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).

Canonical Wnt signaling in the visceral muscle is required for left-right asymmetric development of the Drosophila midgut

Many animals develop left-right (LR) asymmetry in their internal organs. The mechanisms of LR asymmetric development are evolutionarily divergent, and are poorly understood in invertebrates. Drosophila has several organs that show directional and stereotypic LR asymmetry, including the embryonic gut, which is the first organ to develop LR asymmetry during Drosophila development. This study found that genes encoding components of the Wnt-signaling pathway are required for LR asymmetric development of the anterior part of the embryonic midgut (AMG). frizzled 2 and Wnt4, which encode a receptor and ligand of Wnt signaling respectively, are required for the LR asymmetric development of the AMG. arrow, an ortholog of the mammalian gene encoding low-density lipoprotein receptor-related protein 5/6, which is a co-receptor of the Wnt-signaling pathway, was also essential for LR asymmetric development of the AMG. These results are the first demonstration that Wnt signaling contributes to LR asymmetric development in invertebrates, as it does in vertebrates. The AMG consists of visceral muscle and an epithelial tube. Genetic analyses revealed that Wnt signaling in the visceral muscle but not the epithelium of the midgut is required for the AMG to develop its normal laterality. Furthermore, fz2 and Wnt4 are expressed in the visceral muscles of the midgut. Consistent with these results, it was observed that the LR asymmetric rearrangement of the visceral muscle cells, the first visible asymmetry of the developing AMG, did not occur in embryos lacking Wnt4 expression. These results also suggest that canonical Wnt/β-catenin signaling, but not non-canonical Wnt signaling, is responsible for the LR asymmetric development of the AMG. Canonical Wnt/β-catenin signaling is reported to have important roles in LR asymmetric development in zebrafish. Thus, the contribution of canonical Wnt/β-catenin signaling to LR asymmetric development may be an evolutionarily conserved feature between vertebrates and invertebrates (Kuroda, 2012).

This study found that Wnt-signaling components Wnt4 and Fz2 are required for LR asymmetric development of the AMG, although contribution of other Wnt ligands and receptors to this process could not be excluded. For example, it is known that Wnt4 binds to Fz and Fz2, and that fz and fz2 function redundantly in the segmentation of Drosophila embryos. This study found that the AMG of embryos homozygous for fz showed similar LR defects to those of fz2, although at a lower frequency. Therefore, it is possible that Fz acts redundantly as the receptor for canonical Wnt/β-catenin signaling, although the expression of fz in the midgut could not be detected by anti-Fz antibody staining. In contrast, analysis of embryos homozygous for derailed (drl) suggested that Wnt5 may not be involved in the LR asymmetric development of the AMG. Drl is a member of the RYK subfamily of receptor tyrosine kinases and is a receptor for Wnt5. The laterality of the AMG was normal in embryos homozygous for drl (Kuroda, 2012).

Wnt4 is one of the few Wnt ligands whose function has been revealed in Drosophila. This study found that Wnt4–Fz2 activates the canonical Wnt/β-catenin signaling pathway for normal LR asymmetric development of the AMG. Consistent with this finding, Wnt4 activates the canonical Wnt/β-catenin signaling pathway in salivary glands through Fz or Fz2, which is required for the glands’ proper migration. However, the Wnt4–Fz2 pathway is also known to activate non-canonical Wnt signaling in other systems. Wnt4 plays an essential role in the cell movement required for formation of the ovariolar sheath cells. In addition, Wnt4 expressed in the developing ventral lamina is required for ventral projection of the retinal axon. In both of these cases, Fz2 acts as a receptor of Wnt4, and the Wnt4–Fz2 pathway activates non-canonical Wnt signaling. Therefore, although the same combination of Wnt ligand and receptor, Wnt4–Fz2, is involved, the downstream cascades of Wnt signaling may be context-dependent, although the factors acting as molecular switches for these downstream pathways remain unknown (Kuroda, 2012).

The first indication of LR symmetric morphogenesis in the AMG is observed as the LR asymmetric rearrangement of circular visceral muscle (CVMU) cells. These rearrangements can be monitored by measuring the major axial angle of the nuclei in the CVMU cells to the midline of the AMG (Kuroda, 2012).

This study found that the LR asymmetry of the rearranged CVMU cells in the ventral AMG became bilaterally symmetric in embryos homozygous for a Wnt4 mutation. This result was consistent with the AMG’s random LR laterality in these embryos. However, unexpectedly, the CVMU cells were rearranged LR asymmetrically in the dorsal AMG in Wnt4 mutant homozygotes, even though the arrangement of these dorsal cells is bilaterally symmetric in wild-type embryos. This result suggests that Wnt signaling may counteract the LR asymmetric morphogenesis in the dorsal side of the AMG, in addition to its role in introducing a LR bias by inducing the rearrangement of CVMU cells in the ventral AMG, via the Wnt4–Fz2 pathway. In embryos homozygous for loss-of-function mutations of Wnt4, arr, or fz2, the LR asymmetric development of the posterior embryonic gut was largely normal. Thus, in wild-type embryos, the Wnt4–Fz2 signal may function to suppress the influence of the LR asymmetric morphogenic signals from the posterior midgut on the AMG (Kuroda, 2012).

The present analyses clarified the requirement for Wnt4–Fz2 signaling in the LR asymmetric morphogenesis of the AMG, but the precise molecular functions of this signal are still unclear. Because Wnt4–Fz2 activates canonical Wnt/β-catenin signaling, it will be important to identify the target genes responsible for LR asymmetric morphogenesis of the AMG (Kuroda, 2012).

Effects of Mutation

Transducing properties of Drosophila Frizzled proteins

In Drosophila, two closely related serpentine receptors, Frizzled and Frizzled2 are able to act as receptors for Wingless. In addition to transducing the Wg signal, Fz (but not Fz2) is able to transduce a second, unidentified signal that mediates planar polarity. Much attention has been focused on the structure of the N-termini of the Fz-class receptors and their role in ligand binding. Experiments using techniques of high-level expression have suggested a role for the C-termini in specifying which of the two second messenger systems the receptors are able to activate. It is argued here that experiments involving high level expression of the receptors cannot be adequately interpreted. The ability of the receptors and chimeric forms when driven at moderate levels to rescue loss of function of the fz and fz2 genes has been tested. Under these conditions all receptors tested will function as Wg receptors, but only a subset show the ability to rescue the polarity pathway. The presence of this subset implies that the N terminus is necessary but not sufficient and suggests that the ability to transduce the polarity signal is widely distributed throughout the protein (Strapps, 2001).

From the rescue experiments it is inferred that all chimeras assayed behaved as functional Wg receptors but only a subset was able to rescue polarity signaling in fz mutant tissue. Comparison of the chimeras that rescue polarity signaling with those that do not suggests that the N-terminal domain of Fz is critical for the transduction of the polarity signal but that alone it is not sufficient. In addition to the N terminus, one of the two other Fz domains is required. Thus the specificity for signal transduction appears spread through the three domains of the protein. The simple way to view this is that ligand binding requires the N terminus, and that transmission of the signal to intracellular molecular machinery can be achieved by at least one of two distinct sites in the remainder of the protein. It is noted that only low levels of Fz are normally required for polarity transduction and that at least two of the constructs that failed to rescue polarity transduction resulted in strong polarity phenotypes when over-expressed in the wing (Strapps, 2001).

Interactions between Wingless and DFz2 during Drosophila wing development

Mutations in the wg gene disrupt the patterning of embryonic segments and their adult derivatives. Wg protein has been shown in cell culture to functionally interact with DFz2, a receptor that is structurally related to the tissue polarity protein Frizzled (Fz). However, it has not been determined if DFz2 functions in the Wg signaling pathway during fly development. Overexpression of DFz2 is shown to increase Wg-dependent signaling to induce ectopic margin bristle formation in developing Drosophila wings. Alongside the anterior margin, supernumerary stout mechanosensory bristles are often observed one or more cell diameters away from the normal stout bristle row. Within the wing, supernumerary chemosensory and slender mechanosensory bristles are detected. The bristle types (stout, slender and chemosensory) are located on the appropriate dorsal or ventral surface of the wing, and are restricted to the anterior region of the wing. Alongside the posterior wing margin, supernumerary non-sensory bristles are observed several cell diameters distant from the margin where these bristles are normally located. The formation of supernumerary bristles by DFz2 does not appear to result from a disturbance in lateral inhibition between neighboring bristle precursor cells. Contrary to what is typically seen when lateral inhibition is perturbed, there is no overall change in margin bristle density. The spacing between bristles within the three regular rows of anterior bristles and within the single row of posterior bristles of wings overexpressing DFz2 is indistinguishable from that of wild type. No ectopic bristles are observed when Fz2 is overexpressed. However, the polarity of margin bristles is abnormal, and wing hair polarity is deranged throughout the plane of the wing blade. It is suggested that the results of overexpression reflect an independence between Fz and DFz2 pathways during wild-type wing development (Zhang, 1998).

Overexpression of a truncated form of DFz2 acts in a dominant-negative manner to block Wg signaling at the wing margin, and this block is rescued by co-expression of full-length DFz2 but not full-length Fz. These results suggest that DFz2 and not Fz acts in the Wg signaling pathway for wing margin development. However, a truncated form of Fz also blocks Wg signaling in embryo and wing margin development; the truncated form of DFz2 affects ommatidial polarity during eye development. These observations suggest that a single dominant-negative form of Fz or DFz2 can block more than one type of Wnt signaling pathway and imply that truncated proteins of the Fz family lose some aspect of signaling specificity (Zhang, 1998).

