Requirement for an Otopetrin-like protein for acid taste in Drosophila

Receptors for bitter, sugar, and other tastes have been identified in the fruit fly Drosophila melanogaster, while a broadly tuned receptor for the taste of acid has been elusive. Previous work showed that such a receptor was unlikely to be encoded by a gene within one of the two major families of taste receptors in Drosophila, the “gustatory receptors” and “ionotropic receptors.” Here, to identify the acid taste receptor, we tested the contributions of genes encoding proteins distantly related to the mammalian Otopertrin1 (OTOP1) proton channel that functions as a sour receptor in mice. RNA interference (RNAi) knockdown or mutation by CRISPR/Cas9 of one of the genes, Otopetrin-Like A (OtopLA), but not of the others (OtopLB or OtopLC) severely impaired the behavioral rejection to a sweet solution laced with high levels of HCl or carboxylic acids and greatly reduced acid-induced action potentials measured from taste hairs. An isoform of OtopLA that we isolated from the proboscis was sufficient to restore behavioral sensitivity and acid-induced action potential firing in OtopLA mutant flies. At lower concentrations, HCl was attractive to the flies, and this attraction was abolished in the OtopLA mutant. Cell type–specific rescue experiments showed that OtopLA functions in distinct subsets of gustatory receptor neurons for repulsion and attraction to high and low levels of protons, respectively. This work highlights a functional conservation of a sensory receptor in flies and mammals and shows that the same receptor can function in both appetitive and repulsive behaviors.

Humans possess the ability to distinguish among five basic tastes: sweet, bitter, salt, sour, and umami. Interestingly, there is considerable variety in the ability of other mammals to detect these qualities. For example, cats are missing sweet taste (1) and the bottlenose dolphin only detects salt in food (2). Yet the fruit fly, Drosophila melanogaster, responds to a similar repertoire of tastes as humans. This is all the more remarkable given the very distant evolutionary relatedness and the enormous differences in the anatomy of the fly and mammalian taste organs and points to a conserved function of these taste qualities in assessing food quality.Many of the receptors involved in Drosophila taste have been defined (3, 4). Those that contribute to sweet and bitter tastes have been characterized extensively and are members of the “gustatory receptor” (GR) family (3, 4). GRs are unrelated to the G protein–coupled receptors that function in mammalian sweet and bitter taste (3). Therefore, the abilities of insects and humans to respond to similar repertoires of chemicals such as sweet and bitter tastants have emerged independently.In mice, the taste of acids depends on a proton-selective channel, Otopetrin1 (OTOP1), which is expressed in type III taste receptor cells (5–7). OTOP1 was first identified based on its essential role in the vestibular systems of the mouse and zebrafish (8–11) and was found to encode a family of proton-selective ion channels functionally conserved from worms to humans (5, 9, 12). In sea urchins, an Otop channel functions in calcifying primary mesenchymal cells by promoting the removal of protons generated during the production of CaCO₃ (13). Otop family members are structurally unrelated to other ion channels and are composed of 12 transmembrane segments (12, 14, 15), which assemble as a dimer with no obvious permeation pathway (14, 15). Flies, mice, and human genomes each contain three otop genes, although the fly genes are not direct homologs of the vertebrate genes (9).In Drosophila, low or moderate levels of some organic acids are attractive and promote feeding, while the same acids at higher concentrations repress food consumption (16, 17). This rejection contributes to survival as it discourages the animals from eating very acidic foods in the environment that can decrease lifespan. Two members of the large family of “ionotropic receptors” (IRs; IR25a and IR76b) function in GR neurons (GRNs) in the legs for sensing carboxylic acids and HCl (18). Mutation of either of these IRs disrupts the preference to lay eggs on acid-containing substrates (18). Flies prefer consuming lactic acid over water, and this preference is mildly reduced in Ir25a mutants (19).The receptors required for the gustatory rejection of noxious levels of acids have been largely enigmatic. An exception is IR7a, which is needed to suppress feeding on foods laced with acetic acid (17). IR7a is very narrowly tuned, as it does not impair the rejection of foods with HCl or any other carboxylic acid tested. This receptor acts in a subset of GRNs called B GRNs that are also activated by bitter chemicals and certain other aversive compounds (4, 17).Here, we identified a member of the family of Otop channels that in Drosophila is required for the detection of protons in food. Wild-type flies are strongly repelled by high levels of HCl and mildly attracted to a low level of HCl. We found that these responses depend on the Otopetrin-Like protein (OtopLA), which has a common evolutionarily origin with mammalian OTOP channels. By performing cell type–specific rescue experiments, we found that the strong repulsion and mild attraction to different levels of acids depends on expression of OtopLA in distinct subsets of GRNs.     

