InteractiveFly: GeneBrief

Wnt oncogene analog 5: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

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 (Fradkin, 1995). 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 porcupine 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).

REGULATION

Transcriptional Regulation

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).

Protein Interactions

Drosophila segment polarity gene product porcupine stimulates the posttranslational N-glycosylation of wingless in the endoplasmic reticulum

Wnt is a family of cysteine-rich secreted glycoproteins, which controls the fate and behavior of the cells in multicellular organisms. In the absence of Drosophila segment polarity gene porcupine (porc), which encodes an endoplasmic reticulum (ER) multispanning transmembrane protein, the N-glycosylation of Wingless (Wg), one of Drosophila Wnt family, is impaired. In contrast, the ectopic expression of porc stimulates the N-glycosylation of both endogenously and exogenously expressed Wg. The N-glycosylation of Wg in the ER occurs posttranslationally, while in the presence of dithiothreitol, it efficiently occurs cotranslationally. Thus, the cotranslational disulfide bond formation of Wg competes with the N-glycosylation by an oligosaccharyl transferase complex. Porc binds the N-terminal 24-amino acid domain (residues 83-106) of Wg, which is highly conserved in the Wnt family and stimulates the N-glycosylation at surrounding sites. Porc is also necessary for the processing of Drosophila Wnt-3/5 in both embryos and cultured cells. Thus, Porc binds the N-terminal specific domain of the Wnt family and stimulates its posttranslational N-glycosylation by anchoring them at the ER membrane possibly through acylation (Tanaka, 2002).

Porc functions on the N-terminal domain of Wg, which is conserved among Wnt family members. It is therefore possible that Porc functions on the processing of other Drosophila Wnt proteins in addition to Wg. To address this possibility, focus was placed on DWnt-3/5, because its specific antibody was available. In wild type embryos at stage 13, DWnt-3/5 is mainly localized on the commissural axon tracts of the central nervous system. In contrast, DWnt-3/5 appears to be confined in the cell bodies of neurons at the ventral nerve cord of porc embryos, which corresponds to DWnt-3/5 RNA expression domain. The shape and number of axon tracts is somewhat disorganized in porc embryos, but they are clearly present based on the staining pattern by monoclonal antibody BP102. Thus, in porc embryos, both Wg and DWnt-3/5 are not secreted from the synthesizing cells. In addition, Porc binds DWnt-3/5 and stimulates its N-glycosylation in S2 cells. These results therefore demonstrate that Porc can function on the N-glycosylation of multiple Drosophila Wnt proteins (Tanaka, 2002).

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).

The Wnt5 receptor, drl, represses Wnt5 transcription in the wild-type AC

Drl, a RYK family member protein expressed predominantly on the AC, has recently been shown to be a receptor for Wnt5 (Yoshikawa, 2003). Therefore, the possibility was evaluated that the low levels of Wnt5 staining seen on AC neurons reflected Drl-mediated binding and trapping of Wnt5 protein from PC neurons at the AC. Unexpectedly, examination of Wnt5 protein expression in a drl mutant background revealed that AC Wnt5 protein levels increased, resulting in similar levels to those seen on the PC. Western blot analyses of lysates made from wild type vs. drl mutant embryos indicate that overall levels of Wnt5 protein increase in the drl mutant. Quantitation of Wnt5 protein levels revealed that overall Wnt5 protein levels increase 2.2-fold in the drl mutant relative to wild type (Fradkin, 2004).

Since the increase in AC-associated Wnt5 protein could reflect regulation of Wnt5 expression by drl at either transcriptional or post-transcriptional levels, Wnt5 mRNA expression patterns were evaluated by fluorescent double RNA in situ/antibody stainings in the drl null mutant vs. wild-type embryos. mAb BP102 was used to visualize all CNS axon tracts and Wnt5 mRNA was detected using an antisense probe. Comparison of the Wnt5 expression pattern in wild type and the drl mutant indicates that Wnt5 mRNA expression expands into the AC. Thus, the presence of wild-type drl in AC neurons is required for the partial suppression Wnt5 transcription in those neurons, contributing to the marked difference observed between AC and PC Wnt5 protein levels (Fradkin, 2004).

porc is a member of the Wnt5 signaling pathway

To demonstrate the utility of the Wnt5 MG overexpression phenotype as a genetic tool to uncover members of the Wnt5 signaling pathway, the ability of a mutant allele of porc to suppress this phenotype was examined. The porc gene was previously shown to be required for the secretion of the Wnt protein Wg and for wg-dependent signaling . The absence or reduction to single copy of the X-linked porc gene in embryos bearing one copy each of the Sim-GAL4 and UAS-Wnt5 transgenes completely suppressed the Wnt5 midline overexpression phenotype, demonstrating that porc is not only required for Wnt5 protein secretion, but also for Wnt5 signaling (Fradkin, 2004).

WNT5 interacts with the Ryk receptors doughnut and derailed to mediate muscle attachment site selection in Drosophila melanogaster

In recent years a number of the genes that regulate muscle formation and maintenance in higher organisms have been identified. Studies employing invertebrate and vertebrate model organisms have revealed that many of the genes required for early mesoderm specification are highly conserved throughout evolution. Less is known about the molecules that mediate the steps subsequent to myogenesis, e. g. myotube guidance and attachment to tendon cells. This study used the stereotypic pattern of the Drosophila embryonic body wall musculature in genetic approaches to identify novel factors required for muscle attachment site selection. Wnt5 is shown to be needed in this process. The lateral transverse muscles frequently overshoot their target attachment sites and stably attach at novel epidermal sites in Wnt5 mutant embryos. Restoration of WNT5 expression in either the muscle or the tendon cell rescues the mutant phenotype. Surprisingly, the novel attachment sites in Wnt5 mutants frequently do not express the Stripe (SR) protein which has been shown to be required for terminal tendon cell differentiation. A muscle bypass phenotype was previously reported for embryos lacking the WNT5 receptor Derailed (DRL). drl and Wnt5 mutant embryos also exhibit axon path finding errors. DRL belongs to the conserved Ryk receptor tyrosine kinase family which includes two other Drosophila orthologs, the Doughnut on 2 (DNT) and Derailed-2 (DRL-2) proteins. A mutant allele of dnt was generated and it was found that dnt, but not Drl-2, mutant embryos also show a muscle bypass phenotype. Genetic interaction experiments indicate that drl and dnt act together, likely as WNT5 receptors, to control muscle attachment site selection. These results extend previous findings that at least some of the molecular pathways that guide axons towards their targets are also employed for guidance of muscle fibers to their appropriate attachment sites (Lahaye, 2012).

The development of the intricate muscle pattern of higher organisms requires the coordinate expression of numerous cellular factors regulating the specific fate, differentiation, orientation and attachment of the individual muscle fibers. The first steps of muscle formation likely occur autonomously, but guidance of myofibers towards and attachment to their appropriate tendon cells are, at least in part, controlled by secreted and transmembrane proteins emanating from both the target cell and the approaching muscle fiber. This study has shown that, in Drosophila, the secreted WNT5 protein and the Ryk transmembrane receptor family members, DRL and DNT, are essential for guidance of a subset of embryonic body wall muscle fibers to their tendon cells (Lahaye, 2012).

There are three Ryk orthologs in Drosophila, drl, dnt and Drl-2. 36%, 8%, 0% of hemisegments display a lateral transverse muscles (LTM) muscle bypass phenotype when drl, dnt or Drl-2 is absent, respectively. Homozygosity for relatively small deficiencies that uncover both drl and dnt results in the bypass phenotype in virtually all hemisegments (96%). Embryos which completely lack DRL and are heterozygous for a mutant allele of dnt display intermediate penetrance of the phenotype (50%). Embryos lacking DNT and are heterozygous for drl have bypassing muscles in 8% of their hemisegments. These results suggest that the Ryk family members, dnt and drl, coordinately regulate the attachment of the LTM muscle fibers to tendon cells with drl being the dominant player. The decrease in penetrance in the animals lacking both copies of drl and one copy of dnt (50%), relative to those completely lacking both genes (96%), indicates that dnt can at least partially compensate for the absence of drl. Consistent with this is the reported ability of the expression of dnt in the LTMs to partially rescue the drl mutant bypass phenotype (Oates, 1998; Lahaye, 2012 and references therein).

Does WNT5 signal through DNT and DRL? Genetic studies indicate that this is likely the case. Female embryos simultaneously heterozygous for Wnt5 and a deficiency which uncovers both drl and dnt display the bypass phenotype while those heterozygous for either Wnt5 or the deficiency alone do not. Furthermore, male Wnt5 mutant hemizygotes, display increased penetrance when single copies of drl and dnt are removed. Thus, it is concluded that Wnt5 genetically interacts with drl and dnt, likely indicating that the WNT5 protein acts as a ligand for these two Ryk family members during muscle attachment site selection (Lahaye, 2012).

