Neural crest and placode cell interactions during cranial gangliogenesis

描述（申请人提供）：周围神经系统的颅神经节是整合感觉信息和控制细胞运动的关键。神经嵴细胞（NCCs）和基质细胞（PCs）的适当结合是许多神经节正常形成所必需的。迁移性ncc也分化产生其他类型的细胞，包括颅面骨架细胞和皮肤色素细胞。因此，在NCC和PC发育过程中发生的异常直接导致了许多人类先天性和遗传性畸形、疾病和癌症。颅神经节组装涉及在ncc和PCs之间建立新的连接，促进它们彼此之间以及与周围环境的相互作用，但这些连接复合物的分子组成尚不清楚。我们发表的和初步的数据表明，NCC和PCs各自表达了不同的钙粘蛋白和连环蛋白，这些钙粘蛋白可能介导NCC和PC的迁移，以及在脑神经节组装过程中NCC和PCs之间形成新的异亲粘附连接。在不同的细胞类型之间还没有报道过这种异亲性相互作用，并且与其他连接的数据一起，可以证明在定义不同细胞如何结合形成多细胞结构方面是范式转移。此外，我们首次报道了在细胞间通讯中起关键作用的间隙连接的扰动对小鸡NCC迁移的影响。根据我们的数据，我们假设颅神经节的组装依赖于迁移的ncc和PCs之间基于钙粘蛋白的异亲粘附连接的时间形成，随后是其他锚定和通信连接的创建。本应用程序的具体目的是：1)确定钙粘蛋白和连环蛋白在介导NCC和PC迁移中的需求，2)确定钙粘蛋白和连环蛋白在颅神经节形成中的作用，以及3)确定协调颅神经节形成的附加连接复合物。在目标1中，我们将通过在鸡胚中使用光形态聚合物和诱导表达结构来干扰NCC和PC钙粘蛋白和连环蛋白，并通过体外和体内成像分析记录NCC和PC迁移的变化。在Aim 2中，我们将使用上述在鸡胚胎中率先使用的扰动方法和生化分析来评估钙粘蛋白和连环蛋白的功能，以及在颅神经节组装中的异亲相互作用。在Aim 3中，我们将对鸡三叉神经节进行rna测序，以阐明神经节发生的分子特征，促进鉴定介导nc - pc相互作用的其他连接复合物。这项提议的研究具有创新性，因为它采用多学科方法，结合胚胎学、生物化学、细胞和分子生物学来研究不同细胞群在组织形成过程中如何相互作用。这些研究意义重大，因为其结果将增强我们对细胞间相互作用机制的理解，这些相互作用是创造组织和器官所必需的，并可能导致人类疾病新疗法的发展。

DESCRIPTION (provided by applicant): Cranial ganglia of the peripheral nervous system are critical for integrating sensory information and controlling cell movements. The appropriate coalescence of both neural crest cells (NCCs) and placode cells (PCs) is required for the proper formation of many of these ganglia. Migratory NCCs also differentiate to create other cell types, including the craniofacial skeleton and skin pigment cells. Abnormalities that occur during NCC and PC development are thus directly responsible for many human congenital and hereditary malformations, diseases and cancers. Cranial ganglia assembly involves the creation of new junctions between NCCs and PCs, facilitating their interactions with one another and with their surroundings, but the molecular composition of these junctional complexes is not known. Our published and preliminary data indicate that NCCs and PCs each express a different repertoire of cadherins and catenins, which may function to mediate both NCC and PC migration as well as the formation of novel heterophilic adherens junctions between NCCs and PCs during cranial ganglia assembly. Such heterophilic interactions have not been reported between different cell types, and, together with data on other junctions, could prove to be paradigm-shifting in defining how different cells coalesce to form multi-cellular structures. Moreover, we report for the first time the effects of perturbation of gap junctions, which play crucial roles in cell-cell communication, on chick NCC migration. In light of our data, we hypothesize that cranial ganglia assembly is dependent upon the temporal formation of heterophilic cadherin-based adherens junctions between migratory NCCs and PCs, followed by the creation of other anchoring and communicating junctions. The Specific Aims of this application are to: 1) determine the requirement for cadherins and catenins in mediating NCC and PC migration, 2) define the role of cadherins and catenins in cranial ganglia formation, and 3) define additional junctional complexes that orchestrate cranial gangliogenesis. In Aim 1, we will perturb NCC and PC cadherins and catenins by adapting the use of photo morpholinos and inducible expression constructs to the chick embryo and documenting changes in NCC and PC migration through in vitro and in vivo imaging assays. In Aim 2, we will use the above perturbation methods and biochemical assays we have pioneered in the chick embryo to evaluate the function of cadherins and catenins, and heterophilic interactions, in cranial ganglia assembly. In Aim 3, we will perform RNA-sequencing of chick trigeminal ganglia to elucidate a molecular signature for gangliogenesis, facilitating the identification of other junctional complexes that mediate NCC-PC interactions. The proposed research is innovative because it takes a multi-disciplinary approach that combines embryology, biochemistry, and cell and molecular biology to examine how different cell populations interact during tissue formation. These studies are significant because the results will enhance our understanding of the mechanisms underlying the intercellular interactions required to create tissues and organs and could lead to the development of new treatments for human diseases.