InteractiveFly: GeneBrief

Guanylyl cyclase at 76C: Biological Overview | References

BIOLOGICAL OVERVIEW

Cyclic nucleotide levels within extending growth cones influence how navigating axons respond to guidance cues. Pharmacological alteration of cAMP or cGMP signaling in vitro dramatically modulates how growth cones respond to attractants and repellents, although how these second messengers function in the context of guidance cue signaling cascades in vivo is poorly understood. Using a novel Sema-1a-dependent forward genetic screening approach, it was found that Drosophila receptor-type guanylyl cyclase: Gyc76C, a protein possessing a single transmembrane domain, is required for semaphorin-1a (Sema-1a)-plexin A repulsive axon guidance of motor axons in vivo. Genetic analyses define a neuronal requirement for Gyc76C in axonal repulsion. Additionally, it was found that the integrity of the Gyc76C catalytic cyclase domain is critical for Gyc76C function in Sema-1a axon repulsion. These results support a model in which cGMP production by Gyc76C facilitates Sema-1a-plexin A-mediated defasciculation of motor axons, allowing for the generation of neuromuscular connectivity in the developing Drosophila embryo (Ayoob, 2004).

During neural development, axons extend along complex, but precisely defined, routes to contact their appropriate targets and establish the connectivity of the adult nervous system. Guidance cues belonging to several families have been identified that direct axons along these pathways through attractive and repulsive mechanisms. For many of these extracellular cues, including ephrins, netrins, slits, and semaphorins, cell surface receptors have been identified that are required for the establishment of these neuronal trajectories. The signal transduction pathways by which these guidance cue receptors direct the cytoskeletal alterations critical for attractive or repulsive steering events, however, are only now beginning to be understood (Ayoob, 2004).

Two well characterized intracellular effectors that can dictate how an axon responds to extracellular signals are the second messengers cAMP and cGMP. Experiments with cultured Xenopus spinal neurons, in an in vitro growth cone turning assay, showed that changing the intracellular levels of cAMP or cGMP alters how an axon responds to extracellular guidance cues. For example, the attractive response of an axon to the guidance cue netrin-1 can be converted to repulsion by decreasing the effective levels of cAMP within the responding neuron. Conversely, the axonal response to the potent chemorepellent semaphorin 3A (Sema3A) can be converted from repulsion to attraction by increasing cGMP levels within the neuron. cAMP and cGMP have been shown to function together to influence how an axon responds to a particular attractant or repellent. The ratio of cAMP to cGMP determines how extending axons respond to netrin-1 in the growth cone turning assay: high cAMP to cGMP ratios produce an attractive response, whereas low ratios lead to repulsion (Nishiyama, 2003). Related observations have been made for Sema3A. When cAMP levels are raised in cultured neurons, the potent growth cone collapsing effect of Sema3A is neutralized (Dontchev, 2002; Chalasani, 2003). However, in these same cultures, raising the levels of cGMP potentiates the growth cone collapsing effect of Sema3A, suggesting that both cyclic nucleotides can modulate the response to a single axon guidance cue (Ayoob, 2004 and references therein).

The molecular mechanisms underlying axon guidance effects caused by pharmacologically altering cyclic nucleotide levels are still unclear. Insight into how cAMP dictates axonal steering responses has been gained from the identification of Nervy, a protein that couples plexin A (PlexA), the receptor for the invertebrate transmembrane semaphorin-1a (Sema-1a), with the cAMP-dependent protein kinase A (PKA). Sema-1a is present on motor axons in the developing Drosophila nervous system and through its receptor PlexA acts as a repellent and directs individual axons away from the tightly fasciculated bundles in which they travel. Nervy tethers PKA to the PlexA, positioning PKA to antagonize Sema-1a-mediated repulsion in response to local increases in cAMP (Ayoob, 2004).

Several studies provide hints as to which proteins involved in cGMP signaling may be involved in modulating or supporting axon guidance events (Gibbs, 1998; Polleux, 2000; Seidel, 2000; Schmidt, 2002; Nishiyama, 2003). How specific proteins function in particular axon guidance signaling pathways to alter cGMP levels is, however, unknown. Using a novel Sema-1a-dependent forward genetic screening approach, it was found that the Drosophila receptor guanylyl cyclase (rGC) Gyc76C, a member of the phylogenetically conserved family of single transmembrane domain guanylyl cyclases (Wedel, 2001), is necessary for Sema-1a-mediated repulsive signaling in the developing Drosophila embryonic nervous system. Furthermore, the data strongly suggest that cGMP production by Gyc76C is essential for its function in vivo. Together, these findings provide a functional link between local production of cGMP within the growth cone and Sema-1a repulsive axon guidance signaling (Ayoob, 2004).

