Expression of a D1 dopamine receptor dDA1/DmDOP1 in the central nervous system of Drosophila melanogaster

The diverse physiological effects of dopamine are mediated by multiple receptor systems. The dDA1 represents one of the Drosophila dopamine receptors that activate the cAMP cascade. To gain insight into the role of dDA1, we generated a polyclonal antibody against the unique sequence in dDA1 and investigated dDA1 distribution in the central nervous system (CNS) of Drosophila melanogaster. In both larval and adult CNS pronounced dDA1 immunoreactivity was present in the neuropil of the mushroom bodies, a brain structure crucial for learning and memory in insects, and four unpaired neurons in each thoracic segment. In addition, the larval abdominal ganglion contained two dDA1 cells in each segment. This expression pattern appeared to be maintained in the condensed adult abdominal ganglion although the precise number and the intensity of staining were somewhat variable. The adult CNS also exhibited intense dDA1 immunoreactivity in the central complex, a structure controlling higher-order motor function, moderate expression in several neurosecretory cells, and weak staining in two unpaired neurons in the mesothoracic neuromere. The dDA1 expression in these areas was only detected in adult, but not in third instar larval CNS.     

Short neuropeptide F (sNPF) is a stage-specific suppressor for juvenile hormone biosynthesis by corpora allata,and a critical factor for the initiation of insect metamorphosis

Molting and metamorphosis are essential events for arthropod development, and juvenile hormone (JH) and its precursors play critical roles for these events. We examined the regulation of JH biosynthesis by the corpora allata (CA) in Bombyx mori, and found that intact brain-corpora cardiaca (CC)–CA complexes produced a smaller amount of JH than that in CC–CA complexes and CA alone throughout the 4th and 5th (last) instar stadium. The smaller amount of synthesis was due to allatostatin-C (AST-C) produced by the brain. The CC synthesized short neuropeptide F (sNPF) that also suppressed the JH synthesis, but only in day 3 4th stadium and after the last larval ecdysis. For the suppression, both peptides prevented the expression of some of the distinct JH biosynthetic enzymes in the mevalonate pathway. Allatotropin (AT) stimulated sNPF expression in the CC of day 1 5th instar stadium, not of day 3 4th; therefore the stage-specific inhibition of JH synthesis by sNPF was partly due to the stimulative action of AT on the sNPF expression besides the stage-specific expression of the sNPF receptors in the CA, the level of which was high in day 2 4th and day 0 5th instar larvae. The cessation of JH biosynthesis in the last instar larvae is a key event to initiate pupal metamorphosis, and both sNPF and AST-C are key factors in shutting down JH synthesis, along with the decline of ecdysone titer and dopamine.     

Drosophila uses two distinct neuropeptide amidating enzymes, dPAL1 and dPAL2

Neuropeptide alpha-amidation is a common C-terminal modification of secretory peptides, frequently required for biological activity. In mammals, amidation is catalyzed by the sequential actions of two enzymes [peptidylglycine-alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL)] that are co-synthesized within a single bifunctional precursor. The Drosophila genome predicts expression of one monofunctional PHM gene and two monofunctional PAL genes. Drosophila PHM encodes an active enzyme that is required for peptide amidation in vivo. Here we initiate studies of the two Drosophila PAL genes. dPAL1 has two predicted transmembrane domains, whereas dPAL2 is predicted to be soluble and secreted. dPAL2 expressed in heterologous cells is secreted readily and co-localized with hormone. In contrast, dPAL1 is secreted poorly, even when expressed with a cleaved signal replacing the predicted transmembrane domains; the majority of dPAL1 stays in the endoplasmic reticulum. Both proteins display PAL enzymatic activity. Compared to the catalytic core of rat PAL, the two Drosophila lyases have higher K(m) values, higher pH optima and similarly broad divalent metal ion requirements. Antibodies to dPAL1 and dPAL2 reveal co-expression in many identified neuroendocrine neurons. Although dPAL1 is broadly expressed, dPAL2 is found in only a limited subset of neurons. dPAL1 expression is highly correlated with the non-amidated peptide proctolin. Tissue immunostaining demonstrates that dPAL1 is largely localized to the cell soma, whereas dPAL2 is distributed throughout neuronal processes.     

