Fig. 6: Differential methylome and chromatin states modulate the context-dependent response of post-implantation primed epiblast cell populations to gastrulation cues.

a Illustration of epigenetically driven priming in post-implantation epiblast cells toward neuroectoderm, DE, ExM, and NMP lineages. CLDN6^High EpiSCs, with high pERK, resemble anterior epiblast in WNT-inhibited regions and give rise to neuroectoderm without PS ingress. In contrast, CLDN6^Low EpiSCs show low pERK, resemble distal posterior epiblast, and exhibit methylation of neuroectoderm genes (Pax6, Onecut2, Dbx1), restricting neuroectodermal potential. Anterior epiblast cells (CLDN6^High EpiSCs, high ERK) respond to WNT signalling signalling by forming DE via anterior PS differentiation. In contrast, distal posterior epiblast cells (CLDN6^Low EpiSCs, low ERK) possess differential DNA methylation signature over DE genes (Foxa2, Gata6, Sox17), blocking the DE fate but enabling NMP formation upon WNT-induced APS differentiation. DNA methylation safeguards distal posterior epiblast cells from alternative fates, and subsequent chromatin remodelling operated by CDX⁵ and LEF1-PBX1³³ pioneer TFs leading to differentiation NMPs generating PSM. Our data indicate that WNT response in primed epiblast cells is shaped by DNA methylation. Proximal posterior epiblasts, with permissive chromatin/methylome, respond to BMP to form ExM tissues. Pbx1-KO EpiSCs have a permissive methylome and chromatin state over yolk sac and primitive erythroid genes (Tbx4, Gata2, Tal1), but repressed states at allantois/endothelial genes (Hand1, Tbx4, Kdr), failing to generate these fates. In contrast, WT EpiSCs, with hypomethylated and accessible allantois/endothelial loci, readily differentiate into these lineages but have reduced yolk sac/erythroid potential. b Revised Waddington’s landscape model illustrating how ERK dosage–dependent DNA methylation regulates cell fate commitment. DNA methylation modulates the balance between canalization and plasticity across germ-layer development, shaping trajectory paths in distinct signalling contexts (yellow, grey, orange, blue landscapes). During epiblast-to-germ-layer transition, cells (coloured balls) roll down lineage paths. DNA methylation (red gates) restricts certain fates, redirecting cells based on methylation signatures (red/blue marks). Dotted lines show fate trajectories shaped by methylation. Shifting signalling landscapes remodel chromatin (grey, blue, orange blocks), with dynamic methylation driving both canalization and plasticity in tissue development. (Adapted from an image by Conrad H. Waddington, licensed under CC BY-SA 4.0 (https://creativecommons.org/licenses/by-sa/4.0/). Source: https://commons.wikimedia.org/wiki/File:Paisagem_epigenetica.jpg. Modifications made by the author).