ABSTRACT The Cyc8p/Tup1p complex mediates repression of diverse genes in Saccharomyces cerevisiae and is recruited by DNA binding proteins specific for the different sets of repressed genes. By screening the yeast deletion library, we identified Cyc8p as a coactivator for Gcn4p, a transcriptional activator of amino acid biosynthetic genes. Deletion of CYC8 confers sensitivity to an inhibitor of isoleucine/valine biosynthesis and impairs activation of Gcn4p-dependent reporters and authentic amino acid biosynthetic target genes. Deletion of TUP1 produces similar but less severe activation defects in vivo. Although expression of Gcn4p is unaffected by deletion of CYC8, chromatin immunoprecipitation assays reveal a strong defect in binding of Gcn4p at the target genes ARG1 and ARG4 in cyc8Δ cells and to a lesser extent in tup1Δ cells. The defects in Gcn4p binding and transcriptional activation in cyc8Δ cells cannot be overcome by Gcn4p overexpression but are partially suppressed in tup1Δ cells. The impairment of Gcn4p binding in cyc8Δ and tup1Δ cells is severe enough to reduce recruitment of SAGA, Srb mediator, TATA binding protein, and RNA polymerase II to the ARG1 and ARG4 promoters, accounting for impaired transcriptional activation of these genes in both mutants. Cyc8p and Tup1p are recruited to the ARG1 and ARG4 promoters, consistent with a direct role for this complex in stimulating Gcn4p occupancy of the upstream activation sequence (UAS). Interestingly, Gcn4p also stimulates binding of Cyc8p/Tup1p at the 3′ ends of these genes, raising the possibility that Cyc8p/Tup1p influences transcription elongation. Our findings reveal a novel coactivator function for Cyc8p/Tup1p at the level of activator binding and suggest that Gcn4p may enhance its own binding to the UAS by recruiting Cyc8p/Tup1p.

In socially monogamous animals mate choice can be expected when mates vary in quality, degree of parental investment, or ability to defend an important resource. Mate choice was expected in the big-clawed snapping shrimp, Alpheus heterochaelis Say, 1818, as female size is positively correlated with fecundity. In addition, males and females share a shelter, which they defend from intruders, and therefore, resource holding potential could be an important component in mate choice. We tested the effect of body size and shelter ownership on mate choice and on competition in both sexes. As shelter occupancy and defence may have an influence on pair formation and mate choice, we also tested competitive success in obtaining preferred shelters as a variable in mate choice. We did not find evidence that males preferred larger females, but showed that females preferred and competed for larger males. Females paired more frequently with smaller males that occupied a shelter than with smaller males without a shelter. Males were, however, more successful in evicting smaller same-sex conspecifics from preferred shelters than females were. This could indicate that competition for shelter favours males with larger claws, and hence, body size, as these are better able to defend a shelter from other conspecifics. A female may therefore choose larger males, because she is dependent on both a male and a shelter for successful reproduction.

Social monogamy has evolved independently in many taxa, and often involves biparental care of the young. Where it does not, mate guarding and shared territoriality have been invoked as causal factors. We evaluated mate guarding and shared resource defence (a common shelter) as factors that could have led to social monogamy in the snapping shrimp, Alpheus heterochelis . This species is found in male-female pairs that defend a common shelter together. Female receptivity lasts only for a few hours immediately after her periodic moult. Their monogamous pair bond may represent mate guarding or joint defence of a territory. Monogamy in A. heterochelis seems most importantly driven by the cryptic nature of the female's moult cycle. We found that males did not discriminate among females at different intermoult stages for pairing, nor did they modulate their defence of mate and shelter (vs. the risk in finding a new shelter and mate) according to female moult stage. This, together with the short period of female receptivity before her single copulation per cycle, make extended mate guarding the most efficient method for a male to secure a mating opportunity. Comparing eviction rates of paired and unpaired shelter residents by conspecific intruders provided no evidence of enhanced resource defence that would confer a selective advantage to a pair. Male presence during the moult is beneficial for the female, as searching for a male during her soft-bodied receptive phase would put her at mortal risk. Our results show empirically for the first time that guarding may be beneficial, even if males are not able to assess the female's reproductive stage. This extends the theoretical framework for understanding the evolution of social monogamy in taxa without biparental care of young.

