Genome-wide identification of notochord enhancers comprising the regulatory landscape of theBrachyury(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 notochord enhancers. In addition, we established a new model for generating notochordal cells in culture, and found 3728 of these enhancers occupied by the essential notochordal regulators 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 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 switching T expressing cells from a NMP/mesodermal trajectory to the notochordal fate. Summary statement Combining multi-omics assays of purified embryonic and in vitro generated cells we identified thousands of notochord enhancers comprising TNE2 essential for T expression and tail development of the mouse embryo.