InteractiveFly: GeneBrief

G protein α q subunit: Biological Overview | References

BIOLOGICAL OVERVIEW

Heterotrimeric G proteins mediate a variety of signaling processes by coupling G protein-coupled receptors to intracellular effector molecules. In Drosophila, the Gαq gene encodes several Gαq splice variants, with the Gαq¹ isoform protein playing a major role in fly phototransduction. However, Gαq¹ null mutant flies still exhibit a residual light response, indicating that other Gαq splice variants or additional Gq α subunits are involved in phototransduction. This study isolated a mutant fly with no detectable light responses, decreased rhodopsin (Rh) levels, and rapid retinal degeneration. Using electrophysiological and genetic studies, biochemical assays, immunoblotting, real-time RT-PCR, and EM analysis, it was found that mutations in the Gαq gene disrupt light responses, and the Gαq3 isoform protein was demonstrated to be responsible for the residual light response in Gαq¹ null mutants. Moreover, this study reports that Gαq3 mediates rhodopsin synthesis. Depletion of all Gαq splice variants led to rapid light-dependent retinal degeneration, due to the formation of stable Rh1-arrestin 2 (Arr2) complexes. These findings clarify essential roles for several different Gαq splice variants in phototransduction and retinal integrity in Drosophila and reveal that Gαq3 functions in rhodopsin synthesis (Gu, 2020).

Heterotrimeric G proteins and G protein-coupled receptors play pivotal roles in mediating a variety of extracellular signals to intracellular signaling pathways, such as hormones, neurotransmitters, peptides, and sensory stimuli. In the Drosophila visual system, light stimulation activates the major rhodopsin (Rh1) to form metarhodopsin, which in turn activates heterotrimeric G proteins and norpA gene-encoded phospholipase C (PLCβ). Activated PLC catalyzes phosphatidylinositol 4,5-bisphosphate to generate diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3). IP3 induces the release of Ca2+ from intracellular Ca2+ stores, whereas both DAG and IP3 may trigger extracellular Ca2+ influx by opening transient receptor potential (Trp) and transient receptor potential-like (TrpL) channels on the cell membrane. The Gαq gene encodes several Gαq splice variants, among which the Gαq-RD variant generates Gαq¹ isoform protein, and other splice variants generate Gαq3 isoform protein. Although both strong alleles of norpA and trpl;trp double mutants show completely abolished photoresponses, the Gαq¹ null mutant allele (Gαq⁹⁶¹) still displays a residual light response. These data indicate that other Gαq splice variants, or the Gq α subunits encoded by additional genes, contribute to the residual light responses in Gαq¹ null mutants (Gu, 2020).

Intracellular Ca2+ homeostasis controlled by Gq signaling is also essential for photoreceptor cell survival. Mutations in phototransduction cascade components, such as those in trp and norpA, prevent normal light-induced Ca2+ influx, resulting in stable Rh1/Arr2 complex formation and severe rapid light-dependent retinal degeneration. Disruption of stable Rh1/Arr2 complexes by genetic removal of Arr2 or suppression of Rh1 endocytosis can suppress the retinal degeneration either in norpA or trp mutant flies. Rh1/Arr2 complex formation is thought to contribute to impaired Ca2+ influx-activated CaM kinase II, which usually phosphorylates Arr2 to release Arr2 from Rh1. However, neither Gαq¹ nor Gαq⁹⁶¹ mutants undergo rapid retinal degeneration, exhibiting only slight retinal degeneration after keeping them in 12-h light/12-h dark cycles for 21 days. The disparate retinal degeneration phenotype between Gαq and norpA mutant is therefore unclear (Gu, 2020).

This study isolated a mutant fly with no detectable light responses and revealed that mutations in the Gαq gene cause the defective light responses. Gαq3 is responsible for the residual light response in Gαq¹ null mutants, and depletion of all Gαq splice variants results in rapid light-dependent retinal degeneration due to formation of stable Rh1/Arr2 complexes. In addition, this study revealed that Gαq3 plays essential roles in Rh1 synthesis. This study clarifies the essential role of different Gαq splice variants in fly phototransduction, retinal degeneration, and rhodopsin synthesis (Gu, 2020).

In Drosophila photoreceptors, G proteins are essential to activate the phototransduction cascade. The Gαq gene encodes several Gαq splice variants, and Gαq¹ has been shown to function as the predominant G protein in fly phototransduction. This study identified a mutation (5501T/A) in the Gαq gene, which specifically mutates Val to Asp at residue 303 in Gαq¹ but not Gαq3 isoforms. Although Val is replaced with Ile at residue 303 in vertebrate Gαq proteins, the hydrophobicity at this position is evolutionally conserved. Structural analyses have shown that the V303 region localizes to the interface between Gα proteins and its downstream effector PLC. The change of a hydrophobic residue to a polar one may affect the interaction between these two proteins. A recent study has shown that Gαq^V303D mutant protein is unable to activate PLC in vivo (Gu, 2020).

Although the 5501T/A Gαq gene mutation largely contributes to abolished light responses, this mutation is not fully responsible for the abolished light responses in no detectable light response (nlr) mutants because both nlr/Gαq¹ and nlr/Gαq⁹⁶¹ flies still exhibited a residual light response similar to Gαq¹ and Gαq⁹⁶¹ mutants. These data also excluded the possibility that Gαq^V303D mutant protein dominantly suppresses the function of Gαq protein. Gαq¹ expression in nlr mutants largely recovers the light response, further excluding the possibility that abolished light responses in nlr mutants are due to the dominant suppression of Gαq^V303D mutant protein (Gu, 2020).

The Gαq gene encodes several Gαq splice variants, and Gαq^221c mutants disrupt the expression of all Gαq splice variants (21). An ERG recording revealed that Gαq^221c null mutant clones showed no light responses. Previous whole-cell voltage-clamp recordings showed that the photoresponse of Gαq¹ homozygous cells is larger than that of Gαq¹ heterozygous cells. These results indicate that other Gαq splice variants might contribute to the residual light response in Gαq¹ null mutants. This study demonstrates that Gαq3 contributes to the residual light response in Gαq¹ null mutants (Gu, 2020).

The Gαq gene encodes several Gαq splice variants. Originally, two cDNAs resulting from different Gαq gene splicing were isolated. These two cDNAs encode Gαq1 and Gαq2 isoform proteins, respectively. Functional studies demonstrated that Gαq1 mediates the light response, whereas Gαq2 has no effect on phototransduction. Subsequently, two additional Gαq splice variants were isolated. To date, seven total Gαq splice variants have been annotated in Flybase, and these splice variants encode three different isoform proteins, including Gαq1, Gαq3, and Gαq4. This study has demonstrated that Gαq3 also mediates phototransduction. Overexpression of Gαq3 in nlr mutants induced detectable light responses but failed to fully restore the light response. Interestingly, the rescue flies exhibited comparable ERG trace amplitude and dynamics as those of Gαq¹ and Gαq⁹⁶¹ flies. These results indicate that different Gαq isoform proteins play different roles in phototransduction. Gαq mediates retinal degeneration (Gu, 2020).

Mutations in most genes encoding components of the phototransduction cascade result in rapid retinal degeneration, except for Gαq hypomorphic allele Gαq¹ and Gαq¹ isoform null mutant allele Gαq⁹⁶¹. Previous studies have shown that both Gαq¹ and Gαq⁹⁶¹ mutants undergo slow light-dependent retinal degeneration due to slow accumulation of stable Rh1/Arr2 complexes. In these Gαq mutants, the small residual photoresponse may reduce Ca2+ influx, which partially activates CaM kinase II and leads to the slow release of Arr2 from Rh1. This study shows that nlr mutants undergo rapid light-dependent retinal degeneration similar to that observed in norpA mutants. Disruption of stable Rh1/Arr2 complexes formation prevented retinal degeneration in the mutants. Under normal conditions, the interaction between Arr2 and Rh1 is transient, because light-triggered Ca2+ influx may activate CaM kinase II, which subsequently phosphorylates Arr2 to release Arr2 from Rh1. In nlr mutants, photoresponses were completely abolished so that the normal rise in Ca2+ after light stimulation was blocked, causing stable Rh1/Arr2 complex formation and retinal degeneration. These observations and explanations are consistent with mutations such as trp and norpA (Gu, 2020).

This study has shown the first evidence that Gαq3 regulates Rh1 synthesis. Rh1 is transported to the plasma membrane by vesicular transport mechanisms regulated by a large number of trafficking proteins. Previous studies have shown that Gαq homologue CG30054 regulates inositol 1,4,5,-tris-phosphate receptor (IP3R) to mediate calcium mobilization from intracellular stores and promote calcium-regulated secretory vesicle exocytosis. Given that Gαq3 shows high sequence identity to CG30054, they may regulate Rh1 synthesis through promoting calcium-regulated secretory vesicle exocytosis (Gu, 2020).

