Mesenchymal expression of Tbx4 gene is not altered in Adriamycin mouse model

Abstract

Purpose: The Adriamycin mouse model (AMM) is a reproducible teratogenic model of esophageal atresia/tracheo-esophageal fistula (EA/TEF). Tbx4 is a member of the T-box family of transcription factor genes, which is reported to play a key role in separation of the respiratory tract and the esophagus. Up-regulation of Tbx4 is reported to cause TEF in the chick. Optical projection tomography (OPT) is a technique that allows three-dimensional (3D) imaging of gene expression in small tissue specimens in an anatomical context. The aim of this study was to investigate the temporo-spatial expression of Tbx4 during the critical period of separation of the trachea and esophagus in normal and Adriamycin treated embryos using OPT.

Materials and methods: Time-mated CBA/Ca mice received intraperitoneal injections of Adriamycin (6 mg/kg) or saline on days 7 and 8 of gestation. Embryos were harvested on days 9-12, stained following whole mount in situ hybridization with labeled RNA probes to detect Tbx4 transcripts (n = 5 for each treatment/day of gestation). Immunolocalization with the endoderm marker Hnf3beta was used to visualize morphology. Embryos were scanned by OPT to obtain 3D representations of gene expression domains. Animal licence no. B100/4106.

Results: OPT elegantly revealed Tbx4 gene expression in both controls and in the disorganized pulmonary mesenchyme in the treated embryos. Although characteristic morphological abnormalities were observed in Adriamycin treated embryos, there was no significant difference in Tbx4 transcript distribution around lung primordia in comparison with control embryos.

Conclusion: Although previously reported morphological abnormalities of notochord and esophagus were observed in AMM, Tbx4 gene expression was unaltered, suggesting that esophageal anomalies can occur in the presence of normal Tbx4 gene expression in this model.