Msx1 is a key factor for the development of tooth and craniofacial skeleton and has been proposed to play a pivotal role in terminal cell differentiation. In this paper, we demonstrated the presence of an endogenous Msx1 antisense RNA (Msx1-AS RNA) in mice, rats, and humans. In situ analysis revealed that this RNA is expressed only in differentiated dental and bone cells with an inverse correlation with Msx1 protein. These in vivo data and overexpression of Msx1 sense and AS RNA in an odontoblastic cell line (MO6-G3) showed that the balance between the levels of the two Msx1 RNAs is related to the expression of Msx1 protein. To analyze the impact of this balance in the Msx-Dlx homeoprotein pathway, we analyzed the effect of Msx1, Msx2, and Dlx5 overexpression on proteins involved in skeletal differentiation. We showed that the Msx1-AS RNA is involved in crosstalk between the Msx-Dlx pathways because its expression was abolished by Dlx5. Msx1 was shown to down-regulate a master gene of skeletal cells differentiation, Cbfa1. All these data strongly suggest that the ratio between Msx1 sense and antisense RNAs is a very important factor in the control of skeletal terminal differentiation. Finally, the initiation site for Msx1-AS RNA transcription was located by primer extension in both mouse and human in an identical region, including a consensus TATA box, suggesting an evolutionary conservation of the AS RNA-mediated regulation of Msx1 gene expression. M sx genes are homeobox genes related to the Drosophila msh (muscle segment homeobox)-like gene family. Msx homeogenes play an important role in inductive epithelio-mesenchymal interactions leading to vertebrate organogenesis (1). Among this family, Msx1 is a fundamental factor for craniofacial skeleton formation. In mouse, head Msx1 gene expression is located mainly in regions of cephalic neural crest cell migration and differentiation, as well as in the derived mesenchymal cells (2-4). Msx1 also is found in a variety of embryonic tissues requiring epithelio-mesenchymal interactions for their morphogenesis such as limb bud, embryonic tail, hair follicle, and tooth bud.Msx1-deficient mice exhibit dental and craniofacial malformations, such as cleft palate, reduced mandible length, abnormalities of nasal, frontal, and parietal bones, as well as arrested tooth development, suggesting a role of Msx1 in outgrowth of these tissues (5, 6). In humans, mutations in Msx1 gene have been involved in tooth agenesis (7-9) and cleft palate (10), and the phenotype was proposed to be related to a dose effect of Msx1 protein (9). Interestingly, Msx1 down-regulation is associated with the terminal differentiation of several cell types such as cartilage (4,11,12) and muscle (13); indeed, in muscle cells, Msx1-forced expression results in a highly proliferative transformed phenotype and blocks myogenic terminal differentiation (14, 15) through the inhibition of a master gene expression, MyoD, by Msx1 (16). Thus, Msx1 is thought to prevent differentiation and enhance proliferation. Other...

Phenotypes associated with Msx1 mutations have established the prominent role of this divergent homeogene in skeletal patterning. Previous studies have been achieved during antenatal development in relation with the early death of null mutant mice. Therefore, the present study is devoted to Msx1 homeogene in the postnatal craniofacial, axial, and appendicular skeleton. A knock-in transgenic mouse line was studied from the first postnatal week until 15 months. Whole-mount ␤-galactosidase enzymology identified Msx1 protein expression pattern. Maintained expression of Msx1 was observed in growing and adult mice, specifically in the sites where Msx1 plays an early morphogenetic role during initial skeletal patterning. These included the craniofacial sutures, autopodium, mandible, and alveolar bone. Furthermore, active membranous and endochondral bone formation involved Msx1 in the entire skeleton. Histologic sections showed that progenitor as well as differentiating and differentiated cells of all the bone cell lineages could express the Msx1 protein (chondrocytes, osteoblasts, tartrate-resistant acid phosphatase positive osteoclasts and chondroclasts). Recent developments in the genetic and developmental biology of skeletal morphogenesis demonstrate that genes critical for development are jointly expressed in discrete embryonic signalling and growth centers, the enamel knot in teeth, the cranial suture in skull morphogenesis, and the progress zone in the limb buds. The present study suggests that these signalling pathways are jointly important throughout the entire lifetime with an exquisite site-specificity spatially related to early patterning.