Hu Z. et al.
Cranial neural crest (NC) cells, which can migrate, adopt multiple fates, and form most of the craniofacial skeleton, are an excellent model for studying cell fate decisions. Using time-resolved single-cell multi-omics, spatial transcriptomics, and systematic Perturb-seq, we fully deciphered zebrafish cranial NC programs, including 23 cell states and three spatial trajectories, reconstructed and tested the complete gene regulatory network (GRN). Our GRN model, combined with a novel velocity-embedded simulation method, accurately predicted functions of all major regulons, with over a 3-fold increase in correlation between in vivo and in silico perturbations. Using our new approach based on regulatory synchronization, we discovered a post-epithelial-mesenchymal-transition endothelial-like program crucial for migration, identified motif coordinators for dual-fate priming, and quantified lineage-specific cooperative transcription factor functions. This study provides a comprehensive and validated NC regulatory landscape with unprecedented resolution, offering general regulatory models for cell fate decisions in vertebrates.
