Neuronal Migration: Researchers Advance Understanding of Brain's Wiring

Brain development is an awesome feat. Over a trillion neurons make connections with thousands of target cells to create coherent circuits for vision, language, movement, and memory. Most neurons migrate from their birthplace to their permanent home in the developing brain. Once they find a home, some cells send growth cones to different parts of the brain, creating networks with their axons. Corey Goodman, professor of molecular biology at the University of California, Berkeley, elaborates, "There are choice points where certain cells turn left while others turn right. Somehow they are integrating environmental information [in the brain] and deciding which way to grow." Photo: Yi Rao Neurons labeled with a flourescent dye migrate from the anterior subventricular zone in the upper right hand corner towards the olfactory bulb (not pictured, in the lower left hand corner). This experiment was done on a slice of postnatal rat brain. Over the last decade researchers have uncovered the identity of key genes for neuronal guidance molecules. So far four families have been identified: netrins, semiphorins, ephrins, and Slit. These molecules act by ligand-receptor interactions. "There are positive influences pulling things, but negative repulsion plays a major role in the way things are guided," says Goodman. Attraction and repulsion can act in short- and long-range. Short-range requires contact with cell surfaces or extracellular matrix to guide cells, and long-range interactions occur by diffusible factors that set up gradients. Unraveling the brain's wiring mechanism is complicated by the fact that guidance molecules can have bifunctional roles. The same molecule can be attractive or repulsive, short-or long-range. The ligands are simply guidance cues; depending on the receptors that growth cones or neurons express, the signals can be interpreted in different ways. Since March of this year, the Slit protein has entered the limelight as a key player in neuronal migration and growth-cone extension. Three groups have found that Slit acts as a repellent for migrating neurons and extending growth cones, herding them toward the necessary direction for proper wiring. The Goodman group discovered the Slit protein 10 years ago when, during research into axon guidance mutants, a slit mutant materialized in Drosophila. Spyridon Artavanis-Tsakonas, a professor of cell biology presently at Harvard University, cloned the gene while at Yale University. In a fly, Slit is expressed as a large secreted molecule expressed by midline glia. The resulting paper left Slit's function ambiguous.1 Goodman comments, "Although it looked to us like [Slit] was affecting axon guidance, we were concerned that it might also be affecting midline cell fate and survival. We didn't have the markers in 1990 that we have now [to confirm the function of Slit]." Things were left at this stage while they went on to work on other aspects of growth cone guidance. Many growth cones cross the midline of the nervous system during development, but they only cross over once. The Goodman lab did a Drosophila mutant screen to see what controls midline crossing. They identified two key genes, roundabout (robo) and commissureless (comm). In a robo mutant, cones cross the midline back and forth indiscriminately; in comm mutants, cones do not cross the midline at all.2 Several years after identifying the mutants, the Goodman lab cloned the robo gene and identified it as expressing a novel receptor. Comm protein turns out to be a negative regulator of robo. Growth cones keep a low level of Robo until they cross the midline, and then they crank up the level. This causes the midline to be repulsive so the growth cone will not cross back again. As long as Comm is being expressed, Robo is maintained at a low level. When the comm gene is knocked out, cells express too much Robo from the beginning and cannot cross the midline at all. The only thing missing from this picture was the ligand for the Robo receptor. The Human Genome Project provided a clue with the identification of a second robo gene. This allowed the Goodman group to narrow down the candidate ligands to Slit. The researchers showed that slit and robo display dosage-sensitive genetic interactions, indicating that they are likely to function in the same pathway. Slit appears to function as a short-range repellent, controlling axon crossing at the midline. Slit may also function as a long-range chemorepellent, controlling mesodermal migration away from the midline.3 Goodman and Marc Tessier-Lavigne, a professor of anatomy, and biochemistry and biophysics at the University of California, San Francisco, collaborated