DRL is specifically expressed at the muscle tips of fibers 21-23 while they are in the process of extending towards their attachment sites. The protein is also expressed early in development from 6 hours AEL (stage 10) onwards in reiterated stripes in the epidermis and at stage 12 in clusters of epidermal tendon precursor cells, partially overlapping with the SR expression domain. Rescue of the drl mutant LTM bypass phenotype was only achieved when DRL was restored in the muscle and not the attachment sites. At present, the role of the early expression of drl in the tendon precursor cells is not clear (Lahaye, 2012).

dnt mRNA is also expressed in stripes in the epidermis associated with invaginating cells (Oates, 1998; Savant-Bhonsale, 1999). This transcript is also present at a low level in many embryonic tissues including the somatic musculature. Like DRL, DNT is likely required in the muscle fiber since transgenic expression of dnt in the LTMs rescues the drl phenotype. DRL-2 is expressed most predominantly in the central nervous system, suggesting that it was unlikely to have a role in LTM guidance, as was shown in this study. While almost all hemisegments display overshooting LTMs in the absence of DRL and DNT, only one or two of the three LTM fibers, usually muscles 21 and/or 23, exhibit this phenotype. This result indicates that other non Ryk-dependent mechanisms are required to guide these three muscles to their attachment sites. Alternatively, these two muscles may experience fewer physical barriers blocking their ventral extension beyond muscle 12. In addition, the overshooting of the appropriate tendon cells by these muscles is only observed at the ventral and not the dorsal attachment sites, indicating that guidance mechanisms differ for the two ends of the muscle (Lahaye, 2012).

WNT5 has an important role in guidance of embryonic central nervous system commissural axons and acts as a ligand for DRL in these tissues. When LTM trajectories were investigated in Wnt5 mutant embryos it was found that one or more LTMs overshoot their normal tendon cells in only 17% of the hemisegments compared with 36% in the drl mutant. This result suggests that there are likely other DRL ligands in addition to WNT5. Possible other candidates include the other six wnt genes present in Drosophila, wg, Wnt2, Wnt4, Wnt6, Wnt8 and Wnt10 [reviewed at 'The Wnt Home page' (www.stanford.edu/group/nusselab/cgi-bin/wnt)]. Segmentation defects during early embryogenesis in wg mutants and the lack of available mutants for Wnt6 and Wnt10 precludes further analyses of muscle pattern formation in the absence of these genes. Furthermore, Wnt8 is not detectably expressed in the somatic mesoderm. Since both Wnt2 and Wnt4 had been previously implicated in diverse stages of muscle formation and function, this study analyzed LTM trajectories in a Wnt2/Wnt4 double mutant. No bypassing LTMs were observed in the double mutant embryos, nor in the singly homozygous mutants, indicating that these two Wnt genes are not likely involved in regulating LTM attachment. WNT10 is the most probable alternative ligand for DRL and DNT in muscle since its mRNA is expressed in the developing somatic mesoderm (Janson, 2001), however evaluation of its potential roles awaits the generation of a mutant allele (Lahaye, 2012).

In which cells is WNT5 expressed and required? This study found that Wnt5 mRNA and protein are expressed at low levels in all somatic muscles while they are extending, in mature attachment sites and also during early development in a subset of the tendon cell precursors and in the epidermis. Furthermore, rescue of the bypass phenotype is seen when a Wnt5 transgene is expressed in either of these two tissues. Since WNT5 is a secreted factor and rescue of the Wnt5 phenotype is observed with restoration in either the muscle or the tendon cells, it is difficult to conclude unambiguously in which tissue it is needed. Restoring expression of WNT5 in muscle fiber 12 only does not rescue the bypass phenotype. This result suggests that it is not simply sufficient to have a high source of WNT5 in the muscle close to the original attachment sites for appropriate inhibition of LTM extension. It is more likely, that WNT5, which is widely expressed in the epidermis and musculature, is modified in some way to become locally activated as a specific LTM repulsive guidance cue. Support for this hypothesis comes from previous observations that Wnt5 is proteolytically-processed (Fradkin, 1995). Furthermore, WNT5 expressed by anterior commissural midline glial cells, but not in all neurons, blocks anterior commissure formation (Fradkin, 1995) due to the repulsion of DRL⁺ axons, indicating that elevated local expression of WNT5 can have different outcomes depending on the cell types which express it. Finally, although WNT5 is observed to be widely expressed in the larval/adult brain, it acts specifically to guide mushroom body α-lobe axons indicating that an apparently ubiquitously-expressed ligand can act as a directional cue. Alternatively, WNT5 may be sequestered from some regions of the extending muscle fiber by so-called 'extrinsic receptors' which results in a directional cue received by the leading edge of the muscle (Lahaye, 2012).

There is mounting evidence that the final differentiation of the Drosophila tendon cell, in particular the secretion of an elaborate extracellular matrix, is tightly coupled to the arrival of the muscle fiber. The resulting myotendinous junction is essential for force transmission and counteraction of muscle contraction by tendon cells. Similar junctions exist in vertebrates where tendons attach the muscles to the bone. In Drosophila, it consists of hemi-adherens junction formed by the association of integrin receptor heterodimers on the muscle tip and the tendon cell with the intercalating ECM proteins (Schweitzer, 2010) such as Laminin and TSP secreted from the tendon cells and Tiggrin from the muscle cell. The myotendinous junction is not functional when integrin, TSP or laminin are absent resulting in dissociation of fibers from their attachment sites which leads to lethality. The signals allowing recognition of the appropriate tendon cell, arrest of muscle fiber extension and the formation of the myotendinous junction remain unclear. However, genetic phenotypic analyses indicate that changes in local integrin receptor accumulation on muscle tips and differential responses to TSP presented on the tendons might slow down and stop muscle migration prior to the initiation of myotendinous junction formation (Schweitzer, 2010). A functional myotendinous junction is formed at the novel attachment site of Wnt5 and drl mutants as evidenced by the observation that βPS integrin accumulates at this site. βPS integrin expression was not observed at the original attachment site indicating that the interaction of the muscle tip with the bypassed site, if it occurs at all, is not of sufficient duration to initiate attachment site maturation (Lahaye, 2012).

The observation that the initial outgrowth and guidance of the LTMs are normal in Wnt5 and drl mutants suggests that these proteins act during the recognition of the target cell and not earlier during muscle extension. Wnt/Ryk signaling may be required for induction of a localized 'stop' signal for the LTM at its normal attachment site. In this scenario DRL and DNT present on muscle fibers would bind activated WNT5 secreted from their normal attachment sites. This interaction might then result in the transcription of genes encoding extracellular matrix proteins in the muscle fiber which are required to increase adhesiveness between the muscle and tendon cell, slowing down the fibers extension. When either WNT5 or DRL/DNT is absent this signal is not appropriately received by the approaching fiber and it overshoots its target and attaches relatively randomly to a more distant epidermal cell (Lahaye, 2012).

In the Drosophila embryonic CNS, DRL acts as a repulsive guidance receptor on growth cones of anterior commissural axons to steer them away from the posterior commissural axons which express WNT5. It seemed thus possible that DRL/DNT also acts in the muscle as a repulsive receptor upon binding of WNT5. However, no clear muscle guidance defects were observed when WNT5 was ectopically expressed on either specific muscle fibers or in the tendon cells. As mentioned above, it is possible that WNT5 has to be locally modified and activated or differentially sequestered to function as a guidance cue in this tissue (Lahaye, 2012).

It was found that that the novel attachment site for the overshooting muscle in embryos and larvae is an epidermal cell and not another muscle. The normal LTM attachment site that is not recognized by the bypassing muscle is present in Wnt5 and drl mutants as visualized by its ability to express Stripe, a transcription factor that is both necessary and sufficient to drive tendon cell fate. Therefore, this tendon cell follows important early stages of normal tendon cell differentiation, but does not bind the fiber (Lahaye, 2012).

In contrast, only 35% of the ectopic tendon cells express Stripe suggesting that Stripe expression is not obligatorily required for formation of a stable myotendinous junction. At present, it is not known whether the novel attachment site expressed SR earlier in development or whether, despite its stability against contraction-induced damage, the ectopic myotendinous junction is different in some manner from the normal junction as to not allow maintenance of Stripe expression. The FAS2 protein that is normally expressed at the muscle tip and the tendon cell to which it attaches, is present at both the original and the novel attachment sites in drl and Wnt5 mutant larvae. This result indicates that the muscle 'filopodia' likely transiently interact with its normal tendon cell target but does not cease extension. This further supports the notion that Wnt/Ryk signaling may increase the stability of muscle/tendon cell interactions (Lahaye, 2012).

It is too early to evaluate whether the molecular mechanisms of muscle attachment site selection are conserved between vertebrates and invertebrates because of the paucity of knowledge about the molecules required for tendon differentiation and its connections to muscle and skeletal tissues in vertebrates. Components of Integrin-mediated adhesion complexes, e. g., talin 1 and talin 2 and several laminin integrin receptors were, however, recently shown to be essential for the formation of the vertebrate myotendinous junction, as has been observed for their orthologs in Drosophila. In the coming years, as more becomes known about the mechanisms that mediate the connections between muscles and tendons, it will be apparent whether other aspects of muscle guidance and target site selection are also conserved (Lahaye, 2012).

