L1- and NCAM-type cell adhesion molecules represent distinct protein families that function as specific receptors for different axon guidance cues. However, both L1 and NCAM proteins promote axonal growth by inducing neuronal tyrosine kinase activity and are coexpressed in subsets of axon tracts in arthropods and vertebrates. The functional requirements for the Drosophila L1- and NCAM-type proteins, Neuroglian (Nrg) and Fasciclin II (FasII), have been studied during postembryonic sensory axon guidance. The rescue of the Neuroglian loss-of-function (LOF) phenotype by transgenically expressed L1- and NCAM-type proteins demonstrates a functional interchangeability between these proteins in Drosophila photoreceptor pioneer axons, where both proteins are normally coexpressed. In contrast, the ectopic expression of Fasciclin II in mechanosensory neurons causes a strong enhancement of the axonal misguidance phenotype. Moreover, these findings demonstrate that this functionally redundant specificity to mediate axon guidance has been conserved in their vertebrate homologs, L1-CAM and NCAM (Kristiansen, 2005).

This study presents an analysis of the requirements and the functional specificity of Drosophila L1- and NCAM-type proteins during the postembryonic development of the Drosophila peripheral sensory nervous system. The partially penetrant phenotypes, which have been reported for L1- and NCAM-LOF mutants in Drosophila and different vertebrate model systems, suggest that the requirement for these neural CAMs is not absolute and that the lack of either L1- or NCAM-type proteins during nervous system development can be partially compensated for by other gene products. Moreover, considering the unique specificity of L1's and NCAM's homo- and heterophilic adhesive interactions, a molecular redundancy between these protein families may be unexpected. The specificities of the homophilic adhesive interactions within the L1 and the NCAM protein families have undergone considerable evolutionary changes. Drosophila Nrg and FasII exhibit a very low cross-reactivity with their vertebrate homologs, L1-CAM and NCAM. Although only the neuronal isoforms of human (L1-CAM^RSLE+) and of Drosophila Neuroglian (Nrg¹⁸⁰) have been directly tested for their ability to interact with each other, these results indicate that the ability of vertebrate CAMs to rescue the Nrg LOF phenotype most likely relies on homotypic adhesion, rather than on an interaction with endogenous Drosophila CAMs. This conclusion is also supported by the observation that the GOF phenotype in the wing sensory nervous system is only observed when the vertebrate transgene is expressed in both the wing epithelium and the sensory neurons. In addition, endogenous Nrg expression is not required for the production of the GOF axonal misguidance phenotype in the Drosophila wing (Kristiansen, 2005).

Although axonal growth and guidance involve a large array of different neuronal adhesion molecules, there appears to be a limited number of signaling pathways that are shared among structurally different CAM families. The two major signaling pathways, which are triggered by Ig-CAMs, involve nonreceptor tyrosine kinases or receptor tyrosine kinases, such as FGFR and EGFR. Both of these signaling pathways may act synergistically or in a redundant manner. L1-CAM-, NCAM-, as well as N-cadherin-mediated neuronal cell adhesion all activate neuronal FGF receptors and thereby induce neurite outgrowth in vitro. This suggests that structurally different neural CAMs are capable of feeding into the same signaling pathway and that multiple adhesive specificities coordinately influence axonal growth and guidance (Kristiansen, 2005).

Axonal guidance in the Drosophila ocellar sensory system (OSS) and the wing sensory nervous system involves the Nrg-mediated activation of FGF and EGF receptors. Constitutive activation of FGFR or EGFR can rescue the nrg³ LOF phenotype in the OSS, and Nrg GOF axonal misguidance in the developing wing is reversed by a hypomorphic allele of the Drosophila EGF receptor. The two types of neurons in the Drosophila OSS, ocellar pioneer (OP) and bristle mechanosensory (BM) neurons, differ in their expression of Nrg and FasII protein and in their requirement for both proteins during axonal growth and guidance. Whereas the neuron-specific isoforms Nrg¹⁸⁰ and FasII^PEST+ are coexpressed in OP axons, BM axons only express Nrg¹⁸⁰, but not FasII. The surrounding epidermis, which interacts with BM but not with OP axons, expresses the nonneuronal Nrg¹⁶⁷ isoform (Kristiansen, 2005).