The mushroom body D1 dopamine receptor controls innate courtship drive

Mating is critical for species survival and is profoundly regulated by neuromodulators and neurohormones to accommodate internal states and external factors. To identify the underlying neuromodulatory mechanisms, we investigated the roles of dopamine receptors in various aspects of courtship behavior in Drosophila. Here, we report that the D1 dopamine receptor dDA1 regulates courtship drive in naïve males. The wild‐type naïve males actively courted females regardless their appearance or mating status. On the contrary, the dDA1 mutant (dumb) males exhibited substantially reduced courtship toward less appealing females including decapitated, leg‐less and mated females. The dumb male's reduced courtship activity was due to delay in courtship initiation and prolonged intervals between courtship bouts. The dampened courtship drive of dumb males was rescued by reinstated dDA1 expression in the mushroom body α/β and γ neurons but not α/β or γ neurons alone, which is distinct from the previously characterized dDA1 functions in experience‐dependent courtship or other learning and memory processes. We also found that the dopamine receptors dDA1, DAMB and dD2R are dispensable for associative memory formation and short‐term memory of conditioned courtship, thus courtship motivation and associative courtship learning and memory are regulated by distinct neuromodulatory mechanisms. Taken together, our study narrows the gap in the knowledge of the mechanism that dopamine regulates male courtship behavior.     

Mechanisms of midgut remodeling: juvenile hormone analog methoprene blocks midgut metamorphosis by modulating ecdysone action

In holometabolous insects such as mosquito, Aedes aegypti, midgut undergoes remodeling during metamorphosis. Insect metamorphosis is regulated by several hormones including juvenile hormone (JH) and 20-hydroxyecdysone (20E). The cellular and molecular events that occur during midgut remodeling were investigated by studying nuclear stained whole mounts and cross-sections of midguts and by monitoring the mRNA levels of genes involved in 20E action in methoprene-treated and untreated Ae. aegypti. We used JH analog, methoprene, to mimic JH action. In Ae. aegypti larvae, the programmed cell death (PCD) of larval midgut cells and the proliferation and differentiation of imaginal cells were initiated at about 36h after ecdysis to the 4th instar larval stage (AEFL) and were completed by 12h after ecdysis to the pupal stage (AEPS). In methoprene-treated larvae, the proliferation and differentiation of imaginal cells was initiated at 36h AEFL, but the PCD was initiated only after ecdysis to the pupal stage. However, the terminal events that occur for completion of PCD during pupal stage were blocked. As a result, the pupae developed from methoprene-treated larvae contained two midgut epithelial layers until they died during the pupal stage. Quantitative PCR analyses showed that methoprene affected midgut remodeling by modulating the expression of ecdysone receptor B, ultraspiracle A, broad complex, E93, ftz-f1, dronc and drice, the genes that are shown to play key roles in 20E action and PCD. Thus, JH analog, methoprene acts on Ae. aegypti by interfering with the expression of genes involved in 20E action resulting in a block in midgut remodeling and death during pupal stage.     

The Toll immune-regulated Drosophila protein Fondue is involved in hemolymph clotting and puparium formation

Clotting is critical in limiting hemolymph loss and initiating wound healing in insects as in vertebrates. It is also an important immune defense, quickly forming a secondary barrier to infection, immobilizing bacteria and thereby promoting their killing. However, hemolymph clotting is one of the least understood immune responses in insects. Here, we characterize fondue (fon; CG15825), an immune-responsive gene of Drosophila melanogaster that encodes an abundant hemolymph protein containing multiple repeat blocks. After knockdown of fon by RNAi, bead aggregation activity of larval hemolymph is strongly reduced, and wound closure is affected. fon is thus the second Drosophila gene after hemolectin (hml), for which a knockdown causes a clotting phenotype. In contrast to hml-RNAi larvae, clot fibers are still observed in samples from fon-RNAi larvae. However, clot fibers from fon-RNAi larvae are more ductile and longer than in wt hemolymph samples, indicating that Fondue might be involved in cross-linking of fiber proteins. In addition, fon-RNAi larvae exhibit melanotic tumors and constitutive expression of the antifungal peptide gene Drosomycin (Drs), while fon-RNAi pupae display an aberrant pupal phenotype. Altogether, our studies indicate that Fondue is a major hemolymph protein required for efficient clotting in Drosophila.     

