derailed

REGULATION

The derailed (drl) gene encodes a receptor tyrosine kinase (RTK) that governs aspects of axon guidance and muscle-epidermal interactions in the Drosophila embryo. To determine the types of neurons that express drl, a series of drl promoter fusions to axon-targeted reporters have been studied. Described here are intronic enhancers that drive reporter expression in four distinct subtypes of embryonic neurons, all of which project axons in the anterior commissure of the developing nervous system. The first population is defined by drlR, which drives reporter expression in 10 medial interneurons per hemisegment clustered ventral to the anterior commissure. These neurons extend ipsilateral axons in the anterior commissure and ramify local processes near the junction of the anterior commissure and the longitudinal connective. The second population is defined by the expression of drlT, and is composed of 20 lateral interneurons in each hemisegment that project contralaterally in the anterior commissure. A subset of these axons turns anteriorly upon reaching the contralateral connective, and form a single longitudinal fascicle. The third population is defined by drlU expression in a single bilaterally-paired interneuron present in segment A3-A8 that crosses the midline in the anterior commissure and extends anteriorly in the longitudinal connective. The final population is defined by drlZ expression in two VUM motorneurons that bifurcate in the anterior commissure and exit in the intersegmental nerve. The combined expression of these four neuronal populations, appears to give the pattern of CNS expression observed in a drl enhancer trap line (Bonkowsky, 1999).

Enhancers driving expression in the drl-expressing muscles and epidermal attachment cells are also described. These enhancers define the classes of neurons projecting in the anterior commissure and can be used to precisely define axon pathfinding errors in drl and other mutants. drl neurons are a heterogeneous population of neurons that ultimately choose different pathways and synapse with different target cells. However, they all share the common property of projecting axons in the anterior commissure, as well as expressing drl, suggesting a role for drl in axon guidance through the anterior commissure. Isolation of separate enhancers for the drl and their corresponding epidermal attachment cells provides the opportunity to analyze the reciprocal interactions during muscle attachment site selection. Finally, expression of drl in each of the four subclasses of anterior commissural neurons is controlled by separate transcriptional regulatory sequences. This cell-type specific modular nature of drl transcriptional regulation makes it an attractive system for understanding higher order cis-regulation of gene expression in the developing nervous system (Bonkowsky, 1999).

Nerfin-1 is a nuclear regulator of axon guidance required for a subset of early pathfinding events in the developing Drosophila CNS. Nerfin-1 belongs to a highly conserved subfamily of Zn-finger proteins with cognates identified in nematodes and man. The neural precursor gene prospero is essential for nerfin-1 expression. Unlike nerfin-1 mRNA, which is expressed in many neural precursor cells, the encoded Nerfin-1 protein is only detected in the nuclei of neuronal precursors that will divide just once and then transiently in their nascent neurons. Although nerfin-1 null embryos have no discernible alterations in neural lineage development or in neuronal or glial identities, CNS pioneering neurons require nerfin-1 function for early axon guidance decisions. Furthermore, nerfin-1 is required for the proper development of commissural and connective axon fascicles. Nerfin-1 is essential for the proper expression of robo2, wnt5, derailed, G-oα47A, Lar, and futsch<, genes whose encoded proteins participate in these early navigational events (Kuzin, 2005).

Given the axon guidance defects in nerfin-1^null embryos and the fact that Nerfin-1 is a Zn-finger nuclear protein, it was hypothesized that Nerfin-1 may be required for the correct expression of genes involved in axon guidance. Accordingly, the embryonic expression profiles of over 35 genes that have been shown to play important roles in axon guidance were examined. Included in the candidate screen were genes encoding transcription factors, RNA-binding proteins, cell surface receptor proteins, their ligands, signal transduction proteins, and components of the cytoskeleton. Homozygous nerfin-1^null embryos were identified by the absence of Nerfin-1 immunoreactivity. Whole-mount in situ hybridization and/or protein immunostaining for altered spatial or temporal expression in nerfin-1^null embryos identified six genes that require nerfin-1 function to achieve full wild-type expression levels (Kuzin, 2005).

Two genes involved in anterior vs. posterior commissure choice, those encoding the receptor tyrosine kinase Derailed, and its ligand Wnt5, both required nerfin-1 for full expression. In the absence of nerfin-1, ventral cord expression levels of Robo and Robo3 were unaffected; however, Robo2 expression levels were significantly reduced. Expression of Slit, the ligand for Robo receptors, and Commissureless, a factor responsible for clearing Robo receptors from commissural axons, was unaffected in nerfin-1^null embryos (Kuzin, 2005).

Loss of nerfin-1 function also significantly delayed and/or reduced the early expression of the neuron-specific microtubule-associated MAP1B-like gene futsch. futsch expression is normally activated in newborn neurons starting at stage 11; however, in nerfin-1^null embryos expression is first detected only at the stage 13. Not until embryonic stage 15 did the level of futsch expression in mutant embryos approach that of wild type. Reduced mRNA steady state levels for the genes encoding Leukocyte-antigen-related-like (Lar), another receptor tyrosine kinase, and G-oα47A gene, which encodes an alpha subunit of heterotrimeric G proteins, were also detected in nerfin-1^null embryos. The reduced level of gene expression in mutant embryos was nervous system specific. For example, G-oα47A gene expression in mesodermal derived tissues was not altered in nerfin-1^null embryos (Kuzin, 2005).

Intra-axonal patterning: intrinsic compartmentalization of the axonal membrane in Drosophila neurons

In the developing nervous system, distribution of membrane molecules, particularly axon guidance receptors, is often restricted to specific segments of axons. Such localization of membrane molecules can be important for the formation and function of neural networks; however, how this patterning within axons is achieved remains elusive. This study shows that Drosophila neurons in culture establish intra-axonal patterns in a cell-autonomous manner; several membrane molecules localize to either proximal or distal axon segments without cell-cell contacts. This distinct patterning of membrane proteins is not explained by a simple temporal control of expression, and likely involves spatially controlled vesicular targeting or retrieval. Mobility of transmembrane molecules is restricted at the boundary of intra-axonal segments, indicating that the axonal membrane is compartmentalized by a barrier mechanism. It is proposed that this intra-axonal compartmentalization is an intrinsic property of Drosophila neurons that provides a basis for the structural and functional development of the nervous system (Katsuki, 2009).

This study describes a patterning phenomenon that takes place within single axonal processes as a cell-intrinsic event. This patterning involves compartmentalization of the axonal membrane with a diffusion barrier located at a medial point of the axon. The data suggest that this patterning ability is a fundamental property of Drosophila neurons, because the compartment-specific localization of GFP-tagged receptors can be observed in the majority (>90%) of neurons. In the CNS of Drosophila, more than 90% of neurons project their axons to the contralateral side of the nervous system, and the width of the commissural segment or precrossing segment of those neurons is 20-40 μm, which parallels the length of the proximal compartment observed in vitro. This raises the possibility that the intrinsic patterning ability of neurons may serve as the basis of generating the intra-axonal localization of guidance molecules in vivo (Katsuki, 2009).

In addition to these intrinsic abilities of neurons, the results suggest that extrinsic factors may also contribute to the intra-axonal patterning, because not all ROBO receptors examined in this study recapitulated the localization patterns observed in vivo. All three members of ROBO family receptors are localized to distal axon in vivo. Whereas ROBO2 and ROBO3 retained the ability to localize distally when isolated in culture, ROBO was uniformly distributed along axons under such conditions. Localization of ROBO may require extrinsic signals that are absent in the culture system. One of the candidate extrinsic factors are the midline cells, which lie on an axonal region where ROBO expression is downregulated in vivo. It is also possible that the location of the compartment boundary determined by the intrinsic mechanisms is refined by extrinsic signals. It would be interesting to test whether contact with midline cells in culture can induce distal localization of ROBO, or alter the position of the boundary (Katsuki, 2009).

It has been commonly suggested that axon guidance receptors are targeted to the growth cone, and intra-axonal localization patterns of guidance receptors reflect temporal profiles of receptor expression at the growth cone during axonal extension. This study demonstrates that intra-axonal localization patterns that are evident in the culture condition can form regardless of the timing of receptor expression. Although this result does not rule out the involvement of temporal control of expression during axon navigation in vivo, it suggests that critical mechanisms for the intra-axonal localization of receptors described in this study are compartment-specific trafficking pathways. One such trafficking mechanism could involve local translation or targeted membrane transport, which can specifically deposit membrane proteins to either the proximal or distal membrane compartment. It is also possible that membrane proteins are selectively retrieved from one compartment through endocytic pathways (Katsuki, 2009).

A time course experiment in shi^ts1 mutant backgrounds suggests that Derailed (DRL) is preferentially targeted to the proximal compartment. It was also shown that the correct intra-axonal localization of DRL requires Dynamin-dependent endocytosis; however, at present it cannot be distinguished whether or not the endocytosis of DRL is compartment specific. Because fluorescence recovery after photobleaching (FRAP) experiments on CD8-GFP suggest that the barrier between the proximal and distal axon compartments does not completely block the movement of membrane proteins between the compartments, it is possible that the Dynamin-dependent endocytosis is required to remove DRL that leaks into the distal compartment, serving to maintain the pattern generated by targeting. Alternatively, endocytosis itself may be compartment specific, contributing to the establishment of the pattern (Katsuki, 2009).

In contrast to DRL, ROBO3 does not appear to require shibire function for its localization, demonstrating the presence of differential trafficking mechanisms for DRL and ROBO3. Due to this shi-independence of ROBO3, it is not possible to conclusively demonstrate the presence of preferential targeting of ROBO3 by performing a time course experiment. Even if there is preferential targeting, it is likely that ROBO3 also needs to be removed from the incorrect compartment, because ROBO3 shows a level of lateral mobility on the axon similar to that of DRL. Since ROBO3 localization is largely independent of Dynamin function, such a retrieval pathway must be based on Dynamin-independent mechanisms. While the complementary localization patterns of DRL and ROBO3 suggests that intra-axonal compartments are fundamental units for localization of multiple molecules, molecular mechanisms for generating or maintaining their compartmental localization could be diverse (Katsuki, 2009).

Another critical issue raised in the previous studies in vivo is how the intra-axonal localization of guidance receptors is maintained over time. If the guidance receptors are freely diffusible on the axonal membrane, they may spread along the axon, leading to a uniform distribution. FRAP experiments in cultured neurons revealed that localized receptors (ROBO3-EGFP and DRL-EGFP) are indeed mobile within the intra-axonal compartment. Although the mobility of these localized receptors across the compartment boundary was not directly measurable, the mobility of several transmembrane proteins (ROBO-EGFP and CD8-GFP) and lipid-anchored protein (GFPgpi) that distribute along the entire axon length was significantly restricted at the compartment boundary. This restriction is likely due to the diffusion barrier that spans over a 10 μm axon length around the boundary. It is proposed that this barrier is a part of the mechanisms that maintain the pattern of compartment-specific membrane proteins, as shown in different subcellular regions such as the tight junction of epithelial cells, the posterior ring of sperm, the cleavage furrow of dividing yeast and mammalian cells, and the initial segment of mammalian neurons. No significant barrier effect on GAP-GFP, which resides in the inner leaflet of the plasma membrane, was detected. It was also observed that vesicles containing membrane proteins pass through the barrier region. Thus, a model is favored in which the barrier becomes effective only after the molecules are inserted into the axonal membrane. It would be important to test whether or not a diffusion barrier exists in vivo, and whether or not it plays a role in the development of the nervous system (Katsuki, 2009).

