John Flanagan (left) has discovered a protein that appears to help neural maps form in the brain. First author Masaru Nakamoto is poking a hole in the shell of a chicken egg, exposing the developing embryo to label growing axons.

For a scientist, finding a solution to a centuries-old riddle is a rare treat. John Flanagan has done just that by bringing a fresh approach to one of the most longstanding questions in brain research-how neural maps form in the embryonic brain.

In the September 6 issue of Cell, the assistant professor of cell biology at Harvard Medical School and his colleagues describe evidence that a molecule identified earlier by his group can guide the proper wiring of neuronal connections in the embryo's visual system.

"This paper is the first demonstration of a specific topographic effect of a specific molecule," says Flanagan.

This research provides the first conclusive evidence for a theory that dates back to the philosopher RenÚ Descartes and has been a central idea in neurobiology ever since. It addresses the question of how spatial information from the outside world gets laid down in the brain. Descartes envisioned ordered sets of nerve fibers in the retina carrying spatially accurate images into the brain. There they would make connections that precisely mirror the spatial order of the incoming fibers, in essence imprinting a so-called topographic map of the outside world onto the brain.

In the 1940s, neurobiologist Roger Sperry went on to suggest that this map arises because molecular tags on the incoming neurons match up with corresponding tags on the target neurons in the brain. Since not every neuron could possibly carry its own distinctive tag-there are not enough genes in our genome-Sperry proposed another idea. He wrote that the molecular tags were probably spread across the incoming fibers in a concentration gradient, which was again mirrored by an opposite gradient of matching tags inside the brain. This way, each incoming neuron would have precise spatial "coordinates" and could find its place in the brain, enabling points in the outside world to "map" to corresponding points inside the brain.

Eyes on the Prize

Flanagan's team entered this scene from a different direction. Initially unconcerned with neuronal maps, the researchers were led toward them when a new molecular probe Flanagan had devised to detect ligands for receptors in the nervous system turned up an unidentified molecule in an area of the mouse midbrain called the tectum. When the team cloned the molecule, ELF-1, they found it bound to a corresponding molecule on neurons from the retina that map to the tectum. That molecule was the receptor Mek4.

Making the right connections: Axons originating from retinal neurons near the temple normally project to the front end of the tectum. They have large numbers of Mek4 receptors on their surfaces, making them highly sensitive to the ligand ELF-1. In the tectum, a gradient of the repellant ELF-1 builds up from front to back. So temporal axons, entering the tectum from its front end, keep growing in until they meet increasing amounts of ELF-1 and stop. By contrast, axons originating from retinal neurons near the nose normally map to the back of the tectum, where ELF-1 levels are high. These axons have few Mek4 receptors, rendering them nearly oblivious to ELF-1 and allowing them to cruise through increasing concentrations of ELF-1 as they cross the tectum.

As reported in Cell last year, they then discovered that ELF-1 and Mek4 fit the requirements for the much-sought Sperry tags suspiciously well: ELF-1 was spread out over the tectum in a gradient, as was Mek4 in the retina, both gradients recognized each other and emerged just at the point in time in embryonic development when retinal axons travel into the tectum to make their connections.

In the upcoming report, the researchers buttress their case by providing functional evidence that retinal axons can indeed read the ELF-1 gradient, and that they respond to it in a differential way.

To test the effect of ELF-1 on retinal axons, the researchers manipulated the ELF-1 gradient. First they used a viral carrier to slip extraneous ELF-1 genes into the embryo's developing brain. Then they observed how labeled incoming retinal axons reacted upon encountering areas along their path where ELF-1 was overexpressed. They saw that the axons whose proper destination was at the low end of the ELF- 1 gradient avoided those regions, stopping or growing around them. At the same time, axons that normally map to high-ELF- 1 regions of the tectum were unperturbed by the ELF-1 overexpression. Together with other experiments exposing retinal neurons to ELF-1 in petri dishes, Flanagan and his coworkers thus demonstrated that ELF-1 can guide incoming axons by repelling them from regions in which they do not belong.

"The axons grow into the tectum from the low end of the ELF-1 gradient and more and more stop as the gradient rises, and the most resistant ones from cells closest to the nose reach the back," says Flanagan.

ELF-1 may not be the only molecule to guide the formation of this visual map, says Flanagan. A German research group is racing to identify another one and yet others may still await detection. Beyond finding those, Flanagan's team is hoping to learn whether ELF-1 or similar molecules are governing the building of maps for other brain functions. Throughout the brain there are somatosensory, auditory, and other maps-each of them representing the world according to one of the senses.

-Gabrielle Strobel