BIOLOGICAL OVERVIEW

When viewed across phyla from insects to vertebrates, the CNS midline exhibits both attractive and repulsive properties for neuronal growth cones. In vertebrates, the ventral midline contains a specialized group of cells (the floor plate), while in Drosophila midline glia act to attract commissural growth cones while simultaneously presenting a repulsive boundary to axons that do not cross.

The netrins are a family of secreted proteins that provide axon guidance. The first netrin characterized, UNC-6 of the nematode C. elegans, was identified as the product of a gene that when mutated, leads to defects in cell migration and axon guidance. unc-6 is expressed in twelve types of neurons and glia and provides a hierarchy of guidance cues throughout the ectoderm that are used in forming the basic axon scaffold of the nervous system. Although the unc-6 expression pattern is complex, early unc-6 expression is spatially restricted to the ventralmost cells within each region of the nervous system. Such ventral restriction of netrin expression likely extends to all phyla (Wadsworth, 1996 and references).

In the rat Netrin-1 is expressed in the ventral midline of the neural tube. Floor plate cells attract ventrally directed spinal commissural axons cells but have a long-range repulsive effect on dorsally directed trochlear motor axons. Both repulsion and attraction are mediated by Netrin-1 (Colamarino, 1995).

In Drosophila Netrin genes are expressed at the ventral midline of the central nervous system. Both Netrin genes are expressed by midline glial cells, while only one (NetA) is expressed in the midline VUM neuronal cluster. The first axons to pioneer the anterior and posterior commissures first project directly toward these midline glia and VUM growth cones, and subsequently make intimate contact with them. Netrins do not function as permissive agents to promote axon outgrowth, but rather Netrin localization is required for proper guidance; that is, Netrins function as instructive guidance cues. There is some evidence from phenotypes of ectopic Netrin expression to suggest that Netrins repel in Drosophila.

In addition to the role in the midline, Netrins also influence the peripheral projections of motor axons to their target muscles. Netrins are expressed by discrete subsets of muscles. In Netrin double mutants, axons wander over more territory than usual, and can sometimes inappropriately cross the segmental border into neighboring segments. Neurons often appear to branch inappropriately over muscle targets, to stall, to project past targets or to project into adjacent segments (Mitchell, 1996).

What is known regarding receptors for the Netrins? In C. elegans there are two candidate genes. The unc-5 (see Drosophila unc-5) gene encodes a membrane protein of the immunoglobulin family. Misexpression experiments have shown than UNC-5 protein is necessary and sufficient for dorsally oriented cell movements that utilize the UNC-6 protein gradient. UNC-40 is another immunoglobulin superfamily member that is implicated as a receptor component for UNC-5 (Harris, 1996 and references). As to the Drosophila receptor, intersegmental neuron phenotypes are remarkably similar to those observed in embryos mutant for the gene frazzled , the Drosophila homolog of mammalian DCC and a putative Netrin receptor (Kolodziej, 1996).

The molecular mechanisms controlling the ability of motor axons to recognize their appropriate muscle targets were dissected using Drosophila genetics to add or subtract Netrin A, Netrin B, Semaphorin II, and Fasciclin II, either alone or in combination. Discrete target selection by neurons might be specified in a point-to-point fashion such that each motor axon and its appropriate target have unique and complementary molecular labels. Alternatively, specificity might emerge from a dynamic and comparative process in which growth cones respond to qualitative and quantitative molecular differences expressed by neighboring targets and make their decisions based on the relative balance of attractive and repulsive forces. Fas II and Sema II are expressed by all muscles where they promote (Fas II) or inhibit (Sema II) promiscuous synaptogenesis. The level of Sema II expression, while not enough to stop growth cones from exploring their environment, nevertheless provides a threshold that specific attractive signals must overcome in order to permit synapse formation. Decreasing Sema II leads to an increase in innervation. In the absence of Sema II, targeting errors occur, usually in the form of additional ectopic connections to neighboring muscles, although in some cases the absence of the normal connection or inappropriate choice point decisions are observed as well. Increasing Sema II leads to a decrease in innervation. It is concluded that growth cones in this system apparently do not rely solely on single molecular labels on individual targets. Rather, these growth cones assess the relative balance of attractive and repulsive forces and select their targets based on the combinatorial and simultaneous input of multiple cues. Apparently a relative balance model is more valid in this system than a lock-and-key model (Winberg, 1998).

