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).

Netrin and frazzled regulate presynaptic gap junctions at a Drosophila giant synapse

Netrin and its receptor, Frazzled, dictate the strength of synaptic connections in the giant fiber system (GFS) of Drosophila melanogaster by regulating gap junction localization in the presynaptic terminal. In Netrin mutant animals, the synaptic coupling between a giant interneuron and the 'jump' motor neuron was weakened and dye coupling between these two neurons was severely compromised or absent. In cases in which Netrin mutants displayed apparently normal synaptic anatomy, half of the specimens exhibited physiologically defective synapses and dye coupling between the giant fiber (GF) and the motor neuron was reduced or eliminated, suggesting that gap junctions were disrupted in the Netrin mutants. When the gap junctions were examined with antibodies to Shaking-B (ShakB) Innexin, they were significantly decreased or absent in the presynaptic terminal of the mutant GF. Frazzled loss of function mutants exhibited similar defects in synaptic transmission, dye coupling, and gap junction localization. These data are the first to show that Netrin and Frazzled regulate the placement of gap junctions presynaptically at a synapse (Orr, 2014).

The results show for the first time that Netrin-Frazzled signaling is specifically responsible for localizing gap junctions presynaptically at the GF-TTMn synapse. In the absence of Netrin, the gap junctions are not assembled in the presynaptic terminal and dye coupling is weak or absent in otherwise anatomically normal synapses. Similarly, Frazzled LOF mutants disrupted gap junctions and synaptic transmission. Finally, presynaptic expression of the dominant-negative Frazzled construct that is missing the intracellular domain also disrupts gap junction assembly, dye coupling, and synaptic transmission. In Netrin LOF mutants, axonal pathfinding is normal because the GF always projects into the target region and occasionally branches ectopically in the target region. However, dendritic path finding is dependent on Netrin-Frazzled signaling. In Netrin LOF mutants, the TTMn dendrite that normally projects toward the midline is often missing, as observed in other motor neurons. Finally, Netrin-Frazzled signaling is implicated in target selection, because GFs that reach the target area often do not build synapses, as seen in other model systems (Orr, 2014).

It was hypothesized that the physiological defect seen in Netrin and frazzled mutants arises from a reduction in trans-synaptic coupling between presynaptic and postsynaptic Innexins. Similar phenotypes, long latency, and lack of dye coupling have been observed in the shakB² mutant, which lacks gap junctions at the GF-TTMn synapse. The data suggest that when presynaptic and postsynaptic cells make contact, Netrin-Frazzled signaling is instructive for presynaptic localization of Innexins in the GF terminal to form trans-synaptic gap junctions (Orr, 2014).

Two roles were identified for Netrin-Frazzled signaling in assembly of the giant fiber system. Netrin was shown to act as a cue to direct the GF to select a target. Netrin-Frazzled signaling was also a local guidance cue for the GF and the medial dendrite of TTMn. The TTMn medial dendrite grows toward the midline glia, which were shown to be a source of Netrin. Second, it was hypothesized that Netrin bound on the postsynaptic Frazzled receptors serves as a synaptogenic cue for presynaptic Frazzled located on the GF. It is proposed that the bound Frazzled receptors directed presynaptic synaptogenesis and Innexin localization in the presynaptic terminal (Orr, 2014).

The frazzled dominant-negative construct supports the hypothesis that Netrin-Frazzled signaling is instructive in GF-TTMn synaptogenesis and function. Expression of fra-ΔC presynaptically disrupts the circuit by interrupting wild-type Netrin-Frazzled signaling. This was demonstrated through disruption of GF-TTMn synaptogenesis and the absence of gap junctions in the presynaptic terminal. However, the expression of UAS-fra-ΔC postsynaptically did not disrupt function, but did disrupt the morphology of the postsynaptic neuron. Postsynaptic expression of UAS-fra-ΔC disrupted dendritic maturation, resulting in medial dendrite pruning defects and lateral dendrite extension defects. The fra-ΔC experiments are interpreted as providing some evidence for Frazzled's cell autonomous role in building this giant synapse. More direct evidence would require rescue experiments. Unfortunately, the relevant genes are located very close to one another, making it difficult to obtain the appropriate recombination event. Future experiments will use recently acquired GAL4 drivers on the third chromosome to clarify this issue. The Frazzled RNAi experiments were uninformative, possibly because RNAi is not a strong enough disruption of frazzled to cause effects in the GFS. In brief, the cell autonomous function of Frazzled warrants further investigation (Orr, 2014).

