G protein oalpha 47A

REGULATION

Drosophila GoLoco-protein Pins is a target of Galpha(o)-mediated G protein-coupled receptor signaling

G protein-coupled receptors (GPCRs) transduce their signals through trimeric G proteins, inducing guanine nucleotide exchange on their Gα-subunits; the resulting Gα-GTP transmits the signal further inside the cell. GoLoco domains present in many proteins play important roles in multiple trimeric G protein-dependent activities, physically binding Gα-subunits of the Gα_i/o class. In most cases GoLoco binds exclusively to the GDP-loaded form of the Gα-subunits. This study demonstrates that the poly-GoLoco-containing protein Pins of Drosophila can bind to both GDP- and GTP-forms of Drosophila Gα_o. Pins GoLoco domain 1 is identified as necessary and sufficient for this unusual interaction with Gα_o-GTP. A lysine residue located centrally in this domain is pinpointed as necessary for the interaction. These studies thus identify Drosophila Pins as a target of Gα_o-mediated GPCR receptor signaling, e.g., in the context of the nervous system development, where Gα_o acts downstream from Frizzled and redundantly with Gα_i to control the asymmetry of cell divisions (Kopein, 2009).

These observations expand a previous report that Pins could interact with Gα_o in the context of the asymmetric cell divisions during formation of Drosophila adult sensory bristles. In that work, a genetic interaction was demonstated, as well as an ability of both GDP- and GTPγS-loaded forms of recombinant Gα_o to pulldown endogenous Pins from Drosophila extracts. However, when the interaction between purified recombinant Gα_o and Pins proteins was tested, only the GDP-loaded Gα_o revealed the binding to Pins. This discrepancy is interpreted by proposing that certain Drosophila proteins could enhance the interaction between the GTP-loaded Gα_o and Pins, while the interaction between the purified proteins was 'canonical' and only happened in the presence of GDP (Kopein, 2009).

Although the existence of helper proteins enhancing the in vivo interactions between GTP-loaded Gα_o and Pins is still a possibility, this study found that the nontagged or (His)₆-tagged Gα_o-GTPγS efficiently binds purified Pins in multiple experimental setups, while Gα_o used in previous experiments was GST-tagged. It was also found that the point Q205L mutation on Gα_o, rendering it unable to hydrolyze GTP and thus constitutively GTP-bound, allows highly efficient Pins binding comparable to that of the Gα_o[GDP]. Although it cannot be fully explained why the GST-tagged Gα_o-GTPγS is unable to bind purified Pins, it is noted that the bulky GST tag reduces the GTP-binding activity of Gα_o 3-5 times. Thus, it is concluded that the active, GTP-loaded Gα_o binds Pins both in vivo and in vitro (Kopein, 2009).

This unusual interaction of the GTP-loaded Gα_o and Pins is confined to the GoLoco1 domain of Pins. Lys15 of the GoLoco1 domain is necessary for the efficient binding to GTP-loaded Gα_o. Substitution of Lys15 of GoLoco1 domain with Gly located in the identical position of GoLoco3 domain uncouples the interaction with GTP-loaded Gα_o but only moderately affects the binding to GDP-loaded Gα_o, and thus recapitulates the GoLoco3 domain-binding pattern. It is thus proposed that Lys15 of the GoLoco1 domain might stabilize the γ-phosphate of GTP during interaction with GTP-loaded Gα_o (Kopein, 2009).

This work provides the second clear demonstration of the interaction of a GoLoco domain-containing protein with the GTP-loaded form of a Gα-subunit. The only other clearly confirmed case of a similar interaction is the binding of the activated rat Gα_z to Rap1GAP. It is interesting to note that Lys15 of the GoLoco1 domain of Pins is absent from the equivalent position of the Rap1GAP' GoLoco domain. It thus might be proposed that multiple mechanisms stabilizing the GoLoco domain interaction with GTP-loaded Gα may exist. Additional evidence is provided by the current experiments with homologues of Gα_o and Pins. Gα_i, being 69% identical to Gα_o, binds Pins or its domains exclusively in the GDP-conformation. This biochemical result is paralleled with in vivo experiments where only Gα_i[GDP] but not Gα_i[GTP] could affect asymmetric divisions in Drosophila. Furthermore, rat Gα_o, 81% identical to Drosophila Gα_o, shows no ability to interact with Drosophila Pins in the GTPγS-loaded form, but interacts efficiently in the GDP-form. Additionally, both Drosophila and rat Gα_o-GTPγS fail to bind the GoLoco region of mammalian Pins homologues AGS3 and LGN, despite the presence of Lys15 in the GoLoco4 domain of AGS3 and LGN. It is still possible that other Gα_o/Pins homologues may reveal an interaction in the GTP state. For example, efficient binding of C. elegans AGS3 (which has Lys15 in GoLoco1 domain and Arg15 in GoLoco2 domain to GAO-1[GDP] and GAO-1[GTP] was demonstrated in the yeast two-hybrid assay, but the biochemical confirmation of this interaction is missing. The detailed information this study provides on the specificity of GoLoco binding to the GTP-loaded Gα_o (Gα_o, but not Gα_i; Drosophila, but not rat Gα_o; Drosophila Pins, but not its mammalian homologues; GoLoco1 domain of Pins, but not other Drosophila GoLoco domains) will help elucidate the structural mechanism of this interaction (Kopein, 2009).

Pins and its homologues have the conserved activity in the regulation of the asymmetric cell divisions. In Drosophila sensory organ formation, the process of the asymmetric cell divisions appears under the redundant control of Gα_o and Gα_i. Down-regulation of Gα_i alone, either by genetic ablation or by targeted RNAi expression, does not result in any defects in the structure of the adult sensory bristles, unlike same manipulations of Pins. In contrast, loss-of-function or overactivation of Gα_o result in aberrations in the process of asymmetric cell divisions and visible defects in the adult bristle structure. However, this study shows that no apparent defects are induced by targeted expression of pertussis toxin, which uncouples Gα_o (and not any other Gα-protein in Drosophila) from its cognate GPCRs such as Frizzled. This observation is not unexpected, as loss of Frizzled itself leads only to the randomization of the axis of the asymmetric cell divisions, but not to the loss of asymmetry or defects in the adult bristle structure. However, the redundancy between Gα_o and Gα_i is revealed by a concomitant expression of the Gα_i-RNAi and pertussis toxin, as this now phenocopies Pins loss-of-function. The same phenotype is produced by the concomitant down-regulation of Frizzled (acting upstream from Gα_o) and Gα_i. These data suggest that Gα_o and Gα_i act coordinately in the process of the asymmetric cell division of the sensory precursor cells, perhaps similarly to what has been demonstrated for the asymmetric division of the C. elegans zygote. The three individual GoLoco domains of Pins bind Gα_i identically; furthermore, multiple Gα_i molecules can simultaneously bind a single Pins scaffold. Similarly, this study shows that Gα_o and Gα_i can simultaneously bind Pins most likely occupying different GoLoco domains. This study also shows that this trimeric complex exists when the two G proteins are bound to different nucleotides: Gα_o to GTP and Gα_i to GDP. Such a multiprotein complex might allow a more effective regulation of the process of the asymmetric cell division (Kopein, 2009).

The results on the in vivo function of Frizzled, Gα_o, Gα_i, and Pins in the Drosophila sensory organ lineage further support the idea that Pins acts as a target and not as an activator of G protein signaling in this physiological process. Indeed, similarity of the Frizzled-RNAi + Gα_i-RNAi phenotypes on one hand, and the pertussis toxin + Gα_i-RNAi phenotypes on the other hand clearly shows the redundancy of the Frizzled→Gα_o module with the Gα_i function for the process of asymmetric cell divisions. This redundancy implies that both Gα_o and Gα_i act upstream from Pins. While generation of active Gα_o from the trimeric G_o complexes can be achieved by Frizzled receptors, it is not clear how Gα_i is released from the trimeric G_i complexes. Ric-8 (a non-GPCR guanine nucleotide exchange factor) might be implicated in activation of Gα_i. Downstream from Pins, a known regulator of the asymmetry of cell divisions is NuMA (known as Mud in flies) that anchors the mitotic spindle at the correct location within the plasma membrane (Kopein, 2009).

While Pins and its homologues have the conserved activity in the regulation of the asymmetric cell divisions, additional functions of these proteins exist. The Pins homologues AGS3 and LGN are strongly expressed in the brain as is Gα_o, where AGS3 is involved e.g., in drug sensitization and seeking behavior. At the molecular level Pins homologues regulate plasma membrane localization and activity of several transmembrane receptors and channels. Drosophila Pins is also expressed in the larval and adult brain. Additionally, Pins affects motor axon guidance and synaptogenesis in Drosophila. Thus a variety of GPCRs are likely to engage Pins and potentially other GoLoco domain-containing proteins through liberation of Gα_o-subunits from the trimeric G_o protein complexes. In addition, some non-GPCR guanine nucleotide exchange factors such as Ric-8 might be involved in the generation of the Pins-interacting Gα_o-GTP. Although clear data demonstrate that Pins and its homologues can modulate activities of Gα_i, the capacity of the activated Gα_o to bind Pins demonstrated in this study highlights the possible important function of Pins as a general transducer of GPCR signaling. Yeast two-hybrid screens have identified multiple interaction partners of Pins. The multidomain structure of Pins may suggest that this protein serves as a scaffold to organize signal transduction downstream from various GPCRs (Kopein, 2009).

The trimeric G protein Go inflicts a double impact on axin in the Wnt/frizzled signaling pathway

The Wnt/Frizzled signaling pathway plays crucial roles in animal development and is deregulated in many cases of carcinogenesis. Frizzled proteins initiating the intracellular signaling are typical G protein-coupled receptors and rely on the trimeric G protein Go for Wnt transduction in Drosophila. However, the mode of action of Go and its interplay with other transducers of the pathway such as Dishevelled and Axin remained unclear. This study shows that the alpha-subunit of Go directly acts on Axin, the multidomain protein playing a negative role in the Wnt signaling. G alpha o physically binds Axin and re-localizes it to the plasma membrane. Furthermore, G alpha o suppresses Axin's inhibitory action on the Wnt pathway in Drosophila wing development. The interaction of G alpha o with Axin critically depends on the RGS domain of the latter. Additionally, the betagamma-component of Go can directly bind and recruit Dishevelled from cytoplasm to the plasma membrane, where activated Dishevelled can act on the DIX domain of Axin. Thus, the two components of the trimeric Go protein mediate a double-direct and indirect-impact on different regions of Axin, which likely serves to ensure a robust inhibition of this protein and transduction of the Wnt signal (Egger-Adam, 2009).

This study has demonstrated that Gαo can physically bind the RGS domain of Axin and recruit it to the plasma membrane, the action likely leading to the destabilization of the Axin-based β-catenin destruction complex and propagation of the Wnt signal inside the cell. In support of this idea, this study has shown that Gαo can suppress the Wnt loss-of-function phenotypes induced by Axin over-expression in wing imaginal discs. This rescue critically depends on the presence of the RGS domain, reiterating the crucial role of this domain for the interaction with Gαo. While the GTP-bound form of Gαo is unable to change the phenotypes of the AxinΔRGS expression, the GDP-bound forms of Gαo even dramatically enhance these phenotypes. It is hypothesized that this enhancement is due to sequestration of the Gβγ heterodimer by the GDP-forms of Gαo. It was also shown that Gβγ can directly bind and recruit Dsh from the cytoplasm to the plasma membrane, thus possibly contributing to the propagation of the Wnt signal (Egger-Adam, 2009).

The RGS domain of Axin, responsible for the interaction with Gαo, is important for the full range of Axin activity in wing imaginal discs. Indeed, over-expression of the ΔRGS form of Axin only partially suppresses Wnt signaling in this tissue. The RGS domain of Axin is known to bind APC, another component of the β-catenin-destruction complex. The inability of AxinΔRGS to directly interact with APC is the likely reason for the reduced activity of this construct in Drosophila wings and in vertebrates. The Gαo and Gαq proteins were shown to dissociate the Axin-based destruction complexes in mammalian cells. It is proposed that in Drosophila, Gαo leads to a similar dissociation of the destruction complex through direct binding to the RGS domain of Axin, which recruits Axin to the plasma membrane and probably displaces APC from Axin (Egger-Adam, 2009).

In vitro, the purified RGS domain of Axin binds equally well both the GDP- and the GTP-loaded forms of Gαo. It also lacks the GTPase-activating protein (GAP) activity towards Gαo, typical for other RGS domains. These data agree with the absence of some of the conserved residues required for the GAP action in Axin RGS. Thus, biochemically Axin binds Gαo regardless of its nucleotide form. However, in vivo the GDP- and the GTP-loaded forms of Gαo behave differently towards Axin. Only Gαo[GTP] is capable of recruiting Axin-GFP to the plasma membrane in the salivary glands. Similarly, Gαo[GTP] is much more potent in rescuing the Axin full-length over-expression effects in wing imaginal discs and adult wings. This seeming contradiction is explained by the fact that in vivo the GDP-loaded forms of Gαo bind the βγ-subunits, recreating the trimeric Go complexes. Indeed, over-expressed, the wild-type Gαo was shown to compete with other Gα proteins for the βγ-subunits. Only the Gαo[GTP] form can stay free and thus exert its activities on Axin in full (Egger-Adam, 2009).

In contrast, the wild-type Gαo also possesses a capacity of over-activating the Wnt pathway in wing imaginal discs, and can to a certain degree rescue the phenotypes of Axin over-expression in this tissue. This contrasts with its inability to recruit Axin-GFP to the plasma membrane in salivary glands. These differences between the two tissues correlate with the degree of Wnt signal transduction. Indeed, the Wnt pathway is highly active in the wing imaginal discs, and Gαo can further enhance the pathway relying on the activity of Fz receptors. In contrast, in larval salivary glands the Wnt pathway is silent, which is illustrated by the cytoplasmic localization of Dsh in this tissue, expected to be plasma membrane localized when the pathway is on. It thus seems probable that in the salivary glands Gαo, forming trimeric Go complexes with Gβγ, fails to be further converted into the monomeric form due to the absence of the Wnt/Fz activity. In contrast, wing imaginal discs provide enough Wnt/Fz activity to activate endogenous as well as exogenous Go, which can then recruit Axin and thus propagate the signal (Egger-Adam, 2009).

The ability of the GDP-bound forms of Gαo to bind to the βγ-subunits is the likely reason for the aggravation of the AxinΔRGS phenotype induced by Gαo. This form, even upon conversion to the GTP-bound state by the action of the Wnt/Fz complexes, can no longer bind the RGS-lacking Axin and suppress Axin's negative action on the Wnt signal transduction. However, it can bind Gβγ. It is proposed that Gβγ plays, in addition to Gαo-GTP, a positive role in the Wnt signal transduction through its ability to bind and recruit Dsh to the plasma membrane. Over-expression of Gαo reduces the amounts of free Gβγ, reducing the efficiency of Dsh re-localization. It is proposed that when the endogenous full-length Axin is present, over-expression of Gαo has the overall stimulating effect on the Wnt signaling in wing discs due to increased generation of Gαo-GTP, which binds and antagonizes Axin. It is only in the artificial situation of over-expression of AxinΔRGS that the other, negative, effect of Gαo can be revealed. To prove that Gαo aggravates the AxinΔRGS phenotypes due to sequestration of Gβγ, the mutant Gαo[GDP] protein unable to charge with GTP but still capable to bind Gβγ was ested, and this form was found to be similar to Gαo in enhancing the AxinΔRGS phenotypes (Egger-Adam, 2009).

Direct experiments were performed testing the involvement of Gβγ in Wnt signaling. In accordance with predictions, down-regulation of Gβγ results in a clear reduction of the Wnt signaling in Drosophila wings and wing discs, affecting the short-range target genes of the Wnt pathway. As over-expression of Gβ alone leads to trapping Dsh in the cytoplasm, such over-expression also produces drastic dominant effects on Wnt signaling in wing discs. Unfortunately, it was not possible to confirm that Dsh was trapped in the cytoplasm of the epithelial cells of such discs due to the low resolution of the Dsh staining obtained in these thin columnar cells. Additionally, not only localization but also abundance of the components of the Wnt pathway are known to change in cells with high levels of Fz activation as part of the feedback regulation. Thus, interpretation of Dsh localization in wing imaginal discs upon perturbations of the Wnt pathway will be difficult. Instead, analysis of a tissue where the Wnt pathway is endogenously silent, such as salivary glands, allows analysis of the direct influence of the subunits of the trimeric Go complex on cellular localization of the components of the Wnt pathway. This analysis led to the identification of the plasma membrane re-localization of Axin by Gαo and of Dsh by Gβγ as such direct cellular responses. These primary responses are probably then utilized in the physiological context as the basis to build positive and negative feedbacks for the final outcome of Wnt signal propagation (Egger-Adam, 2009).

While the numerous data indicate that Gβγ is necessary for the proper activation of the Wnt pathway, probably through plasma membrane re-localization of Dsh, it was not possible to over-activate the Wnt pathway by over-expression of Gβ and Gγ together. Instead, the pathway was down-regulated, although to a weaker extent than that seen by over-expression of Gβ alone. This observation is not easy to reconcile with the other data. One possible explanation is that in the wing discs, unlike the salivary glands, co-overexpression of Gγ might be insufficient to attract the complete pool of Gβ to the plasma membrane, and significant amounts of Gβ may still remain cytoplasmic and retain Dsh. Along these lines, co-overexpression of Gγ shows a partial 'rescue' of the phenotypes induced by Gβ over-expression. Another possible explanation involves the notion of the negative feedback regulation in the Wnt cascade. Proteosomal degradation of Dsh during Wnt signal transduction has been demonstrated. A recent work has shown that targeted plasma membrane localization of Dsh by the Wnt activation or by the Gβγ subunits also destines it for the lysosomal degradation in vertebrate cells. Thus, the activity of Gβγ in the Wnt signaling may be multistep: the initial recruitment of Dsh from the cytosol may serve to activate the pathway, but the persistent membrane localization will lead to Dsh degradation. While Gβ RNAi targeting shows that the Gβγ complex is necessary for the proper Wnt signaling, activation of such a negative feedback loop may underlie the phenotypes observed upon the persistent over-expression of Gβγ. In this scenario, Gβγ will be added to the growing list of regulators of the Wnt pathway, which have both positive and negative activities in this signaling (Egger-Adam, 2009).

