The role of descendants of <i>Sonic hedgehog</i>-expressing cells in limb development
Sonic hedgehog (Shh) plays an important role in patterning the limb, but much is unknown about its mechanism of action. In this thesis, I use a recombinase-based fate mapping technique to fate map descendants of Shh-expressing cells in the mouse limb. The results suggest new mechanisms for Shh action in anterior-posterior patterning of the limb and the regulation of limb outgrowth. Descendants of Shh-expressing cells expand across the posterior of the limb, giving rise to digits III–V and part of the ulna. As the descendants expand anteriorly, they stop expressing Shh, so that only the most posterior cells express it. This expansion of Shh descendants does not shape the spatial gradient of Shh, but causes a temporal gradient of Shh activity. Posterior digits express and see Shh longer than anterior digits. Gain and loss of function experiments in the chick limb also support the idea that the longer cells are exposed to Shh, the more posterior the tissue they will form. In the mouse, conditional knockouts show that prolonged exposure to low levels of Shh is enough to ensure the patterning of digits IV and V, whereas higher levels of Shh for shorter amounts of time are unable to pattern the posterior digits.
The expansion of Shh descendants also indirectly regulates limb outgrowth, by leading to the shutting down of an Shh-Gremlin-Fgf feedback loop. By misexpressing Shh, Fgf4, and Gremlin after they normally turn off, I find that the loop breaks down between Shh and Gremlin. Further, a gap opens between the expression domains of Shh and Gremlin because Gremlin is not expressed in Shh descendants. Through tissue transplants, I show that this repression is cell autonomous. Thus the expansion of Shh descendants creates a physical barrier between cells that express Gremlin and posterior signals necessary for its upregulation. If this physical barrier is removed, the loop continues until it recreates the barrier and correctly regulates limb size. If the loop is disconnected from this inherent braking mechanism by viral misexpression of Gremlin, there is overgrowth of the digits. The expansion of Shh descendants serves as a mechanism of limb size control.
0307: Molecular biology