DEVELOPMENTAL BIOLOGY

Embryonic

The Wnt gene family encodes highly conserved cysteine-rich proteins which appear to act as secreted developmental signals. Both the mouse Wnt-1 gene and the Drosophila wingless (wg) gene play important roles in central nervous system (CNS) development. wg is also required earlier, in the development of the embryonic metameric body pattern. Another member of the Drosophila Wnt gene family, DWnt-3,.is secreted in vivo. The early protein expression domains include the limb and appendage primordia. Late expression domains comprise the ventral cord and supraesophageal ganglia of the CNS. Notably, DWnt-3 protein accumulates on the commissural and longitudinal axon tracts of the CNS. Ectopic expression of DWnt-3 in transgenic embryos bearing a HS-DWnt-3 construct leads to specific disruption of the commissural axon tracts of the CNS. DWnt-3 does not functionally replace Wg in an in vivo assay. Experiments with a tissue culture cell line transfected with a construct encoding the DWnt-3 gene show that DWnt-3 protein is efficiently synthesized, glycosylated, proteolytically processed, and transported to the extracellular matrix and medium. DWnt-3, therefore, encodes a secreted protein, which is likely to play a role in development of the Drosophila CNS (Fradkin, 1995).

Wnt5 mRNA and protein are expressed predominantly on PC neurons

The Wnt5 expression domains were examined by RNA in situ analyses and antibody stainings using a Wnt5 antibody. Wnt5 is expressed predominantly in the CNS from stage 12 onward throughout embryonic development. Wnt5 mRNA was found in a large subset of presumptive neurons. A double Wnt5 RNA in situ and anti-Myc antibody staining for endogenous Wnt5 mRNA and Elav-GAL4-driven τ-Myc protein demonstrates that Wnt5 is expressed in neurons. To localize Wnt5 mRNA-expressing cells with respect to the commissures, double-fluorescent RNA in situs were performed using Wnt5 and drl antisense probes, the latter labeling most AC neuronal cell bodies. Wnt5 mRNA was found predominantly in cell bodies near to and underlying the PC. Wnt5 RNA was also found in occasional cell bodies near the AC, but no overlap between Wnt5⁺ and drl⁺ cells was observed. A double Wnt5 RNA in situ and antibody labeling for the Repo protein showed that Wnt5 mRNA is not expressed by lateral glia. Likewise, a Wnt5 RNA in situ double staining with the anti-Wrapper antibody, which labels all MG, reveals that Wnt5 mRNA is not expressed by midline glia (Fradkin, 2004).

During early stages of CNS development, Wnt5 protein is observed on cell bodies lateral to the ventral midline, and subsequently, on the axons projecting across the midline. During later stages of embryogenesis, Wnt5 protein accumulates primarily on the commissures with only weak staining apparent on the longitudinal pathways (stages 14, 16). No staining was seen in the Wnt5 null mutant. Wnt5 expression on the PC was consistently higher than that on the AC during all stages of embryonic CNS development (Fradkin, 2004).

Differentially expressed Drl and Drl-2 play opposing roles in Wnt5 signaling during Drosophila olfactory system development

In Drosophila, odor information received by olfactory receptor neurons (ORNs) is processed by glomeruli, which are organized in a stereotypic manner in the antennal lobe (AL). This glomerular organization is regulated by Wnt5 signaling. In the embryonic CNS, Wnt5 signaling is transduced by the Drl receptor, a member of the Ryk family. During development of the olfactory system, however, it is antagonized by Drl. This study identified Drl-2 as a receptor mediating Wnt5 signaling. Drl is found in the neurites of brain cells in the AL and specific glia, whereas Drl-2 is predominantly found in subsets of growing ORN axons. A drl-2 mutation produces only mild deficits in glomerular patterning, but when it is combined with a drl mutation, the phenotype is exacerbated and more closely resembles the Wnt5 phenotype. Wnt5 overexpression in ORNs induces aberrant glomeruli positioning. This phenotype is ameliorated in the drl-2 mutant background, indicating that Drl-2 mediates Wnt5 signaling. In contrast, forced expression of Drl-2 in the glia of drl mutants rescues the glomerular phenotype caused by the loss of antagonistic Drl function. Therefore, Drl-2 can also antagonize Wnt5 signaling. Additionally, genetic data suggest that Drl localized to developing glomeruli mediates Wnt5 signaling. Thus, these two members of the Ryk family are capable of carrying out a similar molecular function, but they can play opposing roles in Wnt5 signaling, depending on the type of cells in which they are expressed. These molecules work cooperatively to establish the olfactory circuitry in Drosophila (Santschi, 2009).

Several lines of evidence support a role for Drl-2 in Wnt5 signaling. First, in drl-2 mutants, two glomeruli shifted to abnormal positions as observed in Wnt5 mutants. Second, when drl-2 was combined with drl, the phenotype more closely resembled that of the Wnt5 mutant. Third, the abnormal commissural distribution of the presynaptic protein Bruchpilot, which was induced by overexpression of Wnt5 in ORNs, was restored in drl-2 mutants. Moreover, the ectopic projection pattern of ORNs, which was caused by Wnt5 overexpression in drl mutants, was mostly ameliorated by the absence of drl-2. Thus, drl-2 is epistatic to both Wnt5 and drl, and Drl-2 mediates Wnt5 signaling. This Drl-2 activity is antagonized by Drl during development of the olfactory system. However, the glomerular defects of drl drl-2 mutants are milder than those of Wnt5 mutants. Additional Wnt5 receptors that have yet to be identified may contribute to these phenotype differences (Santschi, 2009).

Antibody staining revealed that the expression patterns of Drl and Drl-2 differ in the developing olfactory system. Drl is expressed by glia and brain cells extending neurites to the AL but is not detected on ORN axons. In contrast, Drl-2 is predominantly detected on ORN axons as well as in a region adjacent to the exit site of ORN axons on the dorsal side of the AL. Thus, the expression patterns of Drl and Drl-2 are complex and mostly nonoverlapping, suggesting that multiple processes use Wnt5 signaling during olfactory system development. To reveal whether Drl-2 functions in ORNs, clonal analyses was performed in which drl-2 clones were generated in ORNs. Rescue experiments were also conducted by expressing Drl-2 in the ORNs of drl-2 mutants with pebbled-Gal4. However, the mild drl-2 phenotype did not allow a clearly determination of the site(s) where Drl-2 functions (Santschi, 2009).

The genetic analysis suggests that Drl and Drl-2 can both play opposing roles in Wnt5 signaling during olfactory system development. The DA1 glomerulus in drl mutant lNB clones shifts in a pattern similar to that of Wnt5 mutants, suggesting that Drl may transduce Wnt5 signaling in the developing AL. In addition to this possible transducing activity of Drl, the antagonistic action of Drl and the transducing activity of Drl-2 can explain other mutant glomerular phenotypes observed in this study. In drl mutant brains, in which both transducing and antagonistic activities of Drl are lost, an excess amount of Wnt5 ligand signals through Drl-2 and causes several glomeruli to shift in the direction opposite to that in Wnt5 mutants. However, in drl drl-2 mutants, in which transducing activities of both Drl and Drl-2 are lost, the glomerular defects resemble those of Wnt5 mutants. Thus, the opposing actions of Drl may both be essential for olfactory system development. In addition, it was demonstrated that Drl-2 can antagonize Wnt5 signaling when ectopically expressed in glia. Therefore, Drl and Drl-2 can each potentially mediate or antagonize Wnt5 signaling, depending on the cells in which they are expressed (Santschi, 2009).

Wnt–Ryk signaling mediates both repulsion of developing axons and induction of neurite growth in Drosophila and vertebrates. In the Drosophila olfactory system, Drl localized to the neurites of AL neurons may mediate both of these activities, because drl NB clones exhibit two distinct phenotypes. Neurites projecting to VA1 were stunted in drl vNB clones, suggesting that Drl may mediate neurite growth. In drl lNB clones, the position of DA1 was shifted ventrally, which may reflect the loss of neurite repulsion triggered by Wnt5 originating from growing ORN axons. Drl-2 on ORN axons can also mediate either of these two neurite activities to control ORN projections to the AL. Thus, proper AL development may be regulated by Wnt–Ryk signaling that mediates both the repulsion and outgrowth of neurites innervating the AL. The development of the Drosophila olfactory system appears to be controlled by a complex network of Wnt5 signaling among ORNs, interneurons, and glia (Santschi, 2009).

In summary, although each of the drl and drl-2 genes has acquired a specific expression pattern during evolution, both products can either mediate or antagonize Wnt5 signaling in a cell-type specific manner. In this manner, Drl and Drl-2 may regulate either the repulsion or outgrowth of neurites, perhaps in accordance with additional mechanisms. Additional studies will reveal the details of the complex Wnt5 signaling that control the formation of an accurate glomerular map of the olfactory circuitry (Santschi, 2009).