Decoding calcium signaling dynamics during Drosophila wing disc development

The robust specification of organ development depends on coordinated cell-cell communication. This process requires signal integration among multiple pathways, relying on second messengers such as calcium ions. Calcium signaling encodes a significant portion of the cellular state by regulating transcription factors, enzymes, and cytoskeletal proteins. However, the relationships between the inputs specifying cell and organ development, calcium signaling dynamics, and final organ morphology are poorly understood. In this study a quantitative image-analysis pipeline was designed for decoding organ-level calcium signaling. With this pipeline, spatiotemporal features were extracted of calcium signaling dynamics during the development of the Drosophila larval wing disc, a genetic model for organogenesis. Specific classes of wing phenotypes were identified that resulted from calcium signaling pathway perturbations, including defects in gross morphology, vein differentiation, and overall size. Four qualitative classes of calcium signaling activity were found. These classes can be ordered based on agonist stimulation strength Gαq-mediated signaling. In vivo calcium signaling dynamics depend on both receptor tyrosine kinase/phospholipase C gamma and G protein-coupled receptor/phospholipase C beta activities. Spatially patterned calcium dynamics were found to correlate with known differential growth rates between anterior and posterior compartments. Integrated calcium signaling activity decreases with increasing tissue size, and it responds to morphogenetic perturbations that impact organ growth. Together, these findings define how calcium signaling dynamics integrate upstream inputs to mediate multiple response outputs in developing epithelial organs (Brodskiy, 2019).

Organ development requires the coordination of many cells to form a structurally integrated tissue. Important properties of the final organ architecture include its shape, size, and spatial distribution of cell types. Notably, the information processing network required for development resembles a 'bow-tie' network structure with many input signals that are funneled through a limited number of second messengers (The wing disc as a model system of signal integration during organogenesis). Signal integration and pathway crosstalk result in many possible downstream outputs that are determined by effector proteins that regulate cellular processes, including cell division, migration, mechanical properties, death, and cell differentiation state. However, how these diverse input signals regulate the dynamics of second messengers is poorly understood. Further, how organ-level properties, such as size and shape, emerge from the integration of second messenger signaling remains to be fully elucidated (Brodskiy, 2019).

A key second messenger that serves as a central node in the bow-tie structure is the calcium ion (Ca2+). Ca2+ signaling is a ubiquitous transducer of cellular information and plays key roles in regulating cell behaviors, such as cell division, growth, and death. Ca2+ dynamics regulate cellular properties and behavior during animal development, and perturbations to Ca2+ signaling often lead to disease. Cells can encode complex signals into a Ca2+ signaling 'signature,' which includes amplitude, frequency, and integrated intensity of Ca2+ oscillations. Cells decode these signaling signatures by modulating the activities of downstream enzymes and transcription factors (Brodskiy, 2019).

Intercellular Ca2+ signaling is correlated with many developmental processes. For example, they have been found to regulate scale development in the butterfly. Ca2+ waves are indispensable to activate Drosophila egg development, and Ca2+ spikes are important for development of Drosophila and Xenopus embryos. Ca2+ signaling responds to Hedgehog (Hh) signaling in the frog neural cord, correlates with Decapentaplegic (Dpp) secretion in Drosophila imaginal discs, and is indispensable for human neural rosette development. Ca2+ dynamics also are essential for cell migration and tissue contractility in zebrafish, Japanese newt, and chick embryos. Recently, intercellular Ca2+ transients (ICTs) have been observed in the Drosophila wing disc, both in vivo and ex vivo, and have been implicated as a first response to wounding and robustness in regeneration, tissue homeostasis, and mechanotransduction. Inhibition of Ca2+ significantly also rescues cancerous overgrowth of wings, thus showing its regulatory role in tissue growth. However, a quantitative characterization of Ca2+ dynamics in organ development is lacking, in part because of a lack of image-processing methods and a suitable model system to analyze the stochastic nature of the signals. Consequently, there is a need for a systems-level description of Ca2+ signaling dynamics to decode the role of Ca2+ signaling in organ development (Brodskiy, 2019).

The Drosophila wing imaginal disc pouch is a premier model system to study how epithelial cells undergo specific morphogenetic steps to form the intricate structure of an adult wing. The wing disc is a powerful model system because of the availability of tools to perturb gene expression in a specific region of a tissue. Multiple conserved regulatory modules for tissue development have been discovered in the wing disc. In the larval organ, morphogens divide the wing disc pouch into regions that define the differentiation state of cells and coordinate morphogenesis. Morphogen signals that are important for wing disc development include Hh and Dpp, which define the anterior/posterior axis. Wg patterns the dorsal/ventral axis. Widely available genetic tools and simple geometry make the Drosophila wing disc a powerful platform to decode Ca2+ signaling at the systems level (Brodskiy, 2019).

This study has developed an image-processing pipeline to quantitatively investigate the relationships between Ca2+ signaling and organ size. First key components of the core Ca2+ signaling pathway, termed elsewhere as the 'Ca2+ signaling toolkit', were genetically inhibited to define the range of adult wing phenotypes. Next, a dose-response experiment of fly extract (FEX) to order the specific classes of Ca2+ signaling based on the relative concentration of agonist-based stimulation. The term 'Ca2+ signaling activity' is used to collectively refer to these four Ca2+ signaling classes. How these classes of Ca2+ signaling correlate with disc age and size, both in vivo and ex vivo, was investigated. FEX was shown to stimulates Ca2+ through Gαq/phospholipase C (PLC) β signaling through genetic perturbation experiments. Advanced image-analysis tools were developed to handle the large data sets to extract quantitative Ca2+ dynamics measurements. Using this image-analysis pipeline, a negative power-law correlation between integrated Ca2+ signaling activity and wing disc pouch size was identified. How the genetic state of the tissue modulates Ca2+ signaling dynamics was examined through genetic perturbation. Ca2+ signaling activity responds to perturbations that impact the morphogenic state of the tissue, resulting in deviations from the quantitative correlation curve between Ca2+ signaling activity and developmental progression. Together, these trends indicate that Ca2+ signaling provides a biochemical readout of organ size. The results suggest Ca2+ could be involved in modulating cell proliferation activity during larval growth. In sum, this study provides significant evidence that Ca2+ signaling contributes to intercellular consensus-building during organ development. This research paves the road of revealing the quantitative and mechanistic regulation of organ development by Ca2+ signaling in future studies (Brodskiy, 2019).

This work has established multiple inputs and outputs for the calcium bow-tie network during wing development. Four classes of spontaneous Ca2+ signaling activity during in vivo development in the wing disc were identified: (1) cellular Ca2+ spikes; (2) ICTs; (3) intercellular Ca2+ waves (ICWs), and (4) elevated Ca2+ fluttering. Increasing Gαq-mediated signaling with increasing concentrations of FEX leads to a natural progression from low (class 1 and 2) to higher levels of Ca2+ signaling responses (classes 3 and 4). These four signaling classes occur both ex vivo and in vivo. Importantly, it was found that multiple classes of Ca2+ activity occur and are a regulated phenomenon in vivo. These findings contradict previous suggestions that ICWs may be an ex vivo artifact. Future work is needed to specify the full set of specific RTKs, GPCRs, and morphogens that modulate Ca2+ dynamics in vivo (Brodskiy, 2019).

A negative correlation was demonstrated between the stimulated Ca2+ signaling responses and the wing disc age and size for third instar larvae. Overall, these observations provide evidence for Ca2+ signaling as a readout for overall organ size in the developing wing and a regulator of cellular processes during larval wing development. Through linear regression analysis, a negative power-law correlation was demonstrated between larval age/pouch size and integrated Ca2+ signaling activity. These findings suggest that Ca2+ signaling decreases during the latter stages of larval wing disc growth. The maximal log-likelihood estimation of the power exponent occurred when the estimate had a value of -0.8 ± 0.5. This is consistent with many allometric scaling relationships observed in biological systems wherein quarter-power scaling frequently occurs. For example, quarter-power scaling has been observed in the organism metabolic rate, lifespan, growth rate, heart rate, and the concentrations of metabolic enzymes. A -0.75-scaling relationship is consistent, near the maximal log-likelihood estimation, and within the 95% confidence interval of the optimal exponent power. This, in turn, may indicate that the underlying metabolic trajectory of organ growth influences the level of agonist-stimulated calcium signaling activity (Brodskiy, 2019).

Further, anterior-posterior patterning of Ca2+ signaling activity amplitudes was observed in the wing disc. The amplitude is higher in the posterior than in the anterior compartment. As these compartments have been shown to grow at different rates, this result is consistent with the correlation between Ca2+ signaling activity and the growth state of each compartment. There are several possible explanations for why there is an absence of amplitude patterning between anterior and posterior compartments for larger discs in Hh (smoRNAi) or Dpp (dppRNAi) signaling-perturbed discs. First, Hh and Dpp signaling may be directly responsible for patterning the anterior-posterior amplitude difference, perhaps through regulation of cAMP levels. Second, this may be because the sizes of anterior and posterior compartments are similar under those conditions. Identifying the cause of this phenomenon may yield insight into additional patterning roles for Ca2+ signaling in wing development, including the pupal stages when vein differentiation occurs. Recently, Ca2+ signaling has been connected to proper Hh signaling in zebrafish embryo. This work suggests that Ca2+ signaling may generally be involved in modulating morphogenesis mediated by Hh signaling and other morphogen pathways (Brodskiy, 2019).

