Neural crest and placode cell interactions during cranial gangliogenesis

描述（由申请人提供）：周围神经系统的脑神经节对于整合感觉信息和控制细胞运动至关重要。神经嵴细胞（NCCs）和基板细胞（PC）的适当合并是许多神经节正常形成所必需的。迁移性NCC还分化产生其他细胞类型，包括颅面骨骼和皮肤色素细胞。因此，在NCC和PC发育期间发生的异常直接导致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PC之间创建新的连接，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。我们发表的初步数据表明，NCC和PC各自表达不同的钙粘蛋白和连环蛋白，这可能起到介导NCC和PC迁移以及在颅神经节组装过程中NCC和PC之间形成新的嗜异性粘附连接的作用。这种异嗜性相互作用尚未在不同细胞类型之间报道，并且与其他连接点的数据一起，可以证明在定义不同细胞如何合并以形成多细胞结构方面的范式转变。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鸡NCC迁移的影响。根据我们的数据，我们假设颅神经节组装依赖于迁移性NCCs和PC之间的嗜异性钙粘蛋白粘附连接的暂时形成，然后创建其他锚定和通信连接。本申请的具体目的是：1）确定介导NCC和PC迁移对钙粘蛋白和连环蛋白的需求，2）定义钙粘蛋白和连环蛋白在颅神经节形成中的作用，以及3）定义协调颅神经节发生的其他连接复合体。在目的1中，我们将通过适应使用光吗啉代和诱导型表达构建体的鸡胚和记录的变化，在NCC和PC迁移通过在体外和体内成像分析，扰乱NCC和PC钙粘蛋白和连环蛋白。在目标2中，我们将使用上述扰动方法和生化测定，我们已经率先在鸡胚中，以评估钙粘蛋白和连环蛋白的功能，和嗜异性相互作用，在颅神经节组装。在目标3中，我们将进行鸡三叉神经节的RNA测序，以阐明神经节发生的分子特征，促进介导NCC-PC相互作用的其他连接复合物的鉴定。这项研究具有创新性，因为它采用了多学科的方法，将胚胎学、生物化学、细胞和分子生物学结合起来，研究不同细胞群在组织形成过程中如何相互作用。这些研究具有重要意义，因为其结果将增强我们对创造组织和器官所需的细胞间相互作用机制的理解，并可能导致人类疾病新疗法的开发。

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.