Phasic and tonic motor nerves originating from crayfish abdominal ganglia, in 2–3‐day‐old cultured explants, display at their transected distal ends growth zones from which axonal sprouts arise. The subcellular morphology of this regenerative response was examined with thin serial‐section electron microscopy and reveals two major remodeling features. First, the external sprouts that exit the nerve are a very small part of a much more massive sprouting response by individual axons comprising several orders of internal sprouts confined to the nerve. Both internal and external sprouts have a simple construction: a cytoskeleton of microtubules and populations of mitochondria, clear synaptic vesicles, membranous sacs, and extrasynaptic active zone dense bars, features reminiscent of motor nerve terminals. Close intermingling of the sprouts of several axons give rise to a neuropil‐like arbor within the nerve. Thus, extensive sprouting is an intrinsic response of crayfish motor axons to transection. Second, an equally dramatic remodeling feature is the appearance of nuclei, which resemble those of adjacent glial cells, within the motor axons. These nuclei often appear where the adjoining membranes of the axon and glial cell are disrupted and where free‐standing lengths of the double membrane are present. These images signify a breakdown of the dividing membranes and assimilation of the glial cell by the axon, the nucleus being the most visible sign of such assimilation. Thus, crayfish motor axons respond to transection by assimilating glial cells that may provide regulatory and trophic support for the sprouting response. J. Comp. Neurol. 464:449–462, 2003. © 2003 Wiley‐Liss, Inc.

Deep and superficial flexor muscles in the crayfish abdomen are innervated respectively by small populations of physiologically distinct phasic and tonic motoneurons. Phasic motoneurons typically produce large EPSP's, releasing 100 to 1000 times more transmitter per synapse than their tonic counterparts, and exhibiting more rapid synaptic depression with maintained stimulation. Freeze-fracturing the abdominal flexor muscles yielded images of phasic and tonic synapse-bearing terminals. The two types of synapse are qualitatively similar in ultrastructure, displaying on the presynaptic membrane's P-face synaptic contacts recognized by relatively particle-free oval plaques which are often framed by the muscle fiber's E-face leaflet with its associated receptor particles. Situated within these presynaptic plaques are discrete clusters of large intramembrane particles, forming active zone (AZ) sites specialized for transmitter release. AZs of phasic and tonic synapses are similar: 80% had a range of 15–40 large particles distributed in either paired spherical clusters or in linear form, with a few depressions denoting sites of synaptic vesicle fusion or retrieval around their perimeters. The packing density of particles is similar for phasic and tonic AZs. The E-face of the muscle membrane displays oval-shaped receptor-containing sites made up of tightly packed intramembranous particles. Phasic and tonic receptor particles are packed at similar densities and the measured values resemble those of several other crustacean and insect neuromuscular junctions. Overall, the similarity between phasic and tonic synapses in the packing density of particles at their presynaptic AZs and postsynaptic receptor surfaces suggests similar regulatory mechanisms for channel insertion and spacing. Furthermore, the findings suggest that morphological differences in active zones or receptor surfaces cannot account for large differences in transmitter release per synapse.