Future work is needed to identify specific mechanisms connecting signal transduction inputs to phenotypic outputs. In a recent article, cellular Ca2+ spikes were found to correlate with secretion of Dpp, a key regulator of wing disc size and tissue patterning (Dahal, 2017). It is speculated that local cellular spike activity might be connected to the positive regulation of organ growth. smo^RNAi and dpp^RNAi leads to smaller wing discs and higher integrated Ca2+ intensity when Ca2+ signaling is stimulated by agonists. The data points from growth-reducing perturbation (smo^RNAi and dpp^RNAi) lie above the negative correlation curve of the control wing discs. In contrast, genetic perturbations leading to more growth (tkv^CA and Pten^RNAi) result in reduced Ca2+ signaling responses when stimulated (Brodskiy, 2019).

These results imply a common underlying regulatory mechanism. As a launching point for future work, a simple model is proposed that explains the results reported in this study. First, the experiments demonstrate that FEX stimulates Gαq/PLCβ activity, which results in IP3 generation and IP3-regulated Ca2+ release. Sufficient IP3 production may lead to phosphatidylinositol bisphosphate (PIP2) substrate depletion. In other systems, PIP2 is often rate limiting for Ca2+ signaling. PIP2 is also required for phosphatidylinositol trisphosphate generation, which then stimulates cell growth through PI3K/AKT signaling. It follows that reduced PI3K signaling resulting from decreased growth stimulation (indirectly through inhibition of Hh or Dpp signaling in these experiments) will lead to higher PIP2 substrate availability and a stronger Ca2+ response. Conversely, decreased PIP2 availability through the inhibition of PTEN (which converts phosphatidylinositol trisphosphate to PIP2) or through constitutively active Dpp signaling would lead to attenuated Ca2+ signaling responses when stimulated by FEX (Brodskiy, 2019).

This interpretation of the data provides a generalizable and testable hypothesis for future work: if PIP2 levels are more abundant (reduced PI3K signaling and growth activity), more IP3 can be generated, resulting in more Ca2+ signaling for a given agonist response. If PIP2 substrate levels are limiting (as results when PTEN is inhibited or more growth is stimulated), less IP3-stimulated Ca2+ signaling can occur. This hypothetical model would predict that sufficient overexpression of Gαq could lead to reduced organ growth by depleting PIP2 substrate availability for growth stimulation. Future work may identify such relationships across biological systems because all of these molecular components are present in most eukaryotic cells. This hypothetical model is termed the 'Ca2+ shunt' hypothesis of growth control (Brodskiy, 2019).

Ca2+ signaling likely modulates other aspects of growth control during larval development. Ca2+ may integrate signals about the availability of nutrients or about mechanical constraints on the tissue. Several known effectors of size control pathways, such as kibra, a regulator of Hippo signaling, have Ca2+ signaling binding domains as annotated by InterPro (Brodskiy, 2019).

Additionally, this work motivates new questions regarding how gap-junction communication, and by extension, membrane voltage, influences the overall control of organ size. A decrease in cell-cell gap-junction permeability occurs over the course of wing development. As gap junctions become less permeable, Ca2+ and IP3 diffuse a shorter distance before being reabsorbed into the endoplasmic reticulum or decaying, respectively. This would explain the transition from ICWs to ICTs and spikes as well as why amplitude is spatially patterned in large discs as development proceeds. Other studies have also implicated gap-junction communication in organ size control. For example, Inx2^RNAi suppresses growth in the developing eye disc. Connexin43 mutants that disrupt gap-junction communication lead to short fin in zebrafish. Gap-junction communication also regulates cell differentiation as Inx2-mediated Ca2+ flux is essential for border cell specification in Drosophila. These results suggest that part of the role of gap-junction communication in regulating size and influencing tissue patterning is through the regulation of Ca2+ transients across the tissue. Taken together, it is therefore likely that the role of Ca2+ signaling in wing growth is conserved in other organs (Brodskiy, 2019).

This phenotypic analysis provides additional evidence that the Ca2+ signaling module contributes to modulating wing morphogenesis during pupal development and vein cell differentiation. It should be noted that the crossvein defects suggest that these veins are particularly sensitive to levels of morphogen signaling, including Dpp. In particular, Dpp signaling has been linked to Ca2+ signaling in the developing wing. Perturbing Ca2+ signaling may also be enhancing the crossvein defects that can occur in the MS1096-Gal4 line, which impacts Beadex gene function. Future work will need to investigate the mechanisms leading to wing shape and vein differentiation defects, which are specified during pupal development (Brodskiy, 2019).

Computational modeling is essential for future efforts to decode the regulation and function of Ca2+ signaling. Understanding the specific roles of Ca2+ signaling in organ development will require computational models that couple multiple signals of Ca2+ signaling across multiple spatiotemporal scales. For example, computational models are particularly useful at the systems level to understand mechanisms for the coupled transport of Ca2+ and wound healing. Regarding this study, these findings that the integrated Ca2+ intensity decreases with development is consistent with a model from the neocortex being applied to the wing disc, in which Ca2+ signaling dynamics are weakly coupled with cell-cycle progression and can influence cell-cycle synchrony with neighbors. In sum, this effort demonstrates key roles of Ca2+ signaling as a signal integrator in epithelial growth and morphogenesis (Brodskiy, 2019).

Gαq and Phospholipase Cβ signaling regulate nociceptor sensitivity in Drosophila melanogaster larvae

Drosophila melanogaster larvae detect noxious thermal and mechanical stimuli in their environment using polymodal nociceptor neurons whose dendrites tile the larval body wall. Activation of these nociceptors by potentially tissue-damaging stimuli elicits a stereotyped escape locomotion response. The cellular and molecular mechanisms that regulate nociceptor function are increasingly well understood, but gaps remain in knowledge of the broad mechanisms that control nociceptor sensitivity. This study used cell-specific knockdown and overexpression to show that nociceptor sensitivity to noxious thermal and mechanical stimuli is correlated with levels of Gαq and phospholipase Cβ (NorpA) signaling. Genetic manipulation of these signaling mechanisms does not result in changes in nociceptor morphology, suggesting that changes in nociceptor function do not arise from changes in nociceptor development, but instead from changes in nociceptor activity. These results demonstrate roles for Gαq and phospholipase Cβ signaling in facilitating the basal sensitivity of the larval nociceptors to noxious thermal and mechanical stimuli and suggest future studies to investigate how these signaling mechanisms may participate in neuromodulation of sensory function (Herman, 2018).

This study has demonstrated that nociceptor-specific knockdown of Gαq causes Drosophila larvae to respond to noxious thermal stimuli with longer response latencies and to noxious mechanical stimuli with reduced frequency. These results suggest a modest role for Gαq in positively regulating nociceptor sensitivity. This interpretation is supported by the observation that Gαq overexpression in the nociceptors causes faster and more frequent responses to thermal and mechanical stimuli respectively. NorpA is an effector of Gαq in phototransduction and thermotransduction in Drosophila. Given the observation that Gαq and norpA knockdown larvae share similar defective nociception defects, it is hypothesized that NorpA also acts as a Gαq effector in larval nociceptor neurons to regulate sensitivity to noxious thermal and mechanical stimuli. This hypothesis could be formally tested by epistasis experiments using Gαq gain-of-function flies. Loss of Gαq and NorpA function in the nociceptors does not result in gross defects in dendrite development or arborization, suggesting that nociception defects do not arise from defects in multidendritic neuron development. Taken together, these results demonstrate that larval nociception is a promising experimental paradigm for further study of the role of Gαq and NorpA signaling in modulation of nociceptor sensitivity (Herman, 2018).