The modest and dynamic level of Fas II helps adjust the threshold for innervation. Prior to synapse formation, Fas II is expressed at a low level across the entire surface of the muscle, making it permissive for growth cone exploration and synapse formation. As the first synapse forms on a muscle, the Fas II level dramatically plummets over the muscle surface while Fas II clusters under the developing synapse. The first successful synapse leads to a rapid reduction in this general attractant, thereby shifting the relative balance in favor of Sema II-mediated repulsion and thus raising the hurdle over which attractive signals must pass in order to promote further synapse formation. In this way, the innervated muscle becomes more refractory to further innervation. Fas II, as a modulator of the balance of attraction and repulsion, becomes a temporal measure of the muscle's synaptic history (Winberg, 1998).

While Sema II generally prevents exuberant synapse formation, it can also play an important role in patterning connections. For example, the two axons that pioneer the transverse nerve (TN) normally meet and fasciculate near muscle 7. In the absence of Sema II, these axons often innervate muscles 7 and 6, and sometimes fail to fasciculate with one another. In this case, Sema II provides a repulsive force (from muscles 7 and 6) at a specific choice point, and in its absence, the TN growth cones make a different decision. Similarly, as the lateral branch of the segmental nerve branch a (SNa) extends posteriorly, one axon branch innervates muscle 5 while another continues posteriorly to innervate muscle 8. In the absence of Sema II, both sometimes stop and innervate muscle 5. In this case, Sema II provides a key repulsive force (from muscle 5) at a specific choice point, and in its absence, the growth cone that usually innervates muscle 8 instead makes a different decision. Both examples show how Sema II can do more than simply sharpen the pattern of innervation; Sema II can also influence specific targeting decisions in a dosage-dependent fashion. The Sema II experiments show that the pattern of expression (i.e., the differential levels expressed by neighboring muscles) can be more important than the absolute level. Simply increasing Sema II on all muscles has little influence on the SNa. But increasing Sema II expression on muscle 5 and not its neighboring muscles does influence the SNa axons, presumably because it presents these axons with a sharp repulsive boundary. This differential expression prevents the lateral branch of the SNa from extending towards muscles 5 and 8 (Winberg, 1998).

The netrins were initially discovered as long-range chemoattractants that are secreted by midline cells and that attract commissural growth cones toward the midline. Netrins might have another function, and strong evidence is presented supporting this notion. In addition to their CNS midline expression and function in axon guidance, NetA and NetB are also expressed by distinct subsets of muscles where they function as short-range target recognition molecules. Genetic analysis suggests that both types of Netrin-mediated attractive responses (i.e., pathfinding and targeting) require Frazzled, the DCC/UNC-40-like Netrin receptor. In contrast, Fra is not required for NetB-mediated repulsion of the segmental nerve. Even though they are expressed by distinct subsets of muscles and function as target recognition molecules, the two netrins, NetA and NetB, do not act alone in specifying any one of these muscle targets. NetB is expressed by muscles 7 and 6, but NetB is not the sole attractant used by RP3 to innervate these muscles. In the absence of NetB, in 35% of segments RP3 makes the correct pathfinding decisions in the periphery but fails to innervate muscles 7 and 6 properly. However, in the other 65% of segments it does innervate muscles 7 and 6. Clearly, other unknown cues must play a major role in this targeting decision. One potential candidate for an additional targeting cue is the Ig CAM Fasciclin III. However, removal of FasIII does not alter the penetrance of the RP3 phenotype of Netrin or frazzled mutants. NetB functions within the context of the relative balance of general attractants and repellents such as Fas II and Sema II. For example, since the TN axons are attracted by NetB, and muscles 7 and 6 express NetB, why do the TN axons not synapse on muscles 7 and 6? Evidently, they are sufficiently repelled by Sema II to prevent inappropriate synapse formation. Either increasing the level of NetA or NetB or decreasing the level of Sema II leads to ectopic TN synapses. The choice of synaptic partner by TN axons is controlled by the balance of NetB in relation to Sema II and Fas II (Winberg, 1998).

