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New Insight Into Regeneration

While humans can't regenerate limbs and other body parts like some animals can, scientists hope that, by understanding how amphibians like frogs regenerate their body parts, they can develop therapeutic approaches for people. In a recent report, researchers revealed the molecular events behind frog tail regeneration.

Scientists have known for years that mild electrical currents can enhance regeneration — from amphibian limbs, to severed guinea pig spinal cords, to nerves in rats. However, why electric fields had this effect, and the molecular source of these fields, were a mystery. Dr. Michael Levin and his colleagues at the Forsyth Institute's Center for Regenerative and Developmental Biology have been exploring the role of electric currents in tail regeneration in the African clawed frog, Xenopus laevis. Xenopus tadpoles are excellent for such studies because they are amenable to many laboratory techniques and can completely regenerate their tails, restoring their nerves, muscle, skin and blood vessels.

Levin's team, with funding from NIH, had previously identified a protein called V-ATPase that's involved in guiding growth patterns during frog embryo development. V-ATPase maintains voltage gradients across cell membranes by pumping charged hydrogen ions (H+) across the membranes. The team set out to explore whether V-ATPase also plays a role in regeneration. Their work was supported by the U.S. Department of Transportation and NIH.

In a paper published online on February 28, 2007, in the scientific journal Development, they report the discovery that a chemical called concanamycin, which specifically inhibits V-ATPase, blocked tail regeneration without being toxic or causing other abnormalities. Tadpoles, the researchers found, produced more V-ATPase in the cells around their wound within six hours of having their tails amputated. When tadpoles were induced to make a defective version of V-ATPase, however, their tails didn't regenerate.

To confirm that membrane voltage differences drive regeneration, the researchers added a cell-surface H+ pump from yeast that isn't sensitive to concanamycin and then treated the amputated tadpoles with the chemical. They found that, while V-ATPase was shut down, the yeast H+ pump could rescue regeneration by once more pumping H+ ions across the cell membrane. More than twice as many tails regenerated to some degree with the yeast pump added (36%, compared with 15% of controls) and, of the ones that regenerated, those with the yeast pump regenerated to a much greater degree, with 18% showing good or perfect regeneration, compared with 5% of controls.

The researchers were also able to visualize the membrane voltage changes during their manipulations using a dye. They showed that the electrical changes orchestrated by V-ATPase around the wound site guided the subsequent path of regeneration, affecting cell proliferation, gene expression and nerve growth patterns. Since electric currents can affect many different types of regeneration, this insight into tail regeneration in Xenopus may be an early step toward developing therapeutic strategies for people.

— by Harrison Wein, Ph.D.