November 2014

Spotlight on Zebrafish

Spotlight on Zebrafish: Translational Impact

Amatruda, J.F. Dhillon, P., Patton, E. and Ramakrishnan L. (2014) Spotlight on Zebrafish: Translational Impact. Dis. Model. Mech. 7, 731733


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In recent years, the zebrafish has emerged as an increasingly prominent model in biomedical research. To showcase the translational impact of the model across multiple disease areas, Disease Models & Mechanisms has compiled a Special Issue that includes thought-provoking reviews, original research reporting new and important insights into disease mechanisms, and novel resources that expand the zebrafish toolkit. This Editorial provides a summary of the issue’s contents, highlighting the diversity of zebrafish disease models and their clinical applications.

Zebrafish (Danio rerio) have fast made their way from pet stores and home aquaria into research laboratories worldwide. Their weekly matings produce 100 to 200 embryos that rapidly and synchronously march through embryonic development, so that within 5 days of fertilization, they are mature, feeding larvae. Zebrafish are small and inexpensive to maintain in high numbers, facilitating large-scale experimentation and cheap in vivo drug screens. Famously, the fish are transparent during early larval stages, allowing investigators to directly observe internal development and making the fish a favorite of developmental biologists since the 1960s. But in recent years, the utility of zebrafish has been proven beyond developmental fields, and they are now being found in more and more laboratories studying behavior, diabetes, heart disease, regeneration, stem cell biology—and cancer.

Critically, zebrafish can be used to identify the important pathways and processes that cause cancer in people. Common organ systems and cell types are shared between human and zebrafish, and whether induced by transgenesis or carcinogens, cancers arising from the blood (leukemia and lymphoma), pigmented cells of the skin (melanoma), and the cells that line the bile ducts (cholangiocarcinoma) have microscopic features that are essentially indistinguishable between humans and zebrafish.

One aim of a Disease Models & Mechanisms (DMM) ‘Special Issue’ is to highlight how emerging disease models can lead to exceptional growth in particular areas of translational research. This is especially true for this issue, Spotlight on Zebrafish: Translational Impact. The zebrafish has traditionally been used to study developmental biology. Its optical transparency for the first few weeks, high fecundity and ex vivo fertilization have meant that the fundamental processes and mechanisms of vertebrate embryo development from a single cell through to a swimming fish can be studied in exquisite detail. Over the past decade these same features have enabled the zebrafish to become a preeminent disease model and tool for studying disease mechanisms. Importantly, discoveries in zebrafish disease models are leading to new perspectives on human disease and new drugs that are entering the clinic in diverse areas from cancer to tuberculosis.

We are delighted to present an issue packed with reviews, research and resource articles from researchers at the cutting-edge of their respective disease area of interest. The issue also includes a compelling interview with Len Zon, pioneer in the zebrafish disease models community, and a unique poster representation of the translational applications of zebrafish research. Here, we summarize the contents of the issue, and give our views on what makes each article special.

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Unfolded Protein Response

Molecularly defined unfolded protein response subclasses have distinct correlations with fatty liver disease in zebrafish

Vacaru A.M., Di Narzo, A.F., Howarth, D.L., Tsedensodnom, O., Imrie, D., Cinaroglu, A., Amin, S., Hao, K., and Sadler, K.C. (2014) Molecularly defined unfolded protein response subclasses have distinct correlations with fatty liver disease in zebrafish. Dis. Model. Mech. 7, 823-835