These experiments provide an important molecular link between semaphorin-mediated repulsion and cGMP signaling in vivo. Gyc76C is critical for Sema-1a-Plexin A-mediated selective defasciculation of axon bundles in the developing Drosophila neuromuscular system. A conserved amino acid residue within the Gyc76C cyclase domain, a residue required for receptor guanylyl cyclase (rGC) catalytic activity, is also required in Gyc76C for correct motor axon pathfinding. The identification of Gyc76C as an essential component of the Sema-1a-PlexA repulsive axon guidance signaling pathway provides insight into how cyclic nucleotide production is linked to the cascade of events downstream of semaphorin-mediated repulsion. These observations also provide a potential target for modulating repulsive semaphorin signaling by alterations of cGMP levels directly through rGCs (Ayoob, 2004).

These analyses demonstrate a role for the rGC Gyc76C in Sema-1a-mediated axon-axon repulsion. LOF mutations were generated in the Gyc76C gene and highly penetrant phenotypes were observed similar to the motor axon guidance defects observed in sema1a, plexA, and mical mutants. Micals are a family of conserved flavoprotein oxidoreductases that function in Plexin-mediated axonal repulsion (Terman, 2002). Neuronal expression of a Gyc76C cDNA restores the wild-type innervation pattern in gyc76C mutant embryos and also restores viability to the lethal gyc76C mutant line, demonstrating a requirement for Gyc76C in neurons for correct axonal pathfinding. Neuronal overexpression of wild-type Gyc76C also results in phenotypes resembling PlexA GOF phenotypes. The genetic interaction analyses confirm a role for Gyc76C in Sema-1a-PlexA repulsive signaling. Embryos heterozygous for both Gyc76C and other members of this signaling cascade, including Sema-1a, PlexA, and MICAL, display motor axon pathway disruptions. These phenotypes are qualitatively similar to LOF mutant phenotypes observed in sema1a, plexA, and mical LOF mutants and are seen at comparable frequencies. In addition to suppressing the Sema-1a- dependent midline phenotype, loss of Gyc76C function also suppresses a PlexA- dependent phenotype. However, increasing the levels of Gyc76C enhances this PlexA GOF phenotype. Finally, a Gyc76C transgene lacking a key conserved aspartate residue required for cyclase catalytic activity does not rescue either the gyc76C embryonic motor axon guidance defects or the lethality associated with gyc76C mutants and appears to function in a dominant-negative manner. Taken together, these results link Gyc76C to the proper generation of neuromuscular connectivity in Drosophila through its role in mediating semaphorin-plexin signaling events associated with axonal repulsion. In addition, these results strongly suggest that cGMP production is critical for Gyc76C participation in Sema1a neuronal signaling events (Ayoob, 2004).

Initial in vitro observations demonstrating the importance of cGMP levels in semaphorin-mediated repulsion shows that increasing cGMP signaling reverses the repulsive signal from the secreted vertebrate semaphorin Sema3A, resulting in Sema3A acting as an attractant in the single growth cone steering assay. Recent studies show that Sema3A growth cone collapse requires increased cGMP signaling and also that cAMP signaling acts in opposition to cGMP signaling in the modulation of Sema3A-mediated growth cone collapse. Support for cAMP signaling cascades modulating semaphorin-mediated repulsion in vivo is provided by a demonstration that the A-kinase anchoring protein Nervy serves to antagonize Sema-1a-mediated axonal repulsion in Drosophila motor axons. Presumably, Nervy acts by localizing cAMP activation of PKA to the Plexin receptor and decreases Sema-1a repulsive signaling (Terman, 2004). The identification of Gyc76C as a positive effector in vivo of Sema-1a-PlexA-mediated repulsion is consistent with these Sema3A growth cone collapse studies. A model recently proposed for cyclic nucleotide modulation of netrin-1-mediated attraction and repulsion provides insight into how cGMP might effect semaphorin-mediated steering, collapse, and in vivo axonal repulsion. Using the in vitro growth cone steering assay, Nishiyama (2004) has shown that the [cAMP]/[cGMP] ratio determines whether netrin-1 acts in an attractive or a repulsive manner: high ratios promote attraction, whereas lower ratios promote repulsion. Importantly, a basal level of cGMP signaling is required for both netrin-mediated attractive and repulsive responses in this system. Although it remains to be determined, it is tempting to speculate that, like the observations for netrin-1-mediated guidance, the [cAMP]/[cGMP] ratio also serves to modulate semaphorin signaling events. In Drosophila motor axons, Gyc76C and Nervy could function antagonistically to regulate Sema-1a signaling in this manner. Gyc76C production of cGMP would lower a [cAMP]/[cGMP] ratio and thus promote repulsion, whereas increases in cAMP levels would decrease repulsion through PKA tethered to PlexA by Nervy. A loss of Gyc76C altogether would result in abolition of Sema-1a repulsion because of a cGMP requirement for any guidance response, and this is what was observed in the gyc76C mutants. Future experiments will determine how raising or lowering Gyc76C activity affects the guidance response to Sema-1a in vivo (Ayoob, 2004).