Transected crustacean motor axons consist of a soma-endowed proximal segment that regenerates and a soma-less distal segment that survives for up to a year. We report on the anatomical remodeling of the proximal segment of phasic motor nerves innervating the deep flexor muscles in the abdomen of adult crayfish following transection. The intact nerve with 10 phasic axons and its two branches with subsets of 6 and 7 of these 10 axons undergo several remodeling changes. First, the transected nerve displays many more and smaller axon profiles than the 6 and 7 axons of the intact nerve, approximately 100 and 300 profiles in the two branches of a preparation transected 8 weeks previously. Serial images of the transected nerve denote that the proliferation of profiles is due to several orders of axon sprouting primary, secondary, and tertiary branches. The greater proliferation of axon sprouts, their smaller size, and the absence of intervening glia in the one nerve branch compared with the other branch denote that sprouting is more advanced in this branch. Second, the axon sprouts are regionally differentiated; thus, although in most regions the sprouts are basically axon-like, with a cytoskeleton of microtubules and peripheral mitochondria, in some regions they appear nerve terminal-like and are characterized by numerous clear synaptic vesicles, a few dense-core vesicles, and dispersed mitochondria. Both regions possess active zone dense bars with clustered synaptic vesicles found opposite other sprouts, glia, hemocytes, and connective tissue, but because the opposing membranes are not differentiated into a synaptic contact, the active zones are extrasynaptic. Third, some of the transected axons display a glial cell nucleus denoting assimilation of an adaxonal glial cell by the transected axons. Fourth, within the nerve trunk are a few myocytes and muscle fibers. These most likely originate from adjoining and intimately connected hemocytes, because such transformation occurs during muscle repair. In a crustacean nerve, however, where muscle is clearly misplaced, its presence implies an instructive role for motor nerves in muscle formation.

Crustacean motoneurons fall into two broad classes of phasic and tonic, and within each class individual neurons show considerable differentiation of their synapses. Hence, regeneration of adult crustacean neuromuscular systems provides opportunities for assessing the role of the neuron and its target in specifying synapses. The crayfish abdominal superficial flexor muscle (SFM) is a particularly profitable preparation for examining the regeneration of tonic synapses. Here, a small population of tonic axons sprout from their cut proximal ends and reinnervate the linearly arranged glow muscle fibers, with differentiated synapses that resemble those formed by the original innervation. Manipulation of the nerve or of the target muscle revealed that some neurons regenerate synapses which are differentiated in a regional manner similar to the synapses of the intact axon, implying a retrograde signaling mechanism. Other regenerating neurons, however, deviate from their intact regional distribution, implying a cell autonomous mechanism for specification of synaptic properties. Selective operation of either presynaptic or postsynaptic influences may account for synapse specification of individual motoneurons. Conversely, regeneration and differentiation of phasic and tonic synapses appear to be intrinsically regulated, as demonstrated by allotransplanting a donor tonic or phasic nerve with its attendant ganglion onto a denervated host SFM. Compared to normal tonic synapses, regenerated phasic synapses show a greater initial release of transmitter, have thinner, mitochondria-sparse terminals, and exhibit synapses with more active zones. These properties are reminiscent of native phasic synapses. Thus, regeneration of phasic and tonic synapses appears to be regulated by the neuron itself, while synapse differentiation within each neuronal class appears to be regulated by the target muscle. Evidence for both regulatory mechanisms was found inDrosophilamotoneurons, which develop a fixed number of active zones in the absence of a target, suggesting an intrinsic mechanism, while their synaptic transmitter release is heavily modulated by a retrograde signaling mechanism. Such combinatorial effects in synapse specification may permit an optimal match between neuron and target.

Donor nerves of different origins, when transplanted onto a previously denervated adult crayfish abdominal superficial flexor muscle (SFM), regenerate excitatory synaptic connections. Here we report that an inhibitory axon in these nerves also regenerates synaptic connections based on observation of nerve terminals with irregular to elliptically shaped synaptic vesicles characteristic of the inhibitory axon in aldehyde fixed tissue. Inhibitory terminals were found at reinnervated sites in all 12 allotransplanted-SFMs, underscoring the fact that the inhibitory axon regenerates just as reliably as the excitatory axons. At sites with degenerating nerve terminals and at sparsely reinnervated sites, we observe densely stained membranes, reminiscent of postsynaptic membranes, but occurring as paired, opposing membranes, extending between extracellular channels of the subsynaptic reticulum. These structures are not found at richly innervated sites in allotransplanted SFMs, in control SFMs, or at several other crustacean muscles. Although their identity is unknown, they are likely to be remnant postsynaptic membranes that become paired with collapse of degenerated nerve terminals of excitatory and inhibitory axons. Because these two axons have uniquely different receptor channels and intramembrane structure, their remnant postsynaptic membranes may therefore attract regenerating nerve terminals to form synaptic contacts selectively by excitatory or inhibitory axons, resulting in postsynaptic specification.

