Nitric oxide synthase
A requirement for nitric oxide (NO) in visual system development has been demonstrated in many model systems, but the role of potential downstream effector molecules has not been established. Developing Drosophila photoreceptors express a NO-sensitive soluble guanylate cyclase (sGC: Guanyl cyclase alpha-subunit at 99B), whereas the optic lobe targets express NO synthase. Both of these molecules are expressed after photoreceptor outgrowth to the optic lobe, when retinal growth cones are actively selecting their postsynaptic partners. Inhibition of the NO-cGMP pathway in vitro leads to overgrowth of retinal axons. Flies mutant for the alpha subunit gene of the Drosophila sGC (Gcalpha1) have been examined. This mutation severely reduces but does not abolish GCalpha1 protein levels and NO-stimulated sGC activity in the developing photoreceptors. Although few mutant individuals possess a disorganized retinal projection pattern, pharmacological NOS inhibition during metamorphosis increases this disorganization in mutants to a greater degree than in the wild type. Adult mutants lack phototactic behavior, and the off-transient component of electroretinograms was frequently absent or greatly reduced in amplitude. Normal phototaxis and off-transient amplitude are restored by heat shock-mediated Gcalpha1 expression applied during metamorphosis but not in the adult. It is proposed that diminished sGC activity in the visual system during development causes inappropriate or inadequate formation of first-order retinal synapses, leading to defects in visual system function and visually mediated behavior (Gibbs, 2001).
Genetic mutants in Gcalpha1 were produced with EMS mutagenesis. Four alleles of Gcalpha1 were recovered. On the basis of decreases in intensity of GCalpha1 immunoreactivity on a Western blot of adult heads, the allelic series is Gcalpha15 = Gcalpha11 = Gcalpha13 > Gcalpha12. The differences in the levels of GCalpha1 protein are virtually indistinguishable among Gcalpha15, Gcalpha11, and Gcalpha13. Almost all of the following experiments were performed with Gcalpha11, the first to be isolated. In a Western blot of protein extract from wild-type adult heads, the anti-GCalpha1 antibody recognized a band of ~76 kDa. In contrast, GCalpha1 antibody staining was greatly reduced in Gcalpha11 flies. A transgene containing the wild-type GCalpha1 was expressed with a 1 hr heat shock in the Gcalpha11 mutant, which increases the anti-GCalpha1 staining to wild-type levels. The Gcalpha11 mutation not only reduces GCalpha1 staining on a Western blot but also greatly attenuates the synthesis of cGMP in the nervous system in response to NO. The activity of sGC in the developing Drosophila nervous system was visualized by exposing the tissue to the NO donor SNP and the phosphodiesterase inhibitor IBMX, followed by immunocytochemistry with an anti-cGMP antibody. At 24 hr APF, the CNS of the Gcalpha11 mutants showed cGMP immunoreactivity (cGMP-IR) in only a few neurons of the central brain, suboesphageal ganglion, and ventral nerve cord in response to SNP and IBMX. This represents a small subset of the cGMP-positive population of cells observed in the wild-type CNS at 24 hr APF after similar treatment. Most cells that showed cGMP-IR in Gcalpha11 appear to be the same cells that remain cGMP-positive in the wild-type CNS after treatment with an sCG inhibitor and are frequently cGMP-positive in nervous systems that have not been exposed to SNP and IBMX. A receptor-type guanylate cyclase has been cloned from Drosophila (Gibbs, 2001).
The photoreceptors possess NO-sensitive sGC activity during the first half of metamorphic development, after they arrive at their optic lobe targets. cGMP immunoreactivity is observed in the cell bodies of photoreceptors in the developing retina as well as their axons projecting into the lamina and medulla regions of the optic lobe. These represent the axons of both classes of photoreceptors, R1-6 and R7/8. The axons of Bolwig's nerve are also cGMP-positive. In contrast, a very low level of cGMP-IR is observed in the visual system of Gcalpha11 after SNP and IBMX treatment at 24 hr APF, primarily in the cell bodies of the eye imaginal disc. This faint cGMP-IR most likely reflects the activity of remaining low levels of GCalpha1 protein in the Gcalpha11 mutant. Heat shock-induced expression of a wild-type Gcalpha1 transgene restores the strong response of the photoreceptors to NO in Gcalpha11 at 24 hr APF. Two hours after a single 45 min heat shock, cGMP production was observed in the photoreceptor cell bodies and axons and the larval pioneers after treatment with 1 mM SNP and IBMX. The intensity of cGMP-IR induced with heat shock was not observed, however, without SNP and IBMX exposure. Surprisingly, after heat shock expression of Gcalpha1, strong cGMP-IR was also observed in the medulla interneurons of the optic lobe of the Gcalpha11 mutants. This was in contrast to the wild-type visual system, in which NO-induced cGMP accumulation is very low in the medulla at 24 hr APF, becoming more prominent by ~48 hr APF. Because both alpha and ß subunits are required for soluble guanylate cyclase activity in response to NO, it is concluded that the GCß1 protein must already be present in the medulla interneurons at 24 hr APF. The delayed onset of NO sensitivity in these cells may then reflect the regulated expression of GCalpha1 rather than GCß (Gibbs, 2001).
Cells of the lamina and medulla express NOS and pharmacological inhibition of NOS or sGC in vitro causes disorganization and overgrowth of the retinal projections in the wild-type visual system. On the basis of these in vitro results, it was predicted that flies mutant for Gcalpha1 would display similar defects in visual system organization. The retinal projection pattern of R7/8 in the medulla of all four Gcalpha1 alleles was examined throughout metamorphosis and in adults, using whole-mount immunocytochemistry with antibodies to chaoptin and fasciclin II, both of which label photoreceptor axons. In general, the mutants did not show dramatic disruption of the projection pattern of R7/8, although minor defects have been noted in some cases. At 48 hr APF, the chaoptin-stained retinal projections of a few Gcalpha11 mutants were slightly disrupted when compared with the wild type, producing gaps in the pattern and extension of retinal axons slightly beyond the medullar margin and producing a 'ragged' border. Similarly, staining with a fasciclin II antibody at the same stage revealed what appeared to be a few retinal axons projecting well beyond the medulla in some individuals. Although this phenotype was not widely observed, it was never seen in age-matched wild-type flies and may reflect the variable penetrance of the Gcalpha11 mutation (Gibbs, 2001).
The Gcalpha11 flies express low levels of GCalpha1 protein, which is also reflected in very low levels of NO-sensitive sGC activity in the developing visual system. sGC is an enzyme; thus, even a small amount is capable of producing a greatly amplified cGMP signaling cascade in the presence of sufficient ligand. It was therefore predicted that although this residual sGC activity normally prevents retinal axon overgrowth in most of the Gcalpha11 mutants, it would not be enough to compensate for decreases in NO production in vitro. CNSs and attached eye discs from wild-type and mutant white puparia were placed in culture with the hormone 20-hydroxyecdysone, which promotes metamorphic development of the nervous system in vitro, or hormone plus the competitive NOS inhibitor L-NAME. After 96 hr in culture, the tissue was then processed for chaoptin immunocytochemistry. For analysis, focus was placed on the projection pattern of R7/8 in the medulla, which is easily resolved in whole-mount preparations. Nervous systems were assigned a score of 0-4 based on the severity of disorganization in the projection pattern. The scores for all samples were averaged to obtain a mean score for a given treatment, called the disruption index, and the percentages of nervous systems showing any retinal growth beyond the medulla were determined for mutant and wild-type nervous systems for each treatment (Gibbs, 2001).
A delay in neural development occurs with culturing, so that after 96 hr in vitro, the visual system has progressed to a stage comparable to 48-50 hr APF. In control cultures without inhibitor, a normal, well organized projection pattern is typically seen in the medulla, and no retinal fibers are seen growing past medullar targets in either wild-type or mutant visual systems. Although not significantly different, the disruption index for the mutants under control conditions is slightly higher than that for the wild type, suggesting that the Gcalpha11 mutation caused subtle projection pattern defects in a low percentage of individuals, as is observed with whole-mount analysis of noncultured nervous systems. When a low level of L-NAME is added to the cultures, essentially no pattern disruption is observed in the medulla of wild-type nervous systems. However, this concentration of L-NAME causes the growth of many retinal axons beyond the posterior medulla of the Gcalpha11 optic lobe. This effect is more severely pronounced in the mutants with a higher level of L-NAME. Under these conditions, the retinal fibers produce a dense, disorganized tangle in the medulla and extend many projections into the lobula. This is not the case for wild-type nervous systems, in which treatment with higher levels of L-NAME produce a slight disorganization of the projection pattern, but no retinal fibers are observed projecting beyond the medulla (Gibbs, 2001).
To examine visual system function in the Gcalpha1 mutant adults, a standard behavioral assay was used for fast phototaxis, using a countercurrent apparatus. In these experiments, a population of ~100 flies was placed in the first of six clear tubes, laid onto a horizontal surface, and transferred through the tubes at 30 sec intervals as they moved toward a light source. An average of 65% of the total population of wild-type flies consistently moved toward the light after each transfer to end up in the last tube (tube 6) by the end of the trial. All four of the Gcalpha1 mutant strains showed a reduction in positive phototaxis at 1 week after eclosion when compared with the claret1 progenitor strain, but the Gcalpha11 mutants were the most severely compromised. A combined 12%-13% of Gcalpha11 flies made it to the last two tubes (tubes 5 and 6) of the apparatus in repeated trials, compared with 65% of claret1 controls. This behavior is not the result of negative phototaxis, as tested by reversing the orientation of the countercurrent apparatus relative to the light source. The profile of Gcalpha11 also remains unchanged in the absence of light. Although visually mediated, phototaxis requires the integration of many behaviors. However, Gcalpha11 adults show normal performance in a geotaxis assay, demonstrating that these flies do not possess gross motor impairments (Gibbs, 2001).
An investigation was carried out to see whether restoring sGC activity during the period of retinal innervation would improve the phototactic performance of the Gcalpha11 mutants, using a heat shock-inducible Gcalpha1 transgene. In this experiment, a 45 min heat shock was given every 8 hr for the first 48 hr of pupal development, approximately encompassing the window of NO-sensitive cyclase activity observed in the photoreceptors. Phototaxis was tested 1 week after adult eclosion, 9 d after the last heat shock. At the completion of this experiment, 51% of the total population of hs-gc+;Gcalpha11 adults exposed to heat shock during metamorphosis converged in the last two tubes of the countercurrent apparatus. This compares with 13% for the non-heat-shocked hs-gc+;Gcalpha11 adults and 65% for the claret1 progenitor strain. In addition, the percentage of flies in the first two tubes is nearly identical between ca1 controls and heat-shocked hs-gc+;Gcalpha11 adults. Thus positive phototaxis is restored in the Gcalpha11 mutants to near wild-type levels when Gcalpha1 is expressed during the first half of metamorphosis, and the phototactic profile is made to resemble that of the wild type. An improvement in positive phototaxis is not observed when adult hs-gc+;Gcalpha11 flies are tested 3 hr after a single acute heat shock. In addition, increased positive phototaxis is not seen in hs-gc+; Gcalpha11 mutants that were exposed to heat shock every 12 hr for 48 hr as adults and tested 24 hr after the last heat shock. These results strongly support the hypothesis that the function of sGC in the phototactic response is developmental, rather than a physiological requirement for sGC activity at the time the behavior is being performed (Gibbs, 2001).
ERGs were perfomed in a further attempt to examine the effects of the Gcalpha11 mutation on visual system function. The ERG primarily characterizes the electrophysiological response to light of a subset of photoreceptors, R1-6, and their postsynaptic cells in the lamina. The ERG consists of a corneal positive 'on-transient,' followed by a sustained negative wave that lasts throughout the period of illumination, and then a corneal negative 'off-transient.' The off-transient results from the summation of postsynaptic potentials in the monopolar neurons L1 and L2 of the lamina and can thus be used as an indicator of synaptic efficacy (Gibbs, 2001).
82% of the ERGs from the Gcalpha11 flies lack off-transients or have off-transients that are greatly reduced in amplitude. Gcalpha11 flies that had Gcalpha1 supplied with heat shock during the first half of metamorphosis express a mean off-transient amplitude that is comparable with that of wild-type controls. The effect of the Gcalpha11 mutation appears to be limited to the off-transient amplitude, because both the mean duration of the off-transient peak and the mean amplitude of the sustained component are not significantly different among all three populations. Thus, both positive phototaxis and a normal ERG trace can be restored to adult flies expressing very low levels of sGC activity when Gcalpha1 is supplied developmentally as the retinal growth cones are undergoing target selection in the optic lobe (Gibbs, 2001).
