Jae H. Park

Hemolymph sugar homeostasis and starvation-induced hyperactivity affected by genetic manipulations of the adipokinetic hormone-encoding gene in Drosophila melanogaster.

Adipokinetic hormones (AKHs) are metabolic neuropeptides, mediating mobilization of energy substrates from the fat body in many insects. In delving into the roles of the Drosophila Akh (dAkh) gene, its developmental expression patterns were examined and the physiological functions of the AKH-producing neurons were investigated using animals devoid of AKH neurons and ones with ectopically expressing dAkh. The dAkh gene is expressed exclusively in the corpora cardiaca from late embryos to adult stages. Projections emanating from the AKH neurons indicated that AKH has multiple target tissues as follows: the prothoracic gland and aorta in the larva and the crop and brain in the adult. Studies using transgenic manipulations of the dAkh gene demonstrated that AKH induced both hypertrehalosemia and hyperlipemia. Starved wild-type flies displayed prolonged hyperactivity prior to death; this novel behavioral pattern could be associated with food-searching activities in response to starvation. In contrast, flies devoid of AKH neurons not only lacked this type of hyperactivity, but also displayed strong resistance to starvation-induced death. From these findings, we propose another role for AKH in the regulation of starvation-induced foraging behavior.

Developmental Control of Foraging and Social Behavior by the Drosophila Neuropeptide Y-like System

Animals display stereotyped behavioral modifications during development, but little is known about how genes and neural circuits are regulated to turn on/off behaviors. Here we report that Drosophila neuropeptide F (dNPF), a human NPY homolog, coordinates larval behavioral changes during development. The brain expression of npf is high in larvae attracted to food, whereas its downregulation coincides with the onset of behaviors of older larvae, including food aversion, hypermobility, and cooperative burrowing. Loss of dNPF signaling in young transgenic larvae led to the premature display of behavioral phenotypes associated with older larvae. Conversely, dNPF overexpression in older larvae prolonged feeding, and suppressed hypermobility and cooperative burrowing behaviors. The dNPF system provides a new paradigm for studying the central control of cooperative behavior.

Targeted ablation of CCAP neuropeptide-containing neurons of Drosophila causes specific defects in execution and circadian timing of ecdysis behavior.

Insect growth and metamorphosis is punctuated by molts, during which a new cuticle is produced. Every molt culminates in ecdysis, the shedding of the remains of the old cuticle. Both the timing of ecdysis relative to the molt and the actual execution of this vital insect behavior are under peptidergic neuronal control. Based on studies in the moth, Manduca sexta, it has been postulated that the neuropeptide Crustacean cardioactive peptide (CCAP) plays a key role in the initiation of the ecdysis motor program. We have used Drosophila bearing targeted ablations of CCAP neurons (CCAP KO animals) to investigate the role of CCAP in the execution and circadian regulation of ecdysis. CCAP KO animals showed specific defects at ecdysis, yet the severity and nature of the defects varied at different developmental stages. The majority of CCAP KO animals died at the pupal stage from the failure of pupal ecdysis, whereas larval ecdysis and adult eclosion behaviors showed only subtle defects. Interestingly, the most severe failure seen at eclosion appeared to be in a function required for abdominal inflation, which could be cardioactive in nature. Although CCAP KO populations exhibited circadian eclosion rhythms, the daily distribution of eclosion events (i.e., gating) was abnormal. Effects on the execution of ecdysis and its circadian regulation indicate that CCAP is a key regulator of the behavior. Nevertheless, an unexpected finding of this work is that the primary functions of CCAP as well as its importance in the control of ecdysis behaviors may change during the postembryonic development of Drosophila.

