With their feather-like gills and serene smiles, axolotls are masters at regeneration. When these salamanders lose an arm or a leg — or when one munches the limb off another — they can grow a new one.

Jessica Whited, a developmental and stem cell biologist at Harvard University studies these vertebrate models to unlock the secrets of regeneration.

“A lot of people are like, ‘well, tell me what genes I need to add to the person so they can regrow a leg.’ Of course, that would be a fantasia,” Whited said. “I'm not saying it's wrong, but it's really simplistic. I like to imagine that it's going to be two parts of the equation. It's not just activating some things, but it's also going to be repressing some other processes.”

By investigating the molecular mechanisms required for axolotl regeneration, Whited hopes that her findings will translate into new treatments for scarring, neurodegeneration, and perhaps even one day, regrowing amputated human limbs.

How did you become interested in studying regeneration?

I trained as a developmental biologist at the Massachusetts Institute of Technology (MIT), and I was really fascinated by how organisms are built and what happens after development. One of my undergraduate degrees was in philosophy, so I've always had a philosophical bent to these kinds of questions. I asked questions like what are the limits to which an animal can replace something later in life? What is the logic behind calling up the developmental program to replace it? Why can some animals do this and other animals cannot, even though they might have all used similar things to build their bodies in the first place? Once I started thinking in those terms, I realized that I was most interested in regeneration. We've known that salamanders can regenerate limbs for well over 200 years, but it's been a really hard question to crack.

Can axolotls regenerate their limbs forever?

Early on, most of the people in the field would have said yes, but there were a few hints that sometimes axolotls can make mistakes. When I first established the axolotl colony in my lab, we received animals from the stock center where they house axolotls together. Axolotls are incredibly cannibalistic, so when we got them, they didn’t have any arms and legs. It opened my eyes to the fact that they actually have limitations to the number of times that they can regrow limbs.

We proved that naive animals grow back limbs almost perfectly after one amputation (1, 2). After two amputations, they all grow the whole length of the limb back, but there can be some minor mistakes in the fingers, for example. But after three amputations, a fraction of them didn't even grow the bud-like structure called the blastema that forms the rest of the limb. And after five amputations, 60% of them couldn't grow a blastema.

There are a couple of possibilities for why that might happen, and I don't think they're mutually exclusive. There could be certain progenitor cells that serve as the building blocks for the new limb that might get exhausted, or there could be an accumulation of nefarious cells like myofibroblasts, which promote scarring in a variety of model systems, including humans.

How does scarring relate to regeneration?

Humans don’t scar up to 24 weeks of gestation, but as we mature, the balance goes from perfect repair to scarring. Humans have this latent ability, but something changes, and there's a lot of hand waving about what changes during fetal development to lead to this outcome.

Other labs have discovered that in mammals, certain subtypes of fibroblasts promote scarring, and those become more numerous as animals age. If you can shift the balance back to the fibroblasts that don't promote scarring, you can get better outcomes. In the salamander world, we're still trying to get to that kind of granularity in our understanding of the different kinds of cells that are there before the injury, how they get activated after the injury, and what they actually do. 

Why do you think humans can’t regenerate their limbs, but axolotls can?

My favorite hypothesis is that humans actually do have all of the same cell types as axolotls, and that these cells start proliferating in response to an amputation. But in a human, they may be in an antagonistic, pro-scarring environment, so they are not given the additional cues to go on and form a blastema.

Axolotls also form a special kind of skin at the amputation site called the wound epidermis, which interacts with the blastema. In the human, that kind of skin doesn't form because the surgeon pulls a skin flap over the amputation site. It's possible that in humans, the relevant cells start getting activated, but they need cues from the wound epidermis to form that blastema. If that is the case, we could somehow recapitulate that in a dressing or matrix embedded in the stump of the amputee, recreating what the salamander does. I don't think this is going to happen in the next five or ten years, but it’s important to understand how a salamander regenerates its limbs to ever regenerate human legs and arms.

Besides full limb regeneration, how can axolotl biology be applied to human health?

We use axolotls to model nerve regeneration because axolotls do it much better than mammals. It’s going to be a little bit easier to translate some of the axolotl factors into human applications in this area, such as into nerve bridges. A surgeon can place a structure that mimics the conductivity of a nerve between the healthy parts of the nerve and the part that's going to degenerate due to a lesion. This nerve bridge can help the nerves crawl past the lesion.

We have a collaboration with a surgeon at Boston Children's Hospital who does nerve transfer surgeries on babies who have a brachial plexus injury, which is a really devastating birth injury. In the process of coming out of the birth canal, stress tears the nerve in the arm. We have a protocol to get tissues from babies during the operation. We receive the nerve that's injured as well as the nerve from elsewhere in the body that surgeons use to repair it. We plan to compare the gene expression in these tissues with what happens during normal peripheral nerve regeneration in a salamander and mice with our mouse collaborators. We’re hoping to get the human data soon to see how it compares as well. Maybe there are some genes in the human that aren't turned on in the injured nerve that are turned on in the salamander. Not only can salamanders perform total limb regeneration, but there are these other things that they can also shed light on.

Even though we're working with the salamander, it's important to me that the work we're doing might one day be developed as a therapy for a human.

This interview has been edited and condensed for clarity.

References

1. Bryant, D.M. et al. Repeated removal of developing limb buds permanently reduces appendage size in the highly-regenerative axolotl. Developmental Biology  424, 1-9 (2017).
2. Bryant, D.M. et al. Identification of regenerative roadblocks via repeat deployment of limb regeneration in axolotls. npj Regen Med  2, 1-15 (2017).