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The unfolded protein response (UPR) is a complex network of sensors and target genes that ensure efficient folding of secretory proteins in the endoplasmic reticulum (ER). UPR activation is mediated by three main sensors, which regulate the expression of hundreds of targets. UPR activation can result in outcomes ranging from enhanced cellular function to cell dysfunction and cell death. How this pathway causes such different outcomes is unknown. Fatty liver disease (steatosis) is associated with markers of UPR activation and robust UPR induction can cause steatosis; however, in other cases, UPR activation can protect against this disease. By assessing the magnitude of activation of UPR sensors and target genes in the liver of zebrafish larvae exposed to three commonly used ER stressors (tunicamycin, thapsigargin and Brefeldin A), we have identified distinct combinations of UPR sensors and targets (i.e. subclasses) activated by each stressor. We found that only the UPR subclass characterized by maximal induction of UPR target genes, which we term a stressed-UPR, induced steatosis. Principal component analysis demonstrated a significant positive association between UPR target gene induction and steatosis. The same principal component analysis showed significant correlation with steatosis in samples from patients with fatty liver disease. We demonstrate that an adaptive UPR induced by a short exposure to thapsigargin prior to challenging with tunicamycin reduced both the induction of a stressed UPR and steatosis incidence. We conclude that a stressed UPR causes steatosis and an adaptive UPR prevents it, demonstrating that this pathway plays dichotomous roles in fatty liver disease.


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Going Viral

Going Viral

University of Maine. Biology & Biomedical Sciences – Going Viral. UMaine

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Virus Study at UMaine’s Zebrafish Facility

“You can see the bacteria?”

“Do you want to say the quality value of biofilm formation?”

“Why would isolating them give you a different value?”

“Are there any other explanations?”

“How do you have 1.8 fish?”

Things can get pretty intense in the hallway outside Carol Kim’s microbiology lab in Hitchner Hall at the University of Maine. For the casual observer — especially one without a science background — the barrage of questions is overwhelming, like listening to an auctioneer calling in a foreign language.

But the three undergraduates standing there explaining their senior research projects, pointing to printouts of data and microscopic images of zebrafish taped to the walls, are completely unfazed. They answer Kim’s questions almost as quickly as she asks them. When she challenges them, they challenge back. They are confident. Eloquent. They know their stuff.

Which is exactly the way Kim, a professor of biochemistry, microbiology and molecular biology who directs UMaine’s Graduate School of Biomedical Sciences, likes it.

“I want to set up an environment where students feel comfortable and nurtured,” Kim says. “They have to know I’m going to ask tough questions and they have to be prepared. It’s going to be a lot nicer for me to ask them than to have them present in front of five professors cold. If I can be tough on them and they can answer the questions, they’ll have confidence.”

She wants to maintain rigorous and demanding standards, without the fear factor that can sometimes overwhelm students during their training in the sciences.

Kim is emblematic of a major push on campus to involve undergrads in research, and scenes like the one outside her lab play out across campus every day, especially in the weeks leading up to graduation. At UMaine, hundreds of science, humanities and engineering majors are involved in research, and close collaborations with faculty members are common.

Increasingly, UMaine has become a destination for top students interested in pre-med and biomedical studies, in large part because of the mentoring and rigorous preparation that Kim and her colleagues provide. As a result, many undergraduates are working at a graduate level long before they earn their bachelor’s degree. Like the students in the hall, they know the answers. But more important, they know which questions to ask.

hat curiosity is what drives Kim’s own career, both as an educator and a researcher. And the students who cross paths with her during their time at UMaine have a strong, demanding role model.

Kim has been fascinated by the craftiness of viruses — their resourcefulness and resilience — since grade school. But she’s equally crafty. And her research has moved the entire field of virology forward.

She conducts disease studies with zebrafish, a model organism, to better understand the human innate immune response to infection. She’s the driving force behind UMaine’s Zebrafish Facility, and since she arrived at UMaine in 1998, Kim has received continuous funding for her zebrafish research — more than $4 million in federal grants, primarily from the National Institutes of Health. Among her landmark discoveries is a zebrafish gene that produces interferon, which can inhibit the growth of a virus. She and colleague Rob Wheeler recently received a $60,000 NASA planning grant to study the effects of radiation on innate immune response and the progression of cancer.

