The researchers bill the new technique, called a PLAP secretory trap screen, as a tool for finding a needle in a haystack. The trick: attach a molecular tag to the needle. Through the magic of genetic engineering, lines of mice are bred to express telltale mutations. Brain neurons harboring particular wiring molecules are revealed by a blue tint, while their tentacle-like extensions, or axons, are colored purple.
Intricate guidance mechanisms have evolved to insure that the brain gets wired up correctly during critical periods in early development. Mistakes in this process, resulting in circuitry gone awry, are hypothesized to occur in neurodevelopmental disorders like schizophrenia and autism. Like ships wending their way to distant ports, embryonic neurons migrate to their appointed destinations in the brain and spinal column with help from navigation systems in their axons. Axons establish connections, or synapses, with remote target cells, networking some 100 billion neurons in humans. Growth cones at the axon tip steer a weaving course, taking their cues from chemical attractants and repellents secreted by guidance cells that serve as the lighthouses and buoys of central nervous system development. Receptor proteins on the axons act as the growth cones' antennae for receiving these signals. Researchers face a daunting task in identifying such axon guidance system components and the genes that code for them, thought to number in the hundreds or thousands.
"There's such complexity, and many of the standard methods, such as chemically inducing a mutation or molecularly knocking out a single gene, take too long," explained Tessier-Lavigne. "In addition, wiring defects can be very difficult to detect against a background of normal projections." In the same time it takes to perform a single gene knockout study, gene traps might net dozens or hundreds of genes. However, due to randomness integral to the methodology, it's not possible to target a particular molecule in advance.
"It starts out as somewhat of a 'fishing expedition,' but ultimately yields an invaluable molecular map of axonal projections by simultaneously mutating genes of interest and labeling the neurons expressing them," added Skarnes. The two laboratories are participating in an effort to develop a bank of mutant mouse lines accessible at www.genetrap.org expressing particular populations of labeled axons as a resource for the neuroscience community.
In the current study, Tessier-Lavigne, Skarnes and colleagues demonstrated, for the first time in vivo, an axon guidance role for a repellent guidance chemical, Sema6A, and clarified the guidance function of a growth cone receptor, EphA4. They inserted into mouse embryonic stem cells a marker, called a "secretory trap" vector, initially developed by Skarnes, that creates a mutation in genes likely to code for axon guidance molecules. To make the resultant subtle wiring defects stand out against a background of normal axonal projections, they added to the vector a second marker, PLAP (human placental alkaline phosphatase). They then used the genetically modified stem cells to breed lines of mice expressing the marker, which, after chemical staining, turns neuron cell bodies blue and axons purple.
As expected, some PLAP-labeled axons had different projection patterns in animals with the mutation, when compared with normal animals. Of 120 known genes trapped, 13 are thought to play a role in neuronal guidance, and the researchers predict that at least a comparable proportion of new genes netted in future studies likely will similarly code for brain wiring proteins. The technique "makes it possible not only to develop a map of the normal wiring pattern of the brain, but also to screen systematically for changes in this map in mutant animals," note the researchers. They have observed "remarkable diversity" of axonal projection patterns among some 40 lines of mice produced to date. In lines where the mutations do not interfere with the animals' survival, the researchers hope to screen for defects in behaviors, such as learning and memory, stress responses, etc. that might reflect underlying defects in the brain that might be traceable to specific genes. The technique's "full impact will come from its application on a large scale to sample as much of the genome as possible," write the researchers.
Also participating in the study were: Philip Leighton, Kevin Mitchell, Lisa Goodrich and Xiaowei Lu of HHMI / UCSF, and Kathy Pinson and Paul Scherz of UC Berkeley.
The National Institute of Mental Health (NIMH) is part of the National Institutes of Health (NIH), the Federal Government's primary agency for biomedical and behavioral research. NIH is a component of the U.S. Department of Health and Human Services.