Background: The adriamycin mouse model is a well-established teratogenic model of esophageal atresia/tracheoesophageal fistula. Fibroblast growth factor 10 (Fgf10) plays a key role in branching of the lung buds during lung morphogenesis. Fgf10 knockout mice exhibit the absence of the lungs. 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 Fgf10 during the critical period of separation of the trachea and esophagus in normal and adriamycin-treated embryos using OPT.
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 10-13, stained after whole mount in situ hybridization with labeled RNA probes to detect Fgf10 transcripts (n = 5 for each treatment/day of gestation). Immunolocalization with endoderm marker Hnf3 beta was used to visualize morphology. Embryos were scanned by OPT to obtain 3D representations of gene expression domains.
Results: Computer reconstructed specimens allowed precise staging of developing embryos according to Theiler Staging (TS) criteria. OPT elegantly displayed Fgf10 gene expression in the pulmonary mesenchyme around the tip of the lung buds in both controls and treated embryos in the same spatial territory. Fgf10 gene expression was first detected in the control embryos at TS17. However, Fgf10 gene expression in adriamycin-treated embryos was first only observed at TS18 in 67% of the specimens.
Conclusion: Delayed Fgf10 gene expression during the critical period of separation of the trachea and esophagus may affect lung bud formation in the adriamycin model leading to tracheoesophageal malformations.