Genetics of shoulder girdle formation: roles of Tbx15 and aristaless-like genes.

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 Cart1 indicates 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.

The OAR/aristaless domain of the homeodomain protein Cart1 has an attenuating role in vivo.

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.