The proposed roles for Gαq and NorpA signaling in regulating nociceptor sensitivity are largely, but not perfectly, supported by the experimental data. Two of three UAS-Gαq-RNAi transgenes and one of two UAS-norpA-RNAi transgenes produce thermal nociception defects when expressed in the larval nociceptors. Roles for Gαq and NorpA in positive regulation of thermal nociception are further supported, however, by the observations that nociceptor-specific Gαq overexpression produces a hypersensitive thermal nociception phenotype and that a norpA loss-of-function mutant shows a hyposensitive thermal nociception phenotype, as would be predicted by the cell-specific RNAi data. Roles for Gαq and NorpA signaling in mechanical nociception are strongly supported by two of three UAS-Gαq-RNAi lines and one of two UAS-norpA-RNAi lines. The remaining UAS-Gαq-RNAi line and UAS-norpA-RNAi line provide only partial support for roles in mechanical nociception, as the knockdown phenotype produced by each is significantly different from only one of the GAL4-only or UAS-RNAi-only controls. It is also noted that the effects of Gαq and norpA knockdown are relatively modest. These observations might be explained by the vast signal amplification potential of Gαq and NorpA signaling (Hardie, 2002). It is possible that modest residual levels of Gαq and NorpA signaling following knockdown could support unexpectedly high levels of second messenger production. In this scenario, only the strongest UAS-RNAi lines would be expected to produce a significant change in nociceptor sensitivity. This hypothesis could be investigated by further analysis of Gαq and norpA mutants as well as antibody staining experiments to quantify the effects of knockdown on Gαq and NorpA protein levels (Herman, 2018).

While it was hypothesized that NorpA is the major effector of Gαq in larval nociceptors, it is also possible that Gαq signals through other effectors aside from NorpA in the mdIV neurons. In some systems, the Trio rhoGEF is directly activated by Gαq signaling, and recent studies have demonstrated roles for Trio in regulating mdIV morphogenesis. However, Trio loss-of-function larvae do not display mechanical nociception defects, suggesting that Trio may not be an effector of Gαq for the modulation of nociceptor sensitivity. However, more targeted epistasis experiments may be needed to formally investigate this possibility (Herman, 2018).

The downstream effectors of Gαq and NorpA signaling in the mdIV neurons remain to be determined. dTRPA1 ion channel function is required in larval nociceptors for thermal and mechanical nociception, and dTRPA1 is known to be activated downstream of NorpA in Drosophila chemosensory neurons and to support thermotaxis behavior. Thus it is reasonable to hypothesize that dTRPA1 in the mdIV neurons is activated less effectively in the absence of Gαq and NorpA signaling, leading to decreased nociceptor sensitivity. One possible mechanism for the activation of dTRPA1 downstream of NorpA is that dTRPA1 may be activated by depletion of PIP2. With this in mind, it is important to note that PIP2 affects the activity of many types of ion channels. PIP2 hydrolysis by NorpA may regulate the function of any number of ion channels that control nociceptor sensitivity, including voltage-gated calcium channels and small-conductance potassium channels. It also cannot be ruled out that the generation of IP3 and DAG second messengers by PIP2 hydrolysis is the principal mechanism by which NorpA regulates nociceptor sensitivity, as these mechanisms are well known to mediate store-operated calcium release, activation of protein kinases, and regulation of the neurotransmitter release machinery (Herman, 2018).

The observed role of Gαq and NorpA in nociception suggests the existence of a GPCR signaling mechanism that activates this signaling pathway under basal conditions (i.e., in the absence of tissue damage or sensitization). The identity of this GPCR or these GPCRs remains to be discovered. Activation of sNPF receptors on the mdIV neurons facilitates mechanical nociception, presumably via a heterotrimeric G protein signaling mechanism. It is possible that loss of Gαq or NorpA signaling prevents this sNPF facilitation of mechanical nociception, thus producing a defective mechanical nociception phenotype. However, signaling through sNPF receptors was found to facilitate mechanical nociception specifically, while the results suggest that Gαq and NorpA signaling facilitates both thermal and mechanical nociception. Thus, it is hypothesized that additional GPCRs exist to facilitate thermal nociception through heterotrimeric G protein signaling under basal conditions. The identities of these putative receptors and their ligands are a promising subject of further study (Herman, 2018).

These studies demonstrate that Gαq and PLCβ signaling acts in the nociceptors of Drosophila larvae to support wild-type sensitivity to noxious thermal and mechanical stimuli. This conclusion is supported by the fact that nociceptor-specific RNAi knockdown of either Gαq or norpA produces hyposensitive thermal and mechanical nociception phenotypes. Additionally, overexpression of Gαq causes thermal and mechanical hypersensitivity. The behavioral phenotypes observed following RNAi knockdown of Gαq or norpA are unlikely to arise from deficits in sensory neuron morphogenesis, as knockdown animals were found to have dendrites with similar length and branching to wild-type animals (Herman, 2018).

Dopamine receptor DAMB signals via Gq to mediate forgetting in Drosophila

Prior studies have shown that aversive olfactory memory is acquired by dopamine acting on a specific receptor, dDA1, expressed by mushroom body neurons. Active forgetting is mediated by dopamine acting on another receptor, Damb, expressed by the same neurons. Surprisingly, prior studies have shown that both receptors stimulate cyclic AMP (cAMP) accumulation, presenting an enigma of how mushroom body neurons distinguish between acquisition and forgetting signals. This study surveyed the spectrum of G protein coupling of dDA1 and Damb, and it was confirmed that both receptors can couple to Gs to stimulate cAMP synthesis. However, the Damb receptor uniquely activates Gq to mobilize Ca(2+) signaling with greater efficiency and dopamine sensitivity. The knockdown of Gαq with RNAi in the mushroom bodies inhibits forgetting but has no effect on acquisition. These findings identify a Damb/Gq-signaling pathway that stimulates forgetting and resolves the opposing effects of dopamine on acquisition and forgetting (Himmelreich, 2017).

This study provides biochemical and behavioral evidence that the Drosophila DA receptor Damb couples preferentially to Gαq to mediate signaling by Damb for active forgetting. This conclusion offers an interesting contrast to the role of the dDA1 receptor in MBns for acquisition, and it resolves the issue of how MBns distinguish DA-mediated instructions to acquire memory versus those to forget. Prior studies had classified both dDA1 and Damb as cAMP-stimulating receptors, similar to mammalian D1/D5 DA receptors that work through Gαs/olf. The results extend prior studies of dDA1 by examining coupling of this receptor with multiple heterotrimeric G proteins to show that the receptor strongly and preferentially couples to Gs proteins. This affirms the receptor's role in the acquisition of memory, consistent with the tight link between acquisition and cAMP signaling. This study found that the Damb receptor can weakly couple to Gs proteins but preferentially engages Gq to trigger the Ca2+-signaling pathway, a feature not displayed by dDA1. Comparing the two Gαq paralogs of Drosophila (G and D) with a human ortholog shows that Drosophila GαqG and human Gαq share a conserved C terminus, essential for selective coupling to GPCRs, but quite distinct in sequence compared to the GαqD C terminus. Since GαqD is a photoreceptor-selective G protein that couples with rhodopsin, it is proposed that GαqG is the isoform that relays Damb's signals to spur forgetting (Himmelreich, 2017).

It is envisioned that memory acquisition triggered by strong DA release from electric shock pulses used for aversive conditioning drives both cAMP and Ca2+ signaling through dDA1 and Damb receptors in the MBns. Forgetting occurs from weaker DA release after the acquisition through restricted Damb/Gαq/Ca2+ signaling in the MBns. The coupling of Damb to Gs at high DA concentrations also explains why Damb mutants have a slight acquisition defect after training with a large number of shocks. Although the model allows the assignment of acquisition and forgetting to two distinct intracellular signaling pathways, it does not preclude the possibility that other differences in signaling distinguish acquisition from forgetting. These include the possible use of different presynaptic signals, such as a co-neurotransmitter released only during acquisition or forgetting (Himmelreich, 2017).

G-protein alphaq gene expression plays a role in alcohol tolerance in Drosophila melanogaster

Ethanol is a psychoactive substance causing both short- and long-term behavioural changes in humans and animal models. This study used the fruit fly Drosophila melanogaster to investigate the effect of ethanol exposure on the expression of the Gαq protein subunit. Repetitive exposure to ethanol causes a reduction in sensitivity (tolerance) to ethanol, which was measured as the time for 50% of a set of flies to become sedated after exposure to ethanol (ST50). It was demonstrated that the same treatment that induces an increase in ST50 over consecutive days (tolerance) also causes a decrease in Gαq protein subunit expression at both the messenger RNA and protein level. To identify whether there may be a causal relationship between these two outcomes, strains of flies were developed in which Gαq messenger RNA expression is suppressed in a time- and tissue-specific manner. In these flies, the sensitivity to ethanol and the development of tolerance are altered. This work further supports the value of Drosophila as a model to dissect the molecular mechanisms of the behavioural response to alcohol and identifies G proteins as potentially important regulatory targets for alcohol use disorders (Aleyakpo, 2019).

A single residue mutation in the Gαq subunit of the G protein complex causes blindness in Drosophila

Heterotrimeric G proteins play central roles in many signaling pathways, including the phototransduction cascade in animals. However, the degree of involvement of the G protein subunit Gαq is not clear since animals with strong loss of function mutations previously reported remain responsive to light stimuli. This study recovered a new allele of Gαq in Drosophila that abolishes light response in a conventional ERG assay, and reduces sensitivity in whole-cell recordings of dissociated cells by at least 5 orders of magnitude. In addition, mutant eyes demonstrate a rapid rate of degeneration in the presence of light. The new allele is likely the strongest hypomorph described to date. Interestingly, the mutant protein is produced in the eyes but carries a single amino acid change of a conserved hydrophobic residue that has been assigned to the interface of interaction between Gαq and its downstream effector PLC. This study thus uncovered possibly the first point mutation that specifically affects this interaction in vivo (Cao, 2017).