A model ia favored whereby Gβγ-induced plasma membrane re-localization of Dsh serves as an initial positive impact to activate the Wnt signal propagation. If this is correct, what may be the immediate consequences of the Gβγ-induced plasma membrane recruitment of Dsh? This scaffolding protein is known to become hyper-phosphorylated upon plasma membrane localization, which correlates with its activity in the Wnt signal transduction. Dsh is known to directly bind Axin through the DIX domain heterodimerization. Although a direct interaction of Gβγ with Axin's protein phosphatase 2A-binding region (N-terminal to the DIX domain) has recently been demonstrated in mammalian cells, no ability was found of Gβγ to re-localize or directly bind Drosophila Axin. Overall, the data and the above considerations lead to the proposal of the following model of the action of the trimeric Go protein in the Drosophila Wnt/Fz pathway (Egger-Adam, 2009).

The trimeric Go protein is a direct target of the activated Fz receptors. Wnt ligand binding to Fz activates the guanine nucleotide exchange activity of Fz towards Go. This in turn dissociates the trimeric Go complex into Gαo-GTP and Gβγ. It is proposed that both these components of the trimeric complex have the initial positive activity in Wnt signal propagation. Gαo-GTP directly binds to the RGS domain of Axin, recruiting Axin to the plasma membrane and dissociating the Axin-based β-catenin destruction complex. In contrast, Gβγ recruits and contributes to activation of Dsh, which then can bind the DIX domain of Axin and thus also promote dissociation of the destruction complex. These two branches of G protein–mediated signal propagation converge on the Axin complex to cooperatively ensure its efficient inhibition. Such a double effect on Axin emanating from the trimeric Go complex may serve to ensure a robust activation of the Wnt signaling (Egger-Adam, 2009).

Kermit interacts with gαo, vang, and motor proteins in Drosophila planar cell polarity

In addition to the ubiquitous apical-basal polarity, epithelial cells are often polarized within the plane of the tissue - the phenomenon known as planar cell polarity (PCP). In Drosophila, manifestations of PCP are visible in the eye, wing, and cuticle. Several components of the PCP signaling have been characterized in flies and vertebrates, including the heterotrimeric Go protein. However, Go signaling partners in PCP remain largely unknown. Using a genetic screen Kermit, previously implicated in G protein and PCP signaling, was unvcovered as a novel binding partner of Go. Through pull-down and genetic interaction studies, it was found that Kermit interacts with Go and another PCP component Vang (Strabismis), known to undergo intracellular relocalization during PCP establishment. It was further demonstrated that the activity of Kermit in PCP differentially relies on the motor proteins: the microtubule-based dynein and kinesin motors and the actin-based myosin VI (Jaguar). The results place Kermit as a potential transducer of Go, linking Vang with motor proteins for its delivery to dedicated cellular compartments during PCP establishment (Lin, 2013).

At the top of the signaling hierarchy in PCP lies a G protein-coupled receptor Fz. The heterotrimeric Go protein emerged as an immediate transducer of Fz in Drosophila as well as other organisms. One of the mediators of Go signaling in PCP is the endocytic GTPase Rab5 required for the proper Fz internalization and relocalization. During PCP establishment, Fz concentrates at the distal apical position of wing epithelia. This study describes identification of Kermit as another transducer of Go in PCP. kermit downregulation suppresses the Gαo-overexpression phenotypes, and Gαo and kermit co-overexpression results in a prominent synergism in PCP malformations (Lin, 2013).

Kermit and its mammalian homolog GIPC, through their PDZ domain, are known to interact with a number of proteins in various organisms. Observations in Xenopus and mice indicated that Kermit/GIPC could interact with members of the Fz and RGS protein families -- Fz3, Fz7, and RGS19 (De Vries, 1998; Tan, 2001). Since Go also binds Fz and RGS proteins, it was hypothesized that a quaternary complex consisting of Fz, Go, Kermit, and RGS19 could form in Drosophila PCP, with Kermit as a potential organizer of these interactions. However, Drosophila Kermit was found not interact with Fz. Similarly, no binding between Kermit and the Drosophila RGS19 homolog could be seen. Thus Kermit is unlikely to act as a scaffold in Fz-Go signaling, and another mode of action of Kermit in transducing Go signal exists in PCP (Lin, 2013).

In a recent study using mouse genetics and cellular assays, a role of GIPC1 in regulating Vangl2 (a murine homolog of Drosophila Vang) intracellular trafficking has been revealed (Giese, 2012). In Drosophila PCP, Vang relocalizes to the site opposite to Fz at the proximal apical tip of wing epithelia. This study provides genetic evidence placing Vang downstream from Kermit in Drosophila PCP, suggesting that the Kermit-Vang connection is conserved from insects to mammals (Lin, 2013).

kermit expression is strongly upregulated in the developing wing during PCP establishment, and kermit overexpression induces strong PCP phenotypes (Djiane, 2010). In Xenopus, both up- and down-regulation of kermit lead to defective Fz3-dependent neural crest induction. It is thus surprising that Drosophila kermit loss-of-function alleles were homozygous viable and did not reveal PCP phenotypes. It is proposed that Kermit may regulate Drosophila PCP redundantly with some other PDZ domain-containing proteins, such as Scribble or Patj, which genetically interact with PCP components but on their own also produce only mild phenotypes; of those Scribble has been shown to interact with Vang both in Drosophila and mammals. In general, up to 75% of genes Drosophila are estimated to be phenotypically silent in loss-of-function due to redundancy, and the significance of gain-of-function analysis in discovery of novel important pathway components has been highlighted in a recent large-scale Drosophila-based assay. Kermit, based on the presented overexpression and genetic interaction studies, can thus be considered as an important regulator of Drosophila PCP (Lin, 2013).

A genetic and physical interaction between Kermit and the unconventional actin-based motor MyoVI has been described. This study confirmed that the dominant UAS-kermit PCP phenotypes critically depend on the MyoVI activity. MyoVI has been previously shown to mediate removal of endocytic vesicles away from the cell's periphery. The excessive activity of Kermit or MyoVI may thus result in removal of Vang-containing vesicles from the apical membrane, contributing to mislocalization of Vang and appearance of the PCP defects. In contrast, microtubule-based transport along the apical microtubule cables, polarized below the apical plasma membrane in wing epithelia, mediates the correct relocalizations of Fz and Vang in PCP. It is probable that a competition between the actin-based and microtubule-based motors may exist for the endocytic vesicles containing PCP components, and that excessive Kermit activity unbalances this competition in favor of the actin-based transport. Thus whether reduction in the levels of the microtubule-based transport system would further aggravate the dominant UAS-kermit PCP phenotypes was tested. And indeed, reduction in either the minus end-directed motor dynein or the plus end-directed motor kinesin significantly enhances the UAS-kermit effects (Lin, 2013).

The following model is proposed to collectively explain the results. It is speculated that endocytic vesicles containing PCP components can be transported in a planar manner, along the microtubule meshwork underlying the apical plasma membrane -- the mode of transport required for the proper apical relocalizations of these components. Alternatively, the vesicles can be trapped by the actin cables and transported away from the apical membrane, removing them from the active pool of PCP components. In the case of Vang, the choice between these decisions is regulated by the Kermit protein, which favors the actin-based transport (Lin, 2013).

These findings and model shed new light on the mechanisms of complex inter-regulations ensuring the robust epithelial polarization, likely conserved across the metazoans (Lin, 2013).

Presynaptic inhibition of γ lobe neurons is required for olfactory learning in Drosophila

The loss of heterotrimeric G_o signaling through the expression of pertussis toxin (PTX) within either the α/β or γ lobe mushroom body neurons of Drosophila results in the impaired aversive olfactory associative memory formation. This study focused on the cellular effects of G_o signaling in the γ lobe mushroom body neurons during memory formation. Expression of PTX in the γ lobes specifically inhibits G_o activation, leading to poor olfactory learning and an increase in odor-elicited synaptic vesicle release. In the γ lobe neurons, training decreases synaptic vesicle release elicited by the unpaired conditioned stimulus minus, while leaving presynaptic activation by the paired conditioned stimulus plus unchanged. PTX expression in γ lobe neurons inhibits the generation of this differential synaptic activation by conditioned stimuli after negative reinforcement. Hyperpolarization of the γ lobe neurons or the inhibition of presynaptic activity through the expression of dominant negative dynamin transgenes ameliorated the memory impairment caused by PTX, indicating that the disinhibition of these neurons by PTX was responsible for the poor memory formation. The role for γ lobe inhibition, carried out by G_o activation, indicates that an inhibitory circuit involving these neurons plays a positive role in memory acquisition. This newly uncovered requirement for inhibition of odor-elicited activity within the γ lobes is consistent with these neurons serving as comparators during learning, perhaps as part of an odor salience modification mechanism (Zhang, 2013).

Activation of G_o is inhibited by the expression of PTX. The inhibition of G_o activation by the expression of PTX within the γ lobe neurons of the mushroom bodies leads to a significant decrease in short term aversive memories. This study has now shown that this memory defect is caused by the disinhibition of the γ lobe neurons. Inhibition of G_o increases odor-induced presynaptic activity. The inhibition of γ lobe neurons by either hyperpolarization or synaptic vesicle depletion reverses the PTX learning phenotype. It is proposed that the G_o-mediated presynaptic inhibition of γ lobe neurons is required to generate differential conditioned stimulus salience during discriminative leaning (Zhang, 2013).

Dopaminergic Modulation of cAMP Drives Nonlinear Plasticity across the Drosophila Mushroom Body Lobes

Activity of dopaminergic neurons is necessary and sufficient to evoke learning-related plasticity in neuronal networks that modulate learning. During olfactory classical conditioning, large subsets of dopaminergic neurons are activated, releasing dopamine across broad sets of postsynaptic neurons. It is unclear how such diffuse dopamine release generates the highly localized patterns of plasticity required for memory formation. This study has mapped spatial patterns of dopaminergic modulation of intracellular signaling and plasticity in Drosophila mushroom body (MB) neurons, combining presynaptic thermogenetic stimulation of dopaminergic neurons with postsynaptic functional imaging in vivo. Stimulation of dopaminergic neurons generated increases in cyclic AMP (cAMP) across multiple spatial regions in the MB. However, odor presentation paired with stimulation of dopaminergic neurons evoked plasticity in Ca2+ responses in discrete spatial patterns. These patterns of plasticity correlated with behavioral requirements for each set of MB neurons in aversive and appetitive conditioning. Finally, broad elevation of cAMP differentially facilitated responses in the gamma lobe, suggesting that it is more sensitive to elevations of cAMP and that it is recruited first into dopamine-dependent memory traces. These data suggest that the spatial pattern of learning-related plasticity is dependent on the postsynaptic neurons' sensitivity to cAMP signaling. This may represent a mechanism through which single-cycle conditioning allocates short-term memory to a specific subset of eligible neurons (gamma neurons) (Boto, 2014).

Dopaminergic neurons are involved in modulating diverse behaviors, including learning, motor control, motivation, arousal, addiction and obesity, and salience-based decision making. In Drosophila, dopaminergic neurons innervate multiple brain regions, including the mushroom body (MB), where they modulate aversive learning, forgetting, state-dependent modulation of appetitive memory retrieval, expression of ethanol-induced reward memory, and temperature-preference behavior (Boto, 2014).

Dopaminergic circuits play a particularly critical role in memory acquisition. During olfactory classical conditioning, where an odor (conditioned stimulus [CS]) is paired with an aversive event (e.g., electric shock; the unconditioned stimulus [US]), dopaminergic neurons respond strongly to the aversive US (Mao, 2009). Dopamine functions in concert with activity-dependent Ca2+ influx to synergistically elevate cyclic AMP (cAMP) (Tomchik, 2009) and PKA (Gervasi, 2010), suggesting that dopamine is one component of a molecular coincidence detector underlying learning. Proper dopamine signaling is necessary for aversive and appetitive memory. Moreover, driving activity of a subset of TH-GAL4+ dopaminergic neurons that differentially innervates the vertical α/α' MB lobes (with less dense innervation of the horizontal β/β'/γ lobes, peduncle, and calyx), is sufficient to induce behavioral aversion to a paired odorant in larvae and adult flies. Conversely, stimulation of a different set of Ddc-GAL4+ dopaminergic neurons, the PAM cluster that innervates mainly the horizontal β/β'/γ lobes, is sufficient to induce behavioral attraction to a paired odorant. Thus, dopaminergic neurons comprise multiple circuits with distinct roles in memory acquisition (Boto, 2014).

Multiple subsets of MB neurons receive CS and US information and express molecules associated with the coincidence detection, making them theoretically eligible to generate dopamine/cAMP-dependent plasticity. Yet only some subsets are required to support memory at any given time following conditioning, leaving open the question of how spatial patterns of plasticity are generated during conditioning. This question has been approached, by using a technique to probe the postsynaptic effects of neuronal pathway activation. Odor presentation was paired with stimulation of presynaptic dopaminergic neurons via ectopic expression of the heat-sensitive channel TRPA1, while monitoring postsynaptic effects with genetically encoded optical reporters for Ca2+, cAMP, and PKA in vivo (Boto, 2014).

The present data demonstrate four major points about how dopaminergic circuits function in neuronal plasticity underlying olfactory classical conditioning. (1) Stimulation of small subsets of dopaminergic neurons evokes consistent, compartmentalized elevations of cAMP across the MB lobes. (2) Broad stimulation of dopaminergic neurons generates broad postsynaptic elevation of cAMP, but Ca2+ response plasticity occurs in discrete spatial regions. (3) Stimulation of TH-GAL4+ neurons and Ddc/R58E02-GAL4+ neurons, which mediate opposing behavioral responses to conditioned stimuli, generates an overlapping pattern of Ca2+ response plasticity in the γ lobe, with additional regions recruited by Ddc/R58E02-GAL4+ stimulation. Finally, (4) the spatial pattern of plasticity coincides with differential sensitivity to cAMP in the γ lobe. Collectively, these data suggest that different subsets of neurons exhibit heterogeneous sensitivity to activation of second messenger signaling cascades, which might shape their responses to neuromodulatory network activity and modulate their propensity for recruitment into memory traces (Boto, 2014).

The data suggest that dopaminergic neurons mediate Ca2+ response plasticity largely in the γ lobe and suggest a potential mechanism for localization of short-term, learning-related plasticity. These data coincide with multiple previous studies that have demonstrated a critical role of γ neurons in short-term memory. Rescue of Rutabaga (Rut) in the γ lobe of rut mutants is sufficient to restore performance in short-term memory, whereas rescue in α/β lobes supports long-term memory. Rescue of the D1-like DopR receptor in the γ lobe is sufficient to rescue both short- and long-term memory in a mutant background, suggesting that the γ neurons mediate the dopaminergic input during conditioning. In addition, stimulating MP1 dopaminergic neurons innervating the heel of the γ lobe is sufficient as an aversive reinforcer. Finally, learning induces plasticity in synaptic vesicle release from MB γ lobes, which depends in part on G(o) signaling (Zhang, 2013). The data support a critical role for the γ lobe in short-term memory. Furthermore, the observation of differential sensitivity of the γ lobe to cAMP might provide an elegant explanation for why it is specifically recruited into short-term memory traces (Boto, 2014).

Direct elevation of cAMP was sufficient to generate localized, concentration-dependent Ca2+ response plasticity in the MB γ lobe in these experiments. Because applying forskolin in the bath is expected to elevate cAMP across the brain, the spatial specificity of the effect is remarkable. This was not an acute effect, because the forskolin was washed out before imaging the first postconditioning odor response. At the concentrations that were tested, only the γ lobe was facilitated. Therefore, it is concluded that the γ lobe is most sensitive to elevation of cAMP, which has the effect of differentially recruiting γ neurons into the representation of short-term memory via dopamine-mediated neuronal plasticity. It is possible that additional signaling cascades are involved in generating learning-related plasticity in α/β and α'/β' neurons, given that no Ca2+ response plasticity was observed in those neurons following forskolin application (Boto, 2014).

The dominant model for cellular mechanisms of olfactory associative learning is that integration of information about the conditioned and unconditioned stimuli are integrated by Rut, which functions as a molecular coincidence detector. This would suggest that MB neurons, which receive CS and US information, would exhibit at least somewhat uniform Ca2+ response plasticity. From this molecular and cellular perspective, the finding that the α/β and α'/β' neurons did not exhibit Ca2+ response plasticity when an odor was paired with stimulation of dopaminergic neurons is surprising. These neurons are theoretically eligible to encode memory, because they receive information about the CS and US. However, the finding that γ neurons differentially exhibit dopamine-dependent plasticity following single-cycle conditioning is consistent with the data from the behavioral experiments. In summary, the present results suggest that differential cAMP sensitivity provides a potential mechanism allowing specific subsets of eligible neurons in an array (γ neurons) to differentially encode CS-US coincidence relative to other subsets (α/β neurons) that also receive CS/US information (Boto, 2014).