ISOLATION AND CHRONOBIOLOGICAL ANALYSIS OF A NEUROPEPTIDE PIGMENT-DISPERSING FACTOR GENE IN DROSOPHILA MELANOGASTER

In this article, the authors isolate a gene encoding a neuropeptide in Drosophila melanogaster. The substance is called pigment-dispersing factor (PDF), based on one of the roles it plays in crustaceans (the arthropods in which this factor was initially discovered). The PDF-encoding Drosophila gene (pdf) is intronless and present in a single copy per haploid genome. The cytological location of pdf is 97B on the third chromosome. The putative 102-amino-acid precursor (prepro-PDF) consists of a signal peptide and a PDF-associated peptide, followed by the mature PDF. The PDF-associated peptide region of the precursor is highly diverged from those of the crustacean precursors, whereas the primary structure of the mature PDF is conserved in other members of the pigment-dispersing hormone family. A single pdf transcript (ca. 0.8 kb) is expressed predominantly in the head; the expression levels of pdf mRNA are consistently higher in males than in females. Putative pdf homologous transcripts are present in other Drosophila species, which exhibit similar sexual dimorphic expression patterns. Cyclic expression of pdf over the course of the day and night was assessed, but the mRNA exhibited at best very gentle cycling. The pdfexpression in two behaviorally arrhythmic mutants were examined; the expression was intact in a period0 mutant but absent in the disconnected mutant.

Sex- and clock-controlled expression of the neuropeptide F gene in Drosophila

Drosophila neuropeptide F (NPF), a homolog of vertebrate neuropeptide Y, functions in feeding and coordination of behavioral changes in larvae and in modulation of alcohol sensitivity in adults, suggesting diverse roles for this peptide. To gain more insight into adult-specific NPF neuronal functions, we studied how npf expression is regulated in the adult brain. Here, we report that npf expression is regulated in both sex-nonspecific and male-specific manners. Our data show that male-specific npf (ms-npf) expression is controlled by the transformer (tra)-dependent sex-determination pathway. Furthermore, fruitless, one of the major genes functioning downstream of tra, is apparently an upstream regulator of ms-npf transcription. Males lacking ms-npf expression (through traF-mediated feminization) or npf-ablated male flies display significantly reduced male courtship activity, suggesting that one function of ms-npf neurons is to modulate fruitless-regulated sexual behavior. Interestingly, one of the ms-npf neuronal groups belongs to the previously defined clock-controlling dorsolateral neurons. Such ms-npf expression in the dorsolateral neurons is absent in arrhythmic ClockJrk and cycle02 mutants, suggesting that npf is under dual regulation by circadian and sex-determining factors. Based on these data, we propose that NPF also plays a role in clock-controlled sexual dimorphism in adult Drosophila.

DOUBLESEX GENE EXPRESSION IN THE CENTRAL NERVOUS SYSTEM OF DROSOPHILA MELANOGASTER

Despite several behavior-genetic studies that have suggested roles played by doublesex ( dsx ) in neural tissues, it has not been demonstrated that the products of this gene are actually present in the central nervous system (CNS). In this report, we describe the cellular, spatial, and temporal expression patterns of dsx gene products in the developing and adult CNS by applying RT-PCR and immunohistochemical procedures. dsx gene products were detected in the CNS of 3rd-instar larvae, pupae, and adults. DSX-immunoreactive signals were observed within the brain and in both the thoracic plus abdominal ganglia of the ventral nerve cord. Most, but not all, cells inferred to contain DSX proteins (by the results of genetic controls for antibody specificity) were further determined to be neurons (by coexpression of a protein that marks such CNS cell types). Temporally varying expression of DSX was most prominently observed in the rapidly metamorphosing early and mid-pupal stages, suggesting that this gene contributes to establishment of sexually dimorphic neuronal structures which subserve adult sexual behaviors. Elements of the spatial and temporal patterns of DSX immunoreactivity also imply that sexually dimorphic dsx expression in certain neuronal clusters within the adult CNS could participate in ongoing operations of the mature nervous system with respect to the courtship behaviors that are affected by dsx mutations.