This study describes a role for a receptor-type guanylyl cyclase in axon guidance as an effector of transmembrane Sema1a axonal repulsion. Soluble guanylyl cyclases in both vertebrates and invertebrates have been implicated in axonal and dendritic guidance. However, in a GOF genetic screen for Sema-1a signaling components, genomic regions containing genes encoding all of the identified Drosophila soluble guanylyl cyclase subunits were assayed, including one known to be expressed in the nervous system, yet heterozygosity at these loci did not suppress or enhance the Sema-1a GOF phenotype. This may reflect a requirement for cGMP production at or near the PlexA receptor to provide a local increase in cGMP levels essential for semaphorin-mediated axonal repulsion and suggests that basal cGMP signaling provided by soluble gyanylyl cyclases is not essential for semaphorin-mediated repulsion. The initial genetic screen covered an additional two of the seven Drosophila rGCs, however, neither of the deficiencies that remove these rGCs genetically interacted with the Sema-1a GOF phenotype. Taken together, these results from the genetic screen suggest that Gyc76C is an integral component of the semaphorin signaling cascade and that cGMP production by other sources may not contribute to this repulsion. These results also motivate future experiments to investigate specific interactions between Gyc76C and PlexA (Ayoob, 2004).

Vertebrate receptor guanylyl cyclases that have a single transmembrane domain like Gyc76C are best known for their roles as receptors for natriuretic peptides that regulate blood pressure and volume and also for their role in the visual phototransduction cascade. The other vertebrate rGCs, however, have no known ligands or functions. In addition, very little is known about what roles, if any, these vertebrate rGCs play during neural development. It will be of great interest to investigate whether any vertebrate rGCs participate in semaphorin repulsive signaling (Ayoob, 2004).

Because Gyc76C is a multidomain protein, it is likely that regions other than the cyclase domain are important for its function. Interestingly, like the transmembrane protein Off-track, which is also required for Sema1a-mediated motor axon repulsion in Drosophila, Gyc76C contains a catalytically inactive kinase homology domain (KHD). In the vertebrate receptor guanylyl cyclase GC-A, this region has been shown to play a regulatory role by inhibiting the catalytic cyclase domain. The KHD of Gyc76C, or possibly Off-track, may function as an important modulator of cyclase activity (Ayoob, 2004).

The portion of Gyc76C that is C terminal to the conserved cyclase domain is unique among rGC family members; it is much longer than the same region in other rGCs and shares no amino acid similarity with these regions or with sequences of any known proteins. However, the last four amino acids of Gyc76C fit the consensus for a PDZ (PSD-95, Discs-large, zona occludens-1) domain binding motif. A similar motif is also found in MICAL, another component of the Sema-1a signaling cascade (Terman, 2002), raising the possibility that, as has been observed for other assemblages of signaling components, PDZ domain-containing scaffolding proteins may serve an important role in semaphorin signaling (Ayoob, 2004).

Gyc76C may provide a direct physical link between the leading edge of the growth cone and the motile machinery of the actin cytoskeleton. Vertebrate rGCs in photoreceptors are able to bind actin filaments, and the C-terminal domains of intestinal rGCs have also been implicated in interactions with the actin cytoskeleton. Perhaps the large C-terminal extension of Gyc76C functions in a similar manner to bridge the regions of signal reception and output. Whether or not Gyc76C cyclase activity is ligand gated remains unknown, and like all other Drosophila rGCs and the majority of vertebrate rGCs, Gyc76C is an orphan receptor. Future experiments will address whether Sema-1a triggers Gyc76C catalytic activity and also whether Gyc76C is indeed part of the receptor complex for Sema-1a (Ayoob, 2004).

In conclusion, using a novel genetic screening paradigm for identifying semaphorin signaling cascade components, an in vivo link was found between Sema-1a-mediated repulsive guidance and cGMP signaling pathways. Characterization of other candidates from this screen will likely provide additional insight into the mechanisms of repulsive axon guidance signaling (Ayoob, 2004).

Function of the Drosophila receptor guanylyl cyclase Gyc76C in PlexA-mediated motor axon guidance

The second messengers cAMP and cGMP modulate attraction and repulsion mediated by neuronal guidance cues. This study found that the Drosophila receptor guanylyl cyclase Gyc76C genetically interacts with Semaphorin 1a (Sema-1a) and physically associates with the Sema-1a receptor Plexin A (PlexA). PlexA regulates Gyc76C catalytic activity in vitro, and each distinct Gyc76C protein domain is crucial for regulating Gyc76C activity in vitro and motor axon guidance in vivo. The cytosolic protein dGIPC interacts with Gyc76C and facilitates Sema-1a-PlexA/Gyc76C-mediated motor axon guidance. These findings provide an in vivo link between semaphorin-mediated repulsive axon guidance and alteration of intracellular neuronal cGMP levels (Chak, 2013).

