Conditional alleles of Msx1 and Msx2

The msh-related homeobox genes, Msx1 and Msx2, have a variety functions during murine organogenesis, Msx1 in the development of the palate and teeth, Msx2 in the skull, teeth, and skin. Msx1 mutants die perinatally. Compound Msx1-2 mutants do not survive past late gestation. The multiplicity of functions of Msx1 and 2, as well as the lethality of Msx1 and Msx1-2 mutants limits the utility of the conventional knockouts. We therefore produced conditional alleles of Msx1 and Msx2. We constructed targeting vectors with LoxP sites flanking the homeodomain-encoding second exons and Frt sites flanking a neo gene. These vectors were used to produce targeted ES cells and mice with floxed alleles. The functionality of the LoxP sites in the floxed alleles was established by crosses with K14-Cre mice (epidermis-specific), and with an Msx2-Cre line that produces a germline deletion. Analysis of progeny by PCR revealed correct Cre-mediated recombination, as well as expected phenotypes.     

Analysis of Msx1; Msx2 double mutants reveals multiple roles for Msx genes in limb development

The homeobox-containing genes Msx1 and Msx2 are highly expressed in the limb field from the earliest stages of limb formation and, subsequently, in both the apical ectodermal ridge and underlying mesenchyme. However, mice homozygous for a null mutation in either Msx1 or Msx2 do not display abnormalities in limb development. By contrast, Msx1; Msx2 double mutants exhibit a severe limb phenotype. Our analysis indicates that these genes play a role in crucial processes during limb morphogenesis along all three axes. Double mutant limbs are shorter and lack anterior skeletal elements (radius/tibia, thumb/hallux). Gene expression analysis confirms that there is no formation of regions with anterior identity. This correlates with the absence of dorsoventral boundary specification in the anterior ectoderm, which precludes apical ectodermal ridge formation anteriorly. As a result, anterior mesenchyme is not maintained, leading to oligodactyly. Paradoxically, polydactyly is also frequent and appears to be associated with extended Fgf activity in the apical ectodermal ridge, which is maintained up to 14.5 dpc. This results in a major outgrowth of the mesenchyme anteriorly, which nevertheless maintains a posterior identity, and leads to formation of extra digits. These defects are interpreted in the context of an impairment of Bmp signalling.