Cytochrome P450 CYP307A1/Spook: a regulator for ecdysone synthesis in insects

The prothoracic gland (PG) has essential roles in synthesizing and secreting a steroid hormone called ecdysone that is critical for molting and metamorphosis of insects. However, little is known about the genes controlling ecdysteroidogenesis in the PG. To identify genes functioning in the PG of the silkworm, Bombyx mori, we used differential display PCR and focused on a cytochrome P450 gene designated Cyp307a1. Its expression level positively correlates with a change in the hemolymph ecdysteroid titer. In addition, Drosophila Cyp307a1 is encoded in the spook locus, one of the Halloween mutant family members showing a low ecdysone titer in vivo, suggesting that Cyp307a1 is involved in ecdysone synthesis. While Drosophila Cyp307a1 is expressed in the early embryos and adult ovaries, the expression is not observed in the PGs of embryos or third instar larvae. These results suggest a difference in the ecdysone synthesis pathways during larval development in these insects.     

Corazonin reduces the spinning rate in the silkworm,Bombyx mori

The insect neuropeptide, [Arg⁷]-corazonin was injected into larvae of the silkworm, Bombyx mori to investigate its influence on development and behavior. A single injection of 50 pmol of corazonin into the fourth and fifth instar larvae induced prolongation of the spinning period in all experimental groups except for those injected on day 10 of the fifth instar. The injection also caused a prolongation of the pupal period in some experimental groups, while it had no effect on the timing of larval ecdysis and the length of feeding period of the fifth instar. The spinning period was significantly prolonged even at a low dose of 1 pmol. Both the spinning rate and the rate of increase in hemolymph ecdysteroid level during the spinning stage were reduced by injection of corazonin. However, corazonin injection during days 5-7 of the fifth instar reduced the spinning rate without influencing the ecdysteroid level until the end of day 8, thereafter the rate of increase in hemolymph ecdysteroid level was slower in the corazonin-injected larvae than in the control larvae. Therefore, the suppressed ecdysteroid level observed in the corazonin-injected larvae appears to be a result rather than a cause of the reduced spinning rate. This study is the first published report for the corazonin effect on the behavior in insects.     

Dopamine is required for learning and forgetting in Drosophila

Psychological studies in humans and behavioral studies of model organisms suggest that forgetting is a common and biologically regulated process, but the molecular, cellular, and circuit mechanisms underlying forgetting are poorly understood. Here we show that the bidirectional modulation of a small subset of dopamine neurons (DANs) after olfactory learning regulates the rate of forgetting of both punishing (aversive) and rewarding (appetitive) memories. Two of these DANs, MP1 and MV1, exhibit synchronized ongoing activity in the mushroom body neuropil in alive and awake flies before and after learning, as revealed by functional cellular imaging. Furthermore, while the mushroom-body-expressed dDA1 dopamine receptor is essential for the acquisition of memory, we show that the dopamine receptor DAMB, also highly expressed in mushroom body neurons, is required for forgetting. We propose?a dual role for dopamine: memory acquisition through dDA1 signaling and forgetting through DAMB signaling in the mushroom body neurons.     

Pharmacological characterisation of neuropeptide F (NPF)-induced effects on the motility of Mesocestoides corti (syn. Mesocestoides vogae) larvae

Neuropeptide F is the most abundant neuropeptide in parasitic flatworms and is analogous to vertebrate neuropeptide Y. This paper examines the effects of neuropeptide F on tetrathyridia of the cestode Mesocestoides vogae and provides preliminary data on the signalling mechanisms employed. Neuropeptide F (>/=10 microM) had profound excitatory effects on larval motility in vitro. The effects were insensitive to high concentrations (1 mM) of the anaesthetic procaine hydrochloride suggesting extraneuronal sites of action. Neuropeptide F activity was not significantly blocked by a FMRFamide-related peptide analog (GNFFRdFamide) that was found to inhibit GNFFRFamide-induced excitation indicating the occurrence of distinct neuropeptide F and FMRFamide-related peptide receptors. Larval treatment with guanosine 5'-O-(2-thiodiphosphate) trilithium salt prior to the addition of neuropeptide F completely abolished the excitatory effects indicating the involvement of G-proteins and a G-protein coupled receptor in neuropeptide F activity. Addition of guanosine 5'-O-(2-thiodiphosphate) following neuropeptide F had limited inhibitory effects consistent with the activation of a signalling cascade by the neuropeptide. With respect to Ca(2+) involvement in neuropeptide F-induced excitation of M. vogae larvae, the L-type Ca(2+)-channel blockers verapamil and nifedipine both abolished neuropeptide F activity as did high Mg(+) concentrations and drugs which blocked sarcoplasmic reticulum Ca(2+)-activated Ca(2+)-channels (ryanodine) and sarcoplasmic reticulum Ca(2+) pumps (cyclopiazonic acid). Therefore, both extracellular and intracellular Ca(2+) is important for neuropeptide F excitation in M. vogae. With respect to second messengers, the protein kinase C inhibitor chelerythrine chloride and the adenylate cyclase inhibitor MDL-2330A both abolished neuropeptide F-induced excitation. The involvement of a signalling pathway that involves protein kinase C was further supported by the fact that phorbol-12-myristate-13-acetate, known to directly activate protein kinase C, had direct excitatory effects on larval motility. Although neuropeptide F is structurally analogous to neuropeptide Y, its mode-of-action in flatworms appears quite distinct from the common signalling mechanism seen in vertebrates.     

