Sonic Hedgehog (Shh) is a secreted molecule with essential roles in several developmental contexts, and inappropriate activation of the Shh pathway is well documented in human cancers such as basal cell carcinoma (BCC). The developing vertebrate limb is an excellent model for addressing fundamental questions about Shh function. During embryogenesis, Shh is required for proper limb patterning and is involved in the determination of digit identity. While a spatial gradient of Shh is thought to be primarily responsible for digit patterning, more recent studies, in which the fate of descendants of Shh expressing cells were followed, have suggested that the length of time a cell expresses Shh (or temporal gradient) may also be important for determining the most posterior digit identities. Based on this and others' work, it was proposed that patterning across the anterior-posterior (AP) axis of the hand is orchestrated both by differences in concentration of Shh and in the duration of Shh exposure. While some downstream targets of Shh signaling have been identified, it is unknown how different thresholds of Shh activity coordinate limb AP patterning and are molecularly interpreted. This temporal regulation by Shh has only been documented in the developmental context of the limb and has been completely unexplored in human cancers. We hypothesize that specific levels or duration of Shh pathway activation are involved in the determination of AP digit identity and have a role in cancer formation or progression. To address this, we first will create murine immortalized limb bud-derived cell lines and characterize them to ensure their limb identity is retained. To standardize our assay with different concentrations and times of Shh exposure, we will examine the induction of expression of genes known to be direct targets of Shh signaling and others that are known to be sensitive to different durations of Shh. We will use these conditions to perform expression profiling to discover new genes regulated by different thresholds of Shh signaling. These genes will be validated and characterized in mouse and chick embryos to determine their role in digit identity. Also, we will examine the expression of these genes in mouse BCC tumors and compare it to normal skin to ascertain whether their expression is associated with cancer formation. While the role of Shh concentration has been previously studied during development, the effect of duration of Shh exposure has not yet been examined at a genome-wide level. The discovery of novel genes that are differentially regulated by specific thresholds of Shh signaling will serve to expand our understanding of this pathway in the developmental context of the limb, and also provide a framework with which to address possible Shh targets involved in cancer formation. ? ? ?
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