NKX2‐1 directly regulates Efnb2 to generate a boundary that allocates tracheal cells and defines the position of tracheoesophageal separation

Abstract

Tracheoesophageal (TE) fistula is a human congenital condition characterized by an improper connection between the esophagus and trachea. These two tubular organs originate from a single foregut tube and separate from one another beginning at the site of lung bud evagination and progressing rostrally. Genetic studies in mice have identified signaling pathways that initiate dorsoventral patterning of the foregut; NKX2‐1 marks ventral, presumptive tracheal cells whereas high levels of SOX2 expression predominantly mark the dorsal, esophageal‐fated cells. Perturbation of either factor results in loss of that identity, increased expression of the opposing transcription factor, and failure of TE separation. Currently, the downstream mechanisms required to effect morphogenetic separation at the cellular and tissue level remain largely unknown. Here, we show that EPHRIN‐B2, a member of the EPH/EPHRIN signaling family, is restricted to the dorsal, esophageal‐fated cells, and that this domain of expression is required for TE separation and for maintaining the correct allocation of tracheal cells to the nascent trachea. Further, we demonstrate that NKX2‐1 directly binds at the Efnb2 locus and negatively regulates its expression, establishing an NKX2‐1/EPHRIN‐B2 TE boundary. Nkx2‐1 mosaic mutants exhibit ectopic NKX2‐1/EPHRIN‐B2 boundaries which drive ectopic tracheal separation morphogenesis in an EPHRIN‐B2 dependent manner. These results establish NKX2‐1 as a regulator of TE cell sorting and tissue separation, through modulation of EPH/EPHRIN signaling, which then serves to translate tissue identity information into morphogenetic outcomes.