DEVELOPMENTAL BIOLOGY

Embryonic

G-oalpha47A, as described by Yoon (1989), codes for two classes of cDNAs (class I and class II) have been extensively documented (de Sousa, 1989; Schmidt, 1989; Thambi, 1989, and Yoon, 1989). These transcripts arise from an alternative splicing in a unique gene and differ only in the ATG-containing first exon. Three developmentally regulated transcripts (3.4kb of maternal origin; 4.2kb and 6kb, zygotic in origin) have been revealed (Wolfgang, 1991). By using specific probes, it has been shown that the class I cDNA corresponds to the 6-kb mRNA appearing after 12h of development, whereas the class II cDNA corresponds to the 4.2-kb mRNA more abundant in earlier stages. Both cDNAs encode proteins of the same size (354 amino acids) that diverge only by 7 amino acids among their 21 N₂-terminal residues. It is not known if these amino acids exert different functions (Frémion, 1999). Despite the relatively high levels of maternal G-oalpha 47A mRNA the protein cannot be detected in ovaries. Just after the completion of germband retraction, elevated levels of both Gsalpha and G-oalpha 47A are first detected in the forming neuropil of the brain and ventral ganglion. This pattern persists for the duration of embryogenesis. The neuropil staining for both Gsalpha and G-oalpha 47A persists through adult life (Wolfgang, 1991).

By using the class II cDNA as a probe, a strong expression of G-oalpha47A has been observed in preblastoderm embryos due to the presence of the 3.4-kb maternal transcript. In early stage 11 embryos, the zygotic transcript can be detected for the first time in clusters of cells within 11 segments. The cells appear to be both cardial and visceral muscle progenitor cells, because based on G-oalpha47A staining patterns in later stage 12 embryos, the cells become integrated into the conspicuous monocellular layer of cardial and visceral mesoderm cells on each side of the embryo. This assumption is further supported by G-oalpha47A expression in embryonic tissues which are unambiguously constituted of such cells, and also by the pattern of expression in tinman (tin) loss of function mutants. In tin mutants, neither heart nor visceral muscles are formed and, correlatively, G-oalpha47A expression is completely abolished in the territories from which the precursor cells for these two tissues originate. From the middle of stage 11 onwards, neuroblasts of the central nervous system (CNS) become labeled and G-oalpha47A expression persists in the neurons of the CNS in later stages of embryogenesis. In a similar way, all the neurons of the peripheral nervous system (PNS) express the G-oalpha47A mRNA from stage 12 onwards, slightly before the onset of axonogenesis. Probes for either cDNA give identical spatial patterns of expression although class I transcripts are quantitatively less abundant and are expressed later than the class II transcripts. Antibodies directed against a COOH-terminal peptide whose sequence is conserved in the alpha subunit of all G_o proteins shows that the pattern of expression of the protein is superimposable on that of the mRNA during embryogenesis. However, probably due to the presence of the protein of maternal origin, these antibodies are poorly efficient in detecting a significant signal in the cardial cells as early as do mRNA probes (Frémion, 1999).

The alpha subunits of heterotrimeric G proteins are responsible for the coupling of receptors for a wide variety of stimuli to a number of intracellular effector systems. In the nervous system of vertebrates, high levels of a specific class of G protein (Go alpha) are expressed. The alpha subunit of Go serves as a substrate for modification by pertussis toxin (PTX). Drosophila heads contain high levels of a 40-kDa PTX substrate. Modification of this protein by PTX is modulated in a manner similar to that observed for vertebrate G proteins. The PTX substrate in Drosophila is also recognized specifically by antibodies raised against peptide sequences found specifically in vertebrate Go alpha. Vertebrate Go alpha probes were used to identify a Drosophila cDNA coding for a potential PTX substrate with high sequence identity (82%) to vertebrate Go alpha. An additional cDNA coding for a related Go alpha has also been isolated. The two cDNAs differ only in the 5'-untranslated and amino-terminal regions of the protein. This observation, in addition to Northern analysis, suggests that alternate splicing may generate a variety of Go alpha-like proteins in Drosophila. In situ hybridization of specific probes to tissue sections indicates that the mRNAs coding for Go alpha-like proteins in Drosophila are expressed primarily in neuronal cell bodies and, at lower levels, in the eyes (Thambi, 1989).

Guanine nucleotide-binding proteins (G proteins) mediate signals between activated cell-surface receptors and cellular effectors. A bovine G-protein alpha-subunit cDNA has been used to isolate similar sequences from Drosophila genomic and cDNA libraries. One class, G-oalpha47A, hybridizes to position 47A on the second chromosome of Drosophila. The nucleotide sequence of the protein coding region of one cDNA has been determined, revealing an alpha subunit that is 81% identical with rat alpha 0. The cDNA hybridizes strongly to a 3.8 kb mRNA and weakly with a 5.3 kb message. Antibodies raised against a fusion protein recognize a 39,000 Da protein in Drosophila extracts. In situ hybridization to adult Drosophila sections combined with immunohistochemical studies reveal expression throughout the optic lobes and central brain and in the thoracic and abdominal ganglia. G-oalpha47A message and protein were also detected in the antennae, oocytes, and ovarian nurse cells. The neuronal expression of this gene is similar to mammalian alpha 0, which is most abundantly expressed in the brain (Schmidt, 1989).

In order to uncover the role of G proteins in the integrative functioning and development of the nervous system, a multidisciplinary study of the G proteins present in the fruit fly has been initiated. The distribution of 3 different G protein alpha-subunits in the adult Drosophila CNS is described as determined by immunocytochemical localization using affinity-purified antibodies generated to synthetic oligopeptide sequences unique to each alpha-subunit. Western blot analysis of membranes prepared from Drosophila heads indicates that antibodies specific for the Drosophila Go alpha and Gs alpha homologs recognize the appropriate protein species predicted by molecular cloning. The Gi alpha homolog could not be detected in head membranes by Western blotting, consistent with the negligible levels of expression observed for Gi alpha on Northern blots of head mRNA. However, a Drosophila Gi alpha fusion protein could be detected by these antibodies following expression in E. coli. Immunolocalization studies revealed that the Go alpha and Gs alpha homologs are expressed at highest levels in neuropils and at intermediate levels in the cortex of all brain and thoracic ganglion areas. Only the lamina contains low levels of these alpha-subunits in the CNS. Additionally, Gs alpha appears to be associated with the cell membranes of neuronal cell bodies, while Go alpha has a more diffuse distribution, suggesting its presence in the cytoplasm as well as cell membranes. In contrast to the wide distribution of Go alpha and Gs alpha, Gi alpha has a surprisingly restricted distribution in the CNS. It is present at high levels only in photoreceptor cell terminations, glomerulae of the antennal lobes, and the ocellar retina. Little or no Gi alpha is detected in other brain regions or in the thoracic ganglion. Gi alpha, then, appears to be uniquely associated with some primary sensory afferents and their terminations, suggesting the presence of specific receptor and/or effector systems which mediate the transmission of primary sensory information in Drosophila (Wolfgang, 1990).

G proteins couple receptors for extracellular signals to several intracellular effector systems and play a key role in signaling transduction mechanisms. In particulate preparations of Drosophila melanogaster heads, only one substrate for pertussis toxin at 39-40 kd was detected. This substrate, which shows only one isoform when analysed by isoelectric focusing, was recognized by immunoblotting and immunoprecipitation techniques using a polyclonal antibody against the alpha subunit of the Go protein purified from bovine brain and can be thus considered as a Go-like protein. Antibodies obtained against a carboxy-terminal sequence of the alpha subunit of Go (but not of Gi1 or Gi2) and against an internal sequence shared by all the alpha subunits, were also able to cross-react with the alpha subunit of this protein in insects. The Go-like protein was studied in several D. melanogaster mutants, primarily in memory and learning mutants. In these mutants there is a sex-dependent enhancement in pertussis toxin-catalysed ADP-ribosylation with respect to the wild-type. This increase could be attributed in part to an increase in the alpha subunit of the Go-like protein, as revealed by immunoblotting with anti-Go alpha polyclonal antibody. This report constitutes the first evidence for the participation of a Go protein in learning and memory (Guillen, 1990).

In particulate preparations from Drosophila melanogaster embryos, only one substrate of 39,000-40,000 Da molecular weight could be ADP-ribosylated with pertussis toxin. This substrate reacts in immunoblotting and immunoprecipitation experiments with a polyclonal antibody directed against the carboxy-terminal sequence of the alpha subunit of the mammalian Go protein. The Drosophila Go alpha protein is present at all stages of embryonic development; however, its expression markedly increases after 10 h embryogenesis, a period of time during which there is an active development of axonal tracts. Immunolocalization on whole mount embryos has indicated that this protein is principally localized in the CNS and is mainly restricted to the neuropil without any labelling of the cell bodies. In contrast, all the axon tracts of the CNS appear to be highly labelled. The distribution of the Go alpha protein was also examined in several neurogenic mutants. The Go alpha protein expression is not altered in any of them but the pattern of labelling is disorganized as is the neuronal network. These results suggest a possible role for the Go protein during axonogenesis (Guillen, 1991).

Before the determination of cardiac precursors in the mesoderm, the overlying ectoderm is subdivided in segmentally repeated units partitioned into an anterior compartment (A-compartment) and a posterior compartment (P-compartment). Analysis of the expression of genes involved in the specification of mesodermal derivatives and other observations lend support to the idea that, after gastrulation, the mesoderm also becomes subdivided into segmentally repeated units, each of which consists of two separate domains. The domains that are located below the ectodermal P-compartments are subject to influences from the striped regulators eve and hh and have been termed 'P-domains' or 'eve-domains'. By contrast, the development of the metameric domains that are located below the A-compartments depends largely on the striped regulators wg and slp (sloppy-paired) and these domains have been termed 'A-domains' or 'slp-domains'. In this scheme, three basic groups of genes are at work to pattern the mesoderm either along the dorso-ventral axis (dpp and tinman) to specify the dorsal mesoderm, or along the anterior-posterior axis (wg and slp) to subdivide it into segmental units, or at defined positions to control tissue specification. For example, recent evidence suggests that wg, whose expression is restricted to striped domains in each of the A-compartments and which is required for a variety of inductive signaling events during embryonic development, is directly involved in heart formation: wg is necessary for further subdividing of the dorsal mesoderm and for specifying cardial cell fates. Elimination of the wg function shortly after gastrulation, at a time when tin becomes restricted to the dorsal mesoderm, results in the selective loss of heart progenitor cells with little effect on segmental patterning of the cuticle or other mesodermal derivatives. From these and other observations, a picture has emerged in which specification of precardiac and dorsal somatic muscle precursors requires intersections of the dorsal domains of dpp expression with the transverse stripes of the dorsal expression of wg (Frémion, 1999 and references).

However, the results presented in the Frémion (1999) study are merely consistent with some precursors of the cardial cells originating from the P-domains and subjected to the influence of hh signaling rather than that of wg. This hypothesis is somewhat difficult to verify because wg expression after gastrulation requires hh and vice versa, but, later in embryogenesis, the two signals become independent. Therefore, the expression of Tin in temperature-sensitive hh^ts2 mutant embryos submitted to a temperature shift ~5 h after egg laying (stage 10) was investigated. In these embryos, the wild-type expression of Tin in four cardial and four pericardial cells is expanded to the two cardial cells located in the P-domains of each segment. It is predicted that these two tin-expressing cells are the same as the two G-oalpha47A-expressing cells in this same domain (Frémion, 1999).

The observation that the cardial progenitor cells can be divided into two cell subpopulations is consistent with the situation in the mature heart tube in which two genetically distinct populations of cardial cells have been described (Bodmer, 1997). For example, tin as well as beta3-tubulin and several P-lacZ reporter genes from enhancer trap lines are expressed in only four cell pairs per segment among the six pairs present. In the same line, a D-mef2 enhancer element directs lacZ transcription in four cardial cell pairs per segment, consistent with a direct regulation by tin, which is expressed in these same cells (Gajewski, 1997). Interestingly, these two subpopulations of cardial cells reside, respectively, below the anterior and the posterior ectodermal parasegmental domains (Frémion, 1999). These different observations could mean that the two P-cardioblasts are specified by the inductive instruction of hh rather than by wg. However, it is not known whether the hh pathway provides a direct late cardiogenic signal or exerts its effect via suppressing the wg function in the posterior domain. This hypothesis is unlikely, since at that stage, reducing the function of hh in the epidermis does not lead to any visible effect on the wg signaling pathway. Indeed, wg expression in the dorsal epidermis is not expanded in a hh mutant. Therefore, it is predicted that hh might behave as a repressive signal for tin expression in the two P-cardioblasts. Cell heterogeneity in terms of gene expression could then be achieved along the anterior-posterior axis by an efficient cooperativity of wg and hh signals in the specification of the cardial cells (Frémion, 1999 and references).

Nerfin-1 is a nuclear regulator of axon guidance required for a subset of early pathfinding events in the developing Drosophila CNS. Nerfin-1 belongs to a highly conserved subfamily of Zn-finger proteins with cognates identified in nematodes and man. The neural precursor gene prospero is essential for nerfin-1 expression. Unlike nerfin-1 mRNA, which is expressed in many neural precursor cells, the encoded Nerfin-1 protein is only detected in the nuclei of neuronal precursors that will divide just once and then transiently in their nascent neurons. Although nerfin-1 null embryos have no discernible alterations in neural lineage development or in neuronal or glial identities, CNS pioneering neurons require nerfin-1 function for early axon guidance decisions. Furthermore, nerfin-1 is required for the proper development of commissural and connective axon fascicles. Nerfin-1 is essential for the proper expression of robo2, wnt5, derailed, G-oα47A, Lar, and futsch<, genes whose encoded proteins participate in these early navigational events (Kuzin, 2005).

Given the axon guidance defects in nerfin-1^null embryos and the fact that Nerfin-1 is a Zn-finger nuclear protein, it was hypothesized that Nerfin-1 may be required for the correct expression of genes involved in axon guidance. Accordingly, the embryonic expression profiles of over 35 genes that have been shown to play important roles in axon guidance were examined. Included in the candidate screen were genes encoding transcription factors, RNA-binding proteins, cell surface receptor proteins, their ligands, signal transduction proteins, and components of the cytoskeleton. Homozygous nerfin-1^null embryos were identified by the absence of Nerfin-1 immunoreactivity. Whole-mount in situ hybridization and/or protein immunostaining for altered spatial or temporal expression in nerfin-1^null embryos identified six genes that require nerfin-1 function to achieve full wild-type expression levels (Kuzin, 2005).

Drosophila pacemaker neurons require g protein signaling and GABAergic inputs to generate twenty-four hour behavioral rhythms

Intercellular signaling is important for accurate circadian rhythms. In Drosophila, the small ventral lateral neurons (s-LN_vs) are the dominant pacemaker neurons and set the pace of most other clock neurons in constant darkness. This study shows that two distinct G protein signaling pathways are required in LN_vs for 24 hr rhythms. Reducing signaling in LN_vs via the G alpha subunit Gs, which signals via cAMP, or via the G alpha subunit Go, which signals via Phospholipase 21c, lengthens the period of behavioral rhythms. In contrast, constitutive Gs or Go signaling makes most flies arrhythmic. Using dissociated LN_vs in culture, it was found that Go and the metabotropic GABA(B)-R3 receptor are required for the inhibitory effects of GABA on LN_vs and that reduced GABA(B)-R3 expression in vivo lengthens period. Although no clock neurons produce GABA, hyperexciting GABAergic neurons disrupts behavioral rhythms and s-LN_v molecular clocks. Therefore, s-LN_vs require GABAergic inputs for 24 hr rhythms (Dahdal, 2010).

The long-periods observed with reduced Gs signaling are consistent with four other manipulations of cAMP levels or PKA activity that alter fly circadian behavior. First, long-period rhythms with dnc over-expression complement the short periods of dnc hypomorphs and suggest that the latter are due to loss of dnc from LN_vs. dnc mutants also increase phase shifts to light in the early evening. However, this study found no difference in phase delays or advances between Pdf > dnc and control flies, suggesting that altered light-responses of dnc hypomorphs are due to dnc acting in other clock neurons. The period-altering effects seen when manipulating cAMP levels are also consistent with finding stat expressing the cAMP-binding domain of mammalian Epac1 in LN_vs lengthens period. This Epac1 domain likely reduces free cAMP levels in LN_vs, although presumably not as potently as UAS-dnc. Third, mutations in PKA catalytic or regulatory subunits that affect the whole fly disrupt circadian behavior. Fourth, over-expressing a PKA catalytic subunit in LN_vs rescues the period-altering effect of a UAS-shibire transgene that alters vesicle recycling, although the PKA catalytic subunit had no effect by itself. The long periods observed with reduced Gs signaling in LN_vs also parallel mammalian studies in which pharmacologically reducing Adenylate cyclase activity lengthened period in SCN explants and mice (Dahdal, 2010).

G-proteins typically transduce extracellular signals. What signals could activate Gs in s-LN_vs? PDF is one possibility since PDFR induces cAMP signaling in response to PDF in vitro, indicating that it likely couples to Gs. PDF could signal in an autocrine manner since PDFR is present in LN_vs. However, the long-periods observed with reduced Gs signaling differ from the short-period and arrhythmic phenotypes of Pdf and pdfr mutants. The likeliest explanation for these differences is that the altered behavior of Pdf and pdfr mutants results from effects of PDF signaling over the entire circadian circuit, whereas the current manipulations specifically targeted LN_vs. Indeed, LN_vs are not responsible for the short-period rhythms in Pdf⁰¹ null mutant flies. Other possible explanations for the differences between the long-period rhythms with decreased Gs signaling in LN_vs and the short-period rhythms of Pdf and pdfr mutants are that additional GPCRs couple to Gs in s-LN_vs and influence molecular clock speed and that the current manipulations decrease rather than abolish reception of PDF. In summary, the data shows that Gs signaling via cAMP in s-LN_vs modulates period length (Dahdal, 2010).