An important but yet poorly explored question is the role of the guidance receptors localized on axon shafts. A straightforward explanation can be offered based on non-cell-autonomous functions of guidance receptors or membrane proteins in general; they may 'label' axon pathways through specific adhesion (fasciculation), or through presenting their ligands, thereby providing instructive spatial cues for the navigation of other axons. For example, Fasciclin cell-adhesion molecules have been suggested to provide pathway labels for guiding other growth cones. Drosophila Netrin receptor Frazzled/DCC relocates its ligand Netrin to strategic positions in the nervous system, thereby generating guidance information for a longitudinal pioneer neuron. Other studies reported that guidance receptors can also play non-cell-autonomous roles in cell migration and synaptogenesis. Thus, spatial patterns of molecules on axon shafts likely have direct roles in neuronal circuit formation (Katsuki, 2009).

Lastly, it is proposed that the compartmentalization of the axonal membrane could be a common basis for the structure and function of the nervous system. In the Drosophila ventral nerve cord, formation of the longitudinal axon tracts depends on the expression of ROBO receptors. On the other hand, longitudinal axon tracts are considered as the site for synapse formation, because synaptic proteins such as synaptotagmin and synapsin accumulate on the longitudinal tracts. This study found that in cultured neurons both ROBO receptors and synaptic proteins localize to the distal axon compartment. This may suggest that the spatial distribution of guidance molecules and synaptic proteins can be collectively governed by the compartmentalization of the axonal membrane. Future work to identify the molecular basis of the compartmentalization, and to establish the link between cellular identity and this intracellular pattern, will aid in determining how intra-axonal patterning contributes to tissue organization (Katsuki, 2009).

Protein Interactions

Wnt-mediated axon guidance via the Drosophila Derailed receptor

In nervous systems with bilateral symmetry, many neurons project axons across the midline to the opposite side. In each segment of the Drosophila embryonic nervous system, axons that display this projection pattern choose one of two distinct tracts: the anterior or posterior commissure. Commissure choice is controlled by Derailed, an atypical receptor tyrosine kinase expressed on axons projecting in the anterior commissure. Derailed keeps these axons out of the posterior commissure by acting as a receptor for Wnt5, a member of the Wnt family of secreted signalling molecules. These results reveal an unexpected role in axon guidance for a Wnt family member, and show that the Derailed receptor is an essential component of Wnt signalling in these guidance events (Yoshikawa, 2003).

The growth cones of developing neurons are guided to their targets by attractive and repulsive cues in the extracellular environment. Specific receptors on the growth cones recognize these cues and transduce signals that ultimately lead to changes in direction of growth. The best understood of these cues and their axonal receptors are involved in guidance of the large number of axons that project across the midline to the opposite side of the central nervous system (CNS). Distinct groups of cells at the midline divide the two halves of the CNS and have a critical role in axon guidance. These cells, termed midline glia in Drosophila, secrete diffusible factors, the Netrins, capable of attracting contralaterally projecting axons. They also secrete a repellent factor Slit, which together with its receptor Roundabout (Robo) and an intracellular sorting factor that modulates the delivery of Robo to the cell surface, controls whether or not axons will cross the midline (Yoshikawa, 2003).

Once axons commit to crossing the midline, they do not do so randomly. Instead, they follow particular tracts. In each segment of the Drosophila embryonic ventral nerve cord, crossing axons choose one of two commissural tracts, either the anterior or posterior commissure (AC or PC, respectively), which connect the two sides. This choice of commissure is controlled in part by the Derailed (Drl) guidance receptor. Drl is expressed on the growth cones and axons of all neurons that project through the AC, and seems to act as a receptor for a repellent factor in the PC. In drl mutants, AC axons abnormally cross in the PC of many segments. Conversely, misexpression of Drl in PC neurons switches their axonal projections to the AC. Thus, Drl is both necessary and sufficient for axons to cross the midline in the AC. The behavioral phenotypes of drl mutants suggest that at least some of the neurons that require Drl for their guidance fail to make synaptic connections essential for coordinated locomotion and learning and memory (Yoshikawa, 2003).

Drl is a member of the RYK subfamily of atypical receptor tyrosine kinases (RTKs). All members of this subfamily have unusual, but highly conserved amino acid substitutions in their kinase domains plus relatively short extracellular domains devoid of motifs commonly found in other RTKs. Consistent with the unusual amino acid substitutions, the kinase domain of RYK family members appears to lack catalytic activity. However, whereas catalytic activity of Drl has been shown to be dispensable, its cytoplasmic domain is required to dictate commissure choice, suggesting that Drl transduces a signal within growth cones in an unconventional manner, perhaps together with another catalytically active kinase20. The extracellular domain of each RYK family member contains a Wnt inhibitory factor (WIF) domain. The WIF domain of other molecules has been shown to bind to and inhibit the function of members of the Wnt family of secreted signalling molecules, raising the possibility that members of the RYK receptor family, including Drl, bind to Wnt proteins. The Wnt family is large, consisting of seven members in Drosophila and 19 in humans, and is involved in a diverse array of developmental events. Wnt proteins have well-established roles in early cell fate decisions and embryonic patterning, but have also been implicated in synaptic remodelling and terminal arborization within the developing CNS, as well as in regulating planar cell polarity by virtue of the phenotypes of mutations in Frizzled (Fz), one of the Drosophila members of the Fz family of Wnt receptors (Yoshikawa, 2003).

To identify components of the Drl signalling pathway, a genetic screen was carried out for mutations that suppress the ability of Drl to switch axons to the AC when misexpressed by PC neurons. A set of chromosomal deletions covering approximately 80% of the Drosophila genome was screened. One of the deletions that showed strong dominant suppression of the PC-to-AC switching activity of Drl is Df(1)N19, a deletion that removes the X chromosome interval 17A1 to 18A2. By testing a series of overlapping deletions within the Df(1)N19 region, the interval was narrowed to 17B. One of the genes in this interval is wnt5 (also called Dwnt3), a member of the Wnt gene family in Drosophila. wnt5 is a single-exon gene encoding an unusually large Wnt protein of 1,004 amino acids with a unique amino-terminal domain that seems to be proteolytically cleaved, followed by the Wnt domain common to all members of the family (Yoshikawa, 2003).

Given the possibility that Drl might interact with Wnt proteins by means of its WIF domain, mutations were generated specifically in the wnt5 gene to test whether reduction of Wnt5 itself is responsible for the suppression observed with the larger chromosomal deletions. A P element transposon, BG00642, was identified from the Berkeley Drosophila Genome Project inserted in the 5'-untranslated region (5' UTR) of wnt5, and mobilized to generate deletions of the wnt5 coding region. A deletion, wnt5^D7, was recovered that removes the first 261 amino acids of the Wnt5 protein but does not affect either adjacent gene. In addition, a larger deletion was recovered, wnt5^D84, that removes the entire wnt5 coding region plus part of the 3' end of the adjacent gene encoding a member of a family of gamma-glutamyl transferases. Both wnt5^D7 and wnt5^D84 abolish Wnt5 expression: the phenotypes of wnt5^D7 and wnt5^D84 homozygotes, as well as wnt5^D7/wnt5^D84 individuals, are indistinguishable. Thus, wnt5^D7 acts as a null allele. wnt5^D7 and wnt5^D84 homozygotes are viable and fertile, but similar to drl mutants, adults are uncoordinated (Yoshikawa, 2003).

Tests were performed to see whether mutations in wnt5 could suppress the ability of Drl to switch axons to the AC. Using the Gal4/UAS transactivation system, Drl was misexpressed in PC neurons with eagle-GAL4 (eg-GAL4). This driver expresses Gal4 in a sufficiently small subset of neurons so that their axonal projections could be followed unambiguously with a UAS-tau-myc-green fluorescent protein (GFP) axon-targeted reporter transgene. eg-GAL4 drives expression in two small clusters of Eg interneurons in each hemisegment, both of which project axons across the midline (Yoshikawa, 2003).

One of the clusters projects in the PC and the other in the AC. At the midline, the axons from homologous clusters on either side of each segment fasciculate with one another, forming two distinct axon bundles, one within each of the commissures. When forced to misexpress Drl using a UAS-drl transgene, Eg PC neurons switch their projections to the AC in all segments, whereas Eg AC neurons are unaffected. In 92% of segments, every Eg PC axon was switched to the AC, whereas in 8% of segments some axons remained within the PC. Misexpression of Drl using the same transgenes, but in a wnt5/+ heterozygous background, resulted in significantly fewer PC-to-AC switched axons: 34% of segments had all axons switched; 34% had some switched and 32% had none switched. Notably, when Drl is misexpressed in a wnt5 hemizygous or homozygous mutant background, its ability to switch Eg PC neurons to the AC is completely abolished. Thus, Drl requires Wnt5 to switch axons to a different commissure, suggesting that Wnt5 is an essential component of the Drl signalling pathway (Yoshikawa, 2003).

In situ hybridization of wnt5 probes to wild-type embryos revealed that wnt5 messenger RNA expression in the CNS commences at stage 12, a point in development when differentiating neurons begin to extend axons, and continues throughout embryogenesis. High levels of wnt5 were detected in subsets of neurons restricted to the posterior half of each segment and low levels in neurons located more anteriorly in the segment. The neurons expressing high levels of wnt5 lie adjacent to and ventral to the PC in each segment. Although the precise identity of these neurons is unknown, their proximity to the PC and the fact that most of the CNS neurons project axons across the midline suggest that many, perhaps all, of the wnt5-expressing neurons project axons through the PC (Yoshikawa, 2003).

To examine the extracellular distribution of Wnt5 protein, live embryos were stained with an antibody raised against a unique region of the protein N-terminal to the Wnt domain. Staining was detected on the major axonal tracts within the CNS, with the highest levels on the two commissures, a pattern similar to that described previously (Fradkin, 1995). Staining is abolished in wnt5 mutants, demonstrating the specificity of the antibody. Given that wnt5 mRNA is expressed predominantly by subsets of neurons located posteriorly within the segment, Wnt5 apparently either diffuses to the AC or is picked up by AC growth cones and axons as they project toward the midline. Whether the proteolytic processing of Wnt5 observed in cultured cells (Fradkin, 1995) is involved in its distribution in vivo is not known, since the anti-Wnt5 antibody recognizes both the unprocessed and processed forms of the protein [relative molecular mass (Mr) of 140K and 80K, respectively]. Similarly, it is unknown whether all of the Wnt5 recognized by the antibody is biologically active, since processing may be required for activity (Yoshikawa, 2003).

