Changes in tooth number are associated with orofacial clefting, the most common craniofacial birth defect in humans. Thus, knowledge of the developmental and genetic basis of tooth number regulation is critical for understanding human craniofacial and dental birth defects, as well as for understanding the fundamental process of tooth number specification. Although genetic studies in mice and humans have identified several signaling pathways involved in tooth development, little is known about the developmental and genetic mechanisms regulating tooth number. Most genetic pathways known to control mammalian craniofacial and tooth development are highly conserved and are also involved in craniofacial and tooth development in lower vertebrates, including fish. Genetic variants underlying natural variation can provide valuable insight into developmental processes and complement more traditional genetic studies of induced mutations in model organisms. Here natural variation in tooth number in the threespine stickleback fish (Gasterosteus aculeatus) is proposed as a new model system to learn how genes regulate tooth number. Different stickleback populations adapted to different diets exhibit dramatic changes in tooth number, with freshwater fish having twice the number of teeth as marine fish. The different forms can be crossed in the lab, enabling detailed forward genetic analyses to map factors controlling the changes in tooth number. Genome-wide linkage mapping has identified ten chromosome regions, or quantitative trait loci (QTL), controlling tooth number in sticklebacks, and methods are in place to identify the underlying genes. To test hypotheses about the developmental and molecular genetic basis of the tooth number differences, three specific aims are proposed. First, the developmental time course of the tooth differences seen in marine and freshwater populations will be determined by analyzing skeletal differentiation in embryos, larvae, and juveniles. Second, gene expression correlates of the tooth number differences will be identified by comparing gene expression patterns in developing craniofacial and dental tissues from marine and freshwater fish. Third, the genetic basis of a QTL on stickleback chromosome 21 controlling tooth gains will be identified by mapping, sequencing, gene expression, and transgenic experiments. PUBLIC HEALTH RELEVANCE: This research will provide fundamental knowledge of how genes control tooth number. This knowledge will help efforts to engineer tooth formation in vitro. In addition, since changes in tooth number are associated with common human craniofacial birth defects, this knowledge will lead to a better understanding and prevention of human craniofacial and dental birth defects.