TY - JOUR
AU - Gibbs, Sarah, M.
AB - Abstract Photoreceptors undergoing target selection in the optic lobe of Drosophila express a nitric oxide sensitive soluble guanylate cyclase (sGC). At the same time, cells in the target region of the optic lobe express nitric oxide synthase (NOS). Pharmacological inhibition of NOS, NO or sGC leads to disruption of the retinal projection pattern in vitro, and the extension of individual retinal axons beyond their appropriate targets. The disruptive effects of NOS inhibition in vitro are prevented by adding a cGMP analog. Mutations in the sGC alpha subunit gene, Gcα1, reduce sGC expression and attenuate NO-sensitive retinal cGMP production in the visual system. Although the retinal projection pattern is undisturbed in Gcα1 mutants, they lack positive phototaxis as adults, suggesting inappropriate connections exist between the photoreceptors and optic lobe interneurons in these flies. Preliminary results show that heat-shock expression of wild-type Gcα1 during metamorphosis can restore positive phototaxis in severe Gcα1 mutants. These in vivo results support the in vitro findings that NOS and sGC activity are required to promote the appropriate retinal innervation of the optic lobe. The formation of functionally appropriate synapses presents an important challenge to the developing nervous system, and involves a complex series of cell-cell interactions. Accurate pathfinding by axons, target recognition and synaptogenesis are required of individual neurons to ensure proper function of the nervous system in the adult. The highly organized repetitive architecture of the visual system of the fruitfly, Drosophila melanogaster, presents an ideal model for investigating the molecular mechanisms that regulate each of these developmental events. In addition, the adult fly has been utilized towards understanding the physiological basis of visual system function and visually-mediated behaviors (Benzer, 1967; Alawi and Pak, 1971; Heisenberg, 1972; Inoue et al., 1985; Shieh et al., 1997). Numerous studies have provided valuable insights into the processes of photoreceptor cell identity and pattern formation in the retina (Wolff and Ready, 1993; Wolff et al., 1997). However, what is less well understood is the means by which this same level of organization is preserved in the connections the photoreceptors make in the central nervous system of the animal. The work presented here investigates the role of the nitric oxide signaling pathway in the developing Drosophila visual system. The gas nitric oxide (NO) is produced by the enzyme nitric oxide synthase (NOS) through the conversion of arginine to citrulline. NO can diffuse to stimulate synthesis of the second messenger 3′,5′- cyclic guanosine monophosphate (cGMP) by interacting with soluble guanylate cyclase (sGC) (Arnold et al., 1977). These molecules have been shown to regulate aspects of both vertebrate and invertebrate neuronal development (Inglis et al., 1998; Truman et al., 1996; Scholz et al., 1998), in particular those of sensory patterning (Roskams et al., 1994; Wu et al., 1994; Cramer et al., 1996; Gibbs and Truman, 1998) and growth cone dynamics (Wang et al., 1995; Renteria and Constantine-Patton, 1996; Song et al., 1998). In Drosophila, both sGC and NOS are present in the developing visual system during the period of post-synaptic target selection by the photoreceptors (Gibbs and Truman, 1998). Pharmacological inhibition of NOS and sGC activity in vitro during metamorphosis disrupts the formation of the retinal projection pattern in the optic lobe, such that individual photoreceptors fail to terminate axon outgrowth appropriately, and often extend beyond their normal targets (Gibbs and Truman, 1998). In the intact fly, genetic mutations in the gene Gcα1, which encodes the alpha subunit of the soluble guanylate cyclase, produced adult flies in which positive phototaxis was weak or absent. Positive phototaxis was improved in severe mutants when Gcα1 was expressed with heat shock during metamorphosis. These preliminary behavioral results suggest that decreased sGC activity during development leads to subtle defects in the innervation of the optic lobe by the retina, which subsequently compromises the function of the visual system as a whole. These studies support a model whereby NO and cGMP are required during metamorphosis for maintaining the photoreceptor axons in the appropriate target region of the optic lobe prior to synapse formation, thus facilitating establishment of a functional adult visual system. The adult visual system forms during metamorphosis. Photoreceptors are determined in the developing eye disc and send axonal projections to targets in the optic lobe beginning in the third (final) larval instar, and proceeding through about 12 hr of pupal development. The photoreceptors are organized in the eye disc in 8-cell clusters called ommatidia (reviewed by Wolff and Ready, 1993), and different subsets of photoreceptors terminate in separate layers of the optic lobe (Meinertzhagen and Hanson, 1993; Wolff et al., 1997). Photoreceptors R1–6 project to the first optic ganglion, the lamina, whereas R7–8 synapse in a deeper layer called the medulla. Once the photoreceptor axons reach their target layers growth cone extension ceases, until 10–12 hr of metamorphosis. At this time the retinal growth cones recommence protrusive activity and begin a series of lateral