Requirement of Gab2 for mast cell development and KitL/c-Kit signaling

Mast cells are thought to participate in a variety of immune responses, such as parasite resistance and the allergic reaction. Mast cell development depends on stem cell factor (Kit ligand) and its receptor, c-Kit. Gab2 is an adaptor molecule containing a pleckstrin homology domain and potential binding sites for SH2 and SH3 domains. Gab2 is phosphorylated on tyrosine after stimulation with cytokines and growth factors, including KitL. Gab2-deficient mice were created to define the physiological requirement for Gab2 in KitL/c-Kit signaling and mast cell development. In Gab2-deficient mice, the number of mast cells was reduced markedly in the stomach and less severely in the skin. Bone marrow-derived mast cells (BMMCs) from the Gab2-deficient mice grew poorly in response to KitL. KitL-induced ERK MAP kinase and Akt activation were impaired in Gab2-deficient BMMCs. These data indicate that Gab2 is required for mast cell development and KitL/c-Kit signaling.     

Requirement of central ghrelin signaling for alcohol reward

The stomach-derived hormone ghrelin interacts with key CNS circuits regulating energy balance and body weight. Here we provide evidence that the central ghrelin signaling system is required for alcohol reward. Central ghrelin administration (to brain ventricles or to tegmental areas involved in reward) increased alcohol intake in a 2-bottle (alcohol/water) free choice limited access paradigm in mice. By contrast, central or peripheral administration of ghrelin receptor (GHS-R1A) antagonists suppressed alcohol intake in this model. Alcohol-induced locomotor stimulation, accumbal dopamine release and conditioned place preference were abolished in models of suppressed central ghrelin signaling: GHS-R1A knockout mice and mice treated with 2 different GHS-R1A antagonists. Thus, central ghrelin signaling, via GHS-R1A, not only stimulates the reward system, but is also required for stimulation of that system by alcohol. Our data suggest that central ghrelin signaling constitutes a potential target for treatment of alcohol-related disorders.     

Sexually dimorphic regulation of the Wingless morphogen controls sex-specific segment number in Drosophila

Sexual dimorphism is widespread throughout the metazoa and plays important roles in mate recognition and preference, sex-based niche partitioning, and sex-specific coadaptation. One notable example of sex-specific differences in insect body morphology is presented by the higher diptera, such as Drosophila, in which males develop fewer abdominal segments than females. Because diversity in segment number is a distinguishing feature of major arthropod clades, it is of fundamental interest to understand how different numbers of segments can be generated within the same species. Here we show that sex-specific and segment-specific regulation of the Wingless (Wg) morphogen underlies the development of sexually dimorphic adult segment number in Drosophila. Wg expression is repressed in the developing terminal male abdominal segment by the combination of the Hox protein Abdominal-B (Abd-B) and the sex-determination regulator Doublesex (Dsx). The subsequent loss of the terminal male abdominal segment during pupation occurs through a combination of developmental processes including segment compartmental transformation, apoptosis, and suppression of cell proliferation. Furthermore, we show that ectopic expression of Wg is sufficient to rescue this loss. We propose that dimorphic Wg regulation, in concert with monomorphic segment-specific programmed cell death, are the principal mechanisms of sculpting the sexually dimorphic abdomen of Drosophila.     

