Given the axon guidance defects in nerfin-1null 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-1null 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-1null 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-1null 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-1null 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-1null 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-1null embryos (Kuzin, 2005).
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).
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, Wnt5D7, 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, Wnt5D84, 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 Wnt5D7 and Wnt5D84 abolish Wnt5 expression: the phenotypes of Wnt5D7 and Wnt5D84 homozygotes, as well as Wnt5D7/Wnt5D84 individuals, are indistinguishable. Thus, Wnt5D7 acts as a null allele. Wnt5D7 and Wnt5D84 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).
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).
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).
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