movements within the target ganglia (Meinertzhagen and Hanson, 1993). By 43 hr of pupal development the basic organization of the visual system has been established (Fig. 1). The terminals of R1–6 form synaptic cartridges with laminar monopolar cells, and R7–8 segregate into two distinct layers of the medulla. Once the photoreceptors have selected their post-synaptic partners, synaptogenesis begins and continues until adult eclosion at 100 hr (Meinertzhagen and Hanson, 1993). The temporal window from approximately 10–48 hr of metamorphosis is crucial, as this is when the retinal projection pattern is formed. The extension of photoreceptor axons beyond the target must be prevented while the retinal growth cones maneuver among the cells of the optic ganglia. The presence of NO in the nervous system can be inferred through the expression or activity of NOS. A neuronal-type NOS has been cloned from adult Drosophila (Regulski and Tully, 1995), and contains several domains found in vertebrate NOS isoforms. An antibody made to the conserved NADPH-binding motif labels cells in the developing Drosophila visual system (Fig. 2). At the beginning of pupal development, this NOS antibody labeled a crescent-shaped group of cells in the developing optic lobe, just proximal to the terminals of photoreceptor axons (Fig. 2A). At 24 hr of metamorphosis, the region of NOS-like immunoreactivity (NOS-IR) had expanded to encompass the entire medulla, where the post-synaptic targets for photoreceptors R7&8 are located (Fig. 2B). Faint NOS-IR was also observed in the lamina, where the terminals of photoreceptors R1–6 are located (Fig. 2B). This pattern of NOS-IR persisted through 50 hr of metamorphosis (Fig. 2C), but began to decrease at 72 hr (Fig. 2D). By the end of pupal development, very little NOS-IR was detected in the optic lobe (Fig. 2E). NOS can also be detected with NADPH-diaphorase staining, a histochemical procedure that exploits the NADPH reducing activity of NOS in fixed tissue (Bredt et al., 1991; Hope et al., 1991). In the Drosophila visual system, strong NADPH-diaphorase staining was seen in the lamina and medulla at 24 hr (Fig. 2F) and throughout most of metamorphosis (not shown). While both methods of NOS detection reveal a putative source of NO in the medulla, only NADPH-diaphorase staining was strong in the lamina; NOS-IR was weak. Preliminary in situ hybridization studies with the Drosophila NOS indicate that its expression pattern resembles that of the universal NOS antibody (M. Regulski, personal communication), thus the strong diaphorase staining in the lamina may reflect the activity of other diaphorase-containing enzymes. The identity of these putative NO-producing cells as neuronal or glial has yet to be determined. However, their location within the target regions of the lamina and medulla is appropriate for providing NO to photoreceptor terminals, during a period of development following photoreceptor axon outgrowth but prior to the formation of synapses between the photoreceptors and cells in the optic lobe. NOS-like immunoreactivity and NADPH-diaphorase staining has been reported in the brains of other insects, such as the adult locust (Bicker and Hahnlein, 1995; Elphick et al., 1996), honeybee (Müller, 1996) and the hornworm Manduca sexta (Nighorn et al., 1998), as well as in adult and larval Drosophila (Müller and Buchner, 1993; Wildemann and Bicker, 1999). Indeed, studies in the adult locust suggest that NO may be involved in mediating the physiological response of the visual system to light (Elphick et al., 1996; Schmachtenberg and Bicker, 1999). NADPH-diaphorase activity is also found in the optic tectum of developing chicks (Wu et al., 1994), and the lateral geniculate nucleus of post-natal ferrets (Cramer et al., 1996). Pharmacological inhibition of NOS disrupts the normal patterning of retinal inputs in both of these vertebrate systems (Wu et al., 1994; Cramer et al., 1996), and double eNOS/nNOS knockout mice also show defects in the refinement of visual connections (Wu et al., 2000). These studies all suggest that NO produced in the target can regulate the specificity of visual connectivity. The result that NOS expression appeared to be low in the lamina and high in the medulla may underlie a mechanism to allow segregation of photoreceptor subtypes within the optic lobe. In this model, R1–6 would be sensitive to low levels of NO, whereas R7&8 would require higher levels to respond, allowing them to grow through the lamina to the medulla. Indeed, our observations suggest a difference does exist in the NO-sensitivity of these different subtypes, based on the production of cGMP to NO donors (S. M. G., unpublished data). In many developing invertebrate nervous systems, NO-sensitive sGC activity is correlated with periods of neuronal development or plasticity. This activity can be visualized by exposing the tissue to an exogenous source of NO, combined with an inhibitor of cyclic nucleotide-dependent phosphodiesterases, which normally act to break down these second messengers during intracellular signaling (Lincoln and Cornwell, 1993). The tissue is then stained with an antibody to cyclic GMP, thus immunocytochemically identifying those cells that can produce cGMP in response to NO at a particular point in time. NO-induced cGMP-immunoreactivity (cGMP-IR) has been observed in discreet sets of neurons in embryonic grasshoppers (Ball and