Evidence that the cysteine-rich domain of Drosophila Frizzled family receptors is dispensable for transducing Wingless

Members of the Frizzled family of serpentine transmembrane receptors are required to transduce Wingless/Int (Wnt) signals and contain in their N-terminal regions a conserved Wnt-binding cysteine-rich domain (CRD). Each CRD has specific affinities for particular Wnts, and it is generally believed that signal transduction depends on the strength of this interaction. Here, we report in vivo evidence that the CRD is dispensable for Frizzled family receptors to transduce Wingless (Wg), the primary Wnt signal in Drosophila. Thus, we infer that signal transduction does not require binding of Wg to the CRD, but instead depends on interactions between Wg and other portions of the receptor, or other proteins of the receptor complex.     

Requirement of gp130 signaling for the AGM hematopoiesis

OBJECTIVE: Definitive hematopoiesis starts in the aorta-gonad-mesonephros (AGM) region during mouse development and remarkably expands in the liver at a later stage of ontogeny. gp130 is a signal transducing receptor component shared by all the IL-6 family cytokines, whose gene ablation in mouse results in the significant reduction in the fetal liver hematopoiesis. The present study aims to evaluate the role of gp130 signaling in the fetal mouse AGM hematopoiesis. METHODS AND MATERIALS: Mouse AGM regions from the wild-type and gp130-deficient mice on embryonic day 11.5 were dissociated and cultured with a mixture of cytokines, including one which activates gp130. Wild-type human gp130 and its mutant constructs were introduced into cultured gp130-deficient AGM cells using retrovirus system. To further analyze gp130 downstream signaling, a dominant-negative mutant of STAT3 was also introduced. RESULTS: The gp130 deficiency in the culture of fetal mouse AGM cells resulted in the failure of the expansion of the c-kit(+), Sca-1(+), and lineage markers(-) population. Such failure was rescued by introduction of a wild-type gp130 expression construct but not its mutant constructs having no ability to activate STAT3. In the normal AGM cell culture, introduction of a dominant-negative form of STAT3 in which Y(705) was changed to phenylalanine suppressed the expansion of hematopoietic cell colonies. CONCLUSION: gp130 plays an indispensable role in the expansion of hematopoietic precursor cells in the fetal mouse AGM. In particular, the activation of STAT3 by gp130 is found to be important in this process.     

Functional screening identifies miR-315 as a potent activator of Wingless signaling

The existence of vast regulatory networks mediated by microRNAs (miRNAs) suggests broad potential for miRNA dysfunction to contribute to disease. However, relatively few miRNA-target interactions are likely to make detectable contributions to phenotype, and effective strategies to identify these few interactions are currently wanting. We hypothesized that signaling cascades represent critical points of susceptibility to miRNA dysfunction, and we developed a strategy to test this theory by using quantitative cell-based screens. Here we report a screen for miRNAs that affect the Wingless (Wg) pathway, a conserved pathway that regulates growth and tissue specification. This process identified ectopic miR-315 as a potent and specific activator of Wg signaling, an activity that we corroborated in transgenic animals. This miR-315 activity was mediated by direct inhibition of Axin and Notum, which encode essential, negatively acting components of the Wg pathway. Genetic interaction tests substantiated both of these genes as key functional targets of miR-315. The ability of ectopic miR-315 to activate Wg signaling was not a trivial consequence of predicted miRNA-target relationships because other miRNAs with conserved sites in the Axin 3' UTR neither activated Wg outputs nor inhibited an Axin sensor. In summary, activity-based screening can selectively identify miRNAs whose deregulation can lead to interpretable phenotypic consequences.     

