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Department of Biological Sciences, Kennedy Center for Research on Human Development, Vanderbilt University, Nashville, Tennessee 37235, USA
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[Keywords: Glutamatergic synaptogenesis; post-synaptic density; glutamate receptor; secretion; Drosophila; neuromuscular junction]
Received May 21, 2007; revised version accepted August 27, 2007.
; Malenka and Bear 2004; Perez-Otano and Ehlers 2005). A critical hypothesis, developed largely from the vertebrate neuromuscular junction (NMJ) model (Sanes and Lichtman 2001), is that presynaptic signals trigger post-synaptic assembly and modulation. Proposed secreted signals include Agrin (Sanes and Lichtman 2001; Bezakova and Ruegg 2003; Misgeld et al. 2005), WNTs (Hall et al. 2000; Packard et al. 2002; Salinas 2003), FGFs (Umemori et al. 2004), and Narp (O’Brien et al. 1999, 2002; Mi et al. 2002, 2004). Transmembrane synaptic signaling proteins include SynCAM (Biederer et al. 2002; Sara et al. 2005), EphrinB–EphB (Bruckner et al. 1999; Dalva et al. 2000), and 
-neurexin/neuroligin (Scheiffele et al. 2000; Dean et al. 2003; Scheiffele 2003; Graf et al. 2004; Chih et al. 2005; Levinson et al. 2005; Nam and Chen 2005; Sara et al. 2005). Both classes of signals are proposed to interact intimately with a specialized synaptic cleft extracellular matrix (ECM), which is molecularly distinct from nonsynaptic ECM (Dityatev and Schachner 2003; Martin 2003b; Nishimune et al. 2004; Dityatev and Schachner 2006).
Synaptic cleft ECM components include heterotrimeric (
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) laminin glycoproteins (Nishimune et al. 2004) and heparan sulfate proteoglycans (HSPGs), which bind extracellular glucuronic acid and N-acetyl glucosamine (GlcNAc) polysaccharides (Johnson et al. 2006; Viapiano and Matthews 2006). The secreted HSPGs Agrin and Perlecan are established regulators of NMJ synaptogenesis (Sanes and Lichtman 2001; Bezakova and Ruegg 2003; Kummer et al. 2004, 2006; Misgeld et al. 2005). The HSPG Syndecan (Sdc)-2 is similarly implicated in hippocampal synapse formation and plasticity (Ethell and Yamaguchi 1999; Ethell et al. 2001; Ethell and Pasquale 2005). ECM signaling at the mammalian NMJ also acts via the /-dystroglycan–glycoprotein complex (DGC) (Henry and Campbell 1996; Grady et al. 2000). Numerous integrin receptors localize to mammalian NMJs and central glutamatergic synapses, and integrin–ECM interactions regulate aspects of synaptic development and modulation, including glutamatergic transmission and plasticity (Bahr et al. 1997; Staubli et al. 1998; Bahr 2000; Chavis and Westbrook 2001; Chan et al. 2003, 2006; Kramar et al. 2003, 2006; Lin et al. 2003; Bernard-Trifilo et al. 2005; Shi and Ethell 2006; Webb et al. 2006, 2007).
The Drosophila glutamatergic NMJ contains an ultrastructurally distinctive synaptic cleft ECM present only between pre- and post-synaptic densities (Prokop 1999), but little is known of its molecular composition. HSPGs, including laminin-binding Sdc (Fox and Zinn 2005; Johnson et al. 2006) and GPI-anchored Dallylike (Dlp) (Johnson et al. 2006), and the secreted Hikaru Genki (HIG) (Hoshino et al. 1996, 1999) are localized to the cleft ECM and regulate synaptic differentiation. A post-synaptic Dystrophin scaffold has recently been shown to regulate NMJ maturation (van der Plas et al. 2006). Drosophila WNT Wingless (Wg) is a secreted anterograde synaptic maturation signal that acts via its pre/post-synaptic receptor Frizzled (Dfz2) (Packard et al. 2002; Marques 2005; Mathew et al. 2005; Ataman et al. 2006). Integrin ECM receptors and laminin are localized to NMJ synapses and regulate synapse formation, and both structural and functional development (Prokop et al. 1998; Beumer et al. 1999, 2002; Rohrbough et al. 2000; Hakeda-Suzuki et al. 2002).
The Drosophila NMJ post-synaptic domain contains two subclasses of tetrameric AMPA/kainate-like glutamate receptors (GluRs), composed of either IIA (A-class) or IIB (B-class) subunits, in combination with common required IIC, IID, and IIE subunits (Petersen et al. 1997; DiAntonio et al. 1999; Marrus and DiAntonio 2004; Marrus et al. 2004; Featherstone et al. 2005; Qin et al. 2005). The PDZ-domain scaffold Discs Large (Dlg), a PSD-95/SAP70 homolog (Lahey et al. 1994), plays a key role in localizing post-synaptic proteins (Ashley et al. 2005; Gorczyca et al. 2007) including B-class GluRs (Chen and Featherstone 2005). The Drosophila p21-activated kinase (dPak), a serine threonine kinase activated by GTPases Rac and Cdc42 (Harden et al. 1996; Newsome et al. 2000; Mentzel and Raabe 2005), and its localizing Rho-type GEF, dPix (Werner and Manseau 1997; Hakeda-Suzuki et al. 2002), also play essential roles in the post-synaptic domain. dPAK interacts directly via its kinase domain with Dlg, and is required for Dlg synaptic expression (Parnas et al. 2001; Albin and Davis 2004). In a second pathway, dPak binding to the adaptor Dreadlocks (Dock) (Albin and Davis 2004), a Src homology (SH3 and SH2)-containing Nck homolog (Rao and Zipursky 1998; Ang et al. 2003; Rao 2005), regulates A-class GluR abundance (Albin and Davis 2004). The dPix–dPak–Dock and dPak–Dlg pathways therefore converge to regulate localization of both GluR classes in the post-synaptic domain.