Truman, 1998), locusts (Truman et al., 1996), and in the developing nervous system of larval lobsters (Scholz et al., 1998). It has also been demonstrated in the Manduca nervous system during larval life (Grueber and Truman, 1999; Zayas et al., 2000) and metamorphosis (Schactner et al., 1998) and in the embryonic and larval nervous systems of Drosophila (Wildemann and Bicker, 1999; S.M.G., unpublished data). In the developing Drosophila visual system, NO-sensitive sGC activity was observed in the photoreceptors during the first half of metamorphosis, subsequent to the arrival of retinal axons in the optic lobe but prior to synaptogenesis (Fig. 3). Pupal nervous systems were incubated with the NO donor sodium nitroprusside (SNP) and the phosophodiesterase inhibitor isobutylmethylxanthine (IBMX), prior to processing for cGMP-IR (De Vente et al., 1987; Truman et al., 1996). The untreated visual system showed essentially no cGMP-IR at 12 hr of pupal development (Fig. 3A). In contrast, strong cGMP-IR was observed at the same time point after exposure to SNP and IBMX, primarily in the photoreceptor cell bodies in the developing eye disc and axons extending into the lamina and medulla (Fig. 3B). The pattern of NO-sensitive sGC activity in the photoreceptors persisted through 24 hr (Fig. 3C), and appeared to cease by 48 hr, although other cells in the optic lobe showed cGMP-IR at this time (Fig. 3D). This period of NO-inducible cGMP production encompasses the developmental window when photoreceptors have extended their growth cones and are choosing those cells in the optic lobe with which they will ultimately form synapses (Meinertzhagen and Hanson, 1993; Gibbs and Truman, 1998). The presence of sGC activity in the photoreceptors and the expression of NOS in the target regions of the optic lobe during the first half of metamorphosis suggested the interaction of NO and sGC were involved in establishing the retinal projection pattern. The central nervous system of Drosophila can be placed in culture, and if provided with the hormone ecdysone it undergoes growth and morphological changes mirroring those observed during in vivo metamorphosis (Awad and Truman, 1998). This technique was adapted to investigate the role of NO and cGMP in the developing visual system in vitro (Gibbs and Truman, 1998). Isolated central nervous systems with attached eye imaginal discs from early pupae were placed in culture for 96 hr. When nervous systems from control cultures were stained with an antibody to chaoptin, which labels the photoreceptors and their axons, the retinal projection pattern was revealed (Fig. 4A) (Zipursky et al., 1984). Photoreceptors R1–6 synapse in the lamina, whereas R7&8 form a highly organized pattern of terminals in the medulla. This pattern was severely disrupted when the NOS inhibitor L-NAME was added to the culture medium. The photoreceptors formed a dense, unorganized tangle in the medulla, and retinal axons were seen projecting beyond their normal targets into other regions of the optic lobe (Fig. 4B). A similar sort of effect was observed in the presence of the NO scavenger PTIO (data not shown; Gibbs and Truman, 1998). The compound D-NAME, which does not inhibit NOS activity, did not affect organization of the retinal terminals in vitro (Fig. 4C). A low level of the sGC inhibitor ODQ also produced a significant disruption in the projection pattern in vitro, in a manner similar to that observed with the L-NAME treatment Fig. 4D). These results demonstrate that NOS activity, NO itself, and sGC activity are each necessary for the formation of the retinal projection pattern in vitro. However, the most convincing evidence that cGMP in the photoreceptors is sufficient to promote their appropriate patterning is shown in Figure 4D. In this experiment, the disruptive effects of L-NAME were rescued in vitro with the addition of the cGMP analog 8-Bromo-cGMP. Under these culture conditions a well-organized pattern of terminals is observed in the optic lobe (Fig. 4D). This result demonstrates that the inhibition of NOS is likely not having a secondary effect on the overall health or organization of putative post-synaptic cells in the optic lobe. More importantly, it appears that cGMP alone is sufficient to stabilize retinal axon outgrowth in the absence of NOS activity. Soluble guanylate cyclases function as heterodimers comprised of a large alpha (α) subunit and a small beta (β) subunit. NO is just one potential sGC ligand; others include the gas carbon monoxide (CO) (Miki et al., 1977; Brune and Ullrich, 1987), and products of arachidonic acid metabolism (Ignarro et al., 1985). Three different α subunits and two β subunits have been cloned from vertebrates (Nakane et al., 1990; Harteneck et al., 1991), and information about their structures and activities has come primarily from biochemical and in vitro studies. For example, the α and β subunits both contain putative catalytic domains that are required for sGC activity (Harteneck et al., 1990; Buechler et al., 1991; Yuen et al., 1994). Genes for the α (dgcα1) and β (dgcβ1) subunits of sGC have been cloned from Drosophila (Yoshikawa et al., 1993; Liu et al., 1995; Shah and Hyde, 1995), and the DGCα1/DGCβ1 heterodimer is stimulated to produce cGMP by NO (Shah and Hyde, 1995). DGCα1 expression has been observed in the adult retina, implicating cGMP as a possible mediator of phototransduction in flies (Yoshikawa