Activation of Smad2 but not Smad3 is required for mediating TGF-beta signaling during limb regeneration in axolotls

Abstract

Axolotls are unique amongst vertebrates in their ability to regenerate their tissues (e.g. limbs, tail, skin etc.). The axolotl limb is the most studied regenerating structure. The process is well characterized morphologically; however, it is not well understood at the molecular level. We demonstrate that TGF-β1 is highly regulated during regeneration and that its signaling is necessary. The present study clearly shows that the basement membrane is not prematurely formed in animals treated with the TGF-β antagonist SB-431542. More importantly, it shows that Smad2 and Smad3 are differentially regulated post-translationally during the preparation phase of limb regeneration. Using specific antagonists for Smad2 and Smad3, results indicate that Smad2 is responsible for the action of TGF-β during regeneration and that Smad3 is not required. We also show that Smad2 target genes (MMP2 & 9) are inhibited in SB-431542 treated limbs and non-canonical TGF-β targets are not affected (e.g. MMP13). This is the first study to show that Smad2 and Smad3 are differentially regulated during regeneration and places Smad2 at the heart of TGF-β signaling supporting the regenerative process.