Extensive work in this model system has established that the presynaptic neuron induces post-synaptic differentiation, inducing development and modulation of GluR domains (Broadie and Bate 1993a, b,c; Saitoe et al. 1997, 2001; Featherstone et al. 2002; Sigrist et al. 2003; Chen and Featherstone 2005). GluR domain formation involves lateral membrane receptor diffusion, receptor sequestration/anchoring mechanisms, regulated GluR subunit transcription, and local post-synaptic translation (Broadie and Bate 1993b; Sigrist et al. 2000, 2003; Chen et al. 2005; Rasse et al. 2005). To define the molecular mechanisms regulating functional post-synaptic differentiation, we undertook a systematic mutagenesis screen to isolate mutants with defective post-synaptic assembly. This approach has revealed mind the gap (mtg), which encodes a secreted protein required for the formation of the synaptic cleft matrix, as well as a localization of the signaling pathways regulating GluR domains. Presynaptic mtg knockdown inhibits post-synaptic differentiation, indicating that presynaptically secreted MTG organizes the extracellular cleft domain and is critically required for transsynaptic signaling that induces post-synaptic differentiation.
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Results |
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TopAbstractResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
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The mind the gap (mtg) mutation was isolated from a screen of >6000 lines for mutants with severely disrupted functional differentiation at the glutamatergic NMJ. EMS-mutagenized lines carrying the rucuca third chromosome markers (ru, h, th, st, cu, sr, e, ca) (Fig. 1A) were sequentially screened for mature embryonic/early larval lethality, normal gross anatomy and neuromuscular architecture, paralysis/reduced movement, and lastly, impaired neurotransmission at the embryonic NMJ. Mature mtg1 mutant embryos (21–22 h) are completely paralyzed, exhibiting no spontaneous or touch-evoked movement, and 100% failure to hatch. Mutant embryos have normal body anatomy, and morphologically normal nervous system, musculature and NMJs. Manually hatched mtg1 embryos display weakened cuticle, and phenotypic posterior gut herniation; a few briefly display only slight, uncoordinated movement in saline, but no indication of neurally induced body wall peristalsis. All mtg mutants show severely compromised NMJ function (see below), indicating that mtg is an essential gene required for glutamatergic synapse formation and function.
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To identify the mtg gene, flanking DNA was isolated from both mtgP2 and mtgP3 insertions (see Materials and Methods). The mtgP2 and mtgP3 insertions are located downstream (335 and 380 nucleotides [nt], respectively) from the TAG stop codon of gene CG15864, and immediately upstream of gene CG7549 (Fig. 1B). All three upstream ORFs (CG33722, CG18749, CG15864) were sequenced in mtg1 mutants and rucuca controls, and no mutant nucleotide changes were found. In contrast, the immediately downstream gene CG7549 contains a single nucleotide change in the ORF coding region, converting Q134 to a stop codon resulting in premature termination (Fig. 1B). 5' RACE (rapid amplification of cDNA end) was used to identify the first three exons, which are not predicted in the existing FlyBase annotation and sequence databases for CG7549, and three alternately spliced CG7549 transcripts (Fig. 1B). The mtgP2 insertion lies 23 nt upstream of exon 1, and the mtgP3 insertion is 14 nt into exon 1. The mtgP4 mutation results in a deletion of 95 nt, removing 22 nt of exon 1 and 73 upstream nucleotides. The mtgP5 mutation results in a deletion of 616 nt, including the last 49 nt of exon 1 and 567 nt of intron 1. Part or all of the original element (P3) is also present in both deletion alleles (Fig. 1B). The mtg1 Q134 point mutation is 102 nt into exon 5. RT–PCR confirmed a predicted reduction in CG7549 mRNA levels for mtgP2 (Supplementary Fig. S1), predicting mRNA reductions for the other P-element alleles. The longest mtg transcript encodes a predicted 556-amino-acid, 62.4-kDa protein. The two shorter transcripts lack 5'-untranslated region (UTR) and exon 2, and initiate at the next available methionine downstream from Q134 in exon 5, encoding predicted proteins of 43.4 kDa (Fig. 1B).
