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Msx2 is a homeobox gene with a regulatory role in inductive tissue interactions, including those that pattern the skull. We demonstrated previously that individuals affected with an autosomal dominant disorder of skull morphogenesis (craniosynostosis, Boston type) bear a mutated form of Msx2 in which a histidine is substituted for a highly conserved proline in position 7 of the N-terminal arm of the homeodomain (p148h). The mutation behaves as a dominant positive in transgenic mice. The location of the mutation in the N-terminal arm of the homeodomain, a region which in other homeodomain proteins plays a key part in protein-protein interactions, prompted us to undertake a yeast two hybrid screen for Msx2-interacting proteins. Here we present a functional analysis of one such protein, designated Miz1 (Msx-interacting-zinc finger). Miz1 is a zinc finger-containing protein whose amino acid sequence closely resembles that of the yeast protein, Nfi-1. Together these proteins define a new, highly conserved protein family. Analysis of Miz1 expression by Northern blot and in situ hybridization revealed a spatiotemporal pattern that overlaps that of Msx2. Further, Miz1 is a sequence specific DNA binding protein, and it can function as a positive-acting transcription factor. Miz1 interacts directly with Msx2 in vitro and enhances the DNA binding affinity of Msx2 for a functionally important element in the rat osteocalcin promoter. The p148h mutation in Msx2 augments the Miz1 effect on Msx2 DNA binding, suggesting a reason why this mutation behaves in vivo as a dominant positive, and providing a potential explanation of the craniosynostosis phenotype.

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.