Presynaptic local signaling by a canonical wingless pathway regulates development of the Drosophila neuromuscular junction

Abstract

Wnt/wingless signaling contributes to the development of neuronal synapses, including the Drosophila neuromuscular junction. Loss of wg (wingless) function alters the number and structure of boutons at this model synapse. Examining Wnt/wingless signaling mechanisms, we find that a distinct pathway operates presynaptically in the motoneuron and can account for many of the effects of wingless at this synapse. This pathway includes the canonical elements arrow/LRP (low-density lipoprotein receptor-related protein), dishevelled, and the glycogen synthase kinase shaggy (GSK3) and regulates the formation of microtubule loops within synaptic boutons as well as the number of synaptic boutons. This pathway, however, appears to be independent of beta-catenin signaling and the transcriptional regulation that is most frequently downstream of these components. Instead, inhibition of shaggy is likely to act locally. This pathway thus provides a parallel mechanism to the postsynaptic activation of frizzled receptors and indicates that synaptic development results from the bidirectional influence of wingless on both presynaptic and postsynaptic structures via distinct intracellular pathways.

Figure 1.

Canonical wg signaling components, but not β-catenin/armadillo, localize presynaptically and postsynaptically at the neuromuscular junction. Synapses on muscles 13/12 were stained with anti-HRP (top row, white; bottom row, blue). Threefold magnifications of single boutons (marked with boxes) are shown as insets. a, b, Wild-type synapses stained with anti-Dlg (red) and either anti-wg (a, green) or anti-arr (b, green). Wg and arrow immunoreactivity show punctate distributions in the muscle and synaptic enrichment. Arrow is also detectable in the axon and is quite prominent presynaptically. c, A synapse expressing GFP-tagged dsh under its endogenous promoter (green) and stained with anti-Dlg (red). Dsh is enriched at the synapse and expressed in both the muscle and nerve. d, A wild-type synapse stained with anti-DLG (red) and anti-GSK3/shaggy (green). Sgg is enriched at axons and boutons and is diffusely present in the muscle. e, Anti-armadillo immunostaining (green) localizes to glia in the segmental nerve, marked by expression of UAS mCD8-GFP under control of the glia-specific driver repo-GAL4 (red). Armadillo is also detected in puncta in the muscle, but is generally absent from muscle nuclei or synaptic regions. Scale bar: 15 μm; insets, 5 μm.

Figure 2.

wg and arrow mutants exhibit similar NMJ defects, whereas overexpression of wg has the opposite phenotype. a–d, Synapses of segment A3 on muscles 7/6 stained with anti-HRP (white). Fourfold magnifications of single boutons from boxed areas are shown in insets. a, Wild type. b, UAS-wg drives overexpression of wg in both neurons and muscle under the control of elav-GAL4 and MHC-GAL4. c, wg^null/wg^ts mutant (mut). d, arr²/arr^K08131. e–g, Quantification of bouton number (e), number of satellites (f), and number of enlarged boutons >5 μm (g; arrows in b–d) in segment A3 for the genotypes in a–d. Compared with wild-type synapses, overexpression of wg (b) significantly increased bouton numbers and satellite boutons (arrowheads), but the number of boutons >5 μm (arrows) remained unaffected. Both wg and arr mutants have significantly reduced numbers of boutons and more oversized and irregularly shaped boutons, whereas the number of satellite boutons remained normal. n = 17 larvae (31 A3 NMJs) for wild types, 19 larvae (34 A3 NMJs) for wg-overexpressing animals, 16 larvae (30 A3 NMJs) for wg mutants, and 23 larvae (43 A3 NMJs) for arr mutants. *p < 0.05 relative to wild type. Scale bar: 20 μm; insets, 5 μm.

Figure 3.

Abnormal microtubule structures in enlarged arr boutons. a–p, Wild-type (a–h) and arr²/arr^K08131 mutant (mut; i–p) synapses immunostained with anti-HRP (green) and for presynaptic and postsynaptic proteins as follows: futsch (magenta), mito-GFP (magenta), GluRIIC (red), and bruchpilot (blue). Individual single channels are also shown in gray-scale below (e–h, m–p). Anti-futsch immunoreactivity (a, d, i, l, magenta; e, h, m, p, white) reveals the altered microtubule structure of arr mutant boutons. Although microtubules appear normally bundled within the axons, in the enlarged (>5 μm) boutons of arr mutants, diffuse punctuate labeling replaces the normal bundled and looped structures. Neuronal mitochondria, labeled with GFP (b, j, magenta; f, n, white), are present at arr mutant synapses, but scarce in those mutant boutons that are abnormally large. c, g, k, o, Monoclonal antibody bruchpilot (blue) and anti-GluRIIC (red) indicate that active zones and postsynaptic receptor clusters are present at mutant synapses, with a distribution similar to wild type. Scale bars: o (for a–c, e–g, i–k, m–o), p (for d, h, l, p), 5 μm.

Figure 4.

