frizzled2

DEVELOPMENTAL BIOLOGY

Embryonic

At 2 hours post-fertilization, embryos have low levels of FZ2 presumably of maternal origin. Expression is detected at stage 6 where it is found in all cells between 15% and 70% egg length, including invaginating cells of the ventral furrow. An emerging stripe pattern is evident by early stage 8, and by stage 10 fz2 expression is seen in 15 stripes in the presumptive head and tail regions, in the posterior midgut primordium (around the stomodeum), in a subset of cells at the site of anterior midgut invagination, in the procephalic lobe, and transiently around the primordia of the tracheal pits. Beginning at stage 12, fz2 expression declines in the epidermis and increases in the midgut and visceral mesoderm. Beginning at stage 9, fz2 expression is also seen in the developing CNS. By stage 17, fz2 expression is limited to the CNS, hindgut and dorsal vessel (Bhanot, 1996).

The Drosophila Frizzled (Fz) and Frizzled2 (DFz2) proteins function as receptors for Wingless (Wg) in tissue culture cells. While previous results indicate that loss of function for fz results in tissue polarity defects, the loss-of-function effects of Dfz2 are not known. The requirements of fz and Dfz2 during neurogenesis have now been examined. Both Fz and DFz2 function in Wg signaling, and loss of either of the two affects the same subset of neuroblasts as those affected by loss of wg. While these defects are partially penetrant in embryos lacking either fz or Dfz2, the penetrance is significantly enhanced in embryos lacking both. Since the penetrance of the CNS phenotypes is not complete in double mutants, additional components that allow some degree of Wg signaling must exist in vivo (Bhat, 1998).

In the ventral nerve cord of the Drosophila embryo, wg is expressed in row 5 cells within a segment. It is nonautonomously required for the formation and specification of row 4 neuroblasts as well as for the formation of a few neuroblasts in row 5 and most neuroblasts in row 6. Among those neuroblasts that are affected in wg mutants, NB4-2, a row 4 neuroblast that gives rise to the RP2/sib lineage, has been one of the most studied neuroblasts in the CNS. The RP2 and its sibling cell are formed from the first GMC of NB4-2 (this GMC is known as GMC-1 or GMC4-2a). In this lineage, wg is required for both the formation and specification of this neuroblast. The elimination of maternal and zygotic fz gene products causes loss of NB4-2->GMC-1->RP2/sib lineage and failure in the formation of row 6 neuroblasts in the ventral nerve cord of the Drosophila embryo (Bhat, 1998).

It has been argued that Fz might not function in the Wg signaling during wing and eye development, and that in these tissues, while DFz2 functions to receive the Wg signal, Fz receives the signal from some other Wnt. If DFz2 is solely responsible for receiving the Wg signal in the CNS, elimination of Dfz2 should have eliminated the NB4-2 lineage in a manner similar to loss of function for wg. Given that the wg-like CNS defects in Dfz2 embryos are only partially penetrant, as is the case with fz mutants, the simplest explanation is that there is a genetic redundancy between fz and Dfz2 and both function in the transduction of the Wg signal. The observation that the penetrance of the RP2/sib lineage phenotype is significantly enhanced in embryos lacking both fz and Dfz2 activities certainly reinforces this view. These results are also consistent with the observation that during epidermal patterning, while the intracellular localization of Arm is not significantly affected in embryos missing Dfz2, it is nearly lost in embryos missing both the activities (Bhat, 1998 and references).

Repulsive Wnt signaling determines synaptic target specificity

How synaptic specificity is molecularly coded in target cells is a long-standing question in neuroscience. Whereas essential roles of several target-derived attractive cues have been shown, less is known about the role of repulsion by nontarget cells. Single-cell microarray analysis was conducted of two neighboring muscles (M12 and M13) in Drosophila, that are innervated by distinct motor neurons, by directly isolating them from dissected embryos. A number of potential target cues that are differentially expressed between the two muscles, including M13-enriched Wnt4, were identifed. When the functions of Wnt4, or putative receptors Frizzled 2 and Derailed-2 or Dishevelled were inhibited, motor neurons that normally innervate M12 (MN12s) formed smaller synapses on M12 but instead formed ectopic nerve endings on M13. Conversely, ectopic expression of Wnt4 in M12 inhibits synapse formation by MN12s. These results suggest that Wnt4, via Frizzled 2, Derailed-2, and Dishevelled, generates target specificity by preventing synapse formation on a nontarget muscle. Ectopic expression of five other M13-enriched genes, including beat-IIIc and Glutactin, also inhibits synapse formation by MN12s. These results demonstrate an important role for local repulsion in regulating cell-to-cell target specificity (Inaki, 2007).

