Abstract
Reactive oxygen species (ROS) were recently identified as a requirement for successful tail regeneration in Xenopus larvae and are present throughout the process of tail regeneration. In this study, the link between ROS and regeneration was investigated. This led to the surprising finding that naturally occurring bacterial flora initiate regeneration in tadpole tails. Tadpole tail regeneration was enhanced by the addition of heat-killed E. coli or lipopolysaccharides (LPS), a component of the outer membrane of Gram-negative bacteria. Reducing bacterial load through the use of the antibiotic, gentamicin, or by inhibiting binding of LPS to toll-like receptor (TLR) 4, greatly reduced tail regeneration. The TLR pathways activate the IκB kinase (IKK) complex and chemical activation of IKK improved regeneration of tadpole tails, suggesting that bacteria initiate regeneration through activation of IKK via TLR pathways.
The transcription factor, NF-κB, is the downstream target of IKK and several NF-κB target genes, including those encoding Nox2, Nox4 and cyclin D1, were upregulated in regenerating hindlimbs during a timeframe spanning blastema formation. The onset of a period in tadpole development where, without TLR stimulation, tadpole tails are refractory for regeneration, corresponded with a reduction in a marker of cell cycling. Transgenic activation of the cell cycle regulator, cyclin D1, improved regeneration in refractory stage tadpoles suggesting that a reduction in cyclin D1 may be responsible for the onset of the refractory period.
Highly localised expression of the gene encoding the protein kinase, Akt1, correspond both spatially and temporally with the loss of regenerative capacity of tadpole limbs during metamorphosis. Conversely, chemical inhibition of Akt impaired tadpole tail regeneration, which is consistent with an important role for Akt in limb regeneration which had previously been recognised. Akt was hypothesised to have both negative (early) and positive (late) roles in regeneration through indirect inhibition or activation of cyclin D1, respectively.
A molecular mechanism for activation and maintenance of regeneration, involving a positive feedback loop between NF-κB, Nox, ROS and cyclin D1, was proposed based on the findings of this and previous studies. From this, a treatment regimen was developed which greatly improved regeneration in late-stage tadpole limbs, and treated mammals also showed signs of regeneration pathway activation. The findings presented here provide reason to believe regeneration may be within our genetic capabilities, but simply requires a little encouragement.