The alpha subunit of a soluble guanylate cyclase has been repeatedly cloned from Drosophila. However, there is controversy as to the expression pattern of Gcalpha1. Using Northern blot analysis, Gcalpha1 mRNA has been demonstrated in wild-type but not eyeless adult fly heads, suggesting that Gcalpha1 is expressed primarily in the adult retina. An antibody was employed to localize the GCalpha1 protein to the retina. Although abundant Gcalpha1 mRNA has been seen in a Northern blot of adult heads, the same probe failed to hybridize to adult retinal tissue in situ, and no retinal staining was found with a GCalpha1 antibody. In this study evidence is presented that Gcalpha1 is expressed in the photoreceptors during development. A genetic mutation that diminishes GCalpha1 protein levels, Gcalpha11, reduces NO-induced production of cGMP in developing photoreceptors: cGMP is restored with heat shock-mediated expression of wild-type Gcalpha1. Thus it is concluded that Gcalpha1 is normally expressed in the photoreceptors from ~12 to 48 hr APF, and it is inferred that the ß subunit is also present at this time. It has not been determined whether the observed loss of NO sensitivity in the photoreceptors at ~48 hr APF and into adulthood is attributable to changes in Gcalpha1 expression. However, spatiotemporal changes in alpha and ß subunit expression may provide a molecular basis for regulating the timing of NO-sensitive sGC activity in the photoreceptors and other cells of the developing visual system (Gibbs, 2001).
Reports of Gcalpha1 expression in the adult retina, and cGMP-mediated enhancement of the photoresponse in isolated Drosophila photoreceptors have implicated cGMP as a putative mediator of a phototransduction mediator in flies. Studies show that NO and cGMP can modulate the locust photoresponse and signaling in other insect sensory systems. The Gcalpha1 mutants were initially generated to further establish the role of cGMP in phototransduction; however, both intracellular and extracellular recordings from these flies have revealed a normal response of the photoreceptors themselves to light. In other studies, inositol trisphosphate and diacylglycerol were shown to be primarily responsible for generating the depolarizing potential in the Drosophila retina. The results presented here suggest that the requirement for cGMP in the Drosophila visual system is developmental, rather than physiological, for at least two reasons: (1) cGMP was never observed in the photoreceptors after 48 hr APF, in the presence or absence of NO stimulation; (2) although mutant adults lack positive phototaxis (a basic visually mediated behavior) positive phototaxis is only restored to Gcalpha11 adults when Gcalpha1 is supplied with heat shock during the first 48 hr of metamorphosis, encompassing the period of observed NO-sensitive sGC activity in the photoreceptors. In contrast, phototaxis did not improve when Gcalpha1 was expressed acutely or chronically in adult mutants. The ERG results also support the hypothesis that sGC signaling is required during the first half of metamorphosis, when retinal growth cones are selecting postsynaptic partners in the optic lobe and NO-sensitive sGC activity is observed. The sustained depolarization of the photoreceptors was normal in Gcalpha11 adults, but the off-transients were frequently undetectable or greatly reduced in amplitude. In addition, some off-transients contained two peaks. The postsynaptic responses of the laminar monoplar cells are responsible for generating the off-transient in Drosophila. Thus, the decreased and aberrant off-transients observed in GCalpha11 implicate a defect in the postsynaptic response of laminar monopolar cells to retinal input in these mutants. This abnormal postsynaptic response could arise from disorganized retinal synapses, perhaps as a result of subtle deviations in growth cone behavior in the absence of normal cGMP levels. When Gcalpha1 is expressed with heat shock in Gcalpha11 mutants during the first half of metamorphosis, the off-transient shape and amplitude are indistinguishable from those of the wild type. The behavioral and electrophysiological results support the hypothesis that cGMP signaling is required in the photoreceptors to promote the appropriate wiring of first-order retinal synapses during metamorphosis. However, because the Gcalpha11 mutation and the heat shock Gcalpha1 expression are global and not eye-specific, the possibility cannot be excluded that formation of downstream connections in the optic lobe and brain also require GCalpha1. The behavioral results in particular may reflect the effects of Gcalpha1 expression on these connections. However, from 12 to 48 hr APF NO-sensitive cGMP production is observed almost exclusively in the photoreceptors and not in other visual centers until later in development. This is also the developmental window during which heat shock Gcalpha1 expression rescues both the phototactic and ERG phenotypes, which strongly suggests that the photoreceptors themselves require GCalpha1 to ensure the appropriate wiring of first-order retinal synapses (Gibbs, 2001).
Despite the profound defects in visual system function, no dramatic and consistent disorganization of the retinal projections is observed in Gcalpha11. This contrasts with previous results, in which pharmacological inhibition of NO-sGC signaling caused severe disruption of the wild-type projection pattern in vitro (Gibbs, 1998). There are several possible explanations for these results. (1) The Gcalpha11 mutants are hypomorphs and do show low levels of GCalpha1 protein and NO-sensitive sGC activity in the photoreceptors during metamorphosis. Residual enzymatic activity of these low levels of functional sGC could produce adequate levels of retinal cGMP to prevent significant axon overgrowth provided that the ligand, NO, is present at wild-type levels. This is supported by experiments in which nervous systems from Gcalpha11 pupae were exposed to low levels of a NOS inhibitor. Under these conditions, the resulting disorganization and overgrowth of the retinal axons were much greater than in wild-type controls. These results suggest that residual GCalpha1 activity and production of cGMP by endogenous NO in Gcalpha11 is sufficient to prevent overgrowth of retinal axons in vivo but cannot compensate for suppression of NO signaling in vitro. (2) The Gcalpha11 visual system has been examined only at the whole-mount level. A more detailed analysis of the retinal projections and cartridge organization, perhaps using electron microscopic techniques, may reveal further architectural defects in the Gcalpha11 mutants. (3) Other signaling pathways have been shown to contribute to formation of the Drosophila retinal projection pattern, and these may compensate for decreased sGC activity during visual system development (Gibbs, 2001).
It is proposed that Gcalpha1expression and subsequent sGC- and NO-induced cGMP activity in the photoreceptors regulate synapse formation between photoreceptors and optic lobe neurons by exerting subtle effects on retinal growth cone behavior during cartridge assembly. Expression of NO-sensitive sGC activity was never seen during retinal axon outgrowth but only in photoreceptors that had arrived at their respective optic ganglia. This makes it unlikely that NO and cGMP are acting in a chemoattractive manner to guide retinal growth cones to the optic lobe. The metamorphic period when NOS expression in the optic lobe and NO-sensitive sGC expression in the photoreceptors was observed (12-48 hr APF) correlates temporally with when retinal growth cones are actively seeking out optic lobe cells with which they will form synaptic cartridges. The results support a model wherein NO from the target acts to stimulate cGMP synthesis in newly arrived retinal growth cones, stabilizing them and preventing further axonal extension but still allowing lateral movement within the target region. When NO production or sGC activity is inhibited pharmacologically, this stabilization is lost, and the photoreceptors resume longitudinal growth. The current results show that although genetically reducing sGC activity leads to more subtle defects in visual system architecture, perhaps at the level of cartridge organization, the overall effects of this mutation on adult visual system function are profound. NO and cGMP have been proposed to regulate vertebrate visual system development by acting as effectors of activity-dependent refinement mechanisms. However, this is not likely to be the case in Drosophila, because the visual system can develop normally in the absence of histamine, the primary visual neurotransmitter. Instead, the effects of NO-induced cGMP production may act to regulate growth cone behavior (Gibbs, 2001).
Animal organ development requires that tissue patterning and differentiation is tightly coordinated with cell multiplication and cell cycle progression. Several variations of the cell cycle program are used by Drosophila cells at different stages during development. In imaginal discs of developing larvae, cell cycle progression is controlled by a modified version of the well-characterized mammalian retinoblastoma (Rb) pathway, which integrates signals from multiple effectors ranging from growth factors and receptors to small signaling molecules. Nitric oxide (NO), a multifunctional second messenger, can reversibly suppress DNA synthesis and cell division. In developing flies, the antiproliferative action of NO is essential for regulating the balance between cell proliferation and differentiation and, ultimately, the shape and size of adult structures in the fly. The mechanisms of the antiproliferative activity of NO in developing organisms are not known, however.
Transgenic flies expressing the Drosophila nitric oxide synthase gene (dNOS1) and/or genes encoding components of the cell cycle regulatory pathways (the Rb-like protein RBF and the E2F transcription factor complex components dE2F and dDP) combined with NOS inhibitors were used to address this issue. Manipulations of endogenous or transgenic NOS activity during imaginal disc development can enhance or suppress the effects of RBF and E2F on development of the eye. These data suggest a role for NO in the developing imaginal eye disc via interaction with the Rb pathway (Kuzin, 2000).
To regulate ectopic production of NO during development, transgenic lines of Drosophila were generated in which the expression of dNOS1 cDNA was controlled either by the heat-shock-inducible hsp70 promoter (hs-dNOS1 flies) or by the eye-specific GMR promoter, which functions in all cells of the eye imaginal disc in, and posterior to, the morphogenetic furrow [19] (GMR-dNOS1 flies). Examination of scanning electron micrographs of the eyes of, and thin sections of the retinas of, different transgenic lines did not reveal obvious differences among eyes of wild-type flies, transgenic hs-dNOS1 flies with or without heat-shock and GMR-dNOS1 flies. This indicates that a moderate increase in NO production on its own does not noticeably affect eye development.
To investigate the relationship between NOS activity and cell cycle progression, NOS activity was manipulated in transgenic flies ectopically expressing genes of the Rb pathway in the developing eye. Drosophila RBF is structurally related to the mammalian proteins of the Rb family and, like the Rb proteins, RBF is a negative regulator of cell cycle progression. The RBF transgene was placed under control of the GMR promoter and flies with either two (GMR-RBF2) or four (GMR-RBF4) copies of the transgene were used in these experiments. The eyes of adult flies with two copies of the RBF transgenes (GMR-RBF2) appear normal, indicating that at this dosage the RBF transgene does not noticeably disturb cell division in the eye disc. When GMR-RBF2 flies are crossed to hs-dNOS1 flies and the progeny larvae are treated with heat shock before pupariation, however, the resulting adults have multiple defects in the eyes, including missing bristles and pigment cells. Pigment cells, which comprise the boundaries of each ommatidia, appear as a characteristic honeycomb pattern in thin sections of normal eyes. A lack of the regular number of pigment cells in GMR-RBF2 + hs-dNOS1 flies results in the appearance of many fused ommatidia and a rough eye phenotype. Thus, hs-dNOS1 and GMR-RBF2 flies, both of which do not affect the development of eye structure when overexpressed on their own, nevertheless yield eye defects when overexpressed together. This transgenic interaction suggests that NOS and RBF genes interact synergistically during the development of ommatidia. A similar effect was observed employing a different genetic strategy. This time, the overexpression of dNOS1 was restricted to the developing eye by crossing GMR-RBF2 flies with GMR-dNOS1 flies. These double-transgenic flies also display eye defects similar to those of heat-shocked GMR-RBF2 + hs-dNOS1 flies ó missing pigment and bristle cells and fused ommatidia. Thus, regardless of the promoter that drives the expression of the dNOS1 transgene, elevated levels of NO and RBF synergize to limit cell number in the developing eye, supporting the notion of interaction between dNOS1 and RBF genes (Kuzin, 2000).
Both RBF and NOS act to suppress cell division. If indeed NOS acts in concert with RBF during eye development, then inhibition of NOS might suppress RBF function and restore the normal number and shape of ommatidia to GMR-RBF4 flies. To test this, endogenous NOS activity was blocked in larvae of GMR-RBF4 flies using a specific NOS inhibitor L-nitroarginine methyl ester, L-NAME (which alone did not affect the eye morphology of the wild-type flies. Remarkably, the eyes of these drug-exposed transgenic flies have an almost normal phenotype as regards the number of photoreceptor and accessory cells and the number and shape of the ommatidia; only a few bristles were still missing. The antiproliferative activity of NO results from its ability to suppress DNA synthesis, as BrdU labeling of the eye imaginal discs showed that the number of cells in S phase is decreased after heat shock in flies carrying the hs-dNOS1 transgene and is increased upon inhibition of NOS activity in GMR-RBF4 flies. Thus, the inhibitory effect of RBF overexpression on cell proliferation is almost completely rescued when endogenous NOS activity is inhibited in the developing larvae (Kuzin, 2000).
In mammalian cells, Rb and Rb-related proteins bind to transcription factors of the E2F family and inhibit E2F-dependent transcription. When phosphorylated by cyclin-dependent kinases, Rb does not bind E2F and E2F-dependent transcription of several genes required for the synthesis of DNA and entry into S phase of the cell cycle is induced. Ectopic overexpression of E2F overcomes the Rb-mediated repression and induces quiescent cells to enter S phase. Similarly, in Drosophila cells, RBF is associated with the E2F transcription factor complex. In transgenic flies overexpressing dE2F and dDP under control of the GMR promoter (GMR-dE2FdDP flies), ommatidia form irregular rows and lack their regular hexagonal shape; in addition, many eye bristles are duplicated. This observation indicates that overexpression of RBF and of E2F have reciprocal effects on cell proliferation in the developing eye (Kuzin, 2000).