In each abdominal hemisegment of embryos and larvae of Drosophila, 37 motor neurons innervate 30 muscles in a highly stereotypic manner. Several candidate target recognition molecules that are expressed in different subsets of muscles have been identified, including Connectin, Fasciclin3, Semaphorin2, NetrinB, Toll, and Capricious. Genetic analysis of these molecules suggests that multiple cues expressed on the target muscles determine the target specificity in a combinatorial and overlapping manner. However, this issue has not been fully addressed; previous studies characterized only a small number of molecules that are expressed in different muscles (Inaki, 2007).

Toward more comprehensive understanding of the molecular basis of target specificity, genome-wide expression profiling was conducted of genes specifically expressed in two neighboring ventral muscles, M12 and M13, which are innervated by distinct motor neurons. M12 is innervated by RP5 and V (collectively called MN12s), whereas M13 is innervated by RP1 and RP4. Because these two muscles show similar morphology, run in parallel, and insert at adjacent muscle insertion sites, they are likely to share most functional characteristics other than neural connectivity. It was therefore reasoned that their subtractive expression profiling might lead to the identification of genes encoding target specificity (Inaki, 2007).

Individual M12s and M13s were collected from abdominal segments of dissected embryos during the stage of motor neuron targeting by aspiration with micropipettes. RNA was extracted from the samples of muscles, each containing 200 cells, and the RNA was amplified through two rounds of linear amplification. Affymetrix Drosophila genome chips were then hybridized with multiple samples of cRNA that was isolated and amplified in independent experiments. Comparing gene-expression profiles of M12 and M13, genes were selected that displayed differential expression consistently in two sets of hybridization experiments. This yielded a list of 96 genes predicted to be preferentially expressed in M12 and 77 genes predicted to be preferentially expressed in M13 (hereafter called M12 and M13 candidate genes) (Inaki, 2007).

A focus was placed on genes that encode putative membrane or secreted proteins with potential roles in target recognition. The predicted differential expression of this class of genes was verified by quantitative real-time RT-PCR analysis (qPCR). Twenty-five of the 34 genes examined gave concordant results with the array data, displaying at least 1.5-fold differential expression between the two muscles. RNA in situ hybridization further confirmed the preferential expression of knockout (ko) in M12, and of Wnt4, beat-IIIc, Sulf1, and CG6867 in M13. These results show specific expression of transcripts that encode a variety of candidate target-recognition molecules in these two muscles (Inaki, 2007).

The role of a prominent candidate gene, Wnt4, which encodes the secreted protein Wnt4 of the WNT family, was genetically analyzed. Wnt4 has been shown to function as an attractive guidance molecule that regulates dorsoventral specificity of photoreceptor-cell projection. Wnt4 is also known to regulate cell movement in the ovary. Wnt4 is expressed in ventral muscles M13 and M26. Much weaker expression is seen in other muscles, including M12. Therefore its function was studied in M12 and M13 targeting. In Wnt4 loss-of-function (LOF) mutant embryos, muscles and major motor nerves showed largely normal development. Specification of the Wnt4-expressing muscle, M13, also appears to be normal because the expression pattern of another M13-enriched gene, Toll, was indistinguishable from that of control. However, the innervation pattern of M12 and M13 was specifically altered. Staining with the anti-Fasciclin II monoclonal antibody 1D4, which visualizes all motor axons, revealed that the axon terminals on M12 were greatly reduced. Remarkably, this reduction of the synaptic endings on M12 was accompanied by the expansion of endings on M13. The number of Bruchpilot-positive putative active zones was also decreased in M12 and increased in M13 in Wnt4 mutants compared to control. These results are consistent with the idea that in Wnt4 mutants, MN12s formed smaller synaptic endings on M12 and instead arborized inappropriately on M13 (Inaki, 2007).

To determine whether the expansion of the M13 terminals in Wnt4 mutants is caused by the formation of ectopic arborization by MN12s, these neurons were specifically labeled with diI. DiI was applied on the M12 nerve endings and RP5 and/or V neurons, whose identities were verified by their axon trajectories and cell-body position, were retrogradely labeled. In late stage 16 wild-type embryos, RP5 neurons retained putative transient contacts on M13. In Wnt4 mutants, the length of the arborizations on M13 formed by RP5 neurons was significantly increased. Wild-type V neurons arborized exclusively on M12 and not along M13. In Wnt4 mutants, V neurons occasionally formed ectopic arborizations on M13, although the frequency is too low to be statistically significant. These results indicate that in Wnt4 mutants, MN12s formed larger or ectopic endings on M13, and further argue that Wnt4 is required to repel and/or restrict inappropriate arborization on M13 formed by MN12s (Inaki, 2007).

