Introduction: Oesophageal atresia/tracheo-oesophageal atresia (OA/TOF) frequently arises with associated anomalies and has been clinically linked with 22q11 deletion syndromes, a group of conditions due to Tbx1 gene mutation which include Di George syndrome. Tbx1 and Tbx2 genes modulate pharyngeal and cardiac development, but are also expressed in the developing foregut and are known to interact with key signalling pathways described in oesophageal formation including bone morphogenic proteins. The adriamycin mouse model (AMM) reliably displays OA/TOF-like foregut malformations providing a powerful system for investigating the disturbances in gene regulation and morphology involved in tracheo-oesophageal malformations. We hypothesised that foregut abnormalities observed in the AMM are associated with altered Tbx1 and Tbx2 gene expression.
Methods: Time-mated CBA/Ca mice received intra-peritoneal injection of adriamycin (for treated) or saline (for controls) on embryonic days (E)7 and 8. Untreated Cd1 embryos were used to establish normal expression patterns. Embryos harvested on E9-E11 underwent whole-mount in situ hybridization with labelled RNA probes for Tbx1 and Tbx2. Optical projection tomography was used to visualise expression in whole embryos by 3D imaging.
Results: Tbx1 expression was visualised in a highly specific pattern in the proximal oesophageal endoderm in normal and control embryos. In the AMM, extensive ectopic expression of Tbx1 was detected in the dorsal foregut and adjacent to the TOF. The focally restricted oesophageal expression pattern persisted in the AMM, but was posteriorly displaced in relation to the tracheal bifurcation. Tbx2 was widely expressed in the ventral foregut mesoderm of controls, lacking specific endoderm localisation. In the AMM, altered Tbx2 expression in the foregut was only seen in severely affected embryos.
Conclusion: Highly specific Tbx1 expression in the proximal oesophageal endoderm suggests that Tbx1 may be an important regulator of normal oesophageal development. Altered Tbx1 expression in dorsal foregut and adjacent to the TOF in the AMM suggests that Tbx1 gene disruption may contribute to the pathogenesis of tracheo-oesophageal malformations.