Maintenance of pluripotency signature in the entire ectoderm enables potential for neural crest formation [bulk_RNASeq]

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. Here we show maintenance of pluripotency-signature (Nanog/Oct4-PouV/Klf4-positive) in undecided pan-ectodermal stem-cells spanning the entire ectoderm late during neurulation with ectodermal patterning completed only at the end of neurulation when the pluripotency-signature becomes restricted to NC, challenging our understanding of gastrulation. Furthermore, broad ectodermal pluripotency-signature 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. Samples from the neural crest domain of chick embryos at the midbrain level were collected for bulk RNAseq (HH5, HH6, HH7 (1ss), 2,ss, 3ss, 4ss, 5ss, 6ss, 7ss, 8ss, 9ss, 10ss)