Go signaling via PLC21C constitutes a novel pathway that regulates the s-LN_v molecular clock. This study found that Go and the metabotropic GABA_B-R3 receptor are required for the inhibitory effects of GABA on larval LN_vs, which develop into adult s-LN_vs. The same genetic manipulations that block GABA inhibition of LN_vs in culture (expression of Ptx or GABA_B-R3-RNAi) lengthened the period of adult locomotor rhythms. Furthermore, the molecular clock in s-LN_vs is disrupted when a subset of GABAergic neurons are hyper-excited. Since the LN_vs do not produce GABA themselves, s-LN_vs require GABAergic inputs to generate 24hr rhythms. Thus s-LN_vs are less autonomous for determining period length in DD than previously anticipated (Dahdal, 2010).

Activation of G-proteins can have both short- and long-term effects on a cell. With Go signaling blocked by Ptx, short-term effects on LN_v responses were detected in response to excitatory ACh and longer-term effects on the molecular clock. The latter are presumably explained by PLC activation since the behavioral phenotypes of Pdf > GoGTP flies were rescued by reducing Plc21C expression (Dahdal, 2010).

Since s-LN_v clocks were unchanged even when the speed of all non-LN_v clock neurons were genetically manipulated, it is surprising to find s-LN_v clocks altered by signaling from GABAergic non-clock neurons. Why would LN_vs need inputs from non-clock neurons to generate 24hr rhythms? One possibility is that LN_vs receive multiple inputs which either accelerate or slow down the pace of their molecular clock but overall balance each other to achieve 24hr rhythms in DD. Since reducing signaling by Gs and Go lengthens period, these pathways normally accelerate the molecular clock. According to this model, there are unidentified inputs to LN_vs which delay the clock. Identifying additional receptors in LN_vs would allow this idea to be tested (Dahdal, 2010).

Previous work showed that GABAergic neurons project to LN_vs and that GABA_A receptors in l-LN_vs regulate sleep. The current data show that constitutive activation of Go signaling dramatically alters behavioral rhythms, suggesting that LN_vs normally receive rhythmic GABAergic inputs. But how can s-LN_vs integrate temporal information from non clock-containing GABAergic neurons? s-LN_vs could respond rhythmically to a constant GABAergic tone by controlling GABA_B-R3 activity. Indeed, a recent study found that GABA_B-R3 RNA levels in s-LN_vs are much higher at ZT12 than at ZT0 (Kula-Eversole, 2010). Strikingly, this rhythm in GABA_B-R3 expression is in antiphase to LN_v neuronal activity. Thus regulated perception of inhibitory GABAergic inputs could at least partly underlie rhythmic LN_v excitability. GABAergic inputs could also help synchronize LN_vs as in the cockroach circadian system. Thus GABA's short-term effects on LN_v excitability, likely mediated by Gβ/γ, and GABA's longer-term effects on the molecular clock via Go may both contribute to robust rhythms (Dahdal, 2010).

This work adds to the growing network view of circadian rhythms in Drosophila where LN_vs integrate information to set period for the rest of the clock network in DD. The period-altering effects of decreased G-protein signaling in LN_vs point to a less hierarchical and more distributed network than previously envisioned. Since the data strongly suggests that GABA inputs are novel regulators of 24hr rhythms, the GABAergic neurons that fine-tune the s-LN_v clock should be considered part of the circadian network (Dahdal, 2010).

Effects of Mutation or Deletion

In the nervous system of homozygous Df(2R)47A embryos, lacking the G-oalpha47A gene, longitudinal axons are often missing and important modifications are observed in the guidance and axonal growth of motoneurons. It should noted that the lola gene is also deleted in the Df(2R)47A deficiency and that mutations in this gene result in missing longitudinal axons of the CNS. Since the Df(2R)47A phenotype is slightly stronger (in terms of the phenotypic penetration) than that provoked by a loss of function of lola, this suggests a function per se for G_o in axonal growth or guidance. This was confirmed by the analysis of the G-oalpha47A⁰⁰⁷ mutation that fully complements mutations in the lola gene but continues to display a neuronal phenotype. In homozygous G-oalpha47A⁰⁰⁷ mutants, axons of the motoneurons are clearly misrouted. However, in contrast to the Df(2R)47A mutation, the longitudinal axons in the CNS are less frequently missing, but are often pinched (Frémion, 1999).

The formation of the dorsal vessel or heart in a Drosophila melanogaster embryo can be divided into three main steps: (1) the determination step allows individualization of heart precursor cells from the dorsal mesoderm. They are arranged in clusters of seven to nine cells, located in each of the eleven segments of the trunk. Preliminary observations suggest that the gene Notch could participate in the choice of fate that the cardioblasts and the pericardial cells will adopt within the cardiogenic region. In the same line, a new gene, whose expression, as revealed by a P-lacZ insertion, is initiated at gastrulation in the developing mesoderm and becomes restricted within the mesoderm to the myogenic lineages, could participate in the determination of the cardioblasts identity; (2) once the cardioblasts have separated from the dorsal mesoderm, they reorganize to form an epithelial monolayer. The gene coding for the alpha-subunit of the transduction protein Go, which is expressed in the cardioblasts shortly before this step, could be involved in this process. Indeed, mutants in the Go alpha gene are affected in the formation of the cardiac endothelium; and (3) the last step consists of the migration of the cardiac epithelium towards the dorsal midline of the embryo to form the dorsal vessel by apposition of the two layers of cardioblasts. An extracellular matrix component is specifically expressed at the surface of the dorsal vessel and could participate in the interaction between the dorsalmost ectodermal cells and the heart during this migration step (Zaffran, 1995).

Trimeric G protein-dependent Frizzled signaling in Drosophila

Frizzled (Fz) proteins are serpentine receptors that transduce critical cellular signals during development. Serpentine receptors usually signal to downstream effectors through an associated trimeric G protein complex. However, clear evidence for the role of trimeric G protein complexes for the Fz family of receptors has hitherto been lacking. This study documents roles for the Galpha_o subunit (G_o) in mediating the two distinct pathways transduced by Fz receptors in Drosophila: the Wnt and planar polarity pathways. G_o is required for transduction of both pathways, and epistasis experiments suggest that it is an immediate transducer of Fz. While overexpression effects of the wild-type form are receptor dependent, the activated form (G_o-GTP) can signal when the receptor is removed. Thus, G_o is likely part of a trimeric G protein complex that directly tranduces Fz signals from the membrane to downstream components (Katanaev, 2005).

The evidence that Go transduces Wg signaling comes from the analysis of Go mutants, from overexpression studies, and from the epistasis experiments. These are addressed in the following discussion (Katanaev, 2005).

The inherent subviability of G_o clones prevented a frank assessment of their loss-of-function effects on Wg transduction: surviving cells likely carried perduring wild-type transcripts or protein. This offers a simple explanation for why not all G_o cells showed effects on Wg targets -- many cells still carried enough G_o function to transduce Wg. However, even given the lack of penetrance of the clones, there was a striking correspondence between G_o mutant clones and the loss of expression of Wg targets, thereby arguing that G_o gene function is critically required for Wg signal transduction (Katanaev, 2005).

Further evidence for the role of G_o in transducing Wg comes from the overexpression experiments. When G_o is overexpressed in the wing disc, clear upregulation of Wg targets is evident. If G_o achieves the upregulation of the target genes by hyperactivating the intracellular Wg transduction machinery, then abrogation of transduction downstream of G_o should nullify its effects. To this end, it was shown that the upregulation of Wg targets is arm and dsh dependent and is abolished by overexpression of sgg. Furthermore, G_o overexpression in embryos gives gain-of-function wg phenotypes that are arm dependent (Katanaev, 2005).

In arm and dsh clones (and fz, fz2 clones described below), residual Dll expression was sometimes found. This occurs in otherwise wild-type tissues and in both anterior and posterior domains of hh-Gal4; UAS-G_o wing discs and is most noticeable with dsh known for strong perdurance. However, arm and dsh clones in the regions of G_o overexpression lose Dll expression to a level comparable with clones in which G_o is not overexpressed. Thus, it is inferred that the upregulation of Wg targets induced by overexpression of G_o requires the Wg transduction pathway utilizing Dsh, Sgg, and Arm (Katanaev, 2005).

Upon activation of serpentine receptors, GDP is exchanged for GTP on Galpha, and the complex dissociates, leaving Galpha-GTP and ßγ free to signal to downstream components. To test whether G_o-GTP is able to activate the transduction pathway, a form of G_o containing an inactive GTPase was overexpressed. Overexpression of G_o-GTP induces Wg targets, indicating that G_o-GTP is a positive transducer of the pathway and that one function of Fz activation is to catalyze the release of G_o-GTP. Any signaling role of the ßγ moiety remains to be investigated. Overexpression of the G_o-GDP mutant form did not produce any effect. This form has a low affinity for GTP and could be expected to have dominant-negative effects. However, this form may not be sufficiently inactive to allow any effects on Wg transduction (and the PCP pathway) to be detected (Katanaev, 2005).

The epistasis experiments provide two key indications that G_o represents an immediate transducer of Fz signaling. (1) Dsh (previously the highest element of the transduction cascade identified downstream of the receptors) is necessary for the effects of G_o overexpression. (2) Since serpentine receptors act as exchange factors for trimeric G proteins, the effects of overexpression of a wild-type form should require the presence of the exchange factor to load and subsequently reload GTP. Conversely, once loaded with GTP, the form lacking GTPase activity (G_o-GTP) will be a long-lived activated subunit. Thus, if Fz acts as the exchange factor for G_o, then it would be expected that wild-type G_o would require Fz for its overexpression effects but that the activated form would be significantly less dependent. This is what was observed: Wg signaling is significantly rescued in fz, fz2 cells concomitantly expressing G_o-GTP as compared to those expressing wild-type G_o (Katanaev, 2005).

Given that G_o functions in the Wg transduction pathway, given that its overexpression effects require Dsh, and given that its activated form is receptor independent, the simplest explanation is that G_o functions in a trimeric G protein complex that relays signals from Fz receptors. These data do not necessarily suggest that G_o is the exclusive transducer of Wg signals: other trimeric complexes may be involved, and non-G protein-mediated signaling may also occur (Katanaev, 2005).

In the wing, the key molecular events associated with PCP occur by 30 hr APF, when Fz becomes specifically localized to the distal membrane of the cell. The localization of Fz appears to require its own signaling, since, in dsh mutants, Fz localization does not occur. A similar effect occurs when Fz is overexpressed: Fz is no longer restricted to the distal membrane. Given this complexity, the following feature of G_o can be predicted if it indeed acts as a transducer of Fz signaling. (1) Loss of G_o activity should induce PCP phenotypes; (2) Fz localization should not occur correctly when G_o signaling is compromised. In regard to these two predictions, it has been shown that (1) reduction of G_o function or G_o overexpression induces clear PCP defects and (2) Fz localization is aberrant when G_o function is down- or up-regulated. Furthermore, it has been shown that G_o itself undergoes a striking asymmetric redistribution in a fz-dependent manner (Katanaev, 2005).

G_o clones can show nonautonomous polarity defects on their proximal side, whereas fz clones show effects on their distal sides. This may indicate that G_o relays a negative signal in PCP transduction. G_o localizes proximally in polarizing cells, as does Strabismus/van Gogh, which also shows proximal nonautonomous effects. Hence, the proximal nonautonomous effects of G_o may result from it functioning negatively in the PCP pathway, from it becoming localized proximally, or from some combination of the two. A further aspect of G_o clones is the inappropriate localization of Fz at the interface of mutant and wild-type cells. It is not clear if this protein is derived from the wild-type cells, the mutant cells, or both. But it implies that the cells are in communication, and again a similar phenomenon has been described for Strabismus/van Gogh clones that may relate to the nonautonomous effects (Katanaev, 2005).

Overexpression of either G_o or G_o-GTP causes PCP defects, suggesting that one function of Fz signaling in the PCP pathway is the generation of free G_o-GTP. However, given the difficulty in distinguishing gain-of-function from loss-of-function effects, it is not possible to say whether G_o-GTP acts positively (as in the Wg pathway) or negatively. Any role for the ß/gamma dimer in transducing PCP signals remains to be established. The secreted multiple wing hairs produced by overexpression of wild-type G_o or G_o-GTP show a marked difference: the effects of wild-type G_o require the presence of the receptor (Fz), whereas the activated form does not. As for the Wg pathway described above, the most likely explanation of this observation is that Fz functions as an exchange factor for G_o (Katanaev, 2005).

GPCR signaling is required for blood-brain barrier formation in Drosophila

The blood-brain barrier of Drosophila is established by surface glia, which ensheath the nerve cord and insulate it against the potassium-rich hemolymph by forming intercellular septate junctions. The mechanisms underlying the formation of this barrier remain obscure. The G protein-coupled receptor (GPCR) Moody, the G protein subunits Gαi and Galphao, and the regulator of G protein signaling Loco are required in the surface glia to achieve effective insulation. The data suggest that the four proteins act in a complex common pathway. At the cellular level, the components function by regulating the cortical actin and thereby stabilizing the extended morphology of the surface glia, which in turn is necessary for the formation of septate junctions of sufficient length to achieve proper sealing of the nerve cord. This study demonstrates the importance of morphogenetic regulation in blood-brain barrier development and places GPCR signaling at its core (Schwabe, 2005).

The Drosophila nerve cord is ensheathed by a thin single-layer epithelium, which in turn is surrounded by an acellular layer of extracellular matrix material. Ultrastructural analysis has revealed that septate junctions (SJs) between the epithelial cells are responsible for the insulation of the nerve cord. Fate-mapping studies have shown that the nerve cord is enveloped by glia expressing the glial-specific marker Repo, but to date there has been no direct proof that it is these surface glia that form intercellular SJs and thus the insulating sheath. Moreover, the time course for the formation of the sheath and of the SJ-mediated seal has not been established (Schwabe, 2005).

Several assays were developed to follow the morphogenesis of the surface glial sheath. Due to the onset of cuticle formation, immunohistochemistry becomes unreliable after 16 hr of development. Live imaging of GFP-tagged marker proteins was therefore used to visualize cell shapes, in particular the actin cytoskeleton marker GFP/RFP-Moesin and the SJ marker Neuroglian (Nrg)-GFP. Nrg-GFP expressed under its own promoter and RFP-Moesin driven by repo-Gal4 are colocalized in the same cells, establishing that the SJ-forming cells are repo positive and thus conclusively demonstrating the insulating function of the surface glia. To probe the permeability of the transcellular barrier, fluorescent dye was injected into the body cavity and dye penetration into the nerve cord was quantified by determining mean pixel intensity in sample sections (Schwabe, 2005).

The surface glia are born in the ventrolateral neuroectoderm and migrate to the surface of the developing nerve cord, where they spread until they touch their neighbors (17 hr of development). The glia then join to form a contiguous sheet of square or trapezoidal cells, tiled to form three-cell corners. SJ material is visible as a thin contiguous belt by 18 hr but continues to accumulate until the end of embryogenesis. Similar to other secondary epithelia, the surface glia do not form a contiguous adherens-junction belt (zonula adherens), but only spotty, inconsistent adherens junctions were seen, as visualized by Armadillo-GFP (driven by own promoter). At 16 hr, the fluorescent dye freely penetrates into the nerve cord, but by 20 hr the nerve cord is completely sealed. The completion of the seal thus coincides with the onset of visible movements in the late embryo (Schwabe, 2005).

To further gauge the dye-penetration assay, embryos mutant for known septate-junction components were examined: Neurexin IV, which is required for blood-nerve barrier formation in the PNS; Neuroglian, and the sodium-pump component Nervana 2, for which only a role in the earlier formation of the ectodermal seal has been demonstrated. In all three mutants, severe penetration of dye was found, well after the nerve cord is sealed in wild-type (22 hr). These findings provide further evidence that the sealing of the nerve cord is achieved by SJs and suggest that the components of the ectodermal SJs are required for the function of surface glial SJs as well (Schwabe, 2005).

In a genome-wide screen for glial genes, using FAC sorting of GFP-labeled embryonic glia and Affymetrix microarray expression analysis, two novel GPCRs, Moody (CG4322) and Tre1 (CG3171) were identified. Both are orphan receptors belonging to the same novel subclass of Rhodopsin-family GPCRs. Their expression was examined by RNA in situ hybridization; different subtypes of glia in the embryonic nerve cord can be distinguished based on their position and morphology. In the CNS, moody is expressed in surface glia from embryonic stage 13 onward (10 hr); in addition to cells surrounding the nerve cord (subperineurial glia), this includes cells lining the dorsoventral channels (channel glia). moody is also expressed in the ensheathing glia of the PNS (exit and peripheral glia). Both CNS and PNS expression of moody are lost in mutants for the master regulator of glial fate, glial cells missing (gcm^N17), confirming that they are indeed glial. tre1 is expressed in all longitudinal glia and a subset of surface glia, as well as in cells along the midline. As expected, the (lateral) glial expression is lost in gcm mutants, while midline expression is not. Both moody and tre1 are also expressed outside the nervous system in a largely mutually exclusive manner, specifically in the germ cells, the gut, and the heart (Schwabe, 2005).

Several additional G protein signaling components are found in the surface glia. The six extant Gα genes show broad and overlapping expression in embryogenesis, with three of them (Go, Gq, and Gs) expressed throughout the nervous system and Gi expressed more specifically in surface glia. Gβ13F and Gγ1 are ubiquitously expressed during embryogenesis. Finally, the RGS loco is uniformly expressed in early embryos due to a maternal contribution but is then transcriptionally upregulated in surface and longitudinal glia, as well as in other tissues outside the nervous system. The nervous-system expression of loco is lost in gcm mutants. The presence of both Moody and Loco protein in the surface glia is confirmed using immunohistochemistry, but at 17 hr of development, when staining is feasible, the protein levels are still quite low (Schwabe, 2005).

In sum, the GPCR Moody, the RGS Loco, and Gi are differentially expressed in surface glia. This expression precedes and accompanies the morphogenesis and sealing of the surface glial sheath (Schwabe, 2005).

