Activation of Smad2 but not Smad3 is required to mediate TGF-β signaling during axolotl limb regeneration

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

Transforming Growth Factor: β Signaling Is Essential for Limb Regeneration in Axolotls

Axolotls (urodele amphibians) have the unique ability, among vertebrates, to perfectly regenerate many parts of their body including limbs, tail, jaw and spinal cord following injury or amputation. The axolotl limb is the most widely used structure as an experimental model to study tissue regeneration. The process is well characterized, requiring multiple cellular and molecular mechanisms. The preparation phase represents the first part of the regeneration process which includes wound healing, cellular migration, dedifferentiation and proliferation. The redevelopment phase represents the second part when dedifferentiated cells stop proliferating and redifferentiate to give rise to all missing structures. In the axolotl, when a limb is amputated, the missing or wounded part is regenerated perfectly without scar formation between the stump and the regenerated structure. Multiple authors have recently highlighted the similarities between the early phases of mammalian wound healing and urodele limb regeneration. In mammals, one very important family of growth factors implicated in the control of almost all aspects of wound healing is the transforming growth factor-beta family (TGF-β). In the present study, the full length sequence of the axolotl TGF-β1 cDNA was isolated. The spatio-temporal expression pattern of TGF-β1 in regenerating limbs shows that this gene is up-regulated during the preparation phase of regeneration. Our results also demonstrate the presence of multiple components of the TGF-β signaling machinery in axolotl cells. By using a specific pharmacological inhibitor of TGF-β type I receptor, SB-431542, we show that TGF-β signaling is required for axolotl limb regeneration. Treatment of regenerating limbs with SB-431542 reveals that cellular proliferation during limb regeneration as well as the expression of genes directly dependent on TGF-β signaling are down-regulated. These data directly implicate TGF-β signaling in the initiation and control of the regeneration process in axolotls

La guerre d'Italie, ou Mémoires du Comte D*** . Contenant quantité de choses particulières & secrètes, qui se sont passées dans les cours d'Allemagne, de France, d'Espagne, de Savoie & d'Italie. Quatrième édition, augmentée des événemens de cette guerre jusques à présent. Tome premier [-second]

Appartient à l’ensemble documentaire : RhoneAlp

Histological analysis of regenerating limbs treated with SB-431542.

<p>Control limbs were treated with DMSO from time of amputation and fixed A) 45 minutes, C) 2 hours, E) 6 hours, G) 48 hours, I) 72 hours and K) 7 days after amputation. Samples treated with 25 µM SB-431542 from time of amputation were fixed at B) 45 minutes, D) 2 hours, F) 6 hours, H) 48 hours, J) 72 hours and L) 7 days after amputation. Masson's trichrome staining method was used to stain cell cytoplasm in red, collagen in blue and nuclei in black. Note the delayed closure of the wound epithelium in SB-431542 treated limbs at 45 minutes and 2 hours post-amputation. Also note that there is no blastema formation or accumulation of cells between the tip of the bone and the wound epithelium in SB-431542 treated limbs.</p

Primers used in RT-PCR reactions

<p>Primers used in RT-PCR reactions</p

Inhibition of limb regeneration by SB-431542.

<p>A) Morphology of axolotl regenerating forelimbs treated with SB-431542. Top row: morphology of a regenerating control limb treated with DMSO. All limbs were distally amputated on the same day. Limbs were photographed when control limbs reached each of the stages indicated on top: early bud (EB), late bud (LB), early differentiation (ED). Complete: regeneration of control limb ended after 30 days. Middle row: morphology of an axolotl limb treated with 25 µM SB-431542 from the time of amputation until the control had regenerated (30 days). Complete inhibition of regeneration and absence of blastema formation were observed in these limbs. Bottom row: morphology of an axolotl limb treated with 25 µM SB-431542 from early bud stage until the control had regenerated. Growth of the blastema was observed in these limbs until it resembled a late bud blastema. B) Inhibition of regeneration with SB-431542 cannot be rescued after 7 or 14 days of treatment. In this panel, limbs were treated with DMSO or SB-431542 from the moment of amputation for the first 7 or 14 days only. Results show that limbs treated with SB-431542 for the first 7 or 14 days after amputation do not regenerate even if treatment was stopped. Control limbs treated for 7 and 14 days with DMSO regenerated normally.</p

Axolotl TGF-β1 protein sequence and domains.

<p>Alignment of axolotl (<i>Ambystoma mexicanum</i>) TGF-β1 amino acid sequence with TGF-β1 sequences of human (<i>Homo sapiens</i>), mouse (<i>Mus musculus</i>) and TGF-β5 of Xenopus (<i>Xenopus laevis</i>). In blue are residues of the axolotl sequence that are conserved in the other three sequences. In red are the 9 conserved cysteine residues found in every TGF-β1 sequence. The red box identifies the pro-domain of the protein. The green box identifies the mature TGF-β domain of the protein.</p

Inhibition of TGF-β1 target genes expression in regenerating limbs treated with SB-431542.

<p>A) RT-PCR showing expression of fibronectin and Runx 2 in axolotl regenerating forelimbs. RT-PCR reactions were performed on at least 4 separate RNA samples extracted from pools of 6 blastemas of animals treated with DMSO or SB-431542. Fibronectin and Runx 2 were strongly expressed in control limbs and significantly down-regulated in limbs treated with SB-431542. GAPDH was used as a control. B) Graph representing the relative value of fibronectin/GAPDH and Runx 2/GAPDH expression in control and SB-431542 treated limbs. Fibronectin and Runx 2 relative expression in control limbs were fixed to 1±0.37 and 1±0.09 respectively. The relative expression values in SB-431542 treated limbs were 0.29±0.23 with a p<0.05 (*) for fibronectin and 0.07±0.01 with a p<0.001 (***) for Runx 2.</p

Inhibition of cellular proliferation in regenerating limbs treated with SB-431542.

<p>A) Control regenerating limb treated with DMSO and assessed for BrdU incorporation at 7 days post-amputation (medium bud stage). Red arrowhead marks a cell positive for BrdU. Note the accumulation of BrdU-positive cells in the regenerating blastema. Cells positive for BrdU are also found (at a lower frequency) in the epidermis of the non-regenerating part of the limb. B) SB-431542 treated limb assessed for BrdU incorporation 7 days post-amputation. No accumulation of BrdU-positive cells at the tip of the limb is observed. Only a few positive cells are found mostly in the epidermis of the limb (red arrowhead). Dotted lines in panels A and B represent the level of amputation. C) Graph comparing percentage of BrdU-positive cells in the regenerating blastema of control limbs (n = 3 animals) and in SB-431542 treated limbs (n = 3 animals). A statistically significant difference in the percentage of BrdU positive cells between control (38%±6.2%) and SB-431542 treated limbs (7%±2.1%) was observed (*** p<0.001).</p