HoxA and HoxD Gene Deletion Effects on the Craniofacial Complex

Introduction: Many genetic diseases involve defects in facial morphogenesis. Craniofacial development involves a complex series of molecular and morphogenetic events generating coordinated patterns of cartilages, bones, and nerves, controlled at gene level. Combinatorial expression of Hox genes (family of master regulatory genes) play a definitive role in hindbrain, branchial arch regions, and consequently the craniofacial complex. Mice carrying targeted gene disruptions provide insights into molecular mechanisms lying behind numerous craniofacial defects; Objectives: To produce conditional inactivation of the entire mouse HoxA cluster in hindbrain neural crest cells, as well as double inactivation of HoxA/HoxD clusters, to precisely assess functions of these genes during facial skeletal development; Methods: Mice were generated as follows. Mice with the HoxA complex flanked with loxP-sites (A^flox) were produced using embryonic-stem cells containing two loxP-sites, one downstream of Hoxa1, one upstream of Hoxa13. The resulting floxed allele was introduced into the germ line of chimaeric males. The conditional deletion in hindbrain was obtained with mice expressing Cre recombinase under the control of Wnt1 promoter. To obtain HoxA conditional mutant mice, a first breeding round was accomplished generating HoxA^flox/+;Wnt1-Cre mice. These animals were in turn mated to HoxA^flox/+ mice. Generating double HoxA/HoxD mutants involved a more lengthy breeding strategy. The HoxD deleted mice were produced with the TAMERE strategy. Genotypes were determined by PCR and Southern-Blot analysis. The resulting phenotypes were analyzed; Results: Conditional HoxA and HoxA/HoxD mutants died at birth. Analysis of stained skeletons revealed that mutant animals contained multiple cranial skeletal defects. In Hoxa2 mouse mutants, the hyoid skeleton is replaced by a duplicated set of mandibular and middle ear structures. Here, more than the Hoxa2 knockout phenotype is seen. Preliminary results will be discussed; Conclusion: These data provide further experimental evidence for the existence of a branchial arch Hox code, and skeletogenic ground pattern.