If Wnt5 were a component of the Drl signalling pathway, then loss-of- function mutations in wnt5 might be expected to exhibit drl-like mutant phenotypes. Using an antibody that labels all CNS axons, it was found that wnt5 mutant embryos, similar to drl mutants, have disorganized commissures. In many segments commissures appear irregular, and there are often abnormal axonal projections between the AC and PC. To determine whether these abnormal projections arise from AC axons projecting to the PC or vice versa, subsets of AC and PC axons were labelled in wnt5 mutants with marker lines used for analysing drl mutants. In all cases it was found that AC axons either wander from the AC into the PC or project entirely through the PC, whereas projections of PC axons appear unaltered. These defects are similar to those seen in drl mutants. For example, in wnt5 mutants assayed with a P{tau-lacZ} marker for AC axons, 80% of segments displayed abnormal projections of AC axons into the PC. Similar results were found using Sema2b-tau-myc, another marker for a subset of AC neurons. In contrast to AC axons, in no segments did PC axons, as assayed with a P{tau-lacZ} marker for the PC, project abnormally into the AC. Thus, Wnt5, similar to Drl, is required for proper projection of AC axons across the midline of the CNS (Yoshikawa, 2003).

It has been proposed that Drl functions to keep axons in the AC by acting as a guidance receptor for a repellent ligand in the PC. The high levels of expression by neurons associated with the PC is consistent with Wnt5 acting as such a repulsive ligand. To examine whether Wnt5 is capable of repelling Drl-expressing axons, it was misexpressed at the midline and the effect on crossing axons was assayed. Misexpression of Wnt5 in midline glia using the sim-GAL4 driver caused a marked reduction or complete loss of AC in 43% of segments, but had no discernible effect on the PC. The affected AC axons appeared to either stall or project ipsilaterally within the longitudinal connectives. This phenotype is interpreted as repulsion of the Drl-expressing AC axons from the ectopic source of Wnt5 at the midline. To determine whether this loss of the AC is dependent on Drl, Wnt5 was misexpressed using the identical combination of transgenes, but in a drl homozygous mutant background. Elimination of Drl completely suppresses the loss of the AC, restoring it in every segment, although as expected, abnormal axonal projections between the AC and PC are detected due to the loss of Drl. Thus, in the absence of Drl, axons are insensitive to Wnt5, a feature that may explain the tight spatial regulation of the Drl receptor during development. Drl is normally expressed on growth cones and axons as they project through the AC, but is rapidly downregulated once these growth cones leave the commissure and begin to project in the longitudinal connectives on the contralateral side. This downregulation of Drl may be required to allow further extension of the AC growth cones as they traverse regions of repellent Wnt5 in the connectives, similar to the downregulation of Robo allowing axons to traverse the Slit-expressing midline (Yoshikawa, 2003).

These results suggest that Drl is a receptor for Wnt5. To test for binding of Drl to Wnt5, an examination was made of the in vivo binding of Drl-Fc, a soluble probe consisting of the Drl extracellular domain epitope-tagged with the human immunoglobulin-g (IgG) Fc fragment. In wild-type embryos, Drl-Fc binding was detected at the PC and to regions at the intersection of the PC and the longitudinal connectives. In wnt5 mutant embryos, binding of Drl-Fc is abolished. Conversely, when Wnt5 is misexpressed by heat-shocking late-stage embryos carrying a heat shock-wnt5 (hswnt5) transgene, followed by incubation with Drl-Fc, binding is markedly expanded to include all axon tracts in the CNS. The observation that Drl-Fc labels only the PC in wild-type embryos, whereas Wnt5 is present on both commissures, suggests that Wnt5 in the AC is bound to endogenous Drl present on the AC growth cones and axons, and that this interaction may block Drl-Fc access to Wnt5. Consistent with this, in drl mutants Drl-Fc labelled both the AC as well as the PC (Yoshikawa, 2003).

SDS-polyacrylamide gel electrophoresis (PAGE), immunoblotted with the anti-Wnt5 antibody was used to further examine the interaction between Wnt5 and Drl. Drl-Fc is able to co-precipitate both the unprocessed and the proteolytically processed forms of Wnt5, as evidenced by the presence of 140K and 80K bands from wild-type extracts, but not from wnt5 mutant extracts. Consistent with the lack of Drl-Fc binding to misexpressed Wg in vivo, Drl-Fc did not co-precipitate Wg, as assayed by immunoblotting with an anti-Wg antibody. Although both forms of Wnt5 present in the extracts are capable of binding to Drl-Fc, it is not known, in vivo, whether both actually have access to the Drl receptor. For example, the 140K form may not be efficiently secreted, as has been observed in cultured cells (Fradkin, 1995). However, regardless of the in vivo distribution of the two forms of Wnt5, this result, together with the genetic evidence, indicates that Wnt5 is the ligand for Drl (Yoshikawa, 2003).

The fact that Wnt proteins are known to signal through Fz receptors raises the possibility that Drl might not be acting as a 'classical' guidance receptor, but as a co-receptor for Wnt5, modulating its signalling through one or more of the Fz proteins in a manner similar to that proposed for Arrow/LRP6. For example, binding of Wnt5 by Drl might modify Wnt5 signalling through Fz and/or Fz2, the two Drosophila Fz family members expressed by embryonic CNS neurons. However, in contrast to wnt5, neither fz;fz2 double mutants, nor mutations in dishevelled (a downstream Fz signalling component) show any effect on Drl-mediated axon switching, and drl/+;fz fz2/+ trans-heterozygous embryos do not show defects in midline crossing. Furthermore, interfering with Fz-mediated Wnt signalling by pan-neuronally expressing a dominant-negative form of Fz2 (GPI-Dfz2) causes no defects in midline crossing (Yoshikawa, 2003).

Although these results do not rule out signalling through Fz proteins, they do advance the idea that Wnt5 might be signalling through the Drl receptor, a possibility consistent with the finding that misexpression of Drl lacking its intracellular domain fails to switch any Eg axons, even when misexpressed at high levels from multiple transgenes. In either event, whether Drl transduces the Wnt5 signal or modulates Wnt5 signalling through Fz proteins, it is an essential component of Wnt5 signalling in the guidance of axons across the midline (Yoshikawa, 2003).

In both drl and wnt5 mutants many axons still project appropriately in the AC, suggesting that Wnt5 and Drl are part of a larger multi-component system to ensure proper sorting of axons as they cross the midline. For example, it seems probable that there are additional attractive cues for AC axons, and that once they are attracted to the AC, Drl functions to prevent them from entering the PC. In addition, there may be a similar mechanism for the guidance of PC axons, whose choice of commissure is unaffected in both drl and wnt5 mutant embryos. Possibilities for additional molecules involved in commissure choice include other members of the Drl and Wnt families, some of which are expressed in the developing CNS. These results in Drosophila suggest that a similar receptor-ligand interaction between RYK and Wnt family members might be functioning in mammalian CNS development. Although nervous system phenotypes have not yet been described for the mouse knockouts of RYK and Wnt5a, the two mutants, although differing in severity, do display qualitatively similar skeletal defects, suggesting the possibility of an interaction. Within the mammalian CNS, Wnt proteins have been implicated in the guidance of commissural axons along the anterior-posterior axis of the spinal cord after they cross the midline. It will be of interest to test whether RYK has a role in these guidance events (Yoshikawa, 2003).

There are at least three distinct guidance mechanisms involved in midline crossing of contralaterally projecting axons within the Drosophila CNS. As in vertebrates, growing axons are attracted to the midline by diffusible cues such as Netrins acting through their receptor Frazzled/Dcc. Once there, the choice of whether or not to cross is controlled by Slit through its receptor Robo. Finally, as shown here, their choice of commissure is controlled by Wnt5 by means of its receptor Drl (Yoshikawa, 2003).

Different Wnt signals act through the Frizzled and RYK receptors during Drosophila salivary gland migration

Guided cell migration is necessary for the proper function and development of many tissues, one of which is the Drosophila embryonic salivary gland. Two distinct Wnt signaling pathways regulate salivary gland migration. Early in migration, the salivary gland responds to a WNT4-Frizzled signal for proper positioning within the embryo. Disruption of this signal, through mutations in Wnt4, frizzled or frizzled 2, results in misguided salivary glands that curve ventrally. Furthermore, disruption of downstream components of the canonical Wnt pathway, such as dishevelled or Tcf, also results in ventrally curved salivary glands. Analysis of a second Wnt signal, which acts through the atypical Wnt receptor Derailed, indicates a requirement for Wnt5 signaling late in salivary gland migration. WNT5 is expressed in the central nervous system and acts as a repulsive signal, needed to keep the migrating salivary gland on course. The receptor for WNT5, Derailed, is expressed in the actively migrating tip of the salivary glands. In embryos mutant for derailed or Wnt5, salivary gland migration is disrupted; the tip of the gland migrates abnormally toward the central nervous system. These results suggest that both the Wnt4-frizzled pathway and a separate Wnt5-derailed pathway are needed for proper salivary gland migration (Harris, 2007).

Salivary gland migration can be separated into three phases. In the first phase, the salivary glands invaginate into the embryo at a 45° angle, moving dorsally until they reach the visceral mesoderm. fkh, RhoGEF2 and 18 wheeler have been shown to regulate apical constriction of the salivary gland cells during this invagination process. In addition, hkb and faint sausage are needed for proper positioning of the site of invagination. No guidance cues have been identified for this first phase of migration; it may be that the patterns of constriction and cell movements at the surface of the embryo are sufficient to direct the invaginating tube (Harris, 2007).

During the second phase of migration, as the salivary gland moves posteriorly within the embryo, two guidance cues, Netrin and Slit, guide salivary gland migration along the visceral mesoderm. Netrin, which is expressed in the CNS and the visceral mesoderm, works to maintain salivary gland positioning on the visceral mesoderm. At the same time, Slit acts as a repellent from the CNS to keep the salivary glands parallel to the CNS. A third guidance signal, WNT4, which acts through FZ or FZ2 receptors, is also required in the second phase of salivary gland migration. Loss of Wnt4, fz or fz2 in the embryo results in salivary glands that are curved in a ventromedial direction. This curving affects a large portion of the salivary gland and may result from the fact that the fz and fz2 receptors, in contrast to drl, are expressed throughout the salivary gland. Furthermore, dominant-negative transgenes that disrupt the function of DSH or TCF cause the same phenotype, suggesting that transcription induced by the canonical Wnt signaling pathway is needed to maintain the proper migratory path of the salivary glands on the circular visceral mesoderm (CVM). The migration along the CVM takes more than 2 hours for completion, which would leave adequate time for a transcriptional response (Harris, 2007).

