Control of epithelial morphology and bioenergetics by Toll receptors during dynamic tissue remodeling

项目摘要-项目负责人Adam Paré 在发育过程中，上皮细胞在形状和相对位置上经历了显著的变化，以建立适当的 组织架构。上皮重塑的错误直接导致一些最常见的类型 先天畸形--神经管缺陷--影响大约1/2000的新生儿。然而， 控制上皮形态的上游信号仍然知之甚少。一种研究上皮细胞的范式 重塑是果蝇神经直肠胚层中的细胞嵌入，研究表明， 高度保守的Toll受体家族以重叠的条纹模式表达，以组织快速细胞 在这个组织中的重排。Toll受体广泛表达于整个人类上皮细胞，并且它们有 在先天免疫信号的背景下被广泛研究。然而，细胞形态的控制是通过 Toll受体几乎没有受到关注。这项建议的重点是了解不统一收费如何 受体表达影响皮质张力、细胞-细胞黏附和线粒体动力学以控制细胞形状 以及在上皮重塑过程中的行为。我们将使用新开发的CRISPR/Cas9衍生基因 背景和抗体以表征Toll受体如何控制细胞极性以触发嵌入性；我们 将应用非破坏性技术来表征完整生命中上皮重组的生物能量学 胚胎；我们将研究Toll受体、Rho和G蛋白偶联受体之间的未寻址联系 发信号。我们的第一个假设是相邻细胞感觉到个体Toll表达的差异 受体类型，可增加皮质张力，减少细胞间黏附。我们已经开发出一种基因系统 用于在单个条带中表达单个受体，我们将使用该条来系统地表征和比较 每种Toll受体类型对细胞形态的影响以及确定Toll受体所需的蛋白质结构域 调节细胞形状。我们的第二个假设是在新直肠胚层中快速的细胞重排 延长需要线粒体结构的特定变化来为细胞骨架和连接提供燃料 重组。为了测试这一点，我们将使用安捷伦海马流量分析仪来测量氧气消耗率 和多光子显微镜观察活体胚胎在上皮重塑期间的细胞氧化还原状态，以及 然后使用功能增强和功能丧失技术来确定线粒体融合和分裂在 上皮性重组。我们的第三个假设是Toll受体和gpr信号融合以激活Rho。 激活剂可以触发细胞嵌入，特别是在神经外胚层。我们将使用增益型和损失型功能 分析以确定这两个信号通路如何相交以控制皮质张力、细胞-细胞黏附、 线粒体在上皮重塑过程中的动态变化。这些实验的成功完成将给我们带来一个 了解Toll受体如何在分子水平上控制细胞形态和生物能量学 在动态组织重塑过程中，这可能有助于了解神经管的细胞生物学基础 缺陷和其他以上皮为基础的疾病。

Project Summary – Project Leader Adam Paré Epithelial cells undergo significant changes in shape and relative position during development to build proper tissue architecture. Errors in epithelial remodeling directly contribute to some of the most common types of congenital abnormalities––neural tube defects––which affect approximately 1 in 2000 births. However, the upstream signals that control epithelial morphology remain poorly understood. A paradigm for studying epithelial remodeling is cell intercalation in the Drosophila neurectoderm, and it was shown that three members of the highly conserved Toll receptor family are expressed in overlapping striped patterns to organize rapid cell rearrangements in this tissue. Toll receptors are widely expressed throughout human epithelia, and they have been extensively studied in the context of innate immune signaling. However, the control of cell morphology by Toll receptors has received very little attention. The focus of this proposal is to understand how non-uniform Toll receptor expression affects cortical tension, cell-cell adhesion, and mitochondrial dynamics to control cell shape and behavior during epithelial remodeling. We will use newly developed CRISPR/Cas9-derived genetic backgrounds and antibodies to characterize how Toll receptors control cell polarity to trigger intercalation; we will apply non-destructive techniques to characterize the bioenergetics of epithelial reorganization in intact living embryos; and we will investigate unaddressed links between Toll receptor, Rho, and G protein-coupled receptor signaling. Our first hypothesis is that neighboring cells sense differences in the expression of individual Toll receptor types to increase cortical tension and decrease cell-cell adhesion. We have developed a genetic system for expressing individual receptors in a single stripe that we will use to systematically characterize and compare the effects of each Toll receptor type on cell morphology and to identify the protein domains necessary for modulating cell shape. Our second hypothesis is that rapid cellular rearrangements during neurectoderm elongation require specific changes in mitochondrial architecture to fuel cytoskeletal and junctional reorganization. To test this, we will use the Agilent Seahorse flux analyzer to measure oxygen consumption rates and multiphoton microscopy to visualize cellular redox state in live embryos during epithelial remodeling, and then use gain- and loss-of-function techniques to determine what role mitochondrial fusion and fission play in epithelial reorganization. Our third hypothesis is that Toll receptor and GPCR signaling converge to activate Rho Kinase to trigger cell intercalation specifically in the neurectoderm. We will use gain- and loss-of-functional analyses to determine how these two signaling pathways intersect to control cortical tension, cell-cell adhesion, mitochondrial dynamics during epithelial remodeling. Successful completion of these experiments will give us an understanding of how Toll receptors function at a molecular level to control cell morphology and bioenergetics during dynamic tissue remodeling, which could shed light on the cell biological underpinning of neural tube defects and other epithelia-based diseases.