So far, we have been unable to rescue mtg mutant phenotypes using the GAL4-UAS system to drive ubiquitous (Uh1 Gal4) or nervous system (elav Gal4) expression of a wild-type mtg transgene. A likely explanation is that the Gal4-UAS approach is unable to correctly replicate the endogenous level and pattern of endogenous mtg gene expression during synaptogenesis. Gal4-driven mtg overexpression may also be deleterious, and therefore prevent mutant rescue. However, the two best alternative proofs provide strong evidence that CG7549 is the mtg gene. First, precise excision of the P-element in CG7549 (mtgP3) completely rescues the mutant, while the adjacent P4H gene shows no mutation in mtg1, strongly indicating that this insertion is solely responsible for mtg phenotypes. Second, a transgenic mtg RNA interference (RNAi) line phenocopies mtg lethality and genetically interacts with mtg1, and produces synaptic defects consistent with mtg mutant phenotypes (see below). In all subsequent studies, Df(CA3) is used as a deletion null, mtg1 as a predicted functional null, and mtgP2-P5 as partial loss-of-function hypomorphic alleles.
MTG is a predicted secreted, N-acetyl-glycosaminoglycan-binding protein
The MTG protein has an N-terminal 19-amino-acid secretion signal peptide, with a cleavage site at T20 (Fig. 1C), and a predicted molecular weight (MW) of 60.2 kDa for the cleaved, secreted product. The N-terminal third of the protein contains a 41-amino-acid, highly glutamine- and leucine-rich domain, predicting an ordered -helical structure (Fig. 1C,D). This domain contains the premature stop codon in mtg1. The C-terminal half of the protein contains a highly structured, 51-amino-acid -helical domain with homology with extracellular carbohydrate-binding (CBM-14/19) and chitin-binding peritrophin-A domains (ChtBD-2) (Fig. 1C–E). Related domains conserved in other extracellular proteins, including the classic synaptic cleft marker wheat germ agglutinin (WGA) (Martin 2002, 2003b), have specific binding affinity for GlcNAc. The structured "ECM-binding" domain contains six conserved cysteines, predicted to form three disulfide bridges stabilizing a folded or knotted structure, and is related to similar extracellular domains within the "cysteine knot"-containing protein superfamily. This family includes carbohydrate-binding lectins (e.g., WGA), knottins, neuronal ion channel-binding toxins, and secreted mucins, glycohormones, and growth factors including EGF and TGF (Vitt et al. 2001; Knottin database, http://knottin.cbs.cnrs.fr). The type 1 EGF domain is also present in laminins, which function as ECM integrin-ligands, and many other EGF signaling proteins.
To directly test the prediction that the MTG protein is secreted, an expression vector containing a mtg-GFP fusion construct was transfected into cultured Drosophila S2 cells. Twenty-four hours to 48 h following induction, MTG-GFP fluorescence was faintly visible in transfected living and fixed cells, compared with more intensely fluorescent Tau-GFP-transfected control cultures. A 105-kDa MTG-GFP protein recognized by anti-GFP antibodies, was found on Western gels of MTG-transfected cultures and was absent from Tau-GFP-transfected control cultures (Supplementary Fig. S2; data not shown). The expected size of GFP-tagged MTG is 89 kDa, suggesting post-translational modification of the expressed MTG protein (see below). A larger band of 
250 kDa may represent a dimer, or a more highly glycosylated form of MTG-GFP, while a smaller 50-kDa band may represent a cleavage product. All three bands are specific to MTG-GFP cultures and are not observed in Tau-GFP-transfected control cultures. The MTG-GFP bands are present in concentrated culture supernatant from MTG-GFP-transfected cultures, consistent with MTG secretion into the extracellular environment (Supplementary Fig. S2). We next used confocal microscopy to image S2 cell MTG-GFP and anti-GFP fluorescence to examine MTG cellular expression, and determine whether secreted protein was localized to the cell surface (Fig. 2A,B). Cultures were processed with detergent (Triton X) or detergent-free conditions to distinguish between total (permeablized) and surface (unpermeablized) protein staining. In detergent-treated cells, MTG-GFP is internally localized in concentrated aggregates, many located peripherally near the cell membrane, as well as diffusely within the cytoplasm (Fig. 2A). With detergent-free staining, MTG-expressing cells were identifiable by internal MTG-GFP expression. Surface anti-GFP staining of MTG-expressing cells typically revealed numerous aggregates distributed to the cell periphery and externally on the cell surface, which could be confirmed by examining Z-axis rotations of imaged staining (Fig. 2B). This protein distribution suggests that intracellular MTG protein is aggregated or packaged near the cell surface and secreted, but that secreted protein can remain bound to the cell surface. In contrast, in detergent-free conditions no external anti-GFP staining was detected for nonpermeablized Tau-GFP-expressing cells, despite much higher levels of internal GFP protein, or in untransfected controls, indicating the specificity of MTG-GFP outer surface localization (Fig. 2C).
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60 kDa, equal to the predicted MW of MTG (Fig. 2F). This band was absent from Df(CA3) bead and eluted fractions, indicating its specificity. These results show that MTG is a secreted glycosylated protein, and binds the extracellular GlcNAc moiety known to be concentrated within the synaptic cleft.