The nrg LOF rescue experiments reveal strikingly different requirements for Nrg and FasII protein in the two neuronal cell populations. The requirement for Nrg in OP axons can be sustained by either the neural Nrg¹⁸⁰ or FasII^PEST+, but not by the nonneuronal Nrg¹⁶⁷ isoform. The two Nrg protein isoforms have identical extracellular domains and only differ in the size of their respective cytoplasmic domain. The capacity of FasII to fulfil the Nrg¹⁸⁰ requirement in OP axon guidance suggests that these structurally different proteins share a redundant function in these axons. This conclusion is further supported by the observation that the partially penetrant nrg LOF OP axonal misguidance phenotype is significantly amplified by a reduction of the fasII gene dosage. Remarkably, ectopic FasII^PEST+ expression in BM neurons enhances the deleterious effect of the Nrg loss, a situation that fits within the concept of antiredundancy or opposing functional capacities (Kristiansen, 2005).

The scenario of cell-specific redundant functions of Nrg¹⁸⁰ and FasII^PEST+ is maintained by their vertebrate homologs L1-CAM/Nr-CAM and NCAM¹⁴⁰, respectively. This indicates that the redundant specificities of L1 and NCAM proteins in neuronal subsets and the corresponding molecular interactions have been conserved in both CAM families over a long evolutionary time period. However, in contrast to the nonneuronal Nrg¹⁶⁷ isoform, which exhibits an antiredundant capacity compared with Nrg¹⁸⁰, the nonneuronal (RSLE−) vertebrate L1-CAM isoform is able to rescue the Nrg deficiency in OP axons. In contrast to Drosophila Nrg, the two vertebrate L1-CAM isoforms differ by the inclusion or exclusion of two small exons. The insertion of the five additional amino acid residues, which are encoded by exon2, into the L1-CAM extracellular domain modifies the homo- and heterophilic functions of vertebrate L1-CAMs. The inability of the human L1-CAM^RSLE+ isoform to efficiently interact with Drosophila Neuroglian suggests that the L1-CAM^RSLE+ GOF phenotype is the result of homotypic molecular interactions. Moreover, the rescue of nrg³ OP axonal phenotype by L1-CAM^RSLE− occurs in an Nrg deficient background, suggesting that vertebrate L1-CAM^RSLE− proteins are able to engage in homotypic molecular interactions in Drosophila. Interestingly, the nonneuronal human L1-CAM^RSLE− protein, for which a lower homophilic interaction capacity has been postulated, causes a much weaker GOF phenotype than the neuronal mouse L1-CAM^RSLE+ isoform. Nevertheless, the results indicate that this lower homophilic binding activity of the RSLE− isoform is sufficient to support the functional replacement of Nrg¹⁸⁰ in OP axons in Drosophila (Kristiansen, 2005).

Inclusion of the cytoplasmic miniexon generates a tyrosine-based endocytosis signal (RSLEY) in the neuronal vetebrate L1-CAM isoform. The AP-2-mediated endocytosis of the neuronal L1-CAM^RSLE+ isoform appears to be an important step in the activation of the MAPK signaling cascade by L1-CAM. Since neither Drosophila Nrg isoform contains an equivalent endocytosis signal in their cytoplasmic domain, Drosophila Nrg function either does not involve endocytosis or uses a different type of sorting signal than vertebrate L1 proteins (Kristiansen, 2005).

