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Can Zebrafish improve human health?

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Ron Lisnet:

This humble creature is known as the zebrafish. It’s a favorite for people with aquariums in their homes because they’re easy to care for, hardy, and fairly low‑maintenance.

What you may not realize is they play a major role in advancing human health in some of our most intractable diseases, everything from cancer and muscular dystrophy to infections. These little guys play a key part in much of the biomedical work being done at 91����, which is taking a major step forward with new investments and facilities to grow and use these zebrafish.

Today on “The Maine Question” podcast, we are going to dive into that work. What makes zebrafish so valuable for research? What special capabilities does it have, and what kind of work is being done here at 91����?

Welcome in everybody. I’m Ron Lisnet. This is The Maine Question podcast, and we’re excited to talk about zebrafish today. Let’s introduce our guest. Rob, maybe let’s start with you.

Rob Wheeler: Sure. My name is Rob Wheeler. I’m a professor of microbiology here. We study a human fungal pathogen. It’s a fungus called Candida albicans. We try to understand how it causes disease and how our immune system protects us against it.

Ron: Ben?

Ben: Yes. I’m Ben King and an associate professor here in the Department of Molecular Biomedical Sciences with my colleagues Robin and Melody. My research focuses on the innate immune response to influenza virus infection.

Ron: This is that time of year, isn’t it?

Ben: It is, unfortunately, but it happens every year.

Ron: Melody?

Melody Neely: I’m Melody Neely. I’m the associate professor and chair of molecular and biomedical sciences. I’m a microbiologist, and I use zebrafish to study infectious disease, specifically streptococci and how the immune system reacts to it.

Ron: Before we get into the science and all that’s going on here at 91����, let’s talk about this little creature, this animal here. What do we know about zebrafish in the wild? Where and how do they live?

Ben King: They are a native fish species to South Asia, so in India, Bangladesh, Pakistan, Nepal, the Ganges Basin and other areas. They are in a fairly warm climate, where it first introduced and started to be used as what we call a genetically tractable organism in the 1980S or so.

Ron: They can tolerate living in not so clean water and less than ideal conditions. Is that right?

Rob: Absolutely. They’re very hardy fish. They’re great for aquarists, and that makes them great for us, too.

Ron: Let’s talk about what this creature allows you as scientists to do on a basic level. What are the most common applications or uses for zebrafish in the science world?

Melody:

I believe that toxicology is a big area since they are an animal that we can look at from pre‑birth. They lay their eggs outside the body and they’re fertilized. From the second they’re fertilized, you can start asking if things are toxic to them.

It can be a really nice first pass for looking at new drugs or new compounds that you might want to use eventually in a human.

The other major thing is as larvae, up to about two weeks old, they are transparent. That allows us to be able to inject fluorescent bacteria and be able to see where they go in the body.

We also have transgenic zebrafish that have fluorescent immune factors. It’s the only animal model in which you can look and see bacteria interacting with an immune cell in a live animal in real time.

Ron: You could see inside without destroying the embryo?

Melody: Exactly. While they’re alive, and then you can do that over time. You can put them back to be incubated and take them out hours later and ask, how has that environment changed?

Ron: Obviously, humans and fish don’t look anything alike and just live completely different worlds, but I was surprised to read that we share 70 to 85 percent of our genetic background with zebrafish? That seems surprising to me for two beings that are so different.

Ben:

The genome, as we call it, the collection of all genes in these organisms, the first report was in 2013 or so. It’s smaller than our genome, but interestingly, there are actually more genes. During the evolution of this species as well as what we call teleost fish, there was what they call a genome duplication event.

All genes that, say, we have, have some kind of a last common ancestor between fish and humans. There was an additional round of whole genome duplication. In many cases, zebrafish have two copies of a particular gene. In some cases, they’ve lost one of those copies and it’s become nonfunctional.

All of the major signaling pathways that are involved with human disease are conserved largely in zebrafish. That makes them a really powerful model.

Ron: When you study, in your case, a fungal disease, Candida, does it affect these fish the same way it affects a human? I know there’s a shared genetic material, but are the ramifications the same?

Rob:

It’s pretty incredible that there are so many things that are the same between the fish and the human. It interacts with the same types of immune cells, and the same types of immune cells are required in zebrafish for resistance to infection as are required in people.