How does the overexpression of DFz2 lead to ectopic bristle induction? One possibility is that cells at some distance from the narrow stripe of Wg-secreting margin cells are normally exposed to a low level of Wg, insufficient to induce bristle determination. Overexpression of DFz2, combined with endogenous receptors, may increase Wg signal transduction in some of these cells to a level comparable to those nearer the wing margin, thus inducing ectopic margin bristles. It is worth noting that at the anterior margin, most ectopic bristles induced close to the margin are of the stout mechanosensory type, which are normally present in a row of cells closest to the stripe of Wg secretion. The ectopic bristles further away from the wing margin are usually of the slender or chemosensory types, which are normally present in two rows of cells more distant from the Wg stripe than the stout row. This suggests that Wg is normally present in the wing as a concentration gradient surrounding the wing margin. Overexpression of DFz2 increases the level of Wg signal transduction in cells that are exposed to a particular concentration of Wg. Consequently, cells adopt fates that are normally induced only at a higher concentration of Wg. Cells that would have normally adopted slender or chemosensory bristle fates instead become stout bristles; cells that would have normally failed to adopt any bristle fate instead become slender or chemosensory bristles. This interpretation is consistent with observations that Wg acts as a gradient morphogen to induce expression of different target genes near the wing margin (Zhang, 1998).

Use of dsRNA-mediated genetic interference to demonstrate that frizzled and frizzled 2 act in the Wingless pathway

Recently, double-stranded RNA (dsRNA) has been found to be a potent and specific inhibitor of gene activity in the nematode Caenorhabditis elegans (Fire, 1998). The potential of dsRNA to interfere with the function of genes in Drosophila, termed RNA inhibition or RNAi) has been investigated. Injection of dsRNA into embryos resulted in potent and specific interference of several genes that were tested. dsRNA corresponding to four genes with previously defined functions was introduced. dsRNA is shown to potently and specifically inhibits the activities of wg, fushi tarazu (ftz), even-skipped (eve), and tramtrack (ttk). The reception mechanism of the morphogen Wingless was determined using dsRNA. Interference of the frizzled and Drosophila frizzled 2 genes together produces defects in embryonic patterning that mimic the loss of wingless function. Interference with the function of either gene alone has no effect on patterning. Epistasis analysis indicates that frizzled and Drosophila frizzled 2 act downstream of wingless and upstream of zeste-white3 in the Wingless pathway. These results demonstrate that dsRNA interference can be used to analyze many aspects of gene function (Kennerdell, 1998).

The potency and specificity of dsRNA interference on gene activity suggests that it might be a useful means to eliminate Frizzled2 activity. Although the null phenotype of Fz2 is unknown, it was reasoned that if Fz2 encodes the Wg receptor, then its mutant phenotype should resemble wg loss-of-function mutants. Larvae that lack wg activity are completely covered with denticles on the ventral cuticle, unlike wild-type larvae in which ventral cuticle is an alternating pattern of naked cuticle and denticles. When dsRNA corresponding to the wg gene is injected, the region around the site of injection exhibits a wg-like mutant phenotype, and the remainder of the embryo was wild type. Surprisingly, no animals exhibit a null phenotype despite the injection of twice as much dsRNA as for other genes. The RNAi (RNA inhibition) effect is localized, and the range of phenotypes is limited by the size of the region with ectopic denticles. When dsRNA corresponding to the 5' UTR of Fz2 is injected, no effect on denticle patterning is observed. Ectopic denticles are not observed in embryos injected with dsRNA corresponding to the 5' UTR of the fz gene. In contrast, an equimolar mixture of ds-fz and ds-Fz2 RNAs causes localized transformation of naked cuticle into denticles. The RNAi effect is limited to the site of injection even at high doses of dsRNA, and its potency is highly similar to the potency of ds-wg RNA. Denticles in the affected regions resemble those typical of the fifth row in a wild-type abdominal segment, and the denticles are oriented either toward the midline or along the anteroposterior axis with reversed polarity. These features are precisely those observed in wg mutant embryos and embryos treated with ds-wg RNA (Kennerdell, 1998).

Engrailed expression initiates normally in wg mutants but fails to be maintained. To examine whether Fz and Fz2 have a similar function, embryos were injected with ds-fz and ds-Fz2 RNAs. After further development, the embryos were stained with an anti-Engrailed antibody. Expression of engrailed os absent in lateral ectoderm within the affected region. This discontinuous loss of engrailed expression resembles loss of functional wg (Kennerdell, 1998).

The interfering activity of ds-fz and ds-Fz2 RNA mixtures could mean that the fz and Fz2 genes act redundantly, and the activities of both genes must be blocked before a phenotype is observed. Alternatively, it could reflect some other synergy between the injected RNAs. ds-Fz2 RNA was injected alone into embryos mutant for fz and was found to possess interfering activity that is comparable to the interfering activity of mixed ds-fz and ds-Fz2 RNAs. These data are most consistent with the fz and Fz2 genes acting redundantly to pattern the ventral epidermis (Kennerdell, 1998).

Experiments in cell culture have suggested that Fz2 acts as a receptor in the Wg signal transduction pathway. Do Fz and Fz2 act between Wg and the intracellular components of its signal transduction pathway? Genetic epistasis can determine the order of action of genes in a common pathway. If fz and Fz2 function downstream of wg, then interference of fz and Fz2 activities should suppress activating mutations of wg. A transgenic strain that expresses high levels of Wg in all epidermal cells causes those cells to secrete naked cuticle. This strain was used to determine whether fz and Fz2 are required for wg action. When these animals are injected with ds-fz and ds-Fz2 RNAs, the formation of ectopic naked cuticle is suppressed. The injected transgenic embryos are distinct from wild-type embryos injected with RNA and from the uninjected transgenic strain. They resembled wild-type embryos injected with dsRNA in that they have denticles alternating with naked cuticle plus some localized patches of continuous denticle lawn. However, they do not usually have the complete complement of denticles. This incomplete suppression is attributed to the fact that interference of fz and Fz2 is primarily localized to regions close to the site of injection. Nevertheless, this result provides genetic evidence for a function of fz and Fz2 downstream of wg (Kennerdell, 1998).

Transduction of a Wg signal antagonizes the Shaggy/Zw3 kinase, which functions to modulate levels of Arm. Do fz and Fz2 act between wg and shaggy, as would be predicted for the Wg receptor? Loss of shaggy activity results in all epidermal cells adopting posterior segmental fates, and mutant embryos lack ventral denticles. When shaggy mutant embryos are injected with ds-fz and ds-Fz2 RNAs, there is no change in their phenotype; they resemble shaggy embryos. The similarity of the phenotypes of shaggy with or without fz and Fz2 interference suggests that fz and Fz2 function upstream of zw3 in wg signaling (Kennerdell, 1998).

There are similarities between how Wg signals to cells in the embryonic epidermis and in wing discs. Several studies suggest that Wg acts as a morphogen in both tissues. Moreover, it utilizes the same signal transduction pathway in responding cells. However, there are significant differences between how Wg works in the two tissues. In the wing disc, Wg specifies cell fate decisions but has no apparent role in controlling planar polarity of wing cells. In the embryonic epidermis, Wg specifies cell fate decisions and controls the planar polarity of cells. This planar polarity is manifested by the orientation of denticles along the anteroposterior axis, which is disrupted in wg mutants or can be redirected by wg misexpression. There is a second major difference between wing and embryo: in the wing, DFz2 and not Fz mediates the Wg signal. Misexpression of DFz2 increases the zone of Wg responsiveness in the wing, but Fz misexpression has no effect. Null fz mutants do not perturb cell fate decisions attributable to Wg. In the embryo, both Fz and DFz2 are required to mediate the Wg signal. Inhibition of both genes is sufficient to disrupt planar polarity and epidermal cell differentiation, whereas inhibition of each gene singly has no effect. These data are also consistent with experimental results in which Fz was overexpressed in embryos (Kennerdell, 1998 and references).

How can Wg, Fz, and DFz2 generate both polarity and cell fate responses in embryos and not in wing discs? One possibility is that in embryos they directly specify cell fates and indirectly affect cell polarity. For instance, they specify the diverse pattern of denticle types that might then determine overall denticle polarity. Another possibility is that distinct domains of Wg activate different cell responses by interacting with receptors in qualitatively different ways. Access to some Wg domains might be limiting in some tissues and not others. A third possibility is that Wg ligand-receptor interactions are quantitatively different in various tissues. A fourth possibility is that intrinsic factors couple Wg-bound Frizzled proteins to a particular cell response, and these factors are differentially active in various tissues (Kennerdell, 1998 and references).

Most ligands pair with specific receptors, and each pairing remains fixed for different tissues and different developmental stages. Wg appears to be an exception to this general rule. What is the significance behind Wg's diverse signaling properties? By adding greater flexibility in the competence of cells to respond to Wg, more diverse responses to a single ligand can be generated. Competence may be modified by changing the number of potential receptors and their ability to trigger more than one transduction pathway. Another reason for this diversity might be related to the function of Wg receptors in shaping the concentration gradient of Wg in a tissue. In the wing disc, high levels of Fz2 stabilize extracellular Wg and allow it to range farther from its source than in the absence of Fz2. Thus, if more than a single Frizzled protein can stabilize Wg, the combination of multiple receptor expression patterns might determine the Wg gradient. This simple combinatorial mechanism could potentially generate a broad range of gradient curves for a single ligand (Kennerdell, 1998).