et al., 1993; Shah and Hyde, 1995). Although exogenous cGMP has been shown to induce membrane currents and enhance the photoresponse in isolated photoreceptors (Bacigalupo et al., 1995), inositol trisphosphate (IP3) and diacylglycerol (DAG) appear to be primarily responsible for generating the depolarizing potential in the Drosophila retina (Zuker, 1996; Chyb et al., 1999). Thus while its primary role in phototransduction remains in question, the importance of sGC activity during visual system development, at least under in vitro conditions, appears clear. The genetic tools available in Drosophila provide an opportunity to further examine the developmental role of sGC in vivo. Flies carrying mutations in the Gcα1 gene were produced via EMS mutagenesis, and recovered by western blot analysis for the absence of GCα1 protein in adult heads. No null mutants were recovered, but four alleles showed decreases in the intensity of GCα1 expression (R. Hardy and A. Becker, personal communication). The allelic series was Gcα15>Gcα11>Gcα13>Gcα12, although differences in the levels of Gcα1 expression were virtually indistinguishable between Gcα15, Gcα11 and Gcα13 (R. Hardy and A. Becker, unpublished data). The Gcα11 allele carries a mutation in the splice acceptor site for the gene, leading to a predicted insertion into the Gcα1 protein (R. Hardy and A. Becker, personal communication). To date this is the only allele that has been characterized in any detail, and it is not known if this insertion leads to instability of the protein. The decreased expression of GCα1 observed in the western blot screen was also reflected in a severe reduction in NO-sensitive sGC activity in the developing visual system (Fig. 5). At 24 hr of metamorphosis, strong cGMP-IR was observed in the photoreceptors of wild-type flies after exposure to 1 mM SNP/IBMX (Fig. 5A; Gibbs and Truman, 1998). In contrast, very little cGMP-IR was detected in the photoreceptors of the Gcα13 mutants, even after exposure to higher levels of SNP (10 mM; Fig. 5B). Weak cGMP-IR persisted in the photoreceptor cell bodies of the eye disc (Fig. 5B, arrow), demonstrating that these mutants are not complete functional nulls and low levels of residual sGC activity remain, but no cGMP-IR was detected in the retinal axons. Thus Gcα1 encodes the alpha subunit of the sGC responsible for generating the observed NO-induced rise in cGMP production in the visual system during metamorphosis. It might be expected that the Gcα1 mutants would display defects in visual system organization mirroring those observed with in vitro inhibition of sGC. As revealed by labeling with an antibody to FasII, the terminals of retinal axons R7&8 in the medulla of Gcα13 did appear slightly expanded in size when compared to wild-type controls (Fig. 5D, C). However, gross defects in visual system architecture and inappropriate retinal outgrowth were not observed in any of the four mutants at any age (data not shown). The residual Gcα1 expression detected by western blot (not shown) and NO-sensitive sGC activity in the retina (Fig. 5B), could be sufficient to prevent any gross defects in visual system topography in vivo, but may sensitize the system to further disruption of NO/cGMP signaling. This hypothesis is currently being tested by exposing the visual system of Gcα1 mutants to low levels of NOS and sGC inhibitors during development, using the in vitro culture system described previously (Fig. 3; Gibbs and Truman, 1998). Although the Gcα1 mutants did not show any obvious morphological defects in visual system organization, they showed severe defects in visual system function. In an assay for phototaxis adapted from Benzer (1967), adult flies were tested for positive phototaxis in a counter-current apparatus. A population of 100 flies were placed in the first of a series of six clear tubes, and transferred through the tubes at 30 sec intervals as they moved towards a horizontal light source. Wild-type flies showing normal phototaxis consistently moved towards the light to end up in the last tube (tube #6) by the end of the trial. In contrast, one-week old Gcα1 adults displayed diminished phototactic behavior, ranging from weak phototaxis (Gcα12, Gcα15) to essentially no phototaxis at all (Gcα11, Gcα13), when compared to wild-type Canton-S strain and the claret1 strain from which the mutants were generated (Fig. 6A). This phenotype was not the result of negative phototaxis, or due to defects in locomotion as measured by geotactic behavior (data not shown). Interestingly, two mutant strains, Gcα12 and Gcα13, showed improved positive phototaxis over the first 10 days of adulthood (Fig. 6B, C). Flies from the same population were tested at 3, 6, 7, and 10 days post-eclosion. At three days of age, only 10% of the Gcα13 mutants ended up in the last two tubes, but at 10 days the percentage of total Gcα13 flies in the last two tubes had more than doubled (Fig. 6B). The change in the behavior of Gcα12 was more dramatic. Only 3.5% of these mutants made it to the last two tubes at 3 days of age (Fig. 6C). At 6 days this number had increased to 27%, and was 44% by one week post-eclosion (Fig. 6C). The performance of Gcα12 was stabilized at 10 days, never reaching the levels of the wild-type population. The phototactic profile of the wild-type Canton-S and the claret1 progenitor strains did not change significantly over the 10-day period (data not shown). There