Vwa1 Knockdown Causes Craniofacial Defects via Loss of Kdm6b Function.

Our previous study linked a point mutation in VWA1 to the development of hemifacial microsomia. This study aims to elucidate the molecular mechanism of VWA1 in craniofacial development. We used transgenic technology to generate mice with Vwa1 knockdown specifically in cranial neural crest cells (CNCCs). Craniofacial and jawbone development was analyzed using skeletal staining and morphometric measurements. Primary CNCCs were isolated and manipulated via lentiviral vectors. Cellular proliferation, migration, differentiation, and apoptosis were assessed using a suite of in vitro assays. RNA-sequencing was employed to identify differentially expressed genes downstream of Vwa1 knockdown, and the epigenetic regulation of Runx2 by KDM6B was verified via ChIP-qPCR. Conditional deletion of Vwa1 in CNCCs resulted in craniofacial hypoplasia, impaired jaw development, and increased CNCC apoptosis in mice. In vitro, knockdown of Vwa1 inhibited CNCC proliferation and migration, hindered osteogenic and chondrogenic differentiation, and enhanced apoptosis. Mechanistically, Vwa1 deletion reduced KDM6B expression. Knockdown of Kdm6b phenocopied the inhibitory effects on CNCC osteogenic differentiation. We demonstrated that KDM6B, a histone demethylase, promotes Runx2 transcription by removing H3K27me3 repressive marks from its promoter. Consistent with this, Runx2-deficient mice also developed craniofacial hypoplasia. Importantly, overexpression of Kdm6b or Runx2 rescued the osteogenic differentiation defects caused by Vwa1 or Kdm6b knockdown, respectively. Vwa1 is essential for proper craniofacial development. Its deletion leads to impaired osteogenic differentiation of CNCCs by disrupting KDM6B-mediated epigenetic activation of Runx2, thus establishing a novel regulatory axis in craniofacial morphogenesis.