The roles of the homeobox genes ALX4 and MSX2 in skull development

Heritable ossification defects of the skull vault often present as enlarged parietal foramina (PFM), bilateral oval openings of the posterior parietal bones. Isolated PFM may originate from wider defects in infancy and usually show an autosomal dominant mode of transmission, offering unexplored genetic insights into the molecular pathways of calvarial development. Haploinsufficiency of the homeobox gene MSX2, located at 5q34-q35, underlies a fraction of PFM families but the locus can be excluded in others, indicating heterogeneity. The proximal 11p deletion syndrome (P11pDS), characterised by multiple exostoses (due to haploinsufficiency of EXT2), occasional mental retardation, and PFM, pointed to a second locus at 11p11-p12. The human orthologue of the mouse paired-like homeobox gene Alx4 was identified adjacent to EXT2. ALX4 was structurally characterised and heterozygous loss-of-function mutations were detected in association with skull vault defects in twenty-nine individuals from six families, including a new case of P11pDS. The calvarial phenotype of ALX4 mutations was almost indistinguishable from the MSX2-caused defects and ranged from a midline gap to non-penetrance, nevertheless typified by classical PFM; abnormal morphology of the dural septa was also observed. The mutation spectrum and the subtle genotype-phenotype correlations suggested haploinsufficiency as the predominant pathophysiological mechanism. Interestingly, Alx4-/+ mice manifest polydactyly but no skull defects, illustrating species-specific dosage sensitivity. Two new MSX2 mutations were also ascertained, one of which segregated with PFM and clavicular hypoplasia. The potential contribution of ALX4 and MSX2 to premature fusion of the cranial sutures - craniosynostosis - was investigated, but no unequivocally pathogenic variants were found. To elucidate the functions of Alx4 and Msx2 in skull development, spatial expression analysis was performed in mouse embryos between embryonic days E12.5-E17. Transcripts of both Alx4 and Msx2 were seen in the early calvarial skeletogenic condensations and in later stages their expression displayed a more restricted pattern, overlapping minimally with the domains of mature bone. By assessing expression in embryonic heads of reciprocal knockout mice, activation of Alx4 was found to be independent of functional Msx2 and vice versa. Analysis of compound mutants demonstrated that the two loci exert roughly additive effects on the skull vault while protein interaction assays did not indicate any physiological interaction between Alx4 and Msx2. Hence, Alx4 and Msx2 appear to regulate proliferation, differentiation, or survival of osteoblast precursors and pre-osteoblasts through parallel pathways.