Epithelial microRNA-9a regulates dendrite growth through Fmi-Gq signaling in Drosophila sensory neurons

microRNA-9 (miR-9) is highly expressed in the nervous system across species and plays essential roles in neurogenesis and axon growth; however, little is known about the mechanisms that link miR-9a with dendrite growth. Using an in vivo model of Drosophila class I dendrite arborization (da) neurons, miR-9a, a Drosophila homolog of mammalian miR-9a, was shown to downregulate the cadherin protein Flamingo (Fmi) thereby attenuating dendrite development in a non-cell autonomous manner. In miR-9a knockout mutants, the dendrite length of a sensory neuron ddaE was significantly increased. Intriguingly, miR-9a is specifically expressed in epithelial cells but not in neurons, thus the expression of epithelial but not neuronal Fmi is greatly elevated in miR-9a mutants. In contrast, overexpression of Fmi in the neuron resulted in a reduction in dendrite growth, suggesting that neuronal Fmi plays a suppressive role in dendrite growth, and that increased epithelial Fmi might promote dendrite growth by competitively binding to neuronal Fmi. Fmi has been proposed as a G protein-coupled receptor (GPCR). Neuronal G protein Gαq (Gq), but not Go, may function downstream of Fmi to negatively regulate dendrite growth. Taken together, these results reveal a novel function of miR-9a in dendrite morphogenesis. Moreover, it is suggested that Gq might mediate the intercellular signal of Fmi in neurons to suppress dendrite growth. These findings provide novel insights into the complex regulatory mechanisms of microRNAs in dendrite development, and further reveal the interplay between the different components of Fmi, functioning in cadherin adhesion and GPCR signalling (Wang, 2015).

Gαq, Ggamma1 and Plc21C control Drosophila body fat storage

Adaptive mobilization of body fat is essential for energy homeostasis in animals. In insects, the adipokinetic hormone (Akh) systemically controls body fat mobilization. Biochemical evidence supports that Akh signals via a G protein-coupled receptor (GPCR) called Akh receptor (AkhR) using cyclic-AMP (cAMP) and Ca(2+) second messengers to induce storage lipid release from fat body cells. Recently, genetic evidence has been provided that the intracellular calcium [iCa(2+)] level in fat storage cells controls adiposity in Drosophila. However, little is known about the genes which mediate Akh signalling downstream of the AkhR to regulate changes in iCa(2+). This study used thermogenetics to provide in vivo evidence that the GPCR signal transducers G protein alpha q subunit (Gαq), G protein gamma1 (Ggamma1) and Phospholipase C at 21C (Plc21C) control cellular and organismal fat storage in Drosophila. Transgenic modulation of Gαq, Ggamma1 and Plc21C affected the iCa(2+) of fat body cells and the expression profile of the lipid metabolism effector genes midway and brummer resulting in severely obese or lean flies. Moreover, functional impairment of Gαq, Ggamma1 and Plc21C antagonised Akh-induced fat depletion. This study characterizes Gαq, Ggamma1 and Plc21C as anti-obesity genes and supports the model that Akh employs the Gαq/Ggamma1/Plc21C module of iCa(2+) control to regulate lipid mobilization in adult Drosophila (Baumbach, 2014).

Regulation of dual oxidase activity by the Gαq-phospholipase Cβ-Ca2+ pathway in Drosophila gut immunity

All metazoan guts are in constant contact with diverse food-borne microorganisms. The signaling mechanisms by which the host regulates gut-microbe interactions, however, are not yet clear. This study shows that phospholipase C-β (PLCβ) signaling modulates dual oxidase (DUOX) activity to produce microbicidal reactive oxygen species (ROS) essential for normal host survival. Gut-microbe contact rapidly activates PLCβ through Gαq, which in turn mobilizes intracellular Ca²⁺ through inositol 1,4,5-trisphosphate generation for DUOX-dependent ROS production. PLCβ mutant flies have a short life span due to the uncontrolled propagation of an essential nutritional microbe, Saccharomyces cerevisiae, in the gut. Gut-specific reintroduction of the PLCβ restores efficient DUOX-dependent microbe-eliminating capacity and normal host survival. These results demonstrate that the Gαq-PLCβ-Ca²⁺-DUOX-ROS signaling pathway acts as a bona fide first line of defense that enables gut epithelia to dynamically control yeast during the Drosophila life cycle (Ha, 2009).

All organisms are in constant contact with a large number of different types of microbes. This is especially true in the case of the gut epithelia, which control life-threatening pathogens as well as food-borne microbes. In addition to this microbe-eliminating capacity, gut epithelia also need to protect normal commensal microbes which are in a mutually beneficial relationship. Therefore, gut epithelia must be equipped to differentially operate innate immunity in order to efficiently eliminate life-threatening microbes while protecting beneficial microbes. Studies using Drosophila as a genetic model have greatly enhanced understanding of the microbe-controlling mucosal immune strategy in gut epithelia. Previous studies in a gut infection model using oral ingestion of pathogens revealed that the redox system has an essential role in host survival by generating microbicidal effectors such as reactive oxygen species (ROS). In this redox system, dual oxidase (DUOX), a member of the nicotinamide adenine dinucleotide phosphate (NADP)H oxidase family, is responsible for the production of ROS in response to gut infection. Following microbe-induced ROS generation, ROS elimination is assured by immune-regulated catalase (IRC), thereby protecting the host from excessive oxidative stress. In addition to the redox system, the mucosal immune deficiency (IMD)/NF-κB signaling pathway, which leads to the de novo synthesis of microbicidal effector molecules such as antimicrobial peptides (AMPs), has an essential complementary role to the redox system when the host encounters ROS-resistant pathogenic microbes. These findings indicate that the different spectra of microbicidal activity encompassed by ROS and AMPs may provide the versatility necessary for Drosophila gut immunity to control microbial infections. Furthermore, in the absence of gut infection, a selective repression of IMD/NF-κB-dependent AMPs is mediated by the homeobox gene Caudal, which is required for protection of the resident commensal community and host health. Therefore, fine-tuning of different gut immune systems appears to be essential for both the elimination of pathogens and the preservation of commensal flora (Ha, 2009).

Most studies evaluating gut immunity have been performed in an oral infection model in which the pathogens are ingested. However, the gut epithelia constitute the interface between the host and the microbial environment; therefore, it is likely that animals in nature have already been subjected to continuous microbial contact, even in the absence of oral infection. Thus, it is essential to determine the mechanism by which this natural and continuous microbial interaction produces ROS at a tightly controlled, yet adequate level that allows for healthy gut-microbe interactions and gut homeostasis, because deregulated generation of ROS is believed to lead to a pathophysiologic condition in the gut epithelia. Although the DUOX system is of central importance in gut immunity, the signaling pathway(s) by which gut epithelia regulate DUOX-dependent microbicidal ROS generation are poorly understood (Ha, 2009).

Drosophila feed on microbes, and one of their most essential microbial food sources is baker's yeast, Saccharomyces cerevisiae. As early as 1930, yeast was discovered to be an essential nutrient source for Drosophila and is now used as a major ingredient in standard laboratory Drosophila food recipes. Further, Drosophila-Saccharomyces interaction occurs in wild-captured Drosophila, which suggests that this interaction is an evolutionarily ancient natural phenomenon. Although many studies have investigated the effect of yeast on Drosophila metabolism and aging, very few works have been reported on the effect of yeast in terms of the host immunity. Specifically, it has previously been shown that dietary yeast contributes to the cellular immune responsiveness of Drosophila against a larval parasitoid, Leptopilina boulardi. However, the relationship between yeast and Drosophila gut immunity during the normal life cycle has never been closely examined. Therefore, in this study, a Drosophila-yeast model was used to investigate the intracellular signaling pathway by which the host mounts mucosal antimicrobial immunity, as well as the in vivo value of this pathway in the host's natural life. Through biochemical and genetic analyses, this study revealed that the Gαq-mediated phospholipase C-β (PLCβ) pathway is involved in the routine control of dietary yeast in the Drosophila gut. PLCβ is dynamically activated in the presence of ingested yeast and subsequently mobilizes the intracellular Ca²⁺ to produce ROS in a DUOX-dependent manner. The presence of all of these signaling components of the Gαq-PLCβ-Ca²⁺-DUOX-ROS pathway in the gut is essential to ensure routine control of dietary yeast and host fitness, highlighting the importance of this immune signaling as a bona fide first line of defense in Drosophila (Ha, 2009).