For many years, the mouse has been a premier model for understanding infectious disease, but we always were pretty sure that mice are not people. In some recent work, it’s clear that in important ways, they’re different, and the ways they respond to infection are different.

One of the current projects we have going on in our lab right now, there’s a gene that people don’t require for resistance to fungal infection. Mice do require it for fungal infection resistance, and zebrafish also don’t require it.

Maybe in some cases, zebrafish may be a better system to understand how our immune system protects us against fungal disease.

Ron: We have more in common with fish in some cases than mice, which are mammals.

Rob:

In some cases, yeah. In some cases, even though we all have our inner fish.

[laughter]

Rob:

Many years ago, it was appreciated that all vertebrates have a very similar developmental pathway, and that’s what we have. When you look at a very small human embryo, it looks a lot like any other vertebrate, including fish.

Then believe it or not, these fish have a liver, so you can study fatty liver disease. They have kidneys, you can study kidney disease. They have pancreas. They have brains. You can study lots about both simple and very complex behaviors in those fish.

Ron: It goes without saying, they’re probably a little less work than taking care of mice, right?

Melody: Yes. Definitely.

Rob: About 100 times less work.

Ron: One of the other fascinating things I came across was the fact that it can regenerate body parts. Talk about that. What’s going on there?

Ben:

They can regenerate essentially any tissue after injury. There are some things that have been first characterized or described in the scientific literature by folks even working with other fish species back in the early 1900s.

They can regenerate their fins quite readily. In about two weeks after, if part of their fin was removed, it will completely regenerate in that time. Their hearts, if they’re injured, they’ll regenerate.

Ron: Really?

Ben: There are scientists that study also how the zebrafish regenerates its spinal cord. You can sever the spinal cord and it will regenerate. It’s quite a very powerful model.

Ron: If there was any way to replicate those features in a human, heart disease, people that are not able to walk, that’s the big‑picture goal out there, isn’t it?

Ben:

Yeah. Certainly, a lot of individuals work on tissue regeneration. It’s a very powerful model. There are other organisms that also have tremendous capacity for regeneration, like the axolotl and other models.

Using a comparative approach, one can try to tease apart what are the genes because we share the genes. It’s a matter of trying to reactivate some of these genes maybe in humans in order to have more regenerative capacity.

Ron: How close is that to crossing over to humans? Are we a long way away from humans being able to repair a damaged heart or anything?

Rob:

Every year, the money that NIH spends to run basic science labs leads to development of new potential drugs. It may not be that we transform people into fish, which we don’t want to do, but we may be able to find molecules that allow fish to do it.

Then instead of putting a regular Band‑Aid on, put a Band‑Aid with one of those molecules on and your cut heals way faster.

You put a gauze around or you put something around the spinal cord that’s been severed, and maybe we’re able to do that 10, 15, 20 years from now. Understanding those basic aspects of normal growth can really help us.

Ron: I know you each described what you’re looking at, but maybe just a big‑picture look. What’s the big question each of you are trying to answer with your research? You have to put on a cocktail napkin or tell somebody in an elevator, “This is my mission. This is what my life’s work is at the moment,” Melody, how would you describe that?

Melody:

I would say that I’m working with streptococcal diseases to determine how the immune system responds. We know that streptococci have the ability to inhibit the immune system to cause disease.

If we can determine what those factors are that strep is causing the immune system to change, we can then augment the immune system in humans to combat that.

Ron: Strep throat that we’re talking, correct?

Melody: Strep throat, necrotizing fasciitis, rheumatic fever, and meningitis from group B strep. Lots of diseases.

Ron: Ben, what’s the long term, what’s the big picture of what you’re working on?

Ben:

Looking at how the immune system responds to influenza virus, and as we’ve all experienced probably influenza infections, one thing that you might remember is a lot of inflammation, so high fever and the like. Looking at how to control that inflammatory response.

If that inflammatory response goes unchecked, then it can eventually lead to tissue damage in the lung and the like. Trying to find ways of trying to control that inflammation so that the response is optimized. There’s enough inflammation to clear the infection, but not too much inflammation to end up with damaged tissues.

There are all of the pathways that we’re studying that we’re interested in in terms of the human immune response are conserved in the zebrafish. We can introduce the virus to the zebrafish and study that response.

As Melody was talking about earlier, one thing we’re currently doing is to look at different small molecules that could be potentially antiviral therapies.