Requirement of ATR for maintenance of intestinal stem cells in aging Drosophila

The stem cell genomic stability forms the basis for robust tissue homeostasis, particularly in high-turnover tissues. For the genomic stability, DNA damage response (DDR) is essential. This study was focused on the role of two major DDR-related factors, ataxia telangiectasia-mutated (ATM) and ATM- and RAD3-related (ATR) kinases, in the maintenance of intestinal stem cells (ISCs) in the adult Drosophila midgut. We explored the role of ATM and ATR, utilizing immunostaining with an anti-pS/TQ antibody as an indicator of ATM/ATR activation, γ-irradiation as a DNA damage inducer, and the UAS/GAL4 system for cell type-specific knockdown of ATM, ATR, or both during adulthood. The results showed that the pS/TQ signals got stronger with age and after oxidative stress. The pS/TQ signals were found to be more dependent on ATR rather than on ATM in ISCs/enteroblasts (EBs). Furthermore, an ISC/EB-specific knockdown of ATR, ATM, or both decreased the number of ISCs and oxidative stress-induced ISC proliferation. The phenotypic changes that were caused by the ATR knockdown were more pronounced than those caused by the ATM knockdown; however, our data indicate that ATR and ATM are both needed for ISC maintenance and proliferation; ATR seems to play a bigger role than does ATM.     

Cytokine signaling through the JAK/STAT pathway is required for long-term memory in Drosophila

Cytokine signaling through the JAK/STAT pathway regulates multiple cellular responses, including cell survival, differentiation, and motility. Although significant attention has been focused on the role of cytokines during inflammation and immunity, it has become clear that they are also implicated in normal brain function. However, because of the large number of different genes encoding cytokines and their receptors in mammals, the precise role of cytokines in brain physiology has been difficult to decipher. Here, we took advantage of Drosophila's being a genetically simpler model system to address the function of cytokines in memory formation. Expression analysis showed that the cytokine Upd is enriched in the Drosophila memory center, the mushroom bodies. Using tissue- and adult-specific expression of RNAi and dominant-negative proteins, we show that not only is Upd specifically required in the mushroom bodies for olfactory aversive long-term memory but the Upd receptor Dome, as well as the Drosophila JAK and STAT homologs Hop and Stat92E, are also required, while being dispensable for less stable memory forms.     

Secreted Wingless-interacting molecule (Swim) promotes long-range signaling by maintaining Wingless solubility

Lipid-modified Wnt/Wingless (Wg) proteins can signal to their target cells in a short- or long-range manner. How these hydrophobic proteins travel through the extracellular environment remains an outstanding question. Here, we report on a Wg binding protein, Secreted Wg-interacting molecule (Swim), that facilitates Wg diffusion through the extracellular matrix. Swim, a putative member of the Lipocalin family of extracellular transport proteins, binds to Wg with nanomolar affinity in a lipid-dependent manner. In quantitative signaling assays, Swim is sufficient to maintain the solubility and activity of purified Wg. In Drosophila, swim RNAi phenotypes resemble wg loss-of-function phenotypes in long-range signaling. We propose that Swim is a cofactor that promotes long-range Wg signaling in vivo by maintaining the solubility of Wg.     