Her current research focus provides a better understanding of how bacteria infect and cause inflammation in cystic fibrosis patients. Kim’s studies shed light on the connection between the cystic fibrosis transmembrane conductance regulator, or CFTR, and the innate immune response. That connection may someday be used as the basis for therapeutics that combat bacterial infections in cystic fibrosis.

“I love doing the brainstorming, working with a student to figure out the best question to ask.”
Carol Kim

“Clinical researchers are trying to develop therapeutics for the immediate needs of these patients, and as a result, we’ve seen significant increases in their quality of life. We’re on the other end, with basic research, trying to figure out what’s happening at the molecular and cellular level with the hope of developing those therapies. This project will be ongoing until CF is completely cured, until it’s no longer a problem. It’s going to take a while.”
Another recent collaboration with UMaine physicist Sam Hess and GSBS student Kristin Gabor focuses on immune response to viral infection — not necessarily in CF patients. By using super-resolution microscopy, the researchers are the first ever to view the single-molecule cellular interactions involving antiviral signaling in caveolae, which are flask-like invaginations in the cell membrane. While previous research has shown that viruses exploit caveolae to enter host cells, Kim took it a step further by demonstrating that viruses can evade host cell defenses by disrupting clusters of signaling molecules within the caveolae. Through a combination of fluorescent tagging and super-resolution imaging of viruses and zebrafish cells, Hess’ FPALM (Fluorescence Photoactivation Localization Microscopy) system has allowed Kim and her team to see how individual molecules and clusters move during a viral infection.

“No one has ever really looked at this,” Kim says. “No one has been able to see it the way we’ve been able to see it.”

To the uninitiated, these may seem like disparate projects, but they all have two things in common: zebrafish and the innate immune response, the body’s first line of defense against infection. Innate immunity deals with how the body reacts immediately after it comes into contact with a pathogen. This happens daily, almost constantly, and it’s why healthy people don’t get sick every time they encounter a new pathogen. It’s why your skin swells when you get a splinter or a paper cut. This is not to be confused with adaptive immunity, which is acquired through vaccination or prior infection. Zebrafish are the ideal model for this research for several reasons, including the fact that they develop rapidly and their embryos are clear, allowing researchers to see the infection as it happens.

Innate immunity is pivotal to understanding how the body defends itself against infection, how viruses and bacteria adapt to the body’s defenses, and how more effective treatments might be developed.

Even one of these accomplishments would be noteworthy. Together, they’re huge. But when asked if there is a single moment that has defined her time at UMaine, she doesn’t miss a beat.

“Every year, when students in our department get into the top graduate schools, the top medical schools, the top dental schools, when they get great jobs it makes me think, ‘Wow, that’s why we’re here.’ UMaine is only the first step, but we hope we had an impact on their lives. We’re very proud of our students”

Working in Kim’s lab has been the highlight of Walter Mowel’s four years at UMaine. Mowel is a Biology major and Pre-Med student from Montpelier, Vt., whose scientific interest lies in infectious diseases. He spent his final semester researching a virus that hasn’t been studied in zebrafish before. And when he stood up for Kim’s hallway inquisition, his enthusiasm for the project outweighed any fear.

“Before this, I spent a lot of time working on an experiment that didn’t work out, but with this project, I was able to get a lot of data that has me really excited,” Mowel says. “It’s so much fun to essentially look at something for the first time. A lot of times, when you set out to conduct a study, you’ll go to a scientific journal or you’ll go online to see what other scientists have done, but in this case, we couldn’t. We’re basically finding something new and that’s been outstanding.”

Aaron Perreault, a Biochemistry major and Honors student from Northfield, N.H., spent his junior and senior years in Kim’s lab studying Pseudomonas aeruginosa, a bacterium that regularly infects cystic fibrosis patients — one of many pathogens that the innate immune response normally quells.

