Restricting cell movement: the role of Tspan18 in neural crest migration

Abstract

The neural crest is a unique population of stem cells that arise from the developing central nervous system of vertebrate embryos. Unlike surrounding neuroepithelial cells, neural crest cells undergo an epithelial-to-mesenchymal transition (EMT) and migrate into the periphery of the embryo where they contribute to diverse adult lineages, including the central nervous system, craniofacial skeleton and melanocytes. Despite the fundamental importance of the neural crest, our understanding of the molecular events controlling neural crest migration is incomplete. My thesis focuses on how the transmembrane scaffolding protein, Tetraspanin18 (Tspan18) regulates neural crest EMT. Here I show that Tspan18 is expressed in premigratory, cranial neural crest cells, but is downregulated prior to migration. Sustained expression of Tspan18 maintains the epithelial cell adhesion molecule cadherin-6B (Cad6B) post-translationally, without affecting Cad6B mRNA levels, and in turn inhibits cranial neural crest migration. In contrast, morpholino-mediated knockdown of Tspan18 reduces Cad6B protein levels. Unexpectedly, this does not lead to precocious migration, emphasizing that although loss of Tspan18 is required for migration, loss of Tspan18 alone is not sufficient to trigger neural crest migration. This is, at least in part, because EMT is a multiple step process, and the other steps of EMT are not affected by Tspan18 knockdown. Together these findings suggest that Tspan18 antagonizes neural crest EMT by supporting cell adhesion. Additionally, I show here that Tspan18 downregulation at the onset of migration is achieved by the winged-helix transcription factor, FoxD3. FoxD3 knockdown sustains Tspan18 mRNA levels and inhibits neural crest formation and migration. Importantly, Tspan18 knockdown rescues the FoxD3 loss-of-function migration defect without rescuing the reduction in neural crest cell number. This suggests that FoxD3 independently regulates neural crest formation and migration, and effects migration through its regulation of Tspan18. Overall, the work in this thesis has defined a novel, Tspan18-dependent maintenance of Cad6B protein levels in epithelial cranial neural crest cells that is relieved, via FoxD3, to support migration. This work provides important insight into how cell adhesion molecules are regulated during EMT and increases our understanding for how neural crest transcription factors, like FoxD3, impact the cellular events that lead to migration.