SMAD6 is required for normal foregut development in humans and frogs.

Esophageal atresia and tracheoesophageal fistula (EA/TEF) are congenital malformations of the foregut. We identified two distinct variants in the BMP/TGFβ repressor SMAD6 in two individuals exhibiting EA/TEF. We investigated the function of SMAD6 in tracheoesophageal development using two orthogonal approaches in Xenopus embryos, both resulting in foregut malformations similar to those observed in EA/TEF. We then used human pluripotent stem cell-derived foregut epithelium and mesenchyme to explore the separate roles of SMAD6 in these germ layers. CRISPR-mediated disruption of human SMAD6 caused an increase in BMP signaling in foregut epithelium and mesenchyme consistent with its role as a BMP repressor. Loss of SMAD6 caused patterning defects in both tissue types; SMAD6-/- endoderm showed increased expression of distal gut tube markers and SMAD6-/- mesenchyme showed increased expression of ventral and posterior markers, including markers of cardiac and liver mesenchyme lineages. Furthermore, SMAD6-/- mesenchyme had decreased ability to form CD31-positive endothelial cells. Our results demonstrate that SMAD6 is required for foregut development and that rare variants in this gene are likely causative for foregut malformations in EA/TEF.