This study demonstrates that the Gαq-PLCβ-Ca²⁺ signaling pathway controls the mucosal gut epithelial defense system through DUOX-dependent ROS generation, which is responsible for routine microbial interactions in the gut epithelia in the absence of infection. The PLCβ pathway impacts a wide variety of biological processes through the generation of a lipid-derived second messenger. In this process, the hydrolysis of a minor membrane phospholipid, phosphatidylinositol 4,5-bisphosphate, by PLCβ generates two intracellular messengers, IP₃ and diacylglycerol. This process is one of the earliest events through which more than 100 extracellular signaling molecules regulate functions in their target cells. It has been shown that Gαq-PLCβ signaling is essential for the activation of the phototransduction cascade in Drosophila. This study revealed a physiological role of PLCβ wherein it is involved in the regulation of DUOX enzymatic activity, which leads to the generation of microbicidal ROS in the mucosal epithelia (Ha, 2009).

PLCβ signaling is very rapid, with only a few seconds necessary to activate Ca²⁺ release and ROS production. This rapid response may be advantageous for the host and may be the mechanism by which dynamic and routine control of microbes in the gut epithelia is achieved. Because the gut is in continuous contact with microbes such as dietary microorganisms, it is conceivable that under normal conditions routine microbial contact dynamically induces a certain level of basal Gαq-PLCβ activity that varies depending on the local microbe concentration. This basal Gαq-PLCβ-DUOX activity seems to be sufficient for host survival. In such conditions of low bacterial burden, NF-κB-dependent AMP expression is known to be largely repressed by Caudal repressor for the preservation of commensal microbiota (Ryu, 2008). However, in the case of high bacterial burden (e.g., gut infection condition), the DUOX-ROS system would be strongly activated for full microbicidal activity. Furthermore, all of the flies that contained impaired signaling potentials for the Gαq-PLCβ-Ca²⁺-DUOX pathway were totally intact following septic injury but short-lived under natural rearing conditions or under gut infection conditions, indicating that the mucosal immune pathway is distinct from the systemic immune pathway (Ha, 2009).

It is not clear how Gαq- and PLCβ-induced Ca²⁺ modulates DUOX enzymatic activity. Because the DUOX lacking Ca²⁺-binding EF hand domains is unable to rescue the DUOX-RNAi flies, it is plausible that Ca²⁺ directly modulates the enzymatic activity of DUOX through binding to the EF hand domains (Ha, 2009).

It is also important to determine what pathogen-associated molecular patterns (PAMPs) are responsible for the activation of PLCβ signaling. In Drosophila, peptidoglycan and β-1,3-glucan are the only two PAMPs known to induce the NF-κB signaling pathway in the systemic immunity. The results showed that neither peptidoglycan nor β-1,3-glucan was able to induce ROS in S2 cells, which suggests that a previously uncharacterized type(s) of PAMP is involved in the mucosal immunity. Because the Gαq protein acts as an upstream signaling component of the PLCβ-Ca²⁺ pathway, a microbe-derived ligand capable of activating G protein coupled receptor(s) and/or Gαq protein may be the best candidate for the Gαq-PLCβ-Ca²⁺-DUOX signaling pathway. Given the broad spectrum of microbes that activate the response, it remains possible that the unknown upstream sensors resemble a stress response more than a PAMP response. Elucidation of the molecular nature of such agonists will greatly enhance understanding of bacteria-modulated redox signaling in the gut epithelia. In conclusion, this study demonstrates that mucosal epithelia have evolved an innate immune strategy, which is functionally distinct from the NF-κB-dependent systemic innate immune system. The rapid Gαq-PLCβ-Ca²⁺-DUOX signaling is adapted to the routine and dynamic control of gut-associated microbes and may impact the long-term physiology of the intestine and host fitness (Ha, 2009).

Altered levels of Gq activity modulate axonal pathfinding in Drosophila.

A majority of neurons that form the ventral nerve cord send out long axons that cross the midline through anterior or posterior commissures. A smaller fraction extend longitudinally and never cross the midline. The decision to cross the midline is governed by a balance of attractive and repulsive signals. This study has explored the role of a G-protein, Gαq, in altering this balance in Drosophila. Dgq was originally identified from a head cDNA library as a homolog of mammalian Gαq. Initial functional characterization had suggested that it was a visual-specific G-protein essential for Drosophila visual transduction. A splice variant of Gαq, dgqalpha3, is expressed in early axonal growth cones, which go to form the commissures in the Drosophila embryonic CNS. Misexpression of a gain-of-function transgene of dgqalpha3 (AcGq3) leads to ectopic midline crossing. Analysis of the AcGq3 phenotype in roundabout and frazzled mutants shows that AcGq3 function is antagonistic to Robo signaling and requires Frazzled to promote ectopic midline crossing. These results show that a heterotrimeric G-protein can affect the balance of attractive versus repulsive cues in the growth cone and that it can function as a component of signaling pathways that regulate axonal pathfinding (Ratnaparkhi, 2002).

cDNA clones corresponding to the dgq gene were isolated in library screens using a fragment from the eye-specific splice variant dgqalpha1. Libraries derived from either embryo or appendage RNAs were screened and dgq-positive cDNA clones were analyzed by restriction digests and PCR. Three classes of cDNA clones were obtained. In the region of the open-reading frame, one of these classes corresponds to a splice variant transcript of the dgq gene, dgqalpha3, known to be expressed in several adult tissues. This class was isolated repeatedly from the embryo cDNA library, as judged by extensive PCR analysis. dgqalpha3-specific transcripts are present in poly(A⁺) RNA extracted from heads, appendages, male and female bodies, and embryos. Another class of cDNA clones was found only in the appendage library and appeared identical to the adult visual Gαq splice form (dgqalpha1) (Ratnaparkhi, 2002).

The presence of the Dgqalpha3 protein in Drosophila embryos was examined by Western blot analysis of embryo extracts. The antiserum used recognizes the C-terminal end of the mammalian Gq protein. In Drosophila Gq this C-terminal sequence is conserved only in the Dgqalpha3 form. The results obtained indicate that a 39 kDa band, corresponding to the predicted size of the Dgqalpha3 protein, is present in embryos throughout development from as early as 0-8 hr (Ratnaparkhi, 2002).

Presence of dgqalpha3 RNA and protein in embryos suggests an involvement of the dgq gene in Drosophila development. The expression pattern of dgqalpha3 during embryonic development was examined by in situ hybridization with a dgqalpha3 splice variant-specific probe. Although dgqalpha3 RNA is present in earlier stages, tissue-specific expression of dgqalpha3 is first seen in the brain and ventral nerve cord at stage 13. This expression persists until late in development, where in addition, strong expression is seen in an anterior sense organ. This organ corresponds in position to the Bolwig's organ or the larval eye (Ratnaparkhi, 2002).

Expression of Dgqalpha3 during development of the embryonic nervous system was further confirmed by immunohistochemical staining of wild-type embryos with the Gq antiserum. The first indication of Dgqalpha3 expression in the CNS is at early stage 12. This is also the stage at which the pioneer neurons begin formation of axon pathways that give rise to the typical ladder-like appearance of the embryonic CNS, consisting of longitudinal tracts and anterior and posterior commissures that can be visualized with the axonal marker mAb BP102. A similar pattern of expression of anti-Gq and the axonal marker mAb BP102 at early stage 12 suggests that Dgqalpha3 is expressed in the pioneer growth cones that give rise to the commissures. At later stages of development Dgqalpha3 protein expression increases in the axonal tracts of the CNS. In addition, Dgqalpha3 expression was visible in the midgut epithelium at stages 12 (Ratnaparkhi, 2002).

Axonal guidance in the Drosophila CNS requires the interpretation of both attractive and repulsive cues, generated by cells that lie in the midline. The expression pattern of Dgqalpha3 protein suggested that it might be required in early growth cones for the interpretation of these cues. To address this possibility, it was essential to alter Gαq signaling in a tissue and cell-specific manner. Therefore, transgenic strains were created with a dominant active form of Dgqalpha3, in which a glutamine residue at position 203 was mutated to a leucine. The mutation was made based on previous studies on dominant active forms of Gαq from mammalian cells and Drosophila. As controls, transgenic lines carrying the wild-type form of Dgqalpha3 were created. Both activated dgqalpha3 (UAS-AcGq3) and dgqalpha3 (UAS-Gq3) cDNAs were placed under the control of the GAL4-inducible UAS promoter that would allow tissue and cell-specific expression. Initially, the C155-GAL4 line, which expresses in all postmitotic neurons, was used in order to study the effect of UAS-AcGq3 expression on axonal development. When stained with mAb BP102, the CNS of C155-GAL4;UAS-Gq3 embryos looked normal. In embryos expressing AcGq3, the pattern of the CNS appeared mildly deranged in that the commissures were thicker, and the neuropil region was broader than usual. More significant differences between the two genotypes were obvious when a monoclonal antibody against Fasciclin II (mAb 1D4) was used. At stage 13, anti-Fasciclin II (anti-Fas II) marks the pioneer axons that go to form the first longitudinal axon pathway, which by stage 16, defasciculates to form three distinct fascicles. These axons project ipsilaterally and do not cross the midline. In embryos of the genotype C155-GAL4;UAS-Gq3, this projection pattern was identical to wild-type embryos, indicating that overexpression of Dgqalpha3 has no effect on Fas II-expressing axons. However, in embryos expressing AcGq3, Fas II-positive axons appeared abnormal in all the embryos examined with variations in the extent of abnormality. One obvious phenotype observed was that of 'stalling' of Fas II-positive axons, which could be seen clearly at late stage 13. At this stage, minute outgrowths from the cell bodies and axonal tracts were also visible. From stage 15 onward, Fasciclin II-expressing axons could be seen crossing the midline. Occasionally a whirling phenotype similar to that observed in robo mutant alleles was seen (Ratnaparkhi, 2002).

