Genetic fishing expedition yields surprising catch important to mammals

Johns Hopkins investigators report the discovery of master controllers of a gene critical to human and all mammalian development by trawling, implausibly enough, through anonymous genetic sequences using tiny zebrafish embryos.

In an article in the September issue of PLoS Genetics, they and a team of government scientists describe experiments that identified bits of "non-coding" DNA known as enhancer sequences that don't carry a blueprint for a protein but instead, in this case, regulate a very specialized developmental gene.

That gene, SOX10, codes for products essential for making the neural crest, a group of cells that form at the top of a spinal cord-to-be and then migrate throughout the body to form a wide variety of tissues. When the genetic machinery that controls neural crest development goes awry, human diseases can result; for instance, Hirschsprung's, a developmental disorder characterized by an absence of nerve cells in the gut that help move waste through and out of the body.

That the scientists identified mammalian regulatory sequences by testing them in zebrafish - creatures with backbones but separated from humans by several hundred million years of evolution - bodes well, they say, as a much simpler tool than the standard mouse models for investigating the deep nooks and crannies of human gene regulation.

"The zebrafish strategy is, at least for this gene, a high-fidelity surrogate for analyzing hard-to-find mammalian regulatory sequences," says Andy McCallion, Ph.D., an assistant professor in molecular and comparative pathobiology and member of the McKusick-Nathans Institute for Genetic Medicine at the Johns Hopkins University School of Medicine.

"The goal is to read the entire genome like a comprehensible book, not just random letters and words," McCallion says "Granted, the noncoding regulatory sequences read a little bit more like James Joyce than Hemingway, but gaining understanding is far easier in transparent zebrafish because we can actually study embryonic development more readily in short periods of time. Luckily, the regulatory sequences are highly similar from one species to the next, so we get an edge both ways."

Fishy though it may sound, it's not your genes that make you you so much as it is regulatory sequences that control the genes that make the proteins. By turning genes on and off at various times and under changing conditions, these master switches underlie cells' diversity by instructing them, despite identical genetic makeup, to differentiate into all the various kinds of cells that make up a body.

Because a mouse shares more than 95 percent of its genes with a human, it is an obvious model in which to study mammalian genes. But McCallion wasn't convinced that mice were an expedient way to discover SOX10's enhancers, one of countless regulatory sequences that remain below the radar of current computer-based screening analyses. So the team used zebrafish, an abundant, inexpensive and transparent animal that allows easy real-time observation of the neural crest in developing embryos.

Earlier research conducted by William J. Pavan, Ph.D., a senior investigator at the Genetic Disease Research Branch National Human Genome Research Institute, had shown that mice missing a DNA sequence located far from the SOX10 gene showed symptoms that collectively resembled Hirschsprung's disease. "These were indicators that potential sequences residing within that deletion were likely to be critical for directing regulation of the SOX10 gene," McCallion says.

Despite overall differences in the regulatory sequences of mice and zebrafish, when scientists put mouse regulatory sequences tagged with green fluorescent "reporter" proteins into developing fish embyros, they lit up as if they were telling us that "here is where the SOX10 gene is turned on in zebrafish," McCallion says. "It was a complete, highly accurate recapitulation of the expression of SOX10 in zebrafish."

The ability to locate hard-to-find and therefore largely neglected regulatory sequences could help researchers find mutations in patients who, for instance, have neural crest disorders such as Hirschsprung's in which SOX10 is suspected of playing a role.

The research was supported by the Human Genome Research Institute Intramural Research Program and with funding from the National Institute of General Medical Sciences.

Authors on the paper are Anthony Antonellis, Shih-Queen Lee-Lin, Gabriel Renaud, Stacie K. Loftus, Tyra G. Wolfsberg, Eric Green and William J. Pavan of the National Human Genome Research Institute; and Jimmy L. Huynh, Ryan M. Vinton and McCallion of Hopkins.