Specific Aim:
In this experiment, the role of Shh as a survival factor in avian (chick) embryos will be examined. We will inhibit Shh with cyclopamine during specific stages of development, and examine the corresponding patterns of cell death in the tail bud.

Rational:
This investigation will attempt to determine whether Shh acts as a survival factor or a proliferation factor. To accomplish this, we will be treating chick embryos with cyclopamine at 20 hours, thus blocking Shh signaling, which has been shown to result in tail bud malformations. A Propidium iodide stain specific for dead cells will determine whether the malformations occurred because of the lack of a survival factor, increasing cell death, or a lack of cell proliferation. The results of this experiment will tell us much about the various roles of Shh during the early stages of chick development. If increased cell death is seen, then Shh signaling plays a role as a cell survival factor, the inhibition of which causes increased apoptosis. If massive cell death is not seen, then the cells of the tail bud did not proliferate normally due to blocked Shh signaling, causing malformations in later stages. The findings about the role of Shh may have various implications for Shh signaling and gene expression in other vertebrates including mammals.