Determining the role of Raftlin-2 in neural crest cell specification and migration

Cranial neural crest cells are a population of multipotent stem cells essential for craniofacial development that contribute to the sensory ganglia and craniofacial skeleton. Neural crest cells are specified during neurulation before undergoing an epithelial-to-mesenchymal transition (EMT) and migration; while we appreciate the complex network of signaling pathways and transcription factors underlying specification and migration, the regulation of these events remains incompletely understood. We have recently identified that expression of the gene encoding Raftlin-2 is upregulated in neural crest cells during specification and maintained throughout migration. Raftlin proteins localize to the plasma membrane where they are reported to organize lipid raft domains and regulate diverse cell signaling events, leading us to hypothesize that Raftlin-2 contributes to the development of neural crest cells through an impact on cell signaling. In preliminary studies probing Raftlin-2 function in the cranial neural crest of avian embryos using multiple knockdown strategies, we observed reduced expression of several genes required for neural crest specification, suggesting that Raftlin-2 is essential for the establishment of the neural crest domain. Aim 1 will investigate the role of Raftlin-2 in regulating specification by using immunohistochemistry to measure neural crest specifier genes expression. Since specifier gene expression is initiated by cell signaling pathways including Wnts and Bone Morphogenetic Proteins, I will next use signaling activity-sensitive reporter constructs, and fluorescent in situ hybridization to investigate if Raftlin-2 regulates the activity of these pathways to govern specification. Finally, I will use RNA sequencing in Raftlin-2 deficient cranial neural crest cells to determine additional targets and signaling pathways dependent on Raftlin- 2 function. Our preliminary results also showed reduced neural crest migration area after Raftlin-2 knockdown compared to controls in vivo, and we find that Raftlin-2 loss of function resulted in decreased migratory persistence ex vivo, which led us to hypothesize that Raftlin-2 is necessary for effective neural crest migration and adhesion after specification. Aim 2 will test this hypothesis using ex vivo explants to assay neural crest cell directional migration and focal adhesion dynamics during chemotaxis. I will also use quantitative western blot analysis to probe the activation states of molecular effector proteins downstream of chemotactic signals to define and subsequently test the precise mechanisms of Raftlin-2 function during migration. Together these experiments will uncover how Raftlin-2 impacts developmental signaling pathways necessary for cranial neural crest cell specification and migration. These results will advance our understanding of how membrane organization regulates craniofacial development insight and will provide novel insights that can be used to treat craniofacial anomalies. Project Number: 1F31DE035770-01 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of Dental and Craniofacial Research (NIDCR) | Principal Investigator: Mallorie Jenne | Institution: JOHNS HOPKINS UNIVERSITY, BALTIMORE, MD | Award Amount: $50,338 | Activity Code: F31 | Study Section: Special Emphasis Panel[ZRG1 F05-A (20)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11315105