'Behind the Blue': Randal Voss, Jeramiah Smith Discuss Axolotl Salamanders and the Keys to Human Regeneration
Regeneration is one of the most enticing areas of biological research. How are some animals able to regrow body parts? Is it possible that humans could do the same? If scientists could unlock the secrets that confer those animals with this remarkable ability, the knowledge could have profound significance in clinical practice down the road.
Scientists at the University of Kentucky have taken this fantasy one step closer to reality, recently announcing that they have assembled the genome of the axolotl, a salamander whose only native habitat is a lake near Mexico City.
On this week’s episode of “Behind the Blue,” UKPR’s Amy Timoney talks with Randal Voss, a professor in the UK Spinal Cord and Brain Injury Research Center, and Jeramiah Smith, an associate professor in the UK Department of Biology. Organisms with larger genomes than humans have been largely impossible to map, due to the remarkable computational burden posed, but Voss and Smith adapted a classical genetic approach called linkage mapping to put the axolotl genome together in the correct order quickly and efficiently – the first genome of this size to be assembled to date.
As proof of concept, Voss and Smith used the assembled data to rapidly identify a gene that causes a heart defect in an axolotl, thus providing a new model of human disease.
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[BTB intro music]
RANDAL VOSS: I'm Randal Voss, R-A-N-D-A-L, one L, V-O-S-S. I'm a professor in the Department of Neuroscience in the spinal cord and brain injury research center here at the University of Kentucky.
JERAMIAH SMITH: Hi, I'm Jeremiah Smith, J-E-R-A-M-I-A-H S-M-I-T-H. I'm an associate professor in the Department of Biology here at the University of Kentucky.
INTERVIEWER: OK, awesome. So to start things off, and we'll start off with Dr. Smith, tell us a little bit about what you and your team have accomplished.
JERAMIAH SMITH: So what sort of this paper that we're talking about marks is sort of the achievement of a new level of the assembly for axolotl. So we and other groups have been trying for several years to figure out what the structure of the genome looks like. And now we're to the point where most of the sequences that we know about we know them. We know what genes are there. And we know where they are on their chromosomes. So effectively we've been able to string together the full length of all of the chromosomes of axolotl.
INTERVIEWER: Awesome. So Dr. Voss, do you have anything that you would like to add to that?
RANDAL VOSS: No, there was just a great accomplishment for not only us but for the community. Because what this new genome assembly does is it allows us to really study the functions of genes for the first time. We have complete knowledge now of what those genes are. And now we can start to look at the DNA regions that flank these regions to understand how the genes themselves are regulated.
INTERVIEWER: Great. And we should note that there could be some sirens in this because we are next a trauma center, so please bear with us for that. So while we have you here, Dr. Voss, tell us what is an axolotl?
RANDAL VOSS: OK, so an axolotl is the world's greatest salamander. So axolotls are originally from Mexico. They were collected in about 1863 and they were shipped to Europe first, before in the 1900s they were shipped back to the United States. And then over the course of decades a laboratory population was developed. And that population came to the University of Kentucky in 2005.
And so here at the University of Kentucky we have the only kind of federally funded-- this axolotl colony. It's funded by the Office of Research Infrastructure Programs at the National Institutes of Health. And what we do with this population is we make offspring and we grow them up and we ship them out to researchers and educators around the world for their activities, whether they're research activities or whether they're educational activities. But to answer your question the axolotl is a salamander that has become a really important biomedical model.
INTERVIEWER: Dr. Smith, do you have anything you want to add about the world's greatest salamander?
JERAMIAH SMITH: Well, yes they're the world's greatest salamander for sure. They also have really large genomes which was a big part of this-- a big part of what we had to work with. And I imagine we'll cover several of the other things that they're used for in terms of biology and medicine, studying evolution, and things like that.
INTERVIEWER: So we'll stay with you here, Dr. Smith. Why was this such a challenge? Human genomes can be sequenced in just days. So why was this such a challenge to be able to do?
JERAMIAH SMITH: Right. So there are a couple of reasons. First of all, it's important to sort of realize there is a slight distinction between sequencing a genome and assembling and scaffolding a genome. So when we sequence we always sequence short bits. Now new technology allows us to do those longer bits, but those bits are never anywhere near as long as a whole chromosome.