Frizzled and DFrizzled-2 function as redundant receptors for Wingless during Drosophila embryonic developmen

In cell culture assays, Frizzled and Dfrizzled2, two members of the Frizzled family of integral membrane proteins, are able to bind Wingless and transduce the Wingless signal. To address the role of these proteins in the intact organism and to explore the question of specificity of ligand-receptor interactions in vivo, a genetic analysis of frizzled and Dfrizzled2 in the embryo has been conducted. These experiments utilize a small gamma-ray-induced deficiency that uncovers Dfrizzled2. Dfz2-deficiency homozygotes die shortly after hatching and exhibit a subtle disorganization of denticle patterning with occasional ectopic denticles in posterior compartments. These data suggest that Dfz2 and/or other genes removed by the 469-2 deficiency play a minor or largely redundant role in cuticle patterning during embryogenesis. Mutants lacking maternal frizzled and zygotic frizzled and Dfrizzled2 exhibit defects in the embryonic epidermis, CNS, heart and midgut that are indistinguishable from those observed in wingless mutants. Epidermal patterning defects in the frizzled, Dfrizzled2 double-mutant embryos can be rescued by ectopic expression of either gene. In frizzled;Dfrizzled2 double mutant embryos, ectopic production of Wingless does not detectably alter the epidermal patterning defect, but ectopic production of an activated form of Armadillo produces a naked cuticle phenotype indistinguishable from that produced by ectopic production of activated Armadillo in wild-type embryos. These experiments indicate that frizzled and Dfrizzled2 function downstream of wingless and upstream of armadillo, consistent with their proposed roles as Wingless receptors. The lack of an effect on epidermal patterning of ectopic Wingless in a frizzled;Dfrizzled2 double mutant argues against the existence of additional Wingless receptors in the embryo or a model in which Frizzled and Dfrizzled2 act simply to present the ligand to its bona fide receptor. These data lead to the conclusion that Frizzled and Dfrizzled2 function as redundant Wingless receptors in multiple embryonic tissues and that this role is accurately reflected in tissue culture experiments. The redundancy of Frizzled and Dfrizzled2 explains why Wingless receptors were not identified in earlier genetic screens for mutants defective in embryonic patterning (Bhanot, 1999).

In the wild-type epidermis, wg functions in an autocrine pathway to maintain its own expression and in a paracrine regulatory loop to maintain expression of en in adjacent cells. In the epidermis at gastrulation, when wg function is first detected, a stripe of cells in the anterior half of each parasegment expresses wg and an adjacent stripe of cells in the posterior half express en. This pattern is initiated by pair-rule and gap genes, but its maintenance requires paracrine signaling by Wg to the en expressing cells and both paracrine signaling by Hh and autocrine signaling by Wg to the wg expressing cells. Thus, in wg mutant embryos the pattern of wg and en gene expression is initiated correctly but is not maintained. In fz;Dfz2 double-mutant embryos, the En stripes begin to fade at stage 9/10 and are completely absent from the epidermis by mid stage 10, similar to wg mutants. By contrast, en expression within the CNS is maintained as it is in wg mutants. Consistent with a defect in Wg signaling, Wg expression is greatly reduced in fz;Dfz2 mutants (Bhanot, 1999).

At the end of gastrulation, wg participates in the morphogenesis of various embryonic structures. In the embryonic central nervous system, wg is expressed by row 5 neuroblasts (NBs) and its function is required to specify NBs in rows 4 and 6. Null mutants of wg show a loss or duplication of several NBs, the most extensively studied being NB-4. The NB-4 lineage gives rise to two RP2 motoneurons per segment that innervate the dorsal musculature and are missing in wg mutant embryos. RP2 neurons are marked by their expression of even-skipped (eve). Mutant embryos missing maternal fz and zygotic fz and Dfz2 or missing only zygotic Dfz2 were examined using an antibody against Eve. fz;Dfz2 double-mutant embryos show a complete loss of RP2 neurons in all hemisegments. As observed in the epidermis, the fzR52 allele shows residual activity: in fz;Dfz2 double mutants carrying the fzR52 allele, approximately 26% of the double-mutant embryos show Eve-positive RP2 staining in 1-3 hemisegments. Interestingly, 469-2 homozygous embryos also show a weakly penetrant RP2 phenotype. In approximately 21% of the 469-2 homozygous embryos, an RP2 neuron is either missing or misplaced in 1-3 hemisegments. It is concluded that fz and Dfz2 are largely but not entirely redundant in specifying RP2 identity (Bhanot, 1999).

Wingless transduction by the Frizzled and Frizzled2 proteins of Drosophila

Wingless (Wg) protein is a founding member of the Wnt family of secreted proteins that have profound organizing roles in animal development. Two members of the Frizzled (Fz) family of seven-pass transmembrane proteins, Drosophila Fz and Fz2, can bind Wg and are candidate Wg receptors. However, null mutations of the fz gene have little effect on Wg signal transduction and the lack of mutations in the fz2 gene has thus far prevented a rigorous examination of its role in vivo. Here, the isolation of an amber mutation of fz2 is described; this mutation truncates the coding sequence just after the amino-terminal extracellular domain and behaves genetically as a loss-of-function allele. Using this mutation, Wg signal transduction is abolished in virtually all cells lacking both Fz and Fz2 activity in embryos, as well as in the wing imaginal disc. Fz and Fz2 are functionally redundant: the presence of either protein is sufficient to confer Wg transducing activity on most or all cells throughout development. These results extend prior evidence of a ligand-receptor relationship between Wnt and Frizzled proteins and suggest that Fz and Fz2 are the primary receptors for Wg in Drosophila (Chen, 1999).

Wg is normally expressed in a thin stripe of cells straddling the dorsoventral compartment boundary of the mature wing imaginal disc, under the control of the extracellular signals Delta and Serrate. Wg protein emanating from these cells directs the formation of wing margin bristles and organizes gene expression, growth and patterning in surrounding cells of the presumptive wing blade. Hence, mutations that block Wg signal transduction cause a loss of wing margin bristles as well as deletions of nearby portions of the wing. Wg also plays a role in restricting its own expression to cells immediately adjacent to the dorsoventral compartment boundary by down-regulating the transcription of wg itself in neighboring cells that are close to, but not next to, the D/V boundary. When Wg signal transduction is blocked in these cells, they ectopically express Wg. As a consequence, nearby wild-type tissue is induced to form ectopic margin bristles. Approximately 100 mutations were obtained in a screen for mutations that cause wing margin defects in clones of mutant cells that are also homozygous for the fz loss-of-function mutation, fzH51. Of these, only one is associated with the formation of ectopic bristles in neighboring, wild-type wing tissue. This mutation, designated fz2C1, appears to be a loss-of-function mutation in fz2 according to the following criteria: (1) the mutation maps meiotically to a location approximately 1 centiMorgan distal to radius incompletus (ri), the expected map position, given the cytological localization of the fz2 gene; (2) both the wing notching and ectopic bristle phenotypes associated with fzH51; fz2C1 mutant cells are completely rescued when the fz2 coding sequence is expressed in these cells using either alpha Tubulin a1-fz2 transgene (which should be expressed in most or all cells), or a UAS-fz2 transgene driven by a vg-Gal4 transgene. All of the remaining wing notching mutations obtained in the screen fully complement the fz2C1 mutation in a fzH51 mutant background, indicating that they are not in the fz2 gene. (3) The fz2C1 mutation is associated with a single base change in the fz2 gene that changes codon 320 from TGG to TAG. This creates a stop codon located at the junction between the coding sequence of the amino-terminal extracellular domain (which contains the CRD) and the remainder of the protein, which includes all seven transmembrane domains. It is unlikely that the resulting truncated protein, composed of just the extracellular domain, would retain any signal transducing activity (Chen, 1999).

To assay the possible roles of Fz and Fz2 in Wg signal transduction during embryogenesis, embryos were generated homozygous for the fzH51 and fz2C1 mutations that derive from female germ cells that are similarly mutant for the two genes. Such embryos lack the maternal and zygotic contributions of both genes, and hence, should be devoid of Fz and Fz2 activity. These embryos are referred to as fz-fz2- mutant embryos. To assay these embryos for Wg signal transducing activity, an examination was performed in six well defined Wg signaling events: two in the ectoderm, one in the visceral mesoderm, one in the endoderm, one in the central nervous system, and one in the somatic mesoderm. These double mutant embryos appear unable to transduce Wg when assayed for each event. (1) Initially examined was the cuticular pattern formed by such double mutant embryos. The epidermis of wild-type embryos secretes a segmented cuticle, decorated on the ventral side by stereotyped bands of patterned hairs separated by broad swaths of naked cuticle. In embryos devoid of Wg activity, or of Dsh or Arm activity, most signs of segmentation are eliminated and the ventral cuticle forms a 'lawn' of hairs spanning most of the anteroposterior body axis. Embryos devoid of Fz and Fz2 activity show the same characteristic 'lawn' phenotype. (2) The early striped expression of En in the ectoderm is labile, unless maintained by Wg signaling from adjacent cells across the parasegment boundary. In wg-, dsh- and arm- mutant embryos, this expression is lost within 2 hours after the onset of gastrulation. A similar loss of ectodermal En expression occurs in fz-fz2- mutant embryos. (3) Wg signaling is essential in the visceral mesoderm for initiating a series of stereotyped constrictions that partition the midgut. As in embryos lacking Wg, Dsh, or Arm activity, these gut constrictions are absent in fz-fz2- mutant embryos. (4) Wg signaling from the visceral mesoderm of parasegment 7 up-regulates the expression of the homeodomain gene labial in the adjacent endoderm. This up-regulation is not observed in wg-, dsh- or arm- embryos, and similarly, it is not apparent in fz-fz2- mutant embryos. (5) During development of the central nervous system, Wg signaling is essential for specifying the neuroblasts that generate the RP2 neurons in each segment. These neurons can be easily visualized because they express Even-skipped (Eve) protein. These Eve-expressing neurons are not present in the absence of Wg signaling (e.g., in wg- embryos). Similarly, they are absent in fz-fz2- mutant embryos. (6) Finally, during development of the somatic musculature, Eve protein is expressed in a subset of myoblasts that will give rise to the heart and the presence of these Eve-expressing cells is strictly dependent on Wg signaling. These Eve-expressing cells are also absent in fz-fz2- mutant embryos. In sum, embryos devoid of both Fz and Fz2 activity appear unable to transduce Wg in any of the several developmental contexts examined. These results indicate an absolute requirement for these Fz proteins for Wg transduction during embryonic development (Chen, 1999).

