Regulation of the Msx2 homeobox gene during mouse embryogenesis: a transgene with 439 bp of 5' flanking sequence is expressed exclusively in the apical ectodermal ridge of the developing limb.

Msx2, a member of the highly conserved and widely distributed msh homeobox gene family, is expressed in a variety of sites in the vertebrate embryo, including craniofacial structures, heart, limb buds and otic and optic vesicles. In many of these sites, its expression is regulated by tissue interactions. Here we address the cis-trans regulatory interactions that direct Msx2 expression to specific regions of the embryo and enable it to respond to tissue interactions. We created a series of Msx2-lacZ fusion constructs with varying amounts of Msx2 genomic sequences. These were introduced into mouse embryos and their expression monitored by staining for beta-galactosidase activity. A construct bearing 5.2 kb of 5' flanking sequence, the intron, both exons and 3 kb of 3' flanking sequence was expressed in a pattern that closely resembled that of the endogenous Msx2 gene. In the E12.5 embryo, sites of expression included craniofacial mesenchyme, portions of the neural ectoderm, mesoderm in the distal limb bud and the overlying apical ectodermal ridge (AER). Removal of intronic and 3' UTR sequences slightly altered the pattern of Msx2 expression in the neural ectoderm of the E12 embryo. Deletion of 5' flanking sequences to -0.5 kb eliminated Msx2 expression in all sites except the AER. The proximal Msx2 promoter, including sequences required for the AER-specific expression of the -0.5 lacZ transgene, is highly conserved between mouse and human, one stretch exhibiting 100% identity over 72 bp. This conservation suggests that the AER element is under remarkably tight evolutionary constraint.