Tetraspanin18 is a FoxD3-responsive antagonist of cranial neural crest epithelial to mesenchymal transition that maintains Cadherin6B protein

Abstract

During epithelial to mesenchymal transition (EMT), tightly associated, polarized epithelial cells become individual mesenchymal cells capable of migrating. Here, we investigate the role of the transmembrane protein tetraspanin18 (Tspan18) in cranial neural crest EMT. Tspan18 mRNA is expressed in premigratory cranial neural crest cells, but is absent from actively migrating neural crest cells. Tspan18 knock down leads to a concomitant loss of Cadherin6B (Cad6B) protein, while Cad6B protein persists when Tspan18 expression is extended. As the temporal profile of Cad6B mRNA downregulation is unaffected in these embryos, this indicates that Tspan18 maintains Cad6B protein levels and reveals that Cad6B is regulated by post-translational mechanisms. Although downregulation of Tspan18 is necessary, it is not sufficient for neural crest migration: the timing of neural crest emigration, basal lamina break down and Cad7 upregulation proceed normally in Tspan18-deficient cells. This emphasizes the need for coordinated transcriptional and post-translational regulation of Cad6B during EMT and illustrates that Tspan18-antagonized remodeling of cell-cell adhesions is only one step in preparing for cranial neural crest migration. Unlike Cad6B, which is transcriptionally repressed by Snail2, Tspan18 expression is downstream of the winged-helix transcription factor FoxD3, providing a new transcriptional input into cranial neural crest EMT. Altogether our data reveal post-translational regulation of Cad6B protein levels by Tspan18 that must be relieved by a FoxD3-dependent mechanism in order for cranial neural crest cells to migrate. These results offer novel insight into the molecular mechanisms of cranial neural crest EMT and expand our understanding of tetraspanin function relevant to metastasis.