Distinct classes of motor axons respond differentially to NetA and NetB While all motor axons in this system appear to be attracted by Fas II and repelled by Sema II, the different types of motor axons respond differently to NetA and NetB. NetB is expressed by a subset of muscles (7 and 6) where it strongly attracts appropriate (RP3) axons, more weakly attracts certain inappropriate (TN) axons, and repels other inappropriate (SN) axons. RP3 and TN axons can also be strongly attracted by NetA, while SN axons are apparently indifferent to NetA. The TN axons display a stronger responsiveness to NetA than to NetB, as judged by the frequency of ectopic innervation of ventral muscles overexpressing either Netrin. This difference may make biological sense, as TN axons normally extend toward a dorsal stripe of epithelial cells expressing NetA but grow past NetB-expressing ventral muscles without innervating them (Winberg, 1998).

Although all of the molecular signals used for this targeting system are not yet known, four key components have been identified: the pan-muscle expression of Fas II and Sema II and the muscle-specific expression of NetA and NetB. Analysis of these four genes shows that the signals they encode are potent, function as short-range signals in a dosage-dependent fashion, and work in combinations that either amplify or antagonize one another. Fas II and Sema II help control the fidelity and precision of the targeting system, while NetA and NetB provide muscle-specific targeting cues. These results suggest that target selection in this system is not based on absolute attractants or repellents that either ensure or prevent synapse formation, but rather it is based on the balance of attractive and repulsive forces on any given target cell in relationship to its neighboring cells. Targeting molecules such as Netrins, Semaphorins, and IgCAMs sometimes function as antagonists and sometimes as collaborators. This model of target selection is very similar to the current view of axon guidance in terms of a relative balance of attractive and repulsive forces (Winberg, 1998).

GENE STRUCTURE

NetA is located just distal to NetB on the chromosome: both genes are transcribed in the same direction, from proximal to distal relative to the centromere. The two genes occupy a region of approximately 150 kb in total (Harris, 1996 and Mitchell, 1996).

Bases in 5' UTR - 590 for NetA and 340 for NetB

Bases in 3' UTR - 490 for NetA and 1200 for NetB

PROTEIN STRUCTURE

Amino Acids - 727 for NETA and 793 for NETB

Structural Domains

The two fly genes are more similar to one another than either is to netrin genes from other species. This is also true of chick netrin-1 and netrin-2, and suggests that separate gene duplication events occurred in the vertebrate and insect lineages. The two fly proteins share a common domain organization and extensive amino acid sequence similarity over the entire length of their open reading frames. Overall, Drosophila Netrin-A is 41% identical to Netrin-B, 39% identical to UNC-6 and 40% identical to chick netrin-1. The N-terminal two-thirds of the netrins are homologous to the N-termini of the polypepide chains (A, B1 and B2) of laminin, a large (880 kD) heterotrimeric protein of the extracellular matrix. The homologous region corresponds to domains VI and V of the laminin chains. Both Drosophila netrins have an N-terminal signal peptide, followed by the domain VI homology region, the domain V homology region, and the C-terminal domain C. Domain V consists of three EGF repeat structures (Harris, 1996, Mitchell, 1996 and Serafini, 1994)

date revised: 12 March 98

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.