(Lightly edited for readability)
Speakers: Judith Eisen, Sridhar Sivasubbu, Megha Kumar, Vatsala Thirumalai, Anuradha Bhat, Subhra Priyadarshini
Support announcement 00:01: This episode is produced with support from the National Centre for Biological Sciences.
Subhra Priyadarshini 00:30: Welcome to our new podcast series – Biolore – your front row seat to lesser-known stories from the history of biological sciences in India. I am Subhra Priyadarshini and in this audio retrospect series, I am looking forward to digging out interesting nuggets from the past.
History of science is always so interesting. But I won’t just talk about these subjects in the past tense – you will also hear me dwell on how this history relates to what’s happening today, and in some ways, what might happen in the future.
So, as they say, fasten your seat belts (or should I say grab your ear pods) as we travel back in time.
The year is 1972. American molecular biologist George Streisinger is looking at a number of tropical fish species in his laboratory at the University of Oregon in the US. He has medaka, the Japanese rice fish, and then this tiny, lithe-bodied fish, about a finger long, with horizontal blue stripes on each side of its body. And not surprisingly it is called zebrafish. He picks up the zebrafish and without realizing the historic importance of what he is about to do, makes it his model organism for research. There are no written records describing the basis of this choice, but it soon becomes quite obvious why he preferred the zebrafish to others.
Cut to India.
Swarms of zebrafish swim in the bottom waters of Ganga and Brahmaputra rivers, in the many wetlands and paddy fields. Biologists often joke that the next best habitats of these small fish are actually the biomedical research laboratories of the world.
Today, Zebrafish, Danio rerio is the scientific name, is a biologists’ top choice because of its fully sequenced genome. And also, because it is easy to genetically manipulate this fish. It's easy to care for and produces large clutches of eggs. The zebrafish's eggs develop outside the mother's body, and scientists can actually watch an egg grow under a microscope because the embryo is so transparent.
Today’s story is about how this tropical fish from the Ganga river landed in a US lab to become the most favourite organism for biomedical research. Tens of thousands of researchers in more than 31 countries use the zebrafish to understand things like diabetes to rare genetic diseases, neurological disorders like Alzheimer’s and Parkinson’s.
So, what’s the back story? I asked neuroscientist Judith Eisen at the University of Oregon. Judith pioneered zebrafish studies for the nervous system. She worked with George Streisinger for a couple of years before he died.
Judith Eisen 03:52: So, this work all started with Professor George Streisinger. He was a molecular biologist. Back in the late 1960s, and the early 1970s, many of the original questions in molecular biology had come to the point where a number of people, George Streisinger included, decided that they wanted to broaden their scope, and start looking at more complicated organisms. So Streisinger decided that he wanted to be able to apply the kind of molecular genetics that people have been doing on bacteria in phage, in yeast, to vertebrate animals, with the ultimate idea of understanding development and function of the nervous system.
Subhra Priyadarshini 04:43: Streisinger did not really come to India to take the fish home, did he?
Judith Eisen 04:48: Streisinger was actually a fish hobbyist, somebody who liked to rear fish and take care of those fish. And so, he looked around for a variety of fish that he could use for these studies. Some of the attributes are that the fish have to be able to be bred in the laboratory. And you don't want a fish that's a seasonal breeder.
And the truth is, we don't know exactly why he chose zebrafish. There were other fish that were being studied already, for example, fundulus and medaka. So this is just surmise on my part, that Streisinger decided on the zebrafish because it is easy to take out of its egg shell, and because of its optical properties it would be excellent as a model. He didn’t actually go to India to collect the fish. He got them from, we think, the local pet supply store that at least claims that they supplied the first fish that he used, although we haven't been able to verify that.
Subhra Priyadarshini 05:55: But then what makes the zebrafish so unique and versatile?
Sridhar Sivasubbu 06:01: The human genome is about 3.3 billion nucleotides, whereas the zebrafish is about 1.5. That's what makes it very interesting that you have the essential building blocks of a vertebrate animal in the zebrafish minus the complexity that larger vertebrates have. It becomes a sandbox for playing around with your genomes and genes and various subclasses of functional units.
Subhra Priyadarshini 06:26: That was Sridhar Sivasubbu, former scientist at the Institute of Genomics and Integrative Biology in Delhi. The zebrafish lab in this institute is India's largest. Water tanks are stacked up to the ceiling in racks in a room where temperature and light are controlled. Thousands of zebrafish dart around in these tanks.