To determine whether the antiproliferative activity of NO can counteract excessive precursor cell proliferation caused by E2F overexpression, GMR-dE2FdDP flies were crossed with hs-dNOS1 flies. When progeny larvae are treated with heat shock, a normalized adult eye developed; in some cases a revertant (wild-type) pattern of ommatidial rows, regularly shaped ommatidia, and the usual number of bristles are seen. Similarly, progeny of a cross between GMR-dE2FdDP flies and GMR-dNOS1 flies developed more normal eyes, corroborating a specific dNOS1-E2F interaction. Thus, in contrast to GMR-RBF4 flies, in with which inhibition of NOS was needed to rescue the mutant phenotype (underproliferation of precursor cells), overexpression of dNOS1 is needed to rescue the phenotype of GMR-dE2FdDP flies (overproliferation of precursor cells). This reciprocal effect of NO levels on RBF and E2F function in cell cycle control adds considerable genetic strength to the idea that NO acts in concert with the Rb pathway to suppress cell division during eye development (Kuzin, 2000).
RBF blocks E2F-dependent transcription in cotransfection assays, in accordance with its ability to sequester E2F proteins. When expressed in the eye, GMR-RBF suppresses the rough-eye phenotype of the GMR-dE2FdDP transgenic flies. Thus, overexpression of RBF and E2F have opposing effects on the decision of precursor cells to enter the cell cycle. A test was performed to see whether NO modulates the effects of GMR-RBF2 on E2F function by inhibiting NOS activity in GMR-RBF2 + GMR-dE2FdDP flies. Ectopic expression of E2F in the developing eye increases both cell proliferation and programmed cell death; the net effect is the appearance of more cells in the eye, however. To minimize the E2F-induced augmentation of cell death, an effective inhibitor of apoptosis, the baculoviral p35 gene, under control of the GMR promoter, was used. Whereas the combination of GMR-dE2FdDP and GMR-p35 transgenes produce an even more severe phenotype than the GMR-dE2FdDP transgene alone, the GMR-RBF2 + GMR-dE2FdDP + GMR-p35 flies have a normal eye phenotype, confirming that, in the absence of programmed cell death, RBF suppresses the consequences of E2F over-expression and rescues the E2F phenotype. In contrast, inhibition of NOS activity in these GMR-RBF2 + GMR-dE2FdDP + GMR-p35 larvae prevents RBF from rescuing the E2F phenotype. In particular, when endogenous NO production is suppressed, the arrangement of ommatidia is still abnormal, and many additional bristles and pigment cells are still observed. This suggests that the RBF-E2F interaction involves NOS and that RBF requires NO to antagonize the E2F activity (Kuzin, 2000).
This study of the developing Drosophila eye presents a series of reciprocal genetic interactions that consistently suggest that NO modulates a signaling pathway involved with cell cycle control. Specifically, increased production of NO in the developing eye acts as an antiproliferative signal, whereas inhibition of NOS activity promotes additional rounds of cell division. It is considered that the reciprocal effects of E2F and NOS and complementary effects of Rb and NOS are best explained by the hypothesis that NO affects the Rb signaling pathway, thereby regulating entry into the S phase of the cell cycle (Kuzin, 2000).
To investigate the function cAMP-dependent protein kinase (PKA) exerts in the induction of long-term memory, changes in PKA activity induced by associative
learning in vivo were measured in the antennal lobes (ALs) of honeybees. The temporal dynamics of PKA activation depend on both the sequence of conditioned
and unconditioned stimuli and the number of conditioning trials. Only multiple-trial conditioning, which induces long-term memory (LTM), leads to a profound
prolongation of PKA activation mediated by the NO/cGMP system. Imitation of this prolonged PKA activation in the ALs in combination with single-trial
conditioning is sufficient to induce LTM. These findings not only demonstrate the close connection between conditioning procedure and temporal dynamics in PKA
activation but also reveal that already during conditioning a distinct temporal pattern of PKA activation is critical for LTM induction in intact animals (Müller, 2000).
Associative olfactory conditioning of the proboscis extension response (PER) in the honeybee induces different forms of memory, depending on the
number of conditioning trials. The memory induced by a single conditioning trial decays over several days and is sensitive to amnestic treatments. This memory is independent of NO synthase (NOS)
blockers and of protein synthesis. In contrast, multiple conditioning trials induce a stable, long-lasting memory.
This memory is dissectable into two independent, parallel phases. The first phase is a medium-term memory (MTM) in the hours range, which requires a constitutive
PKC activity, and the second phase is an LTM (1 day or more), which requires PKA- and NO-dependent processes. Interestingly, LTM can be divided into an early phase (eLTM, 1-2 days) and a protein synthesis-dependent late phase (lLTM, 3 days or more)(Müller, 2000 and references therein).
Even though it has been demonstrated that the cAMP cascade is important for the induction of long-lasting neuronal and behavioral changes, the findings presented here reveal evidence for a direct connection between conditioning procedure, temporal dynamics in
PKA activation, and their contribution to formation of LTM in intact animals. Direct measurement of changes in PKA activity in the ALs induced by in vivo
stimulation reveals that multiple conditioning trials that induce LTM also induce an extremely prolonged PKA activation. The latter contributes to LTM formation
processes, since imitation of the extended PKA activation in the AL in conjunction with a single conditioning trial induces LTM (Müller, 2000).
Recent findings suggest that a distinct temporal activation of the cAMP cascade, dependent on distinct stimulation parameters, is required for the induction of
long-lasting neuronal and behavioral changes. A close connection has been demonstrated between stimulation parameters and the temporal
dynamics of changing cAMP levels, adenylate cyclase activity, PKA activity, and
CREB phosphorylation (Müller, 2000 and references therein).
During associative conditioning in the honeybee, the temporal dynamics of PKA activation in the ALs depend on both the sequence of CS and US stimulation and the
number of conditioning trials. The mechanism underlying the sequence-dependent PKA activation is distinguishable and independent from that underlying
multiple trial-induced PKA activation, as demonstrated by selective impairment of the latter by blocking NOS activity. Regardless of the number of trials,
US/CS backward pairing and US stimulation induce the same transient PKA activity in the ALs. The US-induced PKA activation in the ALs is mediated by
octopamine. The octopamine in the ALs is most likely released by the VUMmx1 neuron,
which has been shown to substitute for the US function in associative olfactory conditioning. The extensive aborizations of the VUMmx1 neuron
in the ALs suggest that the US-mediated PKA activation occurs within all AL glomeruli. In contrast to this, CS stimulation induces odor-specific changes in Ca2+
levels in distinct subsets of glomeruli. Thus, it is conceivable that the prolonged PKA activation induced by CS/US
forward pairing is due to a sequence-dependent interaction between an odor-specific Ca2+-mediated process in distinct glomeruli and a general
US/octopamine-mediated process (Müller, 2000 and references therein).
Dually regulated enzymes, like the Ca2+/calmodulin-dependent adenylate cyclase, have been suggested as molecular convergence sites of different inputs important
for neuronal plasticity and learning. In membrane fractions of Aplysia neurons, the maximal in vitro activation of
the Ca2+/calmodulin-dependent adenylate cyclase is achieved when the Ca2+ stimulus precedes the transmitter stimulus. Although direct
evidence is lacking, the dually-regulated adenylate cyclase may be implicated in the sequence-specific prolongation of PKA activity induced by a CS/US forward
pairing in the honeybee (Müller, 2000).
Findings from Aplysia and Drosophila assign a critical role for induction of
long-lasting changes to the balance of activator and repressor isoforms of CREB. The results from the honeybee,
however, show that already during the short conditioning time window a distinct temporal pattern of PKA activation is critical for LTM induction.
Assuming a similar connection between the PKA pathway and CREB in the induction of LTM in honeybees, future investigations demand a characterization of
whether and how the multiple trial-induced prolonged PKA activation acts on CREB function. But since regulation of CREB isoforms and their function in long-term
neuronal and behavioral changes are results of a complex interaction of different second messenger systems, it is very likely that different signaling cascades contribute to LTM formation (Müller, 2000 and references therein).
It is conspicuous that both the formation of multiple trial-induced LTM and the prolonged PKA activation in the ALs
require NO-mediated mechanisms. The finding that photorelease of cGMP in the ALs in combination with single-trial conditioning induces LTM
supports the idea that the NO/cGMP system within the ALs mediates the prolongation of PKA activation during conditioning. Although the neurons containing the
NO-activated guanylate cyclase have not been described in the honeybee, it is most likely that the NO-releasing neurons that modulate cGMP levels in the target
cells are located within the ALs. Uncaging NO in the entire AL in combination with single-trial conditioning, however, leads to a significant reduction in conditioned
PER as tested at 3 hr and 3 days. Although the reason for this learning impairment is unknown, photolyzing NO in the entire AL probably interferes with
a specific function of NO in signal processing during olfactory learning. The latter is very likely, since an odor induces changes in Ca2+ concentrations in a subset of
glomeruli only. This in turn results in the activation of the Ca2+-dependent NOS and thus in release of NO in a characteristic subset of
glomeruli only. Possibly such a CS-specific release of NO within a subset of glomeruli contributes to aspects of olfactory signal processing required for learning (Müller, 2000 and references therein).
In this context it is interesting to note that multiple conditioning trials lead to more specific responses and thus may be based on more specific
synaptic plasticity. The latter may be due to a Hebbian mechanism of pre/postsynaptic activity detection. It has been proposed that such a mechanism may also be
essential for invertebrates and that NO may play a central function as a retrograde signaling molecule (Müller, 2000 and references therein).
A series of studies in Drosophila convincingly demonstrate that the mushroom bodies (MBs) are essential for olfactory learning, and that they support context generalization in visual learning, and are required for memory formation of
courtship conditioning. These findings not only support the important role of the MBs as multisensory processing centers but also
demonstrate that the contribution of the MBs differs, depending on the learning paradigm and the sensory modality used. In contrast to the considerable knowledge
with regard to the function of the MBs in Drosophila learning, it was only recently proposed that the ALs contribute to short-term
memory in Drosophila courtship conditioning (Müller, 2000 and references therein).
In honeybees, it has been demonstrated that initial olfactory memory (tested 20 min after conditioning) can be induced independently in either the MBs or the ALs. In contrast to Drosophila, however, the majority of studies focused on the function of the ALs in olfactory learning. It has been
demonstrated that differential olfactory conditioning causes changes in the neural representation of the rewarded and the unrewarded odor in the ALs for up to at
least 30 min after conditioning. Moreover, the requirement of a constitutively active PKC in the ALs for multiple trial-induced MTM suggests
that processes located in the ALs contribute to memory maintenance in the range of hours. The results presented here now
demonstrate that a prolonged PKA activation in the ALs induced by multiple-trial conditioning is implicated in induction of LTM. However, since imitation of
prolonged PKA activation in conjunction with single-trial conditioning does not reach the level of conditioned PER after multiple-trial conditioning, a contribution by
other brain areas must be proposed. Collectively, all these findings provide evidence that the ALs are sites that contribute to processes of associative olfactory
learning during the conditioning procedure itself and in early phases of memory formation for up to several hours. Moreover, the ALs are possibly also sites of
long-lasting structural changes. Activity-dependent changes described for the glomerular volume in the ALs may well be the result of structural
plasticity underlying long-term memory (Müller, 2000 and references therein).
Interestingly, in mice and sheep the accessory olfactory system has also been implicated in the formation of olfactory memory. While female mice form a memory of
the pheromones of the mating male, sheep learn to recognize the odors of their lambs in the first hours after birth. In both cases, NO has
been demonstrated to mediate the formation of this memory. In mice the coincident activation of pheromonal inputs and exogenous administration of NO in the
accessory olfactory system can induce a pheromone-specific olfactory memory without mating. Blocking of NOS activity in the olfactory
system of sheep prevents the formation of olfactory memory. Although the targets of the NO/cGMP system in the olfactory systems of
honeybees, mice, and sheep differ, the conspicuous parallels suggest a conserved function of NO-mediated signaling in the olfactory systems with respect to olfactory
memory formation (Müller, 2000 and references therein).
Nitric oxide has been shown to be implicated in neural plasticity that underlies processes of learning and memory. In the honeybee, studies on the role of nitric oxide in associative olfactory learning reveal its specific function in memory formation. Inhibition of nitric oxide synthase during olfactory conditioning impairs a distinct long-term memory that is formed as a consequence of multiple learning trials. Acquisition or retrieval of memory or memory formation induced by a single learning trial is not affected by blocking of nitric oxide synthase. This finding provides a first step toward dissection of molecular mechanisms involved in memory formation, in general, and the special function of nitric oxide synthase in particular (Muller, 1996).
Drosophila NOS is dependent on exogenous Ca2+/calmodulin and on NADPH, two cofactors necessary for activity of constitutive mammalian NOSs (Regulski, 1995).