To examine protein expression and distribution of the GPCR signaling components in greater detail, third-instar larval nerve cords were examined. By this stage, the surface glia have doubled in size and show robust protein expression of GPCR signaling and SJ components (Schwabe, 2005).

Moody immunostaining is found at the plasma membrane, where it shows strong colocalization with the SJ marker Nrg-GFP. Loco immunostaining is punctate and more dispersed throughout the cytoplasm, with some accumulation at the plasma membrane, where it colocalizes with Moody. To avoid fixation and staining artifacts, fluorescent-protein fusions (Moody-mRFP; Loco-GFP) were generated and expressed using moody-Gal4, which drives weak surface glial expression. In the live nerve-cord preparations, Loco-GFP is much less dispersed and shows strong colocalization with Moody-mRFP at the plasma membrane (Schwabe, 2005).

In the absence of a known ligand, the coupling of G proteins to receptors is difficult to establish, but their binding to RGS proteins is readily determined. Loco physically binds to and negatively regulates Gi, and vertebrate Loco homologs (RGS12/14) have been shown to negatively regulate Gi/Go. In S2 tissue-culture assays, it was found that Loco binds to Gi and Go, but not to Gs and Gq. Double-label immunohistochemistry confirms that both Gi and Go are expressed in the surface glia (Schwabe, 2005).

Thus, Loco physically interacts with Gi and Go and shows subcellular colocalization with Moody, suggesting that the four signaling components are part of a common molecular pathway (Schwabe, 2005).

Using dye penetration as the principal assay, whether the GPCR signaling components that are expressed in surface glia play a role in insulation was examined. moody genomic (Δ17; Bainton, 2005) and RNAi mutants show similar, moderate insulation defects. The embryos are able to hatch but show mildly uncoordinated motor behavior and die during larval or pupal stages. The dye-penetration defect of moody^Δ17 is completely rescued by genomic rescue constructs containing only the moody ORF. Both moody splice forms (α and β; Bainton, 2005) are able to rescue the defect independently, as well as in combination. tre1 genomic (Kunwar, 2003) and RNAi mutants show no significant dye-penetration defect and no synergistic effects when combined with moody using RNAi. Thus, despite the close sequence similarity of the two GPCRs and their partially overlapping expression in surface glia, only moody plays a significant role in insulation. Overexpression of moody causes intracellular aggregation of the protein (Schwabe, 2005).

loco is expressed both maternally and zygotically. loco zygotic nulls are paralytic and, on the basis of an ultrastructural analysis, a disruption of the glial seal, has been suggested. In a dye-penetration assay, loco zygotic null mutants show a strong insulation defect, which can be rescued by panglial expression of Loco in its wt or GFP-tagged form. The extant null allele of loco (Δ13) did not yield germline clones; therefore loco RNAi was used to degrade the maternal in addition to the zygotic transcript. In loco RNAi embryos, dye penetration is indeed considerably more severe. Overall, insulation as well as locomotor behavior is affected much more severely in loco than in moody and is close in strength to the SJ mutants. Overexpression of loco is phenotypically normal (Schwabe, 2005).

Thus, positive (moody) and negative (loco) regulators of G protein signaling show qualitatively similar defects in loss of function, suggesting that both loss and gain of signal are disruptive to insulation. Such a phenomenon is not uncommon and is generally observed for pathways that generate a localized or graded signal within the cell (Schwabe, 2005).

Both Gi and Go have a maternal as well as a zygotic component. Gi zygotic null flies survive into adulthood but show strong locomotor defects. In Gi maternal and zygotic null embryos show a mild dye-penetration defect, which is markedly weaker than that of moody, suggesting redundancy among Gα subunits. To further probe Gi function, the wt protein (Gi-wt) as well as a constitutively active version (Gi-GTP) were overexpressed in glia using repo-Gal4; such overexpression presumably leads to a masking of any local differential in endogenous protein distribution. Expression of Gi-wt results in very severe dye penetration, while overexpression of Gi-GTP is phenotypically normal. Only Gi-wt but not Gi-GTP can complex with Gβγ; overexpression of Gi-wt thus forces Gβγ into the inactive trimeric state. This result therefore suggests that the phenotypically crucial signal is not primarily transduced by activated Gi but rather by free Gβγ. Similar results have been obtained in the analysis of Gi function in asymmetric cell division (Schwabe, 2005).

Go null germline clones do not form eggs and do not survive in imaginal discs, indicating an essential function for cell viability (Katanaev, 2005). Therefore animals with glial overexpression of constitutively active (Go-GTP), constitutively inactive (Go-GDP), and wt (Go-wt) Go (Katanaev, 2005) were examined. Overexpression of Go-GDP, which cannot signal but binds free Gβγ, leads to severe dye penetration, again pointing to a requirement for Gβγ in insulation. However, Go-GTP and Go-wt show a moderate effect, suggesting that signaling by active Go does contribute significantly to insulation, in contrast to active Gi (Schwabe, 2005).

Overall, it was found that all four GPCR signaling components expressed in surface glia are required for insulation, further supporting the notion that the four components are part of a common pathway. The phenotypic data suggest that this pathway is complex: two Gα proteins, Gi and Go, are involved, but with distinct roles: activated Go and Gβγ appear to mediate most of the signaling to downstream effectors, while activated Gi seems to function primarily as a positive regulator of Gβγ. The loss of moody appears much less detrimental than the loss of free Gβγ (through overexpression of Gi-wt or Go-GDP); this is inconsistent with a simple linear pathway and points to additional input upstream or divergent output downstream of the G proteins. Finally, it was consistently observed that both loss (moody, Gi null, and Go-GDP) and gain (loco and Go-GTP) of signal are disruptive to insulation, suggesting that the G protein signal or signals have to be localized within the cell (Schwabe, 2005).

These complexities of G protein signaling in insulation preclude an unambiguous interpretation of genetic-interaction experiments and thus the linking of moody to Gi/Go/loco by genetic means. Double-mutant combinations between moody and loco were generated using genomic mutants as well as RNAi, with very complex results: in moody loco genomic double mutants, the insulation defect is worse than that of loco alone, while in moody loco RNAi double mutants the insulation defect is similar to that of moody alone. This strong suppression of loco by moody is also observed in the survival and motor behavior of the RNAi-treated animals. Thus the phenotype of the double-mutant combination is dependent on the remaining levels of moody and loco, with moody suppressing the loco phenotype when loco elimination is near complete (Schwabe, 2005).

To understand how the GPCR signaling components effect insulation at the cellular level, the distribution of different markers in the surface glia was examined under moody and loco loss-of-function conditions and under glial overexpression of Gi-wt. To rule out cell fating and migration defects, the presence and position of the surface glia were determined using the panglial nuclear marker Repo. In all three mutant situations, the full complement of surface glia is present at the surface of the nerve cord, with the positioning of nuclei slightly more variable than in wt (Schwabe, 2005).

In the three mutants, the SJ marker Nrg-GFP still localizes to the lateral membrane compartment, but the label is of variable intensity and sometimes absent, indicating that the integrity of the normally continuous circumferential SJ belt is compromised. Notably, the size and shape of the surface glia are also very irregular. While qualitatively similar, the phenotypic defects are more severe in loco and under Gi-wt overexpression than in moody, in line with the results of functional assays. When examining the three mutants with the actin marker GFP-Moesin, it was found that the cortical actin cytoskeleton is disrupted in varying degrees, ranging from a thinning to complete absence of marker, comparable to the effects observed with Nrg-GFP. However, GFP-positive fibrous structures are present within the cells, indicating that the abnormalities are largely restricted to the cell cortex. The microtubule organization, as judged by tau-GFP marker expression, appears normal in the mutants. The light-microscopic evaluation thus demonstrates that, in the GPCR signaling mutants, the surface glia are positioned correctly and capable of forming a contiguous epithelial sheet as well as septate junctions. Instead, the defects occur at a finer scale—abnormally variable cell shapes and sizes, and irregular distribution of cortical actin and SJ material (Schwabe, 2005).

The changes in cell shape and actin distribution that were observed in the three mutants might simply be a secondary consequence of abnormalities in the SJ belt; to test this possibility, how a loss of the SJ affects the morphology and the actin cytoskeleton of the surface glia was examined. SJ components are interdependent for the formation and localization of the septa, and lack of a single component, such as Nrg, leads to nearly complete loss of the junction and severe insulation defects. In Nrg mutants, the surface glial cell shape and cortical actin distribution show only mild abnormalities. Thus, in contrast to the GPCR signaling mutants, the complete removal of the SJ causes only weak cytoskeletal defects, strongly arguing against an indirect effect. It is concluded that GPCR signaling most likely functions by regulating the cortical actin cytoskeleton of the surface glia, which in turn affects the positioning of SJ material along the lateral membrane (Schwabe, 2005).

More detailed insight into the nature of the defects in GPCR signaling mutants is afforded by electron microscopy. The surface glia in nerve cords of first-instar wild-type and mutant larvae were examined. Initially, dye penetration into the nerve cord was tested using ruthenium red. In wild-type, the dye diffuses only superficially into the surface glial layer, while in moody and loco mutants the dye penetrates deep into the nerve cord, in concordance with light-microscopic data. Tissue organization and SJ morphology were examined under regular fixation in randomly selected transverse sections. It has been reported that the surface glial sheath is discontinuous in loco mutant nerve cords, but this analysis was carried out at 16 hr of development, i.e., at a time when, even in wild-type, SJs are not yet established and the nerve cord is not sealed. In contrast to these findings, in the current study it was observed that, in loco as well as moody mutants, the glial sheath is in fact contiguous at the end of embryonic development. The ultrastructure of individual septa and their spacing also appear normal, indicating that moody and loco do not affect septa formation per se. However, the global organization of the junctions within the glial sheath appears perturbed: in wild-type, the surface glia form deep interdigitations, and the SJs are extended, well-organized structures that retain orientation in the same plane over long distances. In moody and loco mutants, the SJs are much less organized; they are significantly shorter in length and do not form long planar extents as in wild-type (Schwabe, 2005).

Taken together, the light- and electron-microscopic evaluations of the GPCR signaling mutants both show defects in the organization of the surface glial epithelium. The reduction in SJ length is consonant with the variability and local disappearance of the Nrg-GFP marker. Since the sealing capacity of the junction is thought to be a function of its length, the reduction in mean SJ length in the mutants provides a compelling explanation for the observed insulation defect (Schwabe, 2005).

Therefore, in addition to a reduction of the insulating SJs, this analysis of the GPCR signaling mutants revealed irregular cell shape and size, as well as weaker and variable accumulation of cortical actin in the surface glia. These data suggest that the primary defect in the mutants lies with a failure to stabilize the cortical actin, whose proper distribution is required for the complex extended morphology of the glia, which then affects SJ formation as a secondary consequence. Several lines of evidence exclude the reverse chain of causality, that is, a primary SJ defect resulting in destabilization of cortical actin and cell-shape change. Surface glia coalesce into a contiguous sheath and show strong accumulation of cortical actin before SJ material accumulates and sealing is completed. In the GPCR signaling mutants, there is misdistribution of SJ material along the cell perimeter, but the junctions do form. Finally, the GPCR signaling mutants show cell-shape and cortical actin defects that are much more severe than those observed in the near complete absence of SJ (Schwabe, 2005).

Compared to the columnar epithelia of the ectoderm and the trachea, the surface glial sheath is very thin. Compensating for their lack in height, surface glia form deep “tongue-and-groove” interdigitations with their neighbors. This increases the length of the intercellular membrane juxtaposition and thus of the SJ, which ultimately determines the tightness of the seal. It is proposed that the surface glial interdigitations are the principal target of regulation by GPCR signaling. In GPCR signaling mutants, a loss of cortical actin leads to diminished interdigitation and thus to a shortening of the SJ, resulting in greater permeability of the seal. This model integrates all the observations at the light- and electron-microscopic levels (Schwabe, 2005).

Sex-specific signaling in the blood-brain barrier is required for male courtship in Drosophila

Soluble circulating proteins play an important role in the regulation of mating behavior in Drosophila melanogaster. However, how these factors signal through the blood-brain barrier (bbb) to interact with the sex-specific brain circuits that control courtship is unknown. This study shows that male identity of the blood-brain barrier is necessary and that male-specific factors in the bbb are physiologically required for normal male courtship behavior. Feminization of the bbb of adult males significantly reduces male courtship. The bbb-specific G-protein coupled receptor Moody and bbb-specific Go signaling in adult males are necessary for normal courtship. These data identify sex-specific factors and signaling processes in the bbb as important regulators of male mating behavior (Hoxha, 2013).

It is worth noting that the integrity of the bbb was not affected by feminization or by any other of our manipulations using a standard approach to examine bbb barrier function, although small defects cannot be ruled out. Therefore, the observed effects support the interpretation that feminization affects physiological sex- specific processes within the bbb. This study shows that moody GPCR signaling is one of these processes. Normal courtship requires both moody isoforms, α and β, similar to the previously reported response to alcohol and cocaine. As has been described, moody appears to have two distinct roles: While either one of the moody isoforms is sufficient for a functional and intact barrier, both isoforms are required for adult signaling processes. RNA sequencing data suggest that the two isoforms are not present in equal abundance and that the ratio of the two isoforms is sex-specifically regulated. It is not clear at present why two forms of the moody protein are required in behavior. It is unlikely that a strict stoichiometric ratio of the two isoforms is required, since normal courtship was observed in wild-type flies that express additional Moody-β. The two isoforms differ in their intracellular domain, which could indicate that they interact with different effector molecules that are both contributing to the behavioral response. Courtship defects were observed when either isoform is missing, indicating that both forms have a role in regulating courtship. Interestingly, it was observed that the ratio of the two isoforms is under the control of the sex-specific splicing factor TraF, raising the possibility that the moody pre-mRNA is a target for splicing regulation by TraF or one of its downstream effectors. It will be of interest to identify other sex-specific factors in the bbb and examine their contribution to the regulation of male courtship (Hoxha, 2013).

No courtship phenotype was observed when dominant mutants were expressed for Gs and Gq that have been shown to act as dominant negative mutations in developmental processe. Likewise, expression of Gi-RNAi or concertina-RNAi did not result in reduced courtship either. This suggests that these G proteins do not play a significant role in courtship in this layer. In contrast, courtship defects were observed when Go signaling was compromised. Two approaches were employed to show that the heterotrimeric protein Go is required for male courtship behavior: inhibition by PTX and mRNA reduction by RNAi. The S1 subunit of PTX from Bordetella pertussis catalyzes the transfer of an ADP-ribose onto the Gα subunit of the heterotrimeric G protein. In contrast to mammals, where PTX inhibits both Go and Gi, in Drosophila PTX is a specific inhibitor for Go, since the only Gi present (Gⁱ65A) does not contain the PTX recognition site. PTX will only ribosylate heterotrimers (not individual alpha subunits), and the consequence of this ribosylation is inhibition of the heterotrimer activation. The inhibition of Go signaling by PTX is therefore very specific; since the ADP-ribosylated Go heterotrimers cannot be activated, they do not generate ectopic Gβγ subunits, nor do they sequester free Gβγ subunits away from other Gα subunits. Conditional induction of PTX only in adult mature flies, as well as conditional adult reduction of Gαo by RNAi reduced male courtship. This demonstrates that physiological signaling through Go is an important signaling pathway that regulates courtship in the bbb. Given these findings it is likely that moody signals through Go to exert its function in courtship. In embryos, Go, Gi, moody and loco mutations each disrupt the formation of the bbb and lead to bbb leakiness, as shown by dye penetration. In contrast, dye penetration was not observed in the PTX and Go-RNAi mutants that were generated, further evidence that the developmental and physiological roles of moody and Go signaling differ in their mechanisms (Hoxha, 2013).

It is not knowm what the downstream pathways are that are mediating the Go action. Few Go effectors have been demonstrated and its α or βγ subunits could be mediating the signal. In many cases in vertebrates it is the βγ subunits that are responsible for actuating signaling. In neurons, presynaptic voltage-gated Ca²⁺ channels have been shown to represent an effector for Go. Studies of the role of Go in learning and memory in Drosophila have suggested that Go signaling does not occur through the rut adenylyl cyclase. Signaling through Go is not generally thought to occur through PKA, consistent with the finding that disruption of PKA signaling in the bbb did not affect courtship. It is unknown whether potential downstream signaling molecules like loco, Gγ13F or PKC are sex-specifically expressed in the blood-brain barrier and might have a courtship role in this layer. In whole heads, PKC98E is male-preferentially expressed. It is unknown what the ligand is for Moody and it remains to be seen what the exact role is for moody bbb signaling in courtship. Hemolymph factors that influence courtship could conceivably do so by initiating signaling pathways at the bbb, or by passage and transport through the bbb. Moody could be playing a role in signaling, as well as through a possible effect on transport, perhaps in processes similar to its previously demonstrated effects on the actin cytoskeleton during development (Hoxha, 2013).

This study has demonstrated that sex-specific molecules in the bbb are important regulators of male courtship behavior in Drosophila. The Moody GPCR and Go signaling in this layer are an important part of this regulation. It will be of importance to identify the ligand(s) and downstream signaling events that ultimately interact with the brain circuits that control male courtship behavior (Hoxha, 2013).

The mevalonate pathway controls heart formation in Drosophila by isoprenylation of Gγ1

The early morphogenetic mechanisms involved in heart formation are evolutionarily conserved. A screen for genes that control Drosophila heart development revealed a cardiac defect in which pericardial and cardial cells dissociate, which causes loss of cardiac function and embryonic lethality. This phenotype resulted from mutations in the genes encoding HMG-CoA reductase, downstream enzymes in the mevalonate pathway, and G protein Gγ1, which is geranylgeranylated, thus representing an end point of isoprenoid biosynthesis. These findings reveal a cardial cell-autonomous requirement of Gγ1 geranylgeranylation for heart formation and suggest the involvement of the mevalonate pathway in congenital heart disease (Yi, 2006).