Blue crabs are excellent swimmers, using their highly modified last pereiopods as sculling paddles. Hence, the hypertrophied paddle opener muscle was examined for adaptations of its motor innervation by an excitor and a specific inhibitor axon. The muscle has a uniform composition of slow fibers with long (6–12 µm) sarcomere lengths. Individual fibers are richly innervated with approximately two-thirds excitatory and one-third inhibitory innervation. The profuse excitatory innervation reflects the high activity levels of this motoneuron in swimming. Adaptation to sustained activity associated with swimming is also reflected in the motor nerve terminals by a high concentration of energy source, which is equally divided between glycogen granules and mitochondria, the former providing a more rapid source of energy. The excitor axon makes predominantly neuromuscular synapses, but also a few synapses onto the inhibitor axon. The location of these excitatory axoaxonal synapses suggests regional modulation of the inhibitor axon. The specific inhibitor axon makes less than two-thirds of its synapses with the muscle fiber, regulating contraction via postsynaptic inhibition. The remaining inhibitory synapses are onto the excitor axon, signaling very strong presynaptic inhibition. Such presynaptic inhibition will effectively decouple the opener muscle from the stretcher muscle even though both are innervated by a single excitor axon.

Crustacean muscles are unique in their innervation by both excitatory and inhibitory neurons; therefore, they exhibit polyneuronal and multiterminal innervation. Because excitatory motoneurons are broadly divided into phasic and tonic types, we hypothesized that inhibitory neurons would follow a similar dichotomy. The abdominal extensor muscles in crayfish are separated into parallel deep and superficial bundles; the former has fast muscle fibers innervated by phasic excitatory motoneurons, and the latter has slow fibers supplied by tonic excitatory motoneurons. Each muscle also is innervated by a single, separate inhibitory neuron that uses γ‐aminobutyric acid (GABA) as the inhibitory neurotransmitter. The pattern of axonal branching by the separate inhibitory axons in phasic and tonic abdominal extensor muscles was visualized with confocal microscopy in preparations labeled for GABA‐like immunoreactivity. Initial observations indicated that the phasic muscle was covered by extensive GABAergic, filiform axon terminals, whereas innervation of the tonic muscle was comprised of more localized and varicose terminals. With quantitative analyses, we found that the phasic axon has a more highly branched nature than the tonic in first‐ and second‐order branches. The phasic axon branches also were significantly longer than the tonic branches in the second‐ and third‐order branches. Synaptic varicosities in the phasic branches were smaller and less frequent than those in the tonic branches. The fine structure of the inhibitory nerve terminals near synaptic contacts examined with thin‐serial‐section electron microscopy revealed distinct differences between the phasic system and the tonic system. The phasic terminals were smaller in cross‐sectional area than the tonic terminals, and they had smaller synapses and fewer mitochondria. The presynaptic active zone dense bodies were similar in length and number between phasic and tonic synapses. However, their number per synaptic area was two‐fold higher in phasic synapses compared with tonic synapses because of the smaller size of the phasic synapses. Thus, within the same neuromuscular system, inhibitory synaptic terminals revealed unique phasic and tonic identities similar to those observed for the excitatory axons. J. Comp. Neurol. 435:283–290, 2001. © 2001 Wiley‐Liss, Inc.