Although Wnt4 and slit are both required for the second phase of migration, and their mutants show similar, though distinguishable, phenotypes, they are thought to act independently. While most slit-mutant embryos have medially curving salivary glands, embryos lacking Wnt4 have salivary glands that curved in a distinctly different, ventromedial, direction. Embryos doubly mutant for Wnt4 and slit show predominantly one or the other phenotype and neither phenotype increases in severity. These results suggest, though they do not prove, that Wnt4 and slit act in distinct pathways (Harris, 2007).

After the entire salivary gland has invaginated, migrated posteriorly within the embryo and lies parallel to the anteroposterior axis of the embryo, the distal ends of the salivary glands come into contact with the LVM. drl and Wnt5 are required for this late phase of salivary gland positioning. Loss of either drl in the salivary gland or Wnt5 in the CNS results in the distal tip of the salivary gland being misguided to a more ventromedial position. This change in the shape of the salivary gland is seen only after the salivary glands are no longer in contact with the CVM (after stage 13). Thus it is proposed that drl is required during the third phase of salivary gland migration, as the salivary gland detaches from the CVM and contacts the LVM (Harris, 2007).

The striking expression of drl at the tip of the salivary gland makes the leading cells uniquely different from the rest of the salivary gland cells. These cells project lamellipodia upon reaching the visceral mesoderm and beginning their posterior migration. They may act to both guide and pull the rest of the gland during migration. Cells at the tip of a migrating organ are frequently specialized to guide migration. For example, the coordinated migration of the tracheal branches in Drosophila is achieved by induction of distinct tracheal cell fates within the migrating tips. This is illustrated by the fact that FGF (Branchless) signaling becomes restricted to the tips of the tracheal branches soon after they begin to extend. The migration and growth of Drosophila Malpighian tubules provide another clear example of specialized cells needed at the tip of a migrating tissue. One cell is singled out to become the tip cell, which directs the growth of the Malpighian tubules as well as organizes the mitotic response and migration of the other cells forming each tubule. In other systems, such as Dictyostelium slugs, cells at the tip of a migrating group are required and solely able to guide migration. These results establish that the leading cells of the migrating salivary glands have a specialized role to play in proper salivary gland positioning. First they are required to initiate invagination within the embryo, then they actively participate in migration along the CVM, and finally they ensure that the distal tip of the gland will remain associated with the LVM at the end of the migratory phase (Harris, 2007).

Despite the fact that it has been firmly established that Wnt5 and drl are important for the final placement of salivary glands, the signaling pathways downstream are not well defined. Because salivary-gland expression of full-length drl can rescue the drl-mutant phenotype, but drl lacking the intracellular domain cannot, it is thought that the intracellular domain of DRL is important for signaling. Similarly, misexpression of full-length drl can misguide axons in the ventral nerve cord, but misexpression of drl lacking its intracellular domain cannot (Yoshikawa, 2003). The genetic interactions found in this study between drl and Src64 support recent findings suggesting that Src64 acts downstream of drl in the ventral nerve cord. In addition, the other Drosophila Src kinase, Src42, may be required at two stages, during salivary gland migration along the CVM and downstream of WNT5-DRL signaling as the gland moves onto the longitudinal visceral mesoderm (Harris, 2007).

Another intriguing finding of this study is the involvement of the two remaining Drosophila RYKs, Drl-2 and dnt, in salivary gland development. The phenotypes of Drl-2 and dnt mutants are less penetrant than drl mutants, but they are qualitatively very similar. Furthermore, embryos doubly heterozygous for drl and Drl-2 have salivary glands that resemble those seen in drl mutant embryos. These three RYKs appear to act in a partially redundant fashion in the salivary glands, since none of the single gene mutations leads to completely penetrant phenotypes. However, no increase was seen in penetrance of the drl phenotype in embryos lacking both drl and Drl-2. In addition, it was not possible to detect transcripts for either Drl-2 or dnt in the salivary gland. While it is possible that dnt and Drl-2 are expressed at very low levels in the salivary gland, they might be acting non-autonomously (Harris, 2007).

An interesting dilemma in understanding RYK signaling is how inactive kinases propagate a signal into the cell. Recent mammalian studies have suggested that RYKs may associate with another catalytically active receptor, such as FZ or EPH, at the membrane. In the mouse, the extracellular WIF domain of RYK interacts with FZD8, and it has been proposed that the two proteins may form a ternary complex with WNT1 to initiate signaling. However, data from flies and nematodes support the argument that DRL and its C. elegans homolog LIN-18 act independently of FZ. Genetic studies of cell specification in the nematode vulva suggest that LIN-18 acts in a parallel and separate pathway from the LIN-17/FZ receptor. Similarly, reduction of fz and fz2 gene activity in flies has no effect on a DRL misexpression phenotype in the ventral nerve cord (Yoshikawa, 2003). This study has shown that double mutants for the Wnt4 and Wnt5 ligands and for the fz and drl receptors both show strong enhancements in comparison to the single mutants, reinforcing the conclusion that these two ligands are activating different pathways. In addition, the functions of these two pathways can be separated by phenotype. The Wnt4-fz/fz2 phenotype becomes evident earlier and affects a larger portion of the salivary gland than the Wnt5-drl phenotype. Taken together, these results demonstrate that there are two independent Wnt pathways regulating salivary gland positioning. The early WNT4 signal appears to activate the canonical Wnt pathway, whereas there is a later requirement for WNT5 signaling through DRL and the Src kinases (Harris, 2007).

DEVELOPMENTAL BIOLOGY

Embryonic

With regard to Drl expression in the CNS, Drl is localized to the growth cones and axons of the drl neurons as they cross within the anterior commissure. Interestingly, after the neurons have turned toward the anterior, Drl cannot be detected on the portion of the axons within the connectives, and remains restricted to the axon segments within the anterior commissure. This suggests that Drl does not physically participate in the fasciculation of drl neurons within the DD and DV bundles. Instead, the results suggest the existence of multiple, discrete steps in neuronal pathway recognition, with Drl playing a key controlling role as the following suggests: once activated by its ligand, Drl is thought to control pathway selection by modulating the function of specific cell adhesion molecules that mediate proper axons fasciculation (Callahan, 1996)

In addition to its expression within the CNS, drl is expressed in both the mesoderm and epidermis during embryogenesis. Using antibodies directed against its extracellular domain, Drl protein can be detected in a subset of somatic muscles as they grow and form attachments to the epidermis. Each hemisegment in abdominal segments A2 through A7 contains 30 muscles with predictable orientations and attachments to the epidermis. Beginning at hour 10 of development, low levels of Drl are detected in the precursors for muscles 21-23 as they begin to elongate dorsoventrally along the epidermis. Expression increases as these muscles continue to grow toward their attachment sites, and is still present by hour 13 as assayed by in situ hybridization or antibody staining. During the period of attachment site selection, Drl appears enriched near the tips of muscles 21-23, especially at the locations where they contact their ventral attachment sites. By hour 15, after attachment events have been completed, drl transcripts and protein are no longer detectable (Callahan, 1996).

Drl protein is first detected at approximately hour 6, appearing in stripes 3-4 cells wide in each segment. During segmental groove formation, Drl is restricted to anterior cells of each segment near the segmental grooves. At hour 9.5, expression begins to expand posteriorly from the grooves at lateral positions, resulting in broad patches of Drl-expressing epidermal cells. These lateral Drl patches overlie the differentiating muscle precursors 21-23. By hour 11, the lateral Drl patches become more restricted, forming two smaller clusters of Drl-expressing epidermal cells located near the dorsal and ventral attachment sites for muscles 21-23. This pattern subsequently becomes more refined, such that by hour 12.5, each cluster has become restricted to approximately 15 cells that abut and partially overlap the epidermal attachment cell clusters for muscles 21-23, as revealed by double-labeling for Drl and sr expression. As in muscles 21- 23, Drl ceases to be expressed in the epidermis by hour 15. Despite this pattern of drl epidermal expression, drl null mutants do not show any defects in segmental groove formation or in differentiation of the epidermis (Callahan, 1996) .

Larvae and Adults

In third instar larvae, two areas of Drl RTK expression are revealed in the CNS: the optic ganglion primordium, and a small cluster of cells at the inter-hemispheric region. In the adult brain, a lacZ reporter gene gives a cytoplasmic expression, which allows investigators to follow expression patterns in all the subcellular compartments that express GAL4. A preferential expression of the reporter gene is observed in the mushroom body (MB) and central complex (CX). There is also a strong expression in the optic lobes and a weaker expression in many other substructures in the brain. In the CX, expression is observed at the level of the fan-shaped body in the superior arch. There is also a strong expression in the cell body cluster, which is in contact with the top of the fan-shaped body at the junction between the two hemispheres. In the MB, staining is observed in the entire volume of the MB, from the KC body layer to the tips of the lobes. At the level of the lobes, intense staining is observed in the alpha and beta lobes. Staining is restricted to the circumferential elements of the lobes. There is also a weaker staining in a sub-component of the gamma lobes that corresponds to the most posterior fibers of the gamma lobe. These fibers lie dorsal and parallel to the beta lobe (Moreau-Fauvarque, 1998).

Weak Derailed expression appears in young third instar larvae, which increases until pupariation. Drl is then detected in the optic lobe anlage and in fibrous elements of the interhemispheric area, near the commissure. In the central area of the larval brain, the following dynamic pattern of expression is observed. The protein is first detected at 75 hours after egg laying (young third instar larvae) in two anterior projections that grow in multiple directions. At 85 hours, these anterior projections cross the commissure and diverge in two branches. This expression is clearly visible in late larvae. At 95 hours Drl is also detected more posteriorly in symmetrical fibrous elements that cross the commissure and also diverge in two brances. The position of these latter elements is compatible with the idea that they contact axons of mushroom body Kenyon cells. Expression declines in the early pupa, and in the adult brain no clear expression can be detected. This expression pattern suggests that Drl plays an important role at the late larval/early pupal stage (Simon, 1998).

A complete deletion of Drl activity causes specific structural defects in the adult brain. Gal4 enhancer-trap lines used as cell markers reveal that in drl mutants central brain axons behave as if they are abnormally attracted by the midbrain area. Defects are documented in the mushroom bodies and the central complex. Optic lobes also appear abnormal. The Drl protein is also expressed in third instar larvae in a few cells at the junction of the cerebral hemispheres. These glial cells form a newly described ring structure, showing an invariable fibrous organization. The ring -- which has been named transient interhemispheric fibrous ring or TIFR -- is located in a sagittal section in the middle of the brain. The TIFR appears to be highly organized, with entangled fibers creating a constant pattern of loops and hooks. The four large nuclei revealed with a beta-galactosidase reporter in a drl reporter are localized on the TIFR fibrous path. The TIFR is present in late larvae. It seems to be broken up in midpupae since no such structure has been described in the adult. In the wild-type this ring disappears at midpupation. These results indicate that the Drl putative kinase plays a major role in the modeling of the adult brain by controlling the fate of the transient interhemispheric ring (Simon, 1998).