Mutations in genes controlling heart development frequently cause fatal cardiac malformations, the most common type of birth defect in humans. Because many of the mechanisms involved in heart development are evolutionarily conserved, the fruit fly Drosophila represents a powerful model for genetically dissecting this complex developmental process. The Drosophila heart, or dorsal vessel, which pumps bloodlike cells through an open circulatory system, is composed of parallel rows of contractile cardial cells (cardioblasts) tightly attached to pericardial cells; the latter perform supportive and secretory functions (Yi, 2006).

A P-element genetic screen was performed for Drosophila mutants with heart defecusing transgenic flies harboring a green fluorescent protein (GFP) transgene under control of the Hand enhancer, which is specific for cardial cells, pericardial cells, and the lymph gland—a hematopoietic organ in fruit flies. The Hand-GFP transgene allows visualization of the developing heart at single-cell resolution. Among a collection of mutants with cardiac abnormalities, a heart defect was observed in which pericardial cells dissociated from cardioblasts in the dorsal vessel at the end of embryogenesis. This phenotype was termed 'broken hearted' (bro). Five such mutants of different genetic loci are described in this study. In contrast to the wild-type dorsal vessel in which the pericardial cells are intimately associated with cardioblasts, in each of these mutants, the relative positions of pericardial cells and cardioblasts changed with each heartbeat (Yi, 2006).

The P element in the bro1 locus [l(3)01152] is located in the first exon of the hydroxymethyl-glutaryl (HMG)–coenzyme A (CoA) reductase gene (HMGCR), which is expressed in the dorsal vessel and the gonadal mesoderm, where it is required for migration of primordial germ cells (Van Doren, 1998). Mutants trans-heterozygous for HMGCR⁰¹¹⁵² and a deficiency line Df(3R)Exel9013, in which the HMGCR gene is deleted, or two EMS mutants, HMGCR^clb26.31 and HMGCR^clb11.54, showed similar, but more severe, cardiac defects than homozygous HMGCR⁰¹¹⁵² mutants. Expression of HMGCR in the heart, with the use of a Hand-GAL4 driver and a UAS-HMGCR transgene, rescued the cardiac defects in the HMGCR⁰¹¹⁵² mutant (Yi, 2006).

HMGCR controls a rate-limiting step in the conversion of HMG-CoA into mevalonate, a precursor for the synthesis of cholesterol and isoprene derivatives that modify the C termini of proteins containing a CAAX motif (C, cysteine; A, aliphatic amino acid; X, any amino acid). In contrast to mammalian cells, Drosophila does not use the mevalonate pathway to synthesize cholesterol. Injection of embryos at the syncytial blastoderm stage with 0.1 µM mevinolin, a statin drug that lowers cholesterol level by inhibiting HMGCR activity, caused cardiac defects at stage 17 similar to those of the HMGCR mutants (Yi, 2006).

To investigate whether either of the two major isoprenoids, farnesyl pyrophosphate (farnesyl-PP) and geranylgeranyl pyrophosphate (geranylgeranyl-PP), might be required for heart formation, mutants were examined in the genes encoding geranylgeranyl pyrophosphate synthase (GGPPS) and geranylgeranyl transferase type I ß subunit (ßGGT-I), which act downstream of HMGCR and are required for the biosynthesis of geranylgeranyl-PP or transfer of geranylgeranyl-PP to protein, to find out whether they also cause cardiac defects. Indeed, GGPPS (also called qm) mutant embryos showed 100% penetrance for the bro phenotype, just as HMGCR mutants did, and at least 30% of the ßGGT-I mutants displayed the same phenotype. In contrast, two deficiency lines [Df(2L)Exel6010 or Df(3R)Exel6269] deleting either the farnesyl transferase α (CG2976) or ß (CG17565) subunit did not display similar cardiac defects. These findings suggested that the cardiac defects of HMGCR mutant embryos resulted from a failure of geranylgeranylation of a target substrate protein required for the adhesion between cardioblasts and pericardial cells (Yi, 2006).

Analysis of another bro mutant (bro4) suggested that the G protein γ subunit 1 (Gγ1), which contains a C-terminal CAAX motif, is the substrate of this geranylgeranylation modification required for heart formation. The P element in the bro4 locus l(2)k08017 is inserted into the splice donor site after the first exon of the Gγ1 gene. Gγ1 expression level was reduced by more than 50% in homozygous l(2)k08017 embryos, which suggested that l(2)k08017 is a hypomorphic mutant allele of the Gγ1 gene. Mutants trans-heterozygous for the l(2)k08017 insertion and a deficiency that deletes the Gγ1 gene [Df(2R)H3E1] or for Df(2R)H3E1 and a Gγ1 null allele showed the same cardiac defects as the homozygous l(2)k08017 embryos. Double mutants of the hypomorphic HMGCR and Gγ1 alleles showed a more severe cardiac defect than either single mutant. A fifth bro mutation was mapped to the Sar1 gene, which encodes a guanosine triphosphatase that controls budding of vesicles overlaid with coat protein complex II (COPII) from the endoplasmic reticulum (ER) to the Golgi network (Yi, 2006).

The developmental onset of cardiac defects was identical in the HMGCR, Gγ1, GGPPS/qm, ßGGT-I, and Sar1 mutants. Cardioblasts and pericardial cells were properly specified and aligned until stage 16. However, at stage 17, pericardial cells began to dissociate from the dorsal vessel. These observations suggest that these genes are required to maintain cardiac integrity. The phenotypes of the different mutants were also comparable, except for the two HMGCR EMS mutants or the HMGCR⁰¹¹⁵²/Df(3R)Exel9013 mutant, which was more severe and showed distortion of the shape of the dorsal vessel (Yi, 2006).

The final C-terminal residues of all G protein γ subunits contain a CAAX motif in which the variable amino acid X determines the type of lipid modification: If X is serine, methionine, alanine, or glutamine, the cysteine is modified by farnesylation, whereas if X is leucine or valine, it is modified by geranylgeranylation. Using an in vitro prenylation assay, it was found that Drosophila Gγ1 protein, which contains a CAAX motif of Cys-Thr-Val-Leu (CTVL), was modified by geranylgeranylation, but not by farnesylation, in agreement with the requirement of GGPPS/qm and ßGGT-I for cardiac development (Yi, 2006).

To determine directly if geranylgeranylation of Gγ1 is essential for heart development, whether wild-type and mutant forms of Gγ1 protein could rescue the cardiac defect of the Gγ1 mutant was tested. Targeted expression of wild-type Gγ1 in the heart was sufficient to rescue the cardiac defects of Gγ1 mutants, whereas mutant forms of Gγ1, in which geranylgeranylation was abolished by either a substitution of Ser for Cys⁶⁷ (Gγ1-C67S) in the CAAX box or a deletion of the CAAX box (Gγ1-δCAAX), failed to rescue the cardiac defects in Gγ1 mutants. It is concluded that geranylgeranylation of the CAAX motif of Gγ1 is required for its normal activity during Drosophila heart formation (Yi, 2006).

Lipid modification of the CAAX motif facilitates the association of proteins with membranes. To further explore how geranylgeranylation of Gγ1 affects its biological function, the subcellular localization of the Gγ1 protein was examined in Drosophila S2R⁺ cells. Wild-type Gγ1 protein was always excluded from the nucleus in S2R⁺ cells, whereas the two mutant forms of Gγ1, which were not geranylgeranylated, were located throughout the cytoplasm and nucleus. Because Gγ1 is a small protein and can enter the nucleus freely, the specific localization of wild-type Gγ1 protein to the cytoplasm likely reflects its interaction with membranous structures, which requires modification by geranylgeranylation (Yi, 2006).

In S2R⁺ cells treated with three HMGCR inhibitors (atorvastatin, mevinolin, and simvastatin), as well as HMGCR double-stranded RNA, the wild-type Gγ1 protein displayed the same abnormal subcellular distribution as the two mutant forms of Gγ1. These findings suggest that abnormal subcellular localization of Gγ1 accounts for the cardiac defects in the mevalonate pathway mutants and Gγ1 mutants. Gα has also been shown to be required at an earlier stage of heart development for proper alignment of cardioblasts (Fremion, 1999), which is distinct from the function of Gγ1 revealed here (Yi, 2006).

Cardiac defects of HMGCR or Gγ1 mutants could be completely rescued by targeted expression of UAS-HMGCR and UAS-Gγ1 transgenes, respectively, using a Hand-GAL4 driver, which directs expression in both cardioblasts and pericardial cells, or a Mef2-GAL4 driver, which is expressed in cardioblasts but not in pericardial cells. In contrast, targeted expression of HMGCR or Gγ1 using Dot-GAL4, which drives expression only in pericardial cells, failed to rescue the cardiac defects in either mutant. These results demonstrate that HMGCR and Gγ1 function specifically in cardioblasts to adhere with pericardial cells and exclude the possibility that the bro cardiac phenotype arises secondarily from general metabolic abnormalities (Yi, 2006).

HMGCR and downstream enzymes in the biochemical pathway leading to the synthesis of geranylgeranyl-PP are specifically required in cardioblasts to modify Gγ1. It is proposed that geranylgeranylation, which is required for the proper intracellular localization of Gγ1, is in turn required for generating a signal for pericardial cells to adhere to cardioblasts throughout heart formation. Indeed, Gßγ has been shown to control Golgi apparatus organization and vesicle formation during exocytosis in mammalian cells. The finding that a mutation in Sar1 causes the same cardiac phenotype as the Gγ1 mutation further supports the possibility that this collection of mutations perturbs the secretion of a factor required for maintenance of cardiac integrity. Inhibition of this pathway with statins results in cardiac defects similar to those resulting from mutations in HMGCR and downstream genes required for isoprenoid biosynthesis, which raises the possibility that congenital heart defects reportedly associated with the use of statins, which are contraindicated during pregnancy, may reflect perturbation in a similar developmental pathway (Yi, 2006).

HMGCR has also been shown to be required for recruitment of primordial germ cells (PGCs) to the gonad in Drosophila, but the protein target(s) of the mevalonate pathway that mediate this process have not been identified. Perhaps Gγ1 functions in the gonad mesoderm to guide PGC migration. It is speculated that lipid modifications mediated by the mevalonate pathway contribute to directed cell migration and subsequent cell-cell adhesion in diverse cell types. Given the conservation of cardiac developmental control mechanisms, it will be of interest to investigate the potential involvement of the mevalonate pathway in mammalian heart development and congenital heart disease (Yi, 2006).

G_o signaling is required for Drosophila associative learning

Heterotrimeric G_o is one of the most abundant proteins in the brain, yet relatively little is known of its neural functions in vivo. This study demonstrates that G_o signaling is required for the formation of associative memory. In Drosophila, pertussis toxin (PTX) is a selective inhibitor of G_o signaling. The postdevelopmental expression of PTX within mushroom body neurons robustly and reversibly inhibits associative learning. The effect of G_o inhibition is distributed in both gamma- and alpha/beta-lobe mushroom body neurons. However, the expression of PTX in neurons adjacent to the mushroom bodies does not affect memory. PTX expression also does not interact genetically with a rutabaga adenylyl cyclase loss-of-function mutation. Thus, G_o defines a new signaling pathway required in mushroom body neurons for the formation of associative memory (Ferris, 2006).

Heterotrimeric G proteins regulate a noncanonical function of septate junction proteins to maintain cardiac integrity in Drosophila

The gene networks regulating heart morphology and cardiac integrity are largely unknown. The heterotrimeric G protein gamma subunit 1 (Ggamma1) has been shown to mediate cardial-pericardial cell adhesion in Drosophila. This study shows that G-oalpha47A and Gbeta13F cooperate with Ggamma1 to maintain cardiac integrity. Cardial-pericardial cell (CC-PC) adhesion also relies on the septate junction (SJ) proteins Neurexin-IV (Nrx-IV), Sinuous, Coracle, and Nervana2, which together function in a common pathway with Ggamma1. Furthermore, Ggamma1 signaling is required for proper SJ protein localization, and loss of at least one SJ protein, Nrx-IV, induces cardiac lumen collapse. These results are surprising because the embryonic heart lacks SJs and suggest that SJ proteins perform noncanonical functions to maintain cardiac integrity in Drosophila. These findings unveil the components of a previously unrecognized network of genes that couple G protein signaling with structural constituents of the heart (Yi, 2008).

The results of this study show that the heterotrimeric G proteins G-oα47A, Gβ13F, and Gγ1 function together to maintain CC-PC adhesion during the late stage of heart formation in Drosophila. By mapping a new broken hearted (bro) mutant (Nrx-IV) and characterizing additional candidate genes, a noncanonical role was discovered for SJ proteins in mediating CC-PC and CC-CC adhesion outside SJs. Four SJ proteins, Nrx-IV, Sinu, Cora, and Nrv2, operate in a common pathway with Gγ1 to maintain cardiac integrity; these proteins require Gγ1 for proper subcellular localization in the heart. Mechanistically, the presence of SJ proteins in both CCs and PCs suggests that these proteins act in trans to maintain cell-cell adhesion in the dorsal vessel. A model is favored in which the extracellular domain of Nrx-IV engages in heterophilic interactions with SJ-proteins such as Neuroglian or Contactin, and that these interactions would be stabilized by ECM proteins such as Pericardin (Prc). Alternatively, the SJ proteins may directly interact with ECM proteins to provide a structural basis for cardiac integrity (Yi, 2008).

Heterotrimeric G proteins G-oα47A/G-iα65A, Gβ13F, and Gγ1 function with the GPCR moody and the RGS protein loco to regulate SJ formation in the Drosophila brain-blood barrier (Schwabe, 2005). Although loco mutant embryos show the bro heart phenotype, moody mutations do not induce a heart phenotype. A search of the Drosophila protein interaction map reveals that the GPCR CG32447 interacts with both the SJ protein Sinu and the RGS Kermit. Kermit also interacts with Loco, suggesting that the CG32447 GPCR participates in the control of cardiac integrity. However, a deficiency uncovering CG32447 does not induce the bro phenotype. Since the screen for bro mutants, visualized as a perturbation in the ordered expression pattern of Hand-GFP in cardial and pericardial cells, did not identify a GPCR that maintains cardiac integrity, it is concluded that the GPCR regulating cardiac integrity is either pleiotropic, with an early embryonic function that precludes its identification as a regulator of cardiac integrity, or is redundant to a second GPCR in the dorsal vessel (Yi, 2008).

Alternatively, cardiac integrity may be regulated by a GPCR-independent mechanism. In neuroblasts, G-iα65A, Gβ13F, Gγ1, and loco regulate mitotic spindle orientation, protein localization, and ultimately asymmetric cell division via a GPCR-independent signaling pathway. During neuroblast cell division, heterotrimeric G proteins are activated by the GTPase exchange factor (GEF) Ric-8, but not by GPCRs (see David, 2005). However, the lethal mutation ric-8^G0397 does not induce the bro phenotype (Yi, 2008).

During blood-brain barrier formation, sequestering Gβγ or hyperactivating G-oα47A signaling in glial cells leads to SJ defects, whereas hyperactivating G-iα65A signaling does not affect SJ function. A similar relationship exists among heterotrimeric G proteins during asymmetric cell division in neuroblasts. In contrast, sequestering Gβγ in the dorsal vessel has no effect on cardiac integrity, while hyperactivating G-oα47A in the embryonic heart induces the bro phenotype. It is concluded that the bro phenotype in Gβ13F or Gγ1 mutants is caused by misregulation of G-oα47A signaling. This is in sharp contrast to the G proteins regulating blood-brain barrier formation and asymmetric cell division where Gβγ dimers activate a set of downstream effectors distinct from that of G-oα47A signals (Yi, 2008).

G protein signaling regulates SJ formation in Drosophila and tight junction formation in mammalian cells. Even though SJs are analogous to vertebrate tight junctions, it is striking that G protein signaling components colocalize with both SJ and tight junction proteins. In addition, Gα_s interacts with the tight junction protein ZO-1 throughout junction formation, suggesting that Gα subunits physically regulate tight junction assembly. Thus, septate/tight junction proteins appear to be direct targets of G proteins in both flies and vertebrates (Yi, 2008 and references therein).

Although the embryonic heart lacks SJs, the current results are consistent with the idea that SJ proteins are direct targets of G proteins in the dorsal vessel. G protein mutants phenocopy SJ-protein mutants and G proteins operate in a common pathway with SJ proteins to maintain cardiac integrity. In addition, proper localization of SJ proteins in the embryonic heart requires G protein signaling, and G proteins regulate at least one SJ protein at the posttranscriptional level. Finally, loss of G-oα47A signaling (G-oα47A mutants) and hyperactivation of G-oα47A signaling (overexpressing G-oα47A) both result in the bro phenotype; thus Gα signaling is localized to specific foci in cells of the dorsal vessel. It is proposed that an appropriate level of Gα signaling mediates SJ-protein localization, whereas loss or hyperactivation of Gα signaling mislocalizes SJ proteins leading to a loss in cardiac integrity (Yi, 2008).

Cell-cell adhesion plays an essential role during organ morphogenesis. In the Drosophila heart, cell-cell adhesion along three distinct CC membrane domains is required to maintain cardiac integrity. Medioni (2008) provide a detailed description of two CC domains participating in cell-cell adhesion: the adherent domain, positioned immediately dorsal and ventral to the cardiac lumen, promotes cell-cell adhesion between CCs on opposing sides of the heart, and the basal-lateral adherent domain, positioned along the lateral CC membrane, promotes cell-cell adhesion between neighboring CCs on one side of the heart. These studies suggest that a third CC membrane domain, referred to as the pericardial adherent domain, is positioned opposite to the luminal domain and promotes PC-CC adhesion. The loss of cell-cell adhesion along each of the three CC domains gives rise to a unique phenotype: luminal collapse (referred to hereafter as type-1), breaks between neighboring cardial cells (type-2), and loss of PC-CC adhesion (type-3), respectively. The unique nature of these three phenotypes can provide insight into the molecular pathways regulating cardiac integrity (Yi, 2008).

