Genetic Regulation of Skeletal Patterning, Differentiation, and Growth

The development of the vertebrate skeleton represents an outstanding paradigm for elucidating molecular mechanisms of organogenesis and morphogenesis. It offers exciting problems of spatial specification (patterning) of skeletal elements, it provides an excellent opportunity for studying processes that control organ shape and size, and it includes well-defined problems of cellular determination, differentiation, growth, and apoptosis. During postnatal growth and subsequent aging, skeletal biology offers intriguing questions concerning homeostatic regulation, repair, and (in some species) regeneration. This biological complexity can be studied in the context of a rich tradition of comparative anatomy, descriptive embryology, histology, and pathology, utilizing a variety of well-established noninvasive (X-rays, magnetic resonance imaging) and invasive (histology, in situ hybridization, immunohistochemistry, electron microscopy) techniques. There is also a wealth of biochemical information about the constituent cells and their extracellular matrices. Finally, a large number of genetic abnormalities affecting skeletal development and homeostasis are available both in mice and humans. This allows genetic analyses of mutant phenotypes, identification of the responsible genes, and direct access to processes that are critical for skeletal morphogenesis. This information, combined with data from studies of phenotypes caused by mutated genes in transgenic mice, can be further exploited in analyses of cellular and biochemical pathways. By selecting phenotypes that are most likely to provide insights into processes of patterning, growth, and homeostasis of cartilage and bone, it is also possible to gain information about the basis for major human diseases such as arthritis and osteoporosis. To date, more than 150 human osteochondrodysplasias and dysostoses have been described. In addition, a large number of murine skeletal abnormalities are also available for study. Among such mutations, my laboratory has studied conditions that have a clear relationship to key questions concerning skeletal development. The questions we believe to be critical are: (1) How is differentiation of mesenchymal cells to chondrocytes and osteoblasts regulated? (2) What controls the spatial pattern of mesenchymal condensations during membranous ossification and formation of cartilage anlagen? (3) How is proliferation, differentiation, and apoptosis of growth plate chondrocytes regulated? (4) How are bone remodeling processes controlled? (5) What regulates the formation of ossification centers in cartilage anlagen? Disorders that involve abnormalities in mesenchymal cell condensation and osteoblast/chondrocyte differentiation include cleidocranial dysplasia (CCD) and synpolydactyly type II (SPD). The two conditions are caused by mutations in the transcription factors CBFA1 and HOXD13, respectively. In both cases, genoand phenocopies are available in mice. Humans and mice with cleidocranial dysplasia are heterozygous for loss-offunction mutations in CBFA1, suggesting that the disorder is the result of haploinsufficiency for the transcription factor. Mice that are homozygous for inactivated CBFA1 alleles develop a skeleton consisting entirely of cartilage with no membranous or endochondral bone and no bone marrow. In heterozygous knock-outs or in patients with CCD, there is deficient ossification of membranous bones in the skull, hypoplasia or aplasia of the clavicle, supernumerary teeth (secondary dentition), and other signs of decreased bone formation. On the basis of these findings and the results of in vitro studies of the genetic regulation of osteoblastic differentiation, it is clear that CBFA1 is essential for osteoblastic differentiation. Studies of clavicular development in CCD mice also demonstrate that chondrocyte differentiation in the clavicle is critically dependent on the level of CBFA1 expression. In addition, chondrocyte hypertrophy is significantly delayed in homozygous CBFA1 knock-out mice. It is likely, therefore, that CBFA1 not only plays a critical role in osteoblastic differentiation, but is important for differentiation and maturation of chondrocytes as well. Dominant mutations causing SPD (central polydactyly and syndactyly in hands and feet) are expansions of a polyalanine stretch in the amino-terminal, non-DNA binding region of HOXD13. In individuals who are homozygous for such expansions, there is a shortening of metacarpals and metatarsals to wristand ankle-like bones and a severe shortening of phalangeal bones, but no polydactyly. The mechanism by which the mutations cause these phenotypes is unlikely to be a simple loss of function of HOXD13, because mice that are homozygous for inactivated HOXD13 alleles have a very subtle limb phenotype, completely different from SPD. Also, patients with mutations causing truncations of HOXD13 and loss of DNA binding regions have very subtle limb abnormalities. In contrast, mice with polyalanine expansions in HOXD13 and mice that are homozygous for HOXD11, HOXD12, and HOXD13 knock-out alleles have SPD-like phenotypes. Therefore, it is likely that expansion of the alanine-stretch in HOXD13 leads to a loss of function of all the three HOXD genes that are expressed in developing hands and feet, HOXD11, HOXD12, and HOXD13. What the molecular mechanism may be is not known, but we favor the hypothesis that the three HOXD proteins require interaction with a common critical factor for full function. If the concentration of this factor is limiting and HOXD13 protein with expanded polyalanine regions bind more tightly to the factor, Address for correspondence and reprints: Bjorn R. Olsen, M.D., Ph.D., Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115. Bone Vol. 25, No. 1 July 1999:77–79