“When children with cystic fibrosis are younger, they get infections with pathogens that make sense, such as Haemophilius influenzae, staph infections, more common lung pathogens. But by the time they’re in their teens, patients all have Pseudomonas infections, which are very rare in healthy individuals,” Kim says. “Why Pseudomonas rather than a more common respiratory pathogen?”

The answer to that question may someday be used to create therapeutics, and Perreault, one of five UMaine students to gain admission to Tufts University School of Medicine as a sophomore through the Tufts Maine Track Early Assurance program since 2009, may someday prescribe those therapeutics.

Though Perreault admits that the biggest lesson he’s learned is that his lab skills aren’t particularly strong, the experience has also taught him the value of analytical thinking and keeping an open mind. His work in Kim’s lab was the basis for his Honors thesis, and in the weeks leading up to his defense, he was no stranger to Kim’s hallway inquisitions.

“It can be intimidating at first, but once you figure out that she’s grilling you to make you a better student, it makes you work harder,” Perreault says.

Kim will do whatever it takes to instill confidence in her students. Sometimes that means an informal inquisition like the one in the hall. Other times, it means meeting on nights or weekends to make sure that her students are prepared to consider every angle when defending their theses or dissertations. And sometimes, it just means handing over the reins.

When Steve Altman was at UMaine — he earned a bachelor’s in microbiology in 2002 and a master’s in molecular biology in 2003 — he worked on basic immunology in zebrafish. Kim gave him a lot of wiggle room with his experiments, but she also challenged him to try things that might be outside his comfort level, and that continues to influence the way he does science.

“Some of my friends worked in labs where the principal investigator told them what to do,” recalls Altman, who now conducts Alzheimer’s research for Amgen in Cambridge, Mass. “With Carol, it was a little bit more open-ended. She gave me guidance, but she also allowed me to make decisions on my own. That stuck with me.”

Inspiring the next generation of doctors and researchers is what gets Kim out of bed in the morning. She wants them to get jazzed about how crafty bacteria and viruses are. She wants them to experience the thrill of seeing something for the first time. And the best way to do that is through research.

“I love doing the brainstorming, working with a student to figure out the best question to ask,” Kim says. “Seeing them come in to check on their fish, to see what their results are, that’s exciting to me.”

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Developing Muscles

Biology & Biomedical Sciences – Developing Muscles

Munson, D. Biology & Biomedical Sciences – Developing Muscles. UMaine

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Basic biological research on zebrafish may lead to better treatment of human diseases and injuries

“Tendons are incredibly important structures, but exceedingly little is known about tendon development. It is very understudied, but it has implications in the treatment of a variety of tendon afflictions, from tendinitis to disorders caused by antibiotics or cancer treatments,” Henry says. “Traditionally, tendons have been thought of as uniform, with the same protein structure throughout. We have found that that is decidedly not the case. Tendon structure is spatially and temporally dynamic. We’re very excited about looking further at how the type and location of tendon proteins change over time.”

Staring into the crystal-clear plastic boxes inside the University of Maine’s zebrafish research facility, it can be difficult to focus on the movements of an individual fish. Both its stripes and its size make it hard to distinguish from its brethren in a population that is now more than 40,000 strong. The fish, on the other hand, seem to be very good at tracking the movements of an individual human, darting away in a flurry of fins and sinew at the slightest agitation.

At first glance, it seems as if the frightened fish simply bullet through the water, like tiny torpedoes propelled by some hidden, turbocharged motor. But closer observation reveals the true power behind their movements: a fluttering undulation of the body and tail, dependent, of course, on muscle.

With every flip of its fins, tiny bundles of skeletal muscle extend and contract in precisely coordinated synchrony, leveraging their movements against the fish’s miniature frame.

UMaine researcher Clarissa Henry has always been fascinated by the dynamic processes that shape the complex machinery of movement, and has pioneered a unique new system for studying how muscles and tendons develop inside the zebrafish. With a $1.28 million grant from the National Institutes of Health’s National Institute of Child Health and Human Development, Henry is shining a microscope-mounted light into the darkest corners of developmental biology, revealing new truths about embryonic processes that may lead to better treatment methods for conditions ranging from muscular dystrophy to tendinitis.

