Separation of the trachea and esophagus is coupled to identity specification by NKX2‐1‐mediated regulation of EPH/EPHRIN signaling and cell sorting

Abstract

The mechanisms linking the fate specification of tissues with morphogenetic outcomes generally remain poorly elucidated. One such context is the development of the trachea and esophagus, both of which derive from the definitive foregut endoderm and are specified dorsoventrally in part by the mutually repressive transcription factors NKX2-1 and SOX2, respectively. Following specification, the presumptive trachea and esophagus undergo physical separation from one another, but our understanding of how cells are properly allocated into their specified domains remains limited. Here, we show that tracheoesophageal (TE) separation is regulated by the EPH/EPHRIN signaling gene Efnb2, which is required for the proper ventral allocation of tracheal-fated cells into the presumptive trachea. Loss of EPHRIN-B2 impairs TE separation, producing a single foregut tube. Efnb2 expression, normally complementary to NKX2-1 expression in the foregut, is expanded upon loss of NKX2-1 irrespective of Sox2 perturbation, and through ChIP-seq and ChIP-PCR approaches we find that NKX2-1 binds the Efnb2 gene indicating a likely direct repression. Finally, mosaic deletion of Nkx2-1 produces a robust sorting of NKX2-1-positive and -negative cells, inducing ectopic NKX2-1/EPHRIN-B2 boundaries and ultimately driving ectopic tracheal separation events. Taken together, these data indicate that tracheal specification is coupled by NKX2-1 to separation morphogenesis by regulation of EPHRIN-B2 and TE cell sorting.