Sridhar Sivasubbu 06:49: It is so cheap to maintain zebrafish, it's almost like maintaining your hobby fish tank in your home. Just that the labs would have a few hundreds of such tanks. The genome sequence of zebrafish is available. So that makes life a lot easier to mine the genome to look for your favourite gene.
Subhra Priyadarshini 07:07: Sridhar says this striped fish is an Indian import to the West.
Sridhar Sivasubbu 07:12: This fish, while it has been found in India and Indian subcontinent, has not been exploited for its advantages in developmental biology, or more recently in functional genomics research. Primarily it has been ignored till scientists such as Streisinger and others went ahead and developed it as a model, pioneered it, championed the cause for it. Today it is internationally accepted as a model organism. So rather than India actively developing the model organism, we only contributed to the animal as such, but it was developed elsewhere.
Subhra Priyadarshini 07:47: But there's some landmark science happening with zebrafish in developmental biology, and in functional genomics, in which scientists look at new genetic mutations and how they impact our health.
Sridhar Sivasubbu 07:57: In the developmental biology piece, zebrafish is extensively used to discover new genes, discover new functions of existing genes or non-coding RNAs and attributing them to the development of the organism or development of vertebrate biology, whole body of work in pigmentation. You mentioned zebrafish, a very transparent animal, one could really look at the animal under a dissecting microscope and be able to really study pigmentation. There are labs in India that have really used this very powerfully, both at IGIB and at TIFR. My own lab has concentrated on looking at vascular permeability in zebrafish, we have been successful in finding new non-protein coding RNAs that have contributed to regulation of the vascular homeostasis, that functional genomic space, which is now becoming really popular use of zebrafish. A lot of genome sequencing is happening for humans, for medical diseases or medical conditions. Zebrafish become very handy there because genes found in humans are also found in fish and they're very conserved. So you can almost find the same set of domains, the same protein sequences, so you're able to quickly take the human information, go to zebrafish, knock out that gene or do a site-directed targeted mutagenesis, screen using random mutagenesis and look for this gene.
Subhra Priyadarshini 09:18: Similarly, in the 1980s, Judith was looking at a see-through zebrafish larva under the microscope and to her surprise, she saw a motor neuron grow right in front of her. Motor neurons are cells in the brain and spinal cord that allow us to move, speak, swallow and breathe by sending commands from the brain to the muscles. That sight for her was fantastical.
Judith Eisen 09:44: I remember very clearly the first time I watched one of those motor neurons extend this long process called an axon out to muscle and cause the muscle to start to twitch. And then I realised nobody had ever seen this before. It was just a completely amazing thing, sitting in this small, dark room looking at this little, tiny monitor and being able to see this, and realising we could really capitalise on this to learn all sorts of new things about nervous system development.
Subhra Priyadarshini 10:22: And it was a journey into the unknown.
Judith Eisen 10:26: It was very exciting. In the early days. Many, many people from many places in the world wanted to come and see what the zebrafish was all about. People came, or the sent their technicians, or, you know, they asked that people from Oregon go to their labs to help them get set up. We had a very early meeting in 1990. I think this was the first zebrafish meeting and lots of people were very excited about it. And of course, at that point, we didn't have a genetic map, we didn't have many of the tools that we have now. So everything that we were doing was completely new, whether that was developing tools for actually doing experiments to test a particular question.
Subhra Priyadarshini 11:18: It was only in the early 2000s, following the sequencing of the human genome that the international zebrafish community turned to this genome. Sridhar was a PhD student then and was elated at the zebrafish's recognition. But he wanted to see India getting more involved with this native fish's global recognition. Now, years later, the opportunity arose to set up a functional genomics lab at IGIB with a focus on zebrafish.