From these experiments the fate of the axons that cross the midline was unclear. For this purpose a strain with the Apterous tau-ßgalactosidase (Ap-taußgal) construct was created in which single axons could be observed. Ap-taußgal marks specific Apterous-expressing neurons in each hemisegment of the embryo. Normally these axons project anteriorly on the ipsilateral side to form a distinct Apterous fascicle. In embryos of the genotype C155; UAS-AcGq3, axons from Apterous-expressing neurons no longer remain on the ipsilateral side but are now able to cross the midline. However, unlike axons that crossover in robo mutant embryos, these appear to stall after reaching and crossing the midline (Ratnaparkhi, 2002).

The phenotypes observed in embryos expressing AcGq3 suggest that Gq signaling can drive formation of the commissures and longitudinal tracts. This idea is supported by the phenotype observed in embryos homozygous for Df(2R)vg-C (which uncovers dgq). In these embryos the commissures appear thinner, and there are extensive breaks in the longitudinal tracts. These phenotypes are considerably stronger than those observed for frazzled mutants, which is also uncovered by the same deficiency, indicating that the effect of removing both Dgq and Frazzled is additive. However, these defects could be either caused by erroneous signaling within neurons so that they misinterpret existing cues, or by a non-autonomous mechanism that affects midline guidance cues. The latter would result in misplaced neurons or glia or neurons with changed identity. In Df(2R) vg-C embryos, the pattern of neurons expressing the Even-skipped (Eve) protein appear normal, indicating that the defects seen occur after neuronal patterning is complete (Ratnaparkhi, 2002).

To confirm that the phenotype seen by expression of AcGq3 in the CNS is caused by altered signaling within neurons expressing AcGq3, more restrictive GAL4 drivers were used to express UAS-AcGq3 in specific subsets of neurons of the embryonic CNS. ftz_ng-GAL4 expresses in a small subset of neurons that include mostly motor neurons and some interneurons like vMP2, pCC, dMP2, and MP1. These interneurons pioneer the longitudinal axon tracts, which stain positive for Fasciclin II. In addition, these axons never cross the midline. On expressing UAS-AcGq3 with ftz_ng-GAL4, midline crossing by Fasciclin II-positive axons could be observed. At stage 13, the pCC axon, which normally projects anteriorly on the ipsilateral side, could be seen turning toward the midline. At stage 16, aberrant midline crossing by the medial fascicle could be observed. The number of midline crossovers at this stage is less as compared with C155-GAL4, presumably because of the restricted and comparatively weak expression of the ftz_ng-GAL4 line. Similar results were obtained with eve_ng-GAL4, which expresses in aCC, pCC, and RP2 neurons. The pCC axon can be seen crossing the midline, whereas the aCC and RP2 projections look normal on expression of AcGq3. Axons from Apterous-expressing dorsal cells (dc) can also change their trajectory on expression of AcGq3. Instead of projecting toward the anterior and in an ipsilateral direction as is normal, a fraction of the axons can be seen drifting across the midline. The autonomy of AcGq3 function is further supported by the observation that neurons and glia are patterned normally in C155-GAL4/UAS-AcGq3 embryos, as judged by staining with anti-Eve and anti-Repo antibodies. Taken together these data demonstrate that specific activation of Dgqalpha3 in ipsilaterally projecting neurons causes changes in their axonal trajectories so that they are now able to project across the midline (Ratnaparkhi, 2002).

To understand how Dgqalpha3 acts to change axonal paths, possible interactions with genes known to affect midline guidance were sought. Axons that cross the midline and project along the contralateral longitudinal tract normally need to downregulate expression of Robo, which acts as a receptor for the midline repellant Slit. It is known that Robo downregulation requires Commissureless, but the precise mechanism is not understood. A possible mechanism by which AcGq3 could promote midline crossing was by downregulating Robo. To test this hypothesis, Robo expression was examined in ftz_ng-GAL4;UAS-AcGq3 embryos. Interestingly, Robo is not downregulated visibly in axons that ectopically cross the midline under the influence of AcGq3. The extent of Robo staining seen on these axons that aberrantly cross the midline is comparable with that seen on the longitudinal tracts. Thus, constitutive activation of Dgqalpha3 results in aberrant midline crossing of axons by a mechanism that is independent of Robo downregulation (Ratnaparkhi, 2002).

Another mechanism by which AcGq3 could induce midline crossing is through inhibition of the repulsive signal mediated by Robo. If this were so, then reducing levels of Robo by genetic means should enhance the phenotype of AcGq3. To test this, AcGq3 was expressed using ftz_ng-GAL4 in embryos carrying a single copy of the robo¹ mutant allele. robo¹ is a recessive mutation. However, embryos with one copy of this mutation show midline crossing at a frequency of ~10%. When UAS-AcGq3;robo¹/+;ftz_ng-GAL4 embryos were stained with mAb 1D4, a significant increase in the number of midline crossovers was observed as compared with embryos of the genotype UAS-AcGq3;+/+;ftz_ng-GAL4. This suggests that activation of Dgqalpha3 antagonizes the repulsive output through Robo resulting in excessive midline crossing. The antagonism could be mediated either through phosphorylation of Robo or signaling components that function downstream and/or in parallel with Robo (Ratnaparkhi, 2002).

Phosphorylation of a single tyrosine residue on Robo by Abelson (Abl) tyrosine kinase inhibits Robo repulsive signaling and is needed for normal midline crossing to take place. Expression of a mutant form of Robo in which this tyrosine residue (Y1040) has been replaced with a phenylalanine (in a transgenic strain referred to as UAS-roboY-F), leads to constitutive Robo signaling such that no axons cross the midline, resulting in a complete absence of commissure formation. If AcGq3 acts upstream of Robo, it was predicted that ectopic midline-crossovers, induced by expression of AcGq3, would be reduced in the presence of Robo Y-F. In fact, in embryos expressing both AcGq3 and Robo Y-F, no ectopic crossovers are seen, indicating that AcGq3 could inhibit Robo signaling by promoting Robo phosphorylation. This finding is also supportive of the fact that AcGq3 exerts its effect independent of Commissureless-mediated Robo downregulation. It is possible however, that AcGq3 acts through a parallel pathway that is no longer effective in the presence of Robo Y-F (Ratnaparkhi, 2002).

Both the spatiotemporal pattern of expression and functional analysis of dgq indicate that Gq activation in vivo promotes midline crossing. Axons that cross the midline need to down-modulate their repulsive signaling pathway(s) as well as respond positively to attractive cues. Therefore, whether changes in the levels of 'attractive' signaling such as the Netrin-Frazzled pathway affect the phenotype of AcGq3 was examined. Interestingly, AcGq3 phenotype shows a dosage-dependent interaction with Fra. Removal of a single copy of the Fra gene leads to a threefold reduction in the number of midline crossovers induced by AcGq3. A further reduction was observed on removal of both copies of the Fra gene as seen in embryos of the genotype C155-GAL4/UAS-AcGq3;fra³/fra⁴. Signaling through AcGq3 is thus sensitive to levels of Frazzled in the CNS (Ratnaparkhi, 2002).

To examine the effect, if any, of AcGq3 on the frazzled mutant phenotype, embryos of the genotype C155-GAL4/UAS-AcGq3;fra³/fra⁴ were examined with anti-connectin antibody and BP102. Anti-connectin labels a distinct axon fascicle in the longitudinal connectives, axon projections of SP1 and RP1 neurons that project through the anterior commissure, and a subset of axons that project through the posterior commissure to their contralateral targets. In embryos of the genotype C155-GAL4/+; fra³/fra⁴, breaks were observed in connectin-positive commissural axons and longitudinal tracts. Embryos of the genotype C155-GAL4/UAS-AcGq3;fra³/fra⁴ also show similar breaks, indicating that AcGq3 does not have an effect on the frazzled mutant phenotype. Similar results were obtained by staining with BP102 (Ratnaparkhi, 2002).

