Muffled exclamations escape from a curtain in a corner of the zebrafish genetics lab. Holed up inside, Ichabod, the headless fluorescent microscope, projects an image of a zebrafish brain, its pallid light playing on the faces riveted to it. The lab belongs to molecular biologist Stephen Ekker, Ph.D., and the faces and exclamations to a team of science teachers viewing a new line of zebrafish that they have genetically modified. The turbo-charged Dr. Ekker thrives on the organized chaos that characterizes his lab. Since his arrival at Mayo Clinic in 2007, he has forged new career directions that enhance his research, increase his chances of impacting human health, and have generated a model for our nation’s science teachers.

A career makeover

It was a bitter irony for Dr. Ekker’s cancer-fighting lab at the University of Minnesota (UM), to discover that every lab member had someone close to them dying of cancer. Realizing that none of their research projects could help these people caused Dr. Ekker to reassess his professional goals, an exercise that led him to join the research community at Mayo Clinic.

“I began a national search for a place that could help me make a difference to peoples’ lives within my professional lifetime,” says Dr. Ekker. “Mayo has seven SPOREs (Specialized Programs of Research Excellence grants supported by the National Institutes of Health (NIH)) and has a well-earned reputation for quickly getting research advances into the clinic. If I can’t do it here, it can’t be done.”

Developing a gene knockdown

Using zebrafish as his primary model system, Dr. Ekker developed a gene knockdown technology, which has since become a standard tool for decreasing gene expression and blocking translation (Nature Genetics 2000). Scientists use these morpholinos to alter a gene’s function and analyze the effect on the phenotype. They refer to this type of genetics study as reverse genetics, in contrast to the classic forward genetics studies that start with the phenotype then search for the gene that is causing the change. Morpholinos offer a key to unlocking the function of thousands of genes that have been identified through the sequencing of the zebrafish genome.

Zebrafish: genomics on a fast track

You may have seen zebrafish (Danio rerio) in a home aquarium. The small, striped fish are also available in fluorescent hues. The colorful varieties are created by adding the gene for a fluorescent protein, extracted from a naturally luminescent jellyfish or sea coral, into a zebrafish embryo. Their availability in pet stores demonstrates how easy it is to use zebrafish for genetic modification.

“Our success rate for making transgenic animals with transposons (genetically transposed elements) is approaching 100 percent,” says Dr. Ekker. “There’s no other organism like that.”

What makes zebrafish an ideal model organism for genetic and developmental studies? Hundreds of offspring, rapid development, and cost effectiveness. Plus the transparency of embryos allows researchers to track regulation of gene expression using fluorescent protein genes in real time in living animals. They are also biologically similar to humans, especially the heart, kidney, pancreas, bones and cartilage.

In 2007, Mayo Clinic, under Dr. Ekker’s leadership, established a Zebrafish Core Facility to give Mayo scientists a quick and efficient model for large-scale testing. Dr. Ekker collaborates with many of the researchers who use the facility to investigate the molecular genetics of vascular development, heart disease, cell signaling pathways and cancer prevention.

New insights into cancer

Dr. Ekker’s research is well funded, including multiple grants from the NIH, in a variety of areas. He is using a morpholino-based screen to identify genetic influence on blood vessel, sensory organ and kidney development. He has also developed a second zebrafish genetic tool, a mutagenic transposon.

“We are using it to do classic developmental biology — we want to know if tumor cells that metastasize to the liver use the same homing device as developing cells,” says Dr. Ekker. “Ultimately, we would like to be able to regulate and control angiogenesis. Understanding blood vessel formation would have broad application in cancer therapy.”

At UM, Dr. Ekker began an important collaboration with Debabrata Mukhopadhyay, Ph.D. Their task, a collaborative venture between Mayo, UM, and the State of Minnesota called The Minnesota Partnership for Biotechnology and Medical Genomics, was to study how nanotechnology can develop new therapies for cancer. Dr. Mukhopadhyay is delighted to have Dr. Ekker on campus.

“Having Steve and the zebrafish core at Mayo is really changing the way we understand vascular biology,” says Dr. Mukhophadhyay. “The zebrafish is a model system for learning about angiogenesis because it mimics the human condition so well and important new tools have been developed that make our research much easier.”

Chris Pierret, Ph.D., a postdoctoral research fellow and stem cell biologist, is working with stem cell genes from cord blood and exploring the role of these genes in the production of blood, called hematopoietic stem cells. The lab is collaborating with Steven Leach, M.D., a pancreatic cancer specialist at Johns Hopkins, to research metastases in fish that develop pancreatic cancer. The next step will be to investigate other cancers for shared pathways and molecular mechanisms as they metastasize.

The lab has published papers describing mechanisms that influence how blood vessels form, including the identification of the cell surface protein, syndecan-2, as a key factor in angiogenesis during zebrafish development (Nucleic Acids Research 2004). Dr. Ekker is using the transposon system to identify new genes and genetic networks and the technology has led to an array of new developmental genetic loci.

Nicotine addiction: the Bette Davis and Humphrey Bogart zebrafish

The tools Dr. Ekker developed lent themselves to investigating the genetics of behavior, so he chose to target nicotine addiction.

“We all know how hard it is to quit smoking,” says Dr. Ekker. “What most people don’t know is that genetic differences significantly contribute to the degree of nicotine dependence. We want to understand the genetics behind different responses to nicotine and come up with more effective and individualized treatments for people addicted to nicotine.”

The lab has already developed a novel nicotine behavioral assay in zebrafish and, using the transposon mutagenesis, applied it to a forward genetic screen. The large family size allowed the lab to detect a single gene contribution to multiple loci behavioral response. These are genes that, when expressed, make zebrafish more responsive to nicotine and, importantly, they are similarly active in humans.

For this work, Dr. Ekker teams with Richard Hurt, M.D., a national leader in nicotine dependence, and Jon Ebbert, M.D., associate director of Mayo’s Nicotine Dependence Center Research Program. At Mayo’s Center for Tobacco Free Living, normal zebrafish swim in an aquarium with fish that the Ekker lab has genetically modified to exhibit a markedly reduced nicotine response. The fish all look the same but the transgenic ones have colorful names, such as Bette Davis and Humphrey Bogart, after film stars renowned for nicotine dependence.

Dr. Ekker is now working with the NIH to develop and test new small molecule compounds to reduce nicotine dependence.

If the end goal is making a difference, it appears that Dr. Ekker is already achieving it.

“Coming to Mayo was an opportunity to recharge,” says Dr. Ekker. “The level of collaboration here is helping us reach our goals.”

Editor’s note: To see Dr. Ekker’s impact on science education, check out this issue’s Spotlight.

— Yvonne Hubmayr, September 2009