Axon guidance

Axon guidance  (also called axon pathfinding) is a subfield of neural development concerning the process by which neurons send out axons to reach the correct targets. Axons often follow very precise paths in the nervous system, and how they manage to find their way so accurately remains a major puzzle.

Mechanisms
Growing axons have a highly motile structure at the growing tip called the growth cone, which "sniffs out" the extracellular environment for signals that instruct the axon which way to grow. These signals, called guidance cues, can be fixed in place or diffusible; they can attract or repel axons. Growth cones contain receptors that recognize these guidance cues and interpret the signal into a chemotropic response. The general theoretical framework is that when a growth cone "senses" a guidance cue, the receptors activate various signaling molecules in the growth cone that eventually affect the cytoskeleton. If the growth cone senses a gradient of guidance cue, the intracellular signaling in the growth cone happens asymmetrically, so that cytoskeletal changes happen asymmetrically and the growth cone turns toward or away from the guidance cue.

A combination of genetic and biochemical methods (see below) has led to the discovery of several important classes of axon guidance molecules and their receptors:


 * Netrins: Netrins and their receptors, DCC and UNC5, are secreted molecules that can act to attract or repel axons.
 * Slits: Secreted proteins that normally repel growth cones by engaging Robo (Roundabout) class receptors.
 * Ephrins: Ephrins are cell surface molecules that activate Eph receptors on the surface of other cells. This interaction can be attractive or repulsive. In some cases, Ephrins can also act as receptors by transducing a signal into the expressing cell, while Ephs act as the ligands. Signaling into both the Ephrin- and Eph-bearing cells is called "bi-directional signaling."
 * Semaphorins: The many types of Semaphorins are primarily axonal repellents, and activate complexes of cell-surface receptors called Plexins and Neuropilins.

In addition, many other classes of extracellular molecules are used by growth cones to navigate properly:
 * Developmental morphogens, such as BMPs, Wnts, Hedgehog, and FGFs
 * Extracellular matrix and adhesion molecules such as NCAM, L1, and laminin
 * Growth factors like NGF
 * Neurotransmitters and modulators like GABA

Studying axon guidance
The earliest descriptions of the axonal growth cone were made by the Spanish neurobiologist Santiago Ramón y Cajal in the late 19th century. However, understanding the molecular and cellular biology of axon guidance would not begin until decades later. In the last thirty years or so, scientists have used various methods to work out how axons find their way. Much of the early work in axon guidance was done in the grasshopper, where individual motor neurons were identified and their pathways characterized. In genetic model organisms like mice, zebrafish,nematodes, and fruit flies, scientists can generate mutations and see whether and how they cause axons to make errors in navigation. In vitro experiments can be useful for direct manipulation of growing axons. A popular method is to grow neurons in culture and expose growth cones to purified guidance cues to see whether these cause the growing axons to turn. These types experiment have often been done using traditional embryological non-genetic model organisms, such as the chicken and African clawed frog. Embryos of these species are easy to obtain and, unlike mammals, develop externally and are easily accessible to experimental manipulation.

Axon guidance model systems
Several types of axon pathway have been extensively studied in model systems to further understand the mechanisms of axon guidance. Perhaps the two most prominent of these are commissures and topographic maps. Commissures are sites where axons cross from one side of the nervous system to the other. Topographic maps are systems in which groups of neurons in one tissue project their axons to another tissue in an organized arrangement such that spatial relationships are maintained; i.e. adjacent neurons will innervate adjacent regions of the target tissue.

Commissure formation: attraction and repulsion
As described above, axonal guidance cues are often categorized as "attractive" or "repulsive." This is a simplification, as different axons will respond to a given cue differently. Furthermore, the same axonal growth cone can alter its responses to a given cue based on timing, previous experience with the same or other cues, and the context in which the cue is found. These issues are exemplified during the development of commissures. The bilateral symmetry of the nervous system means that axons will encounter the same cues on either side of the midline. Before crossing (ipsilaterally), the growth cone must navigate toward and be attracted to the midline. However, after crossing (contralaterally), the same growth cone must become repelled or lose attraction to the midline and reinterpret the environment to locate the correct target tissue.

Two experimental systems have had particularly strong impacts on understanding how midline axon guidance is regulated:

The ventral nerve cord of Drosophila
The use of powerful genetic tools in Drosophila led to the identification of a key class of axon guidance cues, the Slits, and their receptors, the Robos (short for Roundabout). The ventral nerve looks like a ladder, with three longitudinal axon bundles (fascicles) connected by the commissures, the "rungs" of the ladder. There are two commissures, anterior and posterior, within each segment of the embryo.

