BMP-2 functions independently of SHH signaling and triggers cell condensation and apoptosis in regenerating axolotl limbs

<p>Abstract</p> <p>Background</p> <p>Axolotls have the unique ability, among vertebrates, to perfectly regenerate complex body parts, such as limbs, after amputation. In addition, axolotls pattern developing and regenerating autopods from the anterior to posterior axis instead of posterior to anterior like all tetrapods studied to date. Sonic hedgehog is important in establishing this anterior-posterior axis of limbs in all tetrapods including axolotls. Interestingly, its expression is conserved (to the posterior side of limb buds and blastemas) in axolotl limbs as in other tetrapods. It has been suggested that <it>BMP-2 </it>may be the secondary mediator of sonic hedgehog, although there is mounting evidence to the contrary in mice. Since <it>BMP-2 </it>expression is on the anterior portion of developing and regenerating limbs prior to digit patterning, opposite to the expression of sonic hedgehog, we examined whether <it>BMP-2 </it>expression was dependent on sonic hedgehog signaling and whether it affects patterning of the autopod during regeneration.</p> <p>Results</p> <p>The expression of <it>BMP-2 </it>and <it>SOX-9 </it>in developing and regenerating axolotl limbs corresponded to the first digits forming in the anterior portion of the autopods. The inhibition of sonic hedgehog signaling with cyclopamine caused hypomorphic limbs (during development and regeneration) but did not affect the expression of <it>BMP-2 </it>and <it>SOX-9</it>. Overexpression of <it>BMP-2 </it>in regenerating limbs caused a loss of digits. Overexpression of <it>Noggin </it>(<it>BMP </it>inhibitor) in regenerating limbs also resulted in a loss of digits. Histological analysis indicated that the loss due to <it>BMP-2 </it>overexpression was the result of increased cell condensation and apoptosis while the loss caused by <it>Noggin </it>was due to a decrease in cell division.</p> <p>Conclusion</p> <p>The expression of <it>BMP-2 </it>and its target <it>SOX-9 </it>was independent of sonic hedgehog signaling in developing and regenerating limbs. Their expression correlated with chondrogenesis and the appearance of skeletal elements has described in other tetrapods. Overexpression of <it>BMP-2 </it>did not cause the formation of extra digits, which is consistent with the hypothesis that it is not the secondary signal of sonic hedgehog. However, it did cause the formation of hypomorphic limbs as a result of increased cellular condensation and apoptosis. Taken together, these results suggest that <it>BMP-2 </it>does not have a direct role in patterning regenerating limbs but may be important to trigger condensation prior to ossification and to mediate apoptosis.</p

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

II. Le cadre institutionnel et réglementaire de la distribution d'eau en Suède

Persson Kenneth M., Finnson Anders. II. Le cadre institutionnel et réglementaire de la distribution d'eau en Suède. In: Droit et gestion des collectivités territoriales. Tome 30, 2010. Les enjeux de la gestion locale de l'eau. pp. 275-286

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