Understanding developmental patterning's influence on morphogenesis

了解发育模式对形态发生的影响

• 批准号: 10389165
• 负责人: Emily Bowie
• 金额: $ 6.76万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10389165
• 关键词: Actins; Actomyosin; Address; Affect; Apical; Area; Biochemical; Biological; Brain; Caenorhabditis elegans; Candidate Disease Gene; Cell Polarity; Cell Shape; Cells; Cellular biology; Complement; Congenital Abnormality; Contracts; Cytoskeleton; Data Set; Defect; Development; Embryo; Endoderm; Event; Failure; Future; Genes; Genetic; Genetic Transcription; Goals; Human; Imaging Techniques; Knowledge; Lead; Link; Live Birth; Mass Spectrum Analysis; Modeling; Molecular; Morphogenesis; Myosin ATPase; Nematoda; Neural Tube Closure; Neural Tube Defects; Neural tube; Nonmuscle Myosin Type IIA; Optics; Organism; Pattern; Phosphotransferases; Principal Investigator; Process; Proteins; RNA Interference; Regulation; Reproducibility; Role; Shapes; Side; Spinal Cord; Surface; Testing; Time; Tissues; Training; Transcript; Translating; United States; Up-Regulation; Work; biochemical tools; career; cell cortex; cell fate specification; constriction; gastrulation; insight; knock-down; mutant; neuroepithelium; novel; peroxisome; precursor cell; recruit; response; screening; spatiotemporal; success; transcription factor; transcriptome; transcriptome sequencing

Abstract Morphogenetic events utilize precisely timed changes in cell shape. One of the fundamental mechanisms cells use to change their shape is apical constriction. Apical constriction relies on the contraction of cortical actomyosin networks that causes the apical side of a cell to shrink, resulting in tissue morphogenesis. In humans, apical constriction aids the internalization of the future spinal cord and brain in a process known as neural tube closure. Failure of apical constriction can lead to neural tube defects, which accounts for birth defects in 1 out of every 3,000 live births. Therefore, uncovering the processes that govern apical constriction will advance our understanding of basic mechanisms underlying cell shape changes, causes, and potential treatments for neural tube defects. Despite current knowledge of developmental patterning of apical constriction, precise genetic mechanisms that govern which cells undergo apical constriction, how the apical surface is determined, and when to constrict, remain only partially understood. I plan to use Caenorhabditis elegans (C. elegans) gastrulation, a morphogenetic event driven by apical constriction, to address these issues. Gastrulation in C. elegans starts with the internalization of the two endodermal precursor cells (EPCs), which depend on the spatial and temporal precision of the expression of cell fate specification factors end-1 and end-3. However, mechanistic links between end-1,3 and the resulting apical constriction remain largely unknown. Using the genetically tractable and optically clear C. elegans, I plan to dissect the cellular mechanisms that translate developmental patterning into specific, localized, and precisely timed cell shape changes. Comparing the transcriptome of wild-type and end-3 null embryos, I identified thirty target genes whose expression depends on end-3. After screening these genes, I identified ten new genes that contribute to C. elegans gastrulation. In Aim 1, I will use a variety of cell biological approaches to identify the mechanisms by which some of these genes couple developmental patterning to changing cell shape. Aim 2 focuses on the myosin-activating kinase MRCK-1 localizes to the apical cell cortex of EPCs and is required for apical constriction. MRCK-1 is dependent on end-1,3 expression and becomes localized apically specifically in only EPCs despite MRCK-1 being present at similar levels in all cells. I will use MRCK-1 localization as a molecular foothold for understanding how a pivotal protein becomes recruited to the apical cortex in only certain cells. Aim 2 will further investigate which domains of MRCK-1 are required for this localization pattern and identify interactors with these domains that function to initiate apical constriction, to better connect cell fate regulators and intracellular localization of a key protein. Overall, I propose the use of genetic, biochemical, and imaging techniques to advance our understanding of how transcriptional networks and other developmental patterning inputs deploy localized factors that influence cell shape changes.

摘要 形态发生事件利用细胞形状的精确定时变化。根本的一 细胞用来改变其形状的机制是顶端收缩。心尖收缩依赖于 皮质肌动球蛋白网络的一部分，导致细胞顶端收缩，导致组织 形态发生在人类中，顶端收缩有助于未来脊髓和大脑的内化， 这一过程被称为神经管闭合。顶端收缩的失败可导致神经管缺陷， 每3 000名活产婴儿中就有1名存在出生缺陷。因此，揭示控制 顶端收缩将促进我们对细胞形状变化的基本机制的理解， 神经管缺陷的原因和潜在的治疗方法。 尽管目前对顶端缢缩的发育模式的了解，精确的遗传学研究还不清楚。 控制哪些细胞经历顶端收缩的机制，如何确定顶端表面，以及 什么时候收缩，仍然只是部分理解。我计划使用秀丽隐杆线虫（C. elegans） 原肠胚形成，由顶端收缩驱动的形态发生事件，以解决这些问题。C. elegans从两个内胚层前体细胞（EPCs）的内化开始，这取决于 细胞命运特化因子end-1和end-3表达的空间和时间精度。然而，在这方面， 末端-1，3和所产生的顶端收缩之间的机械联系仍然很大程度上未知。使用 遗传上易处理的和光学上透明的C.我计划剖析细胞机制， 发育模式转化为特定的、局部的和精确定时的细胞形状变化。比较 通过分析野生型和末端3缺失胚胎的转录组，我确定了30个靶基因，它们的表达依赖于 在第三端。在筛选了这些基因后，我发现了10个新的基因，这些基因对C。原肠形成在 目的1，我将使用各种细胞生物学方法来确定其中一些机制， 基因将发育模式与细胞形状的改变结合起来。 目的2：肌球蛋白激活激酶MRCK-1定位于内皮祖细胞的顶细胞皮层， 是心尖收缩所必需的MRCK-1依赖于末端-1，3的表达，并定位于顶端 特别是仅在EPC中，尽管MRCK-1在所有细胞中以相似的水平存在。我将使用MRCK-1 定位作为理解关键蛋白如何被募集到顶端的分子立足点 只在某些细胞中存在。目的2将进一步研究MRCK-1的哪些结构域是必需的 定位模式，并确定与这些结构域的相互作用，功能启动顶端收缩， 更好地连接细胞命运调节因子和关键蛋白的细胞内定位。总的来说，我建议使用 遗传、生化和成像技术，以促进我们对转录网络如何 和其他发育模式输入部署影响细胞形状变化的局部因素。