Expression of homeotic genes Hoxa3, Hoxb3, Hoxd3 and Hoxc4 is decreased in the lungs but not in the hearts of adriamycin-exposed mice.

Exposure of rat and mouse embryos to adriamycin (doxorubicin chlorhydrate) induces esophageal atresia (EA) and VACTERL association. Sonic hedgehog (Shh) and Gli2/Gli3 pathways are involved in these conditions and knockout mice for homeotic Hox genes Hoxa3, Hoxb3, Hoxc3, Hoxc4 and Hoxa5 show phenotypes with some of the associated VACTERL features. This study aims at evaluating the possible influence of Hoxa3, Hoxb3, Hoxd3 and Hoxc4 as upstream regulators of this complex signalling. Pregnant mice were exposed either to 4 mg/kg of adriamycin (EA group) or vehicle (controls) on embryonic days 7.5 and 8.5. Embryos were recovered at four endpoints (E12.5-E15.5) and randomly assigned for immunohistochemical or molecular biology studies. Lungs and hearts were separately harvested and processed for Hoxa3, Hoxb3, Hoxd3 and Hoxc4 quantitative RT-PCR measurements. Antibodies for Hoxa3, Hoxb3 and Hoxd3 proteins were used for immunohistochemical studies. RT-PCR studies showed a drastic and statistically significant decrease of the four genes in the lungs of EA mice when compared to controls, with a slight recovery from E15.5. Hearts of both groups showed a similar expression of all the genes throughout gestation. Control embryos expressed the hox3 paralogous genes in heart, skin, foregut derivatives and their surrounding mesoderm through E12.5-E15.5 whereas adriamycin-exposed embryos showed a severe decrease in expression of these three proteins in the same tissues but not in the heart. Adriamycin drastically reduced the expression of Hoxa3, Hoxb3, Hoxd3 and Hoxc4 in mice embryonic lungs. Their expression in the heart did not seem to be influenced by adriamycin in this experimental setting.