Cas9-mediated excision of Nematostella brachyury disrupts endoderm development, pharynx formation and oral-aboral patterning.

The mesoderm is a key novelty in animal evolution, although we understand little of how the mesoderm arose. brachyury, the founding member of the T-box gene family, is a key gene in chordate mesoderm development. However, the brachyury gene was present in the common ancestor of fungi and animals long before mesoderm appeared. To explore ancestral roles of brachyury prior to the evolution of definitive mesoderm, we excised the gene using CRISPR/Cas9 in the diploblastic cnidarian Nematostella vectensisNvbrachyury is normally expressed in precursors of the pharynx, which separates endoderm from ectoderm. In knockout embryos, the pharynx does not form, embryos fail to elongate, and endoderm organization, ectodermal cell polarity and patterning along the oral-aboral axis are disrupted. Expression of many genes both inside and outside the Nvbrachyury expression domain is affected, including downregulation of Wnt genes at the oral pole. Our results point to an ancient role for brachyury in morphogenesis, cell polarity and the patterning of both ectodermal and endodermal derivatives along the primary body axis.