Scientists have turned to the zebrafish for clues about congenital disease and cancer because this tiny, striped torpedo from the Ganges has many genes in common with humans. In 1991, Leonard Zon wanted to know how embryos produce blood cells. Since zebrafish have see-through embryos that grow outside the mother's body, every step in development can be observed.
As an embryo develops, genes turn on or off in choreographed fashion. If all goes well, red blood cells and various types of white cells form. But if certain genes are faulty or speak up at the wrong time, genetic diseases, such as anemia, can occur. Finding mutated genes sheds light on the causes of disease and can generate more specific treatments.
In 1996, the prospect of isolating any gene from a fish looked pretty slim. Although several microbial genomes had been sequenced, there was no physical map of the zebrafish genome, very few gene markers, and no gene libraries. But Zon regarded the zebrafish as an inch-long packet of genes. In 1997, he helped launch the Trans-NIH Zebrafish Genome Initiative, which will complete the sequence of the fish's DNA in 2007. "One of the great advantages of zebrafish is the ability to produce, very readily, mutations that are relevant to human health and disease," Zon said at the time. "This genomics initiative will superimpose those mutations on disease loci identified through the work of the Human Genome Project."
Using tools they developed en route, Zon's group isolated a mutated zebrafish called sauternes. Because this strain has less hemoglobin and fewer red blood cells than normal fish, its blood looks like white wine instead of red. The researchers pinpointed the sauternes gene on the zebrafish chromosomes and showed that it encoded an enzyme needed to make heme, the iron-containing part of hemoglobin. Therefore, the sauternes mutant became one of the first fish models of human disease because it mimicked humans with congenital sideroblastic anemia (CSA), who also have trouble making heme. This model is enabling scientists to study the ramifications of the mutation, which cannot be determined from human studies.
Another mutant, with a misshapen tail and fatal anemia, lacked blood-forming stem cells, Zon's group discovered. They eventually identified the faulty gene as cdx4, which controls a set of genes that tells the various parts of the body where to develop. When they prevented cdx4 from functioning in normal embryos, they obtained fish with kinky tails. When they overexpressed cdx4, they got a big surprise: the middle layer of the embryo gave rise to blood cells instead of to normal body parts. "We had discovered a new pathway for making blood cells," Zon says.
Zon's lab was also the first fish group to find a new human disease gene. The weissherbst mutation (again named after a wine) prevents iron that is stored in the egg yolk from being circulated into the embryo. After isolating this gene, the researchers found that the normal version encodes ferroportin, a protein that transports iron. In humans, ferroportin is found in the placenta, where it is thought to carry iron from mother to fetus. After birth, it transports iron from food across the intestinal wall. The weissherbst mutation was recently discovered in people with autosomal-dominant hemochromatosis, an iron-overload disease. "This was the first time a fish mutant predicted a new human disease," Zon says.
Zon's group has now collected more than 40 groups of zebrafish mutations that affect blood development, and their work has spawned new ideas for treating human conditions. For example, they found that Notch, a gene widely involved in development, also helps blood cells renew themselves. Using clever genetic techniques, they made a zebrafish in which Notch could be activated by a heat pulse. Then they pulsed fish whose blood cells had been destroyed with radiation. These fish started making new blood cells much faster than fish that had not received a heat pulse. "This suggests that if we had a pharmaceutical compound to activate Notch transiently, it could restore the blood system more quickly in patients who are given stem cell transplantation," Zon says.
Recently, Zon's group undertook a chemical genetic screen in zebrafish and found that prostaglandins stimulate blood stem cell production. In particular, prostaglandin E2 can increase blood stem cells during embryogenesis and in adulthood in zebrafish and mice. A treatment of prostaglandin to mouse bone marrow leads to a threefold increase in stem cell number. The group is currently investigating clinical trials to treat cord blood samples with prostaglandin E2, in an effort to increase the number of stem cells in that sample.
To more fully understand how mutations discovered in zebrafish affect blood development in humans, Zon wants to study human embryonic stem cells. To give stem cell biologists a voice in this controversial issue, he and two other scientists founded the International Society for Stem Cell Research (ISSCR) in 2002. The organization disseminates information about stem cells, promotes stem cell research, and educates the public about potential applications. In 2007, for example, ISSCR released its Guidelines for the Conduct of Human Embryonic Stem Cell Research. "These guidelines are critical to allowing the international community to move forward with ethical considerations in stem cell research," Zon says.