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 intercellu...