April 08, 2005
Genetic Tool Reaps Rich Harvest
In one fell swoop, scientists have increased from dozens to hundreds
the number of known genes that control crucial steps in the development
of many organisms from fruit flies to humans.
Using the power of a genetic technique known as RNA interference,
the researchers identified some 238 potential regulatory genes in the
Wnt signaling pathway. Understanding this pathway will provide
researchers with new insight into the development of cancers of the
liver, colon, breast, and skin, as well as other genetic diseases.
“Before, it would take years to identify a few genes. But with this technology, we can identify in only weeks all the genes that are functionally involved in a pathway.”
Norbert Perrimon
The scientists published their functional genomic analysis of the
Wnt pathway in the April 8, 2005, edition of Science Express,
which provides rapid electronic publication of selected papers in the
journal Science. The research was conducted by lead authors
Ajamete Kaykas and Ramanuj DasGupta. They work, respectively, in the
laboratories of senior authors Randall Moon at the University of
Washington School of Medicine and Norbert Perrimon at Harvard Medical
School. Both Moon and Perrimon are Howard Hughes Medical Institute
investigators.
 | | | | |  |  | | | | |  |  | Immunostaining for Drosophila Wingless protein
Immunostaining for Drosophila Wingless protein (in green) and its target gene Senseless (in red)... more
Image: Courtesy of Norbert Perrimon, HHMI at Harvard Medical School
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The Wnt signaling pathway, which has been evolutionarily conserved
in organisms, governs an array of processes central to cellular
development and function. These processes include cell proliferation,
differentiation of cells into specialized tissues, and establishment of
distinct regions within the cell. Changes in the Wnt pathway have been
implicated in a range of human disease, including cancer and
Alzheimer's disease.
According to Moon, until the new analysis, identifying the genes
that produce components of the Wnt pathway had been a long, hard,
gene-by-gene slog. Basically, researchers would mutate a single gene in
an organism and analyze whether it affected the pathway.
“At the time this screen was conducted, the total number of
genes implicated in the pathway was probably on the order of forty to
sixty,” said Moon. “But this was in multiple organisms, and
it wasn't really clear whether all of these components functioned in
one organism or whether people were comparing different genes in
different critters. There had been no systematic single-organism
genome-wide screen to ask, `what is an approximation of the number of
genes that can affect this pathway?'” he said.
Attempting to develop a more efficient approach, DasGupta and Kaykas
screened fly cells for Wnt-associated genes using a technology called
high-throughput RNA interference screening, developed in Perrimon's
laboratory. RNA interference (RNAi) is considered one of the most
important new techniques for analyzing gene activity. It relies on the
fact that a short segment of double-stranded RNA with a sequence
identical to that of a specific messenger RNA — copied from a gene as
a template for protein synthesis — can interfere with that messenger
RNA. The interaction essentially shuts down the corresponding gene's
function. Perrimon and his colleagues have created large libraries of
RNA segments that together correspond to the entire genome of the fruit
fly Drosophila.
“Our high-throughput RNAi screening technology can probe gene
activity with far greater sensitivity than has been possible
before,” said Perrimon. “It can detect very subtle activity
to reveal many more genes that function in signaling pathways.”
In their screening analysis, DasGupta and Kaykas used a library of some
22,000 short RNA molecules that corresponded to Drosophila
genes. They treated fly cells with each of the RNA molecules, and
determined the effect of each one on the Wnt pathway. To detect the
effect of shutting down a gene, they measured the telltale fluorescent
glow from a protein produced by a “reporter gene” they had
incorporated into the fly genome in such a way that it was switched on
only when the Wnt pathway was active.
Their screening revealed 238 candidate genes that were involved in
the Wnt pathway — either activating or inhibiting it. These genes
coded for proteins with a wide range of functions, including
controlling other genes, acting as molecular switches for enzymes, and
serving as components of the cell's protein garbage-shredding system,
or proteasome.
The researchers further validated their findings by confirming in
other fly cells, in human cells, and in zebrafish embryos that a
selection of the genes they had identified functioned in the Wnt
pathway. According to Moon, the fact that the results of the screen
included many genes that had already been identified by genetics
studies to be involved in the Wnt pathway was further validation of the
new technique.
“I found the discovery of the involvement of this many genes
in the Wnt pathway, and their diversity, to be quite stunning,”
said Moon.
According to Perrimon, whose laboratory is conducting a broad range
of high-throughput RNAi functional genomic screens, the findings in the
Wnt pathway offer dramatic evidence of the technique's power. “We
have studied the Wnt pathway for many years and used classical genetics
techniques to identify most of the initial components,” said
Perrimon. “RNAi screening is completely changing the way we
approach these problems. Before, it would take years to identify a few
genes. But with this technology, we can identify in only weeks all the
genes that are functionally involved in a pathway.”
Moon said the explosive increase in the number of known
Wnt-associated genes brings with it a major opportunity for
understanding the role of these genes and for developing drugs to treat
cancer and other diseases.
“About eighteen percent of the human genes involved in the Wnt
pathway are linked to diseases, according to genomic databases,”
said Moon. “I think this is an extremely high percentage and
points to the value of doing rapid screens in model organisms such as
the fly.
“It's possible to use the same reporter system to screen small
molecules for their effect on the Wnt pathway,” said Moon.
“Using our new knowledge of the genes that function in the
pathway, we can pinpoint those molecules' targets. And this rapid
identification sets the stage for characterizing small molecules that
are potential lead compounds as treatments for cancers and other
diseases involving this pathway,” he said.
Perrimon said the new technology will enable scientific insights far
beyond that of individual gene function. “For the first time, we
can explore these systems globally, identifying patterns of
correlations among groups of genes, understanding how information flows
within entire cellular signaling networks,” he said.
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