EFFECTS OF MUTATION

Generation of Wnt5 null mutants

Wnt5 mutant alleles were generated by imprecise excision of an adjacent P-element. Two lines, Wnt5⁴⁰⁰ and Wnt5²⁰⁷, lacking Wnt5 protein as determined by anti-Wnt5 immunostaining and whole embryo Western blot analysis, were characterized initially by DNA sequence analysis and found to be lacking large regions of the Wnt5 ORF, suggesting that they are likely null alleles. Wnt5 mutants can be maintained as a homozygous stock; however, 19% of Wnt5 embryos fail to hatch. Those embryos failing to hatch likely represent the subset with the most severe CNS defects. Once they hatch, Wnt5 mutant individuals have survival rates indistinguishable from controls at subsequent developmental stages, suggesting that the lethality is restricted to the embryonic stage (Fradkin, 2004).

Initial path finding across the midline is normal in Wnt5 mutants; however, the AC and PC commissures do not separate appropriately

To understand the function of Wnt5 in CNS development, several cell- or lineage-specific mAbs were used to visualize the CNS axon trajectories in Wnt5 mutants. As visualized by mAb BP102, which stains all CNS neurons, the CNS scaffold in wild-type embryos matures into a characteristic ladder-like pattern with two commissures that cross the ventral midline in each segment and two longitudinal connectives that run along either side of the ventral midline. In Wnt5 mutants, pioneering BP102⁺ PC and AC commissural axons cross the midline at stage 12 as in the wild type. However, the AC and PC axons fail to subsequently separate at stage 13 in Wnt5 mutants. In the majority of Wnt5 embryos, the mature AC at stage 16 is much thinner than normal and several AC axons either do not cross or cross ectopically, projecting between AC and PC. In a more severely affected minority of embryos (approximately 10%), no AC is apparent (Fradkin, 2004).

To examine a specific AC-projecting lineage, axon projections were evaluated in embryos bearing the Sema2b-τ-Myc transgene that labels three axons whose cell bodies lie just lateral to the AC. In wild-type embryos, Sema2b⁺ neurons cross the midline through the AC as late follower axons and subsequently turn anteriorly in the longitudinal connectives to fasciculate with their siblings. In Wnt5 mutant embryos, the majority of Sema2b⁺ axons do not enter the AC, but either extend minimally or inappropriately fasciculate with longitudinal projections and project ipsilaterally. A subset of Sema2b⁺ neurons (22%) cross the midline in a region between AC and PC (Fradkin, 2004).

The intermediate and lateral longitudinal fascicles are not formed correctly in Wnt5 mutants

To evaluate longitudinal projections in the Wnt5 mutant, three lineage-specific antibodies, mAb 1D4 (anti-FasII), mAb 22C10 (anti-Futsch), and anti-Robo2, were used. During early path finding stages, mAb 1D4 labels, among others, the pCC neurons that pioneer the medial pathway, the innermost fascicle of the three FasII⁺ fascicles seen in the mature CNS. Early pioneering of the medial pathway is unaffected in Wnt5 mutant embryos and the mature medial pathway is also not affected. In wild-type embryos, the ascending pCC and vMP2 axons form the first continuous longitudinal projection when they join the descending MP1 and dMP2 axons. Later on, at stage 14, the MP1/dMP2 and the pCC/vMP2 pathways defasciculate and are associated only at the segment border, thereby forming an outer (MP1/dMP2) and an inner (pCC/vMP2) fascicle. This defasciculation fails to occur in the Wnt5 mutant, resulting in a single thick fascicle. Furthermore, MP1 later pioneers the intermediate of the three FasII⁺ fascicles in wild-type embryos, but fails to do so in the Wnt5 mutant, resulting in breaks in the fascicle at stage 16 (Fradkin, 2004).

The MP1 and vMP2 pathways can also be visualized with mAb 22C10 (anti-Futsch). Most of the pioneering MP1 and vMP2 axons extend and form the first longitudinal pathway in Wnt5 mutant embryos, but rare breaks are observed in these pathways resulting from the failure of both the MP1 and vMP2 axons to fully extend. mAb 22C10 also labels the VUM neurons whose cell bodies are located at the PC midline and send their axons out anteriorly to subsequently bifurcate at the AC where they fasciculate with the RP2 and aCC axons to project laterally out of the CNS. In the Wnt5 mutant, VUM axons project incorrectly along the medial longitudinal pathway in 70% of segments, possibly due to inappropriate selective fasciculation. The cell bodies of the projections described (VUMS, MP1, vMP2, pCC, dMP2) were present at their wild-type locations in the Wnt5 mutant (Fradkin, 2004).

The Robo2⁺ axons that project ipsilaterally through the lateral-most of the three FasII⁺ pathways were examined in wild-type embryos. Initially, robo2 is expressed in many neurons (among others pCC, MP1, dMP2, vMP2), but expression ceases in these neurons at stage 14, and from then on is present only in the lateral most FasII⁺ fascicle. Examination of Wnt5 mutants indicates that the Robo2⁺ axons initially extend in the mutant, but then stop in an apparently tightly fasciculated bundle by stage 14 and therefore fail to form the continuous lateral Robo2⁺ fascicle seen at stage 16 (Fradkin, 2004).

The phenotypes observed in Wnt5 mutant embryos are interpreted as resulting from defects in the abilities of the Wnt5-responsive subset of axons to defasciculate sufficiently to extend or enter new pathways. However, they could also result from fate changes in cell lineages due to the absence of Wnt5. To evaluate this possibility, Wnt5 mutant embryos were stained with anti-Repo to label all lateral glia and with several mAbs that label specific neuronal subsets: mAb 1D4, anti-Even-skipped, anti-Engrailed, and mAb 22C10. No obvious changes in the fates or numbers of these glia or neuronal cell bodies were detected in Wnt5 mutants. The MG play important roles in commissural separation; therefore, commissural phenotypes could also result from the failure of the MG to migrate appropriately. Consequently, MG migration was visualized throughout embryogenesis using the anti-Wrapper mAb, which labels all of the MG. No obvious differences from wild-type embryos were found in the numbers and final positions of the MG were observed in Wnt5 mutants. Furthermore, the migration patterns of the MG throughout embryonic CNS development in the Wnt5 mutant appeared indistinguishable from those in the wild type (Fradkin, 2004).

Rescue of the Wnt5 null phenotype by panneural Wnt5 expression

To understand where Wnt5 expression is required, rescue experiments were performed in which Wnt5 expression was restored in the Wnt5 null mutant either in all axons, the MG or the lateral glia through use of the UAS-GAL4 system. When Wnt5 was expressed in all CNS neurons (driven by Elav-GAL4), apparently complete rescue of the Wnt5 mutant longitudinal and commissural phenotypes was observed. In contrast, when overexpressed in the lateral glia (using the Repo-GAL4 driver), no rescue of the Wnt5 null mutant phenotypes was observed using either single or double copies of driver and UAS-Wnt5 (Fradkin, 2004).

No rescue of the aberrant commissural or longitudinal pathways was observed in Wnt5 mutant embryos ectopically expressing Wnt5 protein at the midline (driven by Sim-GAL4). Instead, a striking phenotype was observed: although the PC appears normal, the AC fails to form. This phenotype is highly penetrant: 96% of the embryos show this effect. To investigate whether this phenotype could also be seen when Wnt5 protein is expressed at the midline in a wild-type embryo, embryos with a single copy of Sim-GAL4 and UAS-Wnt5 in an otherwise wild type genetic background were generated. A lower penetrance phenotype, in which 17% of the embryos lose the AC in one or two segments, was observed. This phenotype became increasingly more penetrant and severe when two copies each of Sim-GAL4 and UAS-Wnt5 or two copies each of the stronger Slit-GAL4 driver and UAS-Wnt5 were present, suggesting dose-dependent responses to ectopic midline Wnt5 expression (Fradkin, 2004).

The Sim- and Slit-GAL4 transgenes drive transcription from late stage 11 onward in most midline precursors and later on predominantly in the anterior (MGA), the medial (MGM), and the posterior (MGP) MG. To understand the relative importance of MGA/MGM vs. MGP Wnt5 overexpression in eliciting the midline overexpression phenotype, Wnt5 was expressed in the MGP using the Btl-GAL4 driver. This transgene drives expression from stage late 11 onward in a subset of neurons underlying the PC, including the VUMs, but is not expressed in the MGA and MGM. As Btl-GAL4-driven Wnt5 fails to suppress formation of the AC, it is concluded that Wnt5 ectopically expressed by the MGA and/or MGM likely mediates the Wnt5 midline expression phenotype (Fradkin, 2004).

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).