Nitric oxide (NO) is involved in organ development, synaptogenesis, and response to hypoxia in Drosophila. The only gene in the fly genome that encodes Drosophila nitric-oxide synthase (dNOS) has been cloned and analyzed. It consists of 19 exons and is dispersed over 34 kilobases of genomic DNA. Alternative transcription start sites and alternative splice sites are used to generate a remarkable variety of mRNAs from the dNOS gene. Eight new transcripts have been identified that are widely expressed throughout Drosophila development and encode a family of DNOS-related proteins. Alternative splicing affects both the 5'-untranslated region and the coding region of the dNOS primary transcript. Most of the splicing alterations in the coding region of the gene led to premature termination of the open reading frame. As a result, none of the alternative transcripts encoded an enzymatically active protein. However, some of these shorter DNOS protein products can effectively inhibit enzymatic activity of the full-length DNOS1 protein when co-expressed in mammalian cells, thus acting as dominant negative regulators of NO synthesis. Using immunoprecipitation, it has been demonstrated that these short DNOS protein isoforms can form heterodimers with DNOS1, pointing to a physical basis for the dominant negative effect. These results suggest a novel regulatory function for the family of proteins encoded by the Drosophila NOS gene (Stasiv, 2001).
Drosophila NO synthase combines some of the features of all
three mammalian NOS isoforms. Full-length DNOS1 protein (1350 aa)
reveals 43%, 40%, and 39% aa identity to rat nNOS, bovine eNOS, and mouse iNOS, respectively. However, the central portion of the
coding region shows the highest similarity to the neuronal NOS isoform
of mammals. Furthermore, the distribution of exons in regions of
dNOS that encode the cofactor-binding sites of the enzyme is
highly similar (and for many exons, identical) to that in
nNOS. Seven dNOS exons are identical in size
and intron type to the homologous exons in human nNOS gene,
whereas dNOS exons 10 and 11 appear to be a rearranged
fusion of homologous human nNOS exons 11-13.
Evolutionary conservation is evident mainly in the regions crucial for
catalytic activity of NOS, from the heme-binding site to beyond the
CaM-binding site. This region also exhibits the highest level of
nucleotide homology with the human nNOS, 61% of aa of the
DNOS1 oxygenase domain being identical to the corresponding region in
human nNOS. In contrast, much of the reductase domain of the
Drosophila enzyme is encoded by a single 1142-nt-long exon
16, whereas the homologous region in the
human nNOS gene is dispersed among eight exons (exons
19-26). Together, the structural and sequence homology indicate that dNOS is orthologous to the mammalian nNOS (Stasiv, 2001).
The organization of the region that codes for the DNOS1 oxygenase
domain is also highly similar to the organization of the oxygenase-coding region of the mosquito A. stephensi NOS
gene. Seven of the dNOS exons are identical in size and
intron type to the corresponding AsNOS exons. Exon
11 of the dNOS gene appears to be composed of two homologous
exons (exons 8 and 9) in the mosquito gene, whereas two dNOS
exons (exons 8 and 9) correspond to exon 6 of the AsNOS gene. The overall homology between the fly and mosquito proteins
is 81% (with 69% of the aa identical), whereas the homology within
oxygenase domains and CaM-binding sites reaches 88% (with 78% of the
aa identical). Interestingly, there is no exon-intron conservation between these two insect NOS genes in their reductase
domains. The structural similarity between the mosquito
AsNOS and the human nNOS genes within their
reductase-coding regions is higher than similarity between
corresponding regions of the dNOS gene and its human
counterpart. However, additional studies of NOS
genes from various invertebrate and vertebrate species are needed to
better characterize the evolution of NOS gene (Stasiv, 2001).
The dNOS gene demonstrates a remarkable degree of
transcriptional complexity resembling that of mammalian nNOS. Alternative transcription initiation sites
combined with the alternative usage of splice sites generate a family of dNOS transcripts. Four alternative variants of
the first non-coding exon were found: 1a, 1b, 1c, and 1d. All of them are located within the 6-kb-long 5'-region of the dNOS gene upstream of
exon 2. Most likely these exons begin at alternative transcription start sites; using 5'-RACE, it was not possible to extend these exons farther upstream, and using RT-PCR, it was not possible to detect dNOS transcripts in which exon 1 variants are spliced to each other. Exon 1b is common to several dNOS transcripts (dNOS1, 4, 5, and 6),
whereas exons 1a, 1c, and 1d are present in the dNOS7,
dNOS8, and dNOS3 isoforms, respectively. The fact
that dNOS3 and dNOS8 transcripts are expressed
only during the larval stage of Drosophila development further supports the notion that alternative promoters are used to direct expression of the different
dNOS RNAs in a tissue- and/or development-specific manner.
This mechanism of NOS regulation seems to be evolutionary conserved;
similarly, complex splicing patterns were found in the 5'-regions of
mammalian nNOS genes. For instance, in the human
nNOS gene, nine differentially expressed variants of
non-coding exon 1 were identified. It is unclear, though, whether
these human nNOS transcripts have additional structural
alterations (frameshifts, in-frame deletions, or insertions) within their coding region, similar to those found in the dNOS transcripts (Stasiv, 2001).
Alternative splicing affects the coding region as well as the 5'-UTR of
the dNOS gene. Transcript dNOS3 is an example of
a deleted coding exon (exon 3). This deletion leads to a frameshift and premature termination of the dNOS3 ORF. A transcript similar to the dNOS3 was found in the mosquito A. stephensi, where deletion of exon 2 (translation start codon in the AsNOS is located in exon 1) causes a premature termination of the ORF in the exon 2 mRNA (Stasiv, 2001).
Another transcript, dNOS10, has a novel type cassette
in-frame deletion of three consecutive exons (exons 15-17) that
composes almost 40% of the dNOS-coding region. Thus, DNOS10
protein retains only 57 carboxyl-terminal aa of the reductase domain.
No splicing alterations in this part of NOS gene have been
found in other organisms (Stasiv, 2001).
Several dNOS transcripts arise due to the insertion of extra
exon(s) in their coding regions, resulting in the premature termination of their ORFs. The dNOS4 and dNOS7 transcripts
contain an alternative exon 14a, whereas dNOS5 and
dNOS6 RNAs have two extra exons, either 13a plus 14a or 13b
plus 14a, respectively. Similar types of exon insertions that introduce
premature stop codons have been found in mammalian NOS
genes. Two alternative transcripts arise from the human nNOS
primary transcript after the unusual splicing of intron 16 (33). In the
nNOS+47 RNA, the first 47 nt at the very 5'-end of intron 16 are inserted into the coding region between exons 16 and 17. The
nNOS+67 transcript has an extra 67 nt (derived from the
central part of intron 16) inserted between exons 16 and 17. Both insertions introduce an in-frame stop codon. Although exon 16 of the human nNOS gene is homologous to exon 14 of the dNOS gene, no dNOS RNA species were detected with insertions of an extra exon(s)
downstream of exon 14. Thus, the fruit fly most likely does not have an
RNA isoform corresponding to the mammalian nNOSµ transcript, which contains an in-frame 102-nt-long insertion between exons 16 and 17 and is expressed in various rat and human tissues (Stasiv, 2001).
Alternative splicing of the mosquito AsNOS pre-mRNA
causes an insertion of additional exon (174 nt long) between exons 11 and 12 in the exon 11+ transcript. An
alternative AsNOS exon contains a stop codon, which results
in a premature termination of translation 16 aa downstream of the novel
splice junction. This product of the AsNOS gene resembles
the dNOS4 transcript. Finally, transcript dNOS2 has an in-frame cassette deletion of exons 8 and 9 that is identical to the deletion
of exons 9 and 10 found in the alternative transcript nNOS-2
of the human nNOS gene (Stasiv, 2001).
Individual dNOS isoforms are differentially expressed in the
developing Drosophila according to quantitative RT-PCR
results. Although dNOS1 is a predominant RNA product in
embryo, larva, and imago, it is important to note that these
experiments were performed with RNA pools representing various phases
of each developmental stage. It is possible that individual
dNOS transcripts are transiently induced at selected steps
of the developmental cascade (e.g., before pupariation, when
a strong increase in diaphorase staining is observed) (Stasiv, 2001).
The family of dNOS transcripts encodes a variety of
DNOS-like proteins. Seven of them, DNOS3, -4, -5, -6, -7, -9, and -10 (but not DNOS2), lack either part of or the entire reductase domain, which leads to a loss of enzymatic activity. However, most of the
truncated DNOS proteins (except for DNOS2 and DNOS3) retain almost the
entire oxygenase domain, including the sites that are thought to be
responsible for NOS homodimerization in mammalian cells. Thus, these
truncated forms lack NOS enzymatic activity but may still retain their
ability to dimerize. This notion is supported by experiments in
which interaction between GAL4-DNOS4 hybrid
proteins (DNOS4 was fused to GAL4 binding and to GAL4 activation
domain) was detected using a yeast two-hybrid system. Moreover,
this implies that the truncated DNOS proteins may not only form dimers
with each other but also form heterodimers with the full-length DNOS1.
In experiments with purified nNOS and iNOS homodimers, the flow of
electrons during catalysis has been shown to occur from the flavins in
the reductase domain of one subunit to the heme iron in the oxygenase
domain of the other subunit. This suggests that
heterodimers between the full-length and truncated NOS polypeptides
will have diminished enzymatic activity. Indeed, rat nNOS full-length
polypeptide and a synthetic polypeptide lacking the reductase domain
can form heterodimers in vitro, but these complexes show
drastically decreased enzymatic activity. Furthermore, a fragment
of eNOS that lacks the reductase domain can form complexes with the
full-length eNOS when co-expressed in cultured cells and exhibits a
strong dominant negative effect on eNOS activity. Thus, truncated DNOS proteins capable of forming heterodimers with DNOS1 may act as
dominant negative inhibitors of NO production. This notion was
confirmed in experiments in which DNOS4, DNOS5, and DNOS6, each of
which can form complexes with DNOS1, were able to strongly suppress NOS
activity when co-expressed along with the DNOS1. This
suggests that formation of heterodimers between the full-length DNOS1
and its truncated isoforms may be a basis for a mechanism of regulation
of NO production in Drosophila . It will be interesting to
determine whether truncated DNOS proteins are indeed synthesized in the fruit fly and whether they can serve to modulate NOS activity in vivo (Stasiv, 2001).
In summary, these results demonstrate that the dNOS locus in
Drosophila generates a large family of transcripts, some of
which code for truncated DNOS-like proteins. These proteins are capable of suppressing the enzymatic activity of the full-length DNOS1 protein,
perhaps by disrupting the dimerization of DNOS1 molecules. Because such
truncated NOS proteins have been postulated to exist in mammals, this
novel regulatory function proposed for the truncated Drosophila NOS proteins may apply more widely for NOS regulation (Stasiv, 2001).
Nitric oxide (NO) is an essential regulator of Drosophila development and physiology. A novel mode of regulation of NO synthase (NOS) function is described that uses endogenously produced truncated protein isoforms of Drosophila NOS (DNOS). These isoforms inhibit NOS enzymatic activity in vitro and in vivo, reflecting their ability to form complexes with the full-length DNOS protein (DNOS1). Truncated isoforms suppress the antiproliferative action of DNOS1 in the eye imaginal disc by impacting the retinoblastoma-dependent pathway, yielding hyperproliferative phenotypes in pupae and adult flies. These results indicate that endogenous products of the dNOS locus act as dominant negative regulators of NOS activity during Drosophila development (Stasiv, 2004).
The dNOS locus of Drosophila is subject to complex transcriptional and posttranscriptional regulation. It produces a large variety of mRNA isoforms through the use of multiple promoters and alternative splice sites. Only one of them, dNOS1, codes for the full-length enzymatically active protein. Another abundant alternative transcript of the dNOS gene is the dNOS4 isoform, which retains the entire intron 13 (this 109-nucleotide-long segment is now referred to as exon 14a of dNOS4). The resulting open reading frame is terminated by a stop codon 63 nucleotides into exon 14a. It encodes a protein containing 757 amino acids with a predicted molecular mass of 84 kDa (cf. DNOS1 is 1350 amino acids, 150 kDa). DNOS4 contains a unique 21-amino acid-long C-terminal peptide encoded by exon 14a, whereas the preceding 736 amino acids are identical to those of DNOS1. Thus, the DNOS4 protein is a truncated version of DNOS1; it lacks the entire reductase domain, while retaining the oxygenase domain. Semiquantitative RT-PCR analysis indicates that dNOS4 mRNA is expressed in the embryo at levels comparable to those of dNOS1 mRNA; dNOS4 levels are lower in larvae and in adult flies, whereas dNOS1 levels do not change appreciably (Stasiv, 2004).
Another variant of dNOS mRNA, dNOS7, encodes a protein identical to DNOS4; however, the transcription initiation site for dNOS7 RNA is different from that of dNOS4 (exon 1a vs. exon 1b, respectively). Unlike dNOS4, dNOS7 is exclusively expressed during the larval stage (Stasiv, 2004).