The Gyc76C-Sema-1a gain-of-function genetic interactions observed in this study are consistent with previous observations showing that Gyc76C loss and gain of function modifies aberrant CNS midline crossing by FasII+ longitudinal axons in a PlexA gain-of-function genetic background. Furthermore, this study observed robust physical interactions between Gyc76C and PlexA both in vitro and in vivo, raising the possibility that PlexA regulates Gyc76C-mediated signaling. Co-expressing PlexA at high levels in vitro augments cGMP levels produced by Gyc76C. Future work will establish whether extracellular, intracellular, or both, types of protein-protein associations between Gyc76C and PlexA are essential for regulating Gyc76C enzymatic activity (Chak, 2013).

Gyc76C structure-function analyses are consistent with the idea that PlexA binds to the extracellular and intracellular regions of Gyc76C to relieve inhibitory effects on GC activity from of Gyc76C intramolecular interactions, increasing cGMP levels within extending motor axon growth cones and affecting growth cone guidance. This is reminiscent of Sema-3A bath application increasing intracellular cGMP levels in Xenopus spinal neurons in vitro (Togashi, 2008), and the current results suggest that intracellular cGMP produced by Gyc76C is required for Sema-1a-mediated repulsion. However, it is possible that signaling by intracellular cGMP is coupled with intracellular cAMP in Sema-1a-mediated repulsive guidance events, and future work will determine whether varying the cAMP-to-cGMP ratio modulates Sema-1a-mediated repulsion, or converts it to attraction. Bath application of Sema-1a did not affect Gyc76C-PlexA physical associations or Gyc76C-PlexA-mediated cGMP production, suggesting that Sema-1a-dependent regulation of intracellular cGMP levels could involve ligand-gated, dynamic, spatiotemporal regulation of GC activity; visualizing this signaling event will require real-time imaging of cGMP during repulsive growth cone steering (Chak, 2013).

The small GTPase Rac and its downstream effector p21 activated kinase (PAK) can regulate receptor GCs to raise cellular cGMP levels in fibroblasts in vitro, and the kinase domain of PAK interacts with the cyclase domain of receptor GC-E (Guo, 2007; Guo, 2010); PAK, therefore, may associate with Gyc76C and regulate this receptor GC during axon pathfinding (Chak, 2013).

The deletion of the Gyc76C PDZ-binding motif strongly suppresses cGMP production by full length Gyc76C, and Gyc76C variants lacking the PBM motif exhibit low cell-surface expression levels, suggesting dGIPC (Kermit) regulates Gyc76C cell-surface localization. PDZ-containing proteins could form a protein scaffold required for plasma membrane localization of the Sema-1a-PlexA/Gyc76C signaling complex, and this study finds that the PDZ domain-containing dGIPC protein interacts with Gyc76C. In vertebrates, GIPC regulates protein trafficking, subcellular localization and various signaling events. Gyc76C cell-surface localization is enhanced in vitro in the presence of dGIPC, and this may serve to regulate Gyc76C-mediated signaling. dGIPC genetic analyses show that dGIPC plays a neuronal role in motor axon guidance, and this likely occurs through interactions that modulate Gyc76C-mediated cGMP signaling. Mammalian GIPC forms dimers and multimeric complexes. dGIPC may be a part of a molecular scaffold that couples Gyc76C to cell membrane trafficking machinery or anchors Gyc76C to the plasma membrane. Alternatively, dGIPC may be essential for activating or transducing Gyc76C-mediated cGMP signaling in axon guidance events. Future genetic and biochemical analyses will reveal the downstream signaling components that respond to changes in cGMP levels in vivo and direct discrete neuronal growth cone steering responses (Chak, 2013).