The LOF phenotypes suggest that Wnt4 functions in M13 to prevent synapse formation by MN12s. If so, ectopic Wnt4 expression in M12 may inhibit synapse formation by these neurons. To address this possibility, the Gal4-UAS system was used to induce forced expression of Wnt4 in muscles. Strong expression of Wnt4 was induced in all muscles by using the Gal4 driver 24B or E54. In this situation, MN12s often stalled at the edge of M12 and formed much smaller endings. Misexpression of Wnt4 did not cause targeting or pathfinding defects in other regions of the neuromusculature. Wnt4 therefore specifically inhibits synapse formation by MN12s. Next, expression of Wnt4 was induced only in M12 by using a more specific driver, 5053A-Gal4. Because 5053A-Gal4 induces a much higher level of Wnt4 expression in M12 than that of endogenous Wnt4 in M13, this experimental manipulation reverses the relative levels of Wnt4 expression in these muscles. In 5053A-Wnt4 embryos, the arborizations on M12 were greatly reduced in size, as was observed in 24B-Wnt4 embryos. In addition, unlike in 24B-Wnt4 embryos, the arborizations on M13 were enlarged. These results are consistent with the idea that the M12 motor neurons determine target specificity by detecting the relative levels of Wnt4 expressed by these two muscles. Taken together, LOF and gain-of-function (GOF) analyses indicate that differential expression of Wnt4 in M12 and M13 is critical for their targeting (Inaki, 2007).

Which receptor and signaling pathway in motor neurons mediate muscle-derived Wnt4 repulsion? Wnts bind to Frizzled (Fz) family receptors, and the receptor activation in turn activates the intracellular protein Dishevelled (Dsh). Wnt family proteins also bind to other classes of receptors, including Derailed/Ryk family members, which have been shown to transduce Wnt-mediated attraction or repulsion during axon guidance. Previous studies in the visual system and in the ovary have shown that Fz2 and Dsh are involved in Wnt4 signaling. Therefore whether Fz2 and Dsh are required for proper targeting in the neuromuscular system was investigated. Also the possible involvement of Derailed family members was also studied. When the function of Fz2 or Dsh was inhibited by expressing a dominant-negative form of these molecules, the same defects were observed in the targeting of M12 and M13 as observed in Wnt4 mutants. Similarly, LOF of derailed-2 (drl-2) causes the highly specific phenotype in the targeting of M12 and M13. These results suggest that Fz2, Dsh, and Drl-2 are involved in the signaling of Wnt4 repulsion in motor neurons. Whereas Fz2 is expressed in most or all neurons, drl-2 is expressed in subsets of neurons in the CNS. The specific expression of drl-2 may explain why Wnt4 is repulsive to only subsets of motor neurons (Inaki, 2007).

Systematic GOF analyses of the other candidate genes identified by the expression profiling was performed, and it was found that pan-muscle expression of five other genes, beat-IIIc, Glt, Lsp2, Sulf1, and CG6867, caused a reduction of MN12 nerve terminals similar to that seen when Wnt4 was misexpressed. All of these genes are normally expressed in M13 and thus, like Wnt4, may function as repulsive cues that inhibit synapse formation by MN12s. As in the case of Wnt4, misexpression of these five genes did not cause targeting or pathfinding defects in other regions of the neuromusculature. These results suggest a repulsive role for beat-IIIc, Glt, Lsp2, Sulf1, and CG6867 in specific aspects of target selection (Inaki, 2007).

Several molecules have previously been shown to function as attractive target cues that determine target specificity, including Netrins and Capricious in Drosophila, SYG-1 in C. elegans, and Sidekicks in vertebrates. However, little is known about the role of repulsion during target selection. During axon pathfinding, repulsive cues presented by surrounding tissues restrict the direction of the axons by deflecting or arresting their growth. Axons can also be guided by gradients of repulsive cues. Does repulsion also limit the choice of distinct target cells and thus mediate cell-to-cell specificity? Previous GOF analyses of semaphorin2 and Toll in Drosophila showed that they can inhibit synapse formation of specific motor neurons. However, whether such inhibition is essential for the selection of target cells is unknown. This study shows that Wnt4 is required for target recognition by MN12s. In wild-type, upon entering the M12/M13 target region, MN12s selectively innervate M12 with only a small putative transient contact on M13. In Wnt4 mutants, the target preference of these neurons is shifted to M13. This suggests that Wnt4 normally prevents MN12s from making large synapses on M13, and this Wnt4-mediated repulsion on M13 is required to lead these neurons to an alternative target, M12. Data from GOF analyses further support the notion that differential expression of Wnt4 in these two muscles is critical for target selection by MN12s. These results provide strong evidence that local repulsion plays a major role in target specificity (Inaki, 2007).