MTG expression peaks during embryonic NMJ synaptogenesis
Assays of mtg mRNA levels throughout development show that mRNA abundance is very low during the first half of embryogenesis (0–12 h after fertilization). Expression increases sharply at 12–13 h to a peak level at 16–17 h, then declines sharply from 18 to 20 h (Fig. 2G,H). Message level persists at a low level throughout late embryogenesis and subsequent larval stages, with a small transient peak during L2. Expression again transiently increases on the third day after pupariation, and declines prior to adult eclosion (data not shown). In the adult, the mtg message in the head is elevated relative to the rest of the body, consistent with neuronal function. The developmental mtg expression pattern therefore correlates closely with known periods of synaptogenesis (Broadie and Bate 1993a, c) and the expression profiles of other known synaptic genes, suggesting an important role for mtg in this process. For example, still life (sif), a presynaptic rho-GEF required for synaptogenesis, is expressed from 16 h in the embryo (Sone et al. 1997). Hikaru genki (hig), a secreted synaptic cleft protein important for adult central synaptogenesis, is likewise highly expressed at 16–24 h in embryonic neurons, as well as in the late pupa (Hoshino et al. 1996, 1999).
Dissected control and mutant embryos were assayed with anti-MTG antibodies to examine MTG protein localization, in combination with neuronal and synaptic markers. Mature 20- 22-h mutant embryos [mtg1/mtg1 and mtg1/Df(CA3)] probed with the anti-horsradish peroxidase (HRP) neuronal membrane marker display morphologically normal CNS and ventral nerve cord (VNC), and normal peripheral axon extension, branching, and targeting (Fig. 3A,B; data not shown). NMJ formation and presynaptic terminals appear correctly and stereotypically formed (Fig. 3B; see also Figs. 6, 7, below). In control wild-type embryos, anti-MTG staining shows expression in the CNS, VNC, and body wall musculature (Fig. 3A,B). MTG staining is notably concentrated within embryonic neuronal cell bodies, including identified motor neurons at the VNC midline, and motor neurons in lateral VNC regions (Fig. 3A), and is also detectable in segmental nerves and peripheral nerve branches, consistent with a neuronal expression pattern. MTG muscle staining is present in proximity to NMJs, but is not selectively enriched at embryonic NMJs (Fig. 3B). MTG staining level is clearly reduced in homozygous mtg1 and mtg1/Df(CA3) mutant embryos (Fig. 3A,B). As noted above, mtg1 mutants potentially synthesize a short MTG isoform recognized by anti-MTG Abs, which may account for residual staining in mutants. We further assayed MTG staining in mtg RNAi larvae ubiquitously expressing UAS-mtg-RNAi (UH1-Gal4/UAS mtg RNAi; Fig. 3C). Ubiquitous RNAi animals have significantly reduced mtg transcript levels at embryonic stages (see below), and die as seceond instar larvae. At control larval NMJs double-stained for synaptic markers, MTG staining is clearly concentrated to synaptic or perisynaptic regions at many terminals, and also concentrated at muscle ends and muscle attachment sites, where other synaptic scaffolding, cytoskeletal, and ECM proteins—including dPak, Dlg, and integrins—are developmentally expressed (Fig. 3C). MTG level is clearly reduced throughout mtg RNAi larvae, including neuronal, muscle, and NMJ regions (Fig. 3C). Since mtg mRNA level peaks at 16–17 h, during early–mid stages of embryonic synaptogenesis, these results suggest that MTG protein becomes increasingly concentrated at the NMJ during development.
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MTG staining is reproducibly detected at NMJ boutons in third instar larvae (Fig. 3D,F). At a confocal imaging level, MTG bouton staining partly overlaps with HRP-stained presynaptic terminal membrane, as well as with Dlg-stained post-synaptic subsynaptic reticulum (SSR) membrane domains (Fig. 3D,F; data not shown), in addition to associated perisynaptic regions. The pattern of anti-MTG localization at larval NMJs also resembles the NMJ labeling pattern of the GlcNAc-specific lectin WGA (Fig. 3E), strongly suggesting that Drosophila synaptic extracellular and cleft matrix is concentrated in GlcNAc molecules recognized in common by both MTG and WGA. To more precisely localize MTG synaptic localization, immuno-EM studies were conducted using four anti-MTG sera and secondary immunogold labeling (10 or 25 nm colloidal gold) (Fig. 3G–I). Quantified measurements of labeling incidence and density were made in image profiles that included presynaptic boutons, active zones (AZ), post-synaptic SSR, and an area of surrounding nonsynaptic muscle (25,000x magnification) (Fig. 3G–I; Supplementary Table S1). MTG gold grains were consistently detected in both synaptic and perisynaptic domains, with 60% of label detected in presynaptic boutons and surrounding SSR (n = 52 profiles). At higher magnification, label is detected within several specific synaptic subdomains (Fig. 3G–I). In 35 out of 52 profiles, gold grains were localized within the presynaptic terminal, in many cases (20 out of 35) with grains closely associated with the plasma membrane (Fig. 3G). In several terminals, multiple gold grains were concentrated within an 100 nm radius (Fig. 3H), consistent with packaging of MTG protein within secretory vesicles. In 96% of the sections examined (50 out of 52), gold grains were located within the SSR domain (Fig. 3G). SSR-localized grains made up 36% of the total grains scored/section, and were distributed both singly and as localized clusters, suggesting MTG protein is spatially concentrated within the SSR. Strikingly, in 35% of profiles (18 out of 52) one or more gold grains was located within a defined area (0.098 µm2) encompassing the AZ and synaptic cleft. In the majority (10 out of 18) of these instances, gold label was specifically localized to the extracellular synaptic cleft domain immediately under an AZ T-bar structure (Fig. 3I), confirmed by serial sectioning. Quantified gold mean labeling density within defined AZ areas was threefold higher (7.8 ± 1.2 µm–2) than within presynaptic boutons (2.5 ± 0.5 µm–2) or post-synaptic SSR domains (2.4 ± 0.4 µm–2) (Supplementary Table S2). MTG localization both within presynaptic boutons and the post-synaptic SSR is consistent with its secretion at the NMJ synapse. In particular, MTG localization by immuno-EM to the synaptic cleft domain supports the hypothesis that MTG is secreted to function within the synaptic cleft matrix.
