The Craniofacial Development Section pursues the understanding of molecular determinants of skeletal morphogenesis by investigating growth and transcription factor regulation of cartilage patterning, chondrocyte cell fate determination, differentiation and maturation. The development of the skeleton is regulated by interacting signaling pathways composed of extrinsic and intrinsic factors. These factors function in synergistic or antagonistic combinations, and some act as rate-limiting elements to regulate cellular development. An understanding of the mechanisms by which these multiple and diverse pathways interact as networks will contribute to early gene / biomarker-based detection of diseases and disorders that affect cartilage, such as osteoarthritis, and will provide the necessary foundation for prevention and treatment strategies, such as gene therapy and tissue engineering. Previously, using cell, organ and embryo cultures, as well as transgenic animal models, we described the mechanisms by which bone morphogenetic proteins (BMP), fibroblast growth factors (FGF) and epidermal growth factor (EGF) function in combinations to regulate craniofacial cartilage patterning and differentiation. Furthermore, we elucidated that transcription factors such as Sox9 and Msx2 interact to mediate growth factor signaling and specify temporal and positional information for the determination of chondrocytes derived from cranial neural crest cells.Recently, we have extended our molecular analyses and discovered that during cranial neural crest cell specification, sonic hedgehog (Shh) plays a significant role in restoring crest cell phenotype subsequent to an insult from retinoic acid (RA) exposure. Presomitic stage mouse embryos exposed to a teratogenic dose of RA exhibited craniofacial skeletal malformations, reduced expression of Shh in the embryo and abrogation of Sox9 expression in crest cells, suggesting that chondroprogenitors were disrupted. When RA-exposed embryos were supplemented with Shh, the incidence of malformations was reduced, and the pattern and level of Sox9 expression were rescued. Our data suggest that phenotypic and molecular perturbations of crest cells by RA can be reversed by Shh, thus providing a mechanism for embryonic compensation and an additional growth factor regulator of chondrocyte cell fate determination.We have also broadened our studies on skeletal patterning to the developing limb model and discovered that dachshund (Dach), a nuclear factor we previously cloned, is regulated by FGF signaling. Dach is expressed at the progress zone of the developing limb bud and the distal tips of the developing digits, excluded from mesenchymal condensations destined to be cartilage. Preliminary data from bead implantation and promoter reporter assays suggest that FGF signaling contributes to the regulation of Dach expression. Interestingly, Msx2 which has overlapping expression patterns with Dach in the early limb bud also down-regulated Dach promoter activities. These data suggest that several factors co-regulate the expression of Dach which may serve to delineate the zone of chondrogenesis. Limb malformations are common congenital defects in human. We conducted genetic linkage study in a large Turkish kindred with the limb patterning disorder postaxial polydactyly type A (PAP-A) and provided evidence for a subtype of the disorder; PAP-A2, with a locus residing on chromosome 13q21-q32. This region has been shown to be associated with various digital abnormality disorders, and contains at least three PAP-A2 candidate genes that are involved in developmental patterning pathways, one of which is Dach.Dach is also expressed at other sites of epithelial-mesenchymal interactions such as the developing tooth buds, specifically in the early mandibular mesenchyme, the enamel knot, cells of the stratum intermedium, and the inner enamel epithelium. Using protein-soaked bead implantation strategy, we demonstrated that BMP4 inhibited, whereas various FGFs induced Dach expression within the odontogenic field in mandibular explants. Tooth morphogenesis was arrested in cultures with the presence of antisense oligonucleotides directed against Dach. Histological analyses showed a lack of dental mesenchyme, yet an apparent increase in osteoid material. Consistently, tooth specific marker Pax9 was down-regulated while bone specific marker Cbfa1 was up-regulated. These results suggest that Dach also functions to direct dental mesenchymal cell fates. Further, Dach expression is regulated by FGF and BMP signaling in the craniofacial and limb regions. We have continued our studies of chondrocyte differentiation and maturation in the cranial base, as malformations and hypoplasia of the cranial base are associated with several craniofacial syndromes and maybe a cause of craniosynostosis. As a tool for studying the molecular mechanisms regulating cranial base development and endochondral ossification, we have devised an explant culture model system for perinatal development of the cranial base, which include the basioccipital bone and surrounding cartilaginous synchondroses. We have examined the effects of statins which are candidate drugs for regulating bone growth, on cranial base development in this culture system. Lovastatin inhibited chondrocyte maturation, resulting in a decrease in the expression of prehypertrophic and hypertrophic chondrocyte specific genes including alkaline phosphatase and indian hedgehog. Our results suggest that statins may inhibit bone growth or repair that occurs through endochondral ossification. We are currently using this explant system to further dissect the molecular events governing chondrocyte proliferation, maturation and apoptosis in the developing cranial base.Our research focus is to characterize the mechanisms by which genetic and epigenetic factors regulate the developmental programs of chondrogenesis and chondrocyte maturation. The results of these studies are basis to the understanding of multiple growth factor and transcriptional control regulating cartilage development. The goals of our research program in fundamental cartilage biology are targeted towards prevention and intervention of cartilage diseases and disorders.