DNOS4 lacks the C-terminal reductase domain that participates in electron transfer during catalysis, while it retains the catalytic N-terminal oxygenase domain, including the critical heme-binding site. DNOS4 also retains a long stretch of glutamine (Gln) residues at the N terminus; such regions have been shown to promote multimerization of proteins; note that such Gln-rich region is not present in mammalian NOS proteins. These structural features of DNOS4 predict that (1) DNOS4 itself is incapable of producing NO, (2) it may be capable of forming heterodimers with DNOS1, and (3) heteromers between DNOS1 and DNOS4 will have reduced enzymatic activity. To investigate whether DNOS4 is capable of forming a heteromeric complex with DNOS1 and suppressing NOS activity, and to examine which region of DNOS4 may contribute to its effects on DNOS1, expression plasmids were used for DNOS1 and DNOS4 proteins, each with a short peptide tag fused to its C terminus, the influenza virus hemagglutinin (HA) epitope-tagged DNOS1 (pDNOS1-HA), and the synthetic FLAG epitope-tagged DNOS4 (pDNOS4-FLAG). A plasmid was generated, pDNOSoxy-FLAG, that codes for a shorter version of DNOS4 (residues 214-631) and carries the FLAG epitope at its C terminus. It lacks the Gln-rich region at the N terminus, thus representing the "core" oxygenase domain of DNOS, which is highly similar to previously defined oxygenase domains of mammalian NOS proteins (Stasiv, 2004).
To test the effect of DNOS4 and DNOSoxy proteins on enzymatic activity of the full-length DNOS1, recombinant plasmids were transiently coexpressed in cultured human embryonic kidney cells (293 cells) and NOS enzymatic activity was measured in cell-free extracts. NOS activity was undetectable in lysates of untransfected cells and in cells expressing either DNOS4-FLAG or DNOSoxy-FLAG, whereas lysates from cells transfected with pDNOS1-HA showed significant levels of NOS activity. In contrast, enzymatic activity in cell lysates was decreased when a constant amount of the pDNOS1-HA was cotransfected with increasing amounts of the pDNOS4-FLAG. At a 1:1 molar ratio of plasmids encoding DNOS4 and DNOS1, NOS activity was 57% of the activity seen in lysates from cells transfected by the plasmid encoding full-length DNOS1. At a 3:1 ratio, the activity dropped to 36%, and at 10:1 ratio it was 10% of the control level. pDNOSoxy-FLAG, which lacks both the reductase domain and the Gln-rich region, is as effective at suppressing NOS activity (13.5% of control levels when cotransfected with pDNOS1-HA at 10:1 molar ratio) as pDNOS4-HA (which retains the Gln-rich region). Importantly, expression levels of DNOS1 protein were not affected by coexpressed truncated DNOS variants, as determined by immunoblotting using HA-specific antibodies. This observation indicates that a decrease of NOS activity was not simply due to a decrease in expression of the DNOS1 protein, but rather was caused by the presence of coexpressed shorter DNOS polypeptides. Furthermore, removal of the Gln-rich region did not alter the inhibitory effect of DNOSoxy on the enzymatic activity of DNOS1, indicating that the Gln-rich stretch does not appreciably contribute to the inhibitory action of DNOS4. Together, these results indicate that truncated enzymatically inactive forms of DNOS can suppress the activity of the full-length DNOS1 protein, thereby acting as dominant negative inhibitors of NO production (Stasiv, 2004).
To examine the potential of DNOS1 and DNOS4 to form complexes, immunoprecipitation experiments were performed after coexpressing differentially tagged DNOS1 and DNOS4 in 293 cells. Each tagged DNOS protein was immunoprecipitated from extracts of cotransfected cells with antibody specific to its epitope tag (HA for DNOS1 or FLAG for DNOS4). Subsequently, the formation of heteromeric DNOS complexes was examined by immunoblotting using HA-specific antibody for the samples immunoprecipitated with FLAG-specific antibody and vice versa (Stasiv, 2004).
When HA-tagged DNOS1 is expressed in 293 cells, it can be immunoprecipitated with HA-specific but not with FLAG-specific antibody. Conversely, FLAG-tagged DNOS4 can be immunoprecipitated using FLAG-specific, but not HA-specific antibodies. However, if DNOS1-HA and DNOS4-FLAG are coexpressed, each of them can be immunoprecipitated by antibody to either tag. Furthermore, when DNOSoxy-FLAG was coexpressed with DNOS1-HA, complexes between these two proteins were detected using the same combination of immunoprecipitation and immunoblotting. This suggests that even a part of the oxygenase domain lacking the Gln-rich region, but retaining the heme-binding region, is sufficient to form heteromers with the full-length DNOS1 protein. Finally, when lysates of cells transfected separately with either pDNOS1-HA or pDNOS4-FLAG were combined in vitro, the two proteins did not coimmunoprecipitate with each other, indicating that heteromeric DNOS complexes are not formed after cell lysis (Stasiv, 2004).
Together, these data point to a possible mechanism for the observed dominant negative effect of DNOS4 on DNOS1 activity, in which formation of DNOS1-DNOS4 heterodimers inhibits NOS activity by reducing formation of enzymatically active homodimers of DNOS1 (Stasiv, 2004).
To determine whether DNOS4 can act as a dominant negative regulator of NOS activity in vivo, transgenic flies were generated that express FLAG-tagged DNOS4 under the control either of the heat-shock inducible promoter of the hsp70 gene (hs-DNOS4-FLAG flies), or of the GMR promoter, which is active in all cells of the eye imaginal disc within, and posterior to the morphogenetic furrow (GMR-DNOS4-FLAG flies). It was confirmed that DNOS4-FLAG was expressed in transgenic flies; anti-FLAG antibodies detect a protein with an expected mass of 86 kDa in extracts from heads of GMR-DNOS4-FLAG and from hs-DNOS4-FLAG flies, but not in extracts from wild-type flies. The mobility of this protein was identical to that of a protein produced in 293 cells after transfection with pDNOS4-FLAG. Importantly, the expression levels of endogenously produced DNOS1 were not affected by ectopic expression of DNOS4 in transgenic flies. Furthermore, immunochemical detection with FLAG-specific antibodies showed that DNOS4-FLAG was expressed in the expected pattern within, and posterior to, the morphogenetic furrow in the eye imaginal disc of third-instar GMR-DNOS4-FLAG larvae (Stasiv, 2004).
NOS activity was examined in extracts from heads of adult flies and it was found that in hs-DNOS4-FLAG flies and in GMR-DNOS4-FLAG flies activity was 52% and 60%, respectively, of that in control wild-type flies. This indicates that endogenous NOS activity is inhibited by ectopically expressed DNOS4, paralleling observations with cultured cells (Stasiv, 2004).
To determine whether the observed decrease in NOS activity in transgenic flies is accompanied by in vivo formation of DNOS1-DNOS4 heterodimers, coimmunoprecipitation experiments were performed. Using FLAG-specific antibody, proteins were precipitated from head extracts of adult wild-type or hs-DNOS4-FLAG transgenic flies, and then were analyzed by immunoblotting using anti-DNOS1 or anti-FLAG antibodies. Importantly, full-length DNOS1 protein can be immunoprecipitated with anti-FLAG antibody from extracts of hs-DNOS4-FLAG transgenic flies, but not of wild-type flies. This indicates that ectopically expressed DNOS4 can form heterodimeric complexes with endogenous DNOS1 in vivo and suggests that formation of such complexes may explain the dominant negative effect of DNOS4 on NOS activity (Stasiv, 2004).
NO acts as an antiproliferative factor during eye development; pharmacological inhibition of NOS activity results in an increase in the number of cells in the ommatidia. It was asked whether DNOS4 can act to suppress the antiproliferative action of NO and affect cell division in the developing eye. Ectopic expression of DNOS4 driven by an eye-specific promoter in GMR-DNOS4-FLAG flies results in a distorted eye phenotype with visible rearrangements of the ommatidia lattice and extra bristles. To analyze the changes in more detail, the sections of the retina from wild-type and transgenic pupae were compared. GMR-DNOS4-FLAG flies have extra secondary and tertiary pigment cells as well as extra bristle cells; no changes were detected in the number of photoreceptor cells. These extra cells distort the regular hexagonal shape of the ommatidia and the general lattice pattern of the retina (Stasiv, 2004).
To determine whether the increase in the number of cells in pupae was related to increased proliferation in the imaginal discs of larvae, the nuclei of eye-disc cells were labeled in S phase of the cell cycle with 5-bromo-2'-deoxyuridine (BrdU). There was a 2.13-fold increase in the number of BrdU-positive cells in imaginal discs from GMR-DNOS4-FLAG flies as compared with wild-type flies. Extra dividing cells were localized to the region of GMR-driven transgene expression in the morphogenetic furrow and the area posterior to it. The observed increase in the number of dividing cells and the number of cells per ommatidium suggests that introduction of the dominant negative dNOS4 transgene resulted in inhibition of NOS activity and suppression of the antiproliferative effect of NO in the developing eye (Stasiv, 2004).
Because NO interacts with the retinoblastoma (Rb) pathway to control cell division in the developing Drosophila eye, it was of interest to determine whether DNOS4 can act to affect signaling via the Rb pathway in the eye (Stasiv, 2004).
RBF is the Drosophila ortholog of mammalian Rb proteins; like Rb, RBF acts as a negative regulator of cell cycle progression. GMR-RBF flies that carry four copies of the RBF transgene have a profound eye phenotype, due to suppression of cell division by the elevated levels of RBF; some of the pigment cells and bristles are missing and some ommatidia are fused. The RBF phenotype is strongly enhanced by the ectopic overexpression of the dNOS1 transgene. To test the effects of DNOS4 on the action of RBF, GMR-DNOS4-FLAG flies were crossed and then backcrossed to GMR-RBF flies to generate flies bearing one copy of the dNOS4 transgene along with four copies of the RBF transgene. The eyes of the resulting hybrid flies had an almost normal phenotype, without fused ommatidia, and with the usual set of bristles. Thus, the effect of RBF overexpression was counteracted by DNOS4, indicating that this inhibitor of NOS activity counteracts RBF function in the developing eye (Stasiv, 2004).
In mammalian and Drosophila cells, Rb is associated with the E2F transcription-factor complex, whose activity is required for the cells' entry into S phase. Overexpression of E2F overcomes Rb-mediated G1 arrest and induces quiescent cells to enter S phase. Ectopic expression of dE2F/dDP (Drosophila orthologs of mammalian components of the E2F complex) driven by the GMR promoter during development results in generation of extra cells in the adult eye (e.g., multiple extra bristles), particularly when combined with the p35 gene, a baculoviral inhibitor of apoptosis. GMR-DNOS4-FLAG flies were crossed with GMR-dE2F/dDP/GMR-p35 flies and an even more pronounced eye phenotype was found, with a larger eye surface, extra ommatidia, and severe rearrangements of the ommatidia lattice. This indicates that DNOS4 acts to enhance the function of E2F, consistent with the proposed role for DNOS4 as a suppressor of the inhibitory action of NO on cell cycle progression (Stasiv, 2004).
Together, these genetic data confirm that development of the Drosophila eye depends on NO or NO-activated signaling pathways interacting with the Rb pathway. They suggest that, by suppressing the antiproliferative action of NO, DNOS4 acts in vivo to suppress the effect of RBF, and to enhance the effect of E2F, on cell cycle regulation (Stasiv, 2004).
See the embryonic expression pattern of Nos at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site.
Nitric oxide (NO) is a membrane-permeant signaling molecule that activates soluble guanylyl cyclase and leads to the formation
of cyclic GMP (cGMP). The NO/cGMP signaling system is thought to play essential roles during the development of vertebrate
and invertebrate animals. The cellular expression of this signaling pathway during the development of the
Drosophila melanogaster nervous system has been analyzed. Using NADPH diaphorase histochemistry as a marker for NO synthase,
several neuronal and glial cell types have been identified as potential NO donor cells. To label NO-responsive target cells, the detection of
cGMP by an immunocytochemical technique was used. Incubation of tissue in a NO donor induces cGMP immunoreactivity (cGMP-IR) in
individual motoneurons, sensory neurons, and groups of interneurons of the brain and ventral nerve cord. A dynamic pattern of the
cellular expression of NADPHd staining and cGMP-IR is observed during embryonic, larval, and prepupal phases. The
expression of NADPH diaphorase and cGMP-IR in distinct neuronal populations of the larval central nervous system (CNS)
indicates a role for NO in transcellular signaling within the CNS and as a potential retrograde messenger across the neuromuscular
junction. In addition, the presence of NADPH diaphorase-positive imaginal discs containing NO-responsive sensory neurons
suggests that a transcellular NO/cGMP messenger system can operate between cells of epithelial and neuronal phenotype. The
discrete cellular resolution of donor and NO-responsive target cells in identifiable cell types will facilitate the genetic,
pharmacological, and physiological analyses of NO/cGMP signal transduction in the developing nervous system of Drosophila (Wildemann, 1999a).
The grasshopper embryo has been used as a convenient
system with which to investigate mechanisms of axonal
navigation and pathway formation at the level of individual
nerve cells. The developing antenna of the
grasshopper embryo (Schistocerca gregaria), where two
siblings of pioneer neurons establish the first two axonal
pathways to the CNS (Seidel, 2000).
As the embryonic antennae begin to evaginate from the head,
they are initially devoid of sensory neurons. Unlike in the
thoracic and gnathal segments, where a single sibling pair of
sensory cells serve as pioneers to the CNS, in the antenna, two
sibling pairs of pioneer neurons are born. These are termed ventral and dorsal pioneers. To examine neurogenesis and peripheral pathfinding
in the antenna, the differentiating pioneer neurons were labeled
at various stages with a neuron-specific anti-HRP antiserum. This method allowed
the reliable identification of the pioneer neurons.
