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
components—the 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
in—mainly 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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