Role of Sonic hedgehog Signaling during Regeneration of Muscle and Neural Tissue in "Xenopus laevis"

Sonic hedgehog (Shh) is a signaling molecule that plays a key role in the embryonic development of the nervous system. This protein is secreted from ventral structures to the developing spinal cord, and regulates the differentiation of ventral spinal phenotypes. The action of Shh on skeletal muscle development remains unclear. Xenopus tadpoles exhibit a remarkable capacity to regenerate the amputated tail offering an ideal model for the study of muscle and spinal cord regeneration. Here we tested the hypothesis that Shh signaling is important for muscle and spinal cord regeneration upon injury. Under anesthesia, we amputated tails from stage 37-39 (53-56 h post fertilization) Xenopus laevis tadpoles and incubated them in control saline or cyclopamine, a Smoothened, Shh co-receptor, antagonist, for 24, 48 and 72 h. Samples were then fixed and processed for whole-mount immunostaining in order to evaluate the extent of muscle and spinal cord regeneration in control and experimental groups.  Confocal scanning of stained samples followed by Image J particle analysis reveals that blocking Shh signaling increases the area of regenerated muscle as early as 24 h post amputation. Analysis of the effect of Shh signaling on the regenerating spinal cord is underway. Because blocking Shh signaling leads to faster and more robust regeneration of the muscle, we will test whether enhancing Shh signaling inhibits muscle regeneration by incubating tail-amputated tadpoles with SAG, Smoothened agonist. By better understanding natural muscle and neural tissue regeneration, new therapeutic approaches can be developed to help those with neural and muscle injuries.