That’s important because our go‑to antiviral therapies currently for influenza, there unfortunately are strains of influenza that have over time, because of the use of those antivirals, they’ve acquired resistance. We need new antivirals in the future.

Ron: That’s the goal, is a new, more effective drug?

Ben: Yeah. That would be a way of…

Ron: Now, Rob, Candida fungal infections, I think, are more common than a lot of people know. A lot of people may not have heard of it, but…

Rob: Unfortunately.

Ron: What are you looking at specifically?

Rob:

Candida causes is the fourth most common cause of hospital‑acquired infection in the US. In those infections, it can be quite deadly. Even though we have good drugs against fungal disease, they are not good enough.

Then one of the most pervasive diseases that Candida causes are yeast infections in women. We still don’t really understand why some Candida strains cause disease and some don’t, why some women get disease and some don’t.

These are questions that have bearing for a lot of people. We’d like to understand about how candida causes disease, and then from that, understand how we might be able to help our immune system to deal with that.

One of the things we are interested in for fish is to say OK, well, this patient is taking a drug that helps their autoimmune‑related disease, like arthritis or psoriasis, but they become more susceptible to fungal infection from that. Why is it that they become more susceptible to fungal infection?

Is there a way to find drugs that can treat psoriasis or other autoimmune diseases but leave the rest or the important parts of the immune system intact?

Ron: Making progress?

Rob:

Day by day, yeah. Today was a really fantastic day. I had a nice chat with one of my students, and he found that this drug that affects a really important immune cell, it seems to affect the ability of that immune cell to kill the Candida.

Previously, a bit thought it just blocks the immune cell from getting to the infection, but now, we seem to be able to see by following the same fish, the same immune cells over time in the confocal microscope for 18 hours, now you can see the individual fungal cells be killed, and you can see that the drug makes a difference in the ability of the fish to kill them.

Ron: That’s a big day in the research world.

Rob: Absolutely. That happens once in six months.

Ron: Take them where you can get them, right?

Rob: Absolutely. It’s a good day today.

Ron:

Checking in on our other guests here, and they look like they’re doing OK. I want to thank Mark Nilan, who’s the lab manager for the zebrafish facility, who gave us our extra guests here.

As a matter of fact, now we’re going to step away and we’re going to take a look at the newly‑renovated zebrafish facility that has just come online, and Mark Nilan’s going to give us a little tour of that.

Mark Nilan:

This is our new facility we just put together, and it’s up and running. We do research on human disease with these animals. Everything is updated, better lighting, we have control of our heating and cooling in this room, our water system is state of the art.

We have doubled the space, I would say, and that allows us to expand our colony. They can produce a lot of eggs each time they spawn. They can spawn every two weeks. Each female will give 100 to 300 eggs. They’re easy to keep.

Here at Maine, we’re doing things like muscular dystrophy. We’re doing muscle diseases, and so the muscles are at that early stages. Being a vertebrate is the key. I always ask that on tours when people come in because the first thing I always ask is how can you do research on a fish?

We’re not fish. We’re not even look like a fish, but we’re vertebrates. We share that early development. Exactly.

Other things they do here are the flu. I believe it’s 85 percent genes are shared with us humans. Now we’re in the water system room. This is what you’d see if you went to the 91���� pool in the back room, kind of the same thing.

The difference, however, is you want to kill everything in the pool. You bleach the…Here, we don’t. With all those things, the lighting, the temperature, the water, precise, steady, the fish are locked on. They’re dialed in because that’s what makes them produce.

If they have any fluctuations, it makes them slow down. It is just fish in here. Everything about this room is for the growth and care of the fish.

Ron: Thank you, Mark, for that tour. For you folks that work with zebrafish, this new facility must be quite a revelation. What does this new facility mean for the work you’re all doing. Melody?

Melody:

For the new facility, it’s better water system. It’s less problems with heat. The fish have to be in a very controlled environment with special heat, humidity. It’s an old building, and so we were having lots of problems with pests and things.

With the new facility, we’ve eliminated a lot of that. We’ve also increased the size so that we can now increase the number of fish that we’re producing to be able to use, particularly the mutant fish that need to be maintained over time. It provides more facility to be able to grow those.

Ron: It’s beautiful. They’re natives of Southeast Asia, so they like it warm, I would imagine. How about for you? What’s your ability to leverage this new facility?