The Gyc76C receptor Guanylyl cyclase and the Foraging cGMP-dependent kinase regulate extracellular matrix organization and BMP signaling in the developing wing of Drosophila melanogaster

The developing crossveins of the wing of Drosophila melanogaster are specified by long-range BMP signaling and are especially sensitive to loss of extracellular modulators of BMP signaling such as the Chordin homolog Short gastrulation (Sog). However, the role of the extracellular matrix in BMP signaling and Sog activity in the crossveins has been poorly explored. Using a genetic mosaic screen for mutations that disrupt BMP signaling and posterior crossvein development, this study has identified Gyc76C, a member of the receptor guanylyl cyclase family that includes mammalian natriuretic peptide receptors. Gyc76C and the soluble cGMP-dependent kinase Foraging, likely linked by cGMP, are necessary for normal refinement and maintenance of long-range BMP signaling in the posterior crossvein. This does not occur through cell-autonomous crosstalk between cGMP and BMP signal transduction, but likely through altered extracellular activity of Sog. This study identified a novel pathway leading from Gyc76C to the organization of the wing extracellular matrix by matrix metalloproteinases and shows that both the extracellular matrix and BMP signaling effects are largely mediated by changes in the activity of matrix metalloproteinases. Parallels and differences between this pathway and other examples of cGMP activity in both Drosophila melanogaster and mammalian cells and tissues are discussed (Schleede, 2015).

The vein cells that develop from the ectodermal epithelia of the Drosophila melanogaster wing are positioned, elaborated and maintained by a series of well-characterized intercellular signaling pathways. The wing is easily visualized, and specific mutant vein phenotypes have been linked to changes in specific signals, making the wing an ideal tissue for examining signaling mechanisms, for identifying intracellular and extracellular crosstalk between different pathways, and for isolating new pathway components (Schleede, 2015).

The venation defect, the loss of the posterior crossvein (PCV), is used to identify and characterize participants in Bone Morphogenetic Protein (BMP) signaling. The PCV is formed during the end of the first day of pupal wing development, well after the formation of the longitudinal veins (LVs, numbered L1-L6), and requires localized BMP signaling in the PCV region between L4 and L5. Many of the homozygous viable crossveinless mutants identified in early genetic screens have now been shown to disrupt direct regulators of BMP signaling, especially those that bind BMPs and regulate BMP movement and activity in the extracellular space. The PCV is especially sensitive to loss of these regulators because of the long range over which signaling must take place, and the role many of these BMP regulators play in the assembly or disassembly of a BMP-carrying 'shuttle' (Schleede, 2015).

The BMP Decapentaplegic (Dpp) is secreted by the pupal LVs, possibly as a heterodimer with the BMP Glass bottom boat (Gbb). This stimulates autocrine and short-range BMP signaling in the LVs that is relatively insensitive to extracellular BMP regulators. However, Dpp and Gbb also signal over a long range by moving into the intervein tissues where the PCV forms. In order for this to occur, the secreted BMPs must bind the D. melanogaster Chordin homolog Short gastrulation (Sog) and the Twisted gastrulation family member Crossveinless (Cv, termed here Cv-Tsg2 to avoid confusion with other 'Cv' gene names). The Sog/Cv-Tsg2 complex facilitates the movement of BMPs from the LVs through the extracellular space, likely by protecting BMPs from binding to cell bound molecules such as their receptors. In order to stimulate signaling in the PCV, BMPs must also be freed from the complex. The Tolloid-related protease (Tlr, also known as Tolkin) cleaves Sog, lowering its affinity for BMPs, and Tsg family proteins help stimulate this cleavage. Signaling is further aided in the PCV region by a positive feedback loop, as BMP signaling increases localized expression of the BMP-binding protein Crossveinless 2 (Cv-2, recently renamed BMPER in vertebrates). Cv-2 also binds Sog, cell surface glypicans and the BMP receptor complex, and likely acts as a co-receptor and a transfer protein that frees BMPs from Sog. The lipoprotein Crossveinless-d (Cv-d) also binds BMPs and glypicans and helps signaling by an unknown mechanism (Schleede, 2015).

PCV development takes place in a complex and changing extracellular environment, but while there is some evidence that PCV-specific BMP signaling can be influenced by changes in tissue morphology or loss of the cell-bound glypican heparan sulfate proteoglycans, other aspects of the environment have not been greatly investigated. During the initial stages of BMP signaling in the PCV, at 15-18 hours after pupariation (AP), the dorsal and ventral wing epithelia form a sack that retains only a few dorsal to ventral connections from earlier stages; the inner, basal side of the sack is filled with extracellular matrix (ECM) proteins, both diffusely and in laminar aggregates. As BMP signaling in the PCV is maintained and refined, from 18-30 hours AP, increasing numbers of dorsal and ventral epithelial cells adhere, basal to basal, flattening the sack. The veins form as ECM-filled channels between the two epithelia, while in intervein regions scattered pockets of ECM are retained basolaterally between the cells within each epithelium; a small amount of ECM is also retained at the sites of basal-to-basal contact. This changing ECM environment could potentially alter BMP movement, assembly of BMP-containing complexes, and signal reception, as has been demonstrated in other developmental contexts in Drosophila (Schleede, 2015).

