Demystifying Zebrafish

Despite the vast sequencing data available to study the genetics of human disease, experimental animal models still play a critical role in unraveling the mechanisms of disease and screening for new drug targets. Zebrafish are likely not the first animal that comes to mind as when considering animals that have contributed to biomedical research, but they are rapidly gaining popularity with research scientists.1

“After mice, the zebrafish is the most popular animal model,” Leonard Zon, MD, the Grousbeck Professor of Pediatric Medicine at Harvard Medical School, investigator at the Howard Hughes Medical Institute, and director of the Stem Cell Program at Boston Children’s Hospital, told ASH Clinical News. “More than 1,200 laboratories around the world use the organism to study basic biology about organ development or disease.”

What is it about zebrafish that makes them a versatile model for studying human disease, and how are they being used in hematology/oncology research? ASH Clinical News spoke with researchers specializing in zebrafish for answers.

Zebrafish 101: From the Aquarium to the Research Lab

First described in 1822, zebrafish (Danio rerio) are tropical freshwater fish native to the rivers and flood plains of India.3 “They are vertebrates, which provides them an evolutionary advantage over simpler animal models like yeast and fruit flies,” said Jason Berman, MD, associate chair of research in the Department of Pediatrics at IWK Health Centre and professor in the Departments of Pediatrics, Microbiology & Immunology, and Pathology at Dalhousie University in Halifax, Nova Scotia.

Zebrafish have long been popular as pets in home aquaria, as they are easy to care for and have classic black and white stripes. The modern era of zebrafish genetics began in the 1970s, when George Streisinger, PhD, and colleagues from the University of Oregon established the fish as a vertebrate model organism.1,2

In the 1990s, the value of the zebrafish as a model organism was substantiated when U.S. and German research groups induced genome-wide chemical mutations of adult male zebrafish to produce the first large-scale collections of vertebrates with genomic-induced malformations. They subsequently screened embryos for visible mutant phenotypes, or developmental defects, and mapped the relevant mutations within the genome to reveal approximately 600 genes essential for a wide range of functions in early vertebrate development.2-5

Sequencing of the zebrafish genome was completed in 2013 and has been an invaluable reference tool for scientists. It also demonstrated the extent to which zebrafish and human genes are orthologous, or descended from a common ancestral gene.6 “Human and zebrafish genomes have a high degree of conservation,” Dr. Berman said. “Seventy-one percent of human genes have at least one ortholog in zebrafish, and 69 percent of zebrafish genes have at least one ortholog in humans.”

“While it isn’t always obvious why a zebrafish would be relevant to human disease, their genetic similarity to humans means that things we discover in the fish often have direct translational relevance to what happens in human patients,” explained Richard White, MD, PhD, assistant professor in the Cancer Biology and Genetics Program at Memorial Sloan Kettering Cancer Center in New York.

Zebrafish: The Model Model Organism

In addition to their genetic and functional homology to humans, zebrafish have unique advantages over other model organisms. “The biggest difference from other animal models like mice is that the fish are small and reproduce in [large] numbers,” Dr. White said. “This makes them perfect for conducting large-scale genetic studies.”

“Each mother has between 200 to 300 babies per week and each animal is about 1.5 inches long, so many animals can be housed in a small space,” added Dr. Zon. “For most cancer studies, there are 100 fish in each arm of the study, so statistics [adequate statistical power to find differences] are excellent.”

Dr. Berman noted further advantages behind the rapid expansion of zebrafish in research laboratories, including the transparency of zebrafish embryos. This feature means that developing organ systems and disease processes can be visualized under a microscope.1

“The production of large numbers of transparent embryos facilitates the collection and direct observation of normal and abnormal developmental processes,” he said. “Moreover, the rapid pace of zebrafish development – circulation begins by 24 hours post-fertilization – enables longitudinal studies in real time.”

In 2008, Drs. White and Zon developed a zebrafish mutant called casper that remains transparent into adulthood.7 Now the strain of choice for imaging studies, it “allows us to visualize things like hematopoietic stem cells (HSCs) and cancer cells as they move around the body,” explained Dr. White. “This is extremely difficult to do in mice.”

