Control and Implementation of a Morphogenetic Program

项目概要/摘要 细胞形态发生对于动物发育以及伤口愈合和癌症至关重要。期间 在此过程中，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“效应子”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。性别、组织、位置和时间特异性 形态发生是由“调节器”决定的，例如转录因子。主要有两个知识点 差距：（1）形态发生事件如何在时间上安排，以及（2）转录调控输入如何 与效应器相连。在这里，总体目标是阐明计时机制的一个有趣的部分 涉及翻译后调节中的细胞质长非编码RNA，并描绘“缺失的 调节器和效应器之间的“链接”，用于实验上可访问的结构，即秀丽隐杆线虫的尾尖。 尾尖由 4 个细胞组成，仅在雄性中，尾尖在幼年向成年过渡时会改变形状并迁移 (J/A)，一个称为尾尖形态发生 (TTM) 的过程。先前的研究表明（1）长非编码RNA (lep-5) 是 J/A 处 TTM 起始的指导性开关，并且 (2) 需要转录因子 DMD-3，并且 足以用于 TTM 并协调细胞学机器的多个过程。总体方法是 （目标 1）测试以下假设：lep-5 lncRNA 通过搭建支架来自主调度 TTM 细胞 线虫 Makorin 对异时通路 LIN-28 中的中央保守节点进行泛素化 蛋白质、LEP-2（另一种高度保守但特征不足的蛋白质）和（目标 2）以确定什么是 DMD-3 的直接目标及其在 TTM 中的作用。目标 1 将主要使用免疫共沉淀 (coIP) 和泛素化检测来测试 Lep-5 lncRNA 的结合伴侣。目标 2 将使用染色质 免疫沉淀 (ChIP-seq) 以确定 DMD-3 的候选靶点；这些候选人在 TTM 中的角色 然后将通过标记它们或在带有亚细胞标志标记的菌株中对它们进行基因扰动来进行测试 与不同的荧光蛋白。预期结果包括（1）对小说的机械理解 lncRNA在关键保守调控中的指导性和翻译后作用的范例作用 时间调节器，以及（2）用于形态发生连接的系统网络模型的基础 DMD-3（与人类 DMRT 同源的保守转录因子）对转录进行控制 参与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 通过以下方式对生物学和医学产生积极影响：(a) 通过将性别联系起来确定性二态性如何发生 形态发生效应器的测定； (b) 鉴定控制形态发生的关键保守基因和 它的时机，从而有可能确定治疗青春期时机缺陷、性逆转或癌症的新目标， 或帮助伤口愈合； (c) 提供可能构成进化多样性基础的候选基因。

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.