A signaling network for patterning of neuronal connectivity in the Drosophila brain

The precise number and pattern of axonal connections generated during brain development regulates animal behavior. Therefore, understanding how developmental signals interact to regulate axonal extension and retraction to achieve precise neuronal connectivity is a fundamental goal of neurobiology. This question was investigated in the developing adult brain of Drosophila. Extension and retraction is regulated by crosstalk between Wnt, fibroblast growth factor (FGF) receptor, and Jun N-terminal kinase (JNK) signaling, but independent of neuronal activity. The Rac1 GTPase integrates a Wnt-Frizzled-Disheveled axon-stabilizing signal and a Branchless (FGF)-Breathless (FGF receptor) axon-retracting signal to modulate JNK activity. JNK activity is necessary and sufficient for axon extension, whereas the antagonistic Wnt and FGF signals act to balance the extension and retraction required for the generation of the precise wiring pattern (Srahna, 2006).

Based on the observation that blocking Fz2 results in decreased numbers of dorsal cluster neuron (DCN) axons in the medulla, it was reasoned that Fz2 could be a receptor for a putative stabilization signal. Since Fz2 and Fz are partially redundant receptors for the canonical Wnt signaling pathway, expression of the canonical Wnt ligand Wingless (Wg) was investigated in the brain during pupation. However, no Wg expression was detected in the pupal optic lobes, suggesting that Wg is unlikely to be involved in regulating DCN axon extension. Therefore, the expression of Wnt5, which has been shown to be involved in axon repulsion and fasciculation in the embryonic CNS, was investigated. Anti-Wnt5 staining revealed widely distributed Wnt5 expression domains beginning at PF and lasting throughout pupal development and into adult life. Wnt5 is strongly expressed in the distal medulla and is also present on axonal bundles crossing the second optic chiasm.The number of DCN axons crossing to the medulla was examined in wnt5 mutant flies. The number of DCN axons crossing the optic chiasm is reduced from 11.7 to 7.9 in the absence of wnt5, suggesting that it may play a role in stabilizing DCN axons (Srahna, 2006).

Next, the requirement of the Wnt signaling adaptor protein Dsh was tested. In animals heterozygous for dsh⁶, a null allele of dsh, the average number of DCN axons crossing between the lobula and the medulla is reduced from 11.7 to 7.6 with 78.5% showing less than eight axons crossing. Signaling through Dsh is mediated by one of two domains. Signaling via the DIX (Disheveled and Axin) domain is thought to result in the activation of Armadillo/β-Catenin. DEP (Disheveled, Egl-10, Pleckstrin) domain-dependent signaling results in activation of the JNK signaling pathway by regulation of Rho family GTPase proteins during, for example, convergent extension movements in vertebrates. To uncover which of these two pathways is required for DCN axon extension the dsh¹ mutant, deficient only in the activity of the DEP domain, was tested. Indeed, in brains from dsh¹ heterozygous animals the number of extending axons was reduced from 11.7 to 7.4. In flies homozygous for the dsh¹ allele the average number of axons crossing was further reduced to 4.7, with all the samples having less than six axons crossing. In contrast, the DCN-specific expression of Axin, a physiological inhibitor of the Wnt canonical pathway, did not affect the extension of DCN axons. Similarly, expression of a constitutively active form of the fly β-Catenin Armadillo also had no apparent effect on DCN extension. Finally, whether Wnt5 and Dsh interact synergistically was tested. To this end, wnt5, dsh¹ trans-heterozygous animals were generated. These flies show the same phenotype as flies homozygous for dsh¹, suggesting that Wnt5 signals through the Dsh DEP domain (Srahna, 2006).

To determine if dsh is expressed at times and places suggested by its genetic requirement in DCN axon outgrowth, the distribution of Dsh protein during brain development was examined. Dsh protein is ubiquitously expressed during brain development. High expression of Dsh is detected in the distal ends of DCN axons at about 15% PF shortly before they extend across the optic chiasm toward the medulla. In general, higher levels of Dsh were observed in the neuropil than in cell bodies (Srahna, 2006).

In summary, these data indicate that the stabilization of DCN axons is dependent on the Dsh protein acting non-canonically via its DEP domain. Importantly, the axons that do cross in dsh mutant brains do so along the correct paths. This suggests that, like JNK signaling, Wnt signaling regulates extension, but not guidance, of the DCN axons (Srahna, 2006).

Wnt signaling to Dsh requires the Fz receptors. To examine if the effect of Wnt5 on DCN axon extension is also mediated by Fz receptors, the number of DCN axons crossing the optic chiasm in was counted fz, fz2, and fz3 mutants. There was no significant change in the number of axons crossing in the brain of fz3 homozygous animals. In contrast, in brains heterozygous for fz and fz2, the number of the axons crossing was reduced from 11.7 to 6.6 (fz) and 6.9 (fz2), with 71% and 85.7%, respectively, showing less than eight axons crossing. These data suggest that DCN axons respond to Wnt5 using the Fz and Fz2 receptors, but not Fz3. To determine whether the Fz receptors act cell-autonomously in individual DCNs, single-cell clones doubly mutant for fz and fz2 were generated and the number of DCN axons crossing the optic chiasm was counted. In contrast to wild-type cells, where 37% of all DCN axons cross, none of the fz, fz2 mutant axons reach the medulla. To test whether wnt5, fz, and fz2 genetically interact in DCNs, flies trans-heterozygous for wnt5 and both receptors were examined. Flies heterozygous for both wnt5 and fz mutations show a strong synergistic loss of DCN axons (11.7 to 3.7) and in fact have a phenotype very similar to that of flies homozygous for dsh¹. Flies doubly heterozygous for wnt5 and fz2 also show a significant decrease in DCN axons (5.7), compared with either wnt5 (~8) or fz2 (8.5) mutants. These data indicate that the genetic interaction between wnt5 and fz is stronger than the interaction between wnt5 and fz2 (Srahna, 2006).

Examination of the expression domains of Fz and Fz2 in the developing brain supports the possibility that they play roles in stabilizing DCN axons. Both Fz and Fz2 are widely expressed in the developing adult brain neuropil. In addition, Fz is expressed at higher levels in DCN cell bodies (Srahna, 2006).

The observation that the wnt5 null phenotype can be enhanced by reduction of Fz, Fz2, or Dsh suggests that another Wnt may be partially compensating for the loss of Wnt5. To test this possibility, flies heterozygous for either wnt2 or wnt4 were examined. wnt2 heterozygotes display a reduction of DCN axon crossing from 11.7 to 7.3, whereas no phenotype was observed for wnt4. Thus, wnt2 and wnt5 may act together to stabilize the subset of DCN axons that do not retract during development. In summary, these results support the model that Wnt signaling via the Fz receptors transmits a non-canonical signal through Dsh resulting in the stabilization of a subset of DCN axons (Srahna, 2006).

Data is provided that supports the hypothesis that the regulation of JNK by Rac1 modulates DCN axon extension. As such attempts were made to determine how Wnt signaling might interact with Rac1 and JNK. The opposite phenotypes of dsh and Rac1 loss-of-function suggest that they might act antagonistically. To determine if Rac1 is acting upstream of, downstream of, or in parallel to Dsh in DCN axon extension, dominant-negative Rac1 was expressed in dsh¹ mutant flies. If Rac1 acts upstream of Dsh, the dsh¹ phenotype (i.e., decreased numbers of axons crossing the optic chiasm) is expected. If Rac1 acts downstream of Dsh, the Rac1 mutant phenotype (i.e., increased number of axons crossing) would be expected If they act in parallel, an intermediate, relatively normal phenotype is expected. Increased numbers of axon crossing were observed, suggesting that Rac1 acts downstream of Dsh during DCN axon extension and that Dsh may repress Rac1 (Srahna, 2006).

Next, whether Dsh control of DCN axon extension is mediated by the JNK signaling pathway acting downstream of Wnt signaling was tested, as the similarity of their phenotypes suggests. If this were the case, activating JNK signaling should suppress the reduction in Dsh levels. Conversely, reducing both should show a synergistic effect. Therefore the JNKK hep was expressed in dsh¹ heterozygous flies and it was found that the hep gain-of-function is epistatic to dsh loss-of-function. Furthermore, reducing JNK activity by one copy of BSK-DN in dsh¹ mutant animals results in a synergistic reduction of extension to an average of 0.8 axons with 60% showing no axons crossing and no samples with more than three axons. In summary, the results of genetic analyses suggest that Wnt signaling via Dsh enhances JNK activity through the suppression of Rac1 (Srahna, 2006).

Dsh appears to promote JNK signaling and to be expressed in DCN axons prior to their extension toward the medulla early in pupal development. Since JNK signaling is required for this initial extension, it may be that Dsh also plays a role in the early extension of DCN axons. To test this possibility, DCN axon extension was examined at 30% pupal development in dsh¹ mutant brains. In wild-type pupae, essentially all (~40) DCN axons extend toward the medulla. In contrast, in dsh¹ mutant pupae, a strong reduction in the number of DCN axons crossing the optic chiasm between the lobula and the medulla was observed (Srahna, 2006).