The reason why axolotl is particularly difficult is that the axolotl genome is really big. So the genome itself is 10 times bigger than the human genome. The human genome has about 3 billion base pairs of DNA, which is a lot. The axolotl has over 30 billion base pairs of DNA. And so given that we're sequencing these in little bits, we have to sequence lots of little bits. And then we have to effectively, through the simplest approach, compare all those bits to each other. And as you sort of continue to increase the number of bits, the number of comparisons sort of skyrockets at that level.
INTERVIEWER: Dr. Voss, is there anything you would like to add to that answer?
RANDAL VOSS: Well I think that he hit, Jeramiah hits the nail on the head. It's a very large genome. Eventually as you sequence more and more of those bits, you do build larger pieces of the genome. So larger DNA fragments. And then that leads to the second challenge which is, how do you figure out how to put those larger pieces of DNA in their proper order? And then what the challenges is is axolotls have 14 chromosomes. And so essentially we have to make 14 linear puzzles work from the large fragments that are sequenced and assembled.
INTERVIEWER: Wow. That's a big challenge. Tell us a little bit more about how you addressed that challenge and what the implications are.
RANDAL VOSS: OK, so really I have to go back, way back in time really to talk about this. And go back a little bit into Jeramiah and my history. And so Jeramiah was my first graduate student. And over 10 years ago we started crossing the axolotl to other species which are related to the axolotl. All these species are collectively called tiger salamanders in a sense.
And so we did these crosses because we were very interested in understanding why axolotl don't undergo a metamorphosis and don't seemingly age in some sense. And this approach that we used is called genetic linkage analysis. So we can cross two species, we can get an F1 hybrid, and then cross the hybrid back to an axolotl. That segregates all of their variation, both their physical traits and their DNA variations, in the second generation offspring.
And then what we did at that time was to look at those variations and look for associations to whether or not they metamorphosed, like a tiger salamander, or paedomorphic, like an axolotl. But we developed these crosses and stuck them in a freezer. And then Jeramiah was very clever a couple of years ago and he thought, you know we could probably pull those tissues back out of the freezer and we could use them to assemble the large pieces of DNA that needed to be properly ordered to build a genome assembly.
And that's essentially what we did. We sequenced 48 of the individuals from one of these old back crosses that we generated and looked at their variations and mapped them to the large DNA fragments. And then that allowed us to put these pieces together. And so linkage mapping, which is one of the most fundamental ways of doing genetics, it was introduced at the early 1900s, is really the methodology that we used to build this assembly.
INTERVIEWER: Dr. Smith, is there anything that you would like to add to that?
JERAMIAH SMITH: We can cover this-- potentially we'll cover this a little bit later as well. But the other major challenge is really in order to find these differences that tell you whether you inherit part of the genome from a tiger salamander or part of the genome from axolotl, we have to sequence. And to address that question technically you would have to sequence at least twice to know if they have some axolotl and some tiger salamander. But really in practice you have to sequence much more.
And part of what we did for this study is we figured out a way to sort of sequence much less. So each individual we only sequenced roughly on average one [INAUDIBLE], every base of the genome once. And using some clever approaches that were ideas initially but then brought to reality by a really creative postdoc that works in my lab and in Randal's lab, Natalia, we were able to sort of figure out in a very effective way where these genes live in the genome.
INTERVIEWER: Is there anything else that you want to talk about, the linkage mapping and adapting that?
JERAMIAH SMITH: So I mean the approach we're using is fairly classical. It was generally attributed to having been developed by Thomas Hunt Morgan which is-- spent his early life in Kentucky. And it's basically, it's the idea that things that as an individual makes the gametes that'll go on to produce the next generation, you have re-combinations that mix up that material. But things that are close by tend not to get mixed up. And it's that property, the fact that things that are close by tend to not get mixed up, that really we could leverage in order to figure out where these things are in the genome.
INTERVIEWER: Dr. Voss, you want to--
RANDAL VOSS: Yeah I just want to add one more point to that since we're talking about genetic linkage analysis. And the point is that this isn't just a method you can use with the axolotl. You could use it with any species, with any organism. And what it means is that this is really a cost effective way to assemble a genome. And so I can see now that there could be other salamanders that get sequenced and assembled. And all salamanders have incredibly large genomes and so that's a very interesting avenue of research I think that's opening up because of just the methodology that we've developed.