Locomotor activity is regulated by D2-like receptors in Drosophila: an anatomic and functional analysis

In mammals, dopamine 2-like receptors are expressed in distinct pathways within the central nervous system, as well as in peripheral tissues. Selected neuronal D2-like receptors play a critical role in modulating locomotor activity and, as such, represent an important therapeutic target (e.g. in Parkinson's disease). Previous studies have established that proteins required for dopamine (DA) neurotransmission are highly conserved between mammals and the fruit fly Drosophila melanogaster. These include a fly dopamine 2-like receptor (DD2R; Hearn et al. PNAS 2002 99(22):14554) that has structural and pharmacologic similarity to the human D2-like (D2R). In the current study, we define the spatial expression pattern of DD2R, and functionally characterize flies with reduced DD2 receptor levels. We show that DD2R is expressed in the larval and adult nervous systems, in cell groups that include the Ap-let cohort of peptidergic neurons, as well as in peripheral tissues including the gut and Malpighian tubules. To examine DD2R function in vivo, we generated RNA-interference (RNAi) flies with reduced DD2R expression. Behavioral analysis revealed that these flies show significantly decreased locomotor activity, similar to the phenotype observed in mammals with reduced D2R expression. The fly RNAi phenotype can be rescued by administration of the DD2R synthetic agonist bromocriptine, indicating specificity for the RNAi effect. These results suggest Drosophila as a useful system for future studies aimed at identifying modifiers of dopaminergic signaling/locomotor function.     

PHM is required for normal developmental transitions and for biosynthesis of secretory peptides in Drosophila

To understand the roles of secretory peptides in developmental signaling, we have studied Drosophila mutant for the gene peptidylglycine alpha-hydroxylating monooxygenase (PHM). PHM is the rate-limiting enzyme for C-terminal alpha-amidation, a specific and necessary modification of secretory peptides. In insects, more than 90% of known or predicted neuropeptides are amidated. PHM mutants lack PHM protein and enzyme activity; most null animals die as late embryos with few morphological defects. Natural and synthetic PHM hypomorphs revealed phenotypes that resembled those of animals with mutations in genes of the ecdysone-inducible regulatory circuit. Animals bearing a strong hypomorphic allele contain no detectable PHM enzymatic activity or protein; approximately 50% hatch and initially display normal behavior, then die as young larvae, often while attempting to molt. PHM mutants were rescued with daily induction of a PHM transgene and complete rescue was seen with induction limited to the first 4 days after egg-laying. The rescued mutant adults produced progeny which survived to various stages up through metamorphosis (synthetic hypomorphs) and displayed prepupal and pupal phenotypes resembling those of ecdysone-response gene mutations. Examination of neuropeptide biosynthesis in PHM mutants revealed specific disruptions: Amidated peptides were largely absent in strong hypomorphs, but peptide precursors, a nonamidated neuropeptide, nonpeptide transmitters, and other peptide biosynthetic enzymes were readily detected. Mutant adults that were produced by a minimal rescue schedule had lowered PHM enzyme levels and reproducibly altered patterns of amidated neuropeptides in the CNS. These deficits were partially reversed within 24 h by a single PHM induction in the adult stage. These genetic results support the hypothesis that secretory peptide signaling is critical for transitions between developmental stages, without strongly affecting morphogenetic events within a stage. Further, they show that PHM is required for peptide alpha-amidating activity throughout the life of Drosophila. Finally, they define novel methods to study neural and endocrine peptide biosynthesis and functions in vivo.     

amontillado,the Drosophila homolog of the prohormone processing protease PC2, is required during embryogenesis and early larval development

