Sonic hedgehog (Shh) is a secreted protein involved in many embryonic developmental processes including ventral patterning of the neural tube and anteroposterior patterning of the limbs. Many lines of evidence suggest that Shh is both necessary for and sufficient to mediate these events by acting in a graded manner. How this apparent graded activity of Shh is achieved in vivo, however, is not clear. Even though recent studies have shown that Shh can diffuse, it is not clear whether long range signaling of Shh is actually required in all tissues for proper patterning. In order to understand the mechanism by which Shh acts in a graded manner, it is necessary to know whether a constant or changing population of cells responds to Shh during development. If cells move relative to the sources of Shh, then an apparent gradient of Shh activity could be achieved based on the length of time cells are exposed to Shh signaling. Therefore, I propose to generate in vivo mammalian model systems to address the mechanism by which Shh signaling is regulated over distance and time to generate proper patterning during development. First, I propose to investigate whether secretion/diffusion of Shh is critical for normal patterning of the neural tube and limbs. Next, I propose to address the possibility that a graded Shh signal is achieved, at least in part, through the temporal integration of Shh activity by cells as they proliferate and move away from a Shh source by mapping the fate of cells that respond to Shh. Lastly, I will determine whether the earliest expression of Shh iii a tissue is required for patterning and whether the length of time of Shh exposure contributes to the graded activity of Shh by generating a conditional mutant allele of Shh in which Shh expression is temporally controlled.