Neuronally expressed arrow rescues the NMJ phenotypes of arr. Synapses of segment A3, muscles 7/6, were costained with anti-HRP (white; green in insets) and anti-futsch (magenta). a, Wild type. b, arr²/arr^K08131 mutant (mut). c, Rescue (resc) of arr phenotype by neuronal expression of arrow in genotype arr²/arr^K08131;elan-GAL4/UAS-arr. d, Partial rescue of arr phenotype by muscle expression of arrow in genotype arr²/arr^K08131;MHC-GAL4/UAS-arr. Representative boutons, fourfold enlarged, are shown in insets to illustrate microtubule loops and their disruption by arr. e–i, Quantification of bouton number (e), number of satellites (f), boutons with a diameter >5 μm (g), number of loops (h), and boutons with unbundled futsch (i) in the genotypes shown in a–d. Expression of arrow in neurons fully rescued the arr phenotype for each of these parameters. Expression of arrow in muscles restored normal numbers of boutons, but some oversized boutons remained, and the microtubule structure was not completely restored. n = 12 wild-type larvae (21 NMJs), 15 arr larvae (28 NMJs), 16 neuronal rescue larvae (31 NMJs), and 18 muscle rescue larvae (34 NMJs). *Significant differences (p < 0.05) from wild-type controls. Scale bar: 20 μm; insets, 5 μm.

Figure 5.

Microtubule-loop formation in boutons requires canonical wg signaling components presynaptically, but is independent of armadillo and pangolin. a–h, Muscles 7/6 synapses stained with anti-HRP (green) and anti-futsch (magenta). Futsch-immunoreactive microtubule loops (arrowheads) and boutons with unbundled futsch (arrows) are marked. a, Wild type. b, UAS-dsh^DIX;MHC-GAL4. Normal synaptic architecture persists when the dominant-negative construct dsh^DIX is expressed in the muscles. c, UAS-dsh^DIX;elav-GAL4. Neuronal expression of dsh^DIX causes an arr-like phenotype with enlarged boutons and fewer microtubule loops. d, UAS-sgg^A82T;elav-GAL4. Neuronal expression of a dominant-negative construct of sgg increases the number of satellite boutons. e, UAS-arm^S10;elav-GAL4. f, UAS-pan^ΔN;elav-GAL4. Neither expression of constitutively activated armadillo nor expression of dominant-negative pangolin resulted in an arr-like synaptic phenotype. g, UAS-dsh^DIX/UAS-arm^S10;elav-GAL4. Simultaneously activating armadillo and inhibiting dishevelled in neurons caused the arr-like phenotype. h, UAS-sgg^A82T/UAS-pan^ΔN;elav-GAL4. Simultaneously inhibiting shaggy and armadillo (via a dominant-negative pangolin) in neurons produces the sgg dominant-negative phenotype. i–m, For the genotypes in a–h, quantification of bouton number (i), satellite bouton number (j), number of boutons >5 μm (k), number of boutons with futsch-immunoreactive loops (l), and number of boutons with unbundled futsch (m). n, I, Diagram of the wg signaling pathway showing the two possible branches downstream of the wg-dependent inhibition of shaggy: activation of transcription in the nucleus via armadillo and pangolin (green) or local effects of shaggy on microtubule structure (blue). II, III, Illustration of the genotypes in g and h as a means to discriminate between these branches. In II, UAS dn-dsh^DIX inhibits the regulation of shaggy, but a potential transcriptional branch can be activated by UAS ca-arm^S10 as in g. In III, UAS dn-sgg^A82T/UAS dn-pan ^ΔN leads to the opposite effect of II, blocking the transcriptional branch but activating earlier elements of the wg pathway, including the inhibition of sgg. n = 20 wild-type larvae (34 NMJs), 11 MHC-Gal4 UAS dn-dsh^DIX larvae (18 NMJs), 12 elav-Gal4, UAS dn-dsh^DIX larvae (22 NMJs), 12 elav-Gal4, UAS dn-sgg^A82T larvae (21 NMJs), 10 elav-Gal4, UAS ca-arm^S10 larvae (19 NMJs), 13 elav-Gal4, UAS dn-pan^ΔN larvae (22 NMJs), 12 elav-Gal4, UAS dn-dsh^DIX/UAS ca-arm^S10 larvae (23 NMJs), and 12 elav-Gal4, UAS dn-sgg^A82T/dn-pan^ΔN larvae (20 NMJs). *Statistically different from wild type (p < 0.05). Scale bar, 20 μm.

Figure 6.

A model for wingless actions presynaptically and postsynaptically at the NMJ of Drosophila. a, Without a wingless signal, presynaptic dishevelled remains in an inactive state, and shaggy remains active, thereby preventing assembly of microtubule loops, probably by phosphorylation of futsch. Terminals therefore contain punctate futsch and depolymerized or disordered microtubules. The formation of additional boutons is blocked. b, Wingless signaling at the Drosophila NMJ activates two different intracellular cascades. Postsynaptically, the C-terminal cleavage of the receptor frizzled2 takes place, possibly after wg binds to its receptor-coreceptor complex frizzled2/arrow. The C terminus translocates to the muscle nucleus in a process requiring GRIP (Mathew et al., 2005; Ataman et al., 2006). At the presynaptic arbor, wingless binds to the frizzled2/arrow complex and activates dishevelled. The glycogen synthase kinase shaggy is thereby blocked, which permits futsch to promote the organization of microtubules into loops, the budding of new boutons, and synaptic growth.