Effects of Mutation or Deletion

In drl mutants, drl muscles attach at abnormal locations within the epidermis. These muscle attachment defects are not the result of gross alterations of the epidermis nor loss of epidermal attachment cell precursors. In contrast, analogous to its role in axon pathway selection within the nervous system, Drl participates in a mechanism required for muscle attachment site recognition. Epidermal cells destined to become muscle attachment cells express stripe before muscle attachment events, these epidermal cells to which muscles 21-23 attach were identified by their expression of stripe during myogenesis. At hour 10, a stripe marker begins to be expressed at positions where muscles will eventually insert. This includes sites at segmental boundaries where longitudinal muscles will attach, ventral rows of cells parallel to the segmental grooves where ventral muscles will attach, and in small clusters of cells located laterally between segment boundaries where the 21-23 group will attach. In hour 11.5 embryos, muscles 21-23 have extended dorsoventrally and can be seen forming both their dorsal and ventral attachment sites within the lateral clusters of stripe-expressing epidermal cells. By hour 14, the stripe clusters have become restricted to 6-8 cells, and the ventral attachments for muscles 21-23 lie adjacent to one another within the cluster. Normally, muscles 21-23 extend similar distances ventrally and attach adjacent to the dorsal border of muscle 12, always inserting within the stripe expressing epidermal cluster. In drl mutants, muscles 21-23 are present in their normal locations and elongate dorsoventrally, but have ventral attachment site defects. Muscle morphology was examined in drl null mutants. In 20% of hemisegments of drl mutants, one or more muscles of the 21-23 group pass over their normal ventral attachment sites and appear to attach far more ventrally, beyond muscle 13. This dramatic phenotype is termed the 'bypass' phenotype. An additional 10% of hemisegments have more subtle attachment defects where at least one muscle fails to attach within the ventral stripe-expressing cluster, but does not extend beyond muscle 13. Importantly, both the numbers and locations of the stripe-expressing epidermal cells in drl mutants are indistinguishable from drl + embryos. This indicates that the muscle phenotype seen in drl mutants is not the result of a loss of epidermal attachment cell clusters, but instead results from the inability of the muscles and their ventral tendon cells to coordinate functional attachments. Targeted expression of Drl to muscles is shown to rescue the attachment defects (Callahan, 1996).

These results are consistent with a role for drl in the muscle-epidermal interactions that underlie proper attachment site selection. One possibility is that the Drl RTK participates directly in recognition events between muscles 21-23 and their ventral attachment cells. Drl itself may mediate specific contacts between muscles 21-23 and their appropriate attachment site targets. Alternatively, Drl may be more indirectly involved in muscle-epidermal recognition events. For instance, Drl function may be required in muscles 21-23 before attachment site recognition events, serving to regulate or modify other gene products that are more directly involved in recognition processes. Such a possible modulatory role for Drl in muscle-epidermal recognition would be similar to the proposed role for Drl in neuronal pathway recognition events (Callahan, 1995). A further possibility is that Drl is not required for muscle-epidermal recognition events, but instead is required for the subsequent cytoskeletal changes that must accompany successful attachment. In this scenario, drl mutant muscles would fail to attach at their appropriate sites because they are unable to physically anchor at any location. This possibility seems less likely considering that those muscles displaying a mutant phenotype do appear eventually to attach to the epidermis, albeit at incorrect ventral locations (Callahan, 1996).

The fact that muscle defects are not 100% penetrant in drl null mutants illustrates that there are additional genes involved in muscle 21-23 attachment site selection. At present, it is not known what relationship these additional genes have to drl. One possibility is that the products of these genes are somehow modified by activation of Drl, yet are not completely dependent on Drl for their function. Alternatively, the fidelity of muscle 21-23 attachment site selection may arise from the combinatorial functioning of several distinct recognition processes, of which Drl controls only one. Interestingly, misexpression of Drl in muscles that normally do not express Drl does not cause mistargeting to the 21-23 attachment sites. This may be due to a lack of competence on the part of inappropriate muscles to respond to the Drl signal, or may reflect a restricted localization of the Drl ligand. Misexpression of a constitutively active form of Drl may help to resolve this point. The finding that Drl is expressed both in muscles and at epidermal locations along which the muscles grow and attach suggests that there may be both epidermal and mesodermal components to Drl function. For example, Drl could participate in a homotypic interaction mediating some form of recognition during muscle growth over the epidermis. The results argue against this possibility, since the muscle attachment phenotype could be rescued by targeted expression of Drl in muscles. These results strongly suggest that Drl function in muscles 21-23 is sufficient for proper attachment site selection (Callahan, 1996).

Mutations in Drosophila neurotactin, a gene that encodes a cell adhesion protein widely expressed during neural development, have been isolated and characterized. The lack of widespread axonal defects in the CNS of neurotactin mutants suggests that the function of Nrt in CNS morphogenesis might be largely replaced by functionally related molecules. If so, embryos lacking Nrt as well as one of these other molecules may display synergistic mutant phenotypes. To test this possibility, embryos lacking function of both nrt and one of several genes encoding neural CAMs were examined. Embryos of some double mutant combinations of neurotactin and other genes encoding adhesion/signaling molecules, including neuroglian, derailed, and kekkon1, display phenotypic synergy. This result provides evidence for functional cooperativity in vivo between the adhesion and signaling pathways controlled by neurotactin and the other three genes (Speicher, 1998).

linotte (more properly termed derailed) is a new autosomal gene in Drosophila involved with learning and memory. The linotte mutant was derived from a PlacW transposon mutagenesis and was screened for three-hour memory deficits after classical conditioning of an olfactory avoidance response. Sensory and motor systems (olfactory acuity and shock reactivity) required for the classical conditioning experiments are normal in mutant linotte flies--indicating that the mutation disrupts learning/memory specifically. A chromosomal deficiency of the 37D region, where the linotte P insert is localized in situ, fails to complement linotte's memory defect, and flies from two lines homozygous for independent PlacW excisions show normal memory--indicating that the P insertion is responsible for the mutant phenotype. Additional behavior-genetic data suggest that the linotte gene is non-vital (Dura, 1993).

The Drosophila transmembrane protein Derailed (Drl) is expressed in a glial transient interhemispheric fibrous ring (TIFR), which is hypothesized to serve as scaffold for adult brain formation during metamorphosis. (Interactive Fly editor's note: although this paper refers to drl as linotte, this review refers to the gene as drl, following FlyBase convention). TIFR specific enhancers have been isolated from the Drl locus and it has been shown that only four interhemispheric cells give rise to this complex fibrous structure. drl controls the TIFR differentiation, and the major role played by this structure in central brain metamorphosis has been confirmed because TIFR destruction by apoptosis leads to a pronounced adult phenotype. drl interhemispheric expression is specifically affected in a no bridge mutant context, confirming that nob plays a key role in adult brain development via the TIFR (Hitier, 2000).

To identify postembryonic drl enhancers, restriction fragments spanning the drl locus were fused to a reporter encoding either a nuclear form of beta-galactosidase (PX constructs), a cytoplasmic beta-galactosidase (PZ construct) or the transcription factor Gal4. The PX423 construct leads to no obvious enhancer activity in the CNS. The PX421 construct shows enhancer activity restricted to the midline part of the VNC. The PX422 construct, corresponding to the 5' part of the first intron, exhibits enhancer activity in the primordium of the optic lobes and in four cells at the interhemispheric junction (IJ). The PX432 construct corresponding to the 5' part of the 422 fragment, exhibits strong enhancer activity in the primordium of the optic lobes, the CNS, the lateral parts of the brain and in the four interhemispheric cells. The interhemispheric junction specific enhancer was further restricted to 1.3 kb since the PZ442 construct leads to a TIFR staining. Interestingly, the 425 fragment, which lies 5' from the drl transcription start, also shows specific expression in the four IJ cells. Thus two independent TIFR specific enhancers have been isolated. The alternative use of nuclear or cytoplasmic reporter in combination with TIFR specific enhancers demonstrates that only four glial cells generate the complex fibrous structure of the TIFR. The fragments used are partially redundant with the fragments isolated for drl embryonic enhancer activity analysis (Bonkowsky, 1999). The 425 and the 442 fragment are respectively comprised in the drlU and drlT fragments, showing enhancer activity in midline crossing neurons at the embryonic stage. This observation suggests that the same enhancer elements might control expression at the midline of the embryonic VNC and of the larval brain, even though drl is expressed in neural cells in the embryo and in glial cells in the larval brain (Hitier, 2000).

The drl product is required for shaping the TIFR, not for cell survival The expression of drl is required to build up a correct TIFR (Simon, 1998). However, since the TIFR could be observed only in the hypomorphic lin^drlP mutant it has not been clear if drl expression is required for the formation and survival of the four IJ cells or for the TIFR fasciculation per se. A staining experiment performed on lio²;PX422/+ third instar larvae reveals that the four interhemispheric nuclei are normally present in an amorphic drl background. At the pupal stage, using the 442-gal4 construct and the axon targeted tau-based reporter it has been shown that the TIFR is drastically disorganized in the amorphic context. drl is thus required for glial fibrous fasciculation and/or pathway selection in the interhemispheric junction, as is observed for neurons at the embryonic midline, but not for cell survival (Hitier, 2000).

In order to characterize the TIFR function in adult brain development the brain of 442-gal4/+; UAS-hid/+ flies, in which the TIFR was destroyed by hid-induced apoptosis, was examined. Killing specifically the TIFR cells leads to a late partial pupal lethality. Interestingly, the adult 442-gal4/+;UAS-hid/+ shows a strong drl-like phenotype, i.e. protocerebral bridge disorganization, fibers crossing the midline above the fan-shaped body, flat fan-shaped body and fused MB lobes. This result confirms that the lack of drl expression in the TIFR is responsible for most of the adult phenotype displayed by the drl mutant and that the TIFR is a key cellular structure for the midline choice in the central brain. However, unlike the drl amorphic mutant, the alpha lobes are still present in 442-gal4/+;UAS-hid/+ individuals. In drl mutants the absence of a lobe phenotype could thus be due to the lack of drl expression in the MB itself (Hitier, 2000).

The TIFR destruction leads to a new phenotype compared to the drl mutant: the protocerebral bridge was found to be severely disorganized or absent, and the ellipsoid body ventrally opened and flattened. The fact that the amorphic drl mutant does not exhibit such a drastic phenotype confirms the observation that drl is not required for the existence of the four interhemispheric cells. Interestingly, this additional phenotype partially matches that of the nob mutant phenotype in a Canton-S background. It is hypothesized that the nob gene might play a role in the TIFR and interact with the drl gene (Hitier, 2000).

