The first lineage specification of pluripotent mouse epiblast segregates neuroectoderm (NE) from mesoderm and endoderm (ME) by currently poorly understood mechanisms. Here we demonstrate that the induction of any ME-gene programs critically relies on the T-box (Tbx) transcription factors Eomes and Brachyury that concomitantly repress pluripotency and NE gene programs. Tbx-deficient cells retain pluripotency and differentiate to NE lineages despite the presence of ME-inducing signals TGFb/Nodal and WNT. Pluripotency and NE gene networks are additionally repressed by Tbx-induced ME factors, demonstrating a remarkable redundancy in program regulation to safeguard mutually exclusive lineage specification. Chromatin analyses revealed that accessibility of ME-gene enhancers depends on Tbx-binding, while NE-gene enhancers are accessible and activation-primed already at pluripotency state. This asymmetry of chromatin landscape thus explains the default differentiation of pluripotent cells to NE in the absence of MEinduction mediated through the activating and repressive functions of early Tbx factors Eomes and Brachyury.

The first lineage specification of pluripotent mouse epiblast segregates neuroectoderm (NE) from mesoderm and endoderm (ME) by currently poorly understood mechanisms.Here we demonstrate that the induction of any ME-gene programs critically relies on the T-box (Tbx) transcription factors Eomes and Brachyury that concomitantly repress pluripotency and NE gene programs. Tbx-deficient cells retain pluripotency and differentiate to NE lineages despite the presence of ME-inducing signals TGFb/Nodal and WNT. Pluripotency and NE gene networks are additionally repressed by Tbx-induced ME factors, demonstrating a remarkable redundancy in program regulation to safeguard mutually exclusive lineage specification. Chromatin analyses revealed that accessibility of ME-gene enhancers depends on Tbx-binding, while NE-gene enhancers are accessible and activation-primed already at pluripotency state. This asymmetry of chromatin landscape thus explains the default differentiation of pluripotent cells to NE in the absence of MEinduction mediated through the activating and repressive functions of early Tbx factors Eomes and Brachyury.

Different cell types are determined by cell lineage-specific transcriptional programmes and by epigenetic regulation of chromatin. Yet, the functional relationships between dynamically expressed transcription factors (TFs) and chromatin changes guiding lineage specification often remain elusive. First mammalian embryonic lineages segregate when pluripotent cells become committed to either Mesoderm and Endoderm (ME) or Neuroectoderm (NE). NE forms by default in the absence of signalling-induced ME specification, resulting from global asymmetries in chromatin state favouring NE gene programme activation as recently demonstrated. In this study, we unravel the initiation of ME lineage specification by the genome-wide, de novo formation of chromatin accessibility at ME enhancers that epigenetically deflects pluripotent cells from default NE differentiation. The Tbx TF Eomes, previously considered a transcriptional regulator, acts as global chromatin organizer that establishes ME lineage competence. EOMES recruits the canonical ATP-dependent chromatin remodelling complex SWI/SNF to broadly generate the chromatin-accessible ME enhancer landscape. This lineage competence is generated independently of ME gene transcription that fully depends on ME-inducing signalling pathways including Wnts and TGFβ/NODAL. This study thus resolves the successive steps of ME lineage differentiation by globally establishing chromatin accessibility for lineage competence, followed by signal-encoded transcriptional regulation of different ME lineage-defining gene programmes.

In the version of this article originally published, the labels for the orange and blue colour coding in the keys for Fig. 4b and Extended Data Fig. 4b,e were incorrect. In each panel, the orange rectangle should indicate '>III' , and the blue rectangle should indicate 'I and II'. The errors have been corrected in the HTML and PDF versions of the paper.