Zebrafish in Biomedical Research

These unique qualities make zebrafish an ideal model for investigating human disease, Dr. Berman said. And the “ease of genome manipulation, [means] zebrafish are highly amenable as models for disorders caused by both germline and somatic genetic mutations.”

“In being downstream from cell-based studies and upstream from mouse models, the zebrafish occupies a unique position in the preclinical pipeline to inform disease biology and identify novel therapeutics to improve patient care,” he added.

Forward Genetics: Identifying New Genes

The first large-scale forward genetic screens in zebrafish in the 1990s paved the way for the characterization of hundreds of new genes essential to vertebrate development. This unbiased approach can be extended to any phenotype of interest, without prior knowledge of the genes involved.

Random genome-wide mutagenesis is used to create large libraries of mutant fish. Researchers can then screen for the phenotype of interest and identify the relevant mutations. A fluorescent reporter can be incorporated during the mutagenesis step to allow researchers to track the protein encoded by the disrupted gene.2,3

Reverse Genetics: Discovering New Functions of Known Genes

Now that the human and zebrafish genomes have been completely sequenced, researchers are using reverse genetic approaches to directly investigate the functions of genes and pathways of interest and to validate genetic discoveries in patients.3,4

“As with many areas of biology, we can only answer questions when we have the appropriate technology, and sometimes we have to wait for the technology to catch up,” said Seth Corey, MD, MPH, professor and chief of the Division of Hematology and Oncology and the Children’s Hospital Foundation Endowed Chair in Pediatric Cancer Research at the Children’s Hospital of Richmond at Virginia Commonwealth University. “We proposed using fish to model Shwachman-Diamond Syndrome 10 years ago, when all we had were morpholinos to knock down gene expression. We were only successful when new gene-editing technologies came about. The use of CRISPR/Cas9 editing has revolutionized the way mutant strains can be developed.”

Dr. Berman agreed: “CRISPR/Cas9-based genome editing has greatly facilitated the ability to knock out and knock in genes in the zebrafish genome to more accurately model human diseases.” A gene knock-out is when the gene of interest is inactivated to disrupt protein expression and analyze loss of function; a knock-in involves the insertion or substitution of DNA at a specific locus in the genome to create a disease model and observe new phenotypes. Such manipulations of zebrafish genes can provide useful information about the function or associated phenotypes of similar human genes.1,2

Dr. Berman explained that these approaches are now well established for germline mutations, and recent efforts have applied this technology to induce somatic mutations, such as those found in sporadic cancers.

Animal models are not completely accurate representations of what occurs in humans, but some models can provide novel insights into human disease, Dr. Corey added. “It depends on the gene or pathway you’re interested in,” he said. “When there is high conservation between humans and fish, then the model is likely to be more accurate.”

Chemical Screens

In addition to characterizing the mechanisms behind human diseases, zebrafish models can be used to identify and test new drugs for the treatment of these diseases using chemical screens.

“We can bathe fish in thousands of different chemicals, and then find ones that affect something like stem cells or cancer cells inside the animal,” Dr. White said. “There is no other vertebrate animal that is capable of doing this.”

Dr. Zon, who uses zebrafish to study the developmental biology of hematopoiesis and cancer, agreed. “Zebrafish is the best animal model for chemical genetics,” he said. “With embryos, drugs can be added to the water in well plates, and this allows high-throughput chemical screening of a whole vertebrate live animal.”

“We can bathe fish in thousands of different chemicals, and then find ones that affect … stem cells or cancer cells inside the animal.”

—Richard White, MD, PhD

Adult zebrafish can undergo a similar process: Chemicals are placed in the water or injected intravenously or intraperitoneally. “Since marrow transplants can be done on a large scale [in zebrafish], it is possible to do ex vivo chemical treatments and look for effects in vivo,” Dr. Zon said. “About 75 percent of chemicals from humans work on the zebrafish, and vice versa.”

High-throughput chemical screens “can reveal new potential therapeutic avenues that can be prioritized and subsequently validated in other model systems for their ultimate translation back to the clinic,” added Dr. Berman.

Zebrafish and Hematology

The researchers who spoke with ASH Clinical News agreed that zebrafish provide a powerful tool in hematology/oncology research. “Many laboratories have shown that zebrafish have conservation of all types of blood cells, including erythrocytes, neutrophils, lymphocytes, monocytes, mast cells, and HSCs,” Dr. Berman said. The ability to track the fish’s development in real time “has shed light on the origin and biology of these different hematologic cell populations.”