Sridhar Sivasubbu 11:47: We needed a model organism that's also equally versatile, equally diverse, equally inexpensive to also model the thousands of human genomes that we will eventually sequence. But instead of taking a reference genome where people are looking at lab strains, our strategy was very different. Our strategy was: let's really capture the diversity of this fish in its entirety. So, we actually went after a wild type strain. It was crazy. Initially, people thought what is the use of sequencing a wild type. It was very clear that humans are so diverse, we are not clones of each other. But then why do you really study model organisms that are essentially clones of each other in the lab. I convinced committee people at the Council for Scientific and Industrial Research to sequence a wild zebrafish genome. We picked the fish from Assam, Assam wild type fish. And this was a single male animal that was first allowed to breed so that we had enough progeny created out of it. And then we took a small piece of this fin clip to sequence the animal to the whole genome. And there were two challenges here. One, the reference genome was still not built. So, we really did not know how to put it together. Second, really, nobody had done a vertebrate genome in our country. We were tackling two unknowns here. But now I had fantastic collaborations at IGIB that allowed me to sequence the wild type fish, fantastic students who took up the challenge to really put it together.
Subhra Priyadarshini 13:11: In 2009, Sridhar and his lab presented the sequence at the International Zebrafish Conference on Development and Genetics in Rome, the same conference where its reference genome was announced to the world. India's wild type zebrafish genome sequencing created a global stir.
Sridhar Sivasubbu 13:31: Right after the meeting, we are standing in a queue to pick up our cup of coffee and there are two people behind me, and both of them are international stalwarts. Remember, I'm still a young faculty who had a lab for just two years, not really well-known name in the field. So, they don't they're not seeing my face, they don't know who I am. One of the stalwarts is telling the other guy, "You know, the Indians managed to do it, why can't we do it?". And that was a sense of pride that I carried that yes, for once we have managed to be there on the international stage, prove that we can lead from the front. That was a very satisfying experience.
Subhra Priyadarshini 14:06: Back in India, over 350 scientific papers from 50 labs are credited to zebrafish. The zebrafish facility at the Centre for Cellular and Molecular Biology in Hyderabad is among them. There are multiple racks of fish tanks, like your aquarium at home. This actually looks like a zebrafish condominium.
Megha Kumar 14:27: So, we have a lot of people putting a lot of hard work every day. In fact, there are no holidays for such activities and the facility is running 24/7 irrespective of holidays all through the calendar year because someone needs to feed them.
Subhra Priyadarshini 14:43: That's Megha Kumar, in charge of that facility. Megha studies the fertilised embryos to know more about genetic and cellular causes of birth defects, how she and her teammates precisely time the fertilisation of the females' eggs with the males' sperm is actually quite fascinating.
Megha Kumar 15:05: I keep them together in a tank with a physical barrier, which is transparent, so they can see each other but they cannot physically interact. So typically, zebrafish are designed to spawn very early in the morning. So, we remove the physical barrier, we let the male and the female interact. And that's when the female drops the eggs into the tanks. And the male releases the sperms. The fertilisation happens, and the embryos are very beautiful, and they look like teeny, tiny glass beads. So, what we do is we quickly collect them in 20 minutes, because the first division from fertilisation to forming of cells is exactly 14 minutes by the clock. So, these embryos are really going by the clock, you can actually tell the time by looking at the developmental stage. So, all the manipulation that we like to do in terms of genetics is in that first 40 minutes, because once it becomes two cells, the two cells are biochemically different.
Subhra Priyadarshini 16:00: With embryos and adults side by side, her lab can also tell us how different chemicals in our environment affect the embryos development, and then the adult's health.
Megha Kumar 16:11: The embryo develops really fast. By 24 hours, all the organs of the fish are practically formed. Muscle level, neuronal circuitry, pretty much everything is sorted by five days, they have bone and cartilage also. So, it's a very rapid model to test toxicity in a very short time. And the other good part is that you can have large number of fish and use very little amount of the toxin. Plus, you'll have a high throughput screening platform that you can put because the embryos are just so much in number as compared to any other model. And it's systemically taken in organically by the fish. So, you're not forcing anything down its throat.
Subhra Priyadarshini 16:50: Toxicity tests show that chemicals that we often overlook are impacting human health. Megha says these are less talked about substances, their prevalence in our environment remains largely unknown.
Megha Kumar 17:04: One of the compounds that we looked in the lab is called tetra butyl ammonium bromide. And it's a very common industrial reagent. It's found in hand sanitizers and disinfectants, and it's happily thrown in the water. So, what we did was we put that compound into the water of the fish, and it simply gave it acute and chronic toxicity. And we saw that it's giving us a lot of neurodevelopmental effects. So, the brains of these developing embryos were severely affected. And in addition to that, because it's affecting early development, it's affecting multiple tissues, not just the brain. So that's something that was an eye opener.