During normal development of the embryonic ectoderm, Wg protein moves at least a few cell diameters from secreting cells, as assayed by the accum. Therefore, an investigation was carried out to see whether the movement and apparent uptake of secreted Wg protein depends on Fz and Fz2. The distribution of Wg in fz-fz2- mutant embryos was determined. Wild-type and fz-fz2- mutant embryos show indistinguishable distributions of punctate Wg staining during the first two hours following germ band extension, consistent with the view that neither Fz nor Fz2 protein is required for the movement of secreted Wg during this phase of development. However, the fzH51 mutation is expected to generate a protein that is truncated after the sixth transmembrane domain. Hence, if this protein is stable and reaches the cell membrane, it might be able to bind and regulate the movement of secreted Wg even though it can no longer transduce Wg signal. Wg expression dissipates in fz-fz2- mutant embryos shortly after this early stage, as expected given the loss of En expression in neighboring cells across the AP compartment boundary, preventing the examination of later aspects of Wg movement in these embryos. Fz and Fz2 transduce Wg via the regulation of Armadillo. Most, if not all, Wg signal transducing events involve the modification and up-regulation of Armadillo (Arm) protein. Two experiments were performed to test whether Fz and Fz2 transduce Wg through the regulation of Arm. These experiments establish that Fz and Fz2 act upstream of Arm to transduce Wg. In the first experiment, Arm expression was assayed in fz-fz2- mutant embryos. In wild-type embryos, Wg signaling is associated with stabilization of Arm protein and its consequent accumulation in a distinctive pattern of segmental stripes, each straddling a stripe of Wg-expressing cells. This up-regulation is not observed in wg minus embryos, and similarly, it is absent in fz-fz2- mutant embryos. In the second experiment, it was asked whether expression of a truncated, constitutively active form of Arm could drive the Wg signal transduction pathway in fz-fz2- mutant embryos. In this experiment, constitutively active Arm was expressed with the UAS/Gal4 method using a hairy-Gal4 driver line that is active in alternating segmental primordia. Expression of constitutively active Arm in alternating segmental stripes in fz-fz2- mutant embryos causes them to form corresponding stripes of naked cuticle. This result indicates that the activity of constitutively active Arm bypasses the normal requirements for Fz and Fz2 in activating the Wg transduction pathway (Chen, 1999).

Wg is expressed in the wing pouch of late third instar discs in a thin stripe of cells straddling the interface between the dorsal and ventral compartments. Wg emanating from this stripe acts at short range to induce the formation of bristles that will decorate the wing margin, and at longer range, to activate the expression of a number of genes, including Distalless (Dll) and vestigial (vg), that define the primordium of the wing blade and control aspects of its growth and pattern. Wg signal transduction is abolished in presumptive wing cells lacking both Fz and Fz2 activity. As a consequence, cells that lack both activities cannot proliferate normally and are lost from the epithelium. Wg signaling is required for the control of growth and pattern in portions of the wing disc other than the wing pouch. The wing imaginal discs also give rise to the fuselage of the adult second thoracic segment, the mesonotum, the anterior dorsal surface of which is decorated with a stereotyped pattern of large bristles. Wg is expressed in a longitudinal stripe in the developing half-mesonotum derived from each wing disc and this stripe is positioned just lateral to a line of four large bristles. These are the anterior and posterior dorsocentral bristles and the anterior and posterior scutellar bristles. It appears that zygotic activity of the fz2 gene is not essential for Wg signal transduction, provided that a wild-type allele of fz is present. Either protein can transduce most or all Wg signaling events during embryogenesis. It is concluded that Fz and Fz2 proteins are functionally redundant, with either protein being able to bear the full burden of Wg signal transduction in most, if not all, contexts throughout development (Chen, 1999).

The Drosophila STE20-like kinase Misshapen is required downstream of the Frizzled receptor in planar polarity signaling

Misshapen acts in the Frizzled (Fz) mediated epithelial planar polarity (EPP) signaling pathway in eyes and wings. Both msn loss- and gain-of-function result in defective ommatidial polarity and wing hair formation. Genetic and biochemical analyses indicate that msn acts downstream of fz and dishevelled (dsh) in the planar polarity pathway, and thus implicates an STE20-like kinase in Fz/Dsh-mediated signaling. This demonstrates that seven-pass transmembrane receptors can signal via members of the STE20 kinase family in higher eukaryotes. Msn acts in EPP signaling through the JNK (Jun-N-terminal kinase) module as it does in dorsal closure. Although at the level of Fz/Dsh there is no apparent redundancy in this pathway, the downstream effector JNK/MAPK (mitogen-activated protein kinase) module is redundant in planar polarity generation. To address the nature of this redundancy, evidence is provided for an involvement of the related MAP kinases of the p38 subfamily in planar polarity signaling downstream of Msn (Paricio, 1999).

In the Drosophila eye, EPP is reflected in the mirror-symmetric arrangement of ommatidial units relative to the dorso-ventral midline (the equator). This pattern is generated posterior to the morphogenetic furrow when ommatidial preclusters rotate 90° toward the equator, adopting opposite chirality depending on their dorsal or ventral positions. Polarity defects are manifested in the loss of mirror-image symmetry, with the ommatidia misrotating and adopting random chirality or remaining symmetrical. The gain-of-function dsh phenotype (sev-Dsh) has been successfully used in previous reports to identify new components of the Fz/Dsh planar polarity pathway. This same assay, dominant genetic modification of the sev-Dsh phenotype, was used to screen through a large number of known genes. Among the few mutants that show a specific interaction are two msn alleles. msn102 and msn172 are X-ray-induced inversions with breakpoints in the msn gene. Both loss-of-function alleles of msn act as dominant suppressors of sev-Dsh, comparable to other planar polarity-specific Dsh effectors (Paricio, 1999).

In addition, msn has been isolated in a gain-of-function screen for genes involved in planar polarity generation. Overexpression of genes required in planar polarity signaling at the relevant time often results in defects that are similar to the loss-of-function mutant phenotypes, e.g. with Fz and Dsh. In such a screen, ap-GAL4 flies (ap-GAL4 induces overexpression of the corresponding gene in the notum and the dorsal part of the wing), were crossed to the collection of 2200 E/P lines and the progeny were scored for disarranged microchaetae on the notum. One of the lines isolated in this screen, ep(3)0549, shows an abnormal orientation of the microchaetae similar to phenotypes obtained with ap driven Fz overexpression. Similarly, ap-GAL4, ep(3)0549 flies show typical polarity phenotypes on the dorsal surface of the wing where these are manifest in the presence of multiple wing hairs. In situ hybridization experiments to polytene chromosomes and complementation analyses reveal that the EP-element insertion in line ep(3)0549 is in the msn locus and represents a msn allele. Subsequent sequence analyses confirm that the EP insertion is located 24 bp upstream of the 5'-end of a msn cDNA. Taken together, these results suggest that msn is involved in EPP signaling and possibly acts downstream of Dsh (Paricio, 1999).

To gain further confirmation of the role of Msn in Fz/Dsh-mediated polarity signaling, an in vitro assay was used to determine whether Msn acts downstream of Dsh in JNK pathway activation. Previous experiments have shown that expression of Dsh in NIH 3T3 cells activates JNK and Jun phosphorylation, indicating that Dsh is a potent activator of a Jun-kinase pathway. Using the same assay, it was asked whether co-expression of a dominant-negative (kinase-inactive) Msn protein (DN-Msn) has an effect on Dsh-induced Jun phosphorylation. Significantly, co-expression of DN-Msn in this context causes a dramatic concentration-dependent inhibition of Jun phosphorylation. Taken together with the genetic interactions, these experiments confirm that Msn is acting downstream of Fz/Dsh in planar polarity signaling (Paricio, 1999).

msn mutations affect the morphology of the rhabdomeres in photoreceptors, causing malformed, 'misshapen' rhabdomeres, and also, at lower frequency, the number of photoreceptors. In addition, msn is required for the process of dorsal closure, and embryos mutant for msn display a typical dorsal open phenotype. To analyze its requirements in polarity generation, msn mutant clones in the eye and the wing were examined in detail. A phenotypic analysis of eye clones reveals that msn is required for the generation of planar polarity. msn mutant ommatidia containing the normal complement of photoreceptors are often misrotated and display the wrong chiral form or are symmetrical (non-chiral). To confirm that the polarity defects of msn mutant ommatidia are primary defects, and thus implicate msn in polarity generation, ommatidial polarity was examined in msn mutant clones at the earliest possible stage in third instar larval imaginal discs (when tissue polarity genes are required). Spalt is expressed in the R3/R4 precursor pair for about two columns at this stage. In wild type this reflects the regular arrangement and direction of rotation of the preclusters. In msn mutant tissue, ommatidial rotation, and thus polarity, is randomized (e.g. ommatidia rotate in the opposite direction as their wild-type neighbors) showing that these defects result from an early failure in polarity establishment. Thus in the eye, the msn phenotype (defects in polarity, malformed, misshapen and missing photoreceptors) is very reminiscent of other genes involved in both polarity and terminal photoreceptor differentiation (Paricio, 1999).