INTERVIEWER: Why did you choose an axolotl for this specific work?
RANDAL VOSS: Yeah it goes back to their long history as just an animal model in biomedical research. That axolotl today are used by researchers because they provide the best model for understanding mechanisms of tissue regeneration.
INTERVIEWER: Would you like to add anything?
JERAMIAH SMITH: So in addition to being important for regeneration of biomedical studies it's probably also worth noting that there's a lot of utility of these species in understanding evolution and patterns of evolution. In fact, the reason why we made these crosses was because of Randal's initial interest. And when I came into his lab I started working on this question and understanding why axolotl have these beautiful gills that stick off the side of their head and tail fins that are characteristic of larval amphibians, but they sort of keep them throughout their whole life. So this phenotype called paedomorphosis.
INTERVIEWER: Great. Can you comment about what this all may mean down the road?
JERAMIAH SMITH: [LAUGHS] Oh, it's going to solve everything. But more realistically it really-- this work we think is really going to be useful for a large number of researchers that are interested in a variety of traits. Where axolotl is a useful organism to study those traits. So limb regeneration is very obvious.
There are some other things, we highlighted this gene, this mutation cardiac, in this paper. Which we were able to use the fact that we have whole chromosomes to fairly rapidly figure out what the gene was that causes this mutation. And that mutation has been around for decades. And it has been studied but with this resource it allows a way of figuring those things out much more rapidly. And maybe sets the stage for doing similar experiments sort of at larger scales to address other questions.
INTERVIEWER: Dr. Voss, would you like to add to that?
RANDAL VOSS: Sure. Jeramiah touched a little bit on regeneration. So obviously there is a large number of researchers around the world that use axolotl to study limb regeneration like Jeremiah mentioned, but also a spinal cord, even brain regeneration. And so again we have this ability to really study how the genes are regulated during regeneration, which ones need to be repressed, which ones need to be activated.
It also opens up whole new lines of research and new methods that we can use. So with a complete map of the genome we can perform epigenetic studies, we can start to study those chemical modifications of DNA and proteins that DNA is wound around, the histones and how they change their properties, so how the chromatin opens up and then compresses during the course of regeneration, to ultimately understand some of the mechanisms that regulate these genes.
INTERVIEWER: I've heard a little bit about that the axolotl is endangered and from a certain lake in Mexico. Can you talk a little bit more about that?
RANDAL VOSS: Yeah I'll talk a little bit about that. So the axolotl, it's natal habitat is a water system called Xochimilco. It used to be a large lake several hundred years ago, now it's just a series of canals and they're heavily polluted unfortunately. And so the axolotl is threatened and nearly extinct in nature at this point. So there are some conservation efforts going on by several universities in Mexico City to try to save the last individual axolotl in the remnant population there.
JERAMIAH SMITH: Well so the only thing I might add, really not to the question but to build on that sort of theme would be that axolotl is really, it's a member of this sort of bigger group of salamanders that are called tiger salamanders. Which are can be fairly useful for studying evolution. So it turns out that there are several species of salamanders that have these big gills and tails that sort of have popped up in different lakes, either in Mexico or even in the Great Plains the United States. There is one species that sometimes is one kind of salamander, sometimes it's another. And that was what attracted me to the system initially. And I think now we waited decades for the genome to be available to sort of really understand what is going on in that system, within the evolution of that system. And so now this whole tiger salamander species group I think really opens up to even more studies.
INTERVIEWER: OK. So kind of back to regeneration. Are you saying that we can learn to regrow our legs?
JERAMIAH SMITH: Me? [LAUGHS]
INTERVIEWER: Yeah, I think you said [INAUDIBLE]
JERAMIAH SMITH: OK. I've mean, yeah Randal will figure out how to grow back legs. So that's the ultimate goal. Right? That's an ultimate futuristic goal. Is can figure out how axolotl does all this stuff, a person cuts off their arm or their leg and we give them a Band-Aid that has something in it that allows them to grow it back. But realistically that's not going to happen next week.
And people have recognized that axolotl, that salamanders in general, have this tremendous capacity to grow things back after you cut them off. And I've sort of been jealous or curious of that ability for a long time and I've tried to study it for a long time. So we're not going to immediately solve something that people have been curious about for 150 years, or hundreds of years actually. But I think this reflects an important tool for a community that's going to figure those things out.
