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

Abstract

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.

Macrophages are required for adult salamander limb regeneration

“…S7). TGF-β regulates various stages of mammalian wound healing and constitutes an important signaling pathway in salamander limb regeneration (25). The lack of the appropriate levels of TGF-β1 activation in macrophage-depleted limbs following amputation may therefore contribute to the failure to regenerate in these animals.…”

“…Inhibition of TGF-β1 signaling blocks successful limb regeneration in the salamander (25), and fibronectin forms part of the provisional matrix during scar-free repair (31) that may contribute to a regeneration permissive environment. The stunted activation of MMP9 and MMP3 on macrophage ablation is consistent with an essential role for matrix remodeling in successful limb regeneration (47) and implicates macrophages as a key regulator of matrix degrading enzymes.…”

Looking proximally and distally: 100 years of limb regeneration and beyond

“…The redirected accumulation of blastema cells under an eccentric AEC may be due to the migration of the cells on repositioned adhesive substrates produced by the AEC. TGF-b1 is strongly upregulated during blastema formation in amputated axolotl limbs (Levesque et al, 2007). A target gene of TGF-b1 is fibronectin, a substrate molecule for cell migration that is highly expressed by basal cells of the wound epidermis during blastema formation (Christensen and Tassava, 2000;Rao et al, 2009).…”

“…A target gene of TGF-b1 is fibronectin, a substrate molecule for cell migration that is highly expressed by basal cells of the wound epidermis during blastema formation (Christensen and Tassava, 2000;Rao et al, 2009). Inhibition of TGF-b1 expression by the inhibitor of SMAD phosphorylation SB-431542 reduces fibronectin expression and results in failure of blastema formation (Levesque et al, 2007), suggesting that fibronectin produced by the AEC provides directional guidance for blastema cells.…”

Identification of miRNA profile and in-silico analysis of identified miRNA’s functions at different stages of Axolotl tail regeneration

“…In siliko analizler, mir-3689'in SMAD2 ve SMAD3 RNA'larını hedefleyerek TGFß yolağı üzerinde etkili olduğunu düşündürmektedir [42] . Aksolotl uzuv rejenerasyonunda TGFß sinyal yolağının aktivitesinin artıyor olması [43] , bulgularımızda mir-3689'in miktarının azalmış olmasının Aksolotl kuyruk rejenerasyonu için önemini açıklamaya yardımcı olmaktadır. SMAD2 ve SMAD3 RNA'larının mir-3689 tarafından regüle edilmeyecek olması ile, rejenerasyon sürecinde TGFß sinyal yolağı aktif tutulacak, böylece yenilenme sürecinin başarıyla sonuçlanması mümkün olacaktır.…”