The fz gene has been implicated in the specification of the R3 cell within the R3/R4 pair in the process of chirality generation. The mosaic analysis of both loss-of-function and gain-of-function fz alleles has shown that Fz signaling is required in R3 for correct ommatidial chirality generation and also induces R3 fate. The genetic interactions and cell culture experiments have shown that msn acts downstream of Fz/Dsh, and thus it was asked whether msn is also involved in the selection of R3 in analogy to the fz requirement. The genotypic composition of mosaic ommatidial clusters were examined within the R3/R4 pair. This analysis revealed that, as is the case for fz, the msn+ cell has a strong preference for adopting the R3 photoreceptor fate. This can often lead to chirality inversions, where the msn+ R4 precursor adopts the R3 position and displaces the original msn- R3 precursor. In summary, the genetic requirements of msn in single photoreceptors, in particular the R3/R4 pair, are very similar to those of fz (Paricio, 1999).

arrow encodes an LDL-receptor-related protein essential for Wingless signaling

The mechanism by which the Wingless signal is received and transduced across the membrane is not completely understood. The arrow gene function is essential in cells receiving Wingless input. arrow acts upstream of Dishevelled and encodes a single-pass transmembrane protein; this indicates that it may be part of a receptor complex with Frizzled class proteins. Arrow is a low-density lipoprotein (LDL)-receptor-related protein (LRP), strikingly homologous to murine and human LRP5 and LRP6. Thus, a new and conserved function is suggested for the LRP subfamily in Wingless/Wnt signal reception. Mosaic and epistasis analyses place the requirement for Arrow activity upstream of Dishevelled in responding cells. Because arrow encodes a putative transmembrane protein, epistasis tests between Arrow and Fz proteins would be of interest, but no activating mutations exist in either Fz or Arrow that would lead to signal transduction in the absence of ligand. There are, however, several suggestive similarities between Fz proteins and Arrow. Arrow and DFz2 transcription is modulated similarly, whereas that of the Dsh and Arm signal transducers is not. In addition, ectopic DFz2 expression can mildly sensitize cells to Wg signaling, for example in the wing, where overexpression of DFz2 produces ectopic margin bristles -- a Wg-dependent cell type. This potentiation of Wg signaling is ligand-dependent, and ectopic bristles are only found near the wing margin, which is a source of high levels of Wg. As is expected for a gene essential for Wg signaling, loss of arrow function in clones leads to loss of margin, similar to that observed for fz Dfz2 clones. Overexpression of Arrow also produces ectopic bristles near the wing margin. Thus, arrow is required for Wg-dependent signaling at the margin, and can potentiate Wg signaling in a manner similar to that of DFz2. The potentiation of signaling caused by excess DFz2 depends on arrow function, as shown by inducing arrow mutant clones while overexpressing DFz2. If excess DFz2 were able to bypass Arrow and restore signaling on arrow mutant cells then these cells should retain the ability to form wing margin and produce marked arrow mutant margin bristles, neither of which was observed. This result is consistent either with Arrow functioning after DFz2 engages ligand, or with Arrow and the Fz class proteins functioning as co-receptors, although this epistasis must be confirmed when activating mutations become available (Wehrli, 2000).

The extensive homology between Arrow and both mouse and human LRP5 and LRP6, indicates that the role ascribed to Arrow in Wg signaling may extend to these LRPs for Wnt signaling. Indeed, an insertion in the mouse LRP6 gene has been identified that leads to several Wnt-like phenotypes. Arrow, and by extension LRP5 and LRP6, have specific roles for the Wg/Wnt pathway, as both Dpp and Hh can signal to arrow null cells (Wehrli, 1998 and references therein). arrow null mutant cells do not survive as well as their wild-type sister cells when twin spot clones are made, suggesting some role in viability. Whether this can be attributed to a deficit in Wnt signaling is not known, although cells doubly mutant for fz and dfz2 also do not survive (Wehrli, 2000).

The simplest model is that Arrow and a Fz-class protein act together as a receptor complex. Alternatively, Arrow might assist in recycling the Fz receptors to the plasma membrane after Wg/Wnt ligand binding and internalization, thereby providing unoccupied receptors to allow efficient, extended signaling. This idea is suggested by the fact that Arrow is related to LDL receptors, which are sometimes involved in recycling proteins from the plasma membrane. If this is the case, then overexpression of Fz proteins should suppress the need for arrow function by supplying excess nascent receptors. However, overexpressing DFz2 at the wing margin does, in fact, not suppress the Wg signaling defect caused by loss of arrow function (Wehrli, 2000).

Drosophila Rho-associated kinase (Drok) links Frizzled-mediated planar cell polarity signaling to the actin cytoskeleton

Drosophila Rho-associated kinase (Rok) works downstream of Fz/Dsh to mediate a branch of the planar polarity pathway involved in ommatidial rotation in the eye and in restricting actin bundle formation to a single site in developing wing cells. The primary output of Rok signaling is regulating the phosphorylation of nonmuscle myosin regulatory light chain (Mizuno, 1999 and Winter, 2001), and hence the activity of myosin II. Drosophila myosin VIIA, the homolog of the human Usher Syndrome 1B gene, also functions in conjunction with this newly defined portion of the Fz/Dsh signaling pathway to regulate the actin cytoskeleton (Winter, 2001).

The similarity of the Drok2 clonal phenotype in the wing to aspects of the phenotypes of fz, dsh, and RhoA led to the hypothesis that Rok may act downstream of Fz/Dsh. To assess the genetic interactions among these genes, the multiple hair phenotype was quantitated in a defined region: the ventral surface of the proximal-anterior region of the wing. Use was made of the dsh1 allele, which is defective for PCP function without affecting Wg signaling. In dsh1 hemizygous males, an average of 16.8 cells with multiple hairs (12% of the cells) are present in this region. When Rok is overexpressed via a tubP-Drok transgene in the dsh1 hemizygous background, the average number of cells exhibiting multiple hairs is reduced by more than 7-fold to 2.3 per wing region. tubP-Drok expression in a wild-type genetic background does not give rise to any obvious phenotypes. Suppression of the dsh1 multiple hair phenotype by tubP-Drok expression could also be seen when F-actin-based prehairs were visualized in the pupal wing. In contrast to the suppression of the dsh1 phenotype by overexpression of Rok, reduction of rok dosage by 50% (assuming that Drok2 is null) results in a 2.5-fold increase in the number of multiple hair cells (Winter, 2001).

Genetic interactions between rok and fz were examined in a different assay. The proper level of Fz/Dsh signaling is critical for the generation of wild-type PCP, since both overexpression and loss of function of these genes result in polarity defects in the eye and the wing. Overexpression of Fz 30 hr after puparium formation (APF) produces primarily a multiple hair phenotype that is suppressed by dsh1 heterozygosity. Similarly, reducing the wild-type copy number of RhoA and rok by half suppresses the phenotype by 2- to 2.5-fold. Taken together, these experiments suggest that Rok functions downstream of Fz/Dsh in restricting the number of F-actin-based prehairs (Winter, 2001).

The roles of glypicans Dally and Dally-like protein (Dlp), the Wg receptors Frizzled (Fz) and Fz2, and the Wg co-receptor Arrow (Arr) in Wg gradient formation in the wing disc

During the wing development Wingless acts as a morphogen whose concentration gradient provides positional cues for wing patterning. The molecular mechanism(s) of Wg gradient formation is not fully understood. This study systematically analyzes the roles of glypicans Dally and Dally-like protein (Dlp), the Wg receptors Frizzled (Fz) and Fz2, and the Wg co-receptor Arrow (Arr) in Wg gradient formation in the wing disc. Both Dally and Dlp are essential and have different roles in Wg gradient formation. The specificities of Dally and Dlp in Wg gradient formation are at least partially achieved by their distinct expression patterns. Surprisingly, although Fz2 has been suggested to play an essential role in Wg gradient formation by ectopic expression studies, removal of Fz2 activity does not alter the extracellular Wg gradient. Interestingly, removal of both Fz and Fz2, or Arr causes enhanced extracellular Wg levels, which mainly results from upregulated Dlp levels. It is further shown that Notum, a negative regulator of Wg signaling, downregulates Wg signaling mainly by modifying Dally. Last, it is demonstrated that Wg movement is impeded by cells mutant for both dally and dlp. Together, these new findings suggest that the Wg morphogen gradient in the wing disc is mainly controlled by combined actions of Dally and Dlp. It is proposed that Wg establishes its concentration gradient by a restricted diffusion mechanism involving Dally and Dlp in the wing disc (Han, 2005).

One important finding of this study is that removal of the Wg receptors (Fz and Fz2) and the co-receptor Arr does not lead to a loss of extracellular Wg. Fz2 has been proposed to play a major role in Wg gradient formation in the wing disc by ectopic expression studies. Although the high capacity of Fz2 in stabilizing Wg has been demonstrated, loss-of-function results clearly show that extracellular Wg levels are not reduced in clones mutant for fz2. This is apparently not due to the overlapping function of Fz, since the extracellular Wg level is enhanced, rather than reduced, in the absence of both Fz and Fz2 functions. The results argue that Fz2 is not essential for extracellular Wg gradient formation in vivo. It is important to note that in addition to Fz and Fz2, Fz3 is also expressed in the wing disc and its expression is upregulated by Wg signaling. Although Fz3 has lower affinity than Fz2 in Wg binding and acts as an attenuator of Wg signaling, its role in Wg distribution needs to be determined (Han, 2005).