is a process of synaptic refinement that occurs in wild-type flies after eclosion (Meinertzhagen, 1989; Hirsch and Tompkins, 1994), and this may account for the improved phototactic performance of Gcα12 and Gcα13 as adults. It is unlikely that this is a direct consequence of NO/cGMP signaling in the adult, as NOS-IR is weak at this time and adult photoreceptors do not show NO-sensitive cGMP synthesis (S.M.G., unpublished observations). Rather, diminished sGC signaling in the mutants during visual system development may produce delays in the establishment of synaptic connections between the photoreceptors and cells of the optic lobe. Depending on the severity of the mutation, these delays may or may not be corrected by post-eclosion refinement mechanisms. The Gcα11 mutants showed no positive phototaxis (Fig. 6A), a phenotype that did not improve with age (data not shown). We are performing further experiments to better characterize the role of Gcα1 in visual behavior, using Gcα11 flies carrying a heat-shock inducible Gcα1 gene. A PCR fragment representing the full length gene was cloned into the P{CaSpeR-hs}heat shock transformation vector (Thummel and Pirrotta, 1992; R. Hardy and A. Becker, personal communication). P element-mediated germline transformation and fly manipulations were then performed using standard techniques (Karess and Rubin, 1984; R. Hardy and A. Becker, personal communication). In preliminary studies, expression of the Gcα1—heat shock construct during the first 48 hr of metamorphosis resulted in improved phototactic performance by the Gcα11 adults, such that more than 4 times the number of flies receiving heat shocks ended up in the last tube at the end of the experiment as those not receiving heat shocks (Fig. 6D). These results support the hypothesis that sGC activity is required during establishment of the retinal projection pattern, and that lack of this activity produces defects in visual system organization leading to impairment of visual function. The electrophysiological basis for the behavioral phenotypes of the Gcα11 mutants described here is currently being examined with extracellular recording techniques. In the Drosophila visual system cGMP production appears to primarily regulate retinal growth cone dynamics and axon extension during the period of post-synaptic partner selection by the photoreceptors (Fig. 7). The photoreceptors extend axons towards optic lobe targets beginning in the third larval instar and continuing through about 10 hr of pupal development. During this period of time the optic lobe contains glia and some neurons, although neuronal proliferation and maturation continues in the optic lobe through metamorphosis (Hofbauer and Campos-Ortega, 1990; Selleck and Steller, 1991; Meinertzhagen and Hanson, 1993). Once the retinal axons reach their targets, outgrowth ceases and the growth cones become inactive. This may reflect the fact that retinal axons grow sequentially into the optic lobe as they are determined in a posterior to anterior wave in the eye imaginal disc, thus early-arriving axons may “wait” for the remaining axons to arrive (Wolff and Ready, 1993; Wolff et al., 1997). In addition, putative post-synaptic targets may not be mature at this time. The NO/cGMP signaling pathway does not appear to be active during this phase of visual system development, as NOS expression in the optic lobe is low, and NO-sensitive sGC activity is not detected in the photoreceptors. At around 12 hr of metamorphosis the retinal growth cones become active again, possibly in response to the rising levels of ecdysteroid hormones that drive metamorphic development (Fig. 7, second panel; Truman et al., 1993). The extended growth cones move laterally within the cells of the lamina and medulla, selecting those cells with which they will form synapses (Meinertzhagen and Hanson, 1993). The period of post-synaptic partner selection continues through about 48 hr of metamorphosis, and the components of the NO/cGMP signaling pathway are strongly expressed during this time (Fig. 7, second panel). NOS expression peaks in cells of the lamina and medulla, and NO-sensitive sGC activity is strong in the photoreceptors. By 48 hr of pupal development the photoreceptors have chosen their post-synaptic partners, and synaptogenesis begins. At this time the growth cones collapse, and there is no longer the need for a mechanism preventing further longitudinal extension of the retinal axons. NOS expression begins to decrease in the optic lobe after 48 hr, eventually disappearing by the end of metamorphosis, and there is no longer detectable sGC activity in the photoreceptors (Fig. 7, bottom panel). The data presented here complement the growing body of evidence that NO and cGMP play an important role in the establishment of neuronal connections. Indeed, such a signaling system seems ideally suited for developmental processes occurring after outgrowing axons reach their targets, but before the onset of synaptogenesis. NO is a rapidly synthesized molecule that does not need to be packaged or secreted. Although diffusible, NO is also highly reactive, making its activity in biological systems short-lived (Meulmans, 1994). By interacting with sGC to produce the second messenger cGMP, a brief pulse of NO could have long-lasting and specific effects on the physiology of cells it entered. This type of interaction could modulate growth cone responses