DLX2 acts as a pioneer factor and drives <i>Msx1</i> ⁺ ectomesenchyme formation from embryonic stem cells

The ectomesenchyme generates much of the craniofacial skeleton, sutures, and diverse connective tissues in the mammalian head, yet its derivation from embryonic stem cells (ESCs) and the underlying molecular drivers remain poorly defined. Here, we identified <i>Dlx2</i> as a key regulator that efficiently directed murine ESCs toward <i>Msx1</i>⁺ ectomesenchyme, recapitulating the developmental trajectory. These <i>Msx1</i>⁺ progenitors expressed classical craniofacial markers, exhibited robust osteochondral differentiation potential as a group, and supported craniofacial regeneration. Mechanistically, Distal-less homeobox 2 (DLX2) formed a complex with lamina-associated polypeptide 2, isoform alpha (LAP2α) through a 38-amino-acid homeodomain motif, interacting with nucleosomes to promote chromatin remodeling and activate a procraniofacial ectomesenchymal gene network. Disrupting DLX2-LAP2α interaction or silencing <i>Dlx2</i> targets markedly diminished ectomesenchymal differentiation. Our findings established DLX2 as a pioneer factor in ectomesenchyme specification, offering insights into craniofacial development and stem cell engineering.