Updating lab notes, summer 2018

While it’s always busy in graduate school during the summer (no classes! less traffic! more time for research!) it’s been a particularly busy (and youthful!) summer here at the McElroy lab. PhD students Irvin and Lisa BOTH had high school interns working closely with them on their research.

Tyler Masuyama (c/o 2019 – Trinity School, NY) is a 2019 Simons Summer Research Program (SRP) Fellow and worked on a project with Irvin investigating the effects of different wastewater samples on zebrafish behavior and gene expression. Tyler very quickly learned how to dechorionate zebrafish embryos, prepare wastewater extracts, assay swimming behavior, dissect brains from larval zebrafish (!), and assay gene expression in those brain samples using qPCR, all within just 6 and a half weeks!! Tyler finished off his summer with us at the Simons SRP Symposium where he presented a beautiful poster with all of the data he collected over the summer. Tyler is now off enjoying the rest of his summer before heading back to school and chipping away at his college applications. More than a great student though, Tyler is extremely friendly, curious, and insightful and we’ll miss him in the lab but we’re also very excited to see where Tyler goes!

Bronx High School of Science rising Junior, Michael Clerkin, interned with Lisa this summer to broaden his interests and expertise in marine science. Starting the summer unfamiliar with molecular biology, Michael quickly learned to perform RNA extractions of wild-caught shark tissue, optimize primers, run quantitative PCR, and analyze results in the R programming language. Michael’s life-long experience fishing helped him perfect his otolith dissection technique for fluke, tautog, and weakfish to aid the effort of the DEC inshore fishing survey. Lisa, Michael, and Irvin participated in the Long Island Aquarium’s Meet with an Oceanographer program to share their research with curious Long Island kids and families. To round out the summer, Michael accompanied Lisa on the RV Seawolf to collect muscle tissue samples from several of Long Island’s resident sharks–a once in a lifetime experience for a young aspiring scientist! This school year Michael will be tackling many challenging AP courses and begin touring colleges all over the US. We wish Michael the best of luck with his college search and future research!

Life’s lil’ surprises

After working with the same laboratory animals for years, one gets accustomed to the regularity of the same species, from the same conditions, over and over again. But every once in awhile, those same, highly controlled and selected lab species can still surprise you. Considering how our lab uses thousands of fish embryos for our research, with a little luck, we get surprised more than just occasionally. This is a photo of Lil’ Spencer, a Japanese medaka (Oryzias latipes) larva, just after hatching as a control fish in one of my experiments. It’s immediately obvious what makes Lil’ Spencer so special: her (their?) two, fully formed heads (eyes, brain, mouth and all), emerging from a single body. Also present, but not quite visible from this view, is a single, bulbous and misshapen heart, four pectoral fins (the inner two which constantly knock against each other), and a bent spine.

We weren’t able to pinpoint her/their exact mutation, but based on developmental studies in zebrafish and Xenopus frogs, it’s likely that there was a major mutation in the anteroposterior organizer, which is an important set of cells that controls regional differentiation and body axis patterning during early development. Manipulations of signaling molecules like β-catenin and bone morphogen protein (BMP) and pathways such as canonical and non-canonical Wnt pathways have produced a myriad of altered fish development, including secondary body axes complete with their own head! Unfortunately, Lil’ Spencer died a week after hatching, her body unable to support a heart too large and one head too many. But their short time in our lab served to remind us that despite the expertise you gain, the simplest fish can still surprise you in delightful ways. We’re just waiting to see what life surprises us with next.

A Primer: Behavior as a toxicology research tool

Oftentimes when we think about an organism’s health, we don’t really think about including behavior. Maybe it’s just me, but behavior was something associated with the brain, and that thing is just too complicated to think about that they really should just get their own category (which, to be fair, many research groups do separate neurobiology and behavior into their own group, which might contribute to this way of thinking). But, ever since I’ve started my dissertation work, I’ve definitely come around and seen the light – behavior is an incredibly important aspect of physiology and we should all care about it!

Sure, behavior is extremely important part of social interactions; it doesn’t take a scientist to know that. But it’s not always as obvious how behavior is related to your health. Your body comes equipped with many amazing strategies to deal with stress, but it’s your behavior that determines how much stress your body experiences. Changes in behavior are really easy ways for animals to quickly avoid stress. Think about what you’d do if you were stuck outside on a hot summer afternoon. Sure, your body can deal with that stress through all sorts of neat ways like sweating, changing your breathing and heart rate, making new, more heat-sturdy proteins, etc. Or, you could just go find some shade or go inside where it’s air conditioned. Maybe grab an ice cream cone. Your body is capable of some amazing coping mechanisms, but does that mean you have to always use them? A simple change in how you behave can save you a lot in time and energy when dealing with a stressful environment.