Subhra Priyadarshini 17:46: Zebrafish embryo handling, of course, comes with a lot of trials and errors, and then laughs.
Megha Kumar 17:53: And you know, males and females are slightly different shape and size, but pretty much they both have stripes. So, when they are very young, and they just reach sexual maturity, sometimes it can be tricky to identify a male and a female. And I was a novice too. So, I was putting the males in the tank and then I thought now I'll put the females and accidentally put a female into the male tank and the male tank had 20 males in it. And oh my God! All 20 were circle-swimming just after her and entire tank had a commotion. And I kept looking at it and said, that doesn't look right. Why are they chasing this one? For a second, I didn't know what was going on. And then it flashed to me that "Shoot, I put a female in a male tank." And that's exactly what they do – chase the female. So, then we had to quickly rescue her out of that tank and put her in the other tank, and then there was peace and quiet. So, they're pretty much like us.
Subhra Priyadarshini 18:55: Like Megha, the sight of a fresh batch of zebrafish embryos, brings joy to neuroscientist Vatsala Thirumalai at the National Centre for Biological Sciences. These transparent embryos let her look straight into the zebrafish neurons. She learns how they work together to produce movement. But before zebrafish, she was more interested in lobsters.
Vatsala Thirumalai 19:19: Now, the lobster is also an invertebrate, which means that its brain and the rest of its body are organised a little bit differently from what we are used to as humans. And so looking at an vertebrate model organism was important to me. And looking at something that is not very complicated, like a mammal, was also an issue for me. So, combining these two I looked around and there were several that were available at that time, including the Xenopus tadpole, the zebrafish and the Lamprey, and so on. But the zebrafish was special because not only was it a vertebrate, but lots of information about its genome was available even at that time, mutants were available. The zebrafish larvae were also transparent in their early life stages, which meant that you can peer down and look straight into the brain of the animal.
Subhra Priyadarshini 20:13: But these fish can be quite finicky, as Vatsala discovered when she moved them to a different building. In a few weeks, they were laying fewer eggs, and eventually all of them died. Her anxious lab feverishly checked for water quality, food and nutrition and all other parameters. They even bought fish from outside, but the problem persisted, then the Indian jugaad or quick innovation saved the day.
Vatsala Thirumalai 20:39: It was almost getting to be like a Sherlock Holmes mystery, and we didn't know how to fix this, and it went on for about six months, eight months and finally I, by chance, ran into a Google page talking about aquarium enthusiasts, talking about hardness of water. Calcium carbonate hardness. So, it turns out that water needs to have certain hardness for the fish to lay eggs and in the old animal house the pipes were old and they had scales. The water was automatically coming in hard. Whereas in the new animal house, you know, we had new pipes and therefore the water was extremely soft. So, the way to fix this was of course to buy coral powder, coral gravel and put it in the tanks. But coral is not available in India, it's illegal to buy corals. So how do you fix this problem? And you know, Indian jugaad! So, we just bought a whole box of chalk, put it in one of the tanks and lo and behold, our egg problem was solved.
Subhra Priyadarshini 21:36: Elementary, Watson!
Anuradha Bhat 21:45: Although it is a well-known model system, very little stuff is known about what a natural population of zebrafish looks like. Whether it is the same as what you see in the labs around the world, whether its behaviour is the same or not.
Subhra Priyadarshini 21:57: That's fish ecologist Anuradha Bhat at the Indian Institute of Science, Education and Research, Kolkata. Anuradha has just returned to her lab with a bag full of zebrafish she scooped from West Bengal's paddy fields, where wild zebrafish thrive. Curious local communities often find the zebrafish catching exercise quite amusing.
Anuradha Bhat 22:21: And we've often had a lot of interested bystanders who will look at us while we collect a zebrafish, and they are somewhat disappointed when they see that we end up catching fish which are so tiny. And they ask us, "What will you do with this fish, it's so small, it looks so irrelevant". And for them, it's, you know, the bycatch of the larger fish that they are catching when they would probably just throw them away or just use them as a side dish in that meal. So very often, they kind of ask us and say, "Look, don't catch these fish. These are not relevant. Just come with us, we'll show you a better habitat where you can get bigger fish."