Although the analysis of the two Nrg isoforms indicates a specific requirement for Nrg¹⁸⁰ in OSS neurons, analysis of GOF conditions in the wing peripheral nervous system reveals an underlying common ability to activate RTK signaling. Since the Nrg-mediated activation of EGFR kinase only requires the extracellular Nrg domain for its interaction with the EGFR, both Nrg isoforms are able to exhibit an identical RTK-dependent axonal misguidance GOF phenotype. The ability of homologous vertebrate L1- and NCAM proteins to elicit the same response in Drosophila sensory neurons indicates a common, conserved specificity to influence RTK activity and thereby to regulate axonal growth and guidance. However, the different ability of the neuronal versus the nonneuronal Nrg isoform to rescue the nrg LOF phenotype in the OSS indicates that Nrg-mediated axonal guidance is also regulated by cytoplasmic interactions (Kristiansen, 2005).

Since the separation of arthropods and chordates, there has been an enormous diversification in the size and organization of metazoan nervous systems. At the same time, there has also been an increase in the number of L1- and NCAM-type paralogous genes in vertebrates (but not in Drosophila), as well as structural divergence and acquisition of new specific functions within each protein family. Both types of proteins have conserved an average of 25%–30% amino acid identity between their vertebrate and Drosophila homologues. The two groups of genes are of roughly similar size, and both have undergone independent events that resulted in the generation of different tissue-specific isoforms in Drosophila and vertebrates. Although both the vertebrate and invertebrate proteins are normally coexpressed in specific axonal tracts, their respective realms of expression have shifted in insect versus vertebrate nervous systems. As a result, NCAM expression is more widespread than L1-CAM or Nr-CAM in vertebrates, while FasII is more restricted than Nrg in insects. Therefore, all these genes are evidently highly accessible to mutation and genetic drift, and the current situation most probably reflects a selective pressure to maintain NCAM- and L1-type protein coexpression in specific axonal tracts of the nervous system. Nevertheless, although both L1 and NCAM proteins have acquired many new functions in both arthropod and chordate species, it appears that they initially had at least partially overlapping roles in growth cone signaling during axon guidance. Both CAM families have apparently maintained some of these shared functions and a common specificity, including a basic function as activators of RTK signaling, over a long time period (Kristiansen, 2005).

Therefore, it seems that the functional redundancy between L1- and NCAM-type proteins could constitute an important evolutionary constraint. It prevents the drift of these molecules into completely different functional entities, while at the same time, it allows their structures to further diverge and acquire separate and additional specificities. It has been proposed that functional redundancy is one mechanism for the canalization (stability after developmental perturbation and during evolution) of developmental processes. The requirement for a shared specificity between L1- and NCAM-type proteins in the control of RTK signaling during axon guidance might therefore reflect a requisite for redundancy that is found in any complex communication process. Redundancy is an essential component in any communication process for ensuring reliability by compensating the naturally occurring perturbations. Neuronal wiring is a cell communication-driven process where a highly complex set of signaling systems operates in parallel. As the number of different signals involved in axon guidance enlarged concomitant with an increase in complexity during evolution, the system noise affecting growth cone signal integration during development also increased. Unspecific adhesive interactions may also constitute a major source of noise for navigating growth cones. Therefore, cooperative redundancy might contribute to establishing a “buffered” physiological context required for ensuring process fidelity. It is postulated that this is the reason why the ancestral functional redundancy between L1- and NCAM-type molecules has been conserved over the last 600 million years of evolution (Kristiansen, 2005).

Axonal insulation is critical for efficient action potential propagation and normal functioning of the nervous system. In Drosophila, the underlying basis of nerve ensheathment is the axonal insulation by glial cells and the establishment of septate junctions (SJs) between glial cell membranes. However, the details of the cellular and molecular mechanisms underlying axonal insulation and SJ formation are still obscure. This study reports the characterization of axonal insulation in the Drosophila peripheral nervous system (PNS). Targeted expression of tau-green fluorescent protein in the glial cells and ultrastructural analysis of the peripheral nerves allow visualization the glial ensheathment of axons. Individual or a groups of axons are ensheathed by inner glial processes, which in turn are ensheathed by the outer perineurial glial cells. SJs are formed between the inner and outer glial membranes. Neurexin IV, Contactin, and Neuroglian are coexpressed in the peripheral glial membranes and these proteins exist as a complex in the Drosophila nervous system. Mutations in neurexin IV, contactin, and neuroglian result in the disruption of blood-nerve barrier function in the PNS, and ultrastructural analyses of the mutant embryonic peripheral nerves show loss of glial SJs. Interestingly, the murine homologs of Neurexin IV, Contactin, and Neuroglian are expressed at the paranodal SJs and play a key role in axon-glial interactions of myelinated axons. Together, these data suggest that the molecular machinery underlying axonal insulation and axon-glial interactions may be conserved across species (Banerjee, 2006a).

