Zebrafish Reproductive Development May Hold Insights into Ovarian Cancer
For thousands of years, animals have helped humans advance biomedical research. Early Greeks, such as Aristotle and Galen, studied animals to gain insights into anatomy, physiology and pathology. Today, model organisms, like mice, help researchers understand human diseases, opening the door to potential defenses and new therapies.
With funding from the National Institutes of Health and the National Science Foundation, Associate Professor Bruce Draper, Department of Molecular and Cellular Biology, is studying zebrafish (Danio rerio) to learn about the genetics of sexual reproduction in vertebrates. Draper’s research, published in PLOS Genetics with postdoc and Dena Leerberg, ’17 Ph.D., may advance discoveries into the origins of ovarian cancer.
Zebrafish, a model species
In the basement of the Life Sciences Building, around 1,500 fish tanks, ranging in size from briefcases to crates, are systematically laid out in rows on metal shelves. Viewed from one end of the room, some of the tanks look empty. But they’re filled with zebrafish, some no larger than the length of a fingernail and narrow as twine. You wouldn’t know it from their size, but these tiny fish, flittering about to the hum of the facility, are making a big splash in scientific research.
Zebrafish have become a popular model organism in biomedical research. Humans share some 70 percent of DNA with the fish, and 84 percent of the genes associated with human disease have counterparts in zebrafish. These genetic relationships make zebrafish a valuable tool for researchers.
The fish breed year-round and have a quick generation time of about three months. A single breeding pair is capable of producing three to five hundred fertilized eggs in one day. They also live in freshwater, reducing the care and maintenance required for saltwater species.
But what makes the zebrafish species a truly powerful model organism is its transparency. Zebrafish embryos are clear, providing scientists a window into the biological machinery behind the fish’s formation.
“If you’re interested in studying the process of early development, all those eggs develop outside the mom,” said Draper. “From the very first cell division, you can see them.”
A live window into reproductive growth
A live, 35-day old, transgenic (ziwi:EGFP) juvenile zebrafish expresses green fluorescent protein in all germ cells. Bruce Draper
Zebrafish embryos develop rapidly. Within 24 hours, they have a tail, head, patterned brain and the inkling of ears and eyes. By day two, their hearts start beating, and by day five, the minuscule fishes are swimming and feeding on their own.
Draper and his team study the fish’s early life stages to learn about the development of the vertebrate gonad, the organ responsible for producing sperm and eggs.
While sexual development in zebrafish usually begins around the fifth day of development, Draper and his researchers usually start their observations around day 10. But by that time, the gonad is much more difficult to see. As zebrafish age, their skin loses transparency and develops pigments and stripes.
“Studying gonads at 12 days of development is not for the faint of heart,” said Draper. “The fish are tiny, and the gonads are even tinier.”
To ease visibility, Draper and his researchers breed fluorescent zebrafish with gonads that glow under ultraviolet lights. The ability to fluoresce provides valuable bio-indicators which reveal important information about the fishes’ chemical makeup at the cellular level.
Under the microscope, the gonad glows distinctly. This lighting effect provides an otherworldly view—a living window into zebrafish reproductive development.
Draper and Leerberg focus on reverse genetics, blocking the expression of certain reproductive genes to see how zebrafish development is affected.
“Genetic tools such as CRISPR and Cas9 are now extremely efficient in zebrafish,” said Leerberg, a recipient of an NIH T32 predoctoral training grant in Molecular and Cellular Biology. “If I come up with a hypothesis today that a particular gene is required for a particular process, I can test that hypothesis in a living vertebrate in six months. The process used to take years.”
In one experiment, the researchers switched off the gene responsible for producing immature egg cells.
“The remarkable thing we discovered was once they became sterile, the female’s sex-reversed and became male,” said Draper. “That was completely unexpected because adult zebrafish, normally maintain their sex.”
In the wild, about two percent of teleost fish switch sex naturally. By knocking out the gene associated with egg production, Draper and his researchers discovered the latent ability for sex reversal in zebrafish.
The exploration of changes in gender has led to some novel discoveries with potential implications for cancer research.
Seeking the genetic switch for cancer
For Draper, science can be serendipitous. Sometimes the path to discovery doesn’t make sense until it’s in the rearview.
During his postdoc years, a gene called fgf24 caught Draper’s attention. He discovered when fgf24 was switched off in zebrafish, they wouldn’t develop proper pectoral fins. At the time, he suspected the gene was involved in reproductive development, as the adult zebrafish were also sterile.
Years later, with postdoc and former student Leerberg, Draper confirmed that zebrafish with the gene fgf24 switched off developed defective gonads and limited reproductive abilities.
“If the somatic gonad is defective, then the germ cells that would produce the gametes for the next generation don’t do what they’re supposed to do,” said Draper. “They fail to proliferate, and most of these animals grow up sterile.”
While Fgf signaling isn’t known to be involved in mammalian early gonad development, many aggressive ovarian cancers correlate with an overactive signaling pathway related to the gene. Signaling pathways control functions such as cell division and death. Excessive activation of these pathways can lead to the development and proliferation of cancer cells.
“It is quite common that developmental pathways and mechanisms are co-opted by cancer cells to promote tumor growth,” said Leerberg. “Several Fgfs, their receptors, and their downstream targets are linked to many cancers, including ovarian.”
Draper is interested in exploring whether the pathway is involved in the sexual development of mammals, and looks to explore the relationship in mice models next. One of Draper’s graduate students is breeding zebrafish with an overactive Fgf pathway to see if they can develop an ovarian cancer model in zebrafish.
Switching on Fgf signaling alone may not be enough to cause cancer to develop, but it may provide clues to the complexities of ovarian tumor formations.
“You usually have to have multiple things go wrong before there’s cancer,” said Draper. “So there’s a high probability that making just one thing go wrong won’t do that, but it’d still be a significant result. But if it holds true in fish, more likely than not, it’s going to be true in mammals.”