Axolotl limb regeneration needs nerves

Some animals can regrow amputated body parts, and axolotls are among them. If they lose a limb, for instance, they can generate a fully functioning new one. Scientists are obviously very interested in how this process plays out, because it might give us clues to why this doesn’t happen naturally in humans, and how we could make it happen. But one of the key questions is what controls the process? How does the replacement body part “know” how big it should become? One key part of this, appears to be an intact connection to the nervous system. Speaking with Chris Smith and from the University of Massachusetts, Catherine McCusker…

Catherine - We work with a really amazing model organism known as a Mexican axolotyl. And this animal is capable of regenerating a variety of different complex structures, one of them being their limbs. Lots of people have been studying limb regeneration, all different aspects of limb regeneration, particularly at the really early stages. But nobody had really looked at the later stages of regeneration and the different steps that happen to make a fully functional limb.

Chris - If you take one of these axolotls and you remove a limb, does it grow a new limb and start with a really small one and make it big, or does it just grow a complete blob of tissue, which then moulds from something about the right size into something resembling a limb?

Catherine - Essentially the regenerating limb goes through multiple steps. So the first step is that it forms a blastema and a blastema is actually really similar to a structure that forms in the developing embryo when the embryo is making a limb. And then that blastema goes through the process of making a limb. But, initially, the limb is very small in size proportionally to the rest of the animal. And then that small limb - that we have called the tiny limb - grows very rapidly compared to the rest of the animal until it reaches the appropriate size. And then it slows down.

Chris - So how does it know how big the animal that's attached to it is?

Catherine - That's really kind of the heart of the question that we were trying to answer figuring out, what that source is that controls the growth of regenerating limb. And, essentially, what we discovered was that signals that are coming from the limb nerves are regulating this growth.

Chris - Oh, right! So the nerves talk to the developing tissue and, in some way, promote it to get the right size? So how do the nerves know what messages to send to the tissue? I mean, do we understand how this works?

Catherine - No, we don't. And that's really the future of what we are trying to answer in the lab, how the nerve knows the limb has been amputated and how it knows to control the growth. And when it knows to stop the growth. These are all really important questions that we need to answer. And that's what we're trying to do right now.

Chris - How did you find that the nerve had this role in the first place?

Catherine - It was kind of a serendipitous accident. When I was a postdoc, I had been doing a histological analysis on limbs that had regenerated a long time ago. And one thing that I noticed was that the limbs seemed to have a lot more innervation than a normal, unamputated limb. And so I kind of filed that in the back of my brain saying, "I think that this might be important some day, but I don't know what it will be important for." And so, when we actually started getting into studying this process of growth regulation and sizing, and during these later stages of regeneration, I remembered those studies that I had done and said, "maybe we should look at the nerves!" And then the more we actually dove into the previous literature on neural signaling and any kind of correlation with size, we said, "wait a minute, I think there might be really something here."

Chris - Do you know whether the nerves are the be-all and end-all, or is there another link in the chain you've yet to discover: do the nerves talk directly to the tissue and that makes the effect; or do the nerves talk to something else and that changes the chemical milieu, which then affects the growth?

Catherine - The studies that we have indicated that the nerve is talking with that regenerating tissue. And we don't know if there's an intermediate there, or if it's directly talking with that tissue. I think, once we start to get at the heart of the molecular mechanism of what signals are coming from the nerve, then we can start to get at kind of those downstream signals. But one thing that we do know is that the nerve must be receiving signals from something upstream. And the reason why we know that is because we developed a new kind of test - or assay - to see whether nerves alone, when they're separated from the central nervous system, can still regulate growth. And actually we were really surprised to find that they don't regulate growth all on their own. They have to be connected to the central nervous system.

Chris - Are they motor nerves, or are they sensory nerves, or both; because, obviously, the pathway that they follow is quite different for those two different classes of neuron?

Catherine - Absolutely. Yeah. So, we are not a hundred percent sure yet. The assay that we developed focused on sensory neurons. So we know that the sensory neurons are not capable of regulating size on their own. So it absolutely is possible that the motor neurons actually do have this information, that regulates scaling and size. We have to test that in the future.

Chris - So how are you going to pursue this? Because, obviously, finding those factors is going to be absolutely critical because, not just for informing this study, but the whole kind of understanding of how tissue regeneration might or might not play out in other animals, how are you gonna try to get underneath what the nerves are doing?

Catherine - We're essentially taking two approaches. The first approach is really using a list of candidates that we found in the literature that we know are regulated by the nerves and maybe have been already correlated with growth at earlier stages of regeneration. But we also want to use some NextGen sequencing to really characterise both the expression patterns that are happening in the nerve, as well as the regenerating tissue and better understand the changes in the molecular signatures that are present in those tissues during different stages of growth.