The Zebrafish Exposed

Ralf Dahm. American Scientist. Volume 94, Issue 5. Sep/Oct 2006.

Every once in a while a superstar comes onto the scientific stage and revolutionizes our understanding of the world. Readers might be imagining the likes of Newton, Darwin or even Einstein-worthy candidates to be sure, but human beings aren’t always the heroes when it comes to scientific progress. In the biological sciences, the introduction of a new model organism can often transform a field of study as much as the ideas of a revolutionary thinker. Consider the importance of the bacterium Escherichia coli to molecular biology, the fruit fly Drosophila melanogaster to genetics or the laboratory mouse MMS musculus to biomedical research. These life forms have become household names, and it’s hard to imagine the past half-century of research without them. To that list of “supermodels,” we may soon add a rising star: the zebrafish, Danio rerio.

Although aquarium hobbyists know the zebrafish from its ubiquity in pet stores, this small tropical fish only entered the scientific limelight within the past decade. In that brief time it has become one of the most important vertebrate model organisms in biomedical research. Institutes around the globe have built large facilities to house thousands of zebrafish, and more than 4,000 scientists are focused on the biology of this animal. Indeed, Danio rerio will soon become one of the best-documented creatures on the planet. Why has this little fish attracted so much attention?

Very simply, the zebrafish may be the key to understanding how vertebrates, including human beings, develop from an embryo into a whole organism. Many of the metabolic, regulatory and developmental pathways (and the genes that code for them) are conserved through evolution, so zebrafish and human beings share a considerable amount of genetic information. That’s an essential detail because scientists still don’t understand the functions of many of the genes discovered by the Human Genome Project-or, for that matter, any of the other genome projects involving vertebrates. Understanding the genetic processes involved in development is also a crucial step toward addressing developmental disorders in human beings. The zebrafish may well turn out to be the vertebrate equivalent of the fruit fly, which has played a central role in unraveling invertebrate development and gene function for nearly a century.

Danio rerio has some remarkable properties that make it ideal for this role. Although the zebrafish breeds quickly, and scientists can easily keep thousands of these freshwater fish in the laboratory, the organism’s most striking quality is that the embryo develops outside the mother’s body in a see-through egg and is itself beautifully transparent. Scientists can look inside the developing organism and watch an entire life unfold before their eyes in cellular detail. Moreover, the embryonic development of the zebrafish is very fast. This combination of experimental advantages makes the zebrafish an ideal model to study how internal organs form. Although other fishes may have transparent embryos, they lack other qualities of Danio rerio that make it such an ideal model. Indeed, no other vertebrate model can match the zebrafish when it comes to watching the embryo take shape. Investigations on the zebrafish have already resulted in some fundamental discoveries. Here I provide a brief introduction and review some of the most notable research on this little fish from the Indian subcontinent.

Mutants on the Bounty

The transparency of the embryonic zebrafish allows scientists to watch every developmental process live as it happens-the first beats of a new heart, the formation of muscles, and the growth of blood vessels and nerves throughout the body are all easily observed through the light microscope. Although such observations are crucial to the zebrafish’s success as a model organism, they tell us nothing about how genes control these processes. But there are ways that scientists can disrupt the activity of a gene so that it cannot fulfill its function. The intentional production of such genetic mutants allows scientists to identify the gene’s function by observing the resulting effects on the cells and tissues of the body.

Aquarium hobbyists are already familiar with several zebrafish mutant phenotypes that are available in pet shops. The long-fin mutant grows abnormally long fins, and leopard has spots, rather than the zebra-like stripes that give Danio rerio its common name. The golden mutant lacks some black pigment, and this results in a golden-colored skin. Recently, Keith Cheng and his colleagues at the Pennsylvania State University identified the mutated gene that causes the golden phenotype and showed that differences in this gene also cause variations in the skin color of human beings.