MTG is required for functional post-synaptic differentiation
To determine the cause of mtg mutant paralysis, whole-cell patch-clamp recordings were made from muscle 6 at the mature embryonic (20–22 h) NMJ synapse (Fig. 4). Wild-type NMJs exhibit large amplitude (1–1.5 nA) spontaneous excitatory junctional currents (EJCs) driven by spontaneous neuronal action potentials, and occasional patterned EJC bursts driven by central locomotory generator activity (Broadie and Bate 1993c; Baines et al. 1999, 2001). In mtg1/mtg1 and mtg1/Df(CA3) mutant embryos, this endogenous transmission activity is largely silenced. Occasional isolated mutant EJCs are recorded, demonstrating functional transmission; however, mtg terminals display only small amplitude EJCs, and patterned bursts of large EJCs are not observed. Motor nerve stimulation (1 Hz) evokes robust EJCs with 100% reliability in wild-type and mtg1/+ controls (mean amplitude 1274 ± 169 pA) (Fig. 4A,B). In contrast, homozygous mtg1 and mtg1/Df(CA3) NMJs exhibit severely impaired evoked transmission amplitude and reliability. Approximately 90% of nerve stimuli at mtg1 and mtg1/Df(CA3) mutant NMJs result in transmission failure, even with elevated stimulation intensities, while evoked mutant EJCs have reduced amplitudes and more variable response latencies than at control NMJs (Fig. 4A). Mean mutant evoked transmission amplitude is depressed by >90% [mtg1: 46 ± 23 pA, n = 7; mtg1/Df(CA3): 57 ± 44 pA, n = 7; P < 0.005 vs. control for both alleles] (Fig. 4B). Homozygous mtg1 and mtg1/Df(CA3) transmission levels do not differ significantly from each other, confirming that mtg1 behaves functionally as a null mutant. The mtgP2 allele, which displays movement in the eggcase but fails to hatch, exhibits insignificantly reduced evoked EJC amplitude compared with control (1085 ± 208 pA, n = 5), consistent with a hypomorphic phenotype (see also below). These results show that MTG function is required for the maturation of NMJ synaptic function.
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, 2002) or iontophoresis (Rohrbough et al. 2004), which maximally activates the synaptic GluR population, allowing assessment of overall receptor function. Glutamate response is severely reduced at mtg mutant synapses, indicating that mutant evoked transmission deficits are due largely or entirely to a deficit in post-synaptic GluR function (Fig. 4C). In mtg1 mutants, mean glutamate response amplitude is reduced to 20.8 ± 4.1% of control (Fig. 4D). Glutamate response amplitude is also significantly reduced in mtgP2/Df(CA3) embryos (60.2 ± 5.4% of control; P < 0.02; n = 6) (Fig. 4D), confirming that mtgP2 behaves as a hypomorphic mtg allele. As a further functional post-synaptic assay, miniature EJCs (mEJCs) were recorded in saline containing 0.5 mM external Ca2+ and TTX to block spontaneous action potentials. Under these conditions, mEJCs are presumed to represent the activation of single, distinct post-synaptic GluR aggregates by spontaneous presynaptic vesicle fusion and quantal transmitter release. The mtg1 mEJC mean amplitude is significantly reduced (wild type: 83 ± 9 pA, n = 7; mtg1: 58 ± 9 pA, n = 9; P < 0.01; mtgP2: 110 ± 19 pA, n = 5; P > 0.2), indicating a decrease in functional synaptic GluRs. Mean mEJC frequencies did not vary significantly between wild type (5.0 ± 1.3 min–1), mtgP2 (6.4 ± 1.7 min–1; P > 0.10 vs. wild type), and mtg1 (8.7 ± 1.2 min–1; P > 0.05 vs. wild type). To test whether the mutant post-synaptic impairment is rescuable by an exogenous supply of receptor, dGluRIIA-myc was overexpressed under endogenous genomic control in mtg1 mutants. Glutamate responses in mtg1; GluRIIA-myc embryos remain at the severely impaired amplitudes of mtg1 mutants (Fig. 4D), suggesting a defect in GluR function downstream from receptor gene expression.