The induction of ectopic midline crossing by AcGq3 suggests that Dgqalpha3 function might be required during commissural growth. What activates Dgqalpha3 in vivo? In Drosophila, the only pathway so far known to mediate attraction toward the midline, is the Netrin-Frazzled signaling pathway. However, null mutants for netrins and frazzled continue to show formation of commissures, albeit thin and poorly organized. The failure to show a complete absence of commissures suggests that an alternate signaling pathway or pathways exists at the midline, one that promotes commissural growth. The presence of a second attractive signaling pathway operating at the midline has also been suggested based on analysis of mutants involved in formation of commissures. Dgqalpha3 might act as a component of this alternate pathway to promote commissural growth (Ratnaparkhi, 2002).

Signaling mechanisms involved in DCC/Frazzled-mediated attraction are poorly understood in vertebrates as well as invertebrates. In vitro studies using pharmacology in vertebrate systems have shown that guidance mediated by Netrin-1 is dependent on cAMP levels in the growth cone. Increase in cAMP levels results in attraction, whereas low levels of the cyclic nucleotide causes repulsion. In Xenopus cultured neurons, Netrin-1-induced turning response has also been shown to depend on Ca²⁺ influx through the plasma membrane and Ca²⁺-induced Ca²⁺ release through intracellular stores. The involvement of second messengers such as Ca²⁺ and cAMP suggests that G-protein-coupled signaling pathways might be involved. Heterotrimeric G-proteins are also thought to play a role in neuronal migration and growth cone collapse (Ratnaparkhi, 2002).

The Adenosine A2b receptor has been implicated in Netrin-1 signaling. However, it has been shown that DCC can bind Netrin-1 and signal attraction independent of the Adenosine A2b receptor. DCC undergoes a ligand-dependent dimerization essential for its signaling that remains unaffected even in the presence of antagonists to adenosine receptors, thus providing evidence that DCC alone is central to Netrin-1 signaling. As compared with vertebrates, the mechanism of Netrin signaling in Drosophila is still obscure. Given the evolutionarily conserved nature of both, the ligand and the receptor, similar downstream signaling elements are very likely involved in mediating attraction. It is possible that a seven transmembrane domain receptor activates Dgqalpha3 signaling in response to novel attractive cues or Netrins leading to increase in Ca²⁺ levels and thus promoting attraction (Ratnaparkhi, 2002).

The results from the genetic analysis of AcGq3 and frazzled suggest that Frazzled function is essential for AcGq3-mediated ectopic midline crossing. In addition, they also indicate that Dgqalpha3 does not function downstream of frazzled signaling. A simple explanation for these observations could be that activity of Dgqalpha3 and Frazzled are both essential to promote midline crossing. The effects of the two signaling pathways are additive; activation of Frazzled and Dgqalpha3 are both necessary to elicit attraction. Removal of one or both copies of frazzled in the presence of AcGq3 simply reduces the sum total of attraction sensed by the growth cone, thus inhibiting aberrant midline crossing of ipsilateral axons (Ratnaparkhi, 2002).

The antagonism between AcGq3 and Robo suggests that AcGq3 operates by modulating repulsion from the midline during commissural growth. It has been demonstrated that Robo signaling is negatively modulated by tyrosine phosphorylation by Abelson kinase. AcGq3 could inhibit Robo signaling by a similar mechanism of phosphorylating Robo. It could perhaps do this by activating a kinase cascade involving a nonreceptor tyrosine kinase such as Bruton's tyrosine kinase (BTK or Tec kinase) which, in mammalian cells, has been shown to be a direct effector of Gq signaling. The results are equally consistent with the possibility that AcGq3 and Robo act through parallel pathways, such that AcGq3 induced midline crossing requires downregulation of Robo signaling (Ratnaparkhi, 2002).

Based on the results obtained from genetic analysis of AcGq3 with frazzled and robo, the following models can be proposed to explain the function of Dgqalpha3. In the first, Dgqalpha3 can be thought of as being a component of the attractive signaling pathway alone. Expression of the activated form of the protein functions to override the repulsive cues at the midline and promote ectopic midline crossing. In such a scenario, one would argue that the synergism observed between AcGq3 and robo¹ is a consequence of the combined effect of reduced Robo signaling and excess attractive signaling induced by AcGq3 leading to an increase in the number of midline crossovers. In the presence of UAS-RoboY-F, repulsive signaling increases to a level that cannot be overriden by AcGq3-attractive signaling. A second possibility is that Dgqalpha3 is a component of an attractive signaling pathway, which functions to potentiate Frazzled signaling by negatively modulating the repulsion mediated by Robo signaling. This could be through phosphorylation of Robo. A recent study using spinal axons from stage 22 Xenopus embryos has shown that the repulsive ligand Slit can 'silence' the Netrin-mediated attraction through a direct physical interaction between the cytoplasmic domains of Robo and Frazzled. This ligand-dependent silencing effect serves to promote repulsion of growth cones from the midline during the development of commissures. Dgqalpha3 might function conversely at the level of downstream effector molecules to inhibit repulsion in response to attractive cues to promote midline crossing (Ratnaparkhi, 2002).

In summary, these results predict the involvement of a Gq-mediated signaling pathway in regulating midline crossing in Drosophila. In addition, they also support the notion that balance between attraction and repulsion is a crucial factor that determines the final response of a growth cone to different cues. Inhibition of dgq function specifically in the growth cones should prove useful in dissecting out other components of this pathway which regulates midline crossing (Ratnaparkhi, 2002).

Search PubMed for articles about Drosophila Galphaq

Aleyakpo, B., Umukoro, O., Kavlie, R., Ranson, D. C., Thompsett, A., Corcoran, O. and Casalotti, S. O. (2019). G-protein alphaq gene expression plays a role in alcohol tolerance in Drosophila melanogaster. Brain Neurosci Adv 3: 2398212819883081. PubMed ID: 32166184

Baumbach, J., Xu, Y., Hehlert, P. and Kuhnlein, R. P. (2014). Gαq, Ggamma1 and Plc21C control Drosophila body fat storage. J Genet Genomics 41(5): 283-292. PubMed ID: 24894355

Brodskiy, P. A., Wu, Q., Soundarrajan, D. K., Huizar, F. J., Chen, J., Liang, P., Narciso, C., Levis, M. K., Arredondo-Walsh, N., Chen, D. Z. and Zartman, J. J. (2019). Decoding calcium signaling dynamics during Drosophila wing disc development. Biophys J. PubMed ID: 30704858

Cao, J., Bollepalli, M. K., Hu, Y., Zhang, J., Li, Q., Li, H., Chang, H., Xiao, F., Hardie, R. C., Rong, Y. S. and Hu, W. (2017). A single residue mutation in the Gαq subunit of the G protein complex causes blindness in Drosophila. G3 (Bethesda). PubMed ID: 29158337

Dahal, G. R., Pradhan, S. J. and Bates, E. A. (2017). Inwardly rectifying potassium channels influence Drosophila wing morphogenesis by regulating Dpp release. Development 144(15): 2771-2783. PubMed ID: 28684627 .

Gu, Q., Wu, J., Tian, Y., Cheng, S., Zhang, Z. C. and Han, J. (2020). Gαq splice variants mediate phototransduction, rhodopsin synthesis, and retinal integrity in Drosophila. J Biol Chem. PubMed ID: 32198182

Ha, E. M., et al. (2009). Regulation of DUOX by the Gαq-phospholipase Cβ-Ca2+ pathway in Drosophila gut immunity. Dev. Cell 16(3): 386-97. PubMed Citation: 19289084

Hardie, R. C., Martin, F., Cochrane, G. W., Juusola, M., Georgiev, P. and Raghu, P. (2002). Molecular basis of amplification in Drosophila phototransduction: roles for G protein, phospholipase C, and diacylglycerol kinase. Neuron 36(4): 689-701. PubMed ID: 12441057

Herman, J. A., Willits, A. B. and Bellemer, A. (2018). Gαq and Phospholipase Cβ signaling regulate nociceptor sensitivity in Drosophila melanogaster larvae. PeerJ 6: e5632. PubMed ID: 30258723

Himmelreich, S., Masuho, I., Berry, J. A., MacMullen, C., Skamangas, N. K., Martemyanov, K. A. and Davis, R. L. (2017). Dopamine receptor DAMB signals via Gq to mediate forgetting in Drosophila. Cell Rep 21(8): 2074-2081. PubMed ID: 29166600

Ratnaparkhi, A., Banerjee, S. and Hasan, G. (2002). Altered levels of Gq activity modulate axonal pathfinding in Drosophila. J. Neurosci. 22(11): 4499-4508. 12040057

Wang, Y., Wang, H., Li, X. and Li, Y. (2015). Epithelial microRNA-9a regulates dendrite growth through Fmi-Gq signaling in Drosophila sensory neurons. Dev Neurobiol [Epub ahead of print]. PubMed ID: 26016469

date revised: 22 May 2023

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