The first cells that express HRP-IR at 32% of embryonic
development are the pair of ventral pioneer neurons (vPN) at the distal tip of the antenna. At the same time, a
single immunoreactive neuronal cell body appears at the base
of the antenna. The axon of this base pioneer (BP) is the first peripheral process to reach the
CNS from the antenna. During the next few hours two
additional dorsal pioneer neurons (dPN) also become
immunoreactive. Subsequently, the growth cones of the vPN
extended along the ventral inner surface of the antennal
epithelium with a migration path that runs straight to the BP.
At 34%, the growth cones come into filopodial contact with the
BP. Compared with the axons of the vPN the processes of the
dPN initiate their outgrowth slightly later,
navigating along the dorsal inner surface of the antennal lumen.
The dorsal pioneer neurons do not follow a straight trajectory
towards the CNS. Rather, after growing for a distance of 100
mm in proximal direction, the axons of the dorsal pioneers
performed a prominent turn of about 90°, extending ventrally
towards the BP. Remarkably, the BP loses its HRP
immunoreactivity after the growth cones of the vPN and the
dPN make contact. Subsequently, the processes of
vPN and dPN fasciculate with the axon of the base pioneer,
which, meanwhile, has established the earliest afferent
pathway towards the brain. The axonal projections of the pioneers enter the HRP-stained
CNS at a distance of about 50-80 mm from the base
pioneer (Seidel, 2000).
At 38% of development, additional HRP-IR neurons
differentiate in the antenna. These neurons elaborate
prominent apical dendrites indicative of their phenotype as
sensory cells. During the period 38%-45% of embryogenesis, the
regions of sensory cell formation appear restricted to three
circumferential bands. Initially sensory neurons are
born at the tip of the antenna. A few hours later another two
other regions that initiated sensory cell formation can be
discerned. One region is positioned approximately in the
middle of the antenna slightly more distal to the pioneer
neurons. The third region is found near the base of the
antenna but proximal to the BP cell. In this zone, sensory
cells of the later pedicellar chordotonal organ are generated. Neurogenesis
of numerous sensory cells continues within the three initiating
zones. Parallel to the onset of segmentation at 45%, additional
sensory cell initiating zones differentiate within the frame of
the developing annular segments of the antenna. Taken together, these results indicate
that the vPN and dPN axons prefigure two axonal fascicles to
the brain, which are joined by later-born sensory
neurons to form the bipartite antennal nerve of larval and adult
stages (Seidel, 2000).
About midway through insect embryogenesis, the appearance
of NO-induced cGMP synthesis in selective neuronal cell types
appears to be a common developmental phenomenon.
The peripheral neurons of the antenna express cGMP-IR in a
rather early stage of development. After stimulation with the
NO donor SNP, cGMP-IR can be induced in both pairs of
the pioneer neurons in the antenna. The onset of
the cGMP-IR becomes visible in the 38% stage, and
immunoreactivity persists during the following
developmental period. As has been reported for other
embryonic grasshopper neurons, strong
cGMP-IR is also found in the nuclei of the antennal pioneers.
At a 55% of development stage, a second embryonic cuticle
is secreted that prevents the
access of compounds like SNP or IBMX in embryonic whole
mounts. Therefore, with this experimental approach, how long the NO-induced cGMP-IR
persists during embryogenesis could not be examined. Occasionally, an
additional occurrence of NO induced cGMP-IR was noticed in some distal
sensory neurons in preparations of later stages (50%-55%). Embryos that are exposed to 200 mM ODQ for
at least 20 minutes or during a culture period of 24-30 hours
fail to express cGMP-IR after stimulation with an NO donor. These observations provide evidence that the
inhibitors used to study blocking effects of the cGMP pathway
are suitable to reduce the sGC activity in situ effectively (Seidel, 2000).
To search for potential cellular sources of NO,
NADPH-diaphorase staining of formalin-fixed embryonic
whole mounts was used as a histochemical marker for NOS. On
transverse sections through the antenna, the blue
precipitate of the diaphorase reaction is found concentrated in parts of
the epithelial cells facing the basal lamina. The
staining of the basal parts of epithelial cells is not very
pronounced but there is a striking contrast in staining intensity
compared with the mesodermal tissue bordering the basal
lamina. Diaphorase staining of the epithelial cells becomes
visible during a developmental period ranging from about 32%-35% and disappears at later stages (Seidel, 2000).
To investigate the role of
the NO/cGMP signaling system during pathfinding, the pattern of outgrowing pioneer neurons in
embryo culture was examined. Pharmacological inhibition of soluble
guanylyl cyclase and of NO synthase results
in an abnormal pattern of pathway formation in the
antenna. Axonogenesis of both pairs of pioneers are
inhibited when specific NOS or sGC inhibitors are added
to the culture medium; the observed effects include the loss of
axon emergence as well as retardation of outgrowth, such
that growth cones do not reach the CNS. The addition of
membrane-permeant cGMP or a direct activator of the
sGC enzyme to the culture medium completely rescues the
phenotype resulting from the block of NO/cGMP signaling.
These results indicate that NO/cGMP signaling is involved
in axonal elongation of pioneer neurons in the antenna of
the grasshopper (Seidel, 2000).
To visualize the expression of Drosophila NOS, histochemical staining for the NADPH-diaphorase (reduced nicotinamide adenine dinucleotide phosphate-diaphorase) activity of NOS was used. Beginning in the third instar, NADPH-diaphorase staining is observed in all imaginal discs, imaginal rings, histoblasts, and the larval brain. Staining becomes more intense as development proceeds, and by late third instar (larvae and early pupae), a highly specific and reproducible pattern of very intense staining is evident. In the leg imaginal disc, staining is initially seen at the very beginning of the third instar. Staining is confined to the center of the disc, corresponding to the presumptive distal tip of the leg. As the discs matured, staining intensifies. Later, when the discs begin to evert in the prepupae, staining of the forming leg becomes less intense. Wing, eye, haltere, and genital discs in the third instar have distinct and reproducible patterns of intense staining which gradually decreases in a specific spatial pattern during early pupal development (Kuzin, 1996).
During the development of retinal projections into the optic lobe lamina and medulla, the Nitric oxide synthase produced by the optic ganglia of these optic lobe structures produces NO, which serves to regulate the growth of individual retinal axons. Nitric oxide synthase produces NO through the conversion of arginine to citrulline, using NADPH as a cofactor. This activity can be detected histochemically. From the time of puparium formation and continuing for about forty hours, Drosophila brains show strong staining in regions of the optic ganglia and the lamina and medulla, as well as in Bolwig's nerve (see Bolwig's organ). In the visual system at 24 hours after puparium formation, an anti-NOS antibody labels projections in the neuropilar regions of both the lamina and the medulla, although the staining is most evident in the medulla. No staining is evident in the cell body layers. Double labelling with the NOS antibody and an antibody to chaoptin that recognizes photoreceptor cell bodies and axons shows that the axons of photoreceptors R7 and R8 terminate within a region of the medulla containing processes that stain strongly with the NOS antibody. These patterns of staining strongly suggest that NOS is present in retinal targets of photoreceptor axons (Gibbs, 1998).
Nitric oxide sensitive guanylate activity is present in subsets of photoreceptors during metamorphosis. The isolated CNS with intact eye imaginal discs was incubated with a NO donor and an inhibitor of phosphodiesterases. Many neurons within the brain and optic lobes show the appearance of cGMP after such incubation. At the onset of metamorphosis (at the time of puparium formation) and at least ten hours afterwards, this response is limited to the central brain and does not include the photoreceptor axons. Photoreceptors begin to respond shortly after this time. At 16 hours after puparium formation and continuing for more than 24 hours, cGMP is evident in the photoreceptor cell bodies and along the total length of their axons. cGMP synthesis is most dramatic in photoreceptors R1-R6 that project to the lamina, while R7 and R8 show slightly less cGMP synthesis (Gibbs, 1998).
The isolated CNS of Drosophila has been shown to undergo morphological changes in culture mirroring thos observed during metamorphosis. In culture, visual systems continue to develop. When stained with the chaoptin antibody, the segregation of R1-R6 and R7/8 into the lamina and medulla, respectively, is observed, as is the distinctive retinotopic patterning of R7 and R8 terminals in the medulla. After 96 hours in vitro, R7 and R8 have also segregated into two separate layers of the medulla. Although the retinal axons have arrived at their approximate targets and assembly of an organized optic lobe has commenced by 50 hours after puparium formation, the formation of synaptic connections between the photreceptors and optic lobe neurons occurs during the second half of metamorphosis. Thus, synapse formation has most likely not occurred even after 96 hours in vitro. Nervous system cultured in the presence of a competitive NOS inhibitor displays a disrupted pattern of photoreceptor projections in the medula. Individual axons are observed growing in an abnormal fashion beyond the borders of the medulla into the brain. The disruption of retinal axon projections observed with NOS inhibitior does not appear to be a result of severe degeneration or disorganization of targets in the medulla. The effects of NOS inhibition can be prevented with 8-bromo-cGMP, which appears to antagonize the disruptive effects of the NOS inhibitior. Methylene blue, which inhibits guanylate cyclase by binding to the heme groups and which also inhibits NOS activity through a similar mechanism, affects retinal patterning in the medulla, producing nervous systems with significantly greater disruption indices than does the specific NOS inhibitor (Gibbs, 1998).
What is the function of the NO produced by optic lobe neurons? Photoreceptors grow into the optic neuropil over a period of about 40 hours beginning in the third larval instar. Here, the axons arrange themselves retinotopically within their appropriate neuropils but do not immediately begin the process of assembly of the final connections. The first axons to reach their targest may remain in an arrest period for over a day prior to the time that the photoreceptor axons begin to seek their synaptic partners. Subsequent growth cone spreading and activity thus necessitate a mechanism permitting maintenance and flexibility of the growth cone while preventing outgrowth of the axon beyond the target. It is during this time that the retinal axons beome responsive to NO. When NO/cGMP signaling is disrupted, the retinal axons, rather than expanding to associate with their final targets, resume longitudinal growth to deeper layers of the medulla or into the brain. This aberrant growth suggests that NO provides an arrest signal that is required once the axons emerge from their dormant period. At this time, they are switching from a behavior pattern of responding to pathfinding cues to another pattern of forming interactive associations with potential synaptic partners. It is proposed that NO may serve as a stabilizing influence on the maneuvering growth cone, preventing further extension of the axons beyond the vicinity of appropriate post-synaptic neurons during period before the establishment of permanent connections (Gibbs, 1998).
Two types of cyclic GMP regulated neuronal maturation models exist in the grasshopper. One type, termed ecdysis regulated, is not mediated via NO. In this type, neurons that contain crustacean cardioactive peptide (see Cardioacceleratory peptide) develop in the subesophageal ganglion and in segmental arrays of cells in the CNS. These neurons exhibit an ecdysis-related expression of cGMP that is unresponsive to NO donor compounds. A second type of cGMP regulated maturation, termed developmentally regulated, is found in certain motoneurons, interneurons and sensory neurons late in embryonic development. For example, the RP2 motorneuron is the first in any segment to become responsive to NO generating compounds. Subsequently, within a segment, the motoneurons show a stereotyped order in which they become NO responsive. RP2 is followed by the acquisition of competence in the aCC motoneuron; the motor axons that innervate certain muscles become responsive after that. It would seem that every muscle group in the abdomen receives innervation from an axon that is NO responsive. Most of these muscles also receive innervation from other motor axons that do not exhibit NO sensitivity. In each case, neuron NO sensitivity appears after the growth cone has arrived at its target but before it has
started to send out branches. NO sensitivity typically ends as synaptogenesis is nearing
completion (Truman, 1996b).
Data from interneurons and sensory neurons are also consistent with the
hypothesis that NO sensitivity appears as a developing neuron changes from axonal
outgrowth to maturation and synaptogenesis. A well defined interneuron, the H cell, becomes NO response well after it is born, at a time when axon production is finished and the neuron begins maturation as characterized by soma enlargement, changes in membrane currents and elaboration of dendritic arbors. Mechanoreceptor sensory neurons that become NO sensitive include those of the dorsal body wall chordotonal organs, the segmental and wing-hinge stretch receptors, the abdominal 'ear,' campaniform sensilla and mechanosensory bristles. The photoreceptor neurons of the compound eye also become responsive to SNP. Some proprioceptors, like the dBw chordotonal neurons and the wing-hinge stretch receptors, are the first mechanoreceptors to become responsive to NO donors. Other proprioceptors, the afferents that supple external sensory organs (such as bristle and dome sensilla) are born late and develop NO sensitivity late. Associated glial cells also show a weak response to NO (Truman, 1996b).