to extracellular cues. For example, cGMP can alter the turning response of retinal ganglion cell growth cones to the chemorepellent semaphorin from repulsion to attraction (Song et al., 1998). In addition, cGMP has been shown to produce calcium transients in the growth cones of cultured neurons (Van Wagenen and Rehder, 1999; Kafitz et al., 2000), which could also affect growth cone dynamics. In nematode worms, mutations in a cyclic-nucleotide gated channel affect the outgrowth of sensory axons during development and in the adult (Coburn and Bargmann, 1996; Coburn et al., 1998). For such an intracellular signal to influence growth cone dynamics, it must ultimately affect the cytoskeletal elements that form the filipodial and lammelipodial extensions, and there is some evidence to support a role for cyclic nucleotides in this process. For example, in cultured neuroblastoma cells, intracellular injections of cGMP analogues caused the motile structures of growth cones to freeze and retract, whereas cAMP analogs promoted outgrowth and expansion (Bolsover et al., 1992). More recent studies show that cGMP-dependent protein kinase-mediated phosphorylation of the small GTPase RhoA can inactivate RhoA and prevents it from stimulating myosin light chain phosphatase (Surks et al., 1999; Sauzeau et al., 2000; Sawada et al., 2001). Although this is one mechanism by which cGMP could affect the actin/myosin cytoskeleton, its role in neuronal growth cones has not been demonstrated. There is little direct evidence for cyclic nucleotide-mediated cytoskeletal dynamics in Drosophila photoreceptors. Two transcription factors that regulate the specification of photoreceptor identity, rough and glass, have been shown to genetically interact with a gene similar to the mammalian vasodilator-stimulated protein VASP (DeMille et al., 1996). VASP is a substrate for cAMP and cGMP-dependent protein kinases, and is associated with actin filaments, focal adhesions and other dynamic membrane regions (Haffner et al., 1995). The mutant phenotype of this VASP-like gene has not been described, and its expression in retinal axons or growth cones has not been examined. However, it also contains regions of similarity to enabled, which is required for the formation of proper axonal connections in Drosophila (Gertler et al., 1995). A genetic screen for defects in photoreceptor cell connectivity uncovered five genes directly affecting retinal axon outgrowth (Martin et al., 1995), but to date only one of these has been characterized in detail, dreadlocks (dock). Dock protein is localized to the growth cones of photoreceptors that have reached their targets, and mutations in dock disrupt retinal axon guidance and targeting (Garrity et al., 1996). Interestingly, the retinal projection phenotype of dock mutants strikingly resembles the disruptive effects of NO and sGC inhibition in vitro (Gibbs and Truman, 1998). The dock gene encodes an adapter protein containing src homolgy (SH) domains, proposed to link guidance signaling through phosphotyrosine to downstream effectors of the cytoskeleton and growth cone motility (Garrity et al., 1996; Rao and Zipursky, 1998). These studies complement those demonstrating that transmembrane tyrosine phosphatases play important roles in controlling axon guidance decisions and growth cone behaviors in Drosophila (Krueger et al., 1996; Desai et al., 1997). However, it remains to be determined if one or more of these receptor tyrosine phosphatases act through Dock in regulating axon targeting in the Drosophila visual system. Whether cGMP produced in the retinal growth cones acts directly with or parallel to the tyrosine phosphatase/Dock pathways remains a question for further research. Open in new tabDownload slide Fig. 1. The developing adult visual system of Drosophila is highly organized by 43 hr of pupal development. The nervous system was processed for immunocytochemistry with an antibody to chaoptin (gift of H. Steller and S. L. Zipursky), which labels the photoreceptor cell bodies and axons. (A) Photoreceptor cells are born in the developing eye imaginal disc (ed) and send axonal projections to the lamina (la) and medulla (me) ganglia of the optic lobe. (B) The terminals of R7/8 segregate into two synaptic layers of the medulla (arrow) Open in new tabDownload slide Fig. 1. The developing adult visual system of Drosophila is highly organized by 43 hr of pupal development. The nervous system was processed for immunocytochemistry with an antibody to chaoptin (gift of H. Steller and S. L. Zipursky), which labels the photoreceptor cell bodies and axons. (A) Photoreceptor cells are born in the developing eye imaginal disc (ed) and send axonal projections to the lamina (la) and medulla (me) ganglia of the optic lobe. (B) The terminals of R7/8 segregate into two synaptic layers of the medulla (arrow) Open in new tabDownload slide Fig. 2. The optic lobe contains a potential source of NO during metamorphosis. Nervous systems were processed for immunocytochemistry with antibodies to chaoptin (CHP) and nitric oxide synthase (NOS; Affinity BioReagents Inc.). Pupae were staged from the time of puparium formation. (A) The NOS antibody (red) labels a small set of cells in the optic lobe (arrow) directly adjacent to chaoptin-stained retinal axon terminals (green) at white puparium formation (WP). (B) NOS-IR is weak in the lamina (la) and strong in the medulla (me) at 24 