Neural crest and placode cell interactions during cranial gangliogenesis

描述(申请人提供)：外周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。神经脊细胞(NCC)和胎盘细胞(PC)的适当结合是许多神经节正常形成所必需的。迁移性NCC还分化产生其他类型的细胞，包括头面部骨骼和皮肤色素细胞。因此，在NCC和PC发育期间发生的异常是许多人类先天性和遗传性畸形、疾病和癌症的直接原因。脑神经节组装涉及在NCC和PC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，但这些连接复合体的分子组成尚不清楚。我们已发表的和初步的数据表明，NCC和PC各自表达不同的钙粘附素和连接素，它们可能在调节NCC和PC的迁移以及在脑神经节组装过程中NCC和PC之间形成新的异嗜性黏附连接方面发挥作用。这种异嗜性相互作用在不同类型的细胞之间还没有报道，与其他连接的数据一起，可能被证明是定义不同细胞如何结合形成多细胞结构的范式转变。此外，我们首次报道了缝隙连接的扰动对雏鸡NCC迁移的影响，缝隙连接在细胞间的通讯中起着关键作用。根据我们的数据，我们假设颅神经节的组装依赖于迁移性NCC和PC之间基于钙粘附素的异嗜性连接的时间形成，随后是其他锚定和通信连接的创建。这项应用的具体目的是：1)确定在介导NCC和PC迁移中对钙粘附素和连接素的需求，2)确定钙粘附素和连接素在颅神经节形成中的作用，以及3)定义更多的连接复合体来协调颅神经节的发生。在目标1中，我们将通过将光吗啉和可诱导表达构建物应用于鸡胚胎并通过体外和体内成像分析记录NCC和PC迁移的变化来扰乱NCC和PC钙粘附素和连环素。在目标2中，我们将使用上述扰动方法和我们在鸡胚胎中首创的生化分析方法来评估钙粘附素和连接素的功能以及异嗜性相互作用在脑神经节组装中的作用。在目标3中，我们将对鸡三叉神经节进行RNA测序，以阐明神经节形成的分子特征，便于识别其他介导NCC-PC相互作用的连接复合体。这项拟议的研究具有创新性，因为它采用了一种结合胚胎学、生物化学和细胞分子生物学的多学科方法来研究不同细胞群体在组织形成过程中如何相互作用。这些研究意义重大，因为这些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的潜在机制的理解，并可能导致开发治疗人类疾病的新方法。

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.