MTG is required to form the synaptic cleft matrix
Synaptic ultrastructure was examined by electron microscopy to assess overall pre- and post-synaptic morphology, as well as identify any structural changes correlated with post-synaptic dysfunction. Presynaptic bouton morphology and appearance, plasma membrane topology, and electron-dense AZ with T-bars all appear normal in mtg-null mutants [mtg1/mtg1 and mtg1/Df(CA3)], with no obvious defects (Fig. 5A). Quantified comparisons between control and mutants showed no significant differences (P 
0.3) in mean mutant bouton cross-sectional area, mitochondria size, or number of synaptic vesicles clustered at AZ (Fig. 5A; data not shown). Mutant terminals exhibit a significant increase in the incidence of plasma membrane cisternae structures, a 33% increase in total synaptic vesicle density (P < 0.001 vs. wild type) and an increase in vesicles docked at the presynaptic membrane (mtg: 3.6 ± 0.4, wild type: 2.2 ± 0.3; P < 0.01; n = 48). These presynaptic changes cannot account for the post-synaptic dysfunction, but the increase in vesicle density and docked vesicles could potentially represent an additional deficit in secretory processes, or a compensatory response to a loss of post-synaptic GluR function (DiAntonio et al. 1999), in the absence of MTG.
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). This polyribosome domain lies directly opposite and adjacent to the presynaptic AZ and synaptic cleft (Fig. 5A–C). Post-synaptic polyribosomes are clearly reduced in number and density at >65% of mtg mutant synapses, and instead are typically found in dispersed patches, rather than the tight packing found in normal post-synaptic domains (Fig. 5B). Therefore, loss of the local post-synaptic ribosome population may contribute to the loss of GluR responsiveness in the absence of MTG.
MTG is required for formation of post-synaptic GluR domains
The loss of post-synaptic function in mtg mutants could be due to either a failure to form post-synaptic GluR domains apposing presynaptic AZ or a loss of GluR function, or a combination of both. To address these possibilities, GluR localization and distribution was examined using antibodies against the requisite receptor subunits IIC and IID (Fig. 6). Both IIC and IID are contained in all known GluRs, and therefore reveal the total GluR population in the embryonic muscle and developing NMJ (Featherstone et al. 2005; Qin et al. 2005; DiAntonio 2006). In wild-type control embryonic synapses, GluRs are tightly localized to NMJ domains and present at only a low level in extrasynaptic regions of the muscle (Fig. 6A,B). Synaptic GluRs are aggregated into numerous distinct puncta, closely opposed to individual presynaptic AZ. In mtg1/mtg1 and mtg1/Df(CA3)-null mutants, this characteristic synaptic localization is largely lost. For both GluRIIC (Fig. 6A) and GluRIID staining (Fig. 6B), mtg mutants show a loss of GluR puncta specifically clustered opposite the presynaptic terminal, and GluR staining dispersed in nonsynaptic muscle areas. Mutant GluRs appear to form small aggregates, but there is no preferential synaptic targeting or enrichment of these aggregates. These results suggest that GluRs in mtg mutants are synthesized and assembled, but are either incorrectly localized to extrasynaptic regions, or fail to be retained in post-synaptic domains and so disperse to nonsynaptic areas. Moreover, dispersed receptors are largely nonfunctional, or not located on the cell surface, since neither puff-applied nor iontophoretically applied glutamate evokes a substantial mutant post-synaptic current in physiological recordings (Fig. 4C,D). MTG thus clearly plays a critical role in the post-synaptic localization of GluRs, and the specific targeting of GluR to punctate domains opposite to presynaptic AZ. The loss of GluR localization provides a mechanistic explanation for the functional silencing of the mtg mutant synapse.
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Previous studies have characterized the dPix–dPak–Dlg–Dock molecular pathways regulating GluR localization at the Drosophila NMJ (Parnas et al. 2001; Albin and Davis 2004; Chen and Featherstone 2005; Chen et al. 2005). Our results indicate that MTG is secreted and acts within the synaptic cleft matrix, predicting that MTG should act in, or upstream of, the established post-synaptic GluR localization pathways. To investigate whether the loss of MTG disrupts these signaling pathways, we next examined synaptic expression levels and the spatial distribution of dPix, dPak, Dlg, and Dock in control and mtg mutant embryos (Fig. 7).
