By David N. Leff

Spiffily striped zebrafish, just over an inch in length, have long swarmed in home and office aquaria. But a decade or so ago, these finny pet-shop favorites began swimming into biology research laboratories worldwide.

Developmental biologist Steven Farber, an assistant professor at Thomas Jefferson University in Philadelphia, counts the ways that Brachydanio rerio, the zebrafish, outpoints the mouse as an animal model:

¿The classic advantages of the zebrafish,¿ Farber observed, ¿are optical clarity of the embryos. They¿re abundant, accessible, rapidly developing. By 30 hours, or a little more than a day, you can see their embryonic heart beating.

¿And B. rerio,¿ he continued, ¿is a vertebrate. We now know from the Human Genome Project that for the most part, every gene that¿s in a fish is in a mammal, and vice versa, with similarity at the protein level. That makes the zebrafish a powerful model system for vertebrate biology.

¿Further evidence that there¿s a parallel between fish and people,¿ he recalled, ¿is given by the fact that Lipitor ¿ one of the most widely prescribed drugs for treating hypercholesterolemia ¿ works as well in fish as it does in people. It interfered with the processing of our lipid reporter.

¿Finally,¿ Farber pointed out, ¿it enables one to do genetics ¿ what we refer to as forward genetics. We randomly made mutations throughout the zebrafish genome, not knowing what we¿d mutated, then created families with suites of mutations. We screened through a family by setting up crosses between siblings, then looking for a particular phenotype.

¿Up to this point,¿ he added. ¿most of the screening was what we could see with the naked eye. Especially since the zebrafish [ZF] is optically transparent, we could see if the heart beat correctly, did the organs form ¿ things that are apparent by eye. I think many of those mutations have been identified, but my interest was to visualize biochemical processes in vivo, which is one reason I picked the optically clear ZF.¿

Farber is first author of a paper in the current issue of Science, dated May 18, 2001, titled ¿Genetic analysis of digestive physiology using fluorescent phospholipid reporters.¿

To probe their ZF five-day larvae¿s digestive processes in vivo, he and his co-authors devised a color-coded fluorescent probe. ¿The way we did that,¿ he recounted, ¿was to create phospholipid molecules that change their spectral properties and their colors when they¿re enzymatically processed. To that end we designed two probes. One of them changes from orange to green when it¿s cleaved; the other is completely nonfluorescent, until it¿s cleaved. The enzymes that do this to these lipids are important for digestion,¿ Farber noted, ¿but they also play a role in cancer.¿

Optical Biosensor Lights Up Gut Enzymes

¿If you look at a zebrafish it looks like a fish,¿ he observed. ¿You can see the intestines, the liver. But when we put the fluorescent reagent into the water, they had to swallow it, and absorb it from their intestines. In the process of digesting it, they used enzymes that cleave it or eat it. And when they do that this fluorescent agent lights up. It¿s like an optical biosensor, or a lipid enzyme reporter, visualizing to us the digestion of the molecule by becoming fluorescent.

¿One major result,¿ Farber observed, ¿was that when we put this reagent into the fish tank water, we got a brilliant fluorescence from the gall bladder. We knew immediately this would be a powerful genetic screen, because if fish were anything like mammals, getting lipids into the gall bladder meant cleavage in the intestine, transport of lipoproteins to the liver, which bounces the stuff into the gall bladder, and then the gall bladder squirts it back into the intestinal lumen.

¿So our bottom line was that this would encompass many genes that are clinically relevant, for example, the low-density and high-density lipoproteins ¿ LDL and HDL ¿ which play a role in hypercholesterolemia.

¿When we put a fish that¿s normally nonfluorescent into fluorescent cholesterol,¿ Farber went on, ¿we don¿t see anything under the fluorescent light. When we add the cholesterol, it becomes brilliantly labeled, like glow in the dark. That¿s what a wild-type ZF would do. It would glow in particular places ¿ like the gall bladder, a brilliantly fluorescent ball. The mutant fish just looks as if we didn¿t put in anything fluorescent.

¿When we put it in a fluorescent cholesterol there, too, we didn¿t see normal cholesterol processing, indicating that the fat-free mutation ¿ the fat-free gene ¿ is important for dietary lipid absorption. The fat-free larvae looked normal when we screened them, but by day eight or nine they started to die.

¿In a normal zebrafish, we knew what the pattern of fluorescence looked like. So all we had to do was put our experimental randomly mutated ZF larvae under a sort of glow-in-the-dark situation, and we could see, Aha, here¿s a fish where the fluorescence is not going where it should go.¿ Without it, we didn¿t know anything, because the larvae ¿ especially in the case of the fat-free gene mutation we identified ¿ looked OK. They failed to cleave our special phospholipid.

¿So you can imagine,¿ Farber commented, ¿there are a lot of people interested in knowing what this putative fat-free gene is. Our lab is in the process of racing toward it. Right now we¿re within about 5 million bases to the gene. And in this day and age that is not that far. We don¿t know what it is but we¿re getting close to knowing what it might be.¿

First, Get Fat-Free Gene Out Of Closet

¿We¿ve looked at thousands of mutations and never seen that before. We¿re pretty sure that it could be a problem in the liver; that the gene regulates the lipid secretion, or it could be in the intestine ¿ some kind of a transport molecule, and without it you can¿t move these lipids around. But either way, by impairing this gene, its protein is likely to be a way to regulate lipid processing. And we won¿t know until we¿ve identified the gene.

¿Once we identify fat-free,¿ he pointed out, ¿we¿ll have a gene that powerfully controls cholesterol processing. The big pharmaceuticals would love to know what that gene is, because it could be a very powerful drug target to regulate cholesterol levels in people.

¿The immediate implications,¿ Farber concluded, ¿would be for treating problems in lipid processing ¿ such as hypercholesterolemia, atherosclerosis and obesity, because the gene seems to regulate fat utilization.¿

No Comments