March 28, 2003

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Fruit Fly Cells Reveal Hedgehog's Secrets

A Johns Hopkins-led research team has successfully used a technique to rapidly find fruit fly genes involved in a cell signaling pathway called Hedgehog, which is critical to proper embryo development and a key trigger in some cancers, including the deadly childhood brain cancer medulloblastoma.

By using the technique, called RNA interference, to "knock down" the messages of specific genes in fruit fly cells, researchers from Johns Hopkins and the National Institutes of Health screened 43 percent of the fruit fly's genes and found four new players involved in passing along Hedgehog's signal.

Reporting in the March 28 issue of Science, the scientists also suggest that the human counterparts of the fruit fly genes may be involved in birth defects and cancers characteristic of abnormal Hedgehog activity.

"Thanks to the human genome draft, we can make fairly direct connections between what we find in fruit flies and human disease," says Phil Beachy, Ph.D., a Howard Hughes Medical Institute investigator and a professor of molecular biology and genetics in Johns Hopkins' Institute for Basic Biomedical Sciences. "Our evidence suggests that human versions of these genes may be involved in disease, acting through the Hedgehog pathway."

In developing embryos of many organisms, including fruit flies, mice and humans, the Hedgehog signal ensures appropriate patterns of key proteins and cell types. Without the signal, these early developmental steps are disrupted, and severe -- usually fatal -- birth defects result. In some cancers, unnecessary activity of Hedgehog causes excessive cell growth. Understanding Hedgehog's signal one day may help prevent or correct these situations, say the researchers.

The Hedgehog pathway starts with a protein by the same name and ends with another protein that directly regulates the activity of a host of genes. A few key intermediate players have been identified and studied in depth, but much is still unknown, says Beachy. (Hedgehog protein gets its name from the spiky pattern of certain cells that result in fruit fly embryos without it.)

RNA interference, which "knocks down" a gene's message in cultured cells rather than "knocking out" the gene in a whole organism, can help quickly fill in the gaps in the Hedgehog pathway, the scientists discovered. Unlike creating a gaggle of mutant fruit flies with unknown genetic changes, RNA interference lets the scientists keep track of exactly which gene is being compromised in dishes of cells.
"Mutant screens are very useful, but we've shown that RNA interference in cultured cells can very quickly look for genes involved in a process whose activity can be measured," says Beachy. "If that process is highly conserved between fruit flies and people, like Hedgehog is, it's a quick way to identify human genes that may be of interest."

In RNA interference in fruit fly cells, extra bits of RNA corresponding to a known gene are taken up by cells in laboratory dishes. The added RNA guides the action of an enzyme that destroys the cells' own version of the RNA, preventing it from being used to make a protein.

Postdoctoral researcher Lawrence Lum tested the effects of RNA interference on Hedgehog's signal in cultured fruit fly cells by using RNA bits made by colleagues at the NIH. Along with these, Lum added a "reporter" gene that responds to a complete and functioning Hedgehog pathway by activating its instructions to make a glowing protein. If the added RNA blocks something critical to Hedgehog's signal, production of the glowing protein changes as well.

"Since we use an instrument to determine exactly how bright the sample is, we can measure the cell's response to Hedgehog and compare the effects of blocking different genes," says Beachy. "With the technique, we were able to identify all the genes already known in the Hedgehog pathway, another set of genes that altered expression of the reporter gene but are not specific to Hedgehog, and four genes that had never been named as components of the Hedgehog pathway."

Of these four fruit fly genes, two -- dubbed Dally-like and casein kinase 1alpha (CK1alpha) -- had been associated with signaling by a protein called Wingless, whose effects are similar to Hedgehog. Dally-like protein is actually more critical in the Hedgehog pathway than in Wingless, they report. The third gene, caupolican, was thought to be a target of Hedgehog, not a regulator. The fourth gene, known as CG9211, had never been associated with any developmentally important signal.

It turns out that the human or mouse equivalents of three of the newly identified genes --Dally-like, CK1alpha and CG9211 -- appear to be linked to developmental defects that are characteristic of disruptions in Hedgehog signaling, notes Beachy, who plans to screen the rest of the fruit fly genome and examine closely the roles of the four newly identified genes.

The study was funded by the National Cancer Institute, a Life Sciences Research Foundation Fellowship, and the Howard Hughes Medical Institute. Authors on the study are Lum, Beachy, Shenqin Yao and Doris Von Kessler of Johns Hopkins; and Brian Mozer, Alessandra Rovescalli and Marshall Nirenberg of the National Heart, Lung and Blood Institute.

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