This study demonstrates the strong influence of the pupal ECM on PCV-specific long-range BMP signaling, through the identification of a previously unknown ECM-regulating pathway in the wing. In a screen conducted for novel crossveinless mutations on the third chromosome, a mutation was found in the guanylyl cyclase at 76C (gyc76C) locus, which encodes one of five transmembrane, receptor class guanylyl cyclases in D. melanogaster. Gyc76C has been previously characterized for its role in Semaphorin-mediated axon guidance; Malpighian tubule physiology, and the development of embryonic muscles and salivary glands. Like the similar mammalian natriuretic peptide receptors NPR1 and NPR2, the guanylyl cyclase activity of Gyc76C is likely regulated by secreted peptides, and can act via a variety of downstream cGMP sensors (Schleede, 2015).

The evidence suggests that Gyc76C influences BMP signaling in the pupal wing by changing the activity of the cGMP-dependent kinase Foraging (For; also known as Dg2 or Pkg24A), also a novel role for this kinase. But rather than controlling BMP signal transduction in a cell-autonomous manner, evidence is provided that Gyc76C and Foraging regulate BMP signaling non-autonomously by dramatically altering the wing ECM during the period of BMP signaling in the PCV. This effect is largely mediated by changing the levels and activity of matrix metalloproteinases (Mmps), especially Drosophila Mmp2. Genetic interactions suggest that the ECM alterations affect the extracellular mobility and activity of the BMP-binding protein Sog (Schleede, 2015).

This provides the first demonstration of Gyc76C and For activity in the developing wing, and the first evidence these proteins can act by affecting Mmp activity. Moreover, the demonstration of in vivo link from a guanylyl cyclase to Mmps and the ECM, and from there to long-range BMP signaling, may have parallels with findings in mammalian cells and tissues. NPR and NO-mediated changes in cGMP activity can on the one hand change matrix metalloproteinase expression secretion and activity, and on the other change in BMP and TGFβ signaling (Schleede, 2015).

Mutation 3L043, uncovered by a genetic screen to identify homozygous lethal mutations required for PCV development, is a novel allele of gyc76C, a transmembrane peptide receptor that, like vertebrate NPRs, acts as a guanylyl cyclase. gyc76C is likely linked by cGMP production to the activity of the cGMP-dependent kinase For, and that Gyc76C and For define a new pathway for the regulation of wing ECM. This pathway appears to act largely through changes in the activity of ECM-remodeling Mmp enzymes. Loss of gyc76C or For alter both the organization of the wing ECM and the levels of the two D. melanogaster Mmps, and the gyc76C knockdown phenotype can be largely reversed by knockdown of Mmp2. This is the first indication of a role for cGMP, Gyc76C and For function in the developing wing, and their effects on the ECM provides a novel molecular output for each (Schleede, 2015).

Gyc76C and For are necessary for the normal refinement and maintenance of long-range BMP signaling in the posterior crossvein region of the pupal wing; in fact, crossvein loss is the most prominent aspect of the adult gyc76C knockdown phenotype. The evidence suggests that this effect is also mediated by changes in Mmp activity, and most likely the Mmp-dependent reorganization of the ECM. In fact, analysis using genetic mosaics finds no evidence for a reliable, cell autonomous effect of cGMP activity on BMP signal transduction in the wing. Thus, this apparent crosstalk between receptor guanylyl cyclase activity and BMP signaling in the wing is mediated by extracellular effects (Schleede, 2015).

It is noteworthy that the cGMP activity mediated by NPR or nitric oxide signaling can change also Mmp gene expression, secretion or activation in many different mammalian cells and tissues. Both positive and negative effects have been noted, depending on the cells, the context, and the specific Mmp. Given the strong role of the ECM in cell-cell signaling, the contribution of cGMP-mediated changes in Mmp activity to extracellular signaling may be significant. There is also precedent for cGMP activity specifically affecting BMP and TGFβ signaling in mammals. cGMP-dependent kinase activity increases BMP signaling in C2C12 cells, and this effect has been suggested to underlie some of the effects of nitric oxide-induced cGMP on BMP-dependent pulmonary arterial hypertension. Conversely, atrial natriuretic peptide stimulates the guanylyl cyclase activities of NPR1 and NPR2 and can inhibit TGFβ activity in myofibroblasts; this inhibition has been suggested to underlie the opposing roles of atrial natriuretic peptide and TGFβ during hypoxia-induced remodeling of the pulmonary vasculature. However, unlike the pathway observed in the fly wing, these mammalian effects are thought to be mediated by the intracellular modulation of signal transduction, with cGMP-dependent kinases altering BMP receptor activity or the phosphorylation and nuclear accumulation of receptor-activated Smads. Nonetheless, it remains possible that there are additional layers of regulation mediated through extracellular effects, underscoring the importance of testing cell autonomy (Schleede, 2015).

