Maintenance of pluripotency in the entire ectoderm enables neural crest formation and challenges dogmatic viewpoints of gastrulation

The ability of the pluripotent epiblast to contribute progeny to all three germ layers is thought to be lost after gastrulation. The later-forming neural crest (NC) rises from ectoderm and it remains poorly understood how its exceptionally high stem-cell potential to generate mesodermal- and endodermal-like cells is obtained. We monitored transcriptional changes from gastrulation to neurulation using single-cell-Multiplex-Spatial-Transcriptomics (scMST) complemented with RNA-sequencing. Unexpectedly, we find maintenance of undecided Nanog/Oct4-PouV/Klf4-positive pluripotent-like pan-ectodermal stem-cells spanning the entire ectoderm late in the neurulation process with ectodermal patterning completed only at the end of neurulation when pluripotency becomes restricted to NC, challenging our understanding of gastrulation. Furthermore, broad ectodermal pluripotency is found at all axial levels unrelated to the NC lineage the cells later commit to, suggesting a general role in stemness enhancement and proposing a mechanism by which the NC acquires its ability to form derivatives beyond “ectodermal-capacity” in chick and mouse embryos. Overall design: Chick embryo samples from all tissue at the midbrain axial level from, HH5, 1ss, 4ss, 7ss and HH11 stage were dissociated and analyzed by scRNAseq. SEURAT workflow for scRNAseq analysis was followed by differential expression between ectodermal cell types.