The localization of Nrx IV, Cont, and Nrg in the embryonic nervous system was studied. Nrx IV, Nrg, and Cont show colocalization at the nerve glial membranes. Nrx IV, Cont, and Nrg are interdependent for their epithelial SJ localization. Having established that Nrx IV, Cont, and Nrg colocalize in the peripheral nerves, whether these proteins are interdependent for their localization in the peripheral nerves was studied. The effect of the absence of each of these three proteins on the localization of the other two was studied in the embryonic peripheral nerves using nrx IV, cont, and nrg null mutants. nrx IV mutant embryos were stained with anti-Nrx IV, anti-Cont, and anti-Nrg. Cont and Nrg proteins showed a rather diffused localization when compared with their wild-type localization. In addition, Cont is present as puncta in the cytoplasm of the glial cells because of its failure to get targeted properly to the membrane in the absence of Nrx IV. Similarly, cont null mutant embryos also showed less defined distribution and reduction of Nrx IV and Nrg in the glial membrane. nrg null mutant embryos display significant reduction in Nrx IV and Cont localization in the glial membranes. These results demonstrate that Nrx IV, Cont, and Nrg are interdependent for their proper localization in the embryonic peripheral nerves (Banerjee, 2006a).

Ultrastructural analyses of the embryonic epithelia in nrx IV, cont, and nrg mutants showed that these genes are required for the formation and/or organization of epithelial SJs. Nrx IV, Cont, and Nrg are interdependent for their proper localization both in the epithelia and in the peripheral nerves. These phenotypic similarities raised an interesting possibility that these proteins are part of a macromolecular protein complex that exists in the nervous system. To determine whether these three proteins form a biochemical complex in the nervous system, Drosophila heads, which are a rich source of both neurons and glial cells, were used. Coimmunoprecipitation experiments using Nrx IV, Cont, and Nrg antibodies efficiently coprecipitated Nrx IV, Cont, and Nrg. Interestingly, both isoforms of Nrg (180 kDa neuronal and 167 kDa epithelial) were immunoprecipitated by Nrx IV and Cont antibodies, suggesting that both isoforms are part of a protein complex that includes Nrx IV and Cont. However, at this point, it is not possible to differentiate whether isoform-specific complexes are formed or whether both isoforms are in the same complex. In addition, sucrose density gradient analysis of the fly head lysates was performed to determine how Nrx IV, Cont, and Nrg distribute in buoyant density gradients. Nrx IV and Cont cosediment in overlapping fractions. Nrg shows distribution in the lighter sucrose density fractions that do not overlap with Nrx IV and Cont but partially overlaps with that of Nrx IV and Cont, indicating that these proteins are associated with subcellular structures of the same density and may associate into a biochemical complex that is partially maintained during subcellular fractionation (Banerjee, 2006a).

The immunofluorescence analysis of nrx IV, cont, and nrg null mutants showed that in each of these mutants, the other two proteins show qualitatively reduced fluorescence intensities under identical confocal settings. The possible explanation for reduced fluorescence intensity could be that loss of any of these proteins affects the localization or stability of the other proteins. Whether the levels of the other two proteins had changed in nrx IV, cont, and nrg null mutants was examined using immunoblot analysis. Nrx IV protein levels did not seem to be affected in cont and nrg mutants when compared with wild type. Cont protein levels were severely affected in nrx IV mutants compared with wild-type and nrg mutant embryos. Nrg protein levels were also affected in nrx IV mutants but showed no change in cont mutants. At this stage, it cannot be rule out whether the change in Cont and Nrg protein levels in nrx IV mutants are attributable to reduced stability or less synthesis of these proteins. Together, the biochemical data indicate that Nrx IV, Cont, and Nrg form a protein complex in the nervous system and that, in the absence of Nrx IV, the stability of Cont and Nrg is severely affected (Banerjee, 2006a).

