The Role of the Collagen Receptor, Discoidin Domain Receptor 2 (DDR2) in Craniofacial Development

Abstract

Defects in cranial growth and development cause a wide range of disorders known to dramatically impact physical, social, and emotional development of affected children. An understanding of the critical molecules and pathways necessary for craniofacial development is necessary to successfully treat these patients. Development of craniofacial bones requires interactions between progenitor cells and the collagen-rich extracellular matrix. Cell-matrix interactions are critical for normal proliferation, differentiation, migration and remodeling, and required to maintain the shapes of individual bones and their relative proportions (reviewed in Chapter 1). DDR2 is a receptor tyrosine kinase that is activated by triple helical collagens abundant in the bone extracellular matrix (ECM). Inactivating mutations in DDR2 cause spondylo-meta-epiphyseal dysplasia, short limbs and abnormal calcification, a rare, autosomal inherited human disorder associated with distinct craniofacial defects including prominent forehead, wide open fontanels, hypertelorism, a short nose with a depressed nasal bridge, a long philtrum, micrognathia, and abnormal teeth. Ddr2-deficient mice also exhibited craniofacial abnormalities including eye protrusion, short snout, and impaired intramembranous ossification associated with delayed suture formation. However, the specific functions of DDR2 in craniofacial development have not been previously described. In Chapter 2, a detailed characterization of Ddr2-knockout skulls identified growth defects in calvarial and cranial base bones that were most dramatic in the anterior part of skull. Ddr2-deficient mice also exhibited features of delayed endochondral ossification at the cranial base due to chondrocyte disorganization, deficient chondrocyte proliferation, and abnormalities in cartilage ECM distribution and turnover. As a perquisite for understanding its function, the distribution of Ddr2 expression was examined using a Ddr2-LacZ knock-in mouse model. Using this approach, potential cellular sites of DDR2 action were identified in cranial sutures, periosteum, dura mater, and resting and proliferative chondrocytes of cranial base synchondroses, regions enriched in skeletal stem cells. However, Ddr2-LacZ expression could not be detected in terminally differentiated cells, such as hypertrophic chondrocytes and osteocytes, suggesting a potential role in stem/progenitor cell function. Intriguingly, our experiments showed that Ddr2 and Gli1, a mediator of hedgehog signaling, are in overlapping cell populations. Gli1 is associated with sutural stem cells and mesenchymal metaphyseal osteoprogenitors in long bones. In support of our hypothesis that Ddr2 functions in skeletal stem/progenitor cells, genetic labeling of Ddr2-expressing cells using Ddr2Mer-icre-Mer mice bred to Ai14 tdTomato reporter mice revealed the contribution of Ddr2-expressing cells to chondrocyte, osteoblast, osteocyte, and marrow-lining cell lineages. Additional experiments involving tissue-specific knockout approaches established a critical function of Ddr2 in skeletal progenitors cells (Gli1- and Col2-expressing cells) to control chondrocyte and osteoblast differentiation during postnatal craniofacial bone formation. Collagen-mediated signaling is also critical for tissue differentiation and mineralization in tooth dentin and alveolar bone. In Chapter 3, in vivo and in vitro studies demonstrated the requirement for DDR2 signaling in tooth root development, periodontal ligament (PDL) integrity, and alveolar bone maintenance. Furthermore, Ddr2-LacZ expression identified Ddr2 in dental follicle and dental papilla in developing tooth and in dentin-forming odontoblasts and PDL cells in mature teeth. Additional studies revealed DDR2 regulation of RUNX2 phosphorylation as a potential mechanism for tooth root development. Lastly, Chapter 4 summarizes the main findings of this dissertation and suggests future directions for this research. Together, this dissertation advances our understanding of the roles of cell-matrix interactions in craniofacial development by establishing the functions of a new collagen receptor.