It is further demonstrated that extracellular Wg is enhanced in cells mutant for fz-fz2 or arr, suggesting that Wg receptors (Fz and Fz2) and Arr shape extracellular Wg gradient by downregulating extracellular Wg levels. The data argue that this mainly results from upregulation of Dlp. Consistent with this view, the accumulated extracellular Wg can be eliminated by loss of HSPGs in sfl-fz-fz2 or arr-botv mutant clones. Importantly, it is shown that both extracellular Wg and Dlp levels are upregulated on the cell surface of clones mutant for dsh. These data provide compelling evidence that though a feedback mechanism, Wg signaling can control the Dlp levels to regulate the extracellular Wg gradient (Han, 2005).

Another alternative possibility is that enhanced Wg levels in fz-fz2 or arr clones may be caused by impaired Wg internalization. Although a significant amount of internalized Wg vesicles has been demonstrated in fz-fz2 or arr mutant clones, this possibility cannot be ruled out, since a quantitative comparison of Wg internalization between wild-type cells and fz-fz2 or arr mutant cells is difficult. Furthermore, as mentioned above, Fz3 is expressed in the wing disc and its expression is upregulated by Wg signaling. It is possible that Fz3 may mediate the internalization of Wg in the absence of Fz and Fz2 (Han, 2005).

Evidence has been presented that Wg morphogen movement is regulated by a diffusion mechanism(s) in the wing disc. Does Wg diffuse freely in the extracellular matrix/space? In this work, it is shown that Wg fails to move across a strip of cells mutant for the HSPGs Dally and Dlp. This result suggests that Wg cannot freely diffuse in the extracellular matrix. Instead, the findings are consistent with a model in which Wg movement is mediated by the HSPGs Dally and Dlp through a restricted diffusion along the cell surface. Similar mechanisms have been proposed for Hh and Dpp. In biological systems such as imaginal discs, the restraint of Wg spreading to the surface of the epithelial cell layer is important since the folding of imaginal discs, such as the leg disc, poses a problem if the Wg gradient formation were to occur out of the plane of the epithelial cell layer through free diffusion. In agreement with this view, the model proposes that Wg gradient formation depends on Wg movement through the cell surface of the disc epithelium (Han, 2005).

Wnt4, along with frizzled2 and dishevelled, regulates the dorsoventral specificity of retinal projections in the Drosophila melanogaster visual system

In Drosophila, the axons of retinal photoreceptor cells extend to the first optic ganglion, the lamina, forming a topographic representation. DWnt4, a secreted protein of the Wnt family, is the ventral cue for the lamina. In DWnt4 mutants, ventral retinal axons misproject to the dorsal lamina. DWnt4 is normally expressed in the ventral half of the developing lamina and DWnt4 protein is detected along ventral retinal axons. Dfrizzled2 and dishevelled, respectively, encode a receptor and a signaling molecule required for Wnt signaling. Mutations in both genes caused DWnt4-like defects, and both genes are autonomously required in the retina, suggesting a direct role of DWnt4 in retinal axon guidance. In contrast, iroquois homeobox genes are the dorsal cues for the retina. Dorsal axons accumulate DWnt4 and misproject to the ventral lamina in iroquois mutants; the phenotype is suppressed in iroquois:Dfrizzled2 double mutants, suggesting that iroquois may attenuate the competence of Dfrizzled2 to respond to DWnt4 (Sato, 2005).

The spatial order of projecting neurons is preserved in the spatial order of their targets to establish the topographic maps in the nervous system. In the visual system, precise topographic mapping of photoreceptor neurons to their targets in the brain, termed retinotopic mapping, is necessary for the correct interpretation of visual information received in the retina. The Drosophila visual system includes the retina, the compound eye and the optic lobe, which is the visual center of the brain and is connected to the eye via the optic stalk. Each of the approximately 750 ommatidial units in the retina consists of eight unique types of photoreceptor neurons called R cells (R1-R8). During larval development, R cells sequentially differentiate behind the morphogenetic furrow, progressing in a posterior-to-anterior order in the third larval instar retina, and send their axons through the optic stalk to the most distal part of the optic lobe, the lamina. R1-R6 axons terminate in the lamina layer, whereas R7 and R8 axons project through the lamina to terminate in the medulla layer. Although all the retinal axons pass through the narrow optic stalk, they distribute evenly and project to their correct targets along the anteroposterior and the dorsoventral axes. Thus, R cell axon connections between the retina and the lamina (or the medulla) are precisely retinotopic in the adult. Similarly, R axon connections established during the third instar are anatomically retinotopic (Sato, 2005 and references therein).

Like the retina, the lamina must also be patterned along the dorsoventral axis so that retinal axons can project precisely to their targets. It is assumed that selector genes and genes encoding guidance molecules are asymmetrically expressed along the dorsoventral axis in the developing lamina and it was found that DWnt4, one of seven D. melanogaster Wnt family genes, is specifically expressed in the ventral half of the developing lamina during the third larval instar (Sato, 2005).

The present study investigated the involvement of DWnt4 in D. melanogaster retinotopic mapping along the dorsoventral axis. DWnt4 is normally expressed in the ventral half of the developing lamina and DWnt4 protein has been detected on the surface of ventral retinal axons. In DWnt4 mutant backgrounds, ventral axons misproject to the dorsal lamina. Conversely, ventral axons are redirected by an ectopic source of DWnt4, suggesting that DWnt4 is a ventral cue for retinal axon projections in the lamina. Furthermore, ventral axon projections are regulated by noncanonical Wnt signaling in R cells, which is most likely under the control of DWnt4. These genetic data also suggest the involvement of JNK signaling in this process. Finally, iro may attenuate the competence of Dfz2 in dorsal axons to respond to DWnt4, since dorsal-to-ventral misroutings in iro clones are significantly suppressed in iro:Dfz2 double mutant clones (Sato, 2005).

As a first step to investigating retinotopic mapping in D. melanogaster, focus was placed on the iroquois (iro) complex genes, three related homeobox genes that act as selector genes for the dorsal retina. To test if iro regulates retinotopic mapping, cells homozygous for an iro deficiency were generated and labeled with green fluorescent protein (GFP) to visualize axons using the MARCM system. Dorsal mutant R axons were occasionally observed projecting to the ventral lamina. A similar phenotype was observed by generating large iro clones using ey-flp. Both outer- and inner-photoreceptor axons visualized with ro-lacZ and ato-myc were affected by iro. Thus, iro genes seem to function as dorsal cues for the retina (Sato, 2005).

The lamina, to which the retinal axons project, must also be patterned along the dorsoventral axis. It is assumed that selector genes and genes encoding guidance molecules are asymmetrically expressed along the dorsoventral axis in the developing lamina. Attempts were made to identify genes specifically expressed in either the dorsal or the ventral lamina. It was found that DWnt4, one of seven Wnt family genes in D. melanogaster, is specifically expressed in the ventral half of the lamina during the late third instar. At this stage, the lamina expresses Dachshund (Dac) and forms a characteristic, crescent-like structure. In situ hybridization showed specific expression of DWnt4 in the ventral half of the lamina along the lamina furrow, restricted to the anterior-most two or three rows of Dac-positive cells (Sato, 2005).

Because DWnt4 is a secreted glycoprotein, the distribution of DWnt4 protein was compared with that of DWnt4 mRNA. DWnt4 localization along the lamina furrow of the ventral lamina is indistinguishable from that of DWnt4 mRNA, except for small dots found in the ventral lamina. Notably, DWnt4-positive dots are found on the surface of the anterior-most R axons, labeled by GMR-Gal4 UAS-GFP. Given that R axons sequentially project to the lamina from posterior to anterior, DWnt4 probably accumulates on the most recently arriving axons. In more apical optical sections, DWnt4 accumulation is observed between the optic stalk and the lamina. The source of DWnt4 on R axons is likely to be DWnt4 expression along the ventral lamina furrow, since DWnt4 localization is not detectable on R axons in the dorsal half of the lamina (Sato, 2005).

DWnt4 localization on ventral R axons implies its involvement in R axon guidance. This possibility was tested by investigating the DWnt4 loss-of-function phenotype. In wild-type flies, dorsal and ventral axons project to the dorsal and ventral lamina, respectively. In DWnt4 mutants, however, occasional misroutings of ventral axons toward the dorsal lamina were observed. In extreme cases, ventral axons looked as if they were about to project to ventral lamina but had abruptly reoriented toward the dorsal lamina. omb-lacZ was used to visualize the dorsal- and ventral-most axons and ventral axon misrouting within the optic stalk was noted. Together, these data suggest that DWnt4 influences ventral axon projection at various points along the path from the optic stalk to the lamina (Sato, 2005).

Frizzled family receptors and Dishevelled are required for a wide variety of Wnt signaling cascades7. Dfz2 and dsh mutant flies have ovarian defects similar to those of DWnt4, strongly suggesting that Dfz2 and Dsh are involved in DWnt4 signaling. A ventral-to-dorsal misrouting phenotype was observed in Dfz2 and dsh mutant backgrounds. The results suggest that Dfz2 and Dsh are involved in DWnt4 signaling for R axon guidance. In addition, the greater expressivity and penetrance of Dfz2 and dsh mutants suggests the involvement of other Wnt family ligands in this process. However, after examining mRNA expression of all the known D. melanogaster Wnt genes, no such genes were found acting as ventral cues in concert with DWnt4. DWnt2 is expressed just outside the lamina, but its expression is symmetric along the dorsoventral axis (Sato, 2005).

