July 26, 1999 St. Louis, July 22, 1999 -- When nerve cells migrate from their birthplace to their permanent home in the brain, how do they find their way? Researchers have discovered the first molecular guide, a known protein called Slit. This protein doesn't attract young cells to where they need to go. It repels them, like a dog herding a flock of sheep.
"This is the first demonstration of a diffusible molecule that directs migrating neurons," says Yi Rao, Ph.D., assistant professor of neurobiology at Washington University School of Medicine in St. Louis. "Such a repulsive molecule might be useful for controlling the unwanted migration of tumor cells or for delivering therapeutic cells to specific regions of the brain in patients with Parkinson?s disease or Alzheimer?s."
Rao and Jane Y. Wu, M.B., Ph.D., assistant professor of pediatrics and of molecular biology and pharmacology, directed the research. Visiting research technician Wei Wu, from the Chinese Academy of Sciences in Shanghai, was first author of the paper, which appears in the July 22 issue of Nature.
"This finding has important conceptual implications," says Pasko Rakic, M.D., Ph.D., professor and chair of neurobiology at Yale University School of Medicine. "Although it previously has been suggested that some brain structures may release factors that repulse neurons, Rao and his colleagues are the first to identify specific genes whose products perform this function."
In the early 1970s, Rakic obtained the first definitive electron microscopic evidence of neuronal migration and proposed that interactions among different types of cells help direct it. In the past decade, he and others have identified several families of genes and molecules that recognize the "highways" for cell movement. "The novelty of the finding by the St. Louis group is that their molecules are diffusible and are distributed as concentration gradients that migrating cells can read," Rakic says.
Scientists realized in 1888 that brain cells might migrate. Now it is known that the majority travel, even after birth. In humans, for example, young neurons continue their migration to the cerebellum -- the part of the brain that controls movement -- during the first two months of life.
Nerve cell migration can't be studied directly in humans, so the researchers worked with rats. They looked at the olfactory bulb, which gives rodents their all-important sense of smell. During the first two weeks of life, this structure continues to enlarge as a cell nursery in the brain, the subventricular zone, sends it nerve cell precursors. The subventricular zone and the olfactory bulb are several millimeters apart, so the young neurons have to travel several thousand times their length to reach their target. But until now, the molecular signposts have remained obscure.
During the course of a different study, Rao and Wu became interested in Slit, which is secreted by cells in the midline of the embryo. Since 1996, they have cloned slit genes from several different animals. They also showed that two of the three slit genes are active in the midline of the rat septum. This part of the forebrain was known produce a substance that repels migrating neurons. Studying brain tissue from rats 4 days to 7 days of age, the researchers placed pieces of subventricular zone into gel. This enabled them to observe the neurons that flocked away from the tissue in all directions. But when they also added a piece of septum to the gel, the migration pattern changed as the majority of the migrating cells traveled away from the direction of the septum.
To determine whether this repulsive activity was due to Slit or some other substance from the septum, the researchers cultured pieces of subventricular zone with kidney cells, which normally don't make Slit. The young neurons migrated in a symmetrical pattern. But when they used kidney cells that had been genetically altered to make Slit, the neurons migrated in the opposite direction.
The researchers then placed a piece of subventricular zone midway between two masses of Slit-producing kidney cells. The young neurons migrated symmetrically. But when one mass was farther away than the other, the neurons migrated away from the closest mass. So it appears that a concentration gradient rather than a certain amount of Slit guides the migration of young neurons.
The researchers then studied Slit's effect on neurons in their natural surroundings. Wei Wu isolated sections of forebrain that contained the subventricular zone, the olfactory bulb and the pathway between them - the RMS (rostral migratory stream). By labeling the neuronal precursors with dye, the researchers were able to see them migrating in the RMS. But when they placed Slit-producing kidney cells on the RMS, very few young neurons ventured forth. "This experiment provides strong evidence that Slit can repulse neurons in their natural setting," Jane Wu says.
In the intact rat brain, the subventricular zone lies around the septum. "So neurons run away from it anteriorly into the olfactory bulb, their final destination," says Rao.
In four papers in the March 19 issue of Cell, the Washington University researchers and scientists in California reported that Slit interacts with a cell-surface protein called Roundabout (Robo). This receptor is made by certain neurons in the olfactory bulb. In the present study, the Washington University researchers engineered mammalian cells that produced and secreted RoboN, the extracellular fragment of Robo. Then they used the RoboN to prevent Slit from interacting with the normal Robo receptor.
They placed pieces of septum on top of the kidney cells and cultured them with pieces of subventricular zone. When they used normal kidney cells, the septum repelled migrating neurons. But when they used the kidney cells that made RoboN, the septum had much less effect on neuronal migration. "Again, this suggests that Slit is the substance in the septum that repels migrating neurons," Jane Wu says.
The four papers in Cell revealed that Slit guides the growth of axons, the long cables that extend from nerve cell bodies toward other nerve cells. It functions as a repellent, preventing axons from crossing the body's midline. Now the Washington University researchers have shown that Slit guides the migration of the cell bodies themselves, it appears that the same protein enables nerve cells to reach their correct locations and to link up with each other as they settle in. "There have been two schools of thought," Rao says. "One is that projecting axons and migrating neurons are quite different. The other is that there are similarities between them. Our study supports the latter."
Axon guidance involves molecules that attract axons as well as repulsive molecules. "You can have a mother who is encouraging you to do something or a mother who makes you insecure and drives you away," Rao says. "For neuronal migration, we have found a molecule acting like a mother that drives you away."
The discovery of a repulsive guidance molecule suggests a possible strategy for treating neurodegenerative diseases. "If you wanted to transplant neurons, you might not be able to inject them into the right part of the brain without damaging other cells," says Rao. "But perhaps you could inject them into a less vulnerable part of the brain and use Slit to drive them to the intended region."
Wu W, Wong K, Chen J-H, Jiang Z-H, Dupuis S, Wu JY, Rao Y. Directional guidance of neuronal migration in the olfactory system by the concentration gradient of the secreted protein Slit. Nature, July 22, 1999.
Grants and fellowships from the National Institutes of Health, the National Science Foundation of China, the Shanghai Commission of Science and Technology, the John Merck Fund and the Leukemia Society of America funded this research.
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