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 home obox 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 regulate d by tissue interactions. Here we address the cis-trans regulatory int eractions that direct Msx2 expression to specific regions of the embry o and enable it to respond to tissue interactions. We created a series of Msx2-lacZ fusion constructs with varying amounts of Msx2 genomic s equences. These were introduced into mouse embryos and their expressio n monitored by staining for P-galactosidase activity. A construct bear ing 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 resemble d that of the endogenous Msx2 gene. In the E12.5 embryo, sites of expr ession included craniofacial mesenchyme, portions of the neural ectode rm, mesoderm in the distal limb bud and the overlying apical ectoderma l ridge (AER). Removal of intronic and 3' UTR sequences slightly alter ed the pattern of Msx2 expression in the neural ectoderm of the E12 em bryo. Deletion of 5' flanking sequences to -0.5 kb eliminated Msx2 exp ression in all sites except the AER. The proximal Msx2 promoter, inclu ding sequences required for the APR-specific expression of the -0.5 la cZ 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.