Although the genetic data indicate that Dsh- and Rac-mediated signaling have sensitive and antagonistic effects on the JNK pathway, they do not establish whether the Dsh-Rac interaction modulates JNK's intrinsic activity. To test this, the amount of phosphorylated JNK relative to total JNK levels in fly brains was evaluated by Western blot analysis using phospho-JNK (P-JNK) and pan-JNK specific antibodies. Then it was determined if Dsh is indeed required for increased levels of JNK phosphorylation. Dsh¹ mutant brains showed a 25% reduction in P-JNK consistent with a stimulatory role for Dsh on JNK signaling. The reduction caused by loss of Dsh function is reversed, when the amount of Rac is reduced by half, consistent with a negative effect of Rac on JNK signaling downstream of Dsh. These data support the conclusion that Dsh and Rac interact to regulate JNK signaling by modulating the phosphorylated active pool of JNK (Srahna, 2006).

Taken together, these data suggest that during brain development DCN axons extend under the influence of JNK signaling. A non-canonical Wnt signal acting via Fz and Dsh ensures that JNK signaling remains active by attenuating Rac activity. In contrast, activation of the FGFR activates Rac1 and suppresses JNK signaling. These data support a model whereby the balance of the Wnt and FGF signals is responsible for determining the number of DCN axons that stably cross the optic chiasm. To test this model, FGFR levels were reduced, using the dominant-negative btl transgene, in dsh¹ heterozygous flies. It was found that simultaneous reduction of FGF and Wnt signaling restored the number of axons crossing the optic chiasm to almost wild-type levels (10.2, with 33% of the samples indistinguishable from wild-type, suggesting that the two signals in parallel, act to control the patterning of DCN axon connectivity (Srahna, 2006).

These data suggest the following model of DCN axon extension and retraction. DCN axons extend due to active JNK signal. These axons encounter Wnt5 and probably Wnt2 as well, resulting in activation of Disheveled. Disheveled, via its DEP domain, has a negative effect on the activity of the Rac GTPase, thus keeping JNK signaling active. After DCN axons cross the second optic chiasm they encounter a spatially regulated FGF/Branchless signal that activates the FGFR/Breathless pathway. Breathless in turn activates Rac, which inhibits JNK signaling in a subset of axons. These axons then retract back toward the lobula. The wide expression of the different components of these pathways and the modulation of JNK phosphorylation by Dsh and Rac in whole-head extracts strongly suggests that this model may apply to many neuronal types (Srahna, 2006).

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).

Antagonistic roles of Wnt5 and the Drl receptor in patterning the Drosophila antennal lobe

Numerous studies have shown that ingrowing olfactory axons exert powerful inductive influences on olfactory map development. From an overexpression screen, wnt5 was identified as a potent organizer of the olfactory map in Drosophila. Loss of wnt5 results in severe derangement of the glomerular pattern, whereas overexpression of wnt5 results in the formation of ectopic midline glomeruli. Cell type-specific cDNA rescue and mosaic experiments showed that wnt5 functions in olfactory neurons. Mutation of the derailed (drl) gene, encoding a receptor for Wnt5, resulted in derangement of the glomerular map, ectopic midline glomeruli and the accumulation of Wnt5 at the midline. drl functions in glial cells, where it acts upstream of wnt5 to modulate its function in glomerular patterning. These findings establish wnt5 as an anterograde signal that is expressed by olfactory axons and demonstrate a previously unappreciated, yet powerful, role for glia in patterning the Drosophila olfactory map (Yao, 2007).

The mechanisms by which ingrowing axons sort into precise maps, such as those found in the olfactory glomeruli or the somatosensory barrels, are poorly understood. Deafferentation and transplantation experiments revealed that ingrowing axons are important for specifying the maps in the initially homogenous structures. However, little is known about how the ingrowing axons carry out these feats. This report shows that ingrowing ORN axons express Wnt5, which contributes to organizing the glomerular pattern of the Drosophila olfactory system. The Drl receptor tyrosine kinase acts in glial cells to modulate Wnt5 signaling. This previously unknown interaction between ORN axons and glia reveals an important function of ORN axon-glia interactions in regulating the precise neural circuitry of the Drosophila antennal lobes (Yao, 2007).

The wnt5 mutant has characteristic disruptions of the olfactory map. Many dorsomedial glomeruli are displaced ventrally (resulting in heart-shaped antennal lobes) and the antennal commissure fails to form. In contrast to the loss-of-function defects, overexpression of wnt5 leads to the displacement of glomeruli into the midline. Examination of the ORN axons in the wnt5 mutant showed that they take circuitous paths to their targets and frequently misproject to dorsal regions of the brain. Consistent with a role for wnt5 in antennal lobe development, the antennal lobe defects appears during the pupal stage, when ORN axon targeting and glomerular development occur. Genetic mosaic and cell type-specific rescue experiments indicated that wnt5 is required in the ORNs. Antibody stainings indicated that the Wnt5 protein is enriched on the dendrites of the projection neurons, where it presumably accumulates subsequent to its secretion by ORNs. In addition to the projection neuron dendrites, Wnt5 also accumulates in the antennal commissure in the drl² mutant. It is proposed that Wnt5 is a signal by which ingrowing ORN axons direct the development of their target field (Yao, 2007).

Mutation of the drl gene also produces disruptions of the olfactory map. However, unlike the stereotyped shifts of glomeruli seen in the wnt5 mutant, the glomeruli were randomly positioned in or missing from one antennal lobe in the drl mutant. Furthermore, there was a strong tendency for glomeruli to form at the midline. As in the wnt5 mutant, ORN axons take indirect routes to their targets. That drl functions in development is supported by the observation that antennal lobe defects are visible at 40 hAPF, the time when ORN axon targeting and glomerular development take place (Yao, 2007).

Antibody staining showed that the Drl protein is highly expressed by the projection neurons and TIFR glia, cells that are intimately associated with the ingrowing ORN axons. In the projection neurons, Drl is enriched in the dendrites of nascent glomeruli, four of which also appeared to accumulate Wnt5. The TIFR is a donut-shaped mid-sagittal structure located between the antennal lobes. Histological studies showed that TIFR glial processes are closely associated with ORN axons that project across the midline. Several observations indicated that drl functions in the TIFR to regulate wnt5 function. First, removal of drl from single projection neuron clones does not disrupt the development and morphology of the projection neurons. Second, neuronal expression of drl in the drl² mutant background does not rescue the mutant phenotype. Third, expression of UAS-drl under the control of Repo-Gal4 strongly rescues the drl mutant phenotype, suggesting that drl functions in glial cells. Although roles for Drl in the projection neurons cannot be ruled out, collectively, the observations suggest that drl functions predominantly in glial cells to regulate antennal lobe development (Yao, 2007).

The phenotypic similarities between the drl loss-of-function and the wnt5-overexpressing mutants raise the intriguing possibility that the two genes act antagonistically in antennal lobe development. Indeed, expression of a weak wnt5 transgene in the ORNs, which has no effect in the wild type, triggers the formation of ectopic glomeruli in the drl² mutant. Thus, wnt5 and drl function in opposition to each other in antennal lobe development. To ascertain the relative positions of wnt5 and drl in this signaling pathway, animals carrying null mutations in both genes were generated. The wnt5⁴⁰⁰;drl² double mutants was found to have the characteristic wnt5 phenotype. The wnt5 gene is therefore epistatic to the drl gene, indicating that wnt5 functions downstream of drl in antennal lobe development. This conclusion is also supported by the observation that, although the removal of a copy of the wnt5 gene strongly suppresses the drl homozygous mutant phenotype, the removal of a copy of the drl gene has no effect on the wnt5 homozygous mutant phenotype. The genetic data that drl downregulates wnt5 function is further supported by the observation that the Wnt5 protein significantly accumulates in the commissure in the absence of Drl. Taken together, these genetic and histological data indicate that drl acts to inhibit the activity of wnt5 during antennal lobe development (Yao, 2007).

To probe the molecular mechanisms by which Drl regulates antennal lobe development, the various domains of Drl were mutated. It was observed that neither disruption of the kinase activity nor deletion of the intracellular domain significantly impaired rescue by the drl transgene. In contrast, deletion of the extracellular WIF domain completely abolishes Drl's ability to rescue the mutant phenotype. These results suggest that Drl regulates antennal lobe patterning predominantly through its extracellular WIF domain. How might Drl inhibit the function of Wnt5? One possibility is that Drl inhibits Wnt5 function simply by promoting Wnt5's sequestration or endocytosis, thus limiting its interaction with another as yet unidentified receptor. This receptor might be one of the other Drosophila receptor tyrosine kinases or a member of the Frizzled family, one of which, frizzled 2 (fz2), interacts genetically with wnt5 to stabilize axons of the Drosophila visual system. Alternatively, Drl may directly interact with another receptor and Wnt5, as has been observed previously for its mammalian ortholog Ryk and members of the Wnt and Frizzled families (Lu, 2004). This interaction could inhibit or alter the signal transduced from the membrane. However, no requirement was detected for Drl's cytoplasmic domain, suggesting that transduction of the Wnt5 signal by Drl alone is unlikely to have a major role in patterning the antennal lobes (Yao, 2007).