Loss of heterotrimeric G proteins or SJ proteins induces the type-3 (bro) phenotype, and mutations in at least one SJ-protein gene, Nrx-IV, leads to the type-1 phenotype. In addition, Sinu, Cora, and Nrv2 localize to the luminal and perhaps the adherent domains, suggesting that loss of these proteins will also cause the type 1 phenotype. The type 2 phenotype is observed in a subset of Gγ1 embryos, but not in any other heterotrimeric G protein or SJ-protein mutants. Thus, the pathways regulating cell-cell adhesion along the CC basal-lateral membrane may be distinct from those identified in this study (Yi, 2008).

The guidance ligand Slit has been shown to regulate multiple aspects of cardiogenesis in Drosophila, and mutations in slit induce luminal collapse (referred to hereafter as type-1), breaks between neighboring cardial cells (type-2), and likely loss of PC-CC adhesion (type-3) phenotypes. In addition, slit mutant embryos show mesoderm migration and CC polarity defects, however these defects are genetically separable from cardiac integrity defects. Slit signals through the Robo receptors and mutations in genes encoding downstream components of the Robo signaling pathway do not dominantly enhance slit mutations. In contrast, mutations in genes encoding integrins or integrin ligands, such as scab, mys, and Lan-A, dominantly enhance slit mutations and transheterozygous embryos show the type-2 phenotype. This study suggests that Slit activates two pathways during cardiogenesis: one pathway utilizes typical Robo signaling to regulate mesoderm migration and CC polarity while a second pathway uses atypical, or Robo-independent, signaling to regulate cell adhesion between neighboring CCs and likely between opposing CCs to promote lumen formation. Although the role of Slit in regulating PC-CC adhesion has not been studied in detail, one possibility is that Slit signals through G-oα47A/Gβ13F/Gγ1 to regulate CC-CC and even PC-CC adhesion (Yi, 2008).

SJ proteins are functionally interdependent and localization of Sinu to SJs requires Nrx-IV, Cora, and Nrv2 (Wu, 2004), while Nrx-IV, Cora, Cont, and Nrg are equally interdependent for localization to SJs. In addition, both Nrv2 and Nrx-IV are transmembrane proteins, and the extracellular domain of Nrv2 at least is required for SJ function. Since every SJ-protein mutant examined showed PC-CC adhesion defects, SJ proteins likely form interdependent complexes in PCs and CCs. The extracellular domains of SJ proteins may act in trans, either through direct interactions with SJ proteins along opposing membranes or through indirect interactions with ECM proteins such as Pericardin, to maintain cardiac integrity. A search of the Drosophila protein interaction map reveals an interaction between Pericardin and Sinu, supporting the latter possibility. Alternatively, SJ proteins could be required for the formation or function of adherens junctions in the dorsal vessel (Yi, 2008).

All of the bro genes have close vertebrate orthologs. Since the function of mevalonate pathway genes in heart development is conserved from Drosophila to vertebrates (D'Amico, 2007; Edison, 2005; Yi, 2006), it is speculated that G protein-mediated regulation of SJ proteins is also evolutionarily conserved. To date, the role of heterotrimeric G proteins in regulating vertebrate heart development has not been identified, but heterotrimeric G proteins do play a role in heart disease. In contrast, Sinu is a member of the Claudin protein family and even though this protein family is rather divergent (Wu, 2004), vertebrate Claudin-1 is required for normal heart looping in the chick. In addition, Claudin-5 localizes to the lateral membrane of cardiomyocytes and is associated with human cardiomyopathy. Lastly, mutations in the prc ortholog, collagen alpha-1(IV), cause vascular defects in mice and humans. Taken together, these studies raise the possibility that heterotrimeric G proteins and tight junction proteins ensure proper vertebrate cardiovascular morphogenesis (Yi, 2008).

Competing activities of heterotrimeric G proteins in Drosophila wing maturation

Drosophila genome encodes six α-subunits of heterotrimeric G proteins. The α-subunit termed Gαs is involved in the post-eclosion wing maturation, which consists of the epithelial-mesenchymal transition and cell death, accompanied by unfolding of the pupal wing into the firm adult flight organ. This study shows that another α-subunit, Gαo, can specifically antagonize the Gαs activities by competing for the Gβ13F/Ggamma1 subunits of the heterotrimeric Gs protein complex. Loss of Gβ13F, Gγ1, or Gαs, but not any other G protein subunit, results in prevention of post-eclosion cell death and failure of the wing expansion. However, cell death prevention alone is not sufficient to induce the expansion defect, suggesting that the failure of epithelial-mesenchymal transition is key to the folded wing phenotypes. Overactivation of Gαs with cholera toxin mimics expression of constitutively activated Gαs and promotes wing blistering due to precocious cell death. In contrast, co-overexpression of Gβ13F and Gγ1 does not produce wing blistering, revealing the passive role of the Gβγ in the Gαs-mediated activation of apoptosis, but hinting at the possible function of Gβγ in the epithelial-mesenchymal transition. These results provide a comprehensive functional analysis of the heterotrimeric G protein proteome in the late stages of Drosophila wing development (Katanayeva, 2010).

G protein-coupled receptors (GPCRs) represent the most populous receptor family in metazoans. Approximately 380 non-olfactory GPCRs are encoded by the human genome, corroborated by ca. 250 GPCRs in insect genomes, making 1%-1.5% of the total gene number dedicated to this receptor superfamily in invertebrates and mammals. GPCRs transmit their signals by activating heterotrimeric G protein complexes inside the cell. A heterotrimeric G protein consists of a GDP-bound α-subunit and a βα-heterodimer. Ligand-stimulated GPCR serves as a guanine nucleotide-exchange factor, activating the GDP-to-GTP exchange on the Gα-subunit. This leads to dissociation of the heterotrimeric complex into Gα-GTP and flγ, which transmit the signal further inside the cell (Katanayeva, 2010).

The β- and γ-subunit repertoire of the Drosophila genome is reduced as compared with that of mammals: only two Gγ and three Gβ genes are present in flies. Gγ30A and Gβ76C are components of the fly phototransduction cascade and are mostly expressed in the visual system. Gγ1 and Gβ13F have been implicated in the asymmetric cell divisions and gastrulation, while the function of Gβ5 is as yet unknown (Katanayeva, 2010).

Despite the fact that βγ can activate signal effectors, the main selectivity in GPCR coupling and effector activation is provided by the Gα-subunits. Sixteen genes for the α-subunits are present in the human genome, and six in Drosophila. All human Gαsubunit subgroups are represented in Drosophila: Gαi and Gαo belonging to the Gαi/o subgroup; Gαq belonging to the Gαq/11 subgroup; Gαs belonging to the Gαs subgroup, and concertina (cta) belonging to the Gα12/13 subgroup. Additionally, Drosophila genome encodes for Gαf which probably represents an insect-specific subfamily of Gαsubunits (Katanayeva, 2010).

Multiple functions have been allocated to different heterotrimeric G proteins in humans and flies. For example, in Drosophila development cta is a crucial gastrulation regulator, Gαo is important for the transduction of the Wnt/Frizzled signaling cascade, and Gαi controls asymmetric cell divisions during generation of the central and peripheral nervous system (the later in cooperation with Gαo. Gαq is the Drosophila phototransduction Gαsubunit, but probably has additional functions. Pleotropic effects arise from defects in Gαs function, while the function of Gαf has not yet been characterized (Katanayeva, 2010).

Among the developmental processes ascribed to the control by Gαs are the latest stages of Drosophila wing development. Newly hatched flies have soft and folded wings, which during the 1-2 hours post-eclosion expand and harden through intensive synthesis of components of the extracellular matrix. These processes are accompanied by epithelial-mesenchymal transition and apoptosis of the wing epithelial cells, producing a strong but mostly dead adult wing structure. Expression of the constitutively active form of Gαs leads to precocious cell death in the wing epidermis, which results in failure of the closure of the dorsal and ventral wing sheets and accumulation of the hemolymph inside the wing, producing wing blistering. Conversely, clonal elimination of Gαs leads to autonomous prevention of the cell death. Kimura (2004) has performed an extensive analysis of the signaling pathway controlling apoptosis at late stages of wing development. That study provided evidence suggesting that the hormone bursicon, synthesized in the head of post-eclosion Drosophila and secreted in the hemolymph, activates a GPCR Rickets on wing epithelial cells, which signals through Gαs to activate the cAMP-PKA pathway, culminating at the induction of apoptosis. However, the identity and importance of the &βγ subunits in bursicon signaling, as well as possible involvement of other Ga proteins remained outside of their investigation. There also remain some uncertainties as to the phenotypic consequences of elimination of the bursicon-Gαs-PKA pathway in wings (Katanayeva, 2010).

This study describes a comprehensive functional analysis of the Drosophila heterotrimeric G protein proteome using loss-of-function and overexpression experiments. Loss of Gαs but not any other Gαsubunit leads to the failure of wing expansion after fly hatching. Gαo, but not another Gα, can compete with Gαs and thus antagonize its function. Finally, the Gβ13F and Gγ1 as the βγ subunits of the heterotrimeric Gs complex responding to the epithelial-mesenchymal transition and cell death-promoting signal (Katanayeva, 2010).

The soft folded wings of the young insect freshly hatched from the pupal case within 1-2 hours expand and harden, becoming a robust flight organ. This process is accompanied by epithelial-mesenchymal transition and cell death of the wing epithelial cells. Genetic dissection has revealed the function of the neurohormone bursicon and its wing epithelial receptor rickets in initiation of these processes. The GPCR rickets couples to the heterotrimeric G protein Gs; the Gαs-activated cAMP-PKA pathway culminates at the induction of apoptosis. However, the overall phenotypic consequences of the loss of the Gs signaling pathway in post-eclosion wings were unknown, as well as the nature of the Gβγ subunits of the heterotrimeric Gs complex responding to the bursicon-rickets signaling (Katanayeva, 2010).

This study consisted of an extensive analysis of the heterotrimeric G protein subunits in these post-eclosion stages of wing maturation. The whole-wing down-regulation of Gαs results in the failure of wing expansion, demonstrating that this change in the shape of the wing is the major morphological outcome of the bursicon-rickets-Gs signaling. The Gβ13F and Gγ1 subunits were also identified as the other two constituents of the heterotrimeric Gs complex, as downregulation of Gαs, Gβ13F, or Gγ1, but not any other Ga, Gβ, or Gγ subunits encoded by the Drosophila genome, each leads to the same folded wing phenotype (Katanayeva, 2010).

It was also shown that Gαo, but not any other Gαsubunit, can inhibit the wing expansion program through sequestration of the Gβ13F/Gγ1 heterodimer. The reason for the specificity of Gαo over other Gαsubunits in antagonizing the Gs signaling is unclear. It is unlikely that differences in expression levels of the tested Gαsubunits may account for the selective activity of Gαo. Indeed, most overexpression experiments were done with the X-chromosome-inserted MS1096-Gal4 driver, which results in markedly higher expression levels in males than heterozygous female flies, producing a more penetrant folded wing phenotype in males overexpressing Gαo. However, even in male flies overexpressing other Gαsubunits no instances of the folded wing phenotype could be seen. Furthermore, several independent insertions of the UAS-Ga transgenes were tested; while different Gαo transgenes all produced the folded wing phenotype upon overexpression, other Ga constructs remained ineffective (Katanayeva, 2010).

Similarly, the different Gαsubunits possess a similar affinity towards the interaction with the Gβγ heterodimer, not providing an explanation for a specific ability of Gαo to antagonize the Gs-mediated post-eclosion pathway. It is thus thus tempting to propose that a previously uncharacterized biochemical mechanism may allow for a specific antagonism physiologically existing between the Gs- and Go- mediated signaling pathways. As liberation of high amounts of GDP-loaded Gαo is predicted to be a consequence of activation of multiple Go-coupled GPCRs, and as Go is a heavily expressed G protein representing the major G protein species e.g. in the brain of flies and mammals, this specific ability of Gαo to antagonize the Gs-mediated signaling may have physiological implications in other tissues and organisms than Drosophila wing. However, it is added that these speculations are based on the analysis of the overexpression data and must be treated with caution when translating them into physiological situations (Katanayeva, 2010).

Only the GDP-loaded, but not the activated GTP-loaded form of Gαo is effective in antagonizing Gs. A proteomics analysis was performed of the Drosophila proteins which would discriminate between the two nucleotide forms of Gαo, and surprisingly few targets of this kind were revealed. While the chaperone Hsc70-3 and β1-tubulin preferentially interacted with the GTP-loaded Gαo, Gβ13F was found to specifically interact with Gαo-GDP. These data suggest that many Gαo-interaction partners do not discriminate between the two guanine forms of Gαo. These findings are in agreement with our other experimental findings, as well as mathematical modeling predicting that high concentrations of free (monomeric) signaling-competent Gαo-GDP are produced upon activation of Go-coupled GPCRs (Katanayeva, 2010).

Gαo-mediated sequestration of Gβ13F/Gγ1 depletes the pool of the heterotrimeric Gs complexes. As only heterotrimeric Ga&βγ, but not monomeric Ga proteins can efficiently bind and be activated by their cognate GPCRs, overexpression of Gαo abrogates the rickets-Gs signaling. Phenotypic consequences of this abrogation are the failures of apoptosis and wing expansion. In contrast, expression of the constitutively activated form of Gαs induces premature cell death and wing blistering. This phenotype can be also induced by expression of cholera toxin, revealing that the ability of cholera toxin to specifically overactivate Gαs reported in mammalian systems is reproduced with Drosophila proteins. These data also confirm that not only exogenously overexpressed, but also the endogenous Gαs can induce the precocious cell death upon overactivation (Katanayeva, 2010).

However, prevention of apoptosis is not sufficient to produce the folded wing phenotype. Together with the observation that the constitutively active form of Gαs is ineffective in rescuing the wing expansion defects produced by Gαo overexpression, these data suggest that the Gαs-cAMP-PKA pathway culminating at apoptosis is not the sole signaling branch emanating from the bursicon-rickets GPCR activation. It is proposed that the second signaling branch initiated by the rickets-mediated dissociation of the heterotrimeric Gs complex is represented by the free Gββ subunits, signaling to epithelial-mesenchymal transition. Such a double signaling impact mediated by the two components of the heterotrimeric G protein complex leads to initiation of two cellular programs -- apoptosis and epithelial-mesenchymal transition -- which cumulatively result in wing expansion and solidification, producing the adult flight organ. This two-fold response of the Drosophila wing to the maturation signal, mediated by the two components of the heterotrimeric G protein complex activated by the single hormone-responsive GPCR, provides an elegant paradigm for the coordination of signaling and developmental programs (Katanayeva, 2010).

A role for Drosophila Wnt-4 in heart development

In vertebrates, different Wnt signaling pathways are required in a temporally coordinated manner to promote cardiogenesis. In Drosophila, wingless holds an essential role in heart development. Among the known Drosophila Wnts is DWnt4, the function of which has been studied in various developmental processes except for heart development. This study re-evaluated the expression pattern of DWnt4 during embryogenesis and showed that transcripts are not restricted to the dorsal ectoderm but are also present in the cardiogenic mesoderm. Moreover, DWnt4 mRNA transcripts were detected in myocardial cells by stage 16. The heart phenotype in DWnt4 mutant embryos is characterized by various degrees of disrupted expression of different cardiac markers. Overexpression of Dwnt4 also affects heart marker expression, which can be partially rescued by simultaneous inhibition of PKC. These data reveal a role for DWnt4 in cardiogenesis, however integration of DWnt4 with other known signaling pathways that function in heart development still awaits further investigation (Tauc, 2012).

Previously published data indicate that DWnt4 expression in the visceral mesoderm is regulated by Hox genes, in particular by Ultrabithorax (Ubx) and abdominal A (abd-A). Four Hox genes, Antennapedia (Antp), Ubx, abd-A and abd-B are expressed in the Drosophila heart where they specify different regions along the anterior-posterior axis of the heart tube. Therefore, it may be that members of the Hox gene family regulate DWnt4 expression also in the heart tube. Ubx is expressed in the aorta where low levels of DWnt4 mRNA expression and the higher expression levels of Dwnt4 in the heart proper correlate with the expression of Abd-A. The strong accumulation of DWnt4 transcripts in the heart proper is detected at what appears to be the border between high-level Abd-A and Abd-B expression. Ubx and abd-A were also shown to be involved in the establishment and patterning of alary muscles that project from the dorsal vessel. There are seven pairs of alary muscles that attach the heart to the dorsal epidermis in larvae and in adult flies. The extracellular matrix marker Pericardin (Prc) is not only expressed around pericardial cells and in the basal membrane of myocardial cells but also accumulates along the alary muscles. It was noticed that in DWnt4^EMS23 mutants the number of Prc positive projections is affected. Although the phenotype was not quantified, it was observed that the number of projections varied. For example additional Prc positive projections were seen at positions different from where the seven pairs of alary muscles normally attach. Not much is known about the embryonic origin of the alary muscles and the molecules required for their development. The current data may spur investigations on the role of DWnt4 in the development of these muscles. It is intriguing to hypothesize that DWnt4 acts as a guidance cue (attractive or repulsive) for the alary muscle attachment site. A guidance function for DWnt4 has been previously described in the context of dorsoventral projections of retinal axons, of motor neuron target specificity and in salivary gland migration (Tauc, 2012).