Inner glial membrane processes are involved in the ensheathment of either an individual axon or a group of axons. This glial ensheathment not only provides insulation of axons but also generates unique junctions between either glial membranes or between axons and glial membranes. Nrx IV, Cont, and Nrg are involved in the establishment of glial–glial SJs. Although additional components involved in these interactions need to be identified, these findings provide a basis for additional analysis of neuronal SJs, which would be relevant to the understanding of their vertebrate counterparts: the paranodal axo-glial SJs (Bhat, 2003; Hortsch, 2003; Salzer, 2003; Banerjee, 2006a and references therein).

The fundamental basis of axonal ensheathment in any species is to faithfully transmit neuronal signals along the nerve fibers and optimize desired cellular responses. To maximize the speed of conduction and/or to minimize the loss of nerve signals, many species evolved mechanisms in which axonal lengths remained short (as seen in insects) by increasing the diameter of the axons or by clustering voltage-gated Na⁺ channels to discrete unmyelinated regions of the axon, the node of Ranvier, as seen in myelinated nerve fibers of vertebrates. Most invertebrate species use some type of glial cells to ensheath their axons without generating a myelin sheath. The insulation is contiguous without any breaks, suggesting that primitive nodal structures or clustering of voltage-gated Na⁺ channels may not exist in invertebrates. However, recent reports have challenged some of these notions. In copepod crustaceans, ultrastructural analysis of the first antenna and the CNS has revealed extensive myelination of sensory and motor axons. These studies have raised some fundamental questions about axonal insulation and origins of myelination (Banerjee, 2006a and references therein).

In invertebrates like Drosophila, two types of glial cells are involved in insulation. The inner peripheral glial cells are involved in axonal ensheathment, and the outer (perineurial) glial cells wrap around the inner glial cells to provide another level of ensheathment. This two-cell ensheathment in Drosophila peripheral nerves may be advantageous to ensure that high K⁺ containing hemolymph does not interfere with action potential propagation. Although the cellular aspects of axonal insulation are being unraveled, the molecular mechanisms underlying the axonal ensheathment remain to be investigated. Most importantly, what are the protein constituents of the insulating membranes, and whether some of the vertebrate myelin proteins have their homologs in invertebrates? A detailed molecular analysis of the nature of the glial cells that ensheath axons as in Drosophila or produce myelin-like structures and the type of myelin in copepods may provide insights into whether myelinating glial cells arose from a common ancestor. However, a genetic dissection of the axonal ensheathment in Drosophila will uncover some of the basic aspects of the neuron-glial interactions that lead to ensheathment of nerve fibers across species (Banerjee, 2006a).

Cell adhesion molecules play a pivotal role in establishing intercellular junctions [e.g., cadherins and associated catenins form a protein scaffold that establish adhesion contacts at the adherens junctions and link them to the actin cytoskeleton. Similarly, claudins and associated cellular scaffolding proteins are required for establishing TJs. In both of these examples, transmembrane proteins bring two opposing membranes together to establish junctions through homophilic and/or heterophilic interactions (Banerjee, 2006a and references therein).

