March 08, 2001
Gene-Trapping Method Powers Discovery of New Brain-Wiring Signals
Researchers have
developed a powerful screening method to identify genes that produce proteins
that guide the wiring of the trillions of connections in the mammalian brain. The
technique enables scientists to identify new genes and to determine which genes
are responsible for defects in brain wiring that are observed during
development. The scientists believe that this technique is likely to accelerate
the discovery of new molecules involved in axon guidance.
Neurons
wire themselves into networks by extending cable-like axons that grow toward
specific targets in the nervous system. An axon’s path toward a target
neuron is steered by growth cones in the tip of the axon that receive cues
about the best path to follow from chemical attractants and repellents secreted
by cells in the nervous system. These attractants and repellents are
collectively called axon guidance molecules.
“Up until now, we’ve gone about trying to identify brain wiring mechanisms one guidance event at a time, one molecule at a time. With the gene-trapping approach, we can cast a much wider net, studying a great many genes simultaneously, and then determining the effects of mutating them.”
Marc Tessier-Lavigne
In
an article published in the March 8, 2001, issue of
Nature,
researchers led by Howard Hughes Medical Institute
investigator Marc Tessier-Lavigne at the University of California, San Francisco and William
C. Skarnes at the University of California, Berkeley, unveil their new
technique and discuss some early applications of the method.
The
new “gene-trapping” technique could liberate scientists from
laborious genetic screens and biochemical approaches that are currently used to
identify new molecules involved in axon guidance, the researchers say.
“Up until now, we’ve gone about trying to identify brain wiring
mechanisms one guidance event at a time, one molecule at a time,” said
Tessier-Lavigne. “For example, in past work studying axon guidance in the
spinal cord, we developed an assay to study the growth of one particular class
of axon. And that study then led us, through extensive biochemical work, to
identify a small family of guidance molecules called the netrins.”
Guidance
molecules either attract or repel the growing axons of neurons by plugging into
receptors on the surface of the axon tip. Typically, each guidance molecule or
receptor is identified individually through time-consuming screening of random
chemically induced mutations, Tessier-Lavigne said. “However, with the
gene-trapping approach, we can cast a much wider net, studying a great many
genes simultaneously, and then determining the effects of mutating them.”
The
gene trapping technique was built on a method developed earlier by Skarnes at
the University of California, Berkeley. Skarnes’ technique involved
mutating genes in mouse embryonic stem cells by randomly inserting a complex
genetic marker with two componentsthe first is a marker gene that
produces a blue color in cells carrying the inserted gene and the second is a
drug-resistance gene. Thus, the scientists can easily identify cells of
interest by applying a drug to weed out those that did not take up the
drug-resistance gene and then use the blue color to distinguish them further.
Skarnes’s
method refined this standard “gene-trap vector” to include a gene
segment that would only activate the blue marker if the DNA had fused itself
into a gene for a membrane protein, such as a receptor. With this refinement,
called a “secretory trap,” the researchers were able to narrow down
the trapped genes to those coding for receptors of the kind involved in axon
guidance.
“The
secretory trap vector is a nice bonus because we can focus on exactly the kinds
of molecules we’re interested inmainly receptors and
ligands,” said Tessier-Lavigne. “These genes represent only a small
fraction of the genome, and this trap concentrates on just that
fraction.”
However,
the gene trap still needed further refinement before it was ready for use in
fishing out axon guidance molecules, said Tessier-Lavigne. “In early
studies, we found that mice with ‘trapped’ neuronal genes
didn’t show proper axon staining,” he said. Thus, the researchers
had a difficult time exploring the effects that specific gene mutations had on
brain wiring.
In
trying to fix the problem, Tessier-Lavigne and his colleagues inserted an
additional marker (PLAP ) in the gene-trap system. The presence of PLAP stains
axons purple. “This modified gene-trap strategy enabled us to mutate a
gene for a guidance molecule receptor, and by including the PLAP marker, we
were able to see the purple-stained altered neuronal wiring and rapidly assess
what has gone wrong with the wiring process,” said Tessier-Lavigne.
Using the modified gene-trapping technique, the
researchers produced 46 lines of mice with defined defects in axon guidance
molecules, said Tessier-Lavigne. “With these mice, not only have we
proven that we can trap genes that are specifically expressed in the nervous
system, but we can also see discrete patterns of axonal labeling, and we can
uncover mutant phenotypes,” he said.
Specifically,
studies on genes, called
Sema6A
and
EphA4,
demonstrated that the
trapping method could identify axon guidance mutants.
“With
EphA4,
we showed that we could
re-derive a known mutant, and with
Sema6A
we showed that we could use the technique to discover a new mutant
that affects only a small subset of axons in an otherwise normal nervous
system,” said Tessier-Lavigne.
These
results suggest that the new gene-trapping method will enable a rapid increase
in understanding the strategy neurons use in wiring the developing brain.
“It
has been shown that neurons that project their axons to a particular area
follow a code of transcription factor activation that presumably activates
genes for surface receptors that, in turn, dictate what the axon does,”
said Tessier-Lavigne. “We’re hoping that this method can help
identify the underlying code by focusing very specifically on receptors
involved in axon guidance and finding their expression patterns as well as
their mutant phenotypes.”
Furthermore,
the mutant mouse lines produced by this technique should also aid attempts to
map the normal wiring of the brain. “These mouse lines have very specific
populations of axons that are labeled purple,” said Tessier-Lavigne.
“In some cases it’s the first time that a marker has been
identified for those axons, and those markers provide a valuable resource for
people who want to study the normal wiring pattern of the brain.
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