By David N. Leff

When in the mid-1980s the smartly striped, minnow-sized zebrafish (Danio rerio) swam out of the pet shop into the research lab, the other animal models - yeasts, fruit flies, nematodes, mice, rats and chicks - had to sit up and take notice.

"The zebrafish [ZF] lives in a very unusual niche for a model organism," observed molecular biologist Stephen Ekker, University of Minnesota, Minneapolis. "It's a vertebrate, so it has many of the same biological structures that humans have. Obviously, ZF doesn't have fur or lungs. But it does have gills and all the major mammalian organs. Yet it's an egg-laying animal.

"Zebrafish," Ekker said, "are very easy to work with. You can do classical genetics with it, and it's been used for genetic screening - similar to what's been done with the fruit fly. That established ZF as a genetic model system that people are using to study vertebrate processes that you cannot model in classical genetic organisms such as the nematode or Drosophila. That's ZF's niche role."

Adorning that niche are the many unique pluses that D. danio brings to the human-surrogate party: The tiny fish are cheap and easy to maintain. Their bodies are transparent as glass, making mutants a cinch to spot. It's oviparous - eggs are fertilized and grow outside the mother's body - so embryonic development can be followed with ease. And as a vertebrate, its genetic aberrations closely parallel some that occur in human diseases.

Ekker noted that the Wellcome Trust's Sanger Center, in Britain, "has announced that they're going to do the zebrafish genoma - sequencing it entire. That means that it's likely ZF will be the first non-mammalian vertebrate whose sequence will be completed. That will be done in a couple of years - by 2002." Next month, he added, "the Sanger Center is having a genomics meeting to plan this, and I'll be at it as well as 30 other fish scientists, to outline what will be done over the next two years."

But clouding D. danio's primacy as a model lab model, there's one major hitch in ZF's niche: Unlike the other members of the club, it's devilishly difficult to knock out ZF genes in order to determine the functions of the proteins they encode. And the genomes of all organisms already sequenced list a preponderance of "genes of unknown function."

From Knockout To Knockdown

The favored current method of making transgenic knockout organisms relies on the strategy of reverse genetics. Ekker explained: "Reverse genetics is taking information that you have from the genome sequencing projects, and modify the gene based on that sequence. Then you assay the function, as a result of perturbing the sequence. That's what's done in mouse embryonic stem cells or in yeast to make a transgenic gene knockout that has these perturbations in them."

However, despite the best efforts of fish scientists, including Ekker, D. danio stubbornly declined to play a hand in that game. Now the Minnesota researcher has laid down a wild card to trump ZF's standing pat. It's called the morphelino - a man-made RNA construct that causes a gene of interest to fold.

"Morphelinos are modified nucleic acids," Ekker explained. "They work to take sequence information and remove the gene function by inhibiting the ability of the RNA to translate.

"The advent of morphelinos," Ekker pointed out, gives the zebrafish a new role, in that it's the first vertebrate in which you can consider doing the equivalent of reverse genetics. So now you can do all these things that are difficult or expensive or impossible to do in the mouse."

Ekker is senior author of a paper in the October issue of Nature Genetics, released Sept. 27, 2000, which reports applying this novel technique to two human developmental diseases. Its title: "Effective targeted gene 'knockdown' in zebrafish."

"The first is a very common disease called holoprosencephaly [HPE]," he said. "In fact, one in 16,000 children are born with it. HPE's extreme manifestation is a cyclopic child - with only one eye, in the middle of its forehead. It's associated with mental retardation and other major pediatric problems. We showed that there is a genetic locus that will cause these children to have HPE. And that gene locus is called sonic hedgehog - one of the first HPE loci to be characterized.

"We also showed that zebrafish have two copies of that gene," Ekker added, "and if we remove both of them using morphelinos, we end up with HPE fish, targeting the same gene. And it's the first example of the same gene that when mutated or removed causes the same effect in fish and humans. Understanding the disease at a molecular level," he pointed out, "is the first step in the ability to design treatments.

"A second example cited in our paper," Ekker went on, "is represented by a rather rare but dominant blood ailment called hepatoerythroporphyria (HEP). It turned our ZF into vampire fish, with luminescent blood that is photosensitive. If HEP patients go out in the sun, they may die because their blood will die in the light. They're always anemic and pale because they always have a low blood count from running across light periodically. And always they need iron to replenish their constant loss of red blood cells.

"Porphyria," he continued, "is a classic metabolic disorder of blood formation. There is a mutation in the fish that gives the same effect. Morphelinos allowed us to generate tens of thousands of porphyric fish embryos. These would be readily suitable for screening compounds for drugs to treat prophyria."

Next In Line: Antitumor Anti-Angiogenesis

Since their journal paper was accepted, Ekker continued, "We've targeted the gene that's involved in blood vessel formation in mice, and found that we get the same phenotype in fish. We took out the vascular endothelial growth factor VEGF, which took the vasculature - the blood vessels - completely out of the fish. It was very clear that in order for tumors to grow, zebrafish are a perfect model system for going after anti-angiogenesis. But VEGF is just the beginning. Our concept is to use the morphelinos to go after all the angiogenic factors. Each one of those would now be potentially amenable to drug targeting in humans."

Ekker this month founded a company, Discovery Genomics Inc., also in Minneapolis, with which the university is negotiating to license his invention.

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