This new RO3 application proposes to test the hypothesis that sonic hedgehog (Shh), an important morphogen, plays a key role in tooth development and may act as an early signaling molecule and a regulator of cytodifferentiation events. Shh is the vertebrate homolog of the Drosophila segment polarity gene, hedgehog, and its roles in early patterning and morphogenesis events during chick and mouse development have been extensively studied. Recent reports from this and other laboratories have described the appearance of Shh during odontogenesis. Shh mRNA appears early at the sites of initiation of dental lamina and then persists in the area of the enamel knot during active morphogenesis. At the cytodifferentiation stage, Shh gene expression remains restricted to young secretory ameloblasts at the cusp tip. Preliminary data from this laboratory also show an inhibition of tooth morphogenesis when day 12.5 embryonic mandibles are treated with antibodies against the N-terminal of Shh.
Aim 1 will characterize and correlate the temporal and spatial patterns of Shh expression at both mRNA and protein levels using gene-specific probes and isoform-specific polyclonal antibodies to the N- and C-terminal fragments of Shh that will be generated in the principal investigator's laboratory.
Aim 2 will use these antibody preparations to block Shh activity in mandibular explant and molar organ cultures to establish that the molecule is required for tooth development.
Aim 3 will determine whether ectopic expression of Shh affects tooth development in vitro. It is predicted that both these experimental manipulations of Shh expression in tooth organ cultures should result in abnormal odontogenesis. Results of these investigations should provide timely and significant information on the role of this potent signaling protein in tooth development.