Microarray analysis identified a number of putative cell-surface or secreted proteins, in addition to Wnt4, that were differentially expressed between the two muscles. Furthermore, GOF analysis suggested that at least five of them may function, like Wnt4, as repulsive cues on M13. Some of them encode proteins with domains implicated in axon guidance and synapse formation; Beat-IIIc belongs to the Beat family of proteins with immunoglobulin motifs, and Glt belongs to a family of cell-surface proteins with cholinesterase domains. Identification of such a large number of potential cues in a single target cell is unprecedented and provides a valuable opportunity to study the mechanisms of target recognition. Future genetic analysis of these molecules, alone and in combination, may more clearly elucidate the mechanism of how these multiple target cues coordinate to determine target specificity (Inaki, 2007).

Larval

At vertebrate neuromuscular junctions (NMJs), Agrin plays pivotal roles in synapse development, but molecules that activate synapse formation at central synapses are largely unknown. Members of the Wnt family are well established as morphogens, yet recently they have also been implicated in synapse maturation. Wingless is essential for synapse development. Wg and its receptor are expressed at glutamatergic NMJs, and Wg is secreted by synaptic boutons. Loss of Wg leads to dramatic reductions in target-dependent synapse formation, and new boutons either fail to develop active zones and postsynaptic specializations or these are strikingly aberrant. It is suggested that Wg signals the coordinated development of pre- and post-synaptic compartments (Packard, 2002).

Wg is likely to interact with DFz2, which is clustered around synaptic boutons, either postsynaptically or both pre- and postsynaptically. Studies with shi mutants also suggest that secreted Wg is likely to be endocytosed by muscles. Under low Wg levels, synapse formation is severely impaired, with many boutons lacking active zones and post-synaptic structures. In those mutant boutons that develop active zones, release sites are abnormally shaped and the postsynaptic apparatus is markedly abnormal. It is suggested that Wg provides an essential signal for active zone development, and that this signal is critical for proper development of the postsynaptic apparatus. Two models are proposed by which Wg may signal synaptic development. Secretion of Wg and its interaction with pre- and post-synaptic DFz2 may initiate a cascade of events that signals both pre- and postsynaptic differentiation. Alternatively, Wg secretion initiates a postsynaptic response, which elicits a retrograde signal required for proper formation of active zones and new synaptic boutons. It is further proposed that endocytosis of Wg by muscles might regulate Wg concentration at the synapse. The presence of DFz2 in motorneurons suggests that the receptor is present pre- as well as post-synaptically and lends support to the first model (Packard, 2002).

A subset of boutons in wg mutants appears to have normal active zones, but the morphology of the postsynaptic membrane directly juxtaposed to active zones is dramatically altered. Strikingly, synaptic proteins, such as Dlg and DGluRIIA, are abnormally localized in wg mutants, and this phenotype can be replicated by mutations in porc or by postsynaptic overexpression of either DFz2 or DFz2DN. The aberrant distribution of DGluRIIA and Dlg observed in wg mutants may be the consequence of grossly abnormal postsynaptic structure. Alternatively, the diffuse appearance of glutamate receptors may point to a direct role of Wg in glutamate receptor clustering (Packard, 2002).

Taken together, these results are consistent with a model by which Wg is required for the formation of active zones, and that active zone formation is essential for proper development of the postsynaptic apparatus. In this context, Wg may serve a function similar to Agrin at the vertebrate NMJ, in which an extracellular matrix associated protein secreted by the presynaptic cell signals postsynaptic differentiation. Remarkably, mutant mice lacking Agrin have widespread abnormalities in the presynaptic terminal, and blocking Agrin function by antibodies blocks presynaptic differentiation, providing compelling evidence that Agrin is also required for presynaptic development. An Agrin homolog does not appear to be present in the Drosophila genome, suggesting that a different set of secreted proteins, such as Wg, may subserve Agrin's function (Packard, 2002).

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