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ated attraction to the right target muscle (Fig. 4A; reviewedin Bate and Broadie 1995; Keshishian et al.1996). For example, the RP3 neuron grows past Toll-expressingmuscles VO4 and VO5 to innervate the non-Toll-expressing muscles VL3 and VL4. Taking away expressionof the transmembrane molecule Toll from VO4and VO5 causes some extra branching in that area,whereas misexpression of Toll on VL3 and VL4 will repelthe RP3 motoneuron and retard the neuromuscularcontact (Rose et al. 1997). In contrast, the homophilicCAM Fasciclin 3 (Fas3) is specifically expressed on theRP3 motoneuron and at the future innervation site onmuscles VL3 and 4. Ectopic expression of Fas3 on neighbouringmuscles is sufficient to redirect RP3 innervationto these non-target cells, but not if Fas3-expression iseliminated on the RP3 growth cone (Kose et al. 1997).Thus, Fas3 appears to function as a homophilic synapticrecognition molecule mediating attractive cell-cell interactionsbetween RP3 and muscles VL3/4 (stippled linesin Fig. 4A). However, loss of function of Fas3 has no apparenteffect on targeting and innervation, and this mightbe explained by the fact that the target recognition code isredundant (see, for example, Speicher et al. 1998). Observationssimilar to those of Fas3 have been made for Connectin,another homophilic CAM specifically expressedon a different subset of muscles and neurons (Nose et al.1992; Raghavan and White 1997). Thus, specificity of innervationis believed to involve combinatorial functionsof several cell-specific CAMs or repellents. In addition,this first specific contact requires general factors, such asthe transmembrane protein commissureless on all muscles(not shown in Fig. 4). In the absence of commissurelessthe motoneurons stall in close vicinity of their targetmuscles or project beyond (Wolf et al. 1998). Once a tolerableneuromuscular contact is established, its furtherdifferentiation appears to depend on properties which appearto be common to most or even all muscles and motoneurons.This is suggested by the observation that persistentfunctional NMJs differentiate, even when motoneuronsare misrouted to contact wrong muscles (Keshishianet al. 1996; Kose et al. 1997).Adhesive propertiesat the differentiating neuromuscular contact175If particular combinations of specifically expressedCAMs participate in the neuromuscular recognitioncode, they might also mediate adhesion at newly formedneuromuscular cell junctions (connected rectangles inFig. 4B). Usually these junctions consist of short stretchesof apposed membranes, often interrupted by stretchesof non-connected cell surfaces (Schuster et al. 1996b;Yoshihara et al. 1997; Prokop et al. 1998a). Such contactsdevelop into an extended cell junction at the matureNMJ of late stage 17 embryos (Fig. 4B vs. C), suggestingthat adhesive properties change during NMJ differentiation.Micrographs of vertebrate NMJs in vivo and invitro also show an initially narrow and often discontinuousjunctional cleft of about 10–20 nm width, which lateron widens to about 50 nm and accumulates a basementmembrane (Kullberg et al. 1977; Takahashi et al.1987; Hall and Sanes 1993). This clearly indicates a molecularreorganisation of cell adhesion properties. At theDrosophila NMJ, the width of the junctional cleft doesnot change, but nevertheless molecular changes of adhesiveproperties occur during NMJ maturation, as suggestedby several observations:During NMJ maturation, expression of Fas3, whichpotentially contributes to the early phase of adhesion,vanishes from neurons, muscles and NMJs (Broadie andBate 1993c). Connectin vanishes from the extrajunctionalmuscle surfaces but remains in the neuron, and stainingat the NMJ persists into late larval stages (M. Landgraf,personal communication; own observations). Thiscould either mean a complete downregulation of Connectinpostsynaptically, or that Connectin becomes restrictedto the postsynaptic site (Fig. 4C). Furthermore,the homophilic CAM Fas2 is initially expressed stronglyon the surfaces of all motor axons and at low levels onall muscles but, during NMJ formation, Fas2 is progressivelyrestricted to the NMJ in pre- and postsynapticcells (Schuster et al. 1996b). The late neuromuscular restrictionof Fas2 is mediated by the cytoskeletal elementDiscs large (see later; Thomas et al. 1997a; Zito et al.1997; Fig.4C), which itself is not detectable at the NMJuntil late stage 17 (Guan et al. 1996). Deleting Fas2function does not affect NMJ formation in the embryobut strongly affects postembryonic stabilisation andmaintenance of NMJs, suggesting a late requirement forFas2 3 (Schuster et al. 1996b). Even more, it seems to beof developmental importance that Fas2 function is restrictedto the late phase of NMJ differentiation: Overexpressionof Fas2 in muscles during the early phase ofNMJ formation seems to render muscle surfaces too„sticky“ and interferes with neuromuscular target recognition,in that ectopic motoneuronal branches (whichnormally occur only transiently) become trapped andform NMJs on inappropriate muscles (Davis et al. 1997).Taken together, NMJ differentiation appears to involvedynamic but regulated changes in expression and localisationof CAMs, and different CAMs appear to servedistinct functions at different times during this process.A second argument in favour of a switch in adhesiveproperties at the NMJ comes from analysis of mef2 mutantembryos, where the initial NMJ contact can be geneticallyseparated from the late phase of neuromuscular adhesion.In mef2 mutant embryos, muscle founder cells(FC in Fig. 1) form and express muscle-specific markersincluding the CAMs Connectin and Fas3, and motoneuronsestablish contact with their appropriate target cells(Prokop et al. 1996). However, the muscle founder cellsremain immature and fail to acquire properties of general3 During larval life the adhesion of Fas2 not only stabilises NMJsbut even inhibits their further growth, and postembryonic growth atthe NMJ is promoted by a modest downregulation of Fas2 (Schusteret al. 1996a).