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). Dlg is strongly expressed at wild-type embryonic NMJs in a post-synaptic region overlapping with and surrounding the presynaptic terminal (Fig. 7A). In mtg1/mtg1 and mtg1/Df(CA3)-null mutants, Dlg synaptic levels are substantially and consistently reduced. Costaining with the presynaptic vesicle markers Synaptotagmin and CSP (Fig. 7A,B) shows that mutant NMJ presynaptic morphology and SV levels appear normal, and that the post-synaptic loss of Dlg occurs in the presence of a morphologically normal synapse. The dPak kinase directly interacts with Dlg, and binds Dock to direct GluR localization (Albin and Davis 2004). dPak is tightly localized in wild-type embryos to post-synaptic punctae apposing AZ domains, closely resembling the GluR domains, and also strongly concentrated at muscle attachment sites (Fig. 7B). In mtg mutants, dPak localization is strikingly abnormal, and is typically strongly elevated throughout both synaptic and nonsynaptic regions, with nonsynaptic expression greatly obscuring localized synaptic expression (Fig. 7B). Similarly, Dock is concentrated at wild-type NMJs in a pattern resembling Dlg (Fig. 7C), but is grossly mislocalized throughout mtg mutant muscle, with aberrantly high expression in nonsynaptic regions (Fig. 7C). Finally, dPix is detected at the lowest levels among the PSD proteins examined, but is enriched at NMJ boutons in wild-type terminals (Fig. 7D). dPix synaptic expression is consistently weaker than control in mtg mutants, but without the gross mislocalization characterizing dPak and Dock expression (Fig. 7D). These results show that loss of MTG disrupts the post-synaptic localization of all the known molecular players regulating GluR localization. We therefore next performed Western blot analyses of Dlg, dPak, and Dock protein to determine whether the changes in mtg tissue localization/distribution is correlated with alterations in overall protein levels. An obvious caveat to these studies is that changes in protein expression levels at synapses, or even within the muscle, may not be detectable at the whole embryo level. Protein extracts from mature (20–22 h) wild-type and mtg1-null mutant embryos were probed with Western blots for each of these proteins (Fig. 7E). Strong positive bands were detected for all proteins, with dPak signal present in two bands. There was no detectable change in either Dlg or Dock protein levels in mtg1 mutants compared with control. In contrast, dPak protein levels were clearly and consistently elevated in mtg1 over control (n = 4) (Fig. 7E). These results suggest that the altered post-synaptic expression of Dlg and Dock may represent protein mislocalization, whereas the altered post-synaptic expression of dPak may be caused by both mislocalization and overexpression.
MTG is required presynaptically for post-synaptic differentiation
The presynaptic neuron is both necessary and sufficient to induce post-synaptic differentiation at the Drosophila NMJ synapse (Broadie and Bate 1993a, b). Our results strongly suggest that MTG is localized within the motor nerve terminal, secreted into the synaptic cleft to bind extracellular GlcNAc, and plays a role in forming/stabilizing the cleft matrix, which induces post-synaptic differentiation. This hypothesis is further supported in that the post-synaptic targeting of the entire dPix–dPak–DLG–Dock pathway regulating GluR domain is disrupted in mtg mutants. To directly test this hypothesis, we transgenically expressed the UAS-mtg-RNAi construct under the control of ubiquitous, neuronal-specific, and muscle-specific drivers to determine the presynaptic requirement for MTG in regulating this post-synaptic pathway (Fig. 8).
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To test the prediction that presynaptically secreted MTG is required for post-synaptic differentiation, mtg RNAi was targeted to the presynaptic neuron under the control of the neuronal-specific elav-Gal4 driver in the mtg1/+ heterozygous background (elav-Gal4/+; UAS-mtg-RNAi/mtg1) (Fig. 8C). The presynaptic RNAi animals show reduced larval and adult viability, though substantial numbers of viable adults survive. Examination of dPix, dPak, Dock, Dlg, and GluRs in the third instar larvae shows that NMJ expression levels of each protein is decreased in parallel and to a comparable degree, compared with driver controls (Fig. 8C). Again, the larval presynaptic loss-of-function phenotype is manifested as a loss of post-synaptic expression level for each post-synaptic protein, rather than protein mislocalization. Heterozygote mtg1/TM3 synaptic protein staining was not detectably reduced from control (data not shown), indicating the elav; RNAi phenotype is specific to a neuronal loss of function. The severity of the PSD expression phenotypes is comparable for presynaptic RNAi and ubiquitous RNAi larvae (Fig. 8B,C), a result predicted if MTG’s requirement is in the presynaptic neuron. In contrast, mtg RNAi expression targeted in post-synaptic muscle (MHC-Gal4/+; UAS-mtg RNAi) had no effect on larval or adult viability, and caused no detectable change in any post-synaptic protein expression at the NMJ (Supplementary Fig. S3). These results demonstrate that only presynaptic MTG is required for the post-synaptic localization of GluRs, and all proteins known to function upstream of GluR localization, and indicate that presynaptically released MTG plays a critical role in inducing post-synaptic assembly during embryonic synaptogenesis, and has a maintained role in post-embryonic synapse maturation.
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TopAbstractResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
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The mind the gap (mtg) gene was isolated in an unbiased forward Drosophila genetic screen for novel mutants blocking functional differentiation of the glutamatergic NMJ synapse. This screen has now revealed numerous novel genes and mechanisms regulating pre- or post-synaptic development (Featherstone et al. 2002, 2005; Rohrbough et al. 2004; Huang et al. 2006), including GluR subunits and genes regulating functional receptor expression (Featherstone et al. 2002, 2005). Loss of mtg results in a severe, 80% loss of GluR function at the NMJ. Notably, this phenotype represents the most severe mutant GluR impairment ever reported, with the exception of genetic mutants for the requisite GluR subunits (IIC–IIE) themselves (Featherstone et al. 2005; Qin et al. 2005). MTG is expressed neuronally and localized synaptically, contains a well-conserved secretory signal sequence, and is secreted and binds GlcNAc in vitro. These findings support the working hypothesis that MTG is secreted into the synaptic cleft to bind ECM glycosaminoglycans (GAG) or proteoglycans (PG) during glutamatergic synaptogenesis.
