Posterior enhancer (p-Enh) maintains early neuromesodermal progenitors bi-potency during gastrulation

Vertebrate axis patterning requires precise control of the differentiation of neuromesodermal progenitors (NMPs), which generate spinal cord (SC) and presomitic mesoderm (PSM). Previously, we identified a gastrula-premarked posterior enhancer (p-Enh) that is essential for posterior tissue development by regulating somite and SC in organogenetic embryos, while its role in early NMPs cells remains elusive. Here, using a highly efficient in vitro differentiation system, we found that the genetic removal of p-Enh leads to the aberrantly up-regulated PSM-related genes during both PSM and SC differentiation. Time-resolved transcriptomic analysis and experimental characterization revealed the activated PSM transcriptomic signature arose from disorganized NMPs composition, with an over-representation of the T^highSOX2^low NMPs subtype. Besides, through a newly developed bioinformatic tool, ST-Pheno, which effectively bridges the in vitro samples to in vivo embryonic phenotypes within spatiotemporal context, we determined that the over-produced T^highSOX2^low NMPs subtype is predominantly enriched in the anterior primitive streak and adjacent mesoderm region at E7.5, which may disrupt the proper development of NMPs towards prospective PSM and SC, ultimately leading to the posterior development failure. In summary, this study demonstrates a critical role of p-Enh in regulating NMPs subtype composition, which will broaden the molecular understanding of mammalian embryogenesis.