The fact that Sonic hedgehog is the active agent of the ZPA was confirmed by a remarkable gain-of-function mutation. The hemimelic extra-toes (hx) mutant of mice has extra digits on the thumb side of the paws (Figure 1A and 1B). This phenotype is associated with a single base-pair difference in the limb-specific Shh enhancer, a highly conserved region located a long distance (about 1 million base pairs) upstream from the Shh gene itself (Lettice et al. 2003; Sagai et al. 2005). Maas and Fallon (2005) made reporter constructs by fusing a β-galactosidase gene to this long-range limb enhancer region from both wild-type and hx-mutant genes. They injected these reporter constructs into the pronuclei of newly fertilized mouse eggs to obtain transgenic embryos. In the transgenic mouse embryos carrying the reporter gene with wild-type limb enhancer, staining for β-galactosidase activity revealed a single patch of expression in the posterior mesoderm of each limb bud (i.e., in the ZPA; Figure 1C). However, mice carrying the mutant hx reporter construct showed β-galactosidase activity in both the anterior and posterior regions of the limb bud (Figure 1D). It thus appears that (1) this Shh enhancer has both positive and negative functions, and (2) in the anterior region of the limb bud, some inhibitory factor represses the ability of this enhancer to activate Shh transcription. The inhibitor probably cannot bind to the mutated enhancer; thus, in hx mutant mice, Shh is expressed in both the anterior and posterior regions of the limb bud, and this anterior Shh expression causes extra digits to develop. Similar mutations in the long-range limb enhancer of Shh produce polydactylous phenotypes in humans and other mammals (Figure 1E and 1F; Gurnett et al. 2007; Lettice et al. 2008; Sun et al. 2008).
Further Development 19.5: From Humans to Cats: A Natural Gain of Shh Function: The Extra Toes Mutation
Printed from , all rights reserved. © Oxford University Press, 2022