The mtg developmental expression profile closely parallels the timing of NMJ synapse formation and functional differentiation. Expression increases sharply with initial nerve–muscle contact (12–13 h after fertilization), and peaks at 16–17 h, correlating with post-synaptic GluR domain assembly (15–17 h) (Broadie and Bate 1993c). MTG protein is concentrated in embryonic neurons, and becomes increasingly localized with development to NMJ synaptic domains, and to other cell adhesion sites such as muscle attachment sites, where many synaptic signaling (Pix, Pak, integrins) and scaffold proteins (Dlg) are colocalized. MTG is found in the presynaptic terminal and post-synaptic SSR, clearly detected within the extracellular synaptic cleft domain, consistent with presynaptic secretion as well as a transsynaptic regulatory role. In the absence of MTG, post-synaptic GluR puncta apposed to the presynaptic terminal are lost, and GluRs are dispersed in the nonsynaptic membrane, resulting in profound functional transmission loss. The mislocalized GluRs appear nonfunctional based on the muscle response to exogenously applied glutamate, which demonstrates a dramatic overall loss of functional cell surface GluRs. All PSD proteins known to act in the upstream GluR regulatory pathways (dPix, dPak, Dlg, Dock) (Parnas et al. 2001; Albin and Davis 2004) are severely reduced or mislocalized in mtg mutants, indicating that MTG acts at an upstream organizing step required to establish this cascade of post-synaptic interactions. The loss of synaptic cleft matrix material at mtg mutant synapses indicates first, that MTG is required for this signaling domain to be established, and second, suggests that this domain has an important inductive, instructive role in post-synaptic assembly.
We have not yet succeeded in genetically rescuing the mtg mutant with a wild-type copy of the mtg gene. Since mtg is present at low overall levels during much of development, it is likely that mtg expression timing and/or level must be precisely regulated for normal protein function and animal viability. Transgenic rescue with tissue-targeted Gal4-driven mtg expression may therefore result in a deleterious overexpression condition. Transgenically expressed mtg RNAi, however, phenocopies mtg mutant phenotypes. Ubiquitous mtg RNAi causes early lethality and defective post-synaptic assembly, with reduced synaptic localization of GluRs and other upstream regulatory proteins. These phenotypes are more severe in the mtg1 mutant, which exhibits lethality at the hatching stage, and also nonsynaptic mislocalization of post-synaptic proteins. Therefore, the findings are consistent with the expected effects of RNAi as a partial loss-of-function condition. Most conclusively, targeted RNAi knockdown of MTG in the presynaptic neuron impairs post-synaptic differentiation and the assembly of GluR domains with a similar severity to the ubiquitous RNAi condition. In contrast, muscle-targeted mtg RNAi has no detectable effect on movement or animal viability, and does not cause any detectable synaptic impairment or defects in post-synaptic assembly. Taken together, these results support the identity of the mtg gene and suggest that presynaptically secreted MTG protein is required for post-synaptic development. We conclude that MTG is a critical element in the presynaptic inductive mechanism.
MTG is a secreted protein predicted to bind extracellular synaptic proteoglycans
MTG has a cysteine-rich carbohydrate-binding module (CBM) with homology with ChtBDs found in peritrophic matrix proteins, lectins, and other known ECM proteins. This domain contains six conserved cysteines predicted to form three disulfide bridges within a -folded carbohydrate-binding structure that binds GlcNAc moieties. Lectins that specifically bind GlcNAc (WGA) and GalNAc (DBA, VVA) are commonly used synaptic cleft or post-synaptic markers at the vertebrate NMJ (Martin 2003b). Here we confirm that WGA-binding targets are localized to Drosophila NMJ boutons, and that extracted MTG protein binds GlcNAc in vitro, suggesting that MTG recognizes GlcNAc-containing target(s) in the synaptic cleft. Drosophila S2 cells transformed with MTG-GFP secrete the protein, which accumulates both on the outer surface of the cells and in the medium, supporting its extracellular localization. However, we have not yet succeeded in replicating the GlcNAc binding with purified MTG protein, the ideal experiment to confirm and further probe the binding specificity. GlcNAc-containing carbohydrate and GAG scaffolds (e.g., chitin, hyaluronic acid [HA], heparin) and PG (e.g., heparin sulfate, chondroitin sulfate) are components of neuronal and muscle ECM, and concentrated within the specialized synaptic cleft matrix at the NMJ and other synapses in multiple species (Martin 2003b; Dityatev et al. 2006). It was recently shown that loss of GlcNAc transferase alters In Drosophila NMJ synaptic structure, function, and locomotory behavior (Haines and Stewart 2007), independently demonstrating GlcNAc-mediated interactions have roles in synaptic maturation.
MTG does not share significant overall whole-sequence homology with an identified vertebrate protein. It is increasingly recognized that many Drosophila proteins are conserved