Requirement for AHNAK1-mediated calcium signaling during T lymphocyte cytolysis

Cytolytic CD8⁺ T cells (CTLs) kill virally infected cells, tumor cells, or other potentially autoreactive T cells in a calcium-dependent manner. To date, the molecular mechanism that leads to calcium intake during CTL differentiation and function has remained unresolved. We demonstrate that desmoyokin (AHNAK1) is expressed in mature CTLs, but not in naive CD8⁺ T cells, and is critical for calcium entry required for their proper function during immune response. We show that mature AHNAK1-deficient CTLs exhibit reduced Ca_v1.1 α1 subunit expression (also referred to as L-type calcium channels or α1S pore-forming subunits), which recently were suggested to play a role in calcium entry into CD4⁺ T cells. AHNAK1-deficient CTLs show marked reduction in granzyme-B production, cytolytic activity, and IFN-γ secretion after T cell receptor stimulation. Our results demonstrate an AHNAK1-dependent mechanism controlling calcium entry during CTL effector function.Calcium plays critical roles in T-cell differentiation and function, such as activation, proliferation, and cytokine production (1, 2). Cytotoxic CD8⁺ effector T cells (CTLs) primarily use the perforin/granule exocytosis pathway to kill virus-infected and tumor cells (3). The role of calcium in CTL-mediated cytolysis has been studied extensively, mainly by using calcium antagonists (e.g., see ref. 4). The exact requirements for calcium and its molecular mode of entry during CTL function still are largely uncertain (4, 5).The calcium release-activated calcium channel (CRAC) pathway is the most studied plasma membrane store operated calcium (SOC) channel through which calcium enters after T-cell stimulation. Surprisingly, ORAI1 (also known as “CRACM1” or “TMEM142A”)-deficient CTLs indeed show reduced calcium entry but only partial IFN-γ production and normal granzyme-B expression, suggesting that other pore subunits, possibly ORAI2 or ORAI3, or different channels altogether, are involved in this process (6).In addition to CRAC channels (7), we and others (8, 9), found that L-type calcium channel (Ca_v1) subunits α1, α2, β, γ, and δ, which constitute the major route of calcium entry in excitable cells and take up calcium in response to membrane depolarization, also are expressed by T cells (10). Previously, we have shown that CD4⁺ T cells express α1 subunits of the Ca_v1 family and that functional Ca_v β4 and β3 regulatory subunits are necessary for normal TCR-triggered calcium response, nuclear factor of activated T cells (NFAT) nuclear translocation, and cytokine production (8, 11).The AHNAK family of scaffold PDZ proteins consists of 2 giant proteins (700 kDa), AHNAK1 (desmoyokin) and AHNAK2 (12–14). AHNAK1 is involved with calcium signaling through protein–protein interactions (15–18). Furthermore, in cardiomyocytes, AHNAK1 associates with the β-subunit of cardiac Ca_v channels at the plasma membrane and is phosphorylated by protein kinase A (PKA) in response to β-adrenoreceptor stimulation (19).Using AHNAK1-deficient mice (14), we recently described a novel mechanism for the regulation of calcium signaling through Ca_v1.1 α1 subunits mediated by AHNAK1 in peripheral CD4⁺ T cells. AHNAK1 is associated with the regulatory β2 subunit of Ca_v1 channels and is required for normal expression of the Ca_v1.1 α1 subunit and intact calcium influx following TCR cross-linking (11). Here, we demonstrate that CTLs employ AHNAK1 to mediate calcium entry required for cytolytic activity late in primary TCR stimulation through the regulation of Ca_v1.1 channels.     

Notch signaling in Drosophila long-term memory formation

Notch (N) is a cell surface receptor that mediates an evolutionarily ancient signaling pathway to control an extraordinarily broad spectrum of cell fates and developmental processes. To gain insights into the functions of N signaling in the adult brain, we examined the involvement of N in Drosophila olfactory learning and memory. Long-term memory (LTM) was disrupted by blocking N signaling in conditional mutants or by acutely induced expression of a dominant-negative N transgene. In contrast, neither learning nor early memory were affected. Furthermore, induced overexpression of a wild-type (normal) N transgene specifically enhanced LTM formation. These experiments demonstrate that N signaling contributes to LTM formation in the Drosophila adult brain.     