The early expression pattern of DWnt4 in the cardiac mesoderm and in the overlying ectoderm suggested that DWnt4 could be involved in early steps of cardiogenesis such as cardiac specification and differentiation of cardiac cell types. All cardiac marker genes that were analyzed in DWnt4^EMS23 mutant embryos exhibited a range of degrees of disruption, none of which had serious detrimental effects though. Hence, in contrast to wg, DWnt4 does not appear to be essential for Drosophila cardiogenesis. Nevertheless, DWnt4 does play a role to ensure normal cardiac marker gene expression. Whereas in DWnt4 mutants a mild loss of Svp positive cells (or only Svp expression) was observed, DWnt4 overexpression resulted in the loss and increase of Svp expressing cells. It has been suggested that DWnt4 modulates cell fate specification within the Hedgehog-dependent domain and hedgehog was shown to regulate Svp expression. Hence, it is intriguing to speculate that the Svp phenotype is caused by defective Hh signaling that results from inappropriate amounts of DWnt4. Next attempts were made to investigate which components may mediate the DWnt4 signal. Immunostainings for Prc revealed two phenotypes in DWnt4 mutants. Embryos were characterized by gaps in Prc expression along the heart tube and/or by a detachment of Prc expressing cells from the Prc positive basal membrane, which indicates the detachment of pericardial cells from myocardial cells. These phenotypes are reminiscent of the phenotypes described for embryos that are mutant for the α-subunit of the heterotrimeric Go protein bkh. Gαo was shown to couple to the seven transmembrane Fz receptors and mediate Wnt signaling as well as planar polarity signaling. fz mutants, like bkh mutants, exhibit both phenotypes: gaps in Prc and a detachment of pericardial cells from myocardial cells. Of note, DWnt4 was shown to be able to bind to three Fz receptors Fz, Fz2 and Fz4. Unlike Fz2, which was shown to solely activate the arm-dependent Wg signaling pathway, Fz can also mediate a non-canonical, planar polarity signal (Tauc, 2012).

Since similar phenotypes were observed for Prc in DWnt4, fz and bkh mutants tests were performed to see whether fz and bkh may be components of the DWnt4 signaling pathway. The rationale was that if these molecules act in the same pathway, an increase of severity and/or penetrance of the phenotype in would be expected double heterozygous embryos. The results do not support such a simple linear relationship with respect to Prc expression along the heart tube. Nevertheless, changes were observed in the number of embryos showing a particular phenotype, which suggests that these factors could be genetically interacting. Due to the complexity of the data, a straight-forward interpretation is somewhat difficult at this point. Reasons for such phenotypic changes could be due to the involvement of different molecular mechanisms underlying either the gap or detachment phenotype. For example, gaps in Prc expression could result from a defective mesenchymal-epithelial transition required for proper heart morphogenesis as was shown for bkh mutants. One cause for the detachment phenotype is a misregulation of septate junction proteins present in myocardial and pericardial cells where bkh is also involved. It is feasible that DWnt4 may function as a guidance cue for the proper migration of mesodermal cells. Irregularities in mesoderm migration can also lead to heart defects similar to the ones seen in DWnt4 mutants, which was shown for example in embryos mutant for the FGF receptor heartless or for the proteoglycan syndecan (Tauc, 2012).

In addition to Prc possible genetic interactions between DWnt4 and fz or DWnt4 and bkh were examined with respect to the reduction in Odd expressing pericardial cells that was detected in all single mutants. This analysis indeed revealed that the phenotype increased in embryos that were double heterozygous for DWnt4 and bkh compared to embryos that were single heterozygous for each factor (Tauc, 2012).

As to a possible genetic interaction between DWnt4 and fz, the data is less convincing since the phenotype was already highly penetrant in fz/+ heterozygous embryos. Multiple publications have indicated that DWnt4 acts through a non-canonical, arm-independent Wnt pathway. Therefore whether JNK, a core component of the planar polarity pathway, may be part of the DWnt4 signal transduction machinery was tested. Since ectodermal JNK signaling is essential for the morphogenetic process of dorsal closure, which impinges on normal heart development as a secondary effect, the pathway was interrupted in a tissue-specific manner. Mesodermal inhibition of JNK signaling using the dominant-negative bsk construct did not elicit a heart phenotype. Mesodermal inhibition of the canonical Wg signaling pathway using a dominant-negative construct of TCF was detrimental to heart development as expected. These results exclude a primary function for JNK in cardiogenesis and due to the lack of resemblance to the phenotypes in DWnt4 mutants, it is unlikely that JNK is a component of the DWnt4 signaling pathway in this context. Although inhibition of the canonical Wg pathway has a much more dramatic effect on cardiogenesis than lack of DWnt4, the data still leaves the option that DWnt4 may also act through the canonical pathway. Despite several publications that indicate that DWnt4 activates a non-canonical pathway, Harris and Beckendorf (2007) concluded from their data that DWnt4 acts through a canonical Wnt pathway during salivary gland migration. PKCs were shown to mediate non-canonical Wnt signaling in vertebrates and were implicated in mediating the DWnt4 signal during ovarian morphogenesis. This study performed experiments to determine whether PKCs could function in the DWnt4 signaling pathway in heart development expressing a characterized PKC pseudosubstrate that inhibits all PKCs present in Drosophila. The results show that by inhibiting PKC signaling, the amount of embryos exhibiting a reduction in Svp and Odd expressing cells was decreased after the overexpression of DWnt4. Admittedly this is an indirect indication that PKC may mediate the DWnt4 signal but this piece of data together with previously published results encourages further investigation of this possibility (Tauc, 2012).

In summary, although the definite function of DWnt4 in cardiogenesis still awaits further investigation, the data provides a good platform for subsequent analyses of DWnt4 in heart development. In particular with respect to the newly described cardiac expression pattern of DWnt4, future results can be anticipated that demonstrate a function for DWnt4 in heart tube formation and heart function (Tauc, 2012).

The Drosophila neuropeptides PDF and sNPF have opposing electrophysiological and molecular effects on central neurons

Neuropeptides have widespread effects on behavior, but how these molecules alter the activity of their target cells is poorly understood. A new model system was employed in Drosophila to assess the electrophysiological and molecular effects of neuropeptides, recording in situ from larval motor neurons which transgenically express a receptor of choice. Focus was placed on two neuropeptides, Pigment-dispersing factor (PDF) and short neuropeptide F (sNPF), which play important roles in sleep/rhythms and feeding/metabolism. PDF treatment depolarized motor neurons expressing the PDF receptor (PDFR), increasing excitability. sNPF treatment had the opposite effect, hyperpolarizing neurons expressing the sNPF receptor (sNPFR). Live optical imaging using a genetically encoded FRET-based sensor for cyclic AMP (cAMP) showed that PDF induced a large increase in cAMP, whereas sNPF caused a small but significant decrease in cAMP. Co-expression of pertussis toxin or RNAi interference to disrupt the G-protein Galphao blocked the electrophysiological responses to sNPF, showing that sNPFR acts via Galphao signaling. Using a fluorescent sensor for intracellular calcium, it was observed that sNPF-induced hyperpolarization blocked spontaneous waves of activity propagating along the ventral nerve cord, demonstrating that the electrical effects of sNPF can cause profound changes in natural network activity in the brain. This new model system provides a platform for mechanistic analysis of how neuropeptides can affect target cells at the electrical and molecular level, allowing for predictions of how they regulate brain circuits that control behaviors such as sleep and feeding (Vecsey, 2014).

Heterotrimeric Go protein links Wnt-Frizzled signaling with ankyrins to regulate the neuronal microtubule cytoskeleton

Drosophila neuromuscular junctions (NMJs) represent a powerful model system with which to study glutamatergic synapse formation and remodeling. Several proteins have been implicated in these processes, including components of canonical Wingless (Drosophila Wnt1) signaling and the giant isoforms of the membrane-cytoskeleton linker Ankyrin 2, but possible interconnections and cooperation between these proteins were unknown. This study demonstrates that the heterotrimeric G protein G_o functions as a transducer of Wingless-Frizzled 2 signaling in the synapse. Ankyrin 2 was identified as a target of Go signaling required for NMJ formation. Moreover, the G_o-ankyrin interaction is conserved in the mammalian neurite outgrowth pathway. Without ankyrins, a major switch in the G_o-induced neuronal cytoskeleton program is observed, from microtubule-dependent neurite outgrowth to actin-dependent lamellopodial induction. These findings describe a novel mechanism regulating the microtubule cytoskeleton in the nervous system. This work in Drosophila and mammalian cells suggests that this mechanism might be generally applicable in nervous system development and function (Luchtenborg, 2014).

Ankyrins (Ank) are highly abundant modular proteins that mediate protein-protein interactions, mainly serving as adaptors for linking the cytoskeleton to the plasma membrane. Mammalian genomes encode three Ank genes [AnkR (Ank1), AnkB (Ank2) and AnkG (Ank3)], whereas Drosophila has two [Ank1 (also known as Ank - FlyBase) and Ank2]. Ank2 is expressed exclusively in neurons and exists in several splicing variants. The larger isoforms (Ank2M, Ank2L and Ank2XL) are localized to axons and play important roles in NMJ formation and function. The C-terminal part of Ank2L can bind to microtubules. Despite the well-established role of Ank2 in NMJ formation, its function has been considered somewhat passive and its mode of regulation has not been clarified. This study shows that Gαo binds to Ank2 and that these proteins and the Wg pathway components Wg, Fz2, and Sgg jointly coordinate the formation of the NMJ. The functional Gαo-Ank interaction is conserved from insects to mammals (Luchtenborg, 2014).

Synaptic plasticity underlies learning and memory. Both in invertebrates and vertebrates, activation of Wnt signaling is involved in several aspects of synapse formation and remodeling, and defects in this pathway may be causative of synaptic loss and neurodegeneration. Thus, understanding the molecular mechanisms of synaptic Wnt signaling is of fundamental as well as medical importance. The Drosophila NMJ is a powerful model system with which to study glutamatergic synapses, and the Wnt pathway has been widely identified as one of the key regulators of NMJ formation.

This study provides important mechanistic insights into Wnt signal transduction in the NMJ, identifying the heterotrimeric G_o protein as a crucial downstream transducer of the Wg-Fz2 pathway in the presynapse. It was further demonstrated that Ank2, a known player in the NMJ, is a target of Gαo in this signaling (Luchtenborg, 2014).

This study found that the α subunit of G_o is strongly expressed in the presynaptic cell, and that under- or overactivation of this G protein leads to neurotransmission and behavioral defects. At the level of NMJ morphology, presynaptic downregulation or Ptx-mediated inactivation of Gαo was found to recapitulate the phenotypes obtained by similar silencing of wg and fz2. These data confirm that presynaptic Wg signaling, in addition to the Wg pathway active in the muscle, is crucial for proper NMJ formation, and that G_o is required for this process. Furthermore, neuronal Gαo overexpression can rescue the wg and fz2 loss-of-function phenotypes, demonstrating that, as in other contexts of Wnt/Fz signaling, G_o acts as a transducer of Wg/Fz2 in NMJ formation. In contrast to its evident function and clear localization in the presynapse, Gαo localization on the muscle side of the synapse is much less pronounced or absent. Unlike Gαo, the main Drosophila Gβ subunit is strongly expressed in both the pre- and postsynapse. Thus, a heterotrimeric G protein other than G_o might be involved in the postsynaptic Fz2 transduction, as has been implicated in Fz signaling in some other contexts (Luchtenborg, 2014).

A recent study proposed a role for Gαo downstream of the octopamine receptor Octβ1R. This signaling was proposed to regulate the acute behavioral response to starvation both on type II NMJs (octapaminergic) and on the type I NMJs (glutamatergic) analyzed in this study. In contrast to the current observations, downregulation of Gαo in these NMJs was proposed to increase, rather than decrease, type I bouton numbers. It is suspected that the main reason for the discrepancy lies in the Gal4 lines used. The BG439-Gal4 and C380-Gal4 lines of Koon are poorly characterized and, unlike the well-analyzed pan-neuronal elav-Gal4 and motoneuron-specific OK371-Gal4 and D42-Gal4 driver lines used in the current study, might mediate a more acute expression. In this case, this study reflects the positive role of Gαo in the developmental formation of glutamatergic boutons, as opposed to a role in acute fine-tuning in response to environmental factors as studied by Koon (Luchtenborg, 2014).

Postsynaptic expression of fz2 was found to fully rescue fz2 null NMJs. This study found that presynaptic knockdown of Fz2 (and other components of Wg-Fz2-Gαo signaling) recapitulates fz2 null phenotypes, whereas presynaptic overactivation of this pathway increases bouton numbers; furthermore, presynaptic overexpression of fz2 or Gαo rescues the fz2 nulls, just as postsynaptic overexpression of fz2 does. The current data thus support a crucial role for presynaptic Wg-Fz2-Gαo signaling in NMJ formation. Interestingly, both pre- and postsynaptic re-introduction of Arrow, an Fz2 co-receptor that is normally present both pre- and postsynaptically, as is Fz2 itself, can rescue arrow mutant NMJs. Thus, it appears that the pre- and postsynaptic branches of Fz2 signaling are both involved in NMJ development. A certain degree of redundancy between these branches must exist. Indeed, wild-type levels of Fz2 in the muscle are not sufficient to rescue the bouton defects induced by presynaptic expression of RNAi-fz2, yet overexpression of fz2 in the muscle can restore the bouton integrity of fz2 nulls. One might hypothesize that postsynaptic Fz2 overexpression activates a compensatory pathway - such as that mediated by reduction in laminin A signaling - that leads to restoration in bouton numbers in fz2 mutants. The current data showing that the targeted downregulation of Fz2 in the presynapse is sufficient to recapitulate the fz2 null phenotype underpin the crucial function of presynaptic Fz2 signaling in NMJ formation (Luchtenborg, 2014).

This study found that downregulation of Ank2 produces NMJ defects similar to those of wg, fz2 or Gαo silencing. However, Ank2 mutant phenotypes appear more pronounced, indicating that Wg-Fz2-Gαo signaling might control a subset of Ank2-mediated activities in the NMJ. Ank2 was proposed to play a structural role in NMJ formation, binding to microtubules through its C-terminal region. However, since the C-terminal region was insufficient to rescue Ank2L mutant phenotypes, additional domains are likely to mediate Ank2 function through binding to other proteins. This study demonstrates in the yeast two-hybrid system and in pull-down experiments that the ankyrin repeat region of Ank2 physically binds Gαo, suggesting that the function of Ank2 in NMJ formation might be regulated by Wg-Fz2-Gαo signaling. Indeed, epistasis experiments place Ank2 downstream of Gαo in NMJ formation (Luchtenborg, 2014).

Upon dissociation of the heterotrimeric G_o protein by activated GPCRs such as Fz2, the liberated Gαo subunit can signal to its downstream targets both in the GTP- and GDP-bound state (the latter after hydrolysis of GTP and before re-association with Gβγ). The free signaling Gαo-GDP form is predicted to be relatively long lived, and a number of Gαo target proteins have been identified that interact equally well with both of the nucleotide forms of this G protein. In the context of NMJ formation, this study found that Gαo-GTP and -GDP are efficient in the activation of downstream signaling, and identifies Ank2 as a binding partner of Gαo that interacts with both nucleotide forms. The importance of signaling by Gα-GDP released from a heterotrimeric complex by the action of GPCRs has also been demonstrated in recent studies of mammalian chemotaxis, planar cell polarity and cancer (Luchtenborg, 2014).

Gαo[G203T], which largely resides in the GDP-binding state owing to its reduced affinity for GTP, might be expected to act as a dominant-negative. However, in canonical Wnt signaling, regulation of asymmetric cell division as well as in planar cell polarity (PCP) signaling in the wing, Gαo[G203T] displays no dominant-negative activity but is simply silent, whereas in eye PCP signaling this form acts positively but is weaker than other Gαo forms. Biochemical characterization of the mammalian Gαi2[G203T] mutant revealed that it can still bind Gβγ and GTP, but upon nucleotide exchange Gαi2[G203T] fails to adopt the activated confirmation and can further lose GTP. The current biochemical characterization confirms that Gαo[G203T] still binds GTP. Interestingly, Gαi2[G203T] inhibited only a fraction of Gαi2-mediated signaling, suggesting that the dominant-negative effects of the mutant are effector specific. Thus, it is inferred that a portion of Gαo[G203T] can form a competent Fz2-transducing complex, and a portion of overexpressed Gαo[G203T] resides in a free GDP-loaded form that is also competent to activate downstream targets - Ank2 in the context of NMJ formation (Luchtenborg, 2014).

These experiments place Ank2 downstream of Gαo and also of Sgg (GSK3β). It remains to be investigated whether Ank2 can directly interact with and/or be phosphorylated by Sgg. Meanwhile, it is proposed that the microtubule-binding protein Futsch might be a linker between Sgg and Ank2. Futsch is involved in NMJ formation and is placed downstream of Wg-Sgg signaling, being the target of phosphorylation and negative regulation by Sgg as the alternative target to β-catenin, which is dispensable in Wg NMJ signaling. Abnormal Futsch localization has been observed in Ank2 mutants. In Drosophila wing and mammalian cells in culture, Gαo acts upstream of Sgg/GSK3β. Cumulatively, these data might suggest that the Wg-Fz2-Gαo cascade sends a signal to Futsch through Sgg, parallel to that mediated by Ank2 (Luchtenborg, 2014).

The importance of the Gαo-Ank2 interaction for Drosophila NMJ development is corroborated by findings in mammalian neuronal cells, where it was demonstrated that the ability of Gαo to induce neurite outgrowth is critically dependent on AnkB and AnkG. Knockdown of either or both ankyrin reduces neurite production. Remarkably, upon AnkB/G downregulation, Gαo switches its activity from the induction of microtubule-dependent processes (neurites) to actin-dependent protrusions (lamellopodia). Furthermore, Gαo recruits AnkB to the growing neurite tips. These data demonstrate that the Gαo-ankyrin mechanistic interactions are conserved from insects to mammals and are important for control over the neuronal tubulin cytoskeleton in the context of neurite growth and synapse formation. The novel signaling mechanism that were uncovered might thus be of general applicability in animal nervous system development and function (Luchtenborg, 2014).

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