Sonic hedgehog gene expression in nitrofen induced hypoplastic lungs in mice

Abstract

The pathogenesis of congenital diaphragmatic hernia (CDH) is unknown. The sonic hedgehog (Shh) cascade is crucial for the patterning of the early respiratory system in mice. Optical Projection Tomography (OPT) is a new tool for 3D imaging small biological specimens that enables us to visualise both the anatomy of developing organs and gene expression localised in the context of normal or abnormal anatomy. We visualised Shh gene expression patterns in the nitrofen treated and control mouse lung buds at early stages of lung development. Time-mated CD1 female mice (n = 5) received oral administration of 100 mg of the herbicide 2,4-dichlorophenyl-p-nitrophenylether (nitrofen) (WAKO Chemical, Osaka, Japan) in 1 ml of olive oil or olive oil alone at 7 days of gestation. Embryos were harvested on gestation days 9-12, and stained following whole mount in situ hybridisation with labelled RNA probes to detect Shh transcripts at each stage. Embryos were scanned by OPT to obtain 3D representations of gene expression domains in the context of the changing morphology of the embryo. OPT analysis of Shh transcript distribution clearly revealed gene expression in both groups. In treated embryos, there were no significant changes in Shh transcript distribution in lung buds in comparison with control embryos. Although altered Shh expression in the hypoplastic lung has been reported in late gestation, the present study did not reveal any significant alterations in pulmonary Shh spatial transcript distribution or gene expression level during the early gestation in nitrofen CDH model. It would be of great interest in future studies to use OPT approach to investigate pulmonary expression of Shh and other regulatory genes both during early and late stages of lung development in order to provide new insights into the pathogenesis of pulmonary hypoplasia.