Henry’s current research, aimed at developing a better understanding of tendon formation and attachment in the embryo, is the next step in her pioneering efforts to describe the complexities of early development in vertebrates using zebrafish. Her previous research, funded by the Muscular Dystrophy Association, looked at how embryonic muscle cells transform from relatively stubby globs of cytoplasm into the long, multinucleated fibers of skeletal muscle in fully developed fry.

Skeletal muscles–from the orbicularis oculi to the gluteus maximus–are primarily responsible for movement in vertebrates, and abnormalities that arise during their formation can have dire consequences. For example, muscular dystrophy, one of the most common genetic diseases in humans, is characterized by a loss in muscular function that can manifest in many ways.

“One of the critical questions related to the treatment of muscular dystrophy is: How do humans make muscle during embryonic development?” says Henry. “We were able to make a significant step forward in this area because we were able to use an in vivo model. Prior to our work, no one was able to look into a live vertebrate embryo to see how muscle cells form at high resolution. We were able to do that with the zebrafish, thanks to the MDA.”

The strength of the preliminary data was one of the reasons NIH reviewers expressed such strong support for Henry’s latest project, pointing to her well-established methodology and the work’s potential benefits in the treatment of human disease. In addition to her obvious enthusiasm for the research, Henry has a technological advantage as well, utilizing cutting-edge equipment like a Zeiss ApoTome fluorescence microscope to peer inside the living embryo.

Like their plastic tanks, the developing eggs of the zebrafish are largely transparent, allowing researchers to observe and record changes in the cells as they happen, which is difficult or impossible in other vertebrate research models, such as mice or chickens. The zebrafish model has advantages over cell culture techniques as well, revealing important nuances in growth and development that can only be seen when cells form under the influence and constraints of a living organism.

With the help of the tiny zebrafish, Henry’s early work uncovered “a phenomenal amount of data” regarding muscle cell development, laying the foundation for further research related to tendon attachment and other processes. The new research path has already led Henry and her team to some important discoveries.

“There’s a lot of basic science in this area that we just don’t understand,” says Henry. “We don’t know how these structures grow, how they increase in mass or how the attachment between the tendon and the skeleton is maintained. There’s a real opportunity here to do pioneering work.”

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Battle Lines

Battle Lines

Staples, B. (2013) Battle Lines. UMaine Today

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A UMaine biomedical lab looks for answers in the transformation of a peaceful yeast to fatal fungus

“We’re using zebrafish to ask really specific questions that cannot be
answered another way,” Wheeler says. “These questions have been inaccessible
for a long time. We hope to be able to better utilize existing therapies and be
able to develop better therapies.”

LIFE-AND-DEATH battles rage in Robert Wheeler’s lab at the University of Maine. The combatants — zebrafish and Candida albicans— fight to the bitter end in glass-bottom microplates. Similar perilous battles are being fought inside humans. The C. albicans fungus is a leading cause of hospital-acquired infection that annually kills several thousand patients nationwide. During the staged scuffles in Wheeler’s lab in Hitchner Hall, anesthetized zebrafish are injected with Candida and placed in a gelatinous material called agarose.
A laser microscope captures and magnifies the struggles inside the zebrafish blood vessels in real time in high-definition color detail. The microplate clashes provide the assistant professor of microbiology with the ability to view how immune cells fight the microbe, identify genes involved in virulence, test new drugs and learn how gene perturbations affect hostpathogen interaction.

In March 2012, Wheeler received a three-year, more than $421,600 grant from the National Institutes of Health to ask and answer these questions in the project: “Genetics & Visualization of Innate Host Response to Candida albicans Infection In Vivo.” The goal is that the resulting answers will save human lives.

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