Calcium entry into the presynaptic terminal is the trigger for transmitter release at chemical synapses

Separate phasic or tonic nerves allotransplanted to reinnervate a denervated slow superficial flexor muscle (SFM) in the abdomen of adult crayfish regenerate synaptic nerve terminals with phasic or tonic properties. To test competitive interactions between tonic and phasic axons, we allotransplanted the sixth abdominal ganglion with its third nerve root containing a mixture of phasic and tonic axons onto the denervated SFM. The resulting reinnervation of the SFM was compared to the normal innervation on the contralateral intact SFM, which receives innervation only from tonic motoneurons. Variable sizes of excitatory postsynaptic potentials indicated that 2–3 axons innervated each muscle fiber of the SFM in both the allotransplant and normal preparations. Compared to the normal tonic terminals on the intact contralateral side, the allotransplanted synaptic terminals had more phasic-like properties; specificially, they gave rise to larger synaptic potentials, had a lower mitochondrial content and contained a higher density of active zone dense bars per synapse. Moreover, prolific sprouting of the axons in the regenerated nerve, typical of phasic axons, points to more vigorous regeneration of phasic rather than tonic axons to the denervated SFM. In keeping with this prolific axon sprouting, there was both a much higher density of innervation in the allotransplanted SFM compared to the normal SFM, and a higher frequency of extrasynaptic active zones in regenerated terminals of the mixed nerve compared to those of the tonic nerve. Thus, an allotransplanted mixed nerve regenerates mainly phasic axons and synapses on the slow denervated SFM, demonstrating the instructive nature of the neuron in synapse specification, as well as the permissive nature of the target muscle.

Synaptic differentiation among crustacean phasic motoneurons was investigated by characterizing the synaptic output and nerve terminal morphology of the two axons to the adductor exopodite muscle in the crayfish uropod. The muscle is of the fast type with short sarcomeres (2–3 µm) and a low thin to thick filament number (6:1). On single muscle fibers, excitatory postsynaptic potentials generated by the large-diameter axon are significantly larger than those by the small-diameter axon suggesting a presynaptic origin for these differences. Nerve terminals arising from these two axons have typical phasic features, filiform shape and a low (6–8%) mitochondrial density. Synaptic contacts are similar in size between the two axons as is the length and number of active zone dense bars at these synapses. The large-diameter axon, however, exhibits a twofold larger area of nerve terminal than the small-diameter axon resulting in a higher density of synapses per muscle fiber. Hence, differences in synaptic density may in part account for differences in synaptic output between these paired phasic axons.

Most studied snapping shrimp are found in male‐female pairs, cohabiting a common shelter. Studying Alpheus heterochelis in the laboratory, we determined that both sexes discriminate a former mate, from which they have been separated for 24 h, from a stranger. Strangers are more aggressive towards each other and show significantly lower frequencies of non‐agonistic (stroking and touching) behaviours than do former mates. We also established that the probability of re‐pairing between former mates and the pairing latency are strongly dependent on which sex remained in the home tank. If the female remains resident, she is equally likely to pair with a stranger and with her former mate (when tested separately), and there is no difference in pairing latency. If the male remains resident, the frequency of pairing between former mates is significantly higher than between strangers, and the pairing latency is significantly shorter. From these differences, we infer that the social bond may be adapted to absence from the shelter by the female, but not by the male.

Mutations of the genes rutabaga (rut) and dunce (dnc) affect the synthesis and degradation of cAMP, respectively, and disrupt learning in Drosophila. Combined ultrastructural analysis and focal electrophysiological recording in the larval neuromuscular junction revealed a loss of stability and fine tuning of synaptic structure and function in both mutants. Increased ratios of docked/undocked vesicles and poorly defined synaptic specializations characterized dnc synapses. In contrast, rut boutons possessed fewer, although larger, synapses with lower proportions of docked vesicles. At reduced Ca2+ levels, decreased quantal content coupled with an increase in failure rate was seen in rut boutons and reduced pair-pulse facilitation were found in both rut and dnc mutants. At physiological Ca2+ levels, strong enhancement, instead of depression, in evoked release was observed in some dnc and rut boutons during 10 Hz tetanus. Furthermore, increased variability of synaptic transmission, including fluctuation and asynchronicity of evoked release, paralleled an increase in synapse size variation in both dnc and rut boutons, which might impose problems for effective signal processing in the nervous system. Pharmacological and genetic studies indicated broader ranges of physiological alteration by dnc and rut mutations than either the acute effects of cAMP analogs or the available mutations that affect cAMP-dependent protein kinase (PKA) activity. This is consistent with previous reports of more severe learning defects in dnc and rut mutations than these PKA mutants and allows identification of the phenotypes involving long-term developmental regulation and those conferred by PKA.