Aside from its role in adult Malpighian tubule physiology, Gyc76C was previously shown to have three developmental effects: in the embryo it regulates the repulsive axon guidance mediated by Semaphorin 1A and Plexin A, the proper formation and arrangement of somatic muscles, and lumen formation in the salivary gland. All these may have links to the ECM. Loss of gyc76C from embryonic muscles affects the distribution and vesicular accumulation of the βintegrin Mys, and reduces laminins and the integrin regulator Talin in the salivary gland. The axon defects likely involve a physical interaction between Gyc76C and semaphorin receptors that affects cGMP levels; nonetheless, gyc76C mutant axon defects are very similar to those caused by loss of the perlecan Trol (Schleede, 2015).

The parallels between the different contexts of Gyc76C action are not exact, however. First, only the wing phenotype has been linked to a change in Mmp activity. Second, unlike the muscle phenotype, the wing phenotype is not accompanied by any obvious changes in integrin levels or distribution, beyond those caused by altered venation. Finally, most gyc76C mutant phenotypes are reproduced by loss of the Pkg21D (Dg1) cytoplasmic cGMP-dependent kinase, instead of For (Dg2, Pkg24A) as found in the wing, and thus may be mediated by different kinase targets (Schleede, 2015).

For has been largely analyzed for behavioral mutant phenotypes, and the overlap between Pkg21D and For targets is unknown. While many targets have been identified for the two mammalian cGMP-dependent kinases, PRKG1 (which exists in alpha and beta isoforms) and PRKG2, it is not clear if either of these is functionally equivalent to For. One of the protein isoforms generated by the for locus has a putative protein interaction/dimerization motif with slight similarity to the N-terminal binding/dimerization domains of alpha and beta PRKG1, but all three For isoforms have long N-terminal regions that are lacking from PRKG1 and PRKG2. In fact, a recent study suggested that For is instead functionally equivalent to PRKG2: Like PRKG2, For can stimulate phosphorylation of FOXO, and is localized to cell membranes in vitro. But For apparently lacks the canonical myristoylation site that is thought to account for the membrane localization and thus much of the target specificity of PRKG2. FOXO remains the only identified For target, and foxo null mutants are viable with normal wings (Schleede, 2015).

The loss of long range BMP signaling in the PCV region caused by knockdown of gyc76C can, like the ECM, be largely rescued by knockdown of Mmp2. Two results suggest that it is the alteration to the ECM that affects long-range BMP signaling, rather than some independent effect of Mmp2. First, the BMP signaling defects caused by gyc76C knockdown were rescued by directly manipulating the ECM through the overexpression of the perlecan Trol. Second, when Mmp activity is inhibited by overexpression of the diffusible Mmp inhibitor TIMP, this not only rescued the PCV BMP signaling defects caused by gyc76C knockdown, but also led to ectopic BMP signaling, not throughout the region of TIMP expression, but only in those regions with abnormal accumulation of ECM (Schleede, 2015).

The Mmp2-mediated changes in the ECM likely affect long-range BMP signaling by altering the activity of extracellular BMP-binding proteins, particularly Sog. The BMPs Dpp and Gbb produced in the LVs bind Sog and Cv-Tsg2, shuttle into the PCV region, and are released there by Tlr-mediated cleavage of Sog and transfer to Cv-2 and the receptors. Genetic interaction experiments suggest that knockdown of gyc76C both increases Sog's affinity for BMPs and reduces the movement of the Sog/Cv-Tsg2/BMP complex into the crossvein region (Schleede, 2015).

Collagen IV provides the best-studied example for how the ECM might affect Sog activity. The two D. melanogaster collagen IV chains regulate BMP signaling in other contexts, and they bind both Sog and the BMP Dpp. Results suggest that collagen IV helps assemble and release a Dpp/Sog/Tsg shuttling complex, and also recruits the Tld protease that cleaves Sog cleavage and releases Dpp for signaling. D. melanogaster Mmp1 can cleave vertebrate Collagen IV. Since reduced Gyc76C and For activity increases abnormal Collagen IV aggregates throughout the wing and diffuse Collagen IV in the veins, it ishypothesized that these Collagen IV changes both foster the assembly or stability of Sog/Cv-Tsg2/BMP complexes and tether them to the ECM, favoring the sequestration of BMPs in the complex and reducing thelong-range movement of the complex into the region of the PCV (Schleede, 2015).

While few other D. melanogaster Mmp targets have been identified, it is likely that Mmp1 and Mmp2 share the broad specificity of their mammalian counterparts, so other ECM components, known or unknown, might be involved. For instance, vertebrate Perlecan and can be cleaved by Mmps. Trol regulates BMP signaling in other D. melanogaster contexts, and Trol overexpression rescue gyc76C knockdown's effects on BMP signaling. But while null trol alleles are lethal before pupal stages, normal PCVs were formed in viable and even adult lethal alleles like trol^G0023, and actin-Gal 4-driven expression of trol-RNAi using any of four different trol-RNAi lines did not alter adult wing venation. Loss of the D. melanogaster laminin B chain shared by all laminin trimers strongly disrupts wing venation, and a zebrafish laminin mutation can reduce BMP signaling (Schleede, 2015).