Biosynthesis of most peptide hormones and neuropeptides requires proteolytic excision of the active peptide from inactive proprotein precursors, an activity carried out by subtilisin-like proprotein convertases (SPCs) in constitutive or regulated secretory pathways. The Drosophila amontillado (amon) gene encodes a homolog of the mammalian PC2 protein, an SPC that functions in the regulated secretory pathway in neuroendocrine tissues. We have identified amon mutants by isolating ethylmethanesulfonate (EMS)-induced lethal and visible mutations that define two complementation groups in the amon interval at 97D1 of the third chromosome. DNA sequencing identified the amon complementation group and the DNA sequence change for each of the nine amon alleles isolated. amon mutants display partial embryonic lethality, are defective in larval growth, and arrest during the first to second instar larval molt. Mutant larvae can be rescued by heat-shock-induced expression of the amon protein. Rescued larvae arrest at the subsequent larval molt, suggesting that amon is also required for the second to third instar larval molt. Our data indicate that the amon proprotein convertase is required during embryogenesis and larval development in Drosophila and support the hypothesis that AMON acts to proteolytically process peptide hormones that regulate hatching, larval growth, and larval ecdysis.     

A novel fruitfly protein under developmental control degrades uracil-DNA

Uracil in DNA may arise by cytosine deamination or thymine replacement and is removed during DNA repair. Fruitfly larvae lack two repair enzymes, the major uracil-DNA glycosylase and dUTPase, and may accumulate uracil-DNA. We asked if larval tissues contain proteins that specifically recognize uracil-DNA. We show that the best hit of pull-down on uracil-DNA is the protein product of the Drosophila melanogaster gene CG18410. This protein binds to both uracil-DNA and normal DNA but degrades only uracil-DNA; it is termed Uracil-DNA Degrading Factor (UDE). The protein has detectable homology only to a group of sequences present in genomes of pupating insects. It is under detection level in the embryo, most of the larval stages and in the imago, but is strongly upregulated right before pupation. In Schneider 2 cells, UDE mRNA is upregulated by ecdysone. UDE represents a new class of proteins that process uracil-DNA with potential involvement in metamorphosis.     

Drosophila dopamine synthesis pathway genes regulate tracheal morphogenesis

While studying the developmental functions of the Drosophila dopamine synthesis pathway genes, we noted interesting and unexpected mutant phenotypes in the developing trachea, a tubule network that has been studied as a model for branching morphogenesis. Specifically, Punch (Pu) and pale (ple) mutants with reduced dopamine synthesis show ectopic/aberrant migration, while Catecholamines up (Catsup) mutants that over-express dopamine show a characteristic loss of migration phenotype. We also demonstrate expression of Punch, Ple, Catsup and dopamine in tracheal cells. The dopamine pathway mutant phenotypes can be reproduced by pharmacological treatments of dopamine and a pathway inhibitor 3-iodotyrosine (3-IT), implicating dopamine as a direct mediator of the regulatory function. Furthermore, we show that these mutants genetically interact with components of the endocytic pathway, namely shibire/dynamin and awd/nm23, that promote endocytosis of the chemotactic signaling receptor Btl/FGFR. Consistent with the genetic results, the surface and total cellular levels of a Btl-GFP fusion protein in the tracheal cells and in cultured S2 cells are reduced upon dopamine treatment, and increased in the presence of 3-IT. Moreover, the transducer of Btl signaling, MAP kinase, is hyper-activated throughout the tracheal tube in the Pu mutant. Finally we show that dopamine regulates endocytosis via controlling the dynamin protein level.     

Characterization of the short neuropeptide F receptor from Drosophila melanogaster

A seven transmembrane G-protein coupled receptor has been cloned from Drosophila melanogaster. This receptor shows structural similarities to vertebrate Neuropeptide Y(2) receptors and is activated by endogenous Drosophila peptides, recently designated as short neuropeptide Fs (sNPFs). sNPFs have so far been found in neuroendocrine tissues of four other insect species and of the horseshoe crab. In locusts, they accelerate ovarian maturation, and in mosquitoes, they inhibit host-seeking behavior. Expression analysis by RT-PCR shows that the sNPF receptor (Drm-sNPF-R) is present in several tissues (brain, gut, Malpighian tubules and fat body) from Drosophila larvae as well as in ovaries of adult females. All 4 Drosophila sNPFs clearly elicited a calcium response in receptor expressing mammalian Chinese hamster ovary cells. The response is dose-dependent and appeared to be very specific. The short NPF receptor was not activated by any of the other tested arthropod peptides, not even by FMRFamide-related peptides (also ending in RFamide), indicating that the Arg residue at position 4 from the amidated C-terminus appears to be crucial for the response elicited by the sNPFs.