Functions of Alx3, Alx4 and Cart1 during craniofacial development in the mouse

Abstract

Aristaless-related genes are Paired-related homeobox genes that by definition encode a second strongly conserved domain at their C-terminal end, the aristaless-box . During my Ph. D. project I studied the functions of a specific subset of these highly related genes, Alx3, Alx4 and Cart1, during craniofacial development in the mouse. They are expressed in overlapping patterns in developing limb and craniofacial regions. Mice with mutations in Alx4, among which the Strong's Luxoid (lst) mouse, have preaxial polydactyly, defects in the skull base and vaults and gastroschesis. Cart1 mutant mice lack most of the skull vaults and have abnormalities in the skull base. The acrania phenotype probably arises through apoptosis of mesenchyme cells surrounding the forebrain around embryonic day 9.0 (E9.0). During my project, I generated the Alx3 mutant mouse. This mouse does not have abnormalities. We expected that in this mutant mouse the functions of Alx3 were taken over by the highly related Alx4 and Cart1. Therefore, we generated Alx3/Alx4 and Alx3/Cart1 double mutants and Alx3/Alx4/Cart1 triple mutants. In double and triple mutants lacking at least three alleles severe craniofacial abnormalities were observed. The nose region was completely split and most bones of the facial skeleton and anterior skull base region appeared malformed. In Alx3/Alx4 double mutant mice we studied the aetiology of the phenotype. We found that the craniofacial malformations first become apparent in E10.5 Alx3/Alx4 double mutant embryos. Their nasal processes appear to project extremely laterally, resulting in their failure to fuse around E11.5. In situ hybridisations on sections of E10.5 and E11.5 double mutant embryos showed that the expression patterns of several ectodermal and mesenchymal marker genes had changed in line with the morphological malformations. Moreover, we found that the defects correlate with abnormal and localised apoptosis in the frontonasal processes of E10.0 Alx3/Alx4 double mutant embryos. Overall, we conclude that Alx3, Alx4 and Cart1 have important functions in patterning the craniofacial primordia. We deem it likely that they all regulate craniofacial patterning by controlling survival of mesenchymal cells in the craniofacial primordia