How do glial cells interact with the ORN axons to specify the olfactory map? The data suggest that the ingrowing ORN axons contribute to antennal lobe patterning through secretion of Wnt5 and that glial cells locally regulate Wnt5 actions through Drl. The following working model is proposed for how Wnt5-Drl signaling might regulate glomerular patterning. Ingrowing ORN axons express Wnt5, which is important for the precise organization of the glomeruli and pathfinding of the ORN axons, such as those crossing the midline or projecting to the dorsomedial region of the antennal lobes. Normal antennal lobe development requires that the Wnt5 signal be locally attenuated by the TIFR glial cell-expressed Drl protein. In the wnt5 mutant, the lack of Wnt5 signaling results in the failure of ORN axons to cross the midline and the establishment of glomeruli in more ventral positions. In the drl mutant, Wnt5 accumulates at the midline and presumably inappropriately signals through another receptor, resulting in aberrant ORN axon targeting to the midline and the formation of ectopic glomeruli at the dorsomedial corner of the antennal lobe and at the midline. Further studies will hopefully help to unravel the precise mechanisms by which Wnt5 and Drl act together to specify the patterning of the Drosophila olfactory map (Yao, 2007).

Dendritic refinement of an identified neuron in the Drosophila CNS is regulated by neuronal activity and Wnt signaling

The dendrites of neurons undergo dramatic reorganization in response to developmental and other cues, such as stress and hormones. Although their morphogenesis is an active area of research, there are few neuron preparations that allow the mechanistic study of how dendritic fields are established in central neurons. Dendritic refinement is a key final step of neuronal circuit formation and is closely linked to emergence of function. This is a study of a central serotonergic neuron in the Drosophila brain, the dendrites of which undergo a dramatic morphological change during metamorphosis. Using tools to manipulate gene expression in this neuron, the refinement of dendrites during pupal life was examined. This study shows that the final pattern emerges after an initial growth phase, in which the dendrites function as 'detectors', sensing inputs received by the cell. Consistent with this, reducing excitability of the cell through hyperpolarization by expression of K(ir)2.1 results in increased dendritic length. Sensory input, possibly acting through NMDA receptors, is necessary for dendritic refinement. These results indicate that activity triggers Wnt signaling, which plays a 'pro-retraction' role in sculpting the dendritic field: in the absence of sensory input, dendritic arbors do not retract, a phenotype that can be rescued by activating Wnt signaling. These findings integrate sensory activity, NMDA receptors and Wingless/Wnt5 signaling pathways to advance understanding of how dendritic refinement is established. The maturation of sensory function is shown to interact with broadly distributed signaling molecules, resulting in their localized action in the refinement of dendritic arbors (Singh, 2010).

This study focuses on a specific phase during the metamorphosis of the dendrites of a central serotonergic neuron, in which excess growth is removed by a process that has been termed refinement. Genetic analyses using loss-of-function mutants and RNAi-mediated knockdown of specific genes has led to a postulated a link between neuronal activity, synaptic input and Wnt signaling in this process. The sparse dendrites innervating the adult antennal lobe, present on the wide-field serotonergic neurons (CSDn) during the larval stage, are removed early in pupation by pruning, followed by a period of exuberant growth. The arrival of sensory neurons at the antennal lobe correlates well with when growth of the CSDn dendrites ceases and removal of the excess branches occurs. The CSDn must be active for the refinement process to occur, as refinement fails when neuronal activity is inhibited or when the sensory neurons are absent. Phenotypes observed in the latter case can be rescued by ectopic activation of the neuron using the temperature-sensitive dTrp-A1 channel. It is suggested that activity within the CSDn, possibly together with activity in presynaptic neurons, acts to provide the correlated activity required to trigger activation of NMDARs. Knockdown of NMDARs affects the refinement process, although identifying its specific action requires further study. A possible consequence of the activity-dependent process is activation of the Wg pathway, as the phenotype observed in aristalless mutants can also be rescued by ectopic expression of Dishevelled (Dsh) in the CSDn. It seems unlikely that activity within the CSDn leads to the release of Wnt ligands, but rather that dendrites respond locally to Wnt ligands in the region of a dendrite that is receiving input. Although other interpretations of the data are possible, a hypothesis is favored whereby specific synapses are stabilized as a result of correlated neuronal activity, and that excess dendritic branches are removed by Wnt signaling (Singh, 2010).

Perturbations in neuronal activity can be compensated by changes at multiple levels, including alterations in the expression of ion channels and in synaptic strength. Tripodi (2008) provides evidence for structural homeostasis whereby alterations in afferent input during development can be compensated by changes in dendritic geometry. This suggests that dendritic arbors serve as sensors for input levels, thus allowing the self-organization of circuits that is necessary for robust behavioral outputs (Tripodi, 2008). The current studies in the CSDn support these observations: reduced activation of the cell by targeted expression of K_ir2.1 results in a greatly enlarged dendritic field in the adult. This phenotype can be explained by a mechanism in which the absence of electrical activity results in a failure of the signaling mechanisms that stop growth of the arbors and that remove additional branches. Reduced excitability could also drive the homeostatic mechanisms towards making more arbors and to suppress the refinement program (Singh, 2010).

Dendritic growth and refinement are closely associated with input activity and synapse formation during development. Activity-dependent development of circuits is thought to utilize mechanisms similar to those involved in Hebbian learning and plasticity. NMDARs are ideal candidates for detecting correlated pre- and postsynaptic activity, which is crucial in the Hebbian model of learning and plasticity. Strengthening of synapses, as in this study, leads to the stabilization and extension of dendrites, whereas weakening of synapses leads to the destabilization and elimination of dendritic branches (Espinosa, 2009; Cline, 2008; Constantine-Paton, 1998). During vertebrate hippocampal development, NMDAR activation has been shown to limit synapse number and reduce dendritic complexity. The stabilization of a particular synapse or arbor possibly attenuates the formation of new branches or synapses, thus limiting further dendritic growth. In such a scenario, knocking down NMDAR levels would be expected to result in increased dendritic complexity, as indeed has been observed in this study. The mechanism by which 'appropriately connected' synapses are strengthened, whereas suboptimal contacts are eliminated, needs to be studied in thus system. In other systems, Ca²⁺, which is released upon NMDAR activation, impinges on various intracellular effectors that regulate dendritic morphogenesis. In addition, selective stabilization/destabilization of dendritic arbors could be affected by the local release of growth factors in response to activity (Singh, 2010).

This study shows that activity-dependent activation of the Wnt pathway facilitates retraction of dendritic arbors. Arbors that receive appropriate input are somehow protected and stabilized. These experiments suggest that Wnt-dependent refinement functions through a non-nuclear pathway and could act by impinging directly on cytoskeletal dynamics (Schlessinger, 2009; Salinas, 2008). Disruption of the microtubule cytoskeleton is a key feature of dendritic pruning in Drosophila during metamorphosis. GSK3β (Shaggy in Drosophila) an intracellular inhibitor of the Wnt pathway, has been shown to act as a sensor of inputs for neuronal activity (Chiang, 2009) and a potent regulator of microtubule dynamics in axons. In the Drosophila embryonic CNS, the Src family of tyrosine kinases (SFKs) is required for Wnt5/Drl-mediated signaling. Interestingly, SFKs seem to act as a crucial point of convergence for multiple signaling pathways that enhance NMDAR activity and hence are thought to act as molecular hubs for the control of NMDARs. It is tempting to envisage a scenario in which there is cross-talk between Wnt5/Drl signaling-mediated activation of SFKs and NMDAR signaling during refinement (Singh, 2010).

In summary, this study shows that the dendritic refinement of a central modulatory serotonergic neuron is regulated by electrical activity, NMDAR and Wnt signaling. Similar mechanisms have been implicated in dendritic growth and refinement of excitatory neurons in vertebrates. This study provides a model neuron preparation in which the dendritic growth and refinement of a modulatory neuron can be analyzed genetically. It was demonstrated that the dendrites of CSDn receive input from sensory neurons from the arista, supporting previous suggestions that mechanosensory input could alter sensitivity to odorant stimuli. In both Drosophila (Dacks, 2009) and the mammalian olfactory bulb (Petzold, 2009), serotonin gates the odor-evoked sensory response. CSDn sends projections to higher brain centers and multiglomerular projections to the contralateral antennal lobe and hence it is likely to influence the overall properties of the olfactory circuit. This study suggests that the structural and resulting functional properties of this neuron emerge from an interaction between partner neurons, together with input from intrinsic and extrinsic cues (Singh, 2010).