The finding that nrg null mutant nerves display increased spacing between the outer and inner glial membranes suggest that Nrg may be involved in cell–cell interactions and cell–cell adhesion between glial membranes. In contrast, the observation that loss of nrx IV and cont does not affect the membrane spacing between the inner and outer glial membranes suggests that Nrx IV and Cont are not involved in membrane adhesion or bringing the glial membranes in close apposition. Together, these data suggest that Nrg is critical for both the adhesion and SJ formation, whereas Nrx IV and Cont are critical for the formation of the septa at SJs. In addition, the missing axonal fascicles in nrg mutant nerves could result from axon fasciculation defects or axonal degeneration as a secondary consequence resulting from the loss of glial support. Axonal fasciculation defects have been observed in nrg mutants. Alternatively, axonal loss in nrg mutants might result from a disruption in axonal cytoskeleton, because Nrg possesses domains that could potentially interact with and stabilize the axonal cytoskeleton. Based on Nrg expression in S2 cells, Nrg is predicted to recruit membrane skeleton assembly within specialized domains of neurons in response to cell adhesion. Both Nrg protein forms Nrg¹⁶⁷ and Nrg¹⁸⁰ contain a short cytoplasmic domain as a binding site for ankyrin. Ankyrins are linker proteins that connect various membrane proteins with the actin-spectrin network in the cell. Loss of axons observed in nrg mutants is clearly suggestive of axon-glial interdependence that may alter axonal survival. In vertebrates, axolemmal-myelin interactions are critical for the formation of the paranodal axo-glial SJs (see Garcia-Fresco, 2006). This raises an interesting possibility that axon-glial interactions in Drosophila may use similar interactive mechanism for establishing axo-glial SJs. Thus, the current studies on Nrx IV, Cont, and Nrg suggest that these proteins are critical for the formation and/or organization of the SJs between either glial cells and possibly between axons and glial cells, which remains to be further investigated (Banerjee, 2006a).

Invertebrate axons are insulated from their salt-rich environment through a glial-dependent BBB, which plays a crucial role in electrical and chemical insulation. Ultrastructural studies have demonstrated SJs between perineurial glial cells and inner glial cell membrane to form the structural basis of BBB of some insects. Absence of SJs in nrx IV, cont, and nrg mutants, and a compromised BNB in the PNS as evidenced by dye exclusion analyses, provide additional confirmation in support of SJs as a prerequisite for blood-nerve barrier (BNB) formation. Not just in the PNS, recent reports on the BBB formation and function also in the CNS of Drosophila underscore the importance of SJs in proper sealing and insulation of the nerve cord (Schwabe, 2005), thereby supporting that insulation and establishment of functional SJs in both PNS and CNS go hand in hand. Although, G-protein-coupled receptor signaling pathway members have been identified to establish the BBB in Drosophila CNS, the signaling mechanisms that operate in the PNS still remain to be established (Banerjee, 2006a).

The axo-glial SJs in the myelinated axons share many anatomical features similar to those of invertebrate SJs, especially the electron-dense ladder-like transverse septa (Bhat, 2003; Banerjee, 2006a). Ensheathment of Drosophila axons by perineurial glial cells in the absence of myelin-producing glial cells would have predicted that the molecular components of the invertebrate SJs would be different from those of the vertebrate axo-glial SJs. Surprisingly, the fly SJ molecular components are present at the vertebrate axo-glial SJs and not at the TJs, which serve similar functions. Thus, the molecular similarities reflect an evolutionarily conserved function of creating an ionic barrier both at Drosophila SJs and at axo-glial SJs in the paranodal region. More importantly, Drosophila nerves contain a large number of axons, which are collectively held together by glial membranes not only to maintain nerve fasciculation but also to maintain the insulation of individual axons. It would be of significant interest to establish whether axo-glial SJs are present in Drosophila nerves and to identify downstream components of these junctions in Drosophila that link these junctions to the glial or axonal cytoskeleton. Identification of such molecules will provide insights into whether these junctions play a much broader role in axon-glial signal transduction. In summary, the molecular and functional similarities between the Drosophila SJs and vertebrate axo-glial SJs should allow a genetic and molecular dissection to be undertaken of the formation and function of these junctions in Drosophila (Banerjee, 2006a).