Finally, it was recently shown that Dlp, one of the two D. melanogaster glypicans, can be removed from the cell surface by Mmp2. While gyc76C knockdown did not detectably alter anti-Dlp staining in the pupal wing, it is noteworthy that Dlp and the second glypican Dally are required non-autonomously for BMP signaling in the PCV and that they bind BMPs and other BMP-binding proteins.

Search PubMed for articles about Drosophila Gyc76c

Ayoob, J. C., Yu, H.-H. Terman, J. R. and Kolodkin, A. L. (2004). The Drosophila receptor Guanylyl cyclase Gyc76C is required for Semaphorin-1a-Plexin A-mediated axonal repulsion. J. Neurosci. 24(30): 6639-6649. PubMed ID: 15282266

Chak, K. and Kolodkin, A. L. (2013). Function of the Drosophila receptor guanylyl cyclase Gyc76C in PlexA-mediated motor axon guidance. Development 141(1): 136-47. PubMed ID: 24284209

Chalasani, S. H., et al. (2003). A chemokine, SDF-1, reduces the effectiveness of multiple axonal repellents and is required for normal axon pathfinding. J. Neurosci. 23: 1360-1371. PubMed ID: 12598624

Dontchev, V. D. and Letourneau, P. C. (2002). Nerve growth factor and semaphorin 3A signaling pathways interact in regulating sensory neuronal growth cone motility. J. Neurosci. 22: 6659-6669. PubMed ID: 12151545

Gibbs, S. M. and Truman. J. W. (1998). Nitric oxide and cyclic GMP regulate retinal patterning in the optic lobe of Drosophila. Neuron 20: 83-93. PubMed ID: 9459444

Gibbs, S. M., Becker, A., Hardy, R. W. and, Truman, J. W. (2001). Soluble guanylate cyclase is required during development for visual system function in Drosophila. J. Neurosci. 21: 7705-7714. PubMed ID: 11567060

Guo, D., Tan, Y. C., Wang, D., Madhusoodanan, K. S., Zheng, Y., Maack, T., Zhang, J. J. and Huang, X. Y. (2007). A Rac-cGMP signaling pathway. Cell 128: 341-355. PubMed ID: 17254971

Guo, D., Zhang, J. J. and Huang, X. Y. (2010). A new Rac/PAK/GC/cGMP signaling pathway. Mol Cell Biochem 334: 99-103. PubMed ID: 19937092

Nishiyama, M., et al. (2003). Cyclic AMP/GMP-dependent modulation of Ca2+ channels sets the polarity of nerve growth-cone turning. Nature 424: 990-995. PubMed ID: 12827203

Polleux, F., Morrow, T. and Ghosh, A. (2000). Semaphorin 3A is a chemoattractant for cortical apical dendrites. Nature 404: 567-573. PubMed ID: 10766232

Schleede, J. and Blair, S. S. (2015). The Gyc76C receptor Guanylyl cyclase and the Foraging cGMP-dependent kinase regulate extracellular matrix organization and BMP signaling in the developing wing of Drosophila melanogaster. PLoS Genet 11: e1005576. PubMed ID: 26440503

Schmidt, H., et al. (2002). cGMP-mediated signaling via cGKIalpha is required for the guidance and connectivity of sensory axons. J. Cell. Biol. 159: 489-498. PubMed ID: 12417579

Seidel, C. and Bicker, G. (2000). Nitric oxide and cGMP influence axonogenesis of antennal pioneer neurons. Development 127: 4541-4549. PubMed ID: 11023858

Terman, J. R., et al. (2002). MICALs, a family of conserved flavoprotein oxidoreductases, function in Plexin-mediated axonal repulsion. Cell 109: 887-900. 12110185

Terman, J. R. and Kolodkin, A. L. (2004). Nervy links protein kinase a to plexin-mediated semaphorin repulsion. Science 303: 1204-1207. 14976319

Togashi, K., von Schimmelmann, M. J., Nishiyama, M., Lim, C. S., Yoshida, N., Yun, B., Molday, R. S., Goshima, Y. and Hong, K. (2008). Cyclic GMP-gated CNG channels function in Sema3A-induced growth cone repulsion. Neuron 58: 694-707. PubMed ID: 18549782

Wedel, B. and Garbers, D. (2001). The guanylyl cyclase family at Y2K. Annu. Rev. Physiol. 63: 215-233. PubMed ID: 11181955

date revised: 22 March 2022

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