More than that though, lots of behaviors may seem really simple on the surface actually involve a lot more than we realize. Let’s use another example – this time imagine yourself as a small fish trying to remain uneaten (a pretty important behavior, if you ask me). The obvious thing to do when a fish senses a predator is to quickly swim away and find somewhere safe to hide out in. Not exactly rocket science. But let’s break that down into the various steps it takes to complete that action. First, you need to realize that the predator is there to begin with. You might see the predator, hear it, or maybe even smell its presence. That requires a fully functional sensory system. Your eyes, ears, nose, and touch receptors need to be on point and they need to transmit that message to your brain. Well, that’s a huge complex system right there so you better be sure your brain is working properly as well. But that’s not all, your brain has to tell your muscles which way to move, and how much to move, so your nervous system needs to be in tip top shape. Your body is also going to help you prepare for this escape by changing its hormone balance. It’s fight or flight, is not a great time to be thinking about making babies or putting on fat reserves, so your endocrine system is going to temporarily turn the dial down on things like sex hormones, growth and fat storage, and instead mobilize stored energy to make sure you have enough fuel to get away. So right there, in this little fish swimming away from a predator, you need a fully functioning sensory organ system, nervous system, endocrine system, and who knows what else – all to tell your body to just keep swimming. Those systems are some key places that a pollutant can muck it all up and cause real problems for a fish (or any animal, really). So as a toxicologist, when I see a fish that’s behaving abnormally, that is a clue that at least one of these systems is off in some way, as well as potentially which areas might have been affected.

Behavior can be complex but with that complexity comes with a wealth of potential for researchers to study. It takes a lot of work, for sure, but behavior is such an important part of an animal’s life that we really shouldn’t be leaving it out anymore. There’s not doubt, studying behavior has many challenges (and the more complex the behavior, the more challenging it becomes), but that just means we have to get creative. In future posts, I’ll be introducing you to the different ways I, and other scientists, study animal behavior and show you some really creative solutions people have come up with to tackle these complex behaviors.

XYBRUH GHOTI

As a kid growing up in the suburbs of the San Francisco Bay Area, I didn’t get a lot of firsthand experience with animals – mostly just the neighborhood pets and maybe a bold raccoon every once in awhile. When growing up in an urban area, loving nature requires a lot of imagination, and nothing captures a child’s imagination like Animals. We’re talking capital ‘A’, giant, ferocious, dangerous, mysterious Animals. Neighborhood dogs became vicious wolf packs, cats were solitary and deadly tigers, house sparrows were majestic birds of prey (if you squinted and tilted your head and closed one eye and completely ignored the fact that they look nothing like an eagle at all). Nothing inspired and motivated me like the big, charismatic animals and to me, working with anything smaller or less magnificent would be a waste of my time.

Which is why I think it’s pretty funny that 90% of my time now is spent looking at tiny fish that aren’t even fully developed yet. It’s a rare day when I’m not hunched over a microscope cooing over my baby fish. But to be fair, they’re not just any fish – these are special fish. They’re zebrafish.

zebra-fish

Um… No, not quite what I had in mind.  Picture by CAMEye0170 from FreakingNews.com’s photoshop competition

I use zebrafish in my Ph.D. research. I’m an aquatic toxicologist and I use zebrafish as a model organisms to investigate whether or not psychiatric drugs found in wastewater and sewage can affect the development and behavior of zebrafish, which mostly involves observing their behavior and checking out which genes seem to be important in responding to pollution stress. But that’s only one of the many, many ways scientists can, and do, use zebrafish in research.

zfish

I mean, the adults are kinda pretty? And the embryos are pretty cute. Nothing o write home about, right?

I’m sure many of you have even seen zebrafish before, in a pet store or a home aquarium. Zebrafish are originally from the Himalayan region, native to the streams, ponds, canals, and ditches of India, Pakistan, Bangladesh, Nepal, and Burma. They’re little fish (maybe a couple of inches long), they eat bugs, and sometimes can be kind of shiny. There’s not exactly a lot going for them. So why are they so special? Zebrafish are special because they are an important scientific tool that thousands of researchers around the world use to answer some fundamental questions about the life around us.

