Role of actin cytoskeleton regulators in craniofacial development and disease

描述(申请人提供)：神经脊是一种独特的细胞群体，在脊椎动物胚胎发育过程中广泛迁移。特别值得注意的是，颅神经脊细胞在面部各处迁移，形成了颅面骨骼的大部分。它们在增殖、迁移或存活方面的缺陷会导致许多疾病和出生缺陷，包括唇腭裂、Treacher-Collins综合征和DiGeorge综合征。尽管最近在了解神经脊发育的各个方面取得了进展，但神经脊迁移的机制仍然不清楚。因此，研究它们的迁移行为和其中的肌动蛋白细胞骨架的调控对于理解先天性颅面部疾病的基础至关重要。从微阵列筛选中，我已经确定了多个肌动蛋白细胞骨架调节因子(ACR)基因，它们主要在神经脊细胞中表达。我的初步研究表明，肌球蛋白-X和Caldesmon都是通过调节细胞形态和膜突起而正确地进行细胞迁移的关键。基于这些发现，我假设神经脊细胞通过特定的ACRs实现其独特的迁移能力，从而快速而精确地控制 它们的细胞骨架动力学。在这个计划中，我将通过进一步分析这些ACRs的活动，并建立它们与肌动蛋白结构的物理和功能相互作用，来研究神经脊迁移的机制。具体地说，我将把经典胚胎学技术与现代细胞生物学、生物化学和活体显微镜相结合，研究CNC迁移过程中肌动蛋白细胞骨架的动态排列，并研究神经嵴中的ACRs如何调节这一过程。实验将在青蛙和小鸡身上进行，考虑到 两个模型系统的优点：在青蛙中进行简单的基因和组织操作，在雏鸡中进行高分辨率成像和后期电穿孔。本研究的具体目的如下：1)通过实时成像技术研究正常神经脊游走过程中肌动蛋白细胞骨架的动态变化。2)确定肌球蛋白-X和Caldesmon在cnc迁移过程中调节肌动蛋白动态的作用。3)对在微阵列筛选中确定的CDC42ep1和其他候选基因在神经脊发育中的作用进行功能表征。4)确定数控系统迁移中ACR之间的功能和物理相互作用。 与公共健康相关：从背部神经管迁移的数控细胞对面部骨骼做出了重大贡献。这项拟议的工作旨在研究脊椎动物胚胎发生过程中计算机控制细胞迁移的细胞生物学基础。了解神经脊迁移的机制将有助于了解先天性颅面畸形的病因。

DESCRIPTION (provided by applicant): Neural crest is a unique population of cells that migrate extensively during vertebrate embryogenesis. Of particular interest, cranial neural crest (CNC) cells migrate throughout the face to form most of the craniofacial skeleton. Defects in their proliferation, migration, or survival lead to numerous diseases and birth defects, including cleft lip/palate, Treacher-Collins syndrome, and DiGeorge syndrome. Despite recent progress in understanding aspects of neural crest development, the mechanisms underlying neural crest migration are still unclear. Thus, studying their migratory behavior and the regulation of actin cytoskeleton therein is crucial for understanding the basis of congenital craniofacial disorders. From a microarray screen, I have identified multiple actin cytoskeleton regulator (ACR) genes that are predominantly expressed in neural crest cells. My preliminary studies reveal that Myosin-X and Caldesmon are both critical for proper CNC cell migration via regulating cell morphology and membrane protrusions. Based on these findings, I hypothesis that neural crest cells achieve their unique migratory ability through specific ACRs to rapidly and precisely control their cytoskeletal dynamics. In this proposal, I will investigate the mechanism of neural crest migration by further analyzing the activities of these ACRs, and establishing their physical and functional interactions with actin structures. Specifically, I will combine classical embryology techniques with modern cell biology, biochemistry, and in vivo microscopy to study the dynamic arrangements of actin cytoskeleton during CNC migration and examine how ACRs in the neural crest regulate this process. Experiments will be carried out in frog and chick, taking into account the advantages of both model systems: simple gene and tissue manipulation in frog, and high-resolution imaging and late stage electroporation in chick. The following specific aims will be performed: 1) Examine the dynamics of actin cytoskeleton during normal neural crest migration by real time imaging. 2) Determine the role of Myosin-X and Caldesmon in regulating actin dynamics during CNC migration. 3) Functionally characterize Cdc42ep1 and additional candidate genes identified in the microarray screen for their roles in neural crest development. 4) Determine functional and physical interactions between ACRs in CNC migration. PUBLIC HEALTH RELEVANCE: CNC cells that migrate from dorsal neural tube make a major contribution to the facial skeleton. The proposed work aims to study the cell biological basis underlying the migration of CNC cells during vertebrate embryogenesis. Understanding the mechanism of neural crest migration will shed light on the etiology of congenital craniofacial defects.