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Fig. 2A–U Phenotypes of NMJs, central synapses and other releasesites. All images except E,G,H and N are late-stage 17; A–E lightmicroscopicimages of NMJs stained with synaptic markers (A anti-Kakapo, B–D anti-Synaptotagmin, E anti-cysteine string protein;see Table 1); F–M ultrastructural images of boutons; N–U details ofsynaptic or junctional structures. A,B NMJs (black arrowheads) ondifferent muscles show characteristic shape differences (A dorsal,B ventral muscle field). C,D NMJs in mutant embryos (same area asin B): kakapo mutant NMJs are reduced (C), myoblast city mutantmuscles and NMJs are reduced but ectopic presynaptic terminalsform in the area (D). E NMJ boutons and branches have grown atlate larval stages (same NMJ as in B); unstained line (bent black arrowin close-up) represents most likely vesicle-free axolemma (cf.G). F At late embryonic NMJs, neuronal boutons ( * ) are half coveredby basement membrane (open arrowhead) and half attached tomuscle (mu) via an unspecialised junction (small open arrow),which is interspersed with synapses (black arrow; cf. R). G At latelarval NMJs boutons are completely surrounded by folds of themuscle membrane (SSR) and enriched with vesicles, except in theaxolemma (bent black arrows indicate microtubules in axolemma;cf. E). H At late stage 16, the neuromuscular cell junction (smallopen arrow) consists of only short stretches of apposed neuronaland muscle surfaces and harbours immature synapses (cf. N).J lamininA mutant NMJs lack basement membrane attachment andalso neuromuscular contact is reduced. K At mef2 mutant NMJsboutons are separated from the muscle by basement membrane(open arrowhead) and presynaptic structures are mislocalised (blackarrows). L Classical neurohemal release site (black arrow) of a peptidergicvaricosity with dense-cored vesicles attached to muscle,most likely corresponding to larval-type III boutons (Jia et al. 1993;Martínez-Padrón and Ferrús 1997). M Synapses in the central nervoussystem. N Neuromuscular synapse at late stage 16 exhibiting apresynaptic dense body with clustered vesicles and a stretch of electron-densemembranes. O Neuroneural synapse outside the CNSwith presynaptic T-bar (see also Yoshihara et al. 1997). P Centralsynapse with postsynaptic densities (bent white arrows). Note thatsynapses in N–P lack structured material in the synaptic cleft (incontrast to R). Q Neurohemal active zone in myoblast city mutantembryo covered by basement membrane (open arrowhead; rarecases exist also in wild type; Prokop et al. 1996). R,S Neuromuscularsynapse with structured material in the cleft (between white arrowsin R), which appears as a honeycomb-like pattern in obliquesections (white arrow in S). T,U Ribbons of septa (between whitearrows) in synaptic cleft of epidermal pleated septate junctions(pSJ) are similarly spaced as cleft material at NMJs (cf. T with R),and look similar in oblique sections (cf. U with S) [bent black arrowscore of the bouton (axolemma), bent white arrows postsynapticdensity, DO/DA dorsal oblique and acute muscles, black arrowssynapses or release sites, black arrowheads boutons, asterisks presynapticor releasing boutons, ep epidermis, kak kakapo mutant embryo,L3 third larval instar, mbc, myoblast city mutant embryo, mumuscle, open arrowheads basement membrane, pSJ pleated septatejunction, small open arrows extrasynaptic cell junction, SSR subsynapticreticulum, st.16 embryonic stage 16, VL ventral longitudinalmuscles, VO ventral oblique muscles, white arrows demarcate orindicate material in junctional cleft, wt wild type]. Muscle nomenclatureas in Bate 1993. Scale bars 15 µm (A–E), 480 nm (F–M),255 nm (N–U)ther below the T-bar or extrasynaptically, and both populationscan be distinguished (Koenig and Ikeda 1996). Incontrast to extrasynaptic reformation, the vesicles at T-bars form more rapidly, without cisternal intermediatesand can be blocked by high-Mg 2+ /low-Ca 2+ solution.A further feature of neuromuscular synapses in Drosophilaembryos and larvae is the presence of regularlystructured electron-dense material in the synaptic cleftover a distance of several hundred nanometres (betweenbent arrows in Fig. 3). In transverse sections a continuousfine line can be seen in the middle of the cleft, and athick dashed line closely associated with the postsynapticmembrane (Fig. 3A). Oblique sections through thesynaptic cleft show a patch of honeycomb-like pattern(Fig. 3D). Freeze-fracture analyses of comparable neuromuscularsynapses in the moth Manduca and the lobsterHomarus reveal areas of stippled material in the externalleaflet of the muscle membrane opposite the T-bar, suggestingthat the structured material in the cleft is of postsynapticnature and anchors in the muscle membrane(black arrowheads in Fig. 3B,C,E; Rheuben and Reese1978; Walrond et al. 1993). Synaptic proteins likely toform part of these postsynaptic structures are transmembranemolecules like the ionotropic glutamate receptors,voltage-gated Shaker potassium channels and the cell adhesionmolecule (CAM) Fasciclin 2 (Fas2; Fig. 4C; Broadieand Bate 1993c; Saitoe et al. 1997; Tejedor et al.1997; Thomas et al. 1997a; note that Fas2 is expressedalso on the presynaptic side). Interestingly, the honeycomb-likepattern in oblique NMJ sections is very similarto patterns seen in oblique sections through anothertype of cell junction, the so-called pleated septate junction(Fig. 2S vs. U; Baumgartner et al. 1996). In transversesections of pleated septate junctions transverselines can be seen spanning the cleft every 20–25 nm, andthis frequency of repetition is similar to that of the thickdashed line at NMJs (Fig. 2R vs. T). In spite of a similarorganisation, the extracellular material at pleated septatejunctions looks different from that at NMJs and is likelyto include Neurexin IV, an unusual member of theNeurexin family of proteins which has not been found atNMJs (Baumgartner et al. 1996). However, common cytoskeletalelements might arrange the different structuralelements at both junction types into similar honeycomblikepatterns. A likely candidate for such an organisationalcytoskeletal element is the Discs large (Dlg) protein,which is localised both at NMJs and pleated septatejunctions and has been shown to organize transmembraneproteins at junctional sites (Budnik 1996).Formation of the neuromuscular junctionInitiation of neuromuscular contact173In Drosophila, NMJ formation has been studied mostthoroughly on the ventral longitudinal muscles VL3 andVL4 (nomenclature according to Bate 1993). However,all or most embryonic NMJs might develop in a similarway, as they are glutamatergic and share a commonstructural organisation. At stage 15/16 muscles send outfilopodia preferentially from the future site of innervation(Suzuki et al. 1999), and these are contacted by filopodiasent out from the motoneuronal growth cones in arandom fashion (Fig. 4A). Those motoneuronal processeswhich are in contact with inappropriate muscles aresoon withdrawn (Broadie et al. 1993; Yoshihara et al.1997). Selection for the right muscle is thought to be mediatedby repulsion from inappropriate and contact-medi-