Did you know that the zebrafish was among some of the first vertebrates to ever be cloned? (Eat your heart out, Dolly!) Did you know that zebrafish can regrow parts of its body including its heart and nerves? Did you know that there are literally hundreds (maybe even thousands) of mutant strains of zebrafish kept in scientific laboratories, all around the world? And that’s just the tip of the iceberg – zebrafish are incredibly useful to biologists and have contributed countless discoveries to science.

Sometimes it takes some special effort to see how special something is. In this case, the same cutie from above, while unassuming, becomes a majestic glowing tool of science when exposed to the right kind of light. This is a genetically modified zebrafish that has a special protein called Kaede, which is a protein originally found in stony coral. Kaede normally glows green but when exposed to UV light, becomes red. This fish has had the genetic code for kaede tacked onto it's own genes, and when we shine UV light on certain parts of its body, in this case its eyes, gut, and some parts of its tail, they glow red instead of green. Convertible glowing proteins are useful for tracking cells during development - convert just a few cells and literally follow the red cells and see where they end up!

Sometimes it takes some special effort to see how special something is. In this case, the same cutie from above, while unassuming, becomes a majestic glowing tool of science when exposed to the right kind of light. This is a genetically modified zebrafish that has a special protein called Kaede, which is a protein originally found in stony coral. Kaede normally glows green but when exposed to UV light, becomes red. This fish has had the genetic code for kaede tacked onto it’s own genes, and when we shine UV light on certain parts of its body, in this case its eyes, gut, and some parts of its tail, they glow red instead of green. Convertible glowing proteins are useful for tracking cells during development – convert just a few cells and literally follow the red cells and see where they end up!

Imagine if someone gave you a toaster, no manual, blueprint, or instructions, and told you to figure out what each part of the toaster does. The best way (at least the most fun way) to figure that out is to take it apart and then try to put it back together; take a piece out, move it around and see how that affects the toaster’s performance. That’s what zebrafish are to scientists: a living toaster. We know the different genes that make up a zebrafish, and if we want to figure out what each gene does we can go in, tinker around, and see what happens. We can remove a gene and see what happens to the fish without that gene (we call this a knockout experiment). We can take cells from one developing individual and put them into another (we call this a transplantation experiment). We can label genes, proteins, and enzymes with fluorescent proteins to help us see what’s happening – are they growing the same? Does it have all the same parts? Are there extra parts? Does something completely unexpected happen?

casper

So it turns out when you turn off the ‘roy’ and ‘nacre’ genes, you get a transparent fish. So it’s pretty safe to assume those genes are really important in determining pigment formation. Image from Stoletov and Klemke, 2008 (Oncogene)

This is really useful because despite how different we may seem, there are lots of things that we humans share with zebrafish – many of our genes are very similar (we have about 70% of our genes in common with zebrafish!), lots of the inner workings of the body are similar, and we even develop similarly. If you look at the first couple of stages of development humans, chimps, mice, fish, and many other vertebrates all look very similar. This is a HUGE advantage for scientists since most people, for some reason, are opposed to having their genes manipulated in the name of scientific curiosity. Instead of experimenting on people, we can experiment on these fish, who produce a LOT of eggs and grow VERY quickly, and apply those results to people! We’ve learned so much about complicated conditions like cancers, neurodevelopmental disorders, and infectious diseases by studying them in zebrafish. We’ve used zebrafish to discover hundreds of drugs for countless medical conditions that have had a huge benefit to people. We’ve done so much with zebrafish that I can hardly even begin to describe them all to you in this blog, let alone in this one post!

In transplantation experiments, scientists take cells from one individual and inject them into another individual, and then follow and observe what happens to those transplanted cells. In this experiment, Dr. Lennart Hilbert is transplanting neural cells into a zebrafish to see how they grow with the fish.

In transplantation experiments, scientists use a very fine glass needle to take cells from a donor individual, inject them into an acceptor embryo, and then follow and observe what happens to those transplanted cells. These experiments are really valuable in helping figure out what controls cell behavior. Do developing cells already know what organs they are going to grow into or does the local environment tell them what to grow into?

Like I said earlier, I work with zebrafish and even though they aren’t as big and dangerous as a tiger or a great white shark, they have their own quiet mystery that has definitely captured me. I use them in toxicology research, but I have friends and colleagues who use them to study genetics, medicine, diseases, biophysics, and many, many other fields who all use this amazing fish to do incredible things. Stick around and I guarantee you’ll hear more about these amazing little fish.

And before we go – I’m sure you’re wondering about that weird cryptic title. I’ll just leave this here.

ghoti