Axolotls are unique among vertebrates in their ability to regenerate tissues, such as limbs, tail and skin. 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 upregulated during regeneration and that TGF-β signaling is necessary for the regenerative process. We show that the basement membrane is not prematurely formed in animals treated with the TGF-β antagonist SB-431542. More importantly, Smad2 and Smad3 are differentially regulated post-translationally during the preparation phase of limb regeneration. Using specific antagonists for Smad2 and Smad3 we demonstrate that Smad2 is responsible for the action of TGF-β during regeneration, whereas Smad3 is not required. Smad2 target genes (Mmp2 and Mmp9) are inhibited in SB-431542-treated limbs, whereas non-canonical TGF-β targets (e.g. Mmp13) are unaffected. 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.
The authors declare no competing or financial interests.
J.-F.D. performed 80% of the experiments and helped design, analyze and interpret the data and co-wrote the manuscript. F.S. helped with some of the animal treatments, protein preparation and RT-PCR experiments and helped with the writing of the manuscript. S.G. cloned Smad2 and Smad3. E.V. helped with protein and RNA preparation, TUNEL assay and animal husbandry. A.P. helped interpret some of the results and her lab provided antibodies for some of the western blots. S.R. designed and supervised the project, helped analyze and interpret the data and co-wrote the manuscript.
This research is supported by a grant from the Canadian Institutes of Health Research [MOP: 111013] to S.R. J.-F.D. is supported by a PhD scholarship award from the Réseau de Recherche en Santé Buccodentaire et Osseuse (RSBO).
Supplementary information available online at http://dev.biologists.org/lookup/doi/10.1242/dev.131466.supplemental
- Received October 2, 2015.
- Accepted August 8, 2016.