Axon guidance

Growing axons have a highly motile structure at the growing tip called the growth cone, which "sniffs out" the extracellular activities in the environment for signals that instruct the axon which direction 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.^[1]

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:^[2]

• Netrins: Netrins are secreted molecules that can act to attract or repel axons by binding to their receptors, DCC and UNC5.
• Slits aka Sli: 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.
• Cell adhesion molecules (CAMs): Integral membrane proteins mediating adhesion between growing axons and eliciting intracellular signalling within the growth cone. CAMs are the major class of proteins mediating correct axonal navigation of axons growing on axons (fasciculation). There are two CAM subgroups: IgSF-CAMs (belonging to the immunoglobulin superfamily) and Cadherins (Ca-dependent CAMs).

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 laminin, tenascins, proteoglycans, N-CAM, and L1
• Growth factors like NGF
• Neurotransmitters and modulators like GABA

Integration of information in axon guidance[edit]

Growing axons rely on a variety of guidance cues in deciding upon a growth pathway. The growth cones of extending axons process these cues in an intricate system of signal interpretation and integration, in order to ensure appropriate guidance.^[3] These cues can be functionally subdivided into:

• Adhesive cues, that provide physical interaction with the substrate necessary for axon protrusion. These cues can be expressed on glial and neuronal cells the growing axon contacts or be part of the extracellular matrix. Examples are laminin or fibronectin, in the extracellular matrix, and cadherins or Ig-family cell-adhesion molecules, found on cell surfaces.
• Tropic cues, that can act as attractants or repellents and cause changes in growth cone motility by acting on the cytoskeleton through intracellular signaling. For example, Netrin plays a role in guiding axons through the midline, acting as both an attractant and a repellent, while Semaphorin3A helps axons grow from the olfactory epithelium to map different locations in the olfactory bulb.
• Modulatory cues, that influence the sensitivity of growth cones to certain guidance cues. For instance, neurotrophins can make axons less sensitive to the repellent action of Semaphorin3A.

Given the abundance of these different guidance cues it was previously believed that growth cones integrate various information by simply summing the gradient of cues, in different valences, at a given point in time, to making a decision on the direction of growth. However, studies in vertebrate nervous systems of ventral midline crossing axons, has shown that modulatory cues play a crucial part in tuning axon responses to other cues, suggesting that the process of axon guidance is nonlinear. For example, commissural axons are attracted by Netrin and repelled by Slit. However, as axons approach the midline, the repellent action of Slit is suppressed by Robo-3/Rig-1 receptor.^[4] Once the axons cross the midline, activation of Robo by Slit silences Netrin-mediated attraction, and the axons are repelled by Slit.

Cellular strategies of nerve tract formation[edit]

Pioneer axons[edit]

The formation of a nerve tract follows several basic rules. In both invertebrate and vertebrate nervous systems initial nerve tracts are formed by the pioneer axons of pioneer neurons.^[5] These axons follow a reproducible pathway, stop at intermediate targets, and branch axons at certain choice points, in the process of targeting their final destination. This principle is illustrated by CNS extending axons of sensory neurons in insects.

During the process of limb development, proximal neurons are the first to form axonal bundles while growing towards the CNS. In later stages of limb growth, axons from more distal neurons fasciculate with these pioneer axons. Deletion of pioneer neurons disrupts the extension of later axons, destined to innervate the CNS.^[6] At the same time, it is worth noting that in most cases pioneer neurons do not contain unique characteristics and their role in axon guidance can be substituted by other neurons. For instance, in Xenopus retinotectal connection systems, the pioneer axons of retinal ganglion cells originate from the dorsal part of the eye. However, if the dorsal half of the eye is replaced by less mature dorsal part, ventral neurons can replace the pioneer pathway of the dorsal cells, after some delay.^[7] Studies in zebrafish retina showed that inhibiting neural differentiation of early retinal progenitors prevents axons from exiting the eye. The same study demonstrated aberrant growth trajectories in secondary neurons, following the growth of pioneer neurons missing a guidance receptor.^[8] Thus, while the extent of guidance provided by pioneer axons is under debate and may vary from system to system, the pioneer pathways clearly provide the follower projections with guidance cues and enhance their ability to navigate to target.

Role of glia[edit]

The first extending axons in a pathway interact closely with immature glia cells. In the forming corpus callosum of vertebrates, primitive glia cells first migrate to the ependymal zones of hemispheres and the dorsal septum wall to form a transient structure that the pioneer axons of the callosal fibers use to extend.^[9] The signaling between glia and neurons in the developing nervous system is reciprocal. For instance, in the fly visual system, axons of photoreceptors require glia to exit the eye stalk whereas glia cells rely on signals from neurons to migrate back along axons.^[10]

Guideposts[edit]

The growing axons also rely on transient neuronal structures such as guidepost cells, during pathfinding. In the mouse visual system, proper optic chiasm formation depends on a V-shaped structure of transient neurons that intersect with specialized radial glia at the midline of the chiasm. The chiasm axons grow along and around this structure but do not invade it.^[11] Another example is the subplate in the developing cerebral cortex that consists of transient neuronal layer under the subventricular zone and serves as a guidepost for axons entering permanent cortical layers. The subplate is similar to the chiasmatic neurons in that these cell groups disappear (or transit into other cell types) as the brain matures.^[12] These findings indicate that transitory cell populations can serve an important guidance role even though they have no function in the mature nervous system.

Studying axon guidance[edit]

The earliest descriptions of the axonal growth cone were made by the Spanish neurobiologist Santiago Ramón y Cajal in the late 19th century.^[13]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 of experiments 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[edit]

Several types of axon pathways 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 the midline 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[edit]

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.

https://en.wikipedia.org/wiki/Axon_guidance