To assess the drl-nob interaction, drl expression in the nob mutant was followed in third instar larvae brains. In nob mutants drl expression is not detectable in the anterior fibers crossing the interhemispheric junction. In the optic lobes drl expression does not seem to be affected by the nob mutation and the drl product is still detectable in the posterior fibers of the interhemispheric junction. In nob/Y; lio¹/+ larvae the beta-galactosidase expression driven by the lio¹ PlacZ insertion specifically disappears in the four interhemispheric nuclei corresponding to the TIFR. This effect is nob-specific since drl larval interhemispheric expression is normal in several other central brain mutants, including central body defect (cbd), central complex broad (ccb), central complex deranged (ccd), central brain deranged (ceb), central complex (cex), ellipsoid body open (ebo) and mushroom body deranged (mbd) (Hitier, 2000).

In nob/Y; PX425/+ and nob/Y; PX432/+ larvae the interhemispheric expression is specifically absent, as is the pupal TIFR expression. Altogether these results imply either that the nob gene controls drl expression in the TIFR or that the nob gene is required for the TIFR existence. The gal1916 enhancer-trap line inserted in the brain washing gene was initially proposed to stain the TIFR. Recent confocal microscopy study has indicated that the gal1916 line actually stains a neighboring structure. Thus, the drl expression remains the only marker available for the TIFR, so it is impossible to determine whether nob is required for drl expression in the TIFR or for the TIFR existence. However, the fact that nob flies do not exhibit the complete phenotype of 442-gal4/+;UAS-hid/+ flies, in which interhemispheric cells are missing argues for transcriptional regulation of the drl gene by nob. Such transcriptional regulation could nevertheless be indirect and cloning of the nob gene should help address this issue (Hitier, 2000).

Derailed controls key guidance events in the developing nervous system and mesoderm. Like other members of the 'related to tyrosine kinases' (RYK) subfamily of RTKs, Drl has several highly unusual amino acid substitutions within the catalytic domain. All RYK subfamily members contain the 11 subdomains that are hallmarks of the broad family of protein kinases. Furthermore, each member has the invariant lysine of subdomain II that is essential for the phosphotransfer reaction of active kinases. However, all subfamily members share several unusual amino acid substitutions in regions of the catalytic domain that are normally highly conserved in other RTKs. The most notable of these are substitution of the first glycine within the subdomain I nucleotide binding motif [(Q/M/K)XGXXG for GXGXXG] and substitutions within the canonical activation loop motif of subdomain VII [D(S/N)(A/S) for DFG]. The unusual catalytic domain of Drl raises the possibility that members of this subfamily are catalytically inactive. To test the role of Drl kinase activity in vivo, the invariant lysine required for catalytic activity of known kinases was mutated and the ability of this mutant to function in two assays was examined: a dominant gain-of-function axon switch assay in the nervous system and phenotypic rescue of muscle attachment in drl mutants. This predicted kinase-deficient Drl mutant is capable of functioning in both assays. These results indicate that Drl does not require kinase activity in vivo and suggest that members of the RYK subfamily of RTKs transduce signals unconventionally. Transduction of Drl signals might involve the formation of heterodimers with another active RTK, in which case its signaling could be dependent on transphosphorylation of its cytoplasmic domain by this partner. A precedent for this scenario is the formation of heterodimers between the catalytically inactive ErbB3 RTK and other active members of the ErbB subfamily. An alternative mode for Drl action could involve the recruitment of signaling components independent of its phosphorylation state. For example, ligand binding might induce a conformational change of the Drl cytoplasmic domain that would subsequently allow binding of the appropriate signaling components. In either event, these in vivo results indicate that Drl and possibly the entire RYK subfamily of atypical RTKs transduce signals in an unconventional manner (Yoshikawa, 2001).

castor encodes a zinc finger protein expressed in a subset of Drosophila embryonic neuroglioblasts where it controls neuronal differentiation. cas is expressed at larval and pupal stages in brain cell clusters where it participates in the elaboration of the adult structures. In particular using the MARCM system (mosaic analysis with a repressible cell marker), it has been shown that cas is required postembryonically for correct axon pathfinding of the central complex (CX) and mushroom body (MB) neurons. The derailed gene, alternatively termed linotte (lio) in this study, encodes a transmembrane protein expressed at larval/pupal stage in a glial structure, the TIFR, and interacts with the no-bridge (nob) gene. cas interacts genetically with derailed and nob. These interactions do not involve direct transcription regulation but probably cellular communication processes (Hitier, 2001).

Derailed/Linotte is expressed at the embryonic stage in neurons of the VNC and of the procephalic region, and in the late third instar larvae in a glial transient interhemispheric fibrous ring (TIFR) that persists at the early pupal stages and disappears before adulthood. drl null mutants are viable and drl has been implicated in axon pathway selection in the embryonic VNC, and in adult brain development at metamorphosis. The no-bridge mutant, which exhibits adult brain defects, interacts with drl via the TIFR. Using a genetic screen designed to isolate mutations interacting with drl from a collection of Gal4 lines, a new hypomorphic cas allele (cas³⁹²¹) has been identified. Cas protein is expressed in larval and pupal brain in cell clusters. Analysis of mutant clones generated with the MARCM method demonstrates that cas expression is required during larval life to control axonal outgrowth in CX and MB neurons. Although single mutants show only weak brain defects, double mutants lio;cas³⁹²¹ and lio^drlP;cas³⁹²¹ exhibited strong defects in MB and CX indicating that drl and cas interact to build up the adult brain. cas expressing cells are disorganized in the third instar larva brain of drl mutants, whereas no defect is detected in drl embryos. Moreover, nob also interacts genetically with cas. Altogether these data indicate that cas is involved in postembryonic brain development where it interacts with drl and nob, these interactions probably involve cell/cell communication (Hitier, 2001).

Anti-Cas antibodies have revealed that the Cas protein is present in the CNS during larval and pupal stages confirming the cas³⁹²¹ enhancer trap expression pattern. In larva, cas was found expressed in disseminated cells on the ventral side of the VNC. In the dorsal part of the larval brain, cas is found expressed in five linearly organized cells clusters on both sides of the interhemispheric junction. Twenty-four hours after pupariation, expression progressively disappears and no clear signal is detected in the adult brain. The cas³⁹²¹ line led to adult expression in a subset of ellipsoid body and fan-shaped body fibers, and in the pars intercerebralis. Since the enhancer trap expression of cas³⁹²¹ is very similar to that of Cas expression during embryonic, larval and pupal stages, it is speculated that the adult CX expression displayed by cas³⁹²¹ actually reflects cas expression. The stability of Gal4 and ß-gal protein might allow detection in the adult where Cas might be present at weak level. Alternatively the adult Cas product might not be recognized by the antibody because the protein is modified. Cas expression appears normal in the cas³⁹²¹ mutant, confirming that cas³⁹²¹ is a weak hypomorph allele (Hitier, 2001).

Since viable cas mutants are hypomorphic, to fully assess the postembryonic role played by cas in adult brain development the MARCM method was used in combination with a cas null mutation and the UAS-cd8-GFP reporter. Clones were analyzed in paraffin section with anti-GFP antibody rather than with confocal microscopy in whole amount preparations to allow for the detection of non-autonomous effect of the mutant clones on adjacent brain structures. Mutant cas clones induced during larval stages lead to EB and MB defects, indicating that the postembryonic cas expression is required for CX and MB development. No obvious defects are observed with cas^- clones in the lobula, the medulla and the antennal lobes, all regions, where no cas expression is detected. Multi-cellular clones were observed in the CX and in the MBs, indicating that the cas null mutation is not lethal for neuroblasts or ganglion mother cells. Nevertheless, large cas^- clones in the central brain lead to the death of the individual. This probably prevents the observation of wider brain defects. In particular this could explain why the cas clone experiment did not lead to the defect observed in the brain of cas³⁹²¹/cas²⁹⁰ individuals. When only a small subset of EB neurons are mutant for cas, neither cas^- fibers nor the EB complete structure exhibit any obvious defects. However when larger cas^- clones occur in neurons presumably identified as large field R2 or R4 neurons, the EB exhibits a ventral cleft. Interestingly the defect also affects EB neurons that are not mutant for cas, showing that cas is locally non-autonomous. In the MBs non-autonomous defects are also observed: when a subset of ß or ß' neurons lack the Cas product, MBs exhibit a severe fusion of ß and/or ß' lobes. The fusion comprises cas^- fibers but also cas⁺ fibers. Moreover, although cas clones occurring in gamma neurons do not lead to any obvious intrinsic defects, they nevertheless induce cas⁺ ß lobes to fuse. Since gamma lobes differentiate before ß lobes, one can hypothesize that cas controls the expression of a gamma lobe signal that guides ß fibers during pupal differentiation. Alternatively, since a Elav-gal4 driver was used to detect cas clones, only the neuronal component of clones was observed (Hitier, 2001).

The possibility cannot be excluded that glial cas^- cells are responsible for cas⁺ fibers misrooting. This idea is supported by the fact that during embryogenesis Cas is expressed in midline glial precursor cells (Hitier, 2001 and references therein).

cas is shown to interact genetically with drl to build up the adult brain and drl is required for the correct organization of cas cell clusters. Neither drl nor cas control the expression of the other gene, and neither mutation affects the correct development of the cells expressing the second gene during embryogenesis. However, subtle defects might have escaped analysis. At the third instar larvae the situation is different. The five clusters of Cas positive cells linearly organize in a wild-type context but appear disorganized in derailed mutants. Cluster positioning is disturbed and some clusters appeared 'fused' together. Analysis of cas and drl expression in the double mutant third instar larvae CNS suggests that drl and cas are not expressed in the same cells. In particular cas expressing fibers in the central brain did not include the TIFR where drl is expressed. These results suggest that the drl/cas post embryonic interaction does not involve direct transcriptional regulation but rather cellular interactions, between cas expressing clusters and interhemispheric glial cells expressing Drl (Hitier, 2001).

The Drosophila Wnt5 protein, acting through Derailed, mediates selective axon fasciculation in the embryonic central nervous system

The decision of whether and where to cross the midline, an evolutionarily conserved line of bilateral symmetry in the central nervous system, is the first task for many newly extending axons. Wnt5, a member of the conserved Wnt secreted glycoprotein family, is required for the formation of the anterior of the two midline-crossing commissures present in each Drosophila hemisegment. Initial path finding of pioneering neurons across the midline in both commissures is normal in Wnt5 mutant embryos; however, the subsequent separation of the early midline-crossing axons into two distinct commissures does not occur. The majority of the follower axons that normally cross the midline in the anterior commissure fail to do so, remaining tightly associated near their cell bodies, or projecting inappropriately across the midline in between the commissures. The lateral and intermediate longitudinal pathways also fail to form correctly, similarly reflecting earlier failures in pathway defasciculation. Panneural expression of Wnt5 in a Wnt5 mutant background rescues both the commissural and longitudinal defects. Wnt5 protein is predominantly present on posterior commissural axons and at a low level on the anterior commissure and longitudinal projections. Transcriptional repression of Wnt5 in AC neurons by the Wnt5 receptor, Derailed, contributes to this largely posterior commissural localization of Wnt5 protein (Fradkin, 2004).

