The Why Files The Why Files --

Growing limbs, healing limbs

Salamander under (microscope) glass

Slice the leg off an axolotl salamander, and it will grow an identical replacement, FedEx fast and free of charge. For decades, studies of limb regeneration in this Mexican marvel have explored the genes and chemicals that stimulate and control regeneration in the limb stump.

"Everything we know about limb regeneration we have learned through decades of research on salamanders," says Randall Dahn, a biologist at the Mt. Desert Island Biological Lab in Maine. "It's taught us a great deal about how the tissues communicate with each other to drive the process of regrowing the limb."

Through a complex signaling system, cells in a structure called the wound epithelium become less specialized. Having reverted to something akin to stem cells, these cells then divide to form specialized muscle, bone, skin and nerve cells in the regenerating limb.

Closeup of front of small pink salamander, small dark eyes, tiny limbs, six pink protruding fins Photo: Malcolm Maden, University of Florida The axolotl salamander can regenerate entire limbs, and is the basis for most studies of limb regrowth in animals.

Regeneration of a salamander forelimb

Black and white, top image of salamander arm, next amputated, next six showing arm regrowth
The amputation on the left was made below the elbow; the amputation on the right cut through the humerus. Both limbs were totally regenerated within 72 days.

Some animals less evolved than the salamander have even more impressive talents for regeneration, says Dahn. If you cut planaria worms into a hundred pieces, each one will grow into an adult worm, no problem. But "humans have a very restricted regeneration capacity" when compared to low-down, creepy-crawlies like planaria or the axolotl salamander, Dahn says. "We can regenerate skin, some of the peripheral nervous system, and parts of the liver."

Don't harm an old arm

But Brett Favre and other aging quarterbacks know that humans cannot regenerate arms or legs...

The stakes in understanding limb generation and regeneration are even higher than football, since a better picture of the process might lead to treatments for many diseases and injuries. Duplicating natural regeneration might make it possible to heal tissues damaged by diabetes, cancer, and the crushing injuries that caused so many amputations after Haiti's earthquake.

The new understanding of the genes and the signaling molecules they make during regeneration reminds us that recycling and repurposing are a major story line in genetics. The entire situation starts to remind us of a mechanic scouring garage sales, who may put useful things to work without changes, or adapt other gadgets for reuse: turning a storm window into a cold frame or an anvil into an anchor.

This recycling process has continued over hundreds of millions of years of evolution, as subtle changes in the genes that control their structures caused to fins to evolve into limbs. In fact, the genes that regulate many or even all appendages are closely related, says Sean Carroll, a geneticist at the Howard Hughes Medical Institute at the University of Wisconsin-Madison. Carroll notes that the "distal-less" genes, which create proteins that regulate other gene's activity have undergone a broad repurposing. "We know they are essential to all drosophila [fruitfly] appendages," including wings and legs, Carroll says.

Because "all these different structures were expressing the same or similar regulatory proteins, we began to understand that limbs are not just limbs, but are an outgrowth, an appendage, in general," Carroll says. "There was something fundamental in common with the processes that made limbs, fins and wings."

The link between fins and limbs, and the ancient roots of the genes for complex limb development, also emerges from a 2007 study of the paddlefish, a primitive fish with bony structures in the fins that look inescapably like bones in hands or feet. The same study showed that the Hox genes, which are involved in tetrapod limb development, are also active in paddlefish.

Fin development = hand development?

Bright blue and brown image of fish from bottom side, adjacent illustration of fin structure
Blue stain shows cartilage in the North American paddlefish. Inset shows the skeleton of the pectoral fin, which is analogous to the upper limb of land vertebrates, or tetrapods. Notice those hand-like bones?

The broader picture

Genes do vary over long stretches of evolutionary time, however, and one way to study limb generation is to examine the subtle variations among genes in different species that can replace lost appendages without visiting the vet.

After beating around the phylogenetic bush, Dahn found some fish can regenerate new fins as needed. Last week, at the John Fallon Symposium at the University of Wisconsin-Madison, Dahn described his ongoing comparisons of gene activity in a skate, a bony-finned fish called bichir, and the axolotl salamander.

Brown flat sea animal with black spots, tail, laying on bottom of sea floor camoflauged
Photo: NOAA
Like its relatives, the sharks, the bones of the little skate are made of cartilage. Unlike the shark, these guys can regenerate fins that are cut off.

"My approach, rather than focus on a single organism, is to take a broad, comparative look at how animals that are separated by hundreds of millions years of evolutionary time regenerate their appendages, and to compare and contrast their strategies," Dahn says.

Dahn is focusing on gene networks that initiate regeneration during the first days after amputation, when adult cells revert to a primitive state in the wound epithelium, and then grow back the fin or the limb.

The key genes are those that kick-start regeneration, he says: "The action is in the initiation."

Stem cell shocker!

Although similar similarities among genes are a major reason to hope that the study of the basic biology will eventually produce treatments for injury and disease, the exact trigger for the process of limb regeneration remains frustratingly obscure. Scientists have long thought that cells in the wound epithelium were reverting to some form of highly versatile ("pluripotent") stem cell.

These cells seemed rather similar to induced pluripotent stem cells, which are like embryonic stem cells that are not made from embryos. A 2009 study1, however, showed that the de-differentiated cells in the wound epithelium are less versatile: a cell derived from the muscle cell is able to divide, but only to make muscle cells, and nothing else.

Ditto for bone cells in the regenerating limb. Only a type of skin cell seems able to produce new cell types, says Malcolm Maden of the University of Florida, one author of the above study in Nature.

Regeneration - why not us?

If some primitive animals can regenerate limbs, why would evolution have stripped such a handy ability from higher organisms? "You would think evolution would favor this talent," Maden says, "but in higher vertebrates, a rapid blood invasion of the wound site produces scarring, which can prevent invasion and infection. That seems a pretty reasonable tradeoff: Instead of incurring all the costs of regenerating the whole structure, you repair the damage site quickly so it does not get infected, but that scarring prevents regeneration."

From a medical standpoint, Maden sees a silver lining in the fact that the cells involved in the salamander's "make-a-leg" trick are not reverting into highly versatile stem cells. "People can regenerate muscle and bone, and if muscle makes muscle, and bone makes bone in the salamander, it is ... not doing something completely different from us, like generating an embryonic stem cell, when it regenerates a limb."

Diagram of four species and limb structure, illustration of species and skeleton of limb or fin
Paddlefish fins show an expression pattern for the Hox gene that was thought to exist only in the developing hands and feet of land vertebrates (tetrapods). Because fossil fish apparently had the genetic toolkit to pattern hands, feet, fingers and toes, studies of fin development may also apply to limb generation. (Looks like we've lost a few "fingers" along the way, however.)

People can already regenerate bone and muscle, but not whole legs or arms, Maden notes. "But if we can exploit the capacities that humans already have, we might be able to do something more surprising than just regenerating a fingertip."

That ability, Maden notes, disappears by age 12.


Terry Devitt, editor; Steve Furay, project assistant; S.V. Medaris, designer/illustrator; David Tenenbaum, feature writer; Amy Toburen, content development executive

©2017, University of Wisconsin, Board of Regents.