There are now hundreds of zebrafish mutants with defective genes that are involved in the development of one organ or another, and their names often hint at what is wrong with them. Ikarus has tiny or absent pectoral fins. Macho does not react when touched, and Casanova has two hearts. Spock has pear-shaped ears, whereas van Gogh’s ears are very small. The otoliths-small stones in the equilibrium organ of the ear-are loose and lie in odd positions in the rolling stones mutant. When dracula mutants are exposed to light, their blood cells burst and the fish die. An entire ensemble of mutants with abnormally low numbers of red blood cells (and therefore lighter shades of blood) are named after famous wines and varietals, such as chablis, cabernet, chardonnay, retsina, riesling, sauternes and weiƟherbst.

Despite the playful names, the creation and discovery of a new mutant zebrafish can involve a considerable amount of work. Most mutants are produced by chemical mutagenesis using the substance N-ethyl-N-nitrosourea (ENU). When male zebrafish are exposed to ENU, the chemical acts as a potent mutagen and induces single base-pair changes in the germline DNA. The treated zebrafish are then bred twice with wild-type (normal) fish to isolate individual mutations and to multiply the number of fish that carry them. Finally, these mutants are inbred to produce offspring that are homozygous for the mutation. Homozygous mutant embryos and larvae are then examined for phenotypic peculiarities (see figure 6), There are now also methods to specifically “switch off” genes in zebrafish by preventing the production of the corresponding protein. This has become an important method to test for the functions of newly discovered genes. However, ENU-induced mutations remain indispensable in the study of gene function.

A Mutant Named Odysseus

A mutant zebrafish was recently used to answer one of the most intractable questions in developmental biology: How precursors of the germline cells find their way to the developing sex organs. Germline cells have a special status in the body: They form the sperm and egg cells that pass genetic information from one generation to the next. From a biological perspective, the germline is all that remains of an individual after it dies. This makes these cells especially interesting because in some sense they are “immortal.”

The role of germline cells in the succession of generations is also reflected in how they form during embryonic growth. Early in development, the germline cells migrate away from the other cells in the embryo and follow a special “program.” In mice, for example, the precursors of the germ cells arise in the embryo, but then migrate into the yolk sac. Three weeks later the cells are back in the embryo and settle in the location where the sex organs will form. Something similar happens in the zebrafish, where the precursors of the germ cells must travel from their birthplace to the location of the future sex organs.

These observations raise some fundamental questions. How do the precursor cells find their way through the body? What signal directs the wandering cells? Until recently, these were unsolved problems that revealed major gaps in our understanding of germline cells. The solution arrived with the discovery of a zebrafish mutant dubbed odysseus-named after the Greek hero who wandered the seas for years after the Trojan War in Homer’s great epic, The Odyssey. Like the mythological Odysseus who couldn’t find his way home, the precursors of the germ cells in the odysseus mutant cannot make their way to the developing gonads. They wander aimlessly through the body during development and run aground in strange locations.

It turns out that the precursor germline cells with the odysseus mutation wander because they literally cannot find their target tissues. Holger Knaut, in Christiane NussleinVolhard’s group at the Max Planck Institute for Developmental Biology in Tubingen, discovered that a receptor molecule, the protein CXCR4, was the culprit. CXCR4 belongs to a family of receptors that are known to function in the migration of certain somatic cells, but these molecules were not known to be involved in guiding germline cells. Investigators found that the odysseus mutants carry a mutation in the cxcr4 gene, which destroys the CXCR4 protein. The CXCR4 molecule, located on the membrane of the germline cell, acts as a receptor that detects a directional signal released by the target cells. If the receptor doesn’t function, the cells get lost even if the signal is present.

That solved the first part of the mystery, but scientists still didn’t know the identity of the signal. Yet, here again, the zebrafish came to the rescue. Previous studies on mice had shown that CXCR4 interacts with the molecule SDF-1. When scientists looked at the pattern of SDF-1 activity in the zebrafish embryos, they noticed that the molecule maps out the route for the migrating germline cells. When they deactivated SDF-1 in the embryo, the guiding pattern was lost, and germline cells again wandered throughout the body, just as they had in the odysseus mutant. Another experiment confirmed SDF-I’s role as a signaling molecule. If, through a genetic trick, SDF-I is no longer produced in the sex organs, but somewhere else in the body, the germline cells migrate to the new location!

Such studies are only possible because the transparency of the zebrafish embryos allows scientists to track the germline cells.