The correct wiring of a nervous system requires that a large number of neurons stereotypically extend their axonal processes to make synaptic contacts with their muscle and neuronal targets. The leading portion of the axon, the growth cone, is faced with a bewildering number of routing decisions as it travels, frequently many hundreds of cell body diameters, to its target (Fradkin, 2004).

Path finding relies on the growth cone receiving and interpreting guidance cues presented to it at intermediate points in its journey. While the growth cone likely integrates multiple signals at many points, the initial extension of axons has received the most scrutiny; several of the important attractive and repulsive guidance cues and their neuronal receptors have been identified (Fradkin, 2004).

In Drosophila, the majority of the neurons of the embryonic ventral cord are born near the ventral midline, a morphological and functional line of symmetry whose vertebrate equivalent is the floorplate. Axon tracts in the mature embryo form a characteristic “ladder-like” structure reflecting the presence of two longitudinal tracts that extend in the anterior–posterior axis and two commissural tracts, the anterior (AC) and posterior commissures (PC) that bridge the longitudinal pathways in every segment (Fradkin, 2004).

The first choice for many newly extending axons is whether to cross the ventral midline. The axons of certain neurons, the ipsilaterals, do not cross the midline, but instead project with other longitudinal axons toward the anterior or posterior. Other axonal pathways, the contralaterals, cross the midline, extend along the longitudinal tracts and do not recross. The decision to cross or not and the prevention of repeated midline crossing are regulated by interactions between the extending axons and the midline glia cells (MG), specialized cells that emanate repulsive and attractive signals and underlie both the AC and PC at the ventral midline. The Netrin and Slit proteins are among the best characterized of the midline-derived cues. Netrins, signaling through axonal Frazzled receptors, primarily act as attractants, although repulsive Netrin-dependent signaling has also been reported. Slit protein signaling through the axonal Robo family of receptor proteins repels axons away from the midline, thus preventing them from recrossing. In addition to their midline roles, evidence has been provided that the expression domains of the three Robo proteins also delimit lateral domains within the longitudinal axon tracts (Fradkin, 2004).

Initial outgrowth and path finding of pioneering axons is, at least in part, dependent on both their interactions with the glial cell scaffold and their responsiveness to midline-derived cues. Subsequently, “follower” axons fasciculate with the pioneers to form multiaxon fascicles. Regulation of the relative balance between fasciculation and defasciculation through regulation of cell adhesion molecule activities at specific points allows individual axons to branch off to follow separate trajectories. Several cell adhesion proteins have been shown to act in the fasciculation of embryonic Drosophila axons. Embryos bearing mutations in the Drosophila NCAM ortholog, fasciculin II (fasII), display inappropriately defasciculated axons, whereas axons that overexpress fasII become hyperfasciculated. The Connectin (Conn) protein effects homophilic interactions between motoneurons. The beaten path (beat-Ia) gene encodes an Ig domain-containing protein secreted from axonal growth cones. In beat-Ia mutants, axons become hyperfasciculated in a manner suppressible by fasII and conn mutations, suggesting that Beat-Ia acts as a secreted anti-adhesive factor. There are 13 other Drosophila beat genes; interestingly, those four whose full ORF sequences have been determined encode transmembrane or GPI-linked proteins. Genetic interactions between beat-Ia and beat-Ic (encoding a transmembrane Beat protein) indicate their complementary functions, with beat-Ic and beat-Ia acting to increase and decrease adhesiveness, respectively. The Beat receptor(s) have not yet been identified (Fradkin, 2004).

Most of the mechanisms regulating guidance across the midline that have been uncovered thus far operate in both the AC and PC. Little is known about the mechanisms that underlie the choice of axons to go through either the AC or the PC. One gene implicated in this process is derailed (drl), a member of RYK subfamily of receptor tyrosine kinases. Recent studies have demonstrated that interactions between the Drl and Wnt5 proteins play an important role in preventing AC axons from inappropriately crossing in the PC (Yoshikawa, 2003). AC axons are less tightly fasciculated in the drl mutant, suggesting that Drl may act to regulate interneuronal adhesion (Fradkin, 2004).

Wnt5 is a member of the Wnt gene family, a large group of evolutionarily conserved genes encoding secreted glycoproteins that play roles at many developmental stages in a variety of tissues. Among other roles in the nervous system, Wnt proteins act in cell fate determination, synapse formation and maintenance and as mitogens (Fradkin, 2004).

Evidence has been provided that the Drosophila Wnt5 protein is found on the embryonic CNS axon tracts. The Wnt5 gene encodes a highly unusual Wnt protein that bears a long amino terminal extension to the Wnt-homologous domain that contains no known conserved domains. This report presents a detailed analysis of the Wnt5 mRNA and protein expression domains and demonstrates a crucial role for Wnt5 in formation of the major axon tracts during embryonic CNS development through examination of embryos lacking Wnt5. Wnt5 protein is required for the separation or defasciculation of early axonal projections that subsequently form the mature commissural and longitudinal connectives. Evidence is provided that the porcupine (porc) gene is a member of theWnt5 signaling pathway and that the Wnt5 gene is itself one of the downstream targets (Fradkin, 2004).

Wnt5 mRNA is most highly expressed by a large number of neurons predominantly in lateral and midline clusters underlying the PC, but is also found in a few cell bodies more closely associated with the AC. Wnt5 is not apparently expressed by the midline or lateral glia cells. While Wnt5 protein is detected on both AC and PC axons, as well as the longitudinal axon tracts, Wnt5 protein is expressed at higher levels on the PC as compared with the AC from the earliest stages when axons begin to extend and throughout embryonic CNS development (Fradkin, 2004).

Wnt5 null alleles were generated to determine the role of Wnt5 in CNS development. Early BP102⁺ commissural axons that pioneer first the PC, and subsequently the AC, are unaffected in the Wnt5 mutant. In the wild-type embryo, the BP102⁺ AC and PC pioneers and their early followers are closely associated near the midline, but subsequently separate at stage 13, to begin the formation of the two distinct commissures. This separation does not take place in the majority of Wnt5 mutant segments (Fradkin, 2004).

Visualization of the Sema2b⁺ axons, followers that cross in the wild-type AC at early stage 15, reveals that they largely fail to do so in the Wnt5 mutant and fasciculate inappropriately with ipsilateral sibling longitudinal projections or cross the midline in the region between the AC and PC. The PC axon trajectories were visualized by panneural staining and in the Eg⁺ PC-crossing lineage and no major alterations were seen in the Wnt5 mutant (Fradkin, 2004).

In addition to the commissural defects in Wnt5 mutant embryos, alterations to the longitudinal pathway projections were observed. Wnt5 mutant embryos display differential phenotypes with respect to the FasII⁺ longitudinal pathways: the medial or innermost pathway is largely unaffected; however, breaks are found in the intermediate pathway and the lateral pathways. Supporting these observations, the Ap⁺ projections, which in wild type follow the ipsilateral medial pathway, were unaffected by the absence of Wnt5. Visualization of the intermediate and lateral pathways with anti-Robo3 and anti-Robo2, respectively, further indicated that disruption became more severe in the pathways more lateral to the ventral midline (Fradkin, 2004).

Analyses of the longitudinal pioneer neurons indicate that, when specific axons have to selectively defasciculate to pioneer new pathways, particularly the intermediate and lateral pathways, they do not do so in the absence of Wnt5. Examination of the Robo2⁺ neurons indicates that after a limited period of extension, they stop and fail to form the continuous fascicle seen in the wild-type embryo. The Wnt5 longitudinal phenotypes are unlikely to simply reflect the failure of the axons that normally transit the AC to cross based on the following observations: (1) early defasiculation defects in the longitudinal pioneering projections at times where the commissures themselves are being pioneered, (2) discontinuities in ipsilaterally projecting pathways that do not cross the midline, and (3) apparently normal Fas2⁺ longitudinal pathways in the comm null mutant, where few if any axons cross in either commissure (Fradkin, 2004).

The VUM neurons, which normally aid in separating the commissures by sending their projections in between the PC and AC BP102⁺ pioneers, project abnormally in the Wnt5 mutant. Migration of the VUM cell bodies and the MG, which are also involved in establishing the physical separation between the AC and PC, do, however, occur normally in the Wnt5 mutant. Because the failure to establish the AC in the Wnt5 mutant occurs with a similar frequency (67%) to that observed for the abnormal VUM projections (70%), the VUM abnormalities likely contribute to the later failures of the follower axons to form the AC. The VUMs express Wnt5 mRNA at the time of commissure separationand may therefore represent the chief source of Wnt5 protein mediating separation of the AC and PC (Fradkin, 2004).

How does Wnt5 mediate the formation of the AC and the lateral longitudinal pathways? The data, including the observation that Wnt5 protein is most highly expressed on the PC, support the previously proposed role for PC-expressed Wnt5 as a repellent for Drl+ axons (Yoshikawa, 2003), but reveal the likely nature of Wnt5-mediated repulsion required to effect the formation of the AC and more lateral longitudinal pathways. It is suggested that the major role of Wnt5 is to mediate the selective defasciculation of the early commissural and longitudinal pathways necessary for them to separate into distinct commissures or pioneer new pathways, respectively. As defasciculation may be viewed as local repulsion or decreased attraction between axons, this interpretation of the early commissural defects in the Wnt5 mutant is therefore not at odds with Wnt5 acting as a PC-derived repellent. However, Wnt5 appears to act by facilitating the defasciculation of Drl⁺ axons from their siblings necessary for formation of the AC. The role of drl in the Wnt5 mutant longitudinal pathway defasciculation defects is presently unclear. Interestingly, studies of the drl mutant phenotype described the inappropriate defasiculation of axons lacking drl. Similar drl null phenotypes were observed as previously reported; however, the drl CNS phenotypes are consistently less severe than those seen in the Wnt5 null, suggesting that other genes may also interact with Wnt5 to effect selective fasciculation (Fradkin, 2004).

Tescue experiments demonstrated that the primary requirement for Wnt5 expression during embryonic CNS development is in neurons. Panneural Wnt5 expression rescued both the commissural and longitudinal defects. Strikingly, high levels of Wnt5 secreted throughout the CNS-driven by the Repo-Gal4 lateral glial cell driver in the Wnt5 mutant background fail to rescue, suggesting that neurons may process Wnt5 differently than the lateral glia. In support of this possibility, evidence has been presented that Wnt5 protein secreted from tissue culture cells and produced in embryos, respectively, is proteolytically processed (Fradkin, 2004).

