关键词: Address; Adolescent; Adult; Animals; Binding; Biological; Biological Assay; Biology; Caenorhabditis elegans; Candidate Disease Gene; Cell Polarity; Cell Shape; Cell fusion; Cell physiology; Cells; ChIP-seq; Co-Immunoprecipitations; Congenital Abnormality; Cytology; Cytoskeleton; Data; Data Set; Defect; Development; Developmental Process; Embryo; Event; Evolution; Foundations; Gene Targeting; Genes; Genetic; Genetic Transcription; Genomics; Goals; Growth and Development function; Human; Instruction; Intervention; Knowledge; Label; Laboratories; Lasers; Lead; Link; Lobular Neoplasia; Malignant Neoplasms; Mammals; Medicine; MicroRNAs; Modeling; Molecular; Morphogenesis; Mutation; Nematoda; Outcome; Pathway interactions; Pharmacologic Substance; Play; Positioning Attribute; Post-Translational Regulation; Process; Proteins; Regulation; Regulator Genes; Resolution; Role; Schedule; Shapes; Signal Transduction; Specificity; Structure; System; Tail; Techniques; Testing; Time; Tissues; Transcriptional Regulation; Transduction Gene; Ubiquitination; Untranslated RNA; Vesicle; Work; base; cell behavior; cell motility; chromatin immunoprecipitation; experimental study; gene regulatory network; genetic manipulation; in vivo; innovation; male; network models; novel; programs; pubertal timing; scaffold; sex; sex determination; sexual dimorphism; theories; trafficking; transcription factor; transcriptome sequencing; wound healing

PROJECT SUMMARY / ABSTRACT Cellular morphogenesis is essential to animal development as well as wound healing and cancer. During this process, cells coordinately change shape, migrate, and may fuse. Cellular morphogenesis is executed by molecules called "effectors" involved in junctions, cytoskeleton, cell polarity, vesicular trafficking, and other modules/processes of the cytological machinery. The sex-, tissue-, position-, and time-specificity of morphogenesis is determined by "regulators", such as transcription factors. There are two main knowledge gaps: (1) how morphogenetic events are temporally scheduled, and (2) how the transcriptional regulatory input is linked to the effectors. Here, the overall objective is to elucidate an intriguing part of the timing mechanism that involves a cytoplasmic long-noncoding RNA in post-translational regulation, and to delineate the "missing links" between regulators and effectors for an experimentally accessible structure, the tail tip of C. elegans. The tail tip is made of 4 cells which, in males only, change shape and migrate at the juvenile-to-adult transition (J/A), a process called Tail Tip Morphogenesis (TTM). Prior studies showed that (1) a long non-coding RNA (lep-5) is an instructive switch for TTM onset at the J/A, and (2) transcription factor DMD-3 is required and sufficient for TTM and coordinates several processes of the cytological machinery. The overall approach is to (Aim 1) test the hypothesis that the lep-5 lncRNA acts to schedule TTM cell-autonomously by scaffolding the ubiquitination of a central, conserved node in the heterochronic pathway, LIN-28, by the C. elegans Makorin protein, LEP-2 (another highly conserved but under-characterized protein), and (Aim 2) to determine what are the direct targets of DMD-3 and how they function in TTM. Aim 1 will use primarily co-immunoprecipitation (coIP) and ubiquitination assays to test binding partners of lep-5 lncRNA. Aim 2 will use chromatin immunoprecipitation (ChIP-seq) to identify candidate targets of DMD-3; the roles of these candidates in TTM will then be tested by labeling them or genetically perturbing them in a strain with subcellular landmarks tagged with different fluorescent proteins. The expected outcomes include (1) a mechanistic understanding for a novel paradigmatic role for a lncRNA acting instructively and post-translationally in the regulation of a key conserved temporal regulator, and (2) the foundation of a systems network model for morphogenesis connecting transcriptional control by DMD-3 (a conserved transcription factor homologous to human DMRTs) to the cytological machinery involved in morphogenetic cell behaviors. These results are expected to have additional positive impacts on biology and medicine by (a) determining how sexual dimorphism occurs by linking sex determination to morphogenetic effectors; (b) identifying key conserved genes controlling morphogenesis and its timing, thus potentially identifying new targets for treating defects in pubertal timing, sex-reversal or cancer, or to aid wound healing; and (c) providing candidate genes that may underlie evolutionary diversity.

项目总结/摘要 细胞形态发生对于动物发育以及伤口愈合和癌症是必不可少的。期间 在这个过程中，细胞协调地改变形状，迁移，并可能融合。细胞形态发生是由 称为“效应物”的分子参与连接、细胞骨架、细胞极性、囊泡运输和其他 细胞学机器的模块/过程。性、组织、位置和时间特异性 形态发生由“调节因子”如转录因子决定。主要有两个知识 缺口：（1）形态发生事件是如何在时间上安排的，以及（2）转录调控输入是如何 与效应器相连。在这里，总的目标是阐明一个有趣的部分定时机制 涉及翻译后调节的细胞质长非编码RNA，并描绘“缺失的 调节器和效应器之间的“链接”，实验上可访问的结构，C。优美的 尾尖由4个细胞组成，仅在雄性中，在幼年到成年的过渡期改变形状并迁移 （J/A），这一过程称为尾尖形态发生（TTM）。先前的研究表明，（1）长的非编码RNA （lep-5）是在J/A处TTM发作的指导性开关，和（2）转录因子DMD-3是必需的， 足够用于TTM并协调细胞学机制的几个过程。总的办法是 (Aim 1）测试lep-5 lncRNA通过支架化TTM细胞来自主地调度TTM细胞的假设， 泛素化的一个中心，在异时性途径，LIN-28中的保守节点，由C。秀丽隐翅虫 蛋白质，LEP-2（另一种高度保守但特征不足的蛋白质），和（目的2），以确定什么是 DMD-3的直接靶点以及它们在TTM中的作用。目的1将主要使用免疫共沉淀 （coIP）和泛素化测定以测试lep-5lncRNA的结合配偶体。AIM 2将使用染色质 免疫沉淀（ChIP-seq）来鉴定DMD-3的候选靶点;这些候选靶点在TTM中的作用 然后将通过标记它们或在标记有亚细胞标志的菌株中对其进行遗传干扰来进行测试 不同的荧光蛋白。预期的结果包括：（1）对小说的机械理解 一个lncRNA在调节一个关键的保守的 时间调节器;（2）形态发生连接系统网络模型的建立 DMD-3（一种与人DMRT同源的保守转录因子）的转录控制， 涉及形态发生细胞行为的细胞学机制。这些结果预计将有额外的 对生物学和医学的积极影响，（a）确定性二态性是如何发生的， （B）鉴定控制形态发生的关键保守基因， 它的时机，从而可能确定新的目标，治疗缺陷的青春期时间，性别逆转或癌症， 或帮助伤口愈合;以及（c）提供可能构成进化多样性基础的候选基因。