When UAS-fra-ΔC was expressed in the embryo in the Netrin LOF background, it revealed that the disruption of commissures was Netrin dependent. An interaction experiment (NetA^ΔB^Δ/+; A307/+; UAS-fra-ΔC/+) revealed a different mechanism by which the dominant-negative fra-ΔC obstructed synaptogenesis. In a heterozygous Netrin LOF background, the mutant version of Frazzled was expressed, further knocking down Netrin-Frazzled signaling to disrupt synaptogenesis. The results suggested that fra-ΔC was acting as a Netrin sink by binding to secreted Netrin, limiting the amount of Netrin that could bind to wild-type Frazzled receptors (Orr, 2014).

The chemical synaptic component of the GF-TTMn synapse was observed in the Net LOF mutants using antibodies against the presynaptic density protein Bruchpilot (T-bars) with anti-NC82 staining. However, the Bruchpilot labeling was not informative. No further effort was made because the cholinergic component has no effect on synaptic circuit function in the adult (Orr, 2014).

In contrast to the GF-TTMn synapse, the GF-PSI synapse is unaffected by the absence of Netrin, Frazzled, or the expression of the dominant-negative Frazzled dominant-negative. This shows that the GF-TTMn synapse specifically is dependent on Netrin-Frazzled signaling for function. This mechanism for gap junction insertion is so specific that neighboring electrical synapses that share the same presynaptic terminal (GF) use different mechanisms for gap junction localization (Orr, 2014).

Netrin is secreted from two known sources, the midline glia and the postsynaptic target TTMn. A model is presented for Netrin localization and function in which Netrin is captured on the surface of one neuron (TTMn) by Frazzled and is then presented to Frazzled receptors on another neuron (GF) to transmit signaling. During development, the TTMn extends its medial dendrite toward a source of Netrin, the midline glia. After the TTMn dendrite has grown into the synaptic area by 9% of PD, both the midline glia and TTMn are labeled with Netrin. It is hypothesized that this is important in the induction of synaptic maturation of this synapse (Orr, 2014).

Rescuing Netrin LOF mutants by expressing a secreted form of Netrin specifically in either TTMn or midline glia supports a model that Netrin is presented to the GF to promote synapse formation. The secreted Netrin rescue experiments were effective because Netrin could localize where it would normally as long as it was secreted by a nearby endogenous source. This could explain why it was possible to rescue the Netrin LOF mutants in a non-cell-autonomous fashion by expressing secreted Netrin in either midline glia or the TTMn independently. Postsynaptic expression of the Frazzled dominant-negative also supports the presentation model. When two copies of Frazzled lacking its intracellular domain were expressed on the TTMn, Netrin could bind to the mutant Frazzled, be presented to the GF, and support normal synaptic function regardless of disrupted intracellular signaling in the TTMn by the deletion of the intracellular domain (Orr, 2014).

In contrast, expressing membrane-tethered UAS-NetB^CD8-TM on either the midline glia or TTMn failed to rescue function of the circuit because localization and secretion of Netrin was disrupted. When attempts were made to rescue the Netrin LOF mutants by expressing membrane-tethered NetrinB postsynaptically, the defects were enhanced and the medial dendrite did not extend to the midline in 90% of specimens. However, in the tethered NetB mutant, tethered NetrinB was expressed in both of its endogenous sources, midline glia and TTMn, and the synapse functioned normally. While being expressed under its endogenous promoter, tethered Netrin supported normal synaptogenesis. It is possible that, through the endogenous expression pattern, cells not identified in this study could contribute to the normal phenotype seen in the mutants in a nonlocal manner. However, it is hypothesized that the tethered NetrinB mutant does not behave in a predictable way. It is suggested that this protein is not as tightly membrane bound as the UAS-NetB^CD8-TM protein product due to the added extracellular myc domains in the tethered mutant. The tethered mutant's additional myc domains may account for differences in phenotypes due to increased protein flexibility or possible cleavage and secretion from the cell of origin. Considering this, non-cell-autonomous expression of a secreted Netrin rescued Netrin LOF defects, whereas expression of the tethered version using the same GAL4 drivers could not rescue the defects. This is evidence for the importance of Netrin secretion in GFS synaptogenesis (Orr, 2014).

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: 2 December 2018

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