NEW YORK (GenomeWeb) – A short-lived fish from the ephemeral ponds of southeastern Africa is set join the worm, fly, and mouse as an important model organism in research on the genetics of aging. At least, that's the hope of the authors behind a paper published today in Cell, a proof-of-concept that the African turquoise killifish (Nothobranchius furzeri) could become an experimental platform.
"It's a really exciting animal. They only stay around for four months at a time, [and] they've evolved a short lifespan to adapt to ephemeral conditions," Stanford biologist Anne Brunet, a co-author on the paper, told GenomeWeb. "Despite this short lifespan, it has signs of aging. It recapitulates, in a miniature way, several of the characteristics of human aging."
The scientists present the killifish as a new vertebrate model organism for studying aging and age-associated diseases that, due to its short life span and large brood size, will allow for more rapid and higher-throughput experiments than existing platforms. The fish exhibits nine of the hallmark signs of human aging: genomic instability, telomere attrition, epigenetic alterations, loss of proteostatis, deregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, and altered intercellular communication. Unlike some fish, like clown fish, sex is determined by the X and Y chromosomes, just as it is in humans. Not even mice, which have extremely long telomeres, would allow for so many different kinds of experiments, Brunet said.
As Brunet established her laboratory at Stanford in 2004, she began thinking about adding another model organism that would help increase throughput on aging studies. Mice live two and a half to three and a half years; zebrafish live about five years. "That's almost beyond the lifespan of a grad student in the lab," she said.
Brunet also considered developing the pygmy shrew as a model, but the fish had several features that led it to win out. They live four to six months and reach sexual maturity in just over one month. They're also extremely easy to care for. Killifish are simple creatures: they live at room temp, eat brine shrimp and blood worms, and, unlike zebrafish, can live in stagnant water. Some people in the lab even keep them as pets at their desks, Brunet said.
But attractive or interesting traits do not a model organism make. Bowhead whales, which can live up to 200 years, and naked mole rats, which can live for 30 years and don't get cancer, have been featured in comparative genomics studies, but could never be an experimental platform.
"It becomes a model when you can manipulate it easily and it can be used to mimic some aspects of biology," Brunet said. Turning the killifish into a model organism turned out to be a long and committed process.
Brunet first learned about the killifish around 2005, when she met Dario Valenzano, who became a post-doc in her lab. At the time, he was a graduate student in Italy and had learned about the fish from Alessandro Cellerino, who was already working on the fish and had proposed it as a model for aging research as early as 2003.
"The key step that was missing for it was that we didn't have the ability to manipulate it genetically," Brunet said. To do that, her lab turned to an increasingly important tool in biology, CRISPR/Cas9.
"Without CRISPR/Cas9 we couldn't have done anything," she said. "CRISPR/Cas9 can transform a lot of interesting species into model organisms."
Post-docs in Brunet's lab helped generate a platform for the field to use: the fish itself and the tools needed to employ it in experiments that could include everything from identifying genes, all the way to manipulating them and testing their effect on age-related phenotypes.
The work of turning an ephemeral fish into a useable model organism with genome editing fell to Itamar Harel, a post-doc in Brunet's lab, and a lead author on the Cell paper. It's easy to do in killifish, Brunet said, because the RNA coding for both the enzyme and the guide RNA can be injected straight into the fish's eggs. Harel simply used the Cas9 enzyme developed for zebrafish.
While getting the CRISPR/Cas9 enzyme to work in the fish was relatively easy, designing the guide RNAs took considerable effort. Brunet's lab had to sequence and assemble the 2.2 gigabase killifish genome de novo, with most of the work done by post-doc Bérénice Benayoun, also a lead author on the paper.
Though Brunet and Benayoun are currently writing a separate paper on the genome, they are already releasing it to the research community to help design guide RNA for use with the platform. "Anyone can look at their favorite gene," Brunet said.
The ability to generate a large amount of data using the killifish, especially compared to mice, could make it useful for both academia and industry, Brunet said, "especially since we have the genome and the genome editing tools."
The Cell publication demonstrates the usefulness of this platform, showing how changes in the fish's genome could reproduce one of the nine hallmarks of aging, telomere erosion. The TERT gene encodes for an enzymatic component of telomerase, a complex that ensures that chromosome ends stay intact. When TERT was deleted from the killifish genome, both male and female fishes became sterile and tissues that are quickly regenerated, like in the gut and in the blood, showed defects. "It really mimics some of the features of the disease dyskeratosis congenita," a human disorder characterized by short telomeres, Brunet said.
Brunet said it would take a sustained commitment to make the killifish a more robust model.
"It'd be nice to be able to induce mutations at some specific time in development or in some specific tissue," Brunet said. "It would be nice to be able to freeze them and do in vitro studies. It's nice that we can maintain the line, but it'd be a lot nicer to be able to conserve, long term, lines we create right now."
Brunet has already thought of several experiments she wants to try with the fish, including studies on aging-related genes, testing drugs that could affect aging, and even the interactions between genes and drugs.
Brunet said her lab has already made other lines of the fish exhibiting other aging and age-associated disease traits, which she wants to share with the research community.
The fish already has attracted the attention of top scientists in the field of aging and age-associated diseases.
"I think the fish are great," Cynthia Kenyon, a leading aging researcher at the University of California, San Francisco, told GenomeWeb. Kenyon's lab is currently collaborating with Brunet's lab to evaluate the effect of certain molecules on lifespan in the killifish. "It needs a little more validation to become totally widely accepted," she said, "but I think it fills a real gap in our ability to assess the relevance of various biological pathways and networks in a vertebrate, economically and on a large scale."
Brunet also mentioned discussions she's had with other laboratories from the University of Alabama, the University of Michigan, and the Stowers Institute for Medical Research.