The Pickwick Papers

The systematic search for zebrafish mutations was instigated with the aim of gaining fundamental insights into the embryonic development of vertebrates. Nevertheless, it soon became clear that some of the defects observed in zebrafish are very similar to hereditary diseases in human beings. Since most of the processes (and their genetic control) that take place during embryonic development in the zebrafish closely resemble those found in Homo sapiens, many diseases that cannot be investigated in human can be studied in zebrafish. Mutants play an important role in this research.

One such mutant is pickwick, which was discovered during the early 1990s in the laboratories of Mark Fishman and Wolfgang Driever at the Massachusetts General Hospital in Boston. The pickwick mutant has a weak heart that contracts very poorly and hardly pumps any blood through the body. Eventually, the heart swells and the mutant dies of heart failure. Fishman, along with Xiaolei Xu, at the Mayo Clinic College of Medicine, and their colleagues decided to take a closer look at pickwick’s heart muscle cells.

Normal muscle cells in the heart contain protein filaments that are attached at opposite ends of the spindle-shaped cells. The filaments from either end are interlocked with each other near the middle of the cell. As the filaments slide past each other toward the center, the ends of the cell come closer together and the cell contracts. When the filaments slide back toward their own ends, the cell relaxes.

Fishman’s group discovered that pickwick mutants contain relatively few filaments, so the cells are thin and can only contract very weakly. So why is pickwick different? The scientists tracked the pickwick mutation to a gene that codes for a protein called titin, which is known to be produced in heart and skeletal muscles. Consisting of nearly 27,000 amino acids, titin is the largest protein ever identified in vertebrates. By comparison, the light-sensitive protein rhodopsin is made of only 348 amino acids. Various forms of titin are important for the proper arrangement and anchoring of the filaments in heart muscle and skeletal muscle. Interestingly, the pickwick mutation only affects the titin produced in the heart muscle cells, not the titin variants produced by other muscle cells, which explains why the skeletal muscles of pickwick develop normally.

The pickwick mutant is especially interesting because its fish heart resembles the heart of a human patient who suffers from dilated cardiomyopathy. In both species, the disease is characterized by a heart with thin walls, swelling and weak contractions. It’s been estimated that these pathological changes have a genetic cause in a third of the patients with this disease. Investigations inspired by the pickwick mutant showed that at least some of these heart patients carry a mutation in the titin gene. Because of the similarities, scientists can use the pickwick mutant to screen for drugs that might alleviate or reverse the effects of the genetic alteration in human patients.

A Fish for the Future

I’ve described just a few of the insights provided by research on the genetics and development of the zebrafish. Well over 400 genes that lead to developmental defects and disease have been identified in the zebrafish, and we can be sure that many more will be found. The zebrafish genome project will undoubtedly play a role in the search for altered genes in zebrafish mutants. Consisting of 1.5 billion DNA base pairs, the genome of the Danio rerio is roughly half the size of the human genome, and scientists expect to complete the sequencing by the end of this year. Of course, the identification of all the genes and their functions will take many more years.

Zebrafish researchers are now unraveling the activity of thousands of genes in normal and mutant zebrafish with DNA chips. This will help us to understand how genes regulate each other’s activity during normal development, and how this regulation is disrupted in disease. Other scientists generate thousands of zebrafish with fluorescent proteins that are controlled by the regulatory sequences of different genes. When a gene becomes active, the cells that express the gene light up with the fluorescent protein, allowing investigators to track gene activity in the developing animal. The results of such studies should provide new insights into the genetic control of basic biological processes in all vertebrates. Indeed, zebrafish scientists are now targeting a wide variety of human ailments, including cancer, behavioral disorders, muscular dystrophies, neurodegenerative diseases, infections and wound healing.

The potential of the zebrafish as a model organism is rapidly gaining recognition, and this is reflected by the actions of major funding organizations in the US and Europe. In 2003, the National Institutes of Health awarded $61 million to extramural research on Danio rerio, and the European Commission’s Directorate General for Research is currently spending 612 million on a pan-European zebrafish research project. There is little doubt that more and more scientists will turn to the zebrafish in their research. It is now one of a handful of organisms that we know more deeply than any other-in some respects even better than we know ourselves.