Cyclic GMP likely constitutes part of a
retrograde signalling pathway between a neuron and its synaptic partner. NO sensitivity
also appears in some mature neurones at times when they may be undergoing synaptic
rearrangement. Comparative studies on other insects indicate that the association between
a NO-sensitive guanylate cyclase and synaptogenesis is an ancient one, as evidenced by its
presence in both ancient and more recently evolved insect groups. Although cGMP responses are relatively poorly developed in embryos of Drosophila, a cGMP response to NO donors is very prominent when the larval nervous system goes into its second developmental period at metamorphosis. Although diaphorase staining, indicative of NOS presence, shows developmental regulation, the same kind of dramatic modulation in diaphorase staining is not observed as is observed for NO responsiveness. Double staining of the nervous system for cGMP (after supplying exogenous NO) followed by diaphorase staining to reveal NOS positive cells, shows that most, but not all, of the cell bodies that are diaphorase positive are not responsive to SNP (Truman, 1996b).
It is not known what causes the abrupt appearance of NO responsiveness in these neurons although the most obvious possibility is contact with target. An intriguing feature of soluble guanylate cyclase is that it is inhibited by elevated levels of intracellular Ca2+. Ca2+ transients and Ca2+ spikes are characteristic of extending axons and growth cones in many types of developing neurons, and a reduction in Ca2+ currents often accompanies the transition from the axonal outgrowth phase into the maturational phase. This reduction in intracellular Ca2+ might serve to unmask the guanylate cyclase, thereby allowing it to respond to extracellular signals (Truman, 1996b and references).
Proliferation of neural precursors in the optic lobe of
Manduca sexta is controlled by circulating steroids and by
local production of nitric oxide (NO). Diaphorase staining,
anti-NO synthase (NOS) immunocytochemistry and the
NO-indicator, DAF-2, show that cells throughout the optic
anlage contain NOS and produce NO. Signaling via NO
inhibits proliferation in the anlage. When exposed to low
levels of ecdysteroid, NO production is stimulated and
proliferation ceases. When steroid levels are increased,
NO production begins to decrease within 15 minutes
independent of RNA or protein synthesis and cells rapidly
resume proliferation. Resumption of proliferation is not
due simply to the removal of NO repression though, but
also requires an ecdysteroid stimulatory pathway. The
consequence of these opposing pathways is a sharpening of
the responsiveness to the steroid, thereby facilitating a
tight coordination between development of the different
elements of the adult visual system (Champlin, 2000).
In both Drosophila and Manduca, ingrowing photoreceptor
axons stimulate proliferation in a subset of optic anlage (OA) cells. This process
provides one way of coordinating development of the eye with
the optic lobe. In addition, ecdysteroids
provide a means to coordinate development throughout the
entire visual system including developing layers that are not in
direct contact. For example, ecdysteroid is required to sustain
the movement of the morphogenetic furrow and, hence, the
progressive formation of rows of photoreceptors in the
imaginal eye disc. The same ecdysteroid requirements are found for
proliferation of precursors for the medulla and lobula as well
as the lamina optic lobe neurons. In this context, the function of NO signaling within the
OA appears to be to sharpen the proliferative responsiveness
of cells to the steroid so that optic lobe neuron production is
tightly coordinated along the entire length of the OA and
progresses in unison with photoreceptor production in the eye
disc. When NO signaling is inhibited, a five-fold increase in
the 20E titer (20 to 100 ng/ml) is needed to go from the first
signs of proliferation to full proliferation. In the presence of
NO signaling, this range is reduced to just two-fold. This may be of particular importance in Manduca since
metamorphic development of the visual system is reversibly
interrupted during pupal diapause when environmental cues act
to suppress the ecdysteroid titer (Champlin, 2000).
In controlling proliferation within the OA, the ecdysteroid
and NO pathways intersect at two levels. They both
appear to act at the target cell level in controlling entry into
mitosis with ecdysteroid promoting entry and NO inhibiting it.
However, ecdysteroid also acts upstream by suppressing NO
synthesis. NO production decreases rapidly (within 15
minutes) in response to an increase in 20E and even if RNA or
protein synthesis is blocked. Therefore, ecdysteroid suppression of NO
production appears to occur through a rapid, non-genomic
signaling mechanism (Champlin, 2000).
NO has been shown to inhibit proliferation in a variety of cell
types, the best characterized of which are the endothelium and
smooth muscle of vertebrate blood vessels. NO produced by
endothelial cells inhibits proliferation both in the endothelium
itself and also the
overlying smooth muscle cells. Although it is well established that endothelial-derived
NO acts as a vasodilator on smooth muscle cells via
a cGMP-mediated pathway,
conflicting reports have been published for both endothelial
cells and smooth muscle cells as to whether NO also acts
through cGMP to inhibit proliferation. In the OA,
no evidence has been found for an involvement of cGMP in the inhibition of
proliferation by NO. Ecdysteroid-dependent entry into mitosis
is blocked by incubation with a NO donor even when the
soluble guanylate cyclase inhibitor, ODQ, is included to
block production of cGMP. Furthermore, the cGMP analog, 8-bromo-cGMP, is not able to mimic the inhibitory effect of
NO. Consistent with this, cGMP has not been detected in the
OA during development or in response to NO treatment. An
alternative target for NO signaling within the OA may be
through the nitrosylation of target cell proteins (Champlin, 2000).
The exact relationship between NO repression and steroid
regulation of the cell cycle is not yet known in either
vertebrates or invertebrates. In principle, these opposing
signals could interact at any level between the steroid receptor
and the regulated cell cycle factors. In any case, NO repression
in the OA of Manduca does differ from that seen in vertebrate
in at least one respect. In the latter, the arrest typically occurs
during the G1 phase of the cell cycle while in the OA it is late in the G2
phase (Champlin, 2000).
Nitric oxide synthase is present in olfactory receptor cells throughout development of the adult antennal (olfactory) lobe of the brain of the moth Manduca sexta. The possible involvement of nitric oxide (NO) in antennal-lobe morphogenesis has been investigated. Inhibition of NO signaling with a NO synthase inhibitor or a NO scavenger early in development results in abnormal antennal lobes in which neuropil-associated glia fail to migrate. A more subtle effect is seen in the arborization of dendrites of a serotonin-immunoreactive neuron, that grow beyond their normal range. The effects of NO signaling in these types of cells do not appear to be mediated by activation of soluble guanylyl cyclase to produce cGMP, as these cells do not exhibit cGMP immunoreactivity following NO stimulation and are not affected by infusion of a soluble guanylyl cyclase inhibitor. Treatment with Novobiocin, which blocks ADP-ribosylation of proteins, results in a phenotype similar to those seen with blockade of NO signaling. Thus, axons of olfactory receptor cells appear to trigger glial cell migration and limit arborization of serotonin-immunoreactive neurons via NO signaling. The NO effect may be mediated in part by ADP-ribosylation of target cell proteins (Gibson, 2001).
Key cellular interactions must occur for the antennal lobe of M. sexta to develop normal cellular architecture. The axons of olfactory receptor cells (ORCs) arriving from the antennae form a template of protoglomeruli and induce glial cells in the antennal lobe to migrate to surround these protoglomeruli. The glial cells, in turn, act to stabilize the protoglomeruli and possibly confine subsequent axonal and dendritic branching to the glomerulus. Animals in which the antennae are removed to prevent ORC innervation of the antennal lobes never develop glomeruli. Glial cells remain in a rim around the perimeter of the neuropil, and the dendrites of multiglomerular antennal-lobe neurons branch in a diffuse pattern, rather than in glomerular tufts. The dendrites of uniglomerular projection neurons, which grow into protoglomeruli soon after their formation, are less affected. In animals treated to reduce severely the number of glial cells, ORC axons still grow to the developing antennal lobe, and their terminals coalesce to form protoglomeruli, but, in the absence of a sufficient number of glia to surround these protoglomeruli, the structure is lost and both axon terminals and multiglomerular neurons again form diffuse arborizations throughout the lobe. In short, the ORC axons establish the glomerular architecture via an interaction with antennal-lobe glial cells and possibly, also, via interactions with antennal-lobe neurons. Although intercellular interactions are essential, until the current study, no candidate signaling molecules had been identified (Gibson, 2001).
The profound effects resulting from blockade of NO signaling in the current study suggest that NO is indeed produced by the ORCs during development. It is an important component of the signaling by which the ORCs influence the antennal-lobe glial cells to migrate to surround protoglomeruli, and it also appears to restrict the outgrowth of dendrites of the 5-HT+ neuron to the basal (i.e., ORC-axon-free) half of each glomerulus. These specific effects of NO do not appear to be mediated by sGC, but may be mediated in part by ADP-ribosylation (Gibson, 2001).
When ingrowing ORC axons are prevented from signaling via NO by either of two pharmacological manipulations, the neuropil-associated glial cells of the antennal lobe fail to migrate. The effects of blocking NO signaling closely resemble those of removing ORC axons, suggesting that NO signaling may be a critical mode of communication between ORC axons and glia early in development. The results of L-NAME or CPTIO treatment on migration of peripheral glial cells down the antennal nerve provide additional support for this view. The decreased size of the antennal nerves in many treated animals is highly unlikely to be responsible for the similarity in appearance of treated animals to deafferented animals because as few as 12% of the normal complement of ORC axons are sufficient to produce protoglomeruli and induce the formation of mature glomeruli. Furthermore, the axons in treated animals often form a protoglomerular template (Gibson, 2001).
The simplest explanation for these effects on glia is that NO, produced in ORC axons, diffuses to nearby glia and stimulates them to migrate. Another formal possibility is that NO acts indirectly, causing the early-invading dendrites of uniglomerular projection neurons or the ORC axons themselves to alter expression of some factor necessary for glial cell migration. Involvement of uniglomerular projection neurons appears unlikely, because glomeruli form, with a glial envelope, even when the projection neurons are surgically removed prior to glomerulus development. Dendrites of local interneurons arrive at the glomeruli too late to be likely candidates. Thus, the effect of NO on glial cell migration is probably either direct or via an effect on the ORC axons themselves, enabling them to provide the migration signal to glia (Gibson, 2001).
Injections or infusions of NO-signaling blockers have to be started no later than late stage 2 to produce a strong effect on glial migration. Because antennal-lobe glia do not begin to migrate until stage 5, the results may indicate that NO initiates, at stage 2-3, a developmental program that is necessary to allow glia to migrate several days later. NO has been shown to inhibit the transcription factors AP-1 and NF-kB p50 by S-nitrosylation, and to promote cell migration as well as cell-surface expression of integrin alpahvß3, known to mediate cell migration in gliomas (Gibson, 2001).
It is interesting to note that, while the neuropil-associated glia of the antennal lobe and the glia of the antennae fail to migrate following blockade of NO signaling, the sorting zone glia, which migrate distally from the developing antennal lobe, appear to have migrated normally in all cases. The current results could indicate that the sorting zone glia are induced to migrate by a signal other than NO. They have been shown to differ from neuropil-associated glia of the antennal lobe in other ways; for example, sorting-zone glia require the presence of ORC axons for proliferation while antennal-lobe glia do not. Previous research indicates that sorting-zone glia have responded, via proliferation and migration, to ORC axon arrival by stage 3. It is therefore possible that initiation of the sorting-zone glial response to ORC axons would have occurred prior to the establishment of effective levels of anti-NO-signaling drugs in the test animals. This will be difficult to test, as earlier injections of L-NAME cause developmental arrest (Gibson, 2001).
L-NAME-treated animals in which glomeruli form provide a useful insight into the role of NO in determining outgrowth of the dendrites of the 5HT+ neuron. The 5HT+ dendrites normally ramify in areas of the glomerulus not occupied by the ORC axons, but when NO signaling is blocked, the dendrites are able to arborize throughout the region occupied by the ORC axons. The ability of NO to cause growth-cone collapse and thus halt process extension has been documented and attributed to blockade of growth-cone protein acylation. An attractive hypothesis is that, in M. sexta, such a process serves to prevent the majority of 5HT+ dendrites from entering ORN territory within the glomerulus. An alternative possibility is that an effect of NO on glial cells alters some growth-limiting effect on 5HT+ dendrites by the glia, although this seems less likely because glial cells outline the glomeruli and are not present at the equator, where 5HT+ dendritic branches normally terminate (Gibson, 2001).
Several lines of evidence reported here indicate that the NO-cGMP signaling pathway is not involved in either the glial cell migration or 5HT+ dendrite-outgrowth responses to NO. Previous studies indicate that NO does not induce detectable cGMP production in antennal-lobe glia at the relevant stages. Double labeling for 5HT and cGMP in NO-stimulated control brains at stage 7, when the 5HT+ neuron dendrites extend into the glomeruli, indicates that the 5HT+ neuron does not exhibit cGMP production either. Furthermore, the sGC inhibitor ODQ produces antennal lobes in which the glomeruli appear normal, with typical arborization of the 5HT+ neuron dendrites and the normal arrangement of neuropil-associated glia (Gibson, 2001).