Ben:

As Melody was saying, the current facility before this one was built, or this previous facility, was old. It was, I think, built in maybe 2007 or so. It’s something where having newer environmental systems is really important. Now we’re able to have a backup.

These zebrafish lines that we have are really powerful tools, but in some cases, this is the only place where these fish exist, where we’ve engineered specific mutations and the like.

If something catastrophic were to happen to the heat or something like that, we could lose those lines, but now that we have two rooms, we can have some redundancy. That capacity is important.

Ron: I don’t know if the fish noticed it, but I’m sure that it’s cleaner, the light’s nicer. It’s just a nicer place to live than the old one.

Melody: Yes.

Ron: How about for you? Does the new facility make your job easier?

Rob:

Even though zebrafish are a really powerful model system and they make lots of eggs, they can make lots of eggs, they don’t always do it. The better we can keep them, the happier we can keep them, the more likely that we can count on that.

I used to have a student, she would set up four experiments to make sure that she had one experiment to do, sets up four different crosses of fish. If we can be sure that they’re going to spawn, then she just needs to set up one. Then every time, she can get that experiment done.

It’s an incredible gift to have not only a gifted aquarist like Mark to run the facility, but also a really high‑quality facility that is going to enable us to use the zebrafish to their greatest extent.

Ron: How common are zebrafish labs and the similarities to the work you’re doing among other research universities hospitals or other labs and such? Is this a common tool used in human health research around the world?

Melody:

It has become more so. It didn’t really start to be used until 1999, 2000 for infectious disease, which is what we all work on. Since that time, it has really expanded, and then it’s also expanded into the cancer realm.

There are huge research labs at Harvard, at UT Southwestern that study human cancer mutations and genes using the zebrafish. We have actually come up with a lot of knowledge about how to treat cancer, how cancer develops, what genes are involved in that using the zebrafish that we could not use mice or rats previously to do.

Ron: Is there anything that 91���� has a niche in in terms of zebrafish research, or discoveries, or milestones that have been reached here at 91����?

Rob:

Absolutely. We have one of the higher concentrations of zebrafish researchers in the US. If you think about there are large medical facilities, medical schools that have a number of zebrafish researchers, but that’s within much larger faculty.

We have this great concentration of people that are using this model, and that allows us to work with each other collaboratively to raise the same fish lines, to make new fish lines…

Melody: Share resources.

Rob:

share technologies and so on. This is a really unusual situation where we have several people using zebrafish for infectious disease that are sitting around the table, but also another group that’s interested in using zebrafish for understanding muscular dystrophies as well. It’s a very powerful model for that as well.

When you eat a fillet of salmon, that’s all muscle. The zebrafish is mostly muscle, so that gives you a great opportunity to understand how muscle develops and works.

Ron: Go ahead.

Ben:

The influenza virus model that we use in my laboratory with zebrafish was actually developed here at the 91����. Carol Kim, who was a professor here and moved on to be the provost at University of Albany, she, in her lab, were the first to demonstrate that you could introduce influenza virus infection in the zebrafish.

I carry on that work in my lab. That’s something where now there are groups over the summer. There were two papers, one from University of Toronto in Canada, another from a group in Europe that used this influenza model in the zebrafish.

It’s great to see 91���� make that scientific contribution and have these other labs also be using the model.

Rob:

Melody was the first to do streptococcus infection in zebrafish and our lab, the first to do fungal Candida albicans infection in zebrafish and published that. We are on the cutting edge here, for better or for worse.

[laughter]

Ron: I’m just wondering, when you introduce a flu virus to a zebrafish, does it affect fish like it does humans? Did they swim slower and are achy? How does that look?

Ben: We have to introduce it by injecting the virus into the zebrafish just to establish an infection. There is inflammation, as there are in humans, and their movement does slow because of their immune response to the infection.

Ron: We should mention that the handling of these creatures follows all the protocols, safety, and for the benefit of the individual fish. That is all baked into what you do, I imagine.

Rob: Absolutely. These are with any vertebrate animal that we use for research, and zebrafish are certainly one of them.

Ron: I’m sure you all have students that you work with, whether they’re grads or undergrads. What’s this experience like for them? Is it bring it a little more “to life” other than reading about genes and DNA in a book or doing some sort of test tube type of situation in a lab? Does it bring the knowledge and the education home a little bit more?

