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

Abstract

BackgroundAxolotls 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 BMP-2 may be the secondary mediator of sonic hedgehog, although there is mounting evidence to the contrary in mice. Since BMP-2 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 BMP-2 expression was dependent on sonic hedgehog signaling and whether it affects patterning of the autopod during regeneration.ResultsThe expression of BMP-2 and SOX-9 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 BMP-2 and SOX-9. Overexpression of BMP-2 in regenerating limbs caused a loss of digits. Overexpression of Noggin (BMP inhibitor) in regenerating limbs also resulted in a loss of digits. Histological analysis indicated that the loss due to BMP-2 overexpression was the result of increased cell condensation and apoptosis while the loss caused by Noggin was due to a decrease in cell division.ConclusionThe expression of BMP-2 and its target SOX-9 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 BMP-2 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 BMP-2 does not have a direct role in patterning regenerating limbs but may be important to trigger condensation prior to ossification and to mediate apoptosis.

Full regeneration of the tribasal Polypterus fin

“…2D), thereby giving rise to the fin fold that will develop into the endoskeleton and dermal skeleton. Cartilage differentiation is observed in the expected bone formation area as soon as 9 days postamputation (dpa), closely analogous to what is observed during tetrapod limb regeneration (11). Precartilage condensations can be seen in histological slices, and the chondroblasts underneath can be distinguished by their sparse arrangements and the extracellular matrix surrounding them, whereas the apical mesenchymatous cells of the regenerate remain undifferentiated ( Fig.…”

Salamander limb development: Integrating genes, morphology, and fossils

“…Moreover, the expression of Bmp-2 and Sox-9 in urodeles correlates with chondrogenesis and the appearance of skeletal elements as described for other tetrapod taxa. Expression in later stages of limb development are also very similar to other tetrapods despite the absence of a paddle stage, with Bmp-2 expressed in the interdigital tissues where it was shown to be essential for giving anterior-posterior identity to digit primordia of other tetrapod taxa (Dahn and Fallon, 2000;Suzuki et al, 2008;Guimond et al, 2010).…”

“…Recently, Guimond et al (2010) investigated Bmp-2 expression in developing and regenerating limbs of Ambystoma mexicanum. They demonstrated that Bmp-2 and its target Sox-9 are independent of shh signaling in salamanders, corroborating studies on chicks that Bmp-2 is not a direct downstream target of shh involved in anterior-posterior patterning.…”

Macrophages are required for adult salamander limb regeneration

“…S7). Included among markers that were relatively unperturbed were Bmp2, normally associated with differentiation (28), and its downstream target AmTwist, a marker of dermal differentiation (29 Fig. 1.…”