Although sGC is the best characterized target of NO, several other downstream effectors of NO signaling are known. Direct NO-mediated nitrosylation of proteins, a feature common to these other mechanisms, can in turn lead to direct gating of cyclic nucleotide-gated channels, to modification of properties of extracellular matrix proteins, and to covalent attachment of ADP-ribose or NAD. ADP-ribosylation has been shown to affect the targeting or attachment of proteins to the membrane, the adhesion properties of fibronectin, and polymerization of actin and tubulin, mediated (at least in part) by Ras superfamily GTPases.
When ADP-ribosylation was blocked using Novobiocin, it was found that the neuropil-associated glia do not migrate, yet they extend processes to surround glomeruli. The animals were given only a single Novobiocin dose early in development (late 2 or early 3), well before the time at which glia normally extend processes and migrate. Therefore, these results most likely indicate an uncoupling of glial cell process extension and cell-body migration by Novobiocin. Because L-NAME and CPTIO treatment appear to have blocked both process extension and cell-body migration in these glia, it is possible that NO promotes process extension by one mechanism (not yet identified in this system), while mediating migration via an effect on ADP-ribosylation. Novobiocin was found also to mimic the effect of L-NAME and CPTIO on arborization of the dendrites of the 5HT+ neuron, suggesting that ORC axons restrict the outgrowth of 5HT+ dendrites via an NO-mediated increase in ADP-ribosylation of one or more growth cone proteins (Gibson, 2001).
In summary, this work suggests that NO serves as an important messenger in communication between the ORC axons and the antennal-lobe glia and the 5HT+ neuron of the antennal lobe. The fact that blockade of glial migration at stage 5 requires blockade of NO signaling no later than late stage 2 may indicate that NO works in part by initiating critical developmental programs. Moreover, the ability of Novobiocin to mimic some effects of NO-signaling blockade may indicate a role for NO-induced ADPribosylation (Gibson, 2001).
In both Drosophila and Apis (the honeybee), crude brain tissue contains the highest activities of NOS by far, whereas non-neuronal tissues, like thoracic tissue, show less than
5% of total NOS. Within the brain of adult animals, the dissected antennal lobes show the highest levels of NOS activities; the central brain and intermediate and visual neuropils show the lowest levels by far. The chemosensory neuropil in both Drosophila and Apis shows the highest activity levels. The antennal nerve and the contralateral projecting sensory fibers in the antennal commissure are strongly stained and the glomeruli exhibit a strong degree of labelling, with an non-homogeneous distribution within any given glomerulus. Some of the glomeruli are devoid of staining. The mechanosensory fibers show no labelling. In the central complex, the fan-shaped body and the nodule are stained. Unlike Apis, in which the mushroom bodies display a clearly compartmentalized pattern, in Drosophila no staining is detectable in the calyces (Müller, 1994).
The Malpighian tubule of Drosophila, a model for epithelial fluid transport, is an excellent system in which to study the roles of signal transduction processes in the control of fluid transport. The nitric oxide/cGMP signaling system activates epithelial fluid secretion by Drosophila Malpighian tubules. Exogenous cGMP stimulates fluid secretion through action of apical electrogenic Vacuolar H+ ATPase, a multisubunit complex that functions as a proton pump. V-ATPase was originally characterized as an endo-membrane component that pumps protons from the cytoplasm to the internal space of organelles. It also plays a role in energizing plasma membrane ion transport. V-ATPase drives one or more alkali metal/H+ exhangers. By this function, active transport of alkali metal cations (i.e. Na+ or K+) takes place (Davis, 1997 and references).
Nitric oxide stimulates fluid secretion with an associated elevation in intracellular cGMP levels. It is probable that the NO-induced increase in intracellular cGMP levels is due to an activation of soluble guanylate cyclase NO. The neuropeptide cardioacceleratory peptide (CAP2b) is expressed in Drosophila and has been shown to stimulate fluid secretion in Malpighian (renal) tubules. CAP2b acts to raise intracellular cGMP in tubules but not cAMP. As Malpighian tubules have been shown to exhibit NADPH diaphorase activity, implying the existence of Nitric oxide synthase, CAP2b is implicated in the stimulation of fluid transport via the activation of NOS and thence downstream components of the NO/cGMP signaling pathway. Treatment of Malpighian tubules with methylene blue, an inhibitior of soluble guanylate cyclase, attenuates CAP2b-enhanced secretion of fluid by Malpighian tubules. NOS activity was determined in unstimulated and CAP2b-stimulated tubules. After CAP2b treatment NOS activity rises to 144% of basal levels, and this CAP signal is sensitive to known inhibitors of NOS. This is the first demonstration of neuropeptide activation of a transport process via the NOS/cGMP signaling cascade in an epithelium (Davis, 1997)
Calcium is a ubiquitous second messenger that plays a critical role in both excitable
and non-excitable cells. Calcium mobilization in identified cell types within an intact
renal epithelium (the Drosophila melanogaster Malpighian tubule) was studied by
GAL4-directed expression of an aequorin transgene. Aequorin is a Ca2+ sensitive liminescent protein isolated from the coelenterate Aequorea victoria. It is a complex of apoaequorin, a 21 kDA polypeptide, and coelenterazine, a hydrophobic luminophore. Aequorin is used for monitoring Ca2+ changes. CAP2b, a cardioactive
neuropeptide that stimulates fluid secretion by a mechanism involving nitric oxide,
causes a rapid, dose-dependent rise in cytosolic calcium in only a single,
genetically-defined, set of 77 principal cells in the main (secretory) segment of the
tubule. In the absence of external calcium, the CAP2b-induced calcium response is
abolished. In Ca2+-free medium, the endoplasmic reticulum Ca2+-ATPase inhibitor,
thapsigargin, elevates [Ca2+]i only in the smaller stellate cells, suggesting that principal
cells do not contain a thapsigargin-sensitive intracellular pool. Assays for epithelial
function confirm that calcium entry is essential for CAP2b to induce a physiological
response in the whole organ. The data suggest a role for calcium
signaling in the modulation of the nitric oxide signaling pathway in this epithelium. CAP2b must act to increase fluid secretion rates solely by an initial rise of [CA2+]i in principal cells. CAP2b stimulates tubule Nitric oxide synthase activity. It is probable that the CAP2b induced rise in [CA2+]i is sufficient to trigger the activation of Drosophila calcium sensitive Nos. The maximal CAP2b concentrations employed elevate principal cell calcium levels from 87 to 255 nM, a value close to the EC50 of Drosophila NOS. This implies that Drosophila Nos is responsive over the range of the CAP2b concentrations employed. This may account for the observation that thapsigargin treatment results in increased basal cGMP levels that are not further increased on CAP2b stimulation. Thus the data provide strong evidence for a calcium-mediated link between CAP2b and NOS/cGMP activation of fluid secretion. The
GAL4-targeting system allows general application to studies of cell-signaling and
pharmacology that does not rely on invasive or cytotoxic techniques (Rosay, 1997).
Nitric oxide (NO) diffuses as short-lived messenger through the plasma membrane and serves, among many other functions, as an activator of the cGMP synthesizing enzyme soluble guanylyl cyclase (sGC). In view of recent genetic investigations that have postulated a retrograde signal from the larval muscle fibers to the presynaptic terminals, the presence of an NO/cGMP signaling system at the neuromuscular junction (NMJ) of Drosophila melanogaster larvae was sought. Application of NO donors induce cGMP immunoreactivity in the presynaptic terminals but not the postsynaptic muscle fibers at an identified NMJ. The NO-induced cGMP immunoreactivity is sensitive to a specific inhibitor (ODQ) of the sGC. Since presynaptic terminals that have been surgically isolated from the central nervous system are capable of synthesizing cGMP, it is suggested that a NO-sensitive guanylyl cyclase is present in the terminal arborizations. Using a fluorescent dye that is known to stain recycling synaptic vesicles, it has been demonstrated that NO donors and membrane permeant cGMP analogs cause vesicle release at the NMJ. Moreover, the NO-induced release can be blocked by the specific inhibitor of the sGC. A destaining of synaptic terminals after NO exposure in Ca2+-free solution in the presence of cobalt chloride as a channel blocker suggests that NO stimulates Ca2+-independent vesicle release at the NMJ. The combined immunocytochemical and exocytosis imaging experiments imply the involvement of cGMP and NO in the regulation of vesicle release at the NMJ of Drosophila larvae (Wildemann, 1999b).
Naturally occuring polymorphisms in behavior are difficult to map genetically and thus are refractory to molecular
characterization. An exception is the Drosophila
melanogaster foraging (for) gene, which has two naturally occurring variants relating to food-search behavior: rover and sitter. Molecular mapping placed foraging mutations in the dg2 gene, which
encodes a cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG), a target of NOS signaling. Rovers have higher PKG
activity than sitters, and transgenic sitters expressing a dg2 complementary DNA from rover show transformation of
behavior to rover type. Thus, PKG levels affect food-search behavior, and natural variation in PKG activity accounts for a behavioral polymorphism (Osborne, 1997).
Individuals with a rover allele (forR) move greater distances while feeding
than do those homozygous for sitter alleles
(fors). This difference in
foraging behavior is observed during both the larval and adult stages. Rovers and sitters do not differ in general activity in
the absence of food. Both rovers and sitters are
wild-type forms that exist at appreciable frequencies. Several mutations of the locus map with the naturally occurring alleles in
the 24A3-5 region of the D. melanogaster polytene
chromosomes. This region contains dg2, one of two cGMP-dependent protein kinase (PKG) genes in
Drosophila. The dg2 gene has three major transcripts, T1, T2, and
T3, and the for mutations are localized to
this region. The
P[GAL4] transposable element in 189Y was inserted in
the 5' end of the dg2 T2 transcript. This homozygous viable
insertion identified a new for allele, because P-element excision reverts larval foraging behavior from the sitter to the rover
phenotype. As is the case with
other sitter alleles, locomotion of the 189Y larvae is not reduced in
the absence of food, indicating that the change in behavior is
foraging-specific (Osborne, 1997).
To determine whether PKG is directly responsible for the foraging
polymorphism in Drosophila, dg2 was overexpressed
in sitter larvae. This results in a change of behavior to the rover
phenotype. The transgenic strain contains four copies of a heat
shock-driven dg2-cDNA. The basal level of
PKG expression in this transgenic strain is sufficient to rescue rover larval
behavior, thus eliminating the lethal and sublethal effects of heat on
the dg2-transgenic larvae. As expected, the PKG enzyme activities of the dissected larval central nervous systems (CNSs) show that without heat shock, the dg2-cDNA transgenic
strain have levels of PKG similar to those of
forR and significantly higher than those
of the sitter control strain (Osborne, 1997).
The basis for the dg2 activity difference between
forR and
fors was further addressed by
measurement of RNA levels and PKG protein. Northern (RNA) analysis
revealed that fors and
fors2 show a small but consistent reduction in the abundance of T1 RNA relative to that
in forR. T2 and T3 RNA are also reduced in
these strains, but to a lesser extent. To assess
protein levels, extracts of adult heads were subjected to protein
immunoblot analysis by probing with an antibody to bovine PKG, or the
extracts were affinity-purified by chromatography on cGMP-sepharose,
labeled, and electrophoresed. In both experiments, a
prominent band at a molecular mass of 80,000 Daltons was found. This
is the only band strongly induced by heat shock in the
dg2-cDNA transgenic strain, and it is less intense in
fors than
forR. (This band is
also somewhat less intense in fors2 and
nearly absent in 189Y homozygotes). Taken together, these results argue
that the difference between the naturally occurring alleles
forR and
fors is in the level of expression of
the enzyme (Osborne, 1997).
The assignment of mutations in the for gene to the
dg2 locus not only establishes the identification of PKG
mutations but also implicates the cGMP signal transduction pathway in
the regulation of food-search behavior in D. melanogaster.
Small but significant differences in the levels of this kinase affect
the naturally occurring behavioral polymorphism. These small
differences in PKG are even detectable in homogenates, indicating that
the differences in PKG level in rovers and sitters might be larger in
cells relevant to the expression of the foraging behavior. These results
suggest that the amount of kinase activity affects larval food-search behavior. Indeed, even modest quantitative changes in kinase activity affect behavior. Induced mutations that affect
behavioral phenotypes often lie in signal transduction pathways. For example, the cyclic adenosine monophosphate (cAMP)
system influences associative learning in flies, and genetic variants in two other
serine/threonine kinases: the calcium/calmodulin-dependent protein
kinase II and protein kinase C affect learning and behavioral plasticity in flies and mice. The
finding that for encodes a PKG shows that a naturally
occurring genetic polymorphism in behavior involves these pathways. PKG has a variety of pleiotropic cellular regulatory functions
that are also typical of signal transduction components. Electrophysiological studies have shown that injected kinase affects neuronal membrane conductance in snails and mammals; that inhibitors of PKG block long-term potentiation in mammalian hippocampus and that PKG is involved in
presynaptic long-term potentiation in cultured hippocampal neurons. Outside the nervous system, PKG has also been
implicated in controlling proliferation of smooth muscle cells
and neutrophil degranulation. These findings assign behavioral functions to this relatively scarce member
of the serine/threonine kinases and show that subtle differences in PKG
can lead to naturally occurring variation in behavior (Osborne, 1997 and references).
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Nitric oxide synthase:
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