Understanding trigeminal ganglion development through the lens of Familial Dysautonomia

项目总结 脑神经脊细胞(NCC)和胎盘细胞(PC)分化形成不同类型的细胞，包括 感觉神经元、软骨和骨骼以及皮肤色素细胞。NCC和PC期间发生的异常 因此，发育是许多人类疾病的直接原因，包括家族性自主神经障碍(FD)， 感觉和自主神经障碍的部分特征是三叉神经节(TG)功能障碍。 NCC和PC必须结合在一起以组装TG，TG从 从头到脑。TG的双重细胞起源引发了关于两个不同的细胞如何 人群相互作用形成一个单一的组织，这构成了知识的重大缺口。在这份提案中， 我们将通过对两只小鼠的开创性研究来研究TG发生的分子机制 和小鸡模特系统。我们的初步数据显示TG和Tg的进行性形态缺陷 其在FD小鼠模型中的神经，在该模型中，致病基因Elongator Complex Protein 1(Elp1)缺失 在NCC及其衍生物(Elp1 CKO)中。此外，特定的TG神经元亚群感受疼痛和 温度(伤害性感受器)在Elp1 CKO胚胎中被耗尽，为表型提供了细胞基础 在FD中观察到。总之，这些发现揭示了Elp1在TG发育中的关键作用；然而， Elp1协调TG的正确建立的分子机制在很大程度上仍不清楚。 由于Elp1具有不同的、依赖于上下文的功能，因此需要无偏见的方法来检查 Elp1在TG中的独有作用。为此，我们将在不同的发育阶段使用质谱学(MS) 阶段以解决我们的工作模型，即Elp1调节正确形成所需的下游信号 TG和它的神经。本申请的具体目的是：1)确定NCC Elp1损失对 TG蛋白质组和2)在TG中定义特定细胞类型的Elp1相互作用蛋白。在目标1中，我们将描述 Elp1 CKO与Litterate对照中的TG蛋白质组，识别其表达受 Elp1失败。候选函数将在鼠标和小鸡中求值，后者可能是由于 Elp1表达的保守性，提供快速执行功能实验的能力。在目标2中，我们将 使用基于亲和力的MS来发现TG中特定于PC的Elp1结合伙伴(在Elp1 CKO中揭示)和 NCC衍生品(与Elp1 CKO比较后在对照中确定)，候选评估程序如下 目标1.拟议的研究是创新的，因为它结合了两个互补的 用多学科方法建立发育模型(小鼠和小鸡)，涉及胚胎学、生物化学、 蛋白质组学，以及新的显微镜和微扰分析。这些结果将大大推进我们的 对强调细胞间相互作用所需的分子机制的知识 不仅是TG的功能，还包括其他多细胞组织的功能，并将有助于阐明疾病的病因学 FD和其他由胚胎发育过程中异常的NCC和PC引起的疾病。

PROJECT SUMMARY Cranial neural crest cells (NCCs) and placode cells (PCs) differentiate to form diverse cell types, including sensory neurons, cartilage and bone, and skin pigment cells. Abnormalities that occur during NCC and PC development are thus directly responsible for many human diseases, including Familial Dysautonomia (FD), a sensory and autonomic nerve disorder characterized, in part, by cranial trigeminal ganglion (TG) dysfunction. Both NCCs and PCs must coalesce together to assemble the TG, which relays somatosensory information from the head to the brain. The dual cellular origin of the TG evokes questions regarding how two distinct cell populations interact to form a single tissue, which constitutes a significant gap in knowledge. In this proposal, we will investigate molecular mechanisms underlying TG development through pioneering studies in both mouse and chick model systems. Our preliminary data demonstrated progressive morphological deficits in the TG and its nerves in a mouse model of FD in which the causative gene, Elongator Complex Protein 1 (Elp1), is deleted in NCCs and their derivatives (Elp1 CKO). Moreover, specific TG neuron subpopulations that sense pain and temperature (nociceptors) are depleted in Elp1 CKO embryos, providing a cellular basis for the phenotypes observed in FD. Altogether, these findings reveal a critical role for Elp1 in TG development; however, the molecular mechanisms by which Elp1 orchestrates the proper establishment of the TG remain largely unknown. Since Elp1 possesses diverse, context-dependent functions, unbiased approaches are required to examine the role of Elp1 exclusively in the TG. To this end, we will use mass spectrometry (MS) at distinct developmental stages to address our working model that Elp1 mediates downstream signaling required for proper formation of the TG and its nerves. The Specific Aims of this application are to: 1) determine the effects of NCC Elp1 loss on the TG proteome and 2) define cell-type specific Elp1-interacting proteins in the TG. In Aim 1, we will delineate the TG proteome in Elp1 CKO vs. littermate controls, identifying candidates whose expression is affected by Elp1 loss. Candidate function will be evaluated in the mouse and chick, with the latter possible due to conservation of Elp1 expression, providing the ability to rapidly perform functional experiments. In Aim 2, we will use affinity-based MS to uncover Elp1 binding partners in the TG specific to PCs (revealed in the Elp1 CKO) and NCC derivatives (identified in controls after comparison to Elp1 CKO), with candidate evaluation proceeding as in Aim 1. The proposed research is innovative because it combines the power of two complementary developmental models (mouse and chick) with a multidisciplinary approach involving embryology, biochemistry, proteomics, and novel microscopy and perturbation assays. These results will significantly advance our knowledge of the molecular mechanisms underscoring intercellular interactions required for the formation and function of not only the TG but also other multicellular tissues, and will shed light on the etiology of diseases like FD and others caused by aberrant NCCs and PCs during embryonic development.