Movements of the foregut in crustaceans are produced by striated muscles that are innervated by motor neurons in the stomatogastric ganglion (STG). Firing of the STG motor neurons generates excitatory junctional potentials (EJPs) in the stomach muscles. We now provide evidence for the existence of separate inhibitory and neuromodulatory innervations of some pyloric muscles in the foregut of several crabs, Callinectes sapidus, Cancer magister, and Cancer borealis. Electron microscopic examination of several pyloric muscles revealed three distinct types of nerve terminals. Excitatory terminals were readily identified by the spherical shape of their small, clear synaptic vesicles. These terminals also housed a few large dense core vesicles. Inhibitory nerve terminals were recognized by the elliptical shape of their small, clear synaptic vesicles, and contacted the muscles at well-defined synapses equipped with dense bar active zones. Bath application of GABA reduced the amplitudes of EJPs in a pyloric muscle of C. borealis, consistent with the presence of GABAergic inhibitory innervation. Neuromodulatory terminals were characterized by their predominant population of large dense and dense core vesicles. These terminals formed synapses with presynaptic dense bars on the muscle, as well as on the excitatory and inhibitory nerve terminals. The presence of the inhibitory and neuromodulatory terminals creates a functional context for previously described reports of neuromodulatory actions on stomach muscles and suggests that the transfer function from STG motor patterns to pyloric movement may be orchestrated by a complex innervation from sources outside of the STG itself. J. Camp. Neural. 425:70-81, 2000. (C) 2000 Wiley-Liss, Inc.

Phasic or tonic nerves transplanted onto a denervated slow superficial flexor muscle in adult crayfish regenerated synaptic connections that displayed large or small excitatory postsynaptic potentials (EPSPs), respectively, suggesting that the neuron specifies the type of synapse that forms (Krause et al., J Neurophysiol 80:994-997, 1998). To test the hypothesis that such neuronal specification would extend to the synaptic structure as well, we examined the regenerated synaptic terminals with thin serial section electron microscopy. There are distinct differences in structure between regenerated phasic and tonic innervation. The phasic nerve provides more profuse innervation because innervation sites occurred more frequently and contained larger numbers of synaptic terminals than the tonic nerve. Preterminal axons of the phasic nerve also had many more sprouts than those of the tonic nerve. Phasic terminals were thinner and had a lower mitochondrial volume than their tonic counterparts. Phasic synapses were half the size of tonic ones, although their active zone-dense bars were similar in length. The density of active zones was higher in the phasic compared with the tonic innervation, based on estimates of the number of dense bars per synapse, per synaptic area, and per nerve terminal volume. Because these differences mirror those seen between phasic and tonic axons in crayfish muscle in situ, we conclude that the structure of the regenerated synaptic terminals identify with their transplanted axons rather than with their target muscle. Therefore, during neuromuscular regeneration in adult crayfish, the motoneuron appears to specify the identity of synaptic connections.

