Prx1 and Prx2 (previously called MHox and S8, respectively) are the members of a small subfamily of vertebrate homeobox genes expressed during embryogenesis from gastrulation onwards. We directly compared the expression domains of the Prx genes in detail in mouse and in addition some aspects of these patterns in chicken. In addition to the superficially similar expression patterns of Prx1 and Prx2 in cranial mesenchyme, limb buds, axial mesoderm, and branchial arches and their derivatives, we detect major differences at many sites particularly in heart and brain. Our analysis indicated in several cases a correlation with regions developing into connective tissues. From at least day 8.5, Prx-1 expression was observed in the heart, initially in the endocardial cushions and later in the developing semilunar and atrioventricular valves. Prx2 develops early on a diffuse myocardial expression pattern and is later higher expressed in the ventricular septum and in particular in the ductus arteriosus. Prx2 is never expressed in the brain, whereas Prx1 is expressed, from at least day 9.5 onwards, in a unique distinct domain in the ventral part of the hypothalamus, as well as in a broader region of the telencephalon.

The diverse cellular contributions to the skeletal elements of the vertebrate shoulder and pelvic girdles during embryonic development complicate the study of their patterning. Research in avian embryos has recently clarified part of the embryological basis of shoulder formation. Although dermomyotomal cells provide the progenitors of the scapular blade, local signals appear to have an essential guiding role in this process. These signals differ from those that are known to pattern the more distal appendicular skeleton. We have studied the impact of Tbx15, Gli3, Alx4 and related genes on formation of the skeletal elements of the mouse shoulder and pelvic girdles. We observed severe reduction of the scapula in double and triple mutants of these genes. Analyses of a range of complex genotypes revealed aspects of their genetic relationship, as well as functions that had been previously masked due to functional redundancy. Tbx15 and Gli3 appear to have synergistic functions in formation of the scapular blade. Scapular truncation in triple mutants of Tbx15, Alx4 and Cart1indicates essential functions for Alx4 and Cart1 in the anterior part of the scapula, as opposed to Gli3 function being linked to the posterior part. Especially in Alx4/Cart1 mutants, the expression of markers such as Pax1, Pax3 and Scleraxis is altered prior to stages when anatomical aberrations are visible in the shoulder region. This suggests a disorganization of the proximal limb bud and adjacent flank mesoderm, and is likely to reflect the disruption of a mechanism providing positional cues to guide progenitor cells to their destination in the pectoral girdle.

Mouse Alx3 is a homeobox gene that is related to the Drosophila aristaless gene and to a group of vertebrate genes including Prx1, Prx2, Cart1, and Alx4. The protein encoded contains a diverged variant of a conserved peptide sequence present near the carboxyl terminus of at least 15 different paired-class-homeodomain proteins. Alx3 is expressed in mouse embryos from 8 days of gestation onward in a characteristic pattern, predominantly in neural crest-derived mesenchyme and in lateral plate mesoderm. We detected prominent expression in frontonasal head mesenchyme and in the first and second pharyngeal arches and some of their derivatives. High expression was also seen in the tail and in many derivatives of the lateral plate mesoderm including the limbs, the body wall, and the genital tubercle. aristaless-related genes like Alx3, Cart1, and Prx2 are expressed in overlapping proximodistal patterns in the pharyngeal arches. Similar, but more lateral patterns have been described for the Distal-less-related (Dlx) genes. Intriguingly, expression and to some extent function of aristaless and Distal-less in Drosophila also have overlapping as well as complementary aspects. Alx3 was localized to chromosome 3, near the droopy-ear (de) mutation.

Hoxb8 mutant mice were generated by inserting the lacZ coding sequence in frame with the first exon of Hoxb8. These mice express a fusion protein with a functional beta-galactosidase activity instead of Hoxb8. Mutant embryos were analyzed for anatomical changes. The results indicate that Hoxb8 is not an indispensable regulator of A-P patterning in the forelimb, unlike suggested by our Hoxb8 gain of function experiments (Charité J, DeGraaff W, Shen S, Deschamps J. Cell 1994;78:589-601). The null mutant phenotypic traits include degeneration of the second spinal ganglion (C2), an abnormality opposite to the alteration in the gain of function transgenic mice. Subtle changes in the thoracic part of the vertebral column were observed as well. Adult homozygous mutants exhibit an abnormal clasping reflex of the limbs.

Aristaless-related genes encode a structurally defined group of homeoproteins that share a C-terminal stretch of amino acids known as the OAR- or aristaless domain. Many aristaless-related genes have been linked to major developmental functions, but the function of the aristaless domain itself is poorly understood. Expression and functional studies have shown that a subgroup of these genes, including Prx1, Prx2, Alx3, Alx4 and Cart1, is essential for correct morphogenesis of the limbs and cranium. We now demonstrate the function of the aristaless domain in vivo by ectopically expressing normal and mutated forms of Cart1 and Alx3. Ectopic expression of Cart1 in transgenic mice does not disturb development, whereas expression of a Cart1 form from which the aristaless domain has been deleted results in severe cranial and vertebral malformations. The Alx3 protein contains a divergent aristaless domain that appears not to be functional, as ectopic expression of Alx3 results in an altered phenotype irrespective of the presence of this aristaless domain. Linking the Cart1 aristaless domain to Alx3 extinguishes teratogenicity. We show that, at the molecular level, the most important consequence of deleting the aristaless domain is increased DNA binding to its palindromic target sequence. This demonstrates that the aristaless domain functions as an intra-molecular switch to contain the activity of the transcription factor that it is part of.