The currently accepted model is that Slit, produced by midline cells, repels axons from the midline via Robo receptors. Ipsilaterally projecting (non-crossing) axons always have Robo receptors on their surface, while commissural axons have very little or no Robo on their surface, allowing them to be attracted to the midline by Netrins and, probably, other as-yet unidentified cues. After crossing, however, Robo receptors are strongly upregulated on the axon, which allows Robo-mediated repulsion to overcome attraction to the midline. This dynamic regulation of Robo is at least in part accomplished by a molecule called Comm (short for Commissureless), which prevents Robo from reaching the cell surface and targeting it for destruction.

The spinal cord of mice and chickens
In the spinal cord of vertebrates, commissural neurons from the dorsal regions project downward toward the ventral floor plate. Ipsilateral axons turn before reaching the floor plate to grow longitudinally, while commissural axons cross the midline and make their longitudinal turn on the contralateral side. Strikingly, Netrins, Slits, and Robos all play similar functional roles in this system as well. One outstanding mystery was the apparent lack of any comm gene in vertebrates. It now seems that at least some of Comm's functions are performed by a modified form of Robo called Robo3 (or Rig1).

The spinal cord system was the first to demonstrate explicitly the altered responsiveness of growth cones to cues after exposure to the midline. Explanted neurons grown in culture would respond to exogenously supplied Slit according to whether or not they had contacted floor plate tissue.

Topographic maps: gradients for guidance
As described above, topographic maps occur when spatial relationships are maintained between neuronal populations and their target fields in another tissue. This is a major feature of nervous system organization, particular in sensory systems. The neurobiologist Roger Sperry proposed a prescient model for topographic mapping mediated by what he called molecular "tags." The relative amounts of these tags would vary in gradients across both tissues. We now think of these tags as ligands (cues) and their axonal receptors. Perhaps the best understood class of tags are the Ephrin ligands and their receptors, the Ephs.

In the simplest type of mapping model, we could imagine a gradient of Eph receptor expression level in a field of neurons, such as the retina, with the anterior cells expressing very low levels and cells in the posterior expressing the highest levels of the receptor. Meanwhile, in the target of the retinal cells (the optic tectum), Ephrin ligands are organized in a similar gradient: high posterior to low anterior. Retinal axons enter the anterior tectum and proceed posteriorly. Because, in general, Eph-bearing axons are repelled by Ephrins, axons will become more and more reluctanct to proceed the further they advance toward the posterior tectum. However, the degree to which they are repelled is set by their own particular level of Eph expression, which is set by the position of the neuronal cell body in the retina. Thus, axons from the anterior retina, expressing the lowest level of Ephs, can project to the posterior tectum, even though this is where Ephrins are highly expressed. Posterior retinal cells express high Eph level, and their axons will stop more anteriorly in the tectum.

The retinotectal projection of chickens, frogs and fish
The large size and accessibility of the chicken embryo has made it a favorite model organism for embryologists. Researchers used the chick to biochemically purify components from the tectum that showed specific activity against retinal axons in culture. This led to the identification of Ephs and Ephrins as Sperry's hypothesized "tags."

The retinotectal projection has also been studied in Xenopus and zebrafish. Zebrafish is a potentially powerful system because genetic screens like those performed in invertebrates can be done relatively simply and cheaply. In 1996, large scale screens were conducted in zebrafish, including screens for retinal axon guidance and mapping. Many of the mutants have yet to be characterized.

The Cell Biology of Axon Guidance
Genetics and biochemistry have identified a large set of molecules that affect axon guidance, but how do all of these pieces fit together? It is generally presumed, for good reasons, that most axon guidance receptors activate signal transduction cascades that ultimately lead to reorganization of the cytoskeleton and adhesive properties of the growth cone, which together underly the motility of all cells. However, this raises the question of how the same cues can result in a spectrum of response from different growth cones. It may be that different receptors activate attraction or repulsion in response to a single cue. Another possibility is the receptor complexes act as "coincidence detectors" to modify responses to one cue in the presence of another. Similar signaling "cross-talk" could occur intracellularly, downstream of receptors on the cell surface.

In fact, specific instances of all these kinds have been described, and the picture that is emerging is that growth cone signaling mechanisms are highly complex. In addition to signaling to the cytoskeleton, in the past several years that guidance cues cause growth cones to locally synthesize or degrade specific proteins, independent of changes in nuclear gene expression, which occurs far away in the neuronal cell body. This type of protein regulation is involved in mediating attraction and repulsion, and has also been shown to be involved in regulating the altered responses of, for example, commissural neurons to midline cues.