The Craniofacial Development Section conducts research in the pursuit of understanding the molecular determinants of craniofacial and skeletal morphogenesis. The development of the skeleton is regulated by interacting signaling pathways composed of extracellular ligands, ligand binding proteins, membrane bound receptors, intracellular signal transducing molecules, combinations of transcription factors, and target genes. An understanding of the mechanisms by which these multiple and diverse pathways interact as networks that regulate the biological processes of cell proliferation, cell differentiation and apoptosis of cells will contribute to the early detection, management and ultimate prevention of diseases and disorders that affect cartilage, bones and teeth. Signaling pathways mediated by the bone morphogenetic proteins (BMP) and fibroblast growth factors (FGF) are key regulators of normal and abnormal skeletal development as evidenced by the numerous cases of skeletal malformations or disorders such as craniosynostosis that are caused by gene mutations in these complementary pathways. Molecules within these instructive signaling pathways are now implicated to function in the determination of multiple cell lineages of the craniofacial complex and skeletal system. In our previous investigations, using cell, organ and embryo cultures, as well as transgenic animal models, we analyzed the mechanisms by which BMP and FGF-directed signaling regulates craniofacial patterning. In tandem, we discovered that biomechnical forces also serve to modulate these same growth factor-directed signaling pathways. These studies have provided results that serve as a foundation from which a comprehensive molecular understanding of how multiple combinations of growth factor-directed signaling pathways regulate tissue-specific patterning and cell fate determinations required for craniofacial and skeletal cartilage development. Operationally, our research studies are designed to characterize the mechanisms by which epigenetic or extracellular information is transmitted by growth factor-directed signaling pathways into specific target populations of cells. Thereafter, the cell surface language becomes translated through cytoplasmic networks or pathways into another language that functions to confer specificity and sensitivity at the level of nuclear controls of multiple gene expression. We further assume that the language is combinatorial - - - meaning that unique combinations of growth factor directions, cytoplasmic signaling pathways, and nuclear transcription factors serve to regulate the emergence of the cartilage phenotype. These combinations govern the pattern (time and positional information), cell differentiation of the cartilage phenotype, and subsequent maintenance of chondroblasts, chondrocytes and various physiologically significant cartilaginous tissues. Previously, we described the role of the transcription factor Msx2 as a mediator of bone morphogenetic protein (BMP) signaling in the apoptosis of subpopulations of cranial neural crest cells (CNCC), an early patterning event in the development of the craniofacial skeleton. Recently, we characterized Msx2 functions in lineage specification and cell fate determination of surviving CNCC. During the migratory phase of CNCC, some cells co-expressed Msx2 and Sox9. Using gain- and loss-of-function strategies, we showed Msx2 functions as a repressor of chondrogenic differentiation in migratory CNCC. When crest cells reached the first branchial arch and subsequent mandibular process, Msx2 was down-regulated and Sox9-directed chondrogenesis proceeded. We showed that chondrogenesis is regulated combinatorially at the level of clusters of epigenetic growth factors and their cognate receptors at the cell surface and combinations of transcription factors at the level of the nucleus. We also characterized the convergence of BMP and EGF signaling pathways in the regulation of chondrogenesis. EGF inhibited the induction or up regulation of chondrocyte differentiation by BMP signaling. We correlated the antagonistic effects of these factors to the regulation of Smad1 nuclear localization in chondrocytes. Chondrogenesis is regulated by the nuclear accumulation of the transcription factor Smad1, which is in turn regulated by the integration of combinatorial BMP and EGF signaling in the cytoplasm.A number of scientists have postulated that embryonic and fetal cartilage development associated with the cranium, base of the skull, jaw and other skeletal formations significantly influences subsequent bone patterns and articulations during postnatal development. To pursue this hypothesis, we constructed transgenic mice expressing mutant FGFR2 in chondrocytes, which exhibited craniofacial malformations similar to human Apert syndrome. We characterized these mutant mice using techniques of X-ray imaging, skeletal staining, morphometric and histological analysis. We discovered that the dysmorphogenesis of these mice is due in part to defects in the balance of chondrocyte differentiation and proliferation in synchondroses of the bones comprising the cranial base. Through the molecular analyses of the defects in cartilage development in these mice, we intend to characterize the mechanisms by which abnormal FGFR2 signals are integrated with signals from other growth factor mediated pathways to regulate chondrocyte proliferation and differentiation.We have cloned and sequenced mouse and human cDNAs encoding a novel evolutionarily conserved transcription factor called Dach, which may integrate BMP and FGF signaling in multiple developing tissues. Recently we demonstrated that Dach is expressed in the developing teeth, and is co-regulated by BMP and FGF in the mandible. We are testing the hypothesis that mutations in Dach cause a rare malformation in the patterning of the limb skeleton, called postaxial polydactyly A2 (PAP-A2), which maps to the same region of chromosome 13 as the DACH gene (13q21). Since BMP and FGF signaling contribute to the patterning of the skeleton, and mutations affecting these pathways are known to cause limb defects, we believe that further study of the function of the Dach gene will contribute to an understanding of the convergence of BMP and FGF signaling during normal and abnormal skeletal development. We continue to pursue the hypothesis that the qualitative and quantitative combinations of multiple regulatory molecules such as transcription factors and intracellular signaling proteins determine the specificity for embryonic cell lineages and subsequent morphogenesis. In order to test properties of this postulated combinatorial code, we are studying the molecular interactions of the BMP, FGF and EGF pathways and the regulation of their target genes that are specific to craniofacial, dental and skeletal cell phenotypes. We test the combinatorial code by manipulating cell proliferation and cell differentiation in vitro or explant tissue culture, or in animal transgenic models. Progress in this field has already led to novel gene-based diagnostics, and provides the necessary foundation for biomimetic strategies that can result in the improvement of treatment and gene-based therapeutic strategies for acquired and inherited diseases and disorders that affect craniofacial and skeletal tissues.
