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 notochord 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 notochord cells from TS12-13 mouse embryos to identify 8921 putative notochord enhancers. In addition, we established a new model for generating notochord-like cells in culture, and found 3728 of these enhancers occupied by the essential notochord control factors Brachyury (T) and/or Foxa2. We describe the regulatory landscape of the T locus comprising 10 putative enhancers occupied by these factors and confirmed the regulatory activity of 3 of these elements. Moreover, we characterized 7 new elements via knockout analysis in embryos and identified one new notochord enhancer, termed TNE2. TNE2 cooperates with TNE in the trunk notochord, and is essential for notochord differentiation in the tail. Our data emphasize the essential role of Foxa2 in directing T expressing cells towards the notochord lineage.