Genome-wide identification of notochord enhancers comprising the regulatory landscape of the Brachyury (T) locus in mouse

The node and notochord are important signaling centers organizing dorso-ventral patterning of cells arising from neuro-mesodermal progenitors forming the embryonic body anlage. Due to the scarcity of notochordal progenitors and notochord cells, a comprehensive identification of regulatory elements driving notochord-specific gene expression has been lacking. Here we have used ATAC-seq analysis of FACS-purified notochordal cells from TS12-13 mouse embryos to identify 8921 putative enhancers of notochordal cells. In addition, we established a new model for generating notochordal cells in culture, and identified 3728 enhancers occupied by the essential notochordal regulators Brachyury (T) and/or Foxa2. We describe the regulatory landscape of the T locus comprising 8 putative enhancers occupied by these factors and confirmed the regulatory activity of 3 of these elements. Moreover, we characterized one new notochord enhancer, termed TNE2, in embryos. TNE2 complements the loss of TNE in the trunk notochord, and is essential for notochordal cell proliferation and differentiation in the tail. Our data demonstrate the essential role of Foxa2 in the choice of T expressing cells between the notochordal fate and the NMP/mesodermal trajectory Transcriptomes (RNA-Seq), chromatin accessibility (ATAC-Seq) and chromatin IPs (ChIP-Seq) of histone modifications and the T and Foxa2 transcription factors in various FACS sorted (based on Noto, T and Foxa2 expression) or bulk samples from either E8.5/E9.5 embryos or mESCs differentiated in vitro into neuromesodermal, notochord or mesoderm (progenitor)-like cells.