hr of metamorphosis. The terminals of R7/8 lie at the border of NOS staining in the medulla (arrow). (C) This pattern of NOS-IR remains strong at 50 hr, and begins to decrease by 72 hr (D). (E) There is no detectable NOS-IR in the optic lobe at the end of pupal development (96 hr). (F) NADPH-diaphorase staining is observed in the lamina (la) and medulla (me) as well as Bolwig's nerve (bn) at 24 hr. Figures B, F reprinted from Gibbs and Truman, 1998; copyright Cell Press Open in new tabDownload slide Fig. 2. The optic lobe contains a potential source of NO during metamorphosis. Nervous systems were processed for immunocytochemistry with antibodies to chaoptin (CHP) and nitric oxide synthase (NOS; Affinity BioReagents Inc.). Pupae were staged from the time of puparium formation. (A) The NOS antibody (red) labels a small set of cells in the optic lobe (arrow) directly adjacent to chaoptin-stained retinal axon terminals (green) at white puparium formation (WP). (B) NOS-IR is weak in the lamina (la) and strong in the medulla (me) at 24 hr of metamorphosis. The terminals of R7/8 lie at the border of NOS staining in the medulla (arrow). (C) This pattern of NOS-IR remains strong at 50 hr, and begins to decrease by 72 hr (D). (E) There is no detectable NOS-IR in the optic lobe at the end of pupal development (96 hr). (F) NADPH-diaphorase staining is observed in the lamina (la) and medulla (me) as well as Bolwig's nerve (bn) at 24 hr. Figures B, F reprinted from Gibbs and Truman, 1998; copyright Cell Press Open in new tabDownload slide Fig. 3.  The photoreceptors show NO-sensitive sGC activity during the first half of metamorphosis. Nervous systems were processed for immunocytochemistry with an antibody to cGMP (gift of J. De Vente). (A) No cGMP-IR is observed in the untreated eye disc (ed) or optic lobe (ol) at 12 hr of pupal development (P12). (B) When the P12 nervous system is incubated with 1 mM SNP and 1 mM IBMX for 15 min prior to immunocytochemistry, strong cGMP-IR is seen in the photoreceptors, including the axonal projections of R1–6 to the lamina (large arrow) and R7&8 extending to the medulla (small arrow), as well as the larval optic nerve (Bolwig's nerve, bn). (C) Retinal cGMP-IR persists in response to SNP/IBMX at 24 hr of metamorphosis. (D) By 48 hr the photoreceptors no longer show cGMP-IR in response to SNP/IBMX treatment. Weak cGMP-IR appears in the optic lobe cells of the lamina (small arrow) and medulla (large arrow) Open in new tabDownload slide Fig. 3.  The photoreceptors show NO-sensitive sGC activity during the first half of metamorphosis. Nervous systems were processed for immunocytochemistry with an antibody to cGMP (gift of J. De Vente). (A) No cGMP-IR is observed in the untreated eye disc (ed) or optic lobe (ol) at 12 hr of pupal development (P12). (B) When the P12 nervous system is incubated with 1 mM SNP and 1 mM IBMX for 15 min prior to immunocytochemistry, strong cGMP-IR is seen in the photoreceptors, including the axonal projections of R1–6 to the lamina (large arrow) and R7&8 extending to the medulla (small arrow), as well as the larval optic nerve (Bolwig's nerve, bn). (C) Retinal cGMP-IR persists in response to SNP/IBMX at 24 hr of metamorphosis. (D) By 48 hr the photoreceptors no longer show cGMP-IR in response to SNP/IBMX treatment. Weak cGMP-IR appears in the optic lobe cells of the lamina (small arrow) and medulla (large arrow) Open in new tabDownload slide Fig. 4. Inhibition of NOS or sGC disrupts the retinal projection pattern in vitro. Nervous systems and attached eye discs from white puparia were cultured for 96 hr before being processed for chaoptin immunocytochemistry. The hormone 20-hydroxyecdysone (20-HE; 1 μg/ml) was added to all cultures at 24 hr. (A) The visual system after 96 hr in culture under control conditions (20-HE alone). A well-organized retinal projection pattern is formed in the lamina and medulla. (B) With the addition of 1 mM L-NAME, the projection pattern is severely disrupted. Individual retinal axons are seen extending beyond the medulla and into the lobula (arrows). (C) 1 mM D-NAME has no disruptive effect on the retinal projection pattern. (D) 10 μM ODQ, a sGC inhibitor, also causes severe disorganization of the projection pattern. Retinal axons growing beyond the medulla converge into one large fascicle extending all the way into the brain (arrow). (E) When 100 nM 8-Bromo-cGMP is included with 1 mM L-NAME in culture, a normal, well-organized projection pattern is formed Open in new tabDownload slide Fig. 4. Inhibition of NOS or sGC disrupts the retinal projection pattern in vitro. Nervous systems and attached eye discs from white puparia were cultured for 96 hr before being processed for chaoptin immunocytochemistry. The hormone 20-hydroxyecdysone (20-HE; 1 μg/ml) was added to all cultures at 24 hr. (A) The visual system after 96 hr in culture under control conditions (20-HE alone). A well-organized retinal projection pattern is formed in the lamina and medulla. (B) With the addition of 1 mM L-NAME, the projection pattern is severely disrupted. Individual retinal axons are seen extending beyond the medulla and into the lobula (arrows). (C) 1 mM D-NAME has no disruptive effect on the retinal projection pattern. (D) 10 μM ODQ, a sGC inhibitor, also causes severe disorganization of the projection pattern. Retinal axons growing beyond the medulla converge into one large fascicle extending all