Maintenance of pluripotency signature in the entire ectoderm enables potential for neural crest formation
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: Chicken embryos at HH4 were knocked down three genes Oct4/PouV, Nanog and Klf4, respectively, by electroporating a translation blocking morpholino on one side of the embryo (one MO per experiment). Then they were collected at HH7/HH8- for bulk rnaseq. The transcriprome of the knockdown side will be compared to the contralateral side treated with a control MO. Three embryos (3 halves) are pooled together per experiment (except for one gene that we already prepared 4 samples for). (10 samples)