20 Dec 2013

Salamanders have different ways of regenerating lost limbs

Regenerating complex tissues is an enviable ability. Salamanders have mastered this skill to perfection, but a recent study shows that two closely related species use different molecular strategies to regenerate their lost limbs.

The remarkable ability to regenerate body parts is fairly common amongst invertebrates. If you chop up a flat worm (planarian) in several bits, they will each grow into a tiny worm (scientists have even been able to grow flat worms from single cells!). When spiders (and some insects) amputate their own limbs because of an injury or as a defence against predators, a new limb identical to the original one will grow back. But for vertebrates like us, it’s a whole different story.

It’s well known that lizards and some other reptiles can regrow broken (or accidently squashed) tails. But the new tail isn’t a perfect replica (it doesn’t have bones or nerves), and lizards can’t regenerate limbs. In vertebrates, this kind of regeneration is unique to salamanders, and to some extent to fish and frog tadpoles (but not adults).

Lizards and geckos can regrow their tails, but the new tail 
isn't a perfect replica, and they can't regenerate limbs.

Salamanders are amphibians that live near lakes or in wetlands, but you may sometimes find them in your house or garden. They can regenerate any limb in all its complexity—with bones, nerves, muscle and skin—and no matter where the limb is amputated it will grow back exactly like the original one. And what’s even more amazing: salamanders can regenerate their limbs (and some organs) over and over again.

It’s not surprising then that salamanders are scientists’ favourite model system to study regeneration. But they come with a heavy baggage. Their genome is huge—about 10 times bigger than the human genome—and it has only recently been sequenced, and not completely. On top of this, genetic tools that insert or remove genes in salamanders are still scarce, especially when compared to other model organisms like fruit flies or mouse. Nonetheless, scientists have come a long way and we now have a good understanding of the basic steps of limb regeneration.

How to grow a new leg
Limb regeneration in salamanders (and frog tadpoles and fish) occurs in three main steps. Let’s say a salamander's leg is amputated. First, a thin layer of skin quickly covers the wound, and this is a crucial difference between salamanders and most other vertebrates, which develop thick scars. Second, this skin sends chemical signals to the cells underneath to instruct them to reverse their identity (bone, muscle, nerve…) to a stem cell-like undifferentiated state. Finally, these 'dedifferentiated' cells multiply and form the blastema—a pool of cells capable of turning into any cell type that will build a new, fully functional leg.

Diagram showing the steps in limb regeneration (credit: Whited and Tabin, Journal of Biology 2009)

The blastema is key for regeneration: if a blastema is grafted anywhere on the salamander’s body, on its back for example, it will grow a leg there. About a decade ago scientists discovered that blastema cells can also originate from ‘resident’ stem cells that hang around in tissues—satellite cells. Since then a question lingers: where do blastema cells come from? From dedifferentiated cells, satellite stem cells or both?

To answer this complex question, one would need to somehow track specific cells (like muscle satellite cells, for example) during blastema formation, which is a challenging thing to do in salamanders. But a collaborative research team from the Max Planck Institute in Dresden, Germany, and the Karolinska Institute in Sweden has now succeeded in doing just that, and what they found was quite unexpected.

"We show that in one of the salamander species, muscle tissue is regenerated from specialised muscle cells that dedifferentiate and forget what type of cell they've been, […] as opposed to the other species, in which the new muscles are created from existing [satellite] stem cells," said senior author of the new Cell Stem Cell study András Simon in a press release.

An ever so cute axolotl posing for the camera.

Simon and colleagues used genetic tricks to label muscle cells with a fluorescent marker in two closely related salamander species (newts and axolotls) and then tracked them under the microscope at different time points during limb regeneration. They showed that in newt, all blastema cells that form muscle tissue come from dedifferentiated muscle cells, while in axolotl they originate exclusively from satellite cells.

“It has always been assumed that in these animals muscle is derived from two sources during limb regeneration: satellite cells and dedifferentiation of myofibers [muscle cells]. The authors are making a radical departure from this idea,” says David Stocum, director of the Indiana University Centre for Regenerative Biology and Medicine and an expert in amphibian regenerative biology.

Limb regeneration in humans: fiction or reality?
The new findings imply that different species, even closely related ones, may have evolved slightly different ways to regenerate limbs “even though the process at an anatomical and histological level may look the same”, notes Stocum. But it remains to be understood why, and also whether this is the case for other vertebrate species. “It would be interesting to explore how similar are the mechanisms of muscle cell formation, overall blastema formation, and mechanisms of blastema development in different species” Stocum says “We might find some surprises there.”

So will it ever be possible to regenerate limbs in humans?

Frog tadpoles can regrow their limb buds but they lose this regenerative ability in adulthood, which means that the genes controlling regeneration must be shut down sometime during metamorphosis. So in theory it should be possible to trigger regeneration in frog adult limbs if we knew what’s blocking it (and we could then block that), and the same could be true for humans.

“I’m optimistic that it will eventually be possible, but how long it will take is anyone’s guess. […] Clearly, if species on this planet that have the capacity for appendage regeneration exist, understanding how they do it is a huge step forward in determining what is needed to make it happen in mammals, including humans,” Stocum says.

But keep in mind: you're not a salamander. If you were to cut your own leg off (don’t!), a thick layer of skin would close the wound and form a scar, and this would prevent regeneration (your leg would NOT grow back). Interestingly, when scientists grafted extra skin to a salamander wound after limb amputation, or when scaring was induced with genetic tricks, the limb didn’t grow back. Just as in humans.

Sandoval-Guzmán T., Wang H., Khattak S., Schuez M., Roensch K., Nacu E., Tazaki A., Joven A., Tanaka E. & Simon A. & (2013). Fundamental Differences in Dedifferentiation and Stem Cell Recruitment during Skeletal Muscle Regeneration in Two Salamander Species, Cell Stem Cell, DOI:

A shorter version of this article was originally published in Lab Times on the 10-12-2013. You can read it here.

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