Repression of dMyc expression by Wingless promotes Rbf-induced G1 arrest in the presumptive Drosophila wing margin

Little is known about how patterns of cell proliferation and arrest are generated during development, a time when tight regulation of the cell cycle is necessary. In this study, the mechanism by which the developmental signaling molecule Wingless (Wg) generates G(1) arrest in the presumptive Drosophila wing margin is examined in detail. Wg signaling promotes activity of the Drosophila retinoblastoma family (Rbf) protein, which is required for G(1) arrest in the presumptive wing margin. Wg promotes Rbf function by repressing expression of the G(1)-S regulator Drosophila myc (dmyc). Ectopic expression of dMyc induces expression of Cyclin E, Cyclin D, and Cdk4, which can inhibit Rbf and promote G(1)-S progression. Thus, G(1) arrest in the presumptive wing margin depends on the presence of Rbf, which is maintained by the ability of Wg signaling to repress dmyc expression in these cells. In addition to advancing the understanding of how patterned cell-cycle arrest is generated by the Wg signaling molecule during development, this study indicates that components of the Rbf/E2f pathway are targets of dMyc in Drosophila. Although Rbf/E2f pathway components mediate the ability of dMyc to promote G(1) progression, dMyc appears to regulate growth independently of the RBF/E2f pathway.     

SNMP is a signaling component required for pheromone sensitivity in Drosophila

The only known volatile pheromone in Drosophila, 11-cis-vaccenyl acetate (cVA), mediates a variety of behaviors including aggregation, mate recognition, and sexual behavior. cVA is detected by a small set of olfactory neurons located in T1 trichoid sensilla on the antennae of males and females. Two components known to be required for cVA reception are the odorant receptor Or67d and the extracellular pheromone-binding protein LUSH. Using a genetic screen for cVA-insensitive mutants, we have identified a third component required for cVA reception: sensory neuron membrane protein (SNMP). SNMP is a homolog of CD36, a scavenger receptor important for lipoprotein binding and uptake of cholesterol and lipids in vertebrates. In humans, loss of CD36 is linked to a wide range of disorders including insulin resistance, dyslipidemia, and atherosclerosis, but how CD36 functions in lipid transport and signal transduction is poorly understood. We show that SNMP is required in pheromone-sensitive neurons for cVA sensitivity but is not required for sensitivity to general odorants. Using antiserum to SNMP infused directly into the sensillum lymph, we show that SNMP function is required on the dendrites of cVA-sensitive neurons; this finding is consistent with a direct role in cVA signal transduction. Therefore, pheromone perception in Drosophila should serve as an excellent model to elucidate the role of CD36 members in transmembrane signaling.     

Requirement of type I interferon signaling for arthritis triggered by double-stranded RNA

OBJECTIVE: Arthralgias and overt arthritides are often associated with viral infections. Viral infections expose the infected host to proinflammatory double-stranded RNA (dsRNA), which can cause joint inflammation and is a potent activator of interferon-alpha (IFNalpha). The aim of this study was to determine the role of IFNalpha and dsRNA-related signaling molecules in the onset of joint inflammation induced by viral dsRNA. METHODS: IFNalpha and different forms of RNA were injected into the knee joints of wild-type mice, mice lacking the type I interferon receptor (IFNAR(-/-)), and mice deficient in dsRNA-dependent protein kinase (PKR(-/-)). Histologic evidence of joint damage and the ability of splenocytes to produce cytokines in response to dsRNA or IFNalpha were assessed. RESULTS: Viral dsRNA, but not short single-stranded RNA, induced arthritis. The arthritis was aggravated by intracellular delivery of dsRNA. The expression of PKR was not mandatory for dsRNA-induced joint inflammation. In contrast, IFNalpha/beta signaling was important for dsRNA-induced joint inflammation because IFNAR(-/-) mice did not develop arthritis. Furthermore, intraarticular deposition of IFNalpha induced arthritis in PKR(-/-) and control mice, whereas IFNAR(-/-) mice were protected. The arthritogenic effect of IFNalpha was attenuated by in vivo depletion of monocyte/macrophages. CONCLUSION: Arthritis triggered by dsRNA is not dependent on the expression of the dsRNA-signaling molecule PKR (or Toll-like receptor 3, as previously shown), but is associated with the ability to produce type I IFN and is critically dependent on type I IFN receptor signaling. The intrinsic arthritogenic properties of IFNalpha implicate a role of this cytokine in joint manifestations triggered by various interferogenic stimuli.