Volume 17 Number 3 FALL 2000

zebrafish: a model for life

translucent fish offer advantages over furry mice

By Kristin Weidenbach BY MICHAEL MA

HUMANS HAVE A LOT IN COMMON WITH FRUIT FLIES AND ROUNDWORMS, BUT WE ARE EVEN MORE LIKE ZEBRAFISH – AT LEAST ON THE GENETIC LEVEL. That's one reason the pinky-sized, striped, freshwater fish is the latest entrant in biologists' model-organism menagerie. * Scientists typically turn to model organisms to help answer genetic questions that cannot be easily addressed in humans. Though scientists have learned a lot from worms and flies, model organisms that are only distantly related to humans, some find vertebrate models -- animals with backbones -- more appealing, because of our closer kinship to them. For many of these researchers, the laboratory mouse reigns supreme in the world of vertebrate model organisms: Intricacies of the human immune system were first revealed in mice and cancer is "cured" many times over in mice before promising therapies are ever tested in humans.

But over the past 10 years, the mouse has been facing increasing competition from the zebrafish, a.k.a. Danio rerio, a typical denizen of home aquariums. What's so alluring about these fish? For starters, zebrafish develop quickly. A zebrafish progresses from fertilization to a freely swimming creature in five days; a mouse requires 21 days to advance from fertilization to birth. The fish also have modest housing requirements. For example, developmental biologist William Talbot, PhD, an assistant professor at Stanford's medical school, keeps 10 to 15 thousand zebrafish in his "fish room," where 10 to 12 fish can reside comfortably in each 2-liter tank.

But perhaps most important, zebrafish develop in a transparent egg -- not in the mother's uterus, as mouse embryos do. This means that researchers can create mutant fish with genetic defects and see with their own eyes the resultant changes in the developing fish. They can even study lethal mutations that render the animal unable to survive after it hatches. Lastly, the wealth of data already gathered during previous genetic studies in zebrafish gives them a head start over other small fish such as the Japanese medaka, which are also prolific breeders with transparent embryos and commonly used in research.

TALBOT IS PART OF A SMALL BUT GROWING COMMUNITY OF RESEARCHERS WHO ARE ATTEMPTING TO CHART ALL THE ZEBRAFISH GENES. He believes that the zebrafish species is an excellent model for learning how humans and other animals with backbones develop. Mutant fruit flies and roundworms have taught scientists much about early animal development, he says, but scientists need better models to learn about the organs and systems specific to vertebrate animals.

Talbot is in charge of the National Institutes of Health-funded Stanford Zebrafish Genome Project. Members of his laboratory, in collaboration with colleagues in the lab of John Postlethwait at the University of Oregon, recently completed a genetic map (published in the April 15, 2000, issue of Genome Research) that shows the location of more than 1,300 zebrafish genes and genetic markers -- distinctive regions of DNA that genetic researchers use to orient themselves in the genome. The genes and markers on their genetic map act like reference points on the gene-carrying chromosomes in the same way that small towns help travelers orient themselves on a highway. More landmarks make it easier for travelers to find their way on the road and for gene hunters to find a particular gene or stretch of DNA.

The zebrafish map is coming in handy to scientists around the world who are trying to learn which gene defects cause which abnormalities in the thousands of zebrafish mutants they have created. Matching mutated genes with biological problems reveals the function of those genes in normal, non-mutated individuals. A genetic map like the one created by Talbot's group is then used to isolate the gene. With the gene in hand, it is easier to find and study the counterpart human gene. "One of the first things we do when we find a new zebrafish gene is see if there's a human counterpart -- and if so, where it resides in the human genome," Talbot says. Pairing the zebrafish genes with their human equivalents allows the researchers to suggest likely functions for human genes that may be known only by their DNA sequences.

SCIENTISTS IN TALBOT'S LAB MOSTLY STUDY EMBRYOS THAT HAVE LETHAL MUTATIONS AFFECTING THE BASIC LAYOUT OF THE BODY PLAN, INCLUDING THE DEVELOPMENT OF THE MUSCULAR AND NERVOUS SYSTEMS. In many of these mutants, bodily organs are missing or in the wrong location. Some of the syndromes that Talbot studies in zebrafish resemble severe human abnormalities, such as holoprosencephaly, in which the forebrain fails to divide and the face fails to form properly. According to Talbot, four of the genes that contribute to the condition have been identified but others remain to be found. "Some of our fish genes might give us a clue about the human ones," says Talbot. "There is a lot of interest and enthusiasm."

For Stephen J. Smith, PhD, professor of molecular and cellular physiology, the zebrafish's transparent embryo is a bigger draw than its well-studied genetic makeup. Smith says that he and the members of his lab are trying to figure out how brains develop. "There are billions of neurons and they have to be wired up to their right circuits, and no one knows how they do that, except that it's complicated," Smith says. For Smith's purpose, zebrafish are an excellent model because he can look directly inside the translucent animal and watch neurons growing before his eyes.

According to Smith, the central question of neuroscience is how the brain generates behavior. "Zebrafish provide an outstanding opportunity to study the behavior of a vertebrate animal at an early stage of development. You can study its nervous system, its growth and development patterns at the times when it's having its first and simplest behaviors," he says. "The corresponding behavior in mammalian embryos starts long before birth. By the time a rat is born it has a horrendously complicated nervous system."

Members of Smith's lab currently are focused on understanding how synapses are assembled. The synapse is the junction between the axon -- the portion of a neuron that transmits nerve impulses -- and the dendrite -- the portion of a neuron that extends from the cell body and receives nerve impulses. The particular synapse that Smith's team is studying has a crucial role in early development of a vertebrate animal. This synapse establishes the ability of the fish to move and swim. If it doesn't form correctly, the animal will die.

Using colored dyes and sensitive microscopes, the researchers have recorded how the neurons on each side of this synapse connect to each other in the spinal cord of developing zebrafish embryos. In the March 2000 issue of Nature Neuroscience, the researchers described how ephemeral projections that shoot out from the neuron like spindly arms reach toward each other to establish the synaptic connection. "They can't form a synapse until they touch each other so they're both reaching out like crazy for the other one," says Smith.

Colleagues in his lab first documented the moving projections 10 years ago. "It's been a crusade to find out what they mean," says Smith. He now has time-lapse video images from living zebrafish embryos that clearly show the tiny "arms" embracing as the two neurons encounter each other.

These kinds of images can be obtained only because of the transparency of the fish embryo. "In an undisturbed embryo we can visualize cells at a very high level of resolution," Smith says. "In zebrafish we are able to identify particular neurons from fish to fish. Mammals have a much larger brain and more neurons. You can't really single out one neuron in a mammal." Furthermore, mammalian brains cannot be studied in situ. The experiments that Smith's team does with rats can only be done with brain slices thin enough to allow light to pass through, so that the internal structures can be seen under the microscope. The brain-slice strategy's drawback: "You don't have a whole behaving organism there to study any more," he says.

As scientists at Stanford and other centers of zebrafish research continue learning about the inner workings of the fish, its value as a model organism keeps increasing. And unlike many scientific fields of study, where competition between researchers is intense, the small but rapidly growing group of zebrafish researchers prefers to work together to achieve their scientific goals, according to Smith, who counts himself as a peripheral member of the community. "It hasn't got a competitive flavor to it yet. It is relatively helpful and really quite delightful," he says.

You could say that from Smith and Talbot's perspectives, the advantages of zebrafish research are clear. SM

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