Wnt5 overexpression at the AC midline (Sim-Gal4 driver), but not the PC midline (Btl-Gal4 driver) results in a failure to form the AC without noticeable effects on the PC. The noncrossing axons appear tightly associated in the longitudinals, suggesting that Wnt5 protein levels may be important in the regulation of axonal adhesion, with either too little or too much Wnt5 resulting in overly tight fasciculation. The Sim-Gal4-driven Wnt5 overexpression phenotype has also been interpreted to indicate that ectopic Wnt5 repulses all Drl⁺ AC axons (Yoshikawa, 2003). However, given the current absence of data showing that Wnt5 can directly collapse Drl⁺ growth cones, it is equally possible that the overly tight fasciculation of those axons precludes their midline crossing (Fradkin, 2004).

This study has shown that the differing levels of Wnt5 protein on the PC vs. AC are maintained, at least in part, from repression of Wnt5 transcription in AC neurons by Drl. Furthermore, apparently normal CNS architecture is observed when Wnt5 is pan-neurally expressed in a wild-type background (in Elav-GAL4 X UAS-Wnt5 embryos) resulting in high levels of Wnt5 protein on both commissures. The question arises as to why such regulation of Wnt5 in the AC exists? One possibility is that, although high levels of Wnt5 protein on both commissures result in rescue of the Wnt5 mutant phenotype and a lack of an observable phenotype in the wild-type background, respectively, the active species of Wnt5 protein may be asymmetrically generated with respect to the commissure of origin, reflecting PC- vs. AC-specific Wnt5 protein processing. Alternatively, the Drl-mediated repression of AC Wnt5 expression may reflect a requirement for low levels of Wnt5 in AC axons while precluding the higher levels that effect defasciculation, likely via the Drl receptor (Fradkin, 2004).

This study found that porc gene expression is required for the MG Wnt5 overexpression phenotype. Since porc aids in Wnt5 protein secretion (Tanaka, 2002), these data indicate porc-mediated Wnt5 secretion is required for Wnt5 signaling in this overexpression assay. porc function appears to be limiting for Wnt5 secretion in this assay as reduction of porc gene dosage by half in the porc/+ females is sufficient to suppress the Wnt5 midline overexpression phenotype. Is porc required for wild-type Wnt5-mediated signaling? The BP102⁺ axon tracts are disorganized in porc germline clone mutants lacking both maternal and zygotic porc (Tanaka, 2002); however, interpretation of this phenotype is complicated by the requirement for porc in wg signaling, which in turn plays roles in segmentation and neuroblast specification. In porc zygotic mutants, the BP102⁺ axon scaffold is only slightly affected; however, the maternally contributed porc mRNA may mask a requirement for porc in Wnt5 signaling in the zygotic porc mutant embryo. The demonstration of a requirement for porc in establishing the Wnt5 midline overexpression phenotype suggests that this assay will be a useful tool in uncovering novel members of the Wnt5 signaling pathway and its targets, which likely include cell surface adhesion proteins facilitating selective fasciculation and modulators of their activities (Fradkin, 2004).

The RYK subfamily of receptor tyrosine kinases is characterised by unusual, but highly conserved, amino acid substitutions in the kinase domain. The derailed gene encodes a Drosophila RYK subfamily member involved in embryonic and adult central nervous system development. Previous studies have shown that the kinase activity of this receptor is not required in vivo for its embryonic function. In this study, the role was investigated of the cytoplasmic domain and the kinase activity of the Derailed receptor tyrosine kinase in adult brain development. The results indicate that these domains are not essential for adult brain development but they are required for the proper regulation of the activity of this receptor. This sheds light on a regulatory role for the kinase activity of a RYK subfamily member (Taillebourg, 2005).

Respective roles of the DRL receptor and its ligand WNT5 in Drosophila mushroom body development

In recent decades, Drosophila mushroom bodies (MBs) have become a powerful model for elucidating the molecular mechanisms underlying brain development and function. Derailed receptor tyrosine kinase as an essential component of adult MB development. Using MARCM clones, a non-cell-autonomous requirement has been demonstrated for the Drl receptor in MB development. This result is in accordance with the pattern of Drl expression, which occurs throughout development close to, but not inside, MB cells. While Drl expression can be detected within both interhemispheric glial and commissural neuronal cells, rescue of the drl MB defects appears to involve the latter cellular type. The WNT5 protein has been shown to act as a repulsive ligand for the Drl receptor in the embryonic central nervous system. This study shows that WNT5 is required intrinsically within MB neurons for proper MB axonal growth and probably interacts with the extrinsic Drl receptor in order to stop axonal growth. It is therefore proposed that the neuronal requirement for both proteins defines an interacting network acting during MB development (Grillenzoni, 2007).

This study has shown that a drl LOF mutation affects MB development as early as at the newly hatched first instar larval stage. It is at (or just before) this stage that the axons of the first MB intrinsic neurons to be born form the median and vertical lobes. It can be hypothesized that the MB defects displayed by drl LOF adult flies are at least partially due to aberrant early MB development. The Drl protein is not expressed within the MB intrinsic neurons at any developmental stage analyzed. This result is strengthened by clonal analysis experiments, which showed that the early removal of the wild-type drl gene in a subset of the three classes of MB intrinsic neurons does not alter their axonal morphology. The clonal analysis results demonstrate sensu stricto a non-cell-autonomous requirement for the drl gene in the MB intrinsic neurons; it does not, however, completely exclude the possibility that drl mutant clones develop properly due to the expression of the Drl protein in MB intrinsic neurons outside the clones. This would imply that the Drl expression level in the MBs is below the level required by the detection method used in this study. However, restoring the expression of the drl gene solely in a subpopulation of MB intrinsic neurons with the GAL4-247 line, or even in most if not all MB intrinsic neurons with GAL4-OK107, is insufficient to rescue the MB defects induced by the drl LOF mutation. The partial rescue obtained previously with the GAL4-c739 line is likely to be due to some transient expression outside the MBs during development. The fact that the mutant phenotype cannot be rescued by two other GAL4 lines that are expressed either more specifically (GAL-247) or in more MB neurons (GAL-OK107) is ruling out a role of the MB expression of GAL-c739 in the weak rescuing effect. Based on the overall results obtained, the hypothesis is favored that drl gene function is required extrinsically by MBs for their proper development. Finally, the MARCM technique allowed visualization of the morphology of single-side median MB axons in drl LOF individuals. This analysis revealed that the mutant phenotype is not simply a fusion of the median contralateral lobes at the midline, but rather a real crossing of the axons, which then intermingle with their contralateral equivalents (Grillenzoni, 2007).

The function of the Drl protein is required extrinsically by the MBs for their proper development. The data show that the protein is expressed from the onset of brain commissural formation in a subset of neurons crossing the midline. This pattern is a remnant of Drl expression in the embryonic CNS, although at later stages the Drl-expressing brain commissural axons divide into two tracts. It is important to emphasize that the embryonic brain commissure is not identical to those of the ventral CNS, and that the molecular factors involved in their development, although often conserved, do not necessarily play the same role. Knowing that the Drl receptor is necessary cell-autonomously in the CNS to allow the correct midline crossing of a subset of anterior commissural axons, this study analyzed whether similar defects could be observed in the embryonic brain. Such defects could be the primary cause of the MB observed phenotype. This is not the case, since no embryonic brain commissural tract abnormalities were detected using different axonal markers. It has been previously suggested that Drl expression in interhemispheric glial cells during late third instar larval and pupal stages is necessary for MB axonal development. Although Drl expression could be detected in interhemispheric glial cells of third instar brains, it was not possible to rescue the MB phenotype by specific interhemispheric glial cell expression. Moreover, no glial cells expressed the Drl protein at earlier developmental stages, even though MB defects were already present in drl LOF individuals. In addition, the observed Drl expression in commissural neurons and the positive rescue results using a pan-neuronal driver lead to a postulate that Drl is required in neuronal cells extrinsic to the MBs for the correct axonal development of the latter. In conclusion, this study suggests that in the Drosophila brain, Drl expression in a subpopulation of commissural neurons is necessary not for their own axonal development but rather for the guidance of MB intrinsic neurons that do not express the Drl protein (Grillenzoni, 2007).

This neuronal hypothesis is particularly attractive when the Wnt5 results are taken into account. Wnt5 mutants were tested because Wnt5 was described as being a ligand for the Drl receptor in the ventral CNS. Clear MB phenotypes were found in Wnt5 mutant brains. The Wnt5 MB mutant phenotype is most consistent with Wnt5 being required for neurite outgrowth. It is striking that these mutant phenotypes resemble those described for drl⁺ overexpression. It is proposed that this GOF phenotype is due to drl⁺ expression within or close to MB cells, where the ectopic Drl protein can bind to the Wnt5 protein and prevent its function. Therefore, a general model is proposed for the role of the Wnt5-drl pair in building normal MBs: Wnt5 is expressed and required within MB cells in order to insure proper axonal growth. One can propose that during this process the secreted Wnt5 activates an MB intrinsic receptor, which seems not to be of the fz type, in order to activate axonal growth. When Wnt5 is absent, e.g. in a Wnt5 mutant MB, then the axons fail to grow properly. In the normal situation, these MB intrinsic axons will stop growing at the midline when they reach extrinsic axons expressing Drl, because Wnt5 is trapped by the Drl receptor. In drl mutant individuals, however, the MB axons will continue to grow, because Wnt5 is not trapped by the Drl receptor. Although the biochemical relationship between the ligand and receptor is conserved from the embryonic ventral CNS to the adult brain, it should be stressed that MB development involves neurons that express Wnt5 and not Drl, which is exactly opposite to the case in the embryo, where the mutant phenotype involves neurons that express the drl gene and not Wnt5. This is why drl and Wnt5 mutants have the same phenotype in the embryonic ventral CNS but have opposite phenotypes in adult MBs (Grillenzoni, 2007).

The genetic control of brain development requires both intrinsic and extrinsic clues. The perfect crosstalk between both types of molecular information, coming from neurons of different types of brain substructures, ultimately ensures the development of a harmonious and functional brain. It is central for neurobiology to decipher these interacting and developing neuronal networks at the cellular and molecular levels. This study has describe a clear case in which drl, a receptor tyrosine kinase, is required within the brain for the normal development of MBs, although it is neither expressed nor required intrinsically within the MB neurons. Further, it is proposed that the Wnt5 signaling molecule is the intrinsic MB axon target that needs to interact with the extrinsic Drl receptor in order to construct proper MBs within the brain (Grillenzoni, 2007).

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date revised: 28 March 2010

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