Motor nerve terminals on abdominal body-wall muscles 6A and 7A in larval flesh flies were investigated to establish their general structural features with confocal microscopy, transmission electron microscopy, and freeze-fracture procedures. As in Drosophila and other dipterans, two motor axons supply these muscles, and two morphologically different terminals were discerned with confocal microscopy: thin terminals with relatively small varicosities (Type Is), and thicker terminals with larger varicosities (Type Ib). In serial electron micrographs, Type Ib terminals were distinguished from Type Is terminals by their larger cross-sectional area, more extensive subsynaptic reticulum, more mitochondrial profiles, and more clear synaptic vesicles. Type Ib terminals possessed larger synapses and more synaptic contact area per unit terminal length. Although presynaptic dense bars of active zones were similar in mean length for the two terminal types, there were almost twice as many dense bars per synapse for Type Ib terminals. Freeze-fractures through the presynaptic membrane showed particle-free areas indicative of synapses on the P-face, within which were localized aggregations of large intramembranous particles indicative of active zones. These particles were similar in number to those found at active zones of several other arthropod neuromuscular junctions. In general, synaptic structural parameters strongly paralleled those of the anatomically homologous muscles in Drosophila melanogaster. In live preparations, simultaneous focal recording from identified varicosities and intracellular recording indicated that the two terminals produced excitatory junction potentials of similar amplitude in a physiological solution similar to that used for Drosophila.

Cell types in the regenerating claws of adult snapping shrimps, Alpheus heterochelis, are described, based onelectron microscopy. Following autotomy of a limb, the coxal stump is secured by a membrane lined by a layer of proliferatingepithelial cells. Numerous fibroblasts with long cytoplasmic processes form small fluid-filled compartments that provide astructural framework and are inundated with mostly hemocytes and blood vessels. Agranular hemocytes are uncommoncompared with granular ones, which have prominent pseudopodia, vacuoles, and lysosomes, features that suggest a phagocyticfunction. The cytoplasmic network formed by fibroblasts persists in the regenerating blastema and papilla, together withgranular hemocytes and blastemal cells. Close structural associations were observed amongst all four cell types. Regionalproliferation of epithelial cells subdivides the distal tip of the papilla into the presumptive propus and dactyl and marks thebeginning of segmentation, which proceeds in a distal to proximal direction. This is accompanied by the appearance of firstafferent innervation, also proceeding in a distal to proximal direction, and multinucleate myoblasts identified by fragments ofmyofibrils, then efferent innervation and well-organized muscle. Prominent intercellular contacts between hemocytes and othercell types within the papilla may serve for adhesion as well as for communication. The early and prevalent appearance ofhemocytes in the regenerating limb bud, as well as their pluripotent nature in other regenerating tissues, implicates them as theorigin of blastemal cells.

Synapses at larval neuromuscular junctions of the flies Drosophila melanogaster and Sarcophaga bullata are not distributed randomly. They have been studied in serial electron micrographs of two identified axons (axons 1 and 2) that innervate ventral longitudinal muscles 6 and 7 of the larval body wall. The following fly larvae were examined: axon 1-wild-type Sarcophaga and Drosophila and Drosophila mutants dunce(m14) and fasII(e76), a hypomorphic allele of the fasciclin II gene; and axon 2-drosophila wild-type, dunce(m14), and fasII(e76). These lines were selected to provide a wide range of nerve terminal phenotypes in which to study the distribution and spacing of synapses. Each terminal varicosity is applied closely to the underlying subsynaptic reticulum of the muscle fiber and has 15-40 synapses. Each synapse usually bears one or more active zones, characterized by dense bodies that are T-shaped in cross section; they are located at the presumed sites of transmitter release. The distribution of synapses was characterized from the center-to-center distance of each synapse to its nearest neighbor. The mean spacing between nearest-neighbor pairs ranged from 0.84 mu m to 1.05 mu m for axon 1, showing no significant difference regardless of genotype. The corresponding values for axon 2, 0.58 mu m to 0.75 mu m, were also statistically indistinguishable from one another in terminals of different genotype but differed significantly from the values for axon 1. Thus, the functional class of the axon provides a clear prediction of the spacing of its synapses, suggesting that spacing may be determined by the functional properties of transmission at the two types of terminals. Individual dense bodies were situated mostly at least 0.4 mu m away from one another, suggesting that an interaction between neighboring active zones could prevent their final positions from being located more closely. (C) 1998 Wiley-Liss, Inc.