In living animals, growth cones generally travel toward their targets along pre- dictable, stereotyped routes, exploiting a multitude of different cues to find their way, but always requiring a substratum of extracellular matrix or cell surface to crawl over. Often, growth cones take routes that have been pioneered by other neurites, which they follow by contact guidance. As a result, nerve fibers in a mature animal are usually found grouped together in tight parallel bundles (called fascicles or fiber tracts). Such crawling of growth cones along axons is thought to be mediated by homophilic cell–cell adhesion molecules—mem- brane glycoproteins that help a cell displaying them to stick to any other cell that also displays them. As discussed in Chapter 19, two of the most important
(B) (A)
dendrite cell body axon growth cone
10 mm
Figure 22–101Formation of axon and dendrites in culture. A young neuron has
been isolated from the brain of a mammal and put to develop in culture, where it sends out processes. One of these processes, the future axon, has begun to grow out faster than the rest (the future dendrites) and has bifurcated. (A) A phase-contrast picture; (B) the pattern of staining with fluorescent phalloidin, which binds to filamentous actin. Actin is concentrated in the growth cones at the tips of the processes that are actively extending and at some other sites of lamellipodial activity. (Courtesy of Kimberly Goslin.)
classes of such molecules are those that belong to the immunoglobulin super- family, such as N-CAM, and those of the Ca2+-dependent cadherin family, such as N-cadherin. Members of both families are generally present on the surfaces of growth cones, of axons, and of various other cell types that growth cones crawl over, including glial cells in the central nervous system and muscle cells in the periphery of the body. The human genome contains more than 100 cadherin genes, for example, and most of them are expressed in the brain (see Figure 19–6). Different sets of cell–cell adhesion molecules, acting in varied combina- tions, provide a mechanism for selective neuronal guidance and recognition. Growth cones also migrate over components of the extracellular matrix. Some of the matrix molecules, such as laminin, favor axon outgrowth, while others, such as chondroitin sulfate proteoglycans, discourage it.
Growth cones are guided by a succession of different cues at different stages of their journey, and the stickiness of the substratum is not the only thing that matters. Another important part is played by chemotactic factors, secreted from cells that act as beacons at strategic points along the path—some attracting, oth- ers repelling. The trajectory of commissural axons—those that cross from one side of the body to the other—provides a beautiful example of how a combina- tion of guidance signals can specify a complex path. Commissural axons are a general feature of bilaterally symmetrical animals, because the two sides of the body have to be neurally coordinated. Worms, flies and vertebrates use closely related mechanisms to guide their outgrowth.
In the developing spinal cord of a vertebrate, for example, a large number of neurons send their axonal growth cones ventrally toward the floor plate—a spe- cialized band of cells forming the ventral midline of the neural tube (see Figure 22–100). The growth cones cross the floor plate and then turn abruptly through a right angle to follow a longitudinal path up toward the brain, parallel to the floor plate but never again crossing it (Figure 22–102A). The first stage of the journey depends on a concentration gradient of the protein netrin, secreted by the cells of the floor plate: the commissural growth cones sniff their way toward its source. Netrin was purified from chick embryos, by assaying extracts of neu- ral tissue for an activity that would attract commissural growth cones in a cul- ture dish. Its sequence revealed that it was the vertebrate homolog of a protein already known from C. elegans, through genetic screens for mutant worms with misguided axons—called Unc mutants because they move in an uncoordinated fashion. One of the Unc genes, Unc6, codes for the homolog of netrin. Another, Unc40, codes for its transmembrane receptor; and this too has a vertebrate homolog called DCC that is expressed in the commissural neurons and mediates their response to the netrin gradient.
Localized activation of DCC by netrin leads to opening of a specialized class of ion channels in the plasma membrane. These channels, called TRPC (Tran- sient Receptor Potential C) channels, belong to a large family (the TRP family)
roof plate commissural neuron commissural axon midline floor plate TOWARDS BRAIN (A) (B) commissural neuron approaching
midline wall of neural tube
floor plate on ventral midline
growth cone expressing receptor (DCC) for netrin
attractant (netrin)
growth cone expressing receptor (Roundabout) for Slit and receptors for semaphorin
repellent (semaphorin) repellent
(Slit)
TO BRAIN
Figure 22–102The guidance of commissural axons. (A) The pathway
taken by commissural axons in the embryonic spinal cord of a vertebrate. (B) The signals that guide them. The growth cones are first attracted to the floor plate by netrin, which is secreted by the floor-plate cells and acts on the receptor DCC in the axonal membrane. As they cross the floor plate, the growth cones upregulate their expression of Roundabout, the receptor for a repellent protein, Slit, that is also secreted by the floor plate. Slit, binding to Roundabout, not only acts as a repellent to keep the cells from re-entering the floor plate, but also blocks responsiveness to the attractant netrin. At the same time, the growth cones switch on expression of receptors for another repellent protein, semaphorin, that is secreted by the cells in the side walls of the neural tube. Trapped between two repellent territories, the growth cones, having crossed the midline, travel in a tight fascicle up toward the brain.
that is responsible for many other sensory transduction processes, from mechanosensation to the perception of heat and cold. When open, the TRPC channels allow Ca2+(and other cations) to enter the cell. The localized rise in Ca2+then activates the machinery for extension of filopodia and movement of the growth cone toward the netrin source.
The receptors on each growth cone determine the route it will take: non- commissural neurons in the neural tube, lacking DCC, are not attracted to the floor plate; and neurons expressing a different netrin receptor—called Unc5H in vertebrates (with a counterpart Unc5 in the worm)—are actively repelled by the floor plate and send their axons instead toward the roof plate.