A dual role for DLC1 in regulating avian cranial neural crest cell fate commitment and delamination

Abstract

In response to neural crest inductive signals, cells in the open neural plate border region are bestowed with competence to give rise to neural crest cells. Upon neural tube closure, prospective neural crest cells undergo epithelial-mesenchymal transition (EMT) to delaminate from the dorsal neuroepithelium, migrate and differentiate into various cell types throughout the embryos. These sequential steps of neural crest ontogeny are strictly governed by a gene regulatory network (GRN) comprising of an array of transcription factors. However, the molecule mechanisms leading to the emergence and segregation of definitive neural crest cells from other lineages have remained elusive. Here, this study revealed that a RhoGTPase activating (RhoGAP) protein, deleted in liver cancer 1 (DLC1) exhibits both nuclear and cytoplasmic localization in the prospective avian cranial neural crest cells. Intriguingly, forced expression and knockdown of DLC1 led to loss of neural crest specifier genes, SOX9, SNAIL2 and FOXD3 expression and the expansion of SOX2-positive neural progenitor domain into the neural crest forming territory while PAX7-positive neural plate border region remains unaltered, suggesting that appropriate level of DLC1 expression in the nucleus is required for the segregation of neural crest lineage through regulation of their specifier genes expression. This is accomplished by an essential requirement of DLC1 to recruit PAX7 binding to the cis-regulatory elements of NC specifier genes together with transcription elongation factor B polypeptide 1 (TCEB1) for transcriptional regulation. In addition, this study further showed that cytoplasmic DLC1 recruits a novel partner, Serine-Threonine Kinase Receptor-Associated Protein (STRAP) which is a WD-containing protein through its amino acid 69-322 region. The cooperation of DLC1 and STRAP prevented SNAIL2 from degradation via inhibition of GSK3β activity in an RHOA-independent manner, resulting in repression of its nuclear target Cad6B for the proper onset of neural crest delamination. Together, these findings unravel unprecedented subcellular roles of DLC1 in regulating both avian cranial neural crest cell fate commitment and delamination, providing a new paradigm for our understanding of the neural crest-GRN and also on future neural crest reprograming studies.