The accumulation of DWnt4 on the surface of ventral R axons implies reception of the ligand and subsequent signal activation in R cells. Consistently, Dfz2 is localized on the surface of the anterior-most R axons, but not on surrounding lamina cells. To test whether Wnt signaling autonomously regulates axon guidance, dsh homozygous clones were induced in the retina using ey-Gal4:UAS-flp18. Surprisingly, the axonal misrouting phenotype was rarely observed despite the presence of many dsh mutant clones in the retina. When the retina was entirely dsh homozygous, the same dsh allele showed axonal misrouting. It is suspected that mutant R axons project normally in the presence of surrounding wild-type axons due to axon fasciculation. If this is the case, R axons homozygous for dsh may show a misrouting phenotype in the absence of neighboring, wild-type R cells. To test this idea, GMR-hid was introduced in trans to the dsh mutant chromosome. Wild-type R cells eventually die by programmed cell death triggered by hid expression behind the furrow. In this context, a severe misrouting phenotype was observed that was ventral-to-dorsal. Notably, DWnt4 protein accumulation was observed along ventral axons that were mutant for dsh and had misprojected to the dorsal lamina. Retina-specific Dfz2 clones with GMR-hid also showing a ventral-to-dorsal misrouting phenotype. These observations are consistent with the idea that DWnt4 expressed in the lamina directly regulates R axon projections (Sato, 2005).

There are two Wnt signaling pathways: the canonical and noncanonical pathways. In the former, ß-catenin/Armadillo (Arm) and TCF/Pangolin (Pan) form a complex to activate target gene transcription. In the latter, Wnt signaling is transduced independently of Arm and Pan. Canonical Wnt signaling was manipulated using UAS-panN, which encodes a constitutive repressor form of Pan, and UAS-arm, which encodes a constitutively active form of Arm. Misrouting along the dorsoventral axis was hardly observed in either genotype. Thus it is concluded that canonical Wnt signaling plays a very minor role, if any, in dorsoventral specification of R axon guidance. The above results strongly suggest the involvement of noncanonical Wnt signaling in R cells. To confirm this idea, UAS-dshDEP, which acts as a dominant-negative mutant in noncanonical signaling, was expressed in the retina. Again a strong ventral-to-dorsal phenotype was observed (Sato, 2005).

Although the planar cell polarity (PCP) pathway is categorized as a noncanonical Wnt pathway transduced by the Fz receptor, no PCP defects were observed in DWnt4 and Dfz2 mutant retinae. In addition, the retinotopic phenotype was not observed in fz null mutant backgrounds. These results suggest that DWnt4 regulates R axon projections via a noncanonical Wnt signaling distinct from the PCP pathway. wingless (wg) is involved in the specification of the dorsal retina through the activation of iro expression. The distinct chiral forms of ommatidia in the dorsal and ventral retina reflect the dorsoventral specification of the retina and the PCP signaling. The normal iro expression and the normal ommatidial chirality suggest that axonal misroutings occur independently of the retinal dorsoventral specification in DWnt4 and Dfz2 backgrounds. Since dsh is required for PCP signaling and the specification of the dorsal retina, ommatidial chirality was disorganized and dorsal iro expression was eliminated in dsh retinae. However, the expression of Serrate (Ser), which is specific to the ventral retina in wild-type backgrounds, was not affected, suggesting that the ventral cell fate is correctly specified in dsh homozygotes. Additionally, UAS-dsh and UAS-dshDEP expression under the control of GMR-Gal4 did not affect the dorsoventral specification of the retina as visualized by iro and Ser expression. Note that GMR-Gal4 is expressed behind the morphogenetic furrow well after the dorsoventral specification at earlier stages. The data shown above suggest that dsh also regulates R axon projections independently of the dorsoventral patterning of the retina (Sato, 2005).

JNK signaling is known to act downstream of the noncanonical Wnt pathway in many developmental contexts. The involvement of JNK signaling was examined by expressing puckered (puc), which encodes a JNK phosphatase, and a dominant-negative form of JNK encoded by basket (bsk) to block JNK signaling in the retina. Defects were observed only rarely, and it was next asked whether genetic interactions exist between hemipterous (hep) encoding a JNK kinase and DWnt4 or Dfz2. In a strong hep mutant background, or in DWnt4, DWnt4 or Dfz2 heterozygous backgrounds, little or no ventral-to-dorsal misrouting was observed. However, a reduction in the dosage of DWnt4 or Dfz2 in the hep background resulted in a marked increase in the ventral-to-dorsal phenotype. These findings provide some support for the idea that JNK signaling is involved in the DWnt4/Dfz2 pathway in retinal axon guidance. Since iro expression and ommatidial chirality were normal in retinae expressing the dominant-negative form of bsk and in hep hemizygotes in combination with DWnt4/+ and Dfz2/+, the misrouting of ventral axons observed in the brain mutant for JNK signaling appears to be caused by a failure in axon guidance and independent of the dorsoventral cell specification or PCP signaling in the retina. Note that mutations in JNK pathway components alone have no PCP phenotype (Sato, 2005).

iro is thought to be the dorsal cue in the retina. The dorsal axons project to the ventral lamina in the absence of iro, perhaps because dorsal axons are attracted by ventral cues in the lamina, such as DWnt4. When iro mutant clones were generated under the control of ey-flp, dorsal axons projected to the ventral lamina in 32.4% of them, and ectopic accumulations were observed of DWnt4 on the surface of the dorsal axons misprojecting ventrally. Since Dfz2 was expressed in the dorsal axons, iro may attenuate the competence of Dfz2 in the dorsal axons to respond to DWnt4. If this is the case, simultaneous removal of Dfz2 in iro clones should suppress the iro phenotype, which was indeed observed. In iro:Dfz2 double mutant clones, 'dorsal-to-ventral' misroutings were observed in 3.5% of the cases, and the class III phenotype was no longer observed. Instead, abnormal bundles of dorsal axons were found in iro:Dfz2 clones. This might be because iro:Dfz2 axons do not respond to either dorsal or ventral cues in the lamina. Indeed, no DWnt4 accumulation was found in those abnormal bundles found in iro:Dfz2 clones. The absence of iro expression in differentiated R cells behind the morphogenetic furrow suggests indirect modulation of Dfz2-dependent Wnt signaling by iro (Sato, 2005).

It was hypothesized that three events are required for retinotopic mapping along the dorsoventral axis in D. melanogaster: (1) dorsoventral identity is specified by selector genes expressed in the retina; (2) dorsoventral identity is specified by selector genes in the lamina; (3) guidance molecules recruit R axons to their correct targets in the lamina. The results nicely fit the hypothesis. iro expressed in the dorsal retina specifies the dorsal axon identity, and DWnt4 expressed in the ventral lamina recruits ventral axon projections. The restricted expression of DWnt4 to the ventral lamina suggests there could be unidentified dorsoventral selectors in the lamina (Sato, 2005).

CKIε/discs overgrown promotes both Wnt-Fz/β-catenin and Fz/PCP signaling in Drosophila

The related Wnt-Frizzled(Fz)/β-catenin and Fz/planar cell polarity (PCP) pathways are essential for the regulation of numerous developmental processes and are deregulated in many human diseases. Both pathways require members of the Dishevelled (Dsh or Dvl) family of cytoplasmic factors for signal transduction downstream of the Fz receptors. Dsh family members have been studied extensively, but their activation and regulation remains largely unknown. In particular, very little is known about how Dsh differentially signals to the two pathways. Recent work in cell culture has suggested that phosphorylation of Dsh by Casein Kinase I ε may act as a molecular 'switch', promoting Wnt/β-catenin while inhibiting Fz/PCP signaling (Cong, 2004). This study demonstrates in vivo in Drosophila through a series of loss-of-function and coexpression assays that CKIε acts positively for signaling in both pathways, rather than as a switch. The data suggest that the kinase activity of CKIε is required for peak levels of Wnt/β-catenin signaling. In contrast, CKIε is a mandatory signaling factor in the Fz/PCP pathway, possibly through a kinase-independent mechanism. Furthermore, the primary kinase target residue of CKIε on Dsh has been identified. Thus, the data suggest that CKIε modulates Wnt/β-catenin and Fz/PCP signaling pathways via kinase-dependent and -independent mechanisms (Klein, 2006).

Cell-culture assays have suggested that CKIε positively regulates Wnt-Fz/β-catenin signaling and that it antagonizes Fz/PCP signaling. To confirm that CKIε is required for Wnt/β-catenin signaling in vivo, loss of function (LOF) alleles of discs overgrown (dco/doubletime, the Drosophila CKIε gene) were examined for phenotypes indicative of Wingless signaling defects. Consistent with previous data demonstrating a requirement for dco in disc growth, strong dco/CKIε alleles (dcodbt-P) gave clones too small to analyze for disruption of Wg target-gene expression. Thus dco/CKIε mutant clones were generated with the Minute technique. In these, expression of the Wg target gene senseless (sens) was lost in mutant cells. Accordingly, dco/CKIε adult wing clones show loss of margin bristles and/or parts of the wing margin, consistent with a positive requirement for dco in Wg signaling. However, Wg targets that require lower levels of Wg signaling (e.g., Dll) were not affected, indicating that dco/CKIε is only required for peak Wg signaling levels. Consistent with this finding, genome-wide RNAi screens for Wg signaling components identified dco/CKIε as a factor required for peak levels of β-catenin reporter expression (Klein, 2006).

The Fz/PCP pathway can easily be studied in Drosophila. The precise ommatidial arrangement in the eye and the orientation of hairs on the wing depend on correct Fz/PCP input. The two best-studied PCP signaling factors are Fz and Dsh, which also act in canonical Wnt/β-catenin signaling. To study the role of dco/CKIε in PCP, various heteroallelic dco combinations were analyzed. In several of these (e.g., dcodbt-P/dcodbt-AR), typical PCP defects were seen. Clones of a strong dco/CKIε allele show classical PCP phenotypes in the wing, with reoriented wing hairs, and in the eye, with ommatidal chirality and orientation defects (dco/CKIε clones also displayed ommatidia with photoreceptor loss, likely as a result of the cell viability requirement of Dco/CKIε (Klein, 2006).

