Molecular biologist Ken Muneoka, who studies limb development and regeneration at Tulane University, in New Orleans, is exploring how a digit loss that can be repaired differs from one that can’t. Working with mice, his team has discovered that cells release bone morphogenetic proteins (BMPs) at regeneration-capable amputation sites but not at sites that fail to regenerate. The researchers also determined that treating a normally nonregenerative amputation site with these growth factors enables the wound to grow a replacement digit. And they found that mice in different stages of development—newborn versus embryonic, for example—utilize different BMPs to promote regeneration (Development 2010, 137, 551). BMP production is turned on by MSX genes, whose expression increases during regeneration of mouse digits, as well as during regeneration of tadpole tails and zebrafish fins, according to Muneoka.
It’s still a mystery why some animals can regenerate and others can’t, Brockes adds. Researchers don’t even know whether salamanders are unique among vertebrates in having evolved their extensive regenerative ability, or whether ancestral animals possessed the ability but salamanders are unique in retaining it.
Most scientists favor this second hypothesis, Brockes says. They think that once they identify the factors that stop most mammals from regenerating, such as inflammation caused by immune cells or scar formation caused by connective tissue during wound healing, they can remove that block. In that way, they can access “some ancestral property, some ‘newt within,’ that’s just waiting to be unlocked,” and reactivate regenerative capabilities, he says.
But regeneration involves the same signaling pathways, transcription factors, and other biochemical machinery used in development, tissue turnover, wound healing, and other activities common to all vertebrates, Brockes notes. He believes that the salamander, unlike vertebrates that are incapable of regeneration, underwent evolutionary changes that modified these common pathways “to obtain a regenerative outcome” (Integr. Comp. Biol., DOI: 10.1093/icb/icq022).
Unlike nonregenerative organisms, salamanders that lose a limb maintain “a very strong ‘current of injury’ for weeks at the wound edge” while the new limb grows, Levin explains. The current is created by the movement of sodium, chloride, and potassium ions through the wound epithelium. Researchers have demonstrated that applying a particular electric field to an amputated stump allows normally nonregenerative animals such as adult frogs to start regrowing limbs, he says.
Levin’s team used a different, molecular genetics technique to regenerate tadpole tails—complex appendages that contain muscles and spinal cord. First, the researchers determined that regeneration in tadpoles requires the expression of V-ATPase, an enzyme that pumps protons out of cells at the amputation site. This pumping action alters membrane voltage at the wound and also creates a long-range electric field that promotes nerve growth into the site, Levin says.
Next, the researchers turned to tadpoles that had reached a stage of development when they are normally unable to regrow a tail. Levin’s group showed that V-ATPase is ineffective during this phase. In a striking experiment, the researchers nevertheless induced these tadpoles to regenerate complete tails by inserting proton pumps from yeast into the tadpoles’ cell membranes (Development 2007, 134, 1323). The work showed that “any convenient electrogenic protein can be used for regenerative medicine approaches, not just the ones that are natively expressed in the host,” Levin says.
Levin and Kaplan’s teams are drawing on these previous findings as they work out the optimal conditions for regrowing an amputated rat limb with the help of their new regenerative sleeve. After attaching the device to the stump of a rat’s limb, the researchers hope to create a regenerative current at the stump’s surface by adjusting the ionic composition of the solution inside the sleeve and by adding drugs that open or close ion channels in the membranes of the cells at the wound site.
The sleeve will offer some additional benefits. The aqueous environment it provides will prevent the scarring that normally develops in a mammalian wound exposed to air. The researchers might also use it to bathe the wound with scar-reducing compounds, immune-modulating drugs, and more traditional growth factors, Levin says.