Ectomesenchymal identity emerges via relief of Twist1 transcript destabilisation.

During vertebrate development, a subset of cranial neural crest cells (CNCCs) termed 'ectomesenchyme' differentiates into cell types canonically associated with the mesoderm (cartilage, bone and muscle). While the molecular decisions that guide CNCCs toward ectomesenchymal identity remain incompletely understood, the transcription factor Twist1 plays a central role. Here, we show that while Twist1 transcripts accumulate in late migratory CNCCs as cells enter the pharyngeal arch environment, a Twist1 enhancer within Hdac9 is active in the neural tube and CNCCs. We reconcile the temporal discrepancy between enhancer activity and transcript accumulation by showing that the Twist1 3' UTR from multiple vertebrate species (but not the non-vertebrate chordate Ciona intestinalis) destabilises transcripts in the ectoderm via a conserved AU-Rich Element. Together, these findings reveal a vertebrate-specific, two-tiered regulatory mechanism that uncouples enhancer activity from transcript accumulation, gating the onset of Twist1 expression and the acquisition of ectomesenchymal identity in vertebrate CNCCs.