Subhra Priyadarshini 22:57: Anuradha and her lab members then clarify that they are catching these tiny fish to study them. She studies the behavioural and genetic differences between the raw fish that grow in their natural habitats, and the ones that grow in the lab condominiums.
Anuradha Bhat 23:14: Their behaviour is actually quite unique and different in the sense that these wild populations are under the pressure of natural selection in nature, whereas the populations which are kept in the lab, are not selected for the same reasons as what you see in the wild. In the lab, you keep them under a very safe and secure environment without any predators around. You also have ample food for them. The water is very well regulated, so the natural selective forces are just not there. And all these multiple generations of zebrafish that you kept in the lab have now got tuned to living in laboratory conditions. And my studies in the wild populations showed me in what way this can actually alter the behaviour.
Subhra Priyadarshini 23:55: Changes in water temperatures also affect fish, a study that assumes importance in a warming world.
Anuradha Bhat 24:04: And preliminary studies are showing very clear impacts of this with regards to their shoaling behaviour. These are group living fish and well known to occur in large shoals. And we do see that important shoaling traits like shoal cohesion and integrity of these shoals are impacted by temperature changes.
Subhra Priyadarshini 24:20: But for the future, it also looks like zebrafish is going to be a personal fish for everyone.
Sridhar Sivasubbu 24:28: Even though we are so identical, there is a very small subset of the genome that is different. And that's why we look so different the way we look, the way we talk the food that we like and the diseases that we carry, in a way you're unique. If I could model your personal variation into a zebrafish, which is doable today, and we do that almost on a daily basis, we could then create a personal fish for everybody, that mutation or the variation exists for a person. It's not a variation that's there in everybody else. I still carry this dream that beyond just looking at rare genetic diseases where we have successfully managed to do this for families and individuals where mutations have been created in zebrafish to study them, many people would have a personal model. It could be zebrafish, it could be a cell culture model, it could be some of the other models. But I'm pretty sure, the direction in which the biological sciences are heading, many of us would carry our personal models so that we could have a better health care planning.
Subhra Priyadarshini 25:26: At the University of Oregon, where it all began, Judith is now using zebrafish to probe the relationship between the microbiome and neural development.
Judith Eisen 25:38: And so, in the case of zebrafish, and also of us, most of those bacteria are in the guts. They make a variety of different kinds of molecules that are really important in the process of development of the brain, and then later on in the process of brain function. So, I think you would now imagine that those kinds of signals would be so important. Our research suggests that the signals from the associated bacteria are absolutely required for certain aspects of development to occur.
Subhra Priyadarshini 26:18: Vatsala, however, says there's no perfect model organism.
Vatsala Thirumalai 26:22: So, the latest in this field is right now the Danionella, which is a related species. But the difference between Danionella and Danio is that in Danionella, even the adult fish are transparent whereas in zebrafish, only the early larval forms are transparent. And then later on, it acquires these very nice zebra-like stripes on its body, and therefore it's not transparent. But the Danionella remains transparent, and it has vocal communication, it seems. So people have become very interested in studying this fish.
Subhra Priyadarshini 26:55: The zebrafish's relatives might throw more light on the impacts of global changes on larger vertebrates, according to Anuradha, who told us about its natural history.
Anuradha Bhat 27:05: Similar kinds of organisms, we call them cyprinids, the family to which these fish belong, they are ideal model systems to study impacts of important global phenomena like temperature rise, changes in water quality, effects of pollution, all these things. And as a vertebrate system, it's even more relevant because they can be used to understand how other larger vertebrates might be impacted.
Subhra Priyadarshini 27:34: So there, you just heard Judith Eisen, Sridhar Sivasubbu, Vatsala Thirumalai, Anuradha Bhat and Megha Kumar, all zebrafish fans, regaling us with anecdotes around the fish their science and their take on the model's future. Remember, the next time you see a zebrafish swimming gracefully in a tank, you're looking at a species that has revolutionised the way we understand life itself.
Stay tuned for our next episode in the Biolore series, digging into the history of the much-loved basmati rice. We have just begun.
Support Announcement 28:29: This episode was brought to you with support from the National Centre for Biological Sciences.
Never miss an episode: Subscribe to the Nature India Podcast on Apple Podcasts, Google Podcasts, Spotify, Acast or your favourite podcast app. Head here for the Nature India Podcast RSS feed.