to support the genetic studies on Slit with biochemical evidence.4 They cloned rat and human homologs of slit and demonstrated that the mammalian counterparts to the Drosophila gene have the same function. Coincidentally, Tessier-Lavigne's lab fished out Slit as the active component in extracts from spinal cord and brain tissue that promoted elongation and branching of dorsal root ganglion cells.5 This provides evidence that Slit proteins are bifunctional, affecting different axonal populations in different ways. Yi Rao, assistant professor of neurobiology at Washington University, St. Louis, approached the question of signaling molecules from a different angle. His lab was initially studying molecules involved in eye development. On a hunch, he had his students clone the slit gene in Xenopus in 1996, and he later cloned mouse and chick slit. "When we looked at the expression, we found it was in the midline of the neural tube of the vertebrate embryos, [but] it wasn't clear what function Slit would have," says Rao. The Rao lab later discovered Slit was not only acting on axon guidance, but also neuronal migration. Rao's findings on axon guidance were similar to the Goodman and Tessier-Lavigne results. Slit acts as a chemorepellent for axons extending from the olfactory bulb to the telencephalon, another part of the brain, and Robo is the transmembrane receptor.6 While studying axon guidance, the Rao lab also checked to see if Slit was guiding neuronal migration from the anterior subventricular zone (SVZa) of the brain to the olfactory bulb. They found that the postnatal septum expressed two slit genes encoding proteins that are repulsive guidance cues to SVZa neurons. When Rao provided two sources of Slit with a neuron source in the middle, the neurons migrated without being guided. However, if the neurons were closer to one source, they moved away from the closer source.7 In the field of axon guidance, several other guidance cues have been discovered, but no molecule was directly implicated in guiding neuronal migration previously. Rao says, "This is the first molecule shown to directly guide neuronal migration." This finding indicates migration of neuronal cell bodies and axon projection use similar guidance cues. Further study of Slit will have to extend into the signal transduction mechanism that controls neuronal migration. "Though we know Slit needs a concentration gradient to guide neurons, we don't know the mechanism of how this works at a biochemical level," says Rao. He wants to explore how a neuron 10µm in diameter senses concentration differences and transmits a signal inside the cell. Understanding the mechanism of negative and positive guidance cues will have a tremendous clinical impact in the future. Repulsive cues such as Slit may be able to prevent tumor cells in metastasis from moving into lymphatic ducts and blood. On the other hand, in cases of neurodegeneration caused by disease and injury, Slit may be able to guide transplanted healthy cells to a targeted area. Goodman adds, "Maybe in the future we can come up with therapeutics that will block certain repellents or go into the cell and block an aspect of the repulsive mechanism. [This may allow neurons] to grow again with specificity in cases of damage." Beyond clinical implications, understanding how the brain is wired provides a fundamental insight into what it means to be human. As Descartes said, "I think, therefore I am." Nadia S. Halim can be contacted at nhalim@the-scientist.com. J.M. Rothberg et al., "Slit: an extracellular protein necessary for development of midline glia and commissural axon pathways contains both EGF and LRR domains," Genes and Development, 4:2169-87, 1990. Seeger et al., "Mutations affecting growth cone guidance in Drosophila: genes necessary for guidance toward or away from the midline," Neuron, 10:409-26, 1993. T. Kidd et al., "Slit is the midline repellent for the Robo receptor in Drosophila," Cell, 96:785-94, March 19, 1999. K. Brose et al., "Slit proteins bind Robo receptors and have an evolutionarily conserved role in repulsive axon guidance," Cell, 96:795-806, March 19, 1999. K. H. Wang et al., "Biochemical purification of a mammalian Slit protein as a positive regulator of sensory axon elongation and branching," Cell, 96:771-84, March 19, 1999. H. Li et al., "Vertebrate Slit, a secreted ligand for the transmembrane protein roundabout, is a repellent for olfactory bulb axons," Cell, 96:807-18, March 19, 1999. W. Wu et al., "Directional guidance of neuronal migration in the olfactory system by the protein Slit," Nature, 400:331-6, July 22, 1999.

Neuronal Migration: Researchers Advance Understanding of Brain's Wiring

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