Cell adhesion molecules have long been implicated in the regulation of axon growth, but the precise cellular roles played by individual cell adhesion molecules and the molecular basis for their action are still not well understood. The sensory system of the Drosophila embryo was used to shed light on the mechanism by which the L1-type cell adhesion molecule, Neuroglian regulates axon growth. A highly penetrant sensory axon stalling phenotype was found in neuroglian mutant embryos. Axons stalled at a variety of positions along their normal trajectory, but most commonly in the periphery some distance along the peripheral nerve. All lateral and dorsal cluster sensory neurons examined, except for the dorsal cluster neuron dbd, showed stalling. Sensory axons were never seen to project along inappropriate pathways in neuroglian mutants and stalled axons showed normal patterns of fasciculation within nerves. The growth cones of stalled axons possessed a simple morphology, similar to their appearance in wild type embryos when advancing along nerves. Driving expression of the wild type form of Neuroglian in sensory neurons alone rescued the neuroglian mutant phenotype of both pioneering and follower neurons. A partial rescue was achieved by expressing the Neuroglian extracellular domain. Over/mis-expression of Neuroglian in all neurons, oenocytes or trachea had no apparent effect on sensory axon growth. It is concluded that Neuroglian is necessary to maintain axon advance along axonal substrates, but is not required for initiation of axon outgrowth, axon fasciculation or recognition of correct growth substrates. Expression of Neuroglian in sensory neurons alone is sufficient to promote axon advance and the intracellular region of the molecule is largely dispensible for this function. It is therefore unlikely that Nrg acts as a molecular 'clutch' to couple adhesion of F-actin within the growth cone to the extracellular substrate. Rather, it is suggested that Neuroglian mediates sensory axon advance by promoting adhesion of the surface of the growth cone to its substrate. The finding that stalling of a pioneer sensory neuron is rescued by driving Neuroglian in sensory neurons alone may suggest that Neuroglian can act in a heterophilic fashion (Martin, 2008).

nrg LOF mutants show a highly penetrant sensory axon stalling phenotype. This defect is displayed by all neurons examined, except for the dorsal cluster neuron, dbd, which apparently leads other dorsal cluster axons as it grows along the ISN into the CNS. The fact that this pioneering neuron is unaffected by loss of nrg function may suggest that Nrg is necessary for axon advance only along axonal substrates. This idea is supported by the observation that lateral cluster axons in nrg mutants almost always advance successfully along their initial non-neuronal growth substrate, the spiracular branch of the trachea, and stall only after they have subsequently joined the ISN. As sensory axons in nrg mutants generally advance for some distance along nerves before stalling, Nrg is apparently required to maintain axon growth along axonal substrates, rather than to initiate it. Most axon stalls occur in the periphery, but their locations are quite variable and do not obviously coincide with particular structures, such as nerve branches, pointing to a stochastic component in the stalling process (Martin, 2008).

Stalled sensory axons in nrg mutants are always found in their correct nerve pathways. The absence of axon misprojection phenotypes shows that Nrg is not necessary for recognition of correct growth substrates by sensory axons. It was also observed that sensory axons in nrg mutants fasciculate normally with other axons. The simple growth cone morphology of the stalled axons -- club-shaped with few filopodia -- is similar to that seen in wild-type sensory axons when they are growing along nerves. These observations show that Nrg is not necessary for axon-axon fasciculation in this system. The Ig-CAM Fasciclin2 (Fas2) is a key regulator of motor axon fasciculation in the Drosophila embryo and also acts redundantly to Nrg in the regulation of OP axon growth in the adult OSS. However, Fas2 is unlikely to be acting redundantly with Nrg in regulating sensory axon fasciculation in the embryo, since embryonic sensory axons do not express Fas2. In addition, the pattern of 22C10 sensory nerve staining is normal in fas2 LOF mutants and in embryos over/misexpressing Fas2 in sensory neurons and their growth substrates. Whether Nrg acts redundantly with some other CAM to mediate sensory axon fasciculation remains to be determined (Martin, 2008).