And some of the mid-range lessons of how does the wound healing happen in axolotls, and how is the nerve involved, and what are the cells actually doing, and how do they turn on their genes? Those are questions over the short term that might have implications for not full-blown regeneration but helping the healing process.
RANDAL VOSS: Yeah this line of questioning and answering reminds me to think a little bit more about, it wasn't just the National Institutes of Health that funded a lot of our work. They fund the axolotl facility here at University of Kentucky and they funded some of our sequencing work.
But also the Army Research Office at the Department of Defense helped fund a lot of our research. Because unfortunately soldiers in battle often can lose their digits or even have injuries to their limbs. And so the Army is very interested in therapies down the road that can hopefully be used to repair digit and limb injuries.
And so I would say that what the genome is going to allow us to do is really start to understand the differences between the wound healing response that happens when a mammal, when a human, gets injured versus the wound healing response plus regeneration response which seems to happen coincidentally in the case of the axolotl. And so can we eventually learn about this kind of reparative induction that the axolotl does and somehow engineer that to happen after a human has an injury?
INTERVIEWER: So I guess as you kind of look to the future, what's next for this line of research?
RANDAL VOSS: Well for us it's to pursue these epigenetic studies. So NIH and the Army Research Office are both supporting us right now to perform these kinds of studies of the chromatin so that we can better understand how genes are regulated during regeneration.
INTERVIEWER: Dr. Smith?
JERAMIAH SMITH: Yeah I mean now that we have these tools it really-- I mentioned the sort of interesting evolutionary things. We have a lot of interest in my lab and I think historically understanding sort of how genomes evolve in general, how chromosomes evolve. There's even some interesting things about sex chromosome evolution that these salamanders can teach us.
And then the sort of basic-- more and more interest in the basic fundamental questions of how is it that a cell can be instructed to sort of think that it's doing one thing over [? development, ?] it's going to become a muscle, or it's going to become something specific, and then it has to sort of almost go backwards in development, be reprogrammed, retaught. And then told-- often they're told to make a muscle somewhere else but they have to get there, get in position, and do those sorts of things.
And understanding just how this super complex, super long molecule sort of bends on itself. And it's really these bending events and being wrapped around proteins that instructs the salamander to do something with its genes that we seemingly don't do with our genes.
INTERVIEWER: If you could boil this announcement today down to one key thought that you'd want people to take away from it, what would that be?
JERAMIAH SMITH: I think mine would be that you shouldn't be afraid of big genomes and weird genomes when you're doing science. So I mean, there are lots of animals that do really weird things, really cool things, things that we should be envious of, that have strange or weird or hard to sequence genomes. And technology has moved to the point where we can really tackle some of these things that are really cool and sort of embrace how crazy biology really is.
RANDAL VOSS: Yeah I think I can boil it down. Can I boil it down to two points?
RANDAL VOSS: Yeah. So just following on what Jeramiah just said, I think what's amazing to me is to think that we know so little about genomes, of all the rest of the organisms here on planet Earth, that we've only sequenced relatively few and none of the larger genomes. And so it's really amazing to think about how much diversity there is in genome structure and function.
But the second kind of point is to keep your eyes on the axolotl. Because with this new genomic toolbox, it wouldn't surprise me if there aren't revolutionary advances in our understanding of tissue regeneration in the next few years.
INTERVIEWER: That's great to hear. Anything else that you guys want to say?
JERAMIAH SMITH: Long silence.
RANDAL VOSS: Yeah, long silence. I think I've covered most of the points that I wanted to cover.
JERAMIAH SMITH: Yeah. I think know maybe another lesson is-- oh, sorry.
INTERVIEWER: Hold for the siren.
JERAMIAH SMITH: Ambulance.
JERAMIAH SMITH: Right. So I think another lesson is sort of in part in understanding the history behind what we've done, it sort of shows the winding road that science can take. So this initial path towards studying some weird looking, why a salamander looks weird and looks like it stays like a baby forever, really kind of can move us forward into addressing other questions.
To some degree we might have anticipated the questions of how does a salamander grow its limb back because people notice that. But certainly, probably, we don't even understand-- I mean certainly we don't understand the full implications, or really everything that's ultimately going to come out from sort of being able to do better and better genetic studies in this system.