Understanding cranial neural crest development is important to human biology because many cancers - such as melanoma and neuroblastoma - develop from neural crest-derived cells. Crest cells are also the targets for a large number of inherited mutations as well as environmental insults that produce cleft lip, cleft palate, craniosynostosis, and many other significant craniofacial malformations. Moreover, cranial neural crest cells also give rise to neuronal and muscle cell lineages and may be helpful in promoting the regeneration of damaged muscles and nerves later in life. For these reasons, we study cranial neural crest cells and their derivatives in vertebrate species such as chicken and mouse, at both the cellular and molecular levels. It has long been thought that the fate of neural crest cells is determined by local signals present in the different embryonic microenvironments to which these cells migrate. Previous research in our laboratory has contributed to identifying the microenvironmental cues that determine the differentiation of neural crest. We described the role of the transcription factor Msx2 as a mediator of bone morphogenetic protein (BMP) signaling in the apoptosis of subpopulations of neural crest cells, an early patterning event in the development of the face. We mapped morphogenetic compartments in the developing mandible, with varied capacity for ectopic cartilage formation, by implanting beads soaked in BMP4. Our research further characterized mechanical forces as environmental signals that promote the differentiation of chondrocytes. We are continuing these avenues of research, and opening new avenues, in order to understand how cellular communication contributes to the formation of skeletal and mineralized tissues. Objective: We seek to determine when, where and how specific subpopulations of rhombomere-derived crest cells produce bone, cartilage and tooth cell lineages. We also seek to isolate those genes that are required to transmit or receive informative signals or instructions essential for morphogenesis. Morphoregulatory molecules influence neural crest cell fates by changing programs of gene expression within discrete cells. To do this, these signals must alter the cell's complement of transcription factors, molecules that in turn control the expression of other genes. It is therefore important to identify the combination of transcription factors that specifically regulate the timing and position of crest-derived cell lineages such as bone, cartilage and tooth. With such knowledge, it then becomes possible to determine how specific combinations of transcription factors are controlled by growth factor-mediated signal transduction (external signals), and what genes they in turn regulate within the cell. Past Year's Results: During the past year, we have made significant findings in understanding the propagation and integration of environmental signals that induce the differentiation of cells of skeletal and mineralized tissues. Our research has focused on two of the main signaling pathways that regulate development of these and other tissues, the fibroblast growth factor (FGF) and the BMP pathways. Mutations that affect elements of these signaling pathways are associated with human craniofacial and/or other skeletal malformations. In order to understand the cellular and molecular events that lead from point mutations in the type 2 fibroblast growth factor receptor (FGFR2) to craniofacial malformations such as midface hypoplasia and craniosynostosis present in Apert Syndrome, we have constructed a transgenic mouse model for this human genetic disorder. Transgenic mice expressing the mutated FGFR2 are small and exhibit craniofacial malformations including fusion of craniofacial sutures, bending or shortening of the maxilla, and exopthalmos. Since several of these abnormalities are found in human Apert patients, we will continue to use these transgenic mice to determine how alterations in the FGFR2 signaling pathways regulate normal and abnormal craniofacial and skeletal development. To identify transcription factors in the BMP pathway we and other scientific groups have taken hints from fruit fly development. Through sequence comparisons, we identified a human expressed sequence tag with homology to the Drosophila dachshund gene, which is regulated by decapentaplegic, the drosophila BMP homologue, and is essential for eye and leg patterning in the fly. We have cloned mouse and human genes called Dach that encode nearly identical proteins that exhibit significant homology to Drosophila dachshund. Dach is transiently expressed in mouse embryos at several sites of early skeletal patterning including the limb buds, rib primordia, and at sites of tooth bud formation in the developing mandible and maxilla. Similar to Pax9, an early marker of tooth mesenchyme, the expression of Dach is induced by FGFs, and repressed by BMP4. The strong expression of Dach in the progress zone of early limb buds and later in interdigital regions, along with the mapping of human DACH to 13q22 suggest that it is a candidate gene for the limb developmental disorder Postaxial Polydactyly, type A2. In order to characterize the molecular mechanisms of chondrogenesis, we have extended our studies of BMP4-induced cartilage in mouse embryonic mandibular process explant cultures. We have found that BMP4 soaked beads implanted into these explants induce the expression of the transcription factors Msx2 and Sox9. These transcription factors appear to act antagonistically to regulate the position or rate of chondrogenesis, as we have demonstrated that Msx2 is an inhibitor of chondrocyte differentiation, and Sox9 is known to promote chondrogenesis. We are testing the effects of dominant negative and constitutively active Msx2 mutants on chondrogenesis. Conclusions and Significance: Patterning and cell fate determination are fundamental processes for all tissues and all organisms. Defects in these processes, due to spontaneous or inherited genetic mutations frequently affect the development of the skeleton and the craniofacial region. Several such mutations have been characterized in genes of the FGF and BMP signaling pathways.