In vivo LOF analyses allow led to the conclusion that dco/CKIε is required for peak levels of Wg signaling, but does not appear to be a mandatory Wnt/β-catenin signaling component. In addition, the data identify dco/CKIε as a new factor required in Fz/PCP signaling (Klein, 2006).

To dissect the function of dco/CKIε in Wg and PCP signaling, the effects of overexpressing CKIε were examined. In the eye, dco/CKIε was overexpressed with sevenless(sev)-Gal4 (in R3/R4 cells that are critical for PCP establishment, which causes PCP phenotypes. In the wing, decapentaplegic(dpp)-Gal4-driven expression in a proximal-distal stripe along the A-P compartment boundary can be used to identify positive and negative effects on both Wg and PCP signaling. dpp>CKIε displayed a mild but consistent PCP defect, a characteristic hair swirl near the intersection of the dpp stripe and wing margin. It also caused a small number of extra margin bristles, typical of increased Wg signaling (Klein, 2006).

Given that these phenotypes were mild, attempts were made to enhance them. Because CKIε requires an activating dephosphorylation event, which can be induced by Fz signaling, the effect was tested of coexpressing CKIε with either Fz or Fz2 in the dpp stripe. Overexpression of Fz (dpp>Fz) causes a characteristic reorientation of wing hairs that point away from the expression domain, but does not induce ectopic margin bristles. Strikingly, coexpression of Fz and CKIε leads to a dramatic synergy, with enhanced PCP defects and a large number of extra margin bristles in the expression domain, indicative of a positive CKIε role in both Wg and PCP signaling. Consistently, expression of Fz or CKIε alone is not sufficient to induce visible changes in Wg target-gene expression, but coexpression of CKIε and Fz cell-autonomously induces ectopic Senseless-positive cells (Klein, 2006).

dpp>Fz2 induces many ectopic margin bristles near the intersection of dpp expression and the wing margin (at 25°C;). Because dpp-driven coexpression of Fz2 and CKIε at 25°C is lethal, the dpp>Fz2, CKIε coexpression was examined in flies raised at 18°C (allowing for weaker Gal4-driven expression). dpp>Fz2 at 18°C induces only few margin bristles, a phenotype that is enhanced upon coexpression with CKIε; dpp>Fz2, CKIε coexpression also induces PCP-like hair swirls, although this effect could be indirect given that this combination induces wing-margin-like vein tissue, which could repolarize parts of the wing blade). Taken together with the LOF analyses, these data demonstrate a positive, synergistic role for Dco/CKIε not only in Wg signaling, but also in Fz/PCP signaling (Klein, 2006).

To confirm that dco/CKIε acts positively for both pathways in vivo, genetic interactions of dco with known Wg and PCP signaling factors were examined in the eye. Overexpression of Fz (sevFz) causes strong PCP defects, a phenotype that is significantly suppressed by the removal of a single copy of dco. Overexpression of Strabismus (Stbm; also known as Van Gogh or Vang), an antagonist of Fz/PCP signaling, with sevGal4 (sevStbm) causes mild PCP defects, which are enhanced by removal of a copy of dco, again supporting a positive role for dco in Fz/PCP signaling (Klein, 2006).

The effect of removing a copy of dco in the context of Dsh overexpression (sev>Dsh). sev>Dsh causes both PCP defects and loss of photoreceptors, with the latter resembling the effect of sev>Wg. Removing a copy of dco strongly suppresses the loss-of-photoreceptor phenotype, supporting a positive role for dco for peak Wg signaling. Assessing the effect of the removal of a copy of dco on the PCP in sev>Dsh is not possible, because the loss-of-photoreceptor phenotype masks PCP defects in many ommatidia (Klein, 2006).

In addition, genetic analyses was performed with overexpressed CKIε. sev>CKIε eyes exhibit mild PCP defects and a small percentage of ommatidia with a change in photoreceptor number. Removal of a single copy of dsh suppressed the PCP and photoreceptor number defects, consistent with Dco/CKIε acting positively together with Dsh to elicit both phenotypes. Consistent with the coexpression results in the wing, these interactions support a positive role for dco/CKIε in both Wg and Fz/PCP signaling (Klein, 2006).

On the basis of the genetic interaction data with Dsh and cell-culture and in vitro kinase assays that have shown that CKIε can bind and phosphorylate Dsh, Dsh appears to be a likely phosphorylation target of CKIε. The actual site of phosphorylation on Dsh, however, has not been mapped. To narrow down the region of phosphorylation, a series of GST-Dsh constructs were generated covering all domains. In vitro kinase assays using CKIε showed specific phosphorylation of all Dsh isoforms containing the basic region and PDZ domain (GST-Dsh, GST-bPDZ, GST-ΔC), but not of those containing just the PDZ (GST-PDZ) or other parts of Dsh (Klein, 2006).

To determine the exact site of phosphorylation, the region N terminal to the PDZ domain was analzyed for conserved CKIε consensus sites, and a likely motif was identified. In this motif, S236 is predicted to be the first serine residue phosphorylated by CKIε. When this serine is mutated to alanine (within GST-bPDZ), CKIε is no longer able to phosphorylate Dsh. Interestingly, this residue is in a short region of Dsh that is important for Dsh phosphorylation, activity, and signal specificity (Klein, 2006).

Because previous studies have suggested that CKIε kinase activity is required for its ability to transduce Wnt/β-catenin signals, a kinase-dead isoform of CKIε, Dco/CKIεD132N (which affects the ATP binding site) was tested in vivo. This mutant was unable to transduce Wg signals, but, surprisingly, it induced strong GOF PCP phenotypes in both the eye and wing. sev>CKIεD132N eyes display clean PCP phenotypes. Wings from dpp>CKIεD132N flies also display PCP defects, with wing hairs that point away from the expression domain, demonstrating a GOF Fz/PCP phenotype (Klein, 2006).

To investigate the potential for a kinase-independent role of Dco/CKIε, Fz and CKIεD132N (with dppGAL4) were coexpressed. In contrast to coexpression of Fz and wild-type CKIε, CKIεD132N did not display extra margin bristles, indicating that kinase activity is important for Wg signaling. Similarly, in contrast to coexpression of Fz2 and wild-type CKIε, dpp>Fz2, CKIεD132N (at 18°C) did not cause an increase in margin bristles as compared to dpp>Fz2 alone. At 25°C, dpp>Fz2, CKIεD132N was not lethal, as compared to dpp>Fz2 together wild-type CKIε (Klein, 2006).

In summary, these data demonstrate a requirement for the Dco/CKIε kinase activity in Wg signaling, with CKIεD132N often acting as a dominant negative, but suggest that kinase activity is not required for Dco/CKIε activity during Fz/PCP signaling (Klein, 2006).

The CKIε requirement for Dsh phosphorylation was examined. In an unbiased Drosophila S2-cell-based screen for kinases that are required for the PCP-signaling-associated Dsh phosphorylation, dco/CKIε was identified as a kinase required in this context. Strikingly, the kinase-dead CKIε isoform, CKIεD132N, promotes PCP-signaling-associated Dsh phosphorylation as much as wild-type CKIε. These data suggest that although the presence of CKIε protein is important for this phosphorylation event, its kinase activity is not required, consistent with the in vivo expression data with Dco/CKIεD132N (Klein, 2006).

A possible caveat to these data is that, rather than acting on downstream targets in a kinase-independent manner, CKIεD132N could act to titrate away factors that inhibit the endogenous CKIε. To test this, the effect of CKIεD132N on the phosphorylation of Dsh was examined in S2 cells in the absence of endogenous CKIε. These data show that endogenous CKIε is not mediating the CKIεD132N effect, and thus CKIε is likely to act in a kinase-independent manner in the PCP context (Klein, 2006).

The in vivo data support a positive requirement of dco/CKIε for peak levels of Wnt/β-catenin signaling and a strict requirement in the Fz/PCP pathway. Whereas the kinase activity of CKIε is required for Wnt/β-catenin signaling, the analysis suggests that it is not required for Fz/PCP signaling. These findings differ from the proposed inhibitory effect of CKIε on Fz/PCP signaling in cell culture. It is possible that the PCP readout in cell culture, namely activation of JNK, reflects only a subset of PCP activities of Dsh, not representing an accurate measure of overall PCP activity. Alternatively, CKIε could act as a constitutively active kinase when expressed in cell culture, whereas its activity is regulated in vivo. Thus, for Fz/PCP signaling in vivo, the primary role for CKIε may not be as an active kinase, but rather as a stabilizer of a complex that allows for PCP-specific Dsh phosphorylation. This is supported by the data that the kinase activity of CKIε is not required for Fz/PCP signaling and that kinase-dead CKIε still stimulates phosphorylation of Dsh (Klein, 2006).

CKIε phosphorylates a specific residue in Dsh, S236, in a short region known to be phosphorylated by multiple kinases and suggested to be important in the regulation of Dsh signal specificity. This supports the proposed possibility of in vivo competitive phosphorylation as a mechanism for Dsh regulation. The region upstream of the PDZ domain appears to act as a docking site for Dsh binding proteins. Differential phosphorylation of this region could alter the binding properties of Dsh. In support of this possibility, protein-protein interaction studies have identified a large number of proteins that bind to the Dsh PDZ domain. It is unlikely that all these interactions occur at the same time, and phosphorylation is a potential mechanism to regulate this. Further experiments are needed to finely map the many potential phosphorylation target residues and the corresponding kinases and demonstrate their in vivo significance (Klein, 2006).

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).

frizzled2: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | References

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