Mechanisms of microtubule-mediated cranial neural crest EMT and differentiation

项目摘要/摘要 神经脊(NC)细胞发育缺陷可导致结构性和神经性疾病 对人类健康的长期有害影响。NC细胞形成外周的神经元和神经胶质细胞，内含 除颅面骨和软骨外，还有四肢神经系统。NC的早期形成和分离 来自贴壁神经管的细胞是一个受到细胞信号、表观遗传和转录的严格调控的过程。 通过改变钙粘蛋白蛋白的定位，改变细胞间的黏附能力。从结构上讲，作为 神经皱折在胚胎的中心形成神经管，即非神经性外胚层细胞 与胚胎相遇并覆盖，最终分化成表皮和胎盘。神经管关闭后- 当然，NC细胞经历了上皮到间充质的转变(EMT)，离开了他们的神经上皮细胞- BORS，并在整个胚胎中迁移，侵入各种组织，产生不同的衍生产品。快速 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、附着力和迁移性。几种转录因子是编码基因的调节因子 钙粘蛋白在NC细胞发育过程中的作用，但有一个关键的信息是缺乏的，即更快 在NC EMT过程中改变钙粘素蛋白定位的过程。具体地说，我们的目标是了解 在这个发育阶段，微管和相关的细胞骨架因子调节细胞与细胞的黏附。基座 根据我们已发表的工作和初步数据，-III微管蛋白(TUBB3)在NC细胞中开始上调 NC EMT。我们的目标是了解控制NC EMT及其后续的复杂机制 通过定义微管元件TUBB3如何调节钙粘蛋白蛋白定位来实现NC分化 在NC细胞体内的EMT和分化过程中。此外，我们将确定TUBB3的扰动如何影响 NC EMT过程中其他细胞骨架元件的结构。在目标1中，我们将执行 TUBB3实验随后定量分析了epi-1的表达和定位的变化。 内皮和迁移钙粘附素(CDH2、CDH1和CDH11)、组织特异性标记物(NC和神经管)， 细胞迁移和颅骨NC分化的定量空间方法在转录本和蛋白 鸡(Gallus Gallus)中的Tein水平反映了人类早期的发育。在目标2中，我们 将对TUBB3进行扰动以确定有缺陷的TUBB3介导的微管组装 在NC EMT过程中影响微管、中间丝和肌动蛋白细丝的定位。使用 这些实验，我们期望将传统的和新的方法结合起来，以理解COM- 在NC EMT过程中，细胞黏附和细胞骨架排列之间的复杂联系。我们希望能贡献出一份 缺少推动NC发展的蛋白质网络知识的基本要素 细胞，这对于鉴定正常和异常发育的新的标志物是至关重要的。 衍生成体细胞类型。

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.