Wnt5 and drl/ryk gradients pattern the Drosophila olfactory dendritic map

During development, dendrites migrate to their correct locations in response to environmental cues. The mechanisms of dendritic guidance are poorly understood. Recent work has shown that the Drosophila olfactory map is initially formed by the spatial segregation of the projection neuron (PN) dendrites in the developing antennal lobe (AL). This study reports that between 16 and 30 h after puparium formation, the PN dendrites undergo dramatic rotational reordering to achieve their final glomerular positions. During this period, a novel set of AL-extrinsic neurons express high levels of the Wnt5 protein and are tightly associated with the dorsolateral edge of the AL. Wnt5 forms a dorsolateral-high to ventromedial-low pattern in the antennal lobe neuropil. Loss of Wnt5 prevents the ventral targeting of the dendrites, whereas Wnt5 overexpression disrupts dendritic patterning. Drl/Ryk, a known Wnt5 receptor, is expressed in a dorsolateral-to-ventromedial (DL > VM) gradient by the PN dendrites. Loss of Drl in the PNs results in the aberrant ventromedial targeting of the dendrites, a defect that is suppressed by reduction in Wnt5 gene dosage. Conversely, overexpression of Drl in the PNs results in the dorsolateral targeting of their dendrites, an effect that requires Drl's cytoplasmic domain. It is proposed that Wnt5 acts as a repulsive guidance cue for the PN dendrites, whereas Drl signaling in the dendrites inhibits Wnt5 signaling. In this way, the precise expression patterns of Wnt5 and Drl orient the PN dendrites allowing them to target their final glomerular positions (Wu, 2014).

The amyloid precursor protein is a conserved Wnt receptor

The Amyloid Precursor Protein (APP) and its homologues are transmembrane proteins required for various aspects of neuronal development and activity, whose molecular function is unknown. Specifically, it is unclear whether APP acts as a receptor, and if so what its ligand(s) may be. This study shows that APP binds the Wnt ligands Wnt3a and Wnt5a (see Drosophila Wnt5) and that this binding regulates APP protein levels. Wnt3a binding promotes full-length APP (flAPP) recycling and stability. In contrast, promotes APP targeting to lysosomal compartments and reduces flAPP levels. A conserved Cysteine-Rich Domain (CRD) in the extracellular portion of APP is required for Wnt binding, and deletion of the CRD abrogates the effects of Wnts on flAPP levels and trafficking. Finally, loss of APP results in increased axonal and reduced dendritic growth of mouse embryonic primary cortical neurons. This phenotype can be cell-autonomously rescued by full length, but not CRD-deleted, APP and regulated by Wnt ligands in a CRD-dependent manner (Liu, 2021).

This study has identified a previously unknown conserved Wnt receptor function for APP proteins. APP was shown to bind both canonical and non-canonical Wnt ligands via a conserved CRD and that this binding regulates the levels of full-length APP by regulating its intracellular trafficking from early endosomes to the trans Golgi network versus the lysosome. Finally, it was shown that APP through the CRD regulates neurite growth and axon branching complexity in primary mouse cortical neurons (Liu, 2021).

A function for APP as a cell surface receptor has be proposed for quite a long time. The first strong evidence came from the structure of APP which shares similarity with type I transmembrane receptors. For example the growth factor like domain (GFLD) in the E1 region of APP could act as a ligand-binding site, and disulfide bridges within the same E1 area could further facilitate ligand-induced signal transduction by stabilizing the structure of APP ectodomain. In addition, the site-specific proteolytic processing property of APP resembles several membrane receptors and constitute a second line of evidence. For instance, both APP and Notch are processed by γ-secretase and release the intracellular domain AICD and NICD, respectively, to induce transcriptional activity by nuclear transfer of the ICDs. Several putative ligands have been proposed including proteolytic products of APP itself. Interestingly, a conserved CRD within the E1 region of APP makes APP a putative Wnt receptor as the CRD is required for the binding between Wnt and its receptor and co-receptor like Frizzled Ror-2 and Musk, although the crystal structure of the APP CRD is different from that of classic Wnt receptors (Liu, 2021).

Using immunoprecipitation this study has shown that mouse APP binds in a CRD-dependent manner to Wnt3a and Wnt5a the two well-known ligands for triggering canonical or non-canonical Wnt signaling pathways, respectively. Based on these data, it is speculated that APP might act as a conserved Wnt receptor as its CRD is highly conserved across species, and that the binding may not be limited to Wnt3a or Wnt5a, but also apply to all other Wnt family members. As Wnt signaling is critical for development and tissue homeostasis, the role of APP as a component or putative receptor or co-receptor in Wnt signaling should be carefully studied. The difficulty to explore the exact role of APP may not only come from the complexity of Wnt signaling, but also from the structural properties of APP itself. For example, the extracellular domain of APP harbors several binding site for the molecules in the extracellular matrix (ECM), such as the Heparin binding domain (HBD) is not only exist in E1 area a second HBD has been found in the E2 region. A recent study has shown that LRP6, the co-receptor of canonical Wnt signaling, could directly bind to APP. Using peptide Mapping array that study further revealed several binding sites (20-70 amino acids in length) in the ectodomain of APP, and one of the sites (34 amino acids) is located within the APP CRD. Based on IP results indicating a Wnt receptor function for APP, an obvious question arises as to whether LRP6 and APP compete for Wnt. Before trying to address this problem, several issues need to be resolved. First, since in the IP experiments the whole CRD (150 amino acids) was deleted, it is not clear if specific sequences inside the CRD are critical for Wnt binding that may or may not overlap with the reported LRP6-binding site. A second important point is that the results of the peptide mapping array may not reflect the exact binging site for LRP6 as these short synthesized peptides may lack information embedded in the 3D structure like the disulfide bridges formed by Cysteines that may be critical for maintaining the stability of the APP extracellular domain. Significant further biophysical and biochemical analysis is required to understand the details of the interaction and various components of the Wnt receptor complex (Liu, 2021).

APP has been extensively reported to be involved in regulating neurite outgrowth, with conflicting conclusions as to whether APP promotes or inhibits neurite outgrowth. In the current experiments, it was found that while in Drosophila APPL loss reduced axonal growth, the comparison of axonal outgrowth and branching in primary cortical neuron derived from mAPP wild type or mAPP knock out mice at DIV2, DIV3 and DIV7 showed that loss of mAPP significantly accelerated axonal maturation. Specifically, it was found that the initial phase of axonal growth at DIV2 is unaffected, but that APP mutant axons grow longer at DIV3 and then show increased axon complexity at DIV7. It is therefore suggested that the conflicting data in the literature may arise from examining different types of neurons at different time points, where the requirement of APP may differ in a context-specific manner. It is speculated that this context specificity may in part be due to the levels and types of Wnt ligands present in the environment (Liu, 2021).

Finally, the current findings suggest that in addition to the well-described proteolytic processing of APP, the regulation of its recycling by Wnt ligands may be crucial for its function. It is important to note that, like proteolytic processing, Wnt ligands regulate APP stability post-translationally, as no effect was found on App mRNA levels upon Wnt treatment. With regard to the role of APP processing in Alzheimer's disease, recently published work suggests that an imbalance between Wnt3a/canonical signaling pathway and the Wnt5a/PCP signaling pathway at the initial step of amyloid beta production could trigger a vicious cycle favoring the amyloidogenic processing of APP (Sellers, 2018; Elliott, 2018). The findings of this study, that Wnt3a and Wnt5a have opposite effects on amyloid beta production, provide a mechanistic framework for understanding how the normal physiological function of APP directly impacts the generation of a key marker of AD, and thus potentially links the normal activity of APP to its role in neurodegeneration. Whether and how the modulation of APP's role in Wnt signaling may offer future therapeutic avenues for AD is an exciting venue for future research (Liu, 2021).

Robo and Ror function in a common receptor complex to regulate Wnt-mediated neurite outgrowth in Caenorhabditis elegans

Growing axons are exposed to various guidance cues en route to their targets, but the mechanisms that govern the response of growth cones to combinations of signals remain largely elusive. This study found that the sole Robo receptor, SAX-3 (see Drosophila Robo), in Caenorhabditis elegans functions as a coreceptor for Wnt/CWN-2 (Drosophila homolog: Wnt5) molecules. SAX-3 binds to Wnt/CWN-2 and facilitates the membrane recruitment of CWN-2. SAX-3 forms a complex with the Ror/CAM-1 receptor and its downstream effector Dsh/DSH-1, promoting signal transduction from Wnt to Dsh. sax-3 functions in Wnt-responsive cells and the SAX-3 receptor is restricted to the side of the cell from which the neurite is extended. DSH-1 has a similar asymmetric distribution, which is disrupted by sax-3 mutation. Taking these results together, it is proposed that Robo receptor can function as a Wnt coreceptor to regulate Wnt-mediated biological processes in vivo (Wang, 2018).

REFERENCES

Search PubMed for articles about Drosophila Wnt5

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Wang, J. and Ding, M. (2018). Robo and Ror function in a common receptor complex to regulate Wnt-mediated neurite outgrowth in Caenorhabditis elegans. Proc Natl Acad Sci U S A 115(10): E2254-E2263. PubMed ID: 29463707

Wu, Y., Helt, J. C., Wexler, E., Petrova, I. M., Noordermeer, J. N., Fradkin, L. G. and Hing, H. (2014). Wnt5 and drl/ryk gradients pattern the Drosophila olfactory dendritic map. J Neurosci 34: 14961-14972. PubMed ID: 25378162

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date revised: 22 April 2022

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