the way into the brain (arrow). (E) When 100 nM 8-Bromo-cGMP is included with 1 mM L-NAME in culture, a normal, well-organized projection pattern is formed Open in new tabDownload slide Fig. 5. GCα1 mutants show decreased cGMP-IR, but no obvious disorganization of the retinal projection pattern. (A) cGMP-IR in the wild-type visual system at P24 after 1 mM SNP/IBMX treatment. (B) cGMP-IR is severely reduced in the visual system of the Gcα13 mutant after exposure to 10 mM SNP/IBMX. Faint cGMP-IR is observed in the photoreceptor cell bodies in the eye disc (arrow), but not in the retinal axons. (C) The projection pattern of R7&8 in the medulla of the wild-type visual system at P48, as revealed with Fas II immunocytochemistry, is virtually indistinguishable from that of the Gcα13 mutant (D) Open in new tabDownload slide Fig. 5. GCα1 mutants show decreased cGMP-IR, but no obvious disorganization of the retinal projection pattern. (A) cGMP-IR in the wild-type visual system at P24 after 1 mM SNP/IBMX treatment. (B) cGMP-IR is severely reduced in the visual system of the Gcα13 mutant after exposure to 10 mM SNP/IBMX. Faint cGMP-IR is observed in the photoreceptor cell bodies in the eye disc (arrow), but not in the retinal axons. (C) The projection pattern of R7&8 in the medulla of the wild-type visual system at P48, as revealed with Fas II immunocytochemistry, is virtually indistinguishable from that of the Gcα13 mutant (D) Open in new tabDownload slide Fig. 6. Gcα1 mutants lack positive phototactic behavior. Graphs represent the cumulative results from counter-current assays performed on 6-day-old adults. Phototactic profiles were generated by averaging the results from 3 tests of approximately 100 flies/test. (A) The four Gcα1 mutants lack positive phototaxis when compared to Canton-S and the parent wild-type strain claret1 (ca1). (B) The phototactic profile of Gcα13 improves slightly between 3 and 10 day post-eclosion. (C) The phototactic profile of Gca12 greatly improves between 3 and 10 day post-eclosion. (D) Heat shock-induced expression of Gcα1 every 8 hr for the first 48 hr of metamorphosis improves the phototactic capacity of Gcα11 adults. The mean number of total flies in the last tube of the counter-current apparatus at the end of the experiment is shown (100 flies tested for each group; averaged results from 2 experiments). Error bars = standard error Open in new tabDownload slide Fig. 6. Gcα1 mutants lack positive phototactic behavior. Graphs represent the cumulative results from counter-current assays performed on 6-day-old adults. Phototactic profiles were generated by averaging the results from 3 tests of approximately 100 flies/test. (A) The four Gcα1 mutants lack positive phototaxis when compared to Canton-S and the parent wild-type strain claret1 (ca1). (B) The phototactic profile of Gcα13 improves slightly between 3 and 10 day post-eclosion. (C) The phototactic profile of Gca12 greatly improves between 3 and 10 day post-eclosion. (D) Heat shock-induced expression of Gcα1 every 8 hr for the first 48 hr of metamorphosis improves the phototactic capacity of Gcα11 adults. The mean number of total flies in the last tube of the counter-current apparatus at the end of the experiment is shown (100 flies tested for each group; averaged results from 2 experiments). Error bars = standard error Open in new tabDownload slide Fig. 7. Model for NO and cGMP signaling in the developing Drosophila visual system. NO produced by cells in the target ganglia of the optic lobe diffuses into retinal growth cones and stimulates sGC from approximately P10–P48. The resulting production of cGMP acts to stabilize retina axons, preventing further longitudinal growth during the period of lateral growth cone movement Open in new tabDownload slide Fig. 7. Model for NO and cGMP signaling in the developing Drosophila visual system. NO produced by cells in the target ganglia of the optic lobe diffuses into retinal growth cones and stimulates sGC from approximately P10–P48. The resulting production of cGMP acts to stabilize retina axons, preventing further longitudinal growth during the period of lateral growth cone movement 1 From the Symposium Nitric Oxide in the Invertebrates: Comparative Physiology and Diverse Functions presented at the Annual Meeting of the Society for Integrative and Comparative Biology, 4–8 January 2000, at Atlanta, Georgia. 2 E-mail: sarahmgibb@aol.com The author wishes to thank the following people for their generous contributions to this work: Charles Zuker for donating the Gcα1 mutants; Robert Hardy and Ann Becker of the Zuker lab for sharing their unpublished data; Hermann Steller and S. Larry Zipursky for the chaoptin antibody; Jan De Vente for the cGMP antibody; Tarif Awad, David Baldwin, and Doug Currie for technical input and assistance; James W. Truman and members of the Truman lab for insight and advice, and the Department of Zoology at the University of Washington. 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Crossref Search ADS PubMed The Society for Integrative and Comparative Biology
TI - Regulation of Drosophila Visual System Development by Nitric Oxide and Cyclic GMP
JF - Integrative and Comparative Biology
DO - 10.1093/icb/41.2.268
DA - 2001-04-01
UR - https://www.deepdyve.com/lp/oxford-university-press/regulation-of-drosophila-visual-system-development-by-nitric-oxide-and-m02AfnanCm
SP - 268
VL - 41
IS - 2
DP - DeepDyve
ER -