Investigating the mechanisms by which SMAD6 mutations cause tracheoesophageal birth defects

Esophageal atresia (EA) with or without tracheoesophageal fistula (TEF) are tracheoesophageal (TE) birth defects which affect 1 in 3000 live births in the United States. EA occurs when the esophagus fails to form a continuous tube from the oral cavity to the stomach and TEF occurs when there is an abnormal connection between the trachea and esophagus. The etiology of EA/TEF arises when the common foregut tube fails to properly separate into the esophagus and respiratory tract during early embryogenesis. This process is essential for proper organ function and involves complex molecular and morphological processes. As part of a multi-institutional project aiming to identify genes causing tracheoesophageal defects, we identified two EA/TEF patients who presented with different mutations in SMAD6, a BMP signaling inhibitor. We used a CRISPR-based approach to knock down smad6 in frog embryos and observed separation defects of the esophagus and trachea, supporting the conclusion that SMAD6 mutations in humans may cause EA/TEF. To investigate the role of SMAD6 during human development, we used CRISPR to generate human pluripotent stem cell lines with loss-of-function alleles in SMAD6. The human foregut is comprised of an inner layer of endoderm-derived epithelium surrounded by mesenchyme, and it is not known if SMAD6 functions in either or both layers. To elucidate this, we investigated the role of SMAD6 in either foregut epithelium or mesenchyme, or both in combination. Our analysis of human SMAD6-/- tracheoesophageal epithelium and mesenchyme reveals an increase in BMP/TGFβ signaling, consistent with its role as a repressor of these pathways. We also observed patterning defects in both tissue types; SMAD6 mutant endoderm shows increased expression of distal gut tube markers and SMAD6 mutant mesenchyme shows increased expression of cardiac markers. Furthermore, we observed cell differentiation changes, where SMAD6-/- mesenchyme had decreased ability to form CD31-positive vasculature, as well as changes in genes involved in processes such as cell migration and extracellular matrix deposition, which are critical for mesenchyme morphogenesis and differentiation during the process of TE separation. Our results suggest SMAD6 mutations impact the identity and function of human engineered TE tissue. By identifying pathways significant for TE development during early embryogenesis, our results contribute to understanding how patient mutations cause congenital malformations.