[The etiology of congenital diaphragmatic hernia and esophageal atresia: the Hox genes].
BACKGROUND
Prenatal administration of adriamycin or nitrofen to pregnant mice produce in the embryos, respectively, esophageal atresia/VACTERL association (EA) or congenital diaphragmatic hernia (CDH). Various genes and signalling pathways like sonic hedgehog, Gli family, retinoic acid and homeotic genes have been pointed out in the origin of these malformations. Hox genes are master regulatory genes involved in embryo segmentation and other main development processes. Hoxa3, Hoxb3, Hoxc3, Hoxc4 and Hoxa5 knock-out mice show cardiac, tracheal, lung and diaphragmatic malformations, EA and phenotypes that resemble that of VACTERL syndrome. We present herein some of our findings in the expression of these genes in both experimental models.


METHODS
Pregnant mice were exposed either to 4 mg/kg of adriamycin or vehicle on embryonic days 7,5 and 8,5; embryos were recovered at four endpoints (E13 to 16). On the other hand, nitrofen was given to pregnant mice on embryonic day 8th and embryos were recovered at E14, E16 and E19. The embryos or, separately, their lungs and hearts, were randomly processed for immunohistochemical or molecular biology studies (RT-PCR). We used antibodies for Hoxa3, Hoxb3 and Hoxd3 proteins and specific primers for Hoxa3, Hoxa5, Hoxb3, Hoxb5, Hoxc4 and Hoxd3 genes.


RESULTS
EA: Upon immunohistochemistry, adriamycin-exposed embryos showed a severe decrease in expression of Hoxa3, Hoxb3 and Hoxb3 proteins in heart, skin, foregut but not in the heart. RT-PCR studies showed a statistically significant decrease of the four genes studied in the lungs of OA mice when compared to controls. CDH: Upon RT-PCR assessment the expression of Hoxa5 and Hoxb3 were higher in nitrofen-exposed mice than in controls on E14 and E19 and weaker on E16. As regards immunohistochemical localization, expression of the three genes was similar in nitrofen and control animals.


CONCLUSIONS
Both experimental models exhibit an alteration in the expression of several proximal Hox genes, specially in lung and car- diac tissues. The malformations in these organs associated with CDH and EA could be in part caused by these alterations. Due to their specific participation in lung and foregut morphogenesis, their study could let us to better understand the mechanisms of CDH and EA.
Martínez
L
L
Departamento de Cirugía Pediátrica, Hospital Universitario La Paz, Madrid. lmartinezm.hulp@salud.madrid.org
Martínez-Calonge
W
W
Matesanz
R
R
Fernández-Dumont
V
V
Pederiva
F
F
Vallejo
M T
MT
Salinas
J
J
Tovar
J A
JA
D000818:Animals; D004268:DNA-Binding Proteins / Q000235:genetics; D004933:Esophageal Atresia / Q000235:genetics*; D006548:Hernia, Diaphragmatic / Q000235:genetics*; D065630:Hernias, Diaphragmatic, Congenital; D018398:Homeodomain Proteins / Q000235:genetics; D051379:Mice; D008808:Mice, Inbred CBA; D010750:Phosphoproteins / Q000235:genetics; D014157:Transcription Factors
D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; C080331:Hoxa3 protein, mouse; C059637:Hoxa5 protein, mouse; C503713:Hoxb3 protein, mouse; C053925:Hoxb5 protein, mouse; C490904:Hoxc4 protein, mouse; C484113:Hoxd3 protein, mouse; D010750:Phosphoproteins; D014157:Transcription Factors