Melody:

Absolutely. I think we all have a lot of undergrads in our labs, but also graduate students. The undergrads, it’s something accessible. Working with a zebrafish is something that an undergraduate with less training could work with as opposed to mice, a mammalian model.

Even just collecting embryos that have been bred and fertilized and counting them, that type of thing, they’re fascinated by that.

Looking at a green fluorescent protein that’s in a zebrafish and be able to see that in the microscope, that brings it to life that this is a live animal and that we are actually seeing not only the development, but seeing how other organisms within them are interacting.

Ron: It becomes a little more real, I imagine, for these students the first time they look and say, “This is a live creature I’m dealing with here.” Does that make a difference for your students?

Ben: Yeah. We have a robust set of courses that are around discovery so they’re not just following some kind of a protocol where they…

Ron: A cookbook.

Ben:

Exactly. Our phage discovery courses, which Melody has been and is part of, it’s where they’re taking soil samples, isolating a phage and characterizing that, sequencing its genome, annotating it, in many cases, publishing papers on that new genome. We’re fortunate to have some endowed fellowships that students can compete for.

Because of that first‑year experience, they’re interested in doing more and more research. They’ll pick a lab. If they work with one of us, then obviously they will most likely work with zebrafish and hopefully carry that through their entire time here at the 91����.

That becomes something where they might have initially been inspired to maybe try research, but they can get their hands around what research is all about and learn that there’s potential for great discoveries, but there are other things that make research difficult and frustrating at times.

It’s something where hopefully they can be inspired to go to graduate school or go out into the biomedical workforce in some way. Many of our students are…it’s amazing to watch where they go.

Ron:

Next time any of us visit the pet store, we should have a newfound respect for the guys in the tanks there.

[laughter]

Melody: Exactly.

Ben: Absolutely.

Ron: Talk about where we’re headed with all of this. What’s the next frontier? What are next hopeful steps or advances that we might be looking at in…Pick your time horizon. Anything come to mind?

Melody:

Working with the immune system. That’s what comes to my mind at first because that’s what I work on with infectious diseases, is I think we’re going to find ways.

A lot of this is through Ben’s work and looking at small RNAs and molecules that are involved in the immune system that we don’t know how they’re working in humans, but we can study them individually in the zebrafish.

Previously, if you have a disease or an infection, the idea is you treat it with an antibiotic or some kind of a drug, or go to bed and drink lots of water. Now, instead of trying to treat the infection, we can learn things about the immune system and ask, how can we augment that to make it better at responding to this particular disease?

We’re not just targeting killing the bacteria. We can target by learning more, just the knowledge that we’re getting from learning about the immune system that’s so similar to ours. We can figure out ways in which we can turn that on or turn it off when we need to, to keep from harming the body. That’s what I see.

Rob:

If we think about the evolution of drugs that affect the immune system, you think about steroids, which are still used frequently today and that are a whole body drug. These are drugs that affect every part of the immune system in your whole body.

Now, if you look at the commercials that are coming out, you’ll see that there are many individual biological therapies that are out there, antibodies to one immune molecule or another, now are coming out 30 years after we discovered those molecules and understood what they did in people.

The work that we’re doing now is going to bring that next generation of immune‑modulating drugs to market, I’m sure.

Where now, as I mentioned before, instead of just using this drug which will treat the psoriasis but make you more susceptible to infections, now you find the drug that treats the psoriasis, and then a different drug that maybe makes you less susceptible to those infections.

Ron: Do any of you have aquariums at home?

Melody: Not at the moment. I used to.

Ron: I’ve seen you have a dog in a classroom.

Melody:

Yes.

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Rob: We’ve had a lab aquarium where we get the retired zebrafish and we give them a good home.

Ron: That’s very nice. That’s great. Thank you all so much for coming in. Fascinating work, and best of luck in whatever the next steps are.

Melody: Thank you.

Rob: Thanks, Ron.

Ben: Thank you.

Melody: Thanks for the time.

Ron:

Thanks for checking us out on The Maine Question podcast. You can find all of our episodes on Apple Podcast, on Spotify, on 91����’s YouTube page, as well as our website. If you have questions or comments, you can send them along to mainequestion@maine.edu. This is Ron Lisnet. We’ll catch you next time on The Maine Question.

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