Transcriptional regulation of morphogenetic behavior and invasive cell fate specification in C. elegans

项目摘要/摘要 细胞穿越基底膜(BMS)的能力是生育、发育、免疫和 疾病。BM的侵袭是通过表达细胞外基质蛋白、上调基质 金属蛋白酶，F-肌动蛋白细胞骨架的极化，以及细胞周期停滞。它们之间的精确协调 支持侵入性的程序在很大程度上是通过转录调控实现的；然而，我们对 由于缺乏细胞侵袭的模型系统，涉及的基因调控网络(GRN)有限 被视觉化直播。在这里，我建议通过利用线虫的形态发生来填补这一知识空白 秀丽的子宫-外阴连接作为一种易于处理和视觉上易于观察的模型来检查体内的细胞侵袭。 在两性体细胞性腺的发育过程中，一种特殊的子宫细胞称为锚细胞(AC)。 侵入基底膜以连接子宫和外阴上皮。AC本身在 发育早期的细胞命运决定事件，其中两个初始等价的细胞通过随机Notch发散 不对称，导致推测的AC和增生性腹侧子宫(VU)细胞。我们之前的研究 Lab和其他人已经确定了六个调控AC侵袭的转录因子(TF)。其中包括基本的 亮氨酸拉链Tffos-1(Fos)，基本螺旋-环-螺旋Tf-hlh-2(E/无子)，两种核激素 受体NHR-67(NR2E1/Tillless/TLX)和性别-1(RARB/NR1B2)，以及两个锌指转录因子EGL-43 (EVI1/MEL1)和MEP-1。这些TF似乎在至少三个不同的GRN子回路中起作用 调节AC侵袭，其中之一涉及NHR-67，其作用是在CKI-1(p21/p27)上游诱导G1 细胞周期停滞。值得注意的是，在AC/VU细胞的命运过程中，六个前侵袭性TF中的五个反复发挥作用 决定。其中包括三个转录因子，包括NHR-67/细胞周期依赖的前侵袭途径(EGL- 43S、MEP-1和NHR-67)，以及预测了NHR-67内结合位点的HLH-2和性别-1 推动者。因此，根据文献和我的初步研究，我的中心假设是AC侵袭性 程序依赖于多个GRN子电路的功能，其中一个调节细胞周期停滞和 在交流命运规范中反复使用。在这个项目的目标1，我将剖析顺式和跨式调节 AC侵袭，集中在细胞周期依赖的GRN亚路，涉及前侵袭的TF NHR-67/TLX。在……里面 目标2，我将检查在AC规范中反复发挥作用的前侵入性TF的作用，并调查 如果细胞周期控制是这两个过程的共同点。尖端的功能工具， 包括内源性蛋白质耗尽系统和新的细胞周期状态传感器，与 进行高分辨率亚细胞视觉分析，将允许对这一假设进行彻底和严格的测试。

PROJECT SUMMARY / ABSTRACT The ability of cells to traverse basement membranes (BMs) is a key part of fertility, development, immunity, and disease. BM invasion is facilitated through expression of extracellular matrix proteins, upregulation of matrix metalloproteinases, polarization of the F-actin cytoskeleton, and cell cycle arrest. Precise coordination of these pro-invasive programs is largely achieved through transcriptional regulation; however, our understanding of the gene regulatory networks (GRNs) involved is limited due to the lack of model systems in which cell invasion can be visualized live. Here, I propose to fill this gap in knowledge by utilizing morphogenesis of the Caenorhabditis elegans uterine-vulval connection as a tractable and visually amenable model to examine cell invasion in vivo. During development of the hermaphroditic somatic gonad, a specialized uterine cell called the anchor cell (AC) invades through the underlying BM to connect the uterus to the vulval epithelium. The AC itself is specified in a cell fate decision event earlier in development, in which two initially equipotent cells diverge via stochastic Notch asymmetry, giving rise to the presumptive AC and a proliferative ventral uterine (VU) cell. Prior research by our lab and others has identified six transcription factors (TFs) that regulate AC invasion. These include the basic leucine zipper TF fos-1 (Fos), the basic helix-loop-helix TF hlh-2 (E/Daughterless), two nuclear hormone receptors, nhr-67 (NR2E1/Tailless/TLX) and sex-1 (RARB/NR1B2), as well as two zinc-finger TFs, egl-43 (EVI1/MEL1) and mep-1. These TFs appear to be functioning in at least three distinct GRN sub-circuits to regulate AC invasion, one of which involves NHR-67, which functions upstream of CKI-1 (p21/p27) to induce G1 cell cycle arrest. Remarkably, five of the six pro-invasive TFs function reiteratively during the AC/VU cell fate decision. These include the three TFs comprising the NHR-67/cell cycle-dependent pro-invasive pathway (EGL- 43S, MEP-1, and NHR-67), as well as HLH-2 and SEX-1, which have predicted binding sites within the nhr-67 promoter. Thus, based on the literature and my preliminary studies, my central hypothesis is that the AC invasive program is dependent on the function of multiple GRN sub-circuits, one of which modulates cell cycle arrest and is reiteratively used in AC fate specification. In Aim 1 of this project, I will dissect the cis- and trans-regulation of AC invasion, focusing on the cell cycle-dependent GRN sub-circuit involving the pro-invasive TF nhr-67/TLX. In Aim 2, I will examine the roles of pro-invasive TFs that reiteratively function in AC specification and investigate if cell cycle control is the common denominator underlying these two processes. Cutting-edge functional tools, including an endogenous protein depletion system and a novel cell cycle state sensor, paired with the ability to perform high-resolution subcellular visual analyses, will allow for thorough and rigorous testing of this hypothesis.