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.

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.

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limbs/fins, thereby stimulating and maintaining the proliferation of blastemal cells and up-regulating genes important for an effective regeneration progress. Some candidate molecules have been suggested to play the role of MGFs, however, the cellular and molecular mechanisms involved in the dependence of innervation during regeneration are still largely unknown, due to limitations to molecular and genetic manipulation in the organisms previously studied.To clarify the role of the nerve fibres in the process of regeneration we are using the adult zebrafish fin as a model system. We have chosen the zebrafish because this organism has several experimental advantages, including amenability to molecular and genetic manipulation. We have surgically denervated the pectoral fin before its amputation and our results confirm the necessity of proper innervation for fin regeneration to occur. This procedure will allow us to describe in more detail the dynamics of nerve dependence and investigate the role of putative MGFs and its targets, by studying their expression and function, using the molecular tools available in zebrafish.Photosensitizers are chromophores that generate reactive oxygen species (ROS) upon light irradiation. Prolonged illumination of photosensitizer-expressing cells results in localized tissue damage caused by accumulation of reactive oxygen species. The aim of this study is to generate a set of transgenics with tissuespecific expression of a novel photosensitizer, KillerRed (KR) and to elicit precise killing in a temporally and spatially controlled manner in the optically translucent zebrafish embryos.For that an enhancer trap screen for tissue-specific expression of membrane-tethered KR was done using Tol2 transposon-mediated transgenesis. KR is a genetically encoded photosensitizer derived from hydrozoan chromoprotein anm2Cp. KR is capable of producing both singlet oxygen and superoxide upon irradiation with green light. Twenty transgenic lines with tissue-specific KR expression were generated. We present our preliminary results of spatially and temporally controlled killing of KR-positive cells in one of these transgenic lines and demonstrate for the first time KR-specific phototoxic effect in living transgenic zebrafish embryos. We are confidant that these KR transgenic lines expressing the red fluorescent protein with inducible phototoxic properties will become useful tools for both developmental biology and regeneration studies. Axolotls (urodele amphibians) have the unique ability, among vertebrates, to perfectly regenerate many parts of their body following injury or amputation. The axolotl limb is the most widely studied structure as an experimental model for tissue regeneration. When amputated, the missing or wounded part is regenerated perfectly without scar formation between the stump and the regenerated structure. On the opposite, mammals heal by fibrosis and one very important family of growth factors implicated in the control of almost all aspects of wound healing is the TGF-b family. ...