Project Summary The long-term goal of this project is to identify the genetic circuitry regulating tooth formation and replacement. As 30 percent of people worldwide over the age of 65 have no natural teeth, understanding how teeth regenerate is a major goal in biology. Furthermore, teeth, like most organs, form through repeated reciprocal signaling between epithelia and mesenchyme. Thus, understanding the genetic basis of tooth formation and replacement is important both for understanding organogenesis in general, as well as for understanding how teeth can be regenerated in vitro and ultimately in vivo. Teeth are homologous to other vertebrate skin appendages including mammalian hair, and shared genes regulate both tooth and hair formation. Although genetic studies in humans, mice and other vertebrates have identified signaling pathways involved in tooth formation, less is known about how genes regulate tooth replacement. In contrast, how genes regulate mammalian hair regeneration is much more understood. One parsimonious hypothesis is that teeth and hair regenerate using similar genetic circuits. Fish retain the ancestral jawed vertebrate condition of constant tooth replacement throughout adult life. Fish also fertilize their offspring externally in large numbers, providing powerful systems for developmental biology and genetic analyses. Threespine stickleback fish (Gasterosteus aculeatus) offer a new and powerful system to learn the genetic basis of tooth formation and replacement. Relative to low-toothed marine ancestors, derived freshwater populations evolve major heritable increases in tooth number and tooth replacement rates. The different forms can be crossed in the lab, enabling detailed and unbiased forward genetic analyses to map factors controlling the changes in tooth number. Genetic and genomic experiments have mapped one genomic region controlling tooth number to a cis-regulatory intronic tooth enhancer of the Bone Morphogenetic Protein 6 (Bmp6) gene in one high-toothed population. Relative to the marine enhancer, the freshwater enhancer displays expanded tooth epithelial expression, and reduced tooth mesenchymal expression, suggesting these spatial shifts in enhancer activity underlie evolved increases in tooth number. Furthermore, in mice, BMP signaling negatively regulates hair regeneration, and in fish BMP signaling negatively regulates tooth replacement. Together these data support the hypothesis of shared genetic circuitry regulating tooth and hair regeneration. To test this hypothesis, three specific aims are proposed. First, transgenic and genome editing experiments will determine which mutations in the freshwater Bmp6 enhancer affect expression differences and tooth number. Second, genome editing experiments will determine Wnt ligand function during tooth formation and replacement. Third, vital dye pulse-chase experiments will test whether tooth replacement is coordinated within a tooth field. Together these aims will reve...