Highlighting a “success story” of zebrafish in hematologic research, Dr. Zon described how his group identified genes responsible for congenital sideroblastic anemia via large-scale forward genetic screens in mutant fish. The investigators also found patients with these same genetic mutations, “so we discovered five human diseases as a result of our fish,” he said. Similarly, “ferroportin was first found (and named) in the zebrafish.” This iron transporter was subsequently shown to be mutated in patients with iron overload disorders.8

Dr. Zon’s laboratory also used a zebrafish chemical screen to identify a drug that could enhance hematopoietic cell transplantation engraftment.9 “We found a drug, prostaglandin E2, that can stimulate production of blood stem cells in the fish, and later showed that it worked to enhance stem cell numbers in the mouse,” Dr. Zon said. “The compound is now in its fourth clinical trial, looking at patients receiving cord blood units or mobilized peripheral blood stem cells for leukemia.”

More recently, “transgenic zebrafish models of both acute lymphoid leukemia and acute myeloid leukemia have been used to identify a number of promising therapeutic compounds,” noted Dr. Berman, including cyclooxygenase inhibitors in myeloid disease and phosphoinositide 3-kinase inhibitors in lymphoid malignancies.”10

Transplantation of human cells into zebrafish, a technique known as xenotransplantation, also is being used to model human blood disorders and cancers and to screen human cells for drug susceptibility in vivo.11 “Transplanted larvae are bathed in the compound of interest, and the anti-proliferative response to the drug can be determined within the clinically-relevant timespan of a week,” Dr. Berman explained.

“Zebrafish have also been used to identify safer ways of providing chemotherapy,” he added. “By applying chemical screening to zebrafish larvae transplanted with human leukemia cells, compounds that protect against the cardiac damage caused by anthracyclines were identified, which importantly did not compromise the anti-leukemic effects.”12

With an expanding repertoire of tools available to researchers who use zebrafish as a model organism, many more new discoveries are sure to follow. “What fish offer is the ability to find new and unexpected things that would be tough to find in other models of cancer,” remarked Dr. White. “We validate what we find in the fish using human samples and tissues, making it a really exciting opportunity for discovery.” By Amy Dear, PhD


References

  1. Burke E. “Why Use Zebrafish to Study Human Diseases?” Accessed February 8, 2018, from https://irp.nih.gov/blog/post/2016/08/why-use-zebrafish-to-study-human-diseases.
  2. Clark KJ, Ekker SC. How zebrafish genetics informs human biology. Nature Education. 2015;8:3.
  3. Lawson ND, Wolfe SA. Forward and reverse genetic approaches for the analysis of vertebrate development in the zebrafish. Dev Cell. 2011;21:48-64.
  4. Haffter P, Granato M, Brand M, et al. The identification of genes with unique and essential functions in the development of the zebrafish, Danio rerio. Development. 1996;123:1-36.
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  6. Howe K, Clark MD, Torroja CF, et al. The zebrafish reference genome sequence and its relationship to the human genome. Nature. 2013;496:498-503.
  7. White RM, Sessa A, Burke C, et al. Transparent adult zebrafish as a tool for in vivo transplantation analysis. Cell Stem Cell. 2008;2:183-9.
  8. North TE, Zon LI. Modeling human hematopoietic and cardiovascular diseases in zebrafish. Dev Dyn. 2003;228:568-83.
  9. North TE, Goessling W, Walkley CR, et al. Prostaglandin E2 regulates vertebrate haematopoietic stem cell homeostasis. Nature. 2007;447:1007-11.
  10. Liu W, Wu M, Huang Z, et al. c-myb hyperactivity leads to myeloid and lymphoid malignancies in zebrafish. Leukemia. 2017;31:222-33.
  11. Deveau AP, Bentley VL, Berman JN. Using zebrafish models of leukemia to streamline drug screening and discovery. Exp Hematol. 2017;45:1-9.
  12. Liu Y, Asnani A, Zou L, et al. Visnagin protects against doxorubicin-induced cardiomyopathy through modulation of mitochondrial malate dehydrogenase. Sci Transl Med. 2014;6:266ra170.

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