The sensory axon defects seen in nrg mutant embryos are similar to those reported for motor axons, although motor axons stall at a later stage of growth, close to their synaptic targets, the body wall muscles, and unlike sensory axons, some motor axons misproject in nrg mutants. The nrg LOF phenotype in the embryonic sensory system is much more stereotypic than in the adult OSS. This difference may indicate that Nrg performs more diverse functions in the adult OSS, including regulation of axon fasciculation and guidance in addition to axon advance. While Nrg is expressed in both sensory axons and several of their growth substrates, including trachea and motor axons, mutant rescue experiments demonstrate that expression in sensory axons alone is sufficient to mediate their advance. It is unlikely, therefore, that the sensory axon defects are secondary to other known morphological defects in nrg mutants, such as motor axon stalling or failure of glial cell ensheathment of peripheral nerves (Martin, 2008).

An important, unresolved question is whether the effects of Nrg on axon growth are mediated by homophilic interactions between Nrg on the growth cone and its substrate or whether heterophilic interactions are involved. Substrate-bound Nrg has been shown to promote neurite extension from Nrg-expressing neurons in vitro, but it is unclear whether the same holds true in vivo. Expression of Nrg in sensory neurons completely rescues the axon stalling phenotype of lch5-1 seen in nrg mutants. In wild-type embryos and in nrg mutants, lch5-1 is the pioneer for the lateral cluster fascicle and associates with motor axons, rather than dorsal cluster sensory axons as it grows towards the CNS. Lch5-1 mutant rescue findings might suggest, therefore, that Nrg is acting in a heterophilic fashion to promote advance of this axon. This conclusion rests upon the assumption that, in nrg mutant embryos in which the lch5-1 stall phenotype has been rescued, the lch5-1 growth cone does not employ the dorsal sensory axons as a growth substrate as it advances along the ISN. Observations of the dynamics of lch5-1 growth cone activity, and its association with sensory and motor axons in the ISN as it advances along the nerve in these rescued embryos, would help to resolve this question (Martin, 2008).

Mutant rescue experiments show that the intracellular region of Nrg is largely dispensable for its role in promoting sensory axon advance. It has long been known that Nrg-mediated cell-cell adhesion does not require the intracellular region of the molecule. However, mutation of the intracellular ankyrin-binding domain of L1- type CAMs does have clear effects on their membrane mobility and their coupling to retrograde actin flow in growth cones. Given the linkage between ankyrins and the actin-spectrin cytoskeleton, this has led to suggestions that L1-type CAMs may transmit traction force generated by actin flow to the extracellular substrate, as posited by the 'molecular clutch' hypothesis. The recent finding that MAO kinase phosphorylation of the ankyrin-binding site regulates neurite growth from cerebellar granule neurons on a Ng-CAM coated substrate highlights the potential importance of the intracellular region of L1-type CAMs in mediating their effects on axon growth. However, this view is difficult to reconcile with the current results. One possible explanation is that the in vitro models used in most vertebrate studies to date do not accurately reflect the in vivo functions of L1- type CAMs. Alternatively, Nrg, the Drosophila L1 homologue, may function differently to vertebrate L1-type CAMs in promoting axon growth (Martin, 2008).

The finding that the intracellular region of Nrg is not essential for its ability to promote sensory axon advance raises the question of how Nrg-mediated adhesion of the growth cone to its substrate is coupled to retrograde F-actin flow. Current models assume that such a coupling provides the motive force for growth cone advance. One possibility is that Nrg interacts in cis with another receptor or CAM, which in turn is coupled to the cytoskeleton of the growth cone. A genetic screen, using the sensory axon stalling phenotype described in this study as an assay, would be one way of identifying such a Nrg-interacting molecule (Martin, 2008).

Taken together, these results suggest that Nrg may mediate sensory axon advance along nerves in the Drosophila embryo by promoting adhesion between the surface of the growth cone and its axonal substrates. This adhesive interaction is likely to involve a heterophilic interaction between Nrg on the growth cone and some other, as yet unidentified molecule, on the substrate. Intracellular signaling by the Nrg molecule does not appear to be essential for its function in this context. These findings for Nrg are mirrored by the recent discovery that the adhesive, rather than the signaling activity, of another CAM, Drosophila N-cadherin, is essential for its role in target selection of photoreceptor afferents (Martin, 2008).