Mechanisms of microtubule-mediated cranial neural crest EMT and differentiation

项目总结/摘要 神经嵴（NC）细胞的发育缺陷可导致结构和神经系统疾病， 对人类健康的长期有害影响。NC细胞形成外周和内周的神经元和神经胶质细胞。 除了颅面骨和软骨外，还包括三叉神经系统。NC的早期形成和分离 细胞从贴壁神经管中分离是一个受细胞信号传导、表观遗传和转录密切调控的过程， 通过改变钙粘蛋白的蛋白定位改变细胞-细胞粘附。在结构上，作为 神经褶皱在胚胎的中心上升并汇合，形成神经管，即非神经外胚层细胞 与胚相遇并覆盖胚，最终分化成表皮和基板。神经管闭合后- 当然，NC细胞经历了上皮向间质转化（EMT），留下它们的神经上皮相邻细胞， bors，并在整个胚胎中迁移，侵入各种组织，并产生各种衍生物。快速 在转录、翻译和翻译后水平的变化允许细胞中的动态转换， 极性、粘附和迁移。几种转录因子作为基因编码的调节因子起作用。 钙粘蛋白在NC细胞发育过程中的作用，但目前缺乏关于更快速的钙粘蛋白表达的信息。 在NC EMT期间改变钙粘蛋白蛋白定位的过程。具体来说，我们的目标是了解如何 微管和相关的细胞骨架因子在该发育阶段调节细胞-细胞粘附。基于 根据我们已发表的工作和初步数据，在NC细胞中， NC EMT。我们的目标是了解控制NC EMT的复杂机制， 通过定义微管元件TUBB 3如何调节钙粘蛋白蛋白定位的NC分化 在NC细胞EMT和体内分化期间。此外，我们将确定TUBB 3的扰动如何影响 NC EMT过程中其他细胞骨架元素的结构。在目标1中，我们将执行 TUBB 3实验，然后定量分析表位蛋白表达和定位的变化。 上皮和迁移性钙粘蛋白（CDH 2、CDH 1和CDH 11），组织特异性标志物（NC和神经管）， 细胞迁移和颅NC分化使用定量空间方法在转录本和原， 鸡（Gallus gallus）的蛋白质水平，反映了人类早期发育阶段。在目标2中， 将进行TUBB 3的扰动，以确定缺陷TUBB 3介导的微管组装 影响NC EMT过程中微管、中间丝和肌动蛋白丝的定位。与 这些实验，我们希望整合传统和新颖的方法来理解COM， NC EMT期间细胞粘附和细胞骨架排列之间的复杂联系。我们希望能贡献一份 我们对驱动NC发展的蛋白质网络的知识中缺少的基本元素 细胞，这是至关重要的正常和异常发展的NC- 衍生的成体细胞类型。

Project Summary/Abstract Defective development of neural crest (NC) cells can cause structural and neurological disorders that have long-term deleterious effects on human health. NC cells form the neurons and glia of the peripheral and en- teric nervous systems in addition to craniofacial bone and cartilage. Early formation and separation of NC cells from the adherent neural tube is a process tightly regulated by cell signaling, epigenetic and transcrip- tional changes, and altered cell-cell adhesion via changing cadherin protein localization. Structurally, as the neural folds rise to meet in the center of the embryo to create the neural tube, non-neural ectodermal cells meet and cover the embryo, eventually differentiating into epidermis and placodes. After neural tube clo- sure, NC cells undergo an epithelial to mesenchymal transition (EMT), leave their neuroepithelial neigh- bors, and migrate throughout the embryo, invading various tissues, and creating diverse derivatives. Rapid changes at transcriptional, translational, and post-translational levels allow for dynamic transitions in cell polarity, adhesion, and migration. Several transcription factors function as regulators of genes encoding cadherin proteins during NC cell development, but there is a critical lack of information about the more rapid processes that alter cadherin protein localization during NC EMT. Specifically, we aim to understand how microtubules and related cytoskeletal factors regulate cell-cell adhesion at this developmental stage. Based on our published work and preliminary data, -III tubulin (TUBB3) is upregulated in NC cells at the onset of NC EMT. Our objective is to understand the complex mechanisms that control NC EMT and subsequent NC differentiation by defining how the microtubule element, TUBB3, regulates cadherin protein localization during NC cell EMT and differentiation in vivo. Further, we will identify how perturbation of TUBB3 affects the structure of other cytoskeletal elements during NC EMT. In Aim 1 we will perform gain and loss of TUBB3 experiments followed by quantitative analyses of changes in the expression and localization of epi- thelial and migratory cadherins (CDH2, CDH1, and CDH11), tissue-specific markers (NC and neural tube), cell migration and cranial NC differentiation using quantitative spatial approaches at the transcript and pro- tein levels in the chicken (Gallus gallus), which mirrors human development at early stages. In Aim 2, we will perform perturbations of TUBB3 to determine how defective TUBB3-mediated microtubule assembly affects the localization of microtubules, intermediate filaments, and actin filaments during NC EMT. With these experiments, we expect to integrate both traditional and novel approaches to understanding the com- plex links between cell adhesion and cytoskeletal arrangements during NC EMT. We hope to contribute a missing, fundamental element to our knowledge of the protein network that drives the development of NC cells, which is crucial for the identification of novel markers of normal and abnormal development of NC- derived adult cell types.