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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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 Kir2.1 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).
Reference names in red indicate recommended papers.
Search PubMed for articles about Drosophila Wnt5
Chiang, A., et al. (2009). Neuronal activity and Wnt signaling act through Gsk3-β to regulate axonal integrity in mature Drosophila olfactory sensory neurons. Development 136: 1273-1282. PubMed Citation: 19304886
Cline, H. and Haas, K. (2008). The regulation of dendritic arbor development and plasticity by glutamatergic synaptic input: a review of the synaptotrophic hypothesis. J. Physiol. 586: 1509-1517. PubMed Citation: 18202093
Constantine-Paton, M. and Cline, H. T. (1998). LTP and activity-dependent synaptogenesis: the more alike they are, the more different they become. Curr. Opin. Neurobiol. 8: 139-148. PubMed Citation: 9568401
Dacks A. M., et al. (2009). Serotonin modulates olfactory processing in the antennal lobe of Drosophila. J. Neurogenet. 23. 366-377. PubMed Citation: 19863268
Espinosa, J. S., Wheeler, D. G., Tsien, R. W. and Luo, L. (2009). Uncoupling dendrite growth and patterning: single-cell knockout analysis of NMDA receptor 2B. Neuron 62: 205-217. PubMed Citation: 19409266
Fradkin, L. G., Noordermeer, J. N. and Nusse, R. (1995). The Drosophila Wnt protein DWnt-3 is a secreted glycoprotein localized on the axon tracts of the embryonic CNS. Dev. Biol. 168: 202-213. Medline abstract: 7883074
Fradkin, L. G., van Schie, M., Wouda, R. R., de Jong, A., Kamphorst, J. T., Radjkoemar-Bansraj, M. and Noordermeer, J. N. (2004). The Drosophila Wnt5 protein mediates selective axon fasciculation in the embryonic central nervous system. Dev. Biol. 272: 362-375. Medline abstract: 15282154
Grillenzoni, N., Flandre, A., Lasbleiz, C. and Dura, J. M. (2007). Respective roles of the DRL receptor and its ligand WNT5 in Drosophila mushroom body development. Development 134(17): 3089-97. Medline abstract: 17652353
Harris, K. E. and Beckendorf, S. K. (2007). Different Wnt signals act through the Frizzled and RYK receptors during Drosophila salivary gland migration. Development 134(11): 2017-25. Medline abstract: 17507403
Kuzin, A., Brody, T., Moore, A. W. and Odenwald, W. F. (2005). Nerfin-1 is required for early axon guidance decisions in the developing Drosophila CNS. Dev. Biol. 277: 347-365. Medline abstract: 15617679
Lu, W., Yamamoto, V., Ortega, B. and Baltimore, D. (2004). Mammalian Ryk is a Wnt coreceptor required for stimulation of neurite outgrowth. Cell 119(1): 97-108. PubMed citation: 15454084
Petzold G. C., Hagiwara A. and Murthy V. N. (2009). Serotonergic modulation of odor input to the mammalian olfactory bulb. Nat. Neurosci. 12: 784-791. PubMed Citation: 19430472
Salinas, P. C. and Zou Y. (2008). Wnt signaling in neural circuit assembly. Annu. Rev. Neurosci. 31: 339-358. PubMed Citation: 18558859
Schlessinger, K., Hall A. and Tolwinski, N. (2009). Wnt signaling pathways meet Rho GTPases. Genes Dev. 23: 265-277. PubMed Citation: 19204114
Singh, A. P., VijayRaghavan, K. and Rodrigues, V. (2010). Dendritic refinement of an identified neuron in the Drosophila CNS is regulated by neuronal activity and Wnt signaling. Development 137(8): 1351-60. PubMed Citation: 20223760
Srahna, M., Leyssen, M., Choi, C. M., Fradkin, L. G., Noordermeer, J. N. and Hassan, B. A. (2006). A signaling network for patterning of neuronal connectivity in the Drosophila brain. PLoS Biol. 4(11): e348. Medline abstract: 17032066
Tanaka, K., Kitagawa, Y. and Kadowaki, T. (2002). Drosophila segment polarity gene product porcupine stimulates the posttranslational N-glycosylation of wingless in the endoplasmic reticulum. J. Biol. Chem. 277(15): 12816-23. Medline abstract: 11821428
Tripodi M., et al. (2008). Structural homeostasis: compensatory adjustments of dendritic arbor geometry in response to variations of synaptic input. PLoS Biol. 6: e260. PubMed Citation: 18959482
Yao, Y., et al. (2007). Antagonistic roles of Wnt5 and the Drl receptor in patterning the Drosophila antennal lobe. Nat. Neurosci. 10(11): 1423-32. PubMed citation: 17934456
Yoshikawa, S., McKinnon, R. D., Kokel, M. and Thomas, J. B. (2003). Wnt-mediated axon guidance via the Drosophila Derailed receptor. Nature 422(6932): 583-8. 12660735
date revised: 30 May 2008
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