Abstract 17440: Sonic Hedgehog Signaling Regulates Myogenic Stem Cells and Acts Upstream of Wnt Signaling During Limb Regeneration
Appendage regeneration in urodeles such as the newt occurs through formation and maintenance of multipotent progenitors called the blastema. The blastemal progenitors are regulated by the coordinated activity of transcriptional networks and signaling pathways. In the present study, we have investigated the role of sonic hedgehog (Shh) signaling in myogenic stem/progenitor cells during limb regeneration. We show that Shh signaling is essential for the limb regeneration and inhibition of Shh pathway leads to irreversible perturbation of regeneration. Our histological analysis and reversibility experiments suggest that although inhibition of Shh pathway does not affect the dedifferentiation process, it is required for the initial stages of regeneration. Using BrdU incorporation and immunohistochemical analyses, we show that Shh signaling regulates the blastemal cell proliferation, as inhibition of Shh pathway results in a 4-fold reduced labeling of BrdU positive nuclei and results in G0/G1 cell cycle arrest. We further show that limb regeneration is achieved by activation of Pax7+ myogenic stem cells, and Shh signaling inhibition leads to reduced number of Pax7+ cells. Importantly, we find that Shh inhibition leads to retarded migration of myoblast cells and reduced proliferation of the activated myoblasts (MyoD+ cells). Further, we demonstrate using an inhibitor-activator strategy that activation of Wnt signaling could rescue the Shh inhibition. Analysis of cell cycle kinetics reveals that activation of the Wnt pathway in the Shh inhibition background results in increased G2/M populations, concomitant with a 23% increase in BrdU positive cells suggesting that the Wnt pathway is activated later than Shh signaling and modulates cell cycle regulatory networks. Collectively, our results demonstrate the hierarchical regulation of signaling pathways in the regulation of myogenic stem cell populations and limb regeneration.
- © 2011 by American Heart Association, Inc.