21-P001 Developmental regulation and tissue patterning by Shh in vertebrate limbs

During limb development, Sonic Hedgehog (Shh) is expressed in the posterior limb bud and is critical for the antero-posterio (AP) patterning of the limb. Gain or loss of Shh function affects patterning of limb skeletal elements, muscle, and tendons. However, it remains unknown whether Shh signalling regulates the patterning and development of these tissues in a cell-autonomous and non-cell-autonomous fashion. To address this question, we used the Cre-loxP recombination technique to remove the Shh positive effector Smoothened (Smo)specifically from limb progenitor cells that are destined to become muscle, skeleton, muscle connective tissue (MCT), or tendon. Analyses of mutant embryos lacking Shh signalling within muscle progenitors suggest that Shh does not directly pattern muscles along the AP axis. However, mutant embryos do not develop hand muscles, indicating that in this context Shh signalling is acting cell-autonomously to regulate hand muscle development. In order to understand the underlying mechanism, we looked at apoptosis, proliferation, and evidence of muscle migration defects. While cell proliferation seems to be normal, there is increased apoptosis at E10.5. Apoptosis is restricted in the dermomyotome and is resolved by E11.5. However, based on several chick experiments, this is unlikely to cause hand muscle loss. Finally, we analysed the expression of Cxcr4, a cytokine previously implicated in muscle migration. Cxcr4 expression is downregulated in the mutants and we are currently examining hand muscles in Cxcr4/ mutants. Microarray analysis is also being planned to find additional Shh targets that play important roles during hand muscle development.