Dynamic and non-uniform expression of key transcription factors provides novel insights into the emergence of neural crest cells at the neural plate border.

The neural crest is a vertebrate stem cell population with broad developmental potential whose emergence requires precise regulation of gene expression at the neural plate border (NPB). While a hierarchical gene regulatory network (GRN) describing signaling pathways and transcription factors underpinning the establishment of definitive neural crest cells has been generated by integrating the work of numerous groups, much remains to be learned about the relationships of distinct GRN components to each other. Here, we use high-resolution fluorescent in situ Hybridization Chain Reaction (HCR-FISH) to quantify the spatiotemporal dynamics of neural crest gene expression in Xenopus laevis embryos. We find that the onset of snai2 , sox8 , and foxd3 expression during late gastrulation is broad, heterogeneous, and partially overlapping, with distinct anterior-posterior and medio-lateral biases. By neurulation, these markers converge on a shared neural crest domain but retain relative expression differences along axial levels that persist into migratory stages, producing stream-specific gene expression patterns. Computational surface mapping revealed that these differences correlate with dynamic, layered expression of NPB factors, particularly pax3 and zic1 . Correlating relative intensities of pax3 and zic1 with the presence or absence of nascent neural crest transcripts predicts that these NPB factors can differentially regulate snai2 and sox8 , which we confirm with functional experiments. Strikingly, later stages show an inverse correlation between neural crest and NPB gene expression, suggesting a handoff mechanism in which pax3 and zic1 initially promote neural crest gene activation but are downregulated as neural crest identity emerges and contribute to combinatorial signatures of gene expression along the A-P axis. These finding provide important new insights into the genesis of a centrally important cell type.