Programmed cell death mechanisms of identifiable peptidergic neurons in Drosophila melanogaster

The molecular basis of programmed cell death (PCD) of neurons during early metamorphic development of the central nervous system (CNS) in Drosophila melanogaster are largely unknown, in part owing to the lack of appropriate model systems. Here, we provide evidence showing that a group of neurons (vCrz) that express neuropeptide Corazonin (Crz) gene in the ventral nerve cord of the larval CNS undergo programmed death within 6 hours of the onset of metamorphosis. The death was prevented by targeted expression of caspase inhibitor p35, suggesting that these larval neurons are eliminated via a caspase-dependent pathway. Genetic and transgenic disruptions of ecdysone signal transduction involving ecdysone receptor-B (EcR-B) isoforms suppressed vCrz death, whereas transgenic re-introduction of either EcR-B1 or EcR-B2 isoform into the EcR-B-null mutant resumed normal death. Expression of reaper in vCrz neurons and suppression of vCrz-cell death in a reaper-null mutant suggest that reaper functions are required for the death, while no apparent role was found for hid or grim as a death promoter. Our data further suggest that diap1 does not play a role as a central regulator of the PCD of vCrz neurons. Significant delay of vCrz-cell death was observed in mutants that lack dronc or dark functions, indicating that formation of an apoptosome is necessary, but not sufficient, for timely execution of the death. These results suggest that activated ecdysone signaling determines precise developmental timing of the neuronal degeneration during early metamorphosis, and that subsequent reaper-mediated caspase activation occurs through a novel DIAP1-independent pathway.

Comparative analysis of Corazonin-encoding genes (Crz's) in Drosophila species and functional insights into Crz-expressing neurons.

To gain insight into regulatory mechanisms of tissue‐specific Corazonin (Crz) gene expression and its functions in Drosophila, we cloned the Crz genes from four Drosophila species (D. melanogaster, D. simulans, D. erecta, and D. virilis) and performed comparative analyses of Crz gene sequences and expression patterns using in situ hybridization and immunohistochemistry. Although Crz gene sequences showed a great deal of diversity, its expression patterns in the CNS were highly conserved in the Drosophila species examined here. In D. melanogaster larva, Crz expression was found in four pairs of neurons per cerebral lobe and in eight pairs of bilateral neurons in the ventral nerve cord; in adult, the number of Crz‐producing neurons increased to 6–8 in the pars lateralis of each brain lobe, whereas neurons in the ventral nerve cord were no longer detectable. Crz transcripts were also found in the optic lobes; however, these mRNAs do not seem to be translated. Such adult‐like Crz expression patterns were established within 48 hours after pupation. Somata of Crz‐neurons in the pars lateralis are located in the vicinity of terminals emanating from PDF‐containing pacemaking neurons, indicating a functional connection between the two peptidergic nervous systems. A subset of Crz neurons coexpressed the period clock gene; however, normal Crz transcription was unaffected by central clockworks. Two pairs of ectopic Crz cells were detected in the adult brains of behaviorally arrhythmic ClockJrk or cycle02 mutants, suggesting that CLOCK and CYCLE proteins negatively regulate Crz transcription in a cell‐specific manner. J. Comp. Neurol. 482:372–385, 2005.

Agonist-Induced Endocytosis and Receptor Phosphorylation Mediate Resensitization of Dopamine D2 Receptors

The regulatory mechanisms and functional roles of agonist-induced internalization of G protein-coupled receptors (GPCRs) were analyzed using mutant dopamine D(2) receptors (D(2)Rs) in which all possible GPCR kinase (GRK) phosphorylation sites were mutated or the affinity for beta-arrestins was altered. Agonist-induced internalization of D(2)Rs involved a phosphorylation-dependent component, which was mediated by serine/threonine (S/T) residues in the second loop and T225 in the third loop, and a phosphorylation-independent component. GRK2-mediated enhancement of the internalization and inhibition of D(2)R signaling did not involve receptor phosphorylation, and only the former required the enzymatic activity of GRK2. The phosphorylation-deficient mutant (D(2)R-intracellular loop 2/3) recycled more slowly and showed more agonist-induced desensitization than did the wild-type D(2)R, suggesting that receptor phosphorylation mediates the recycling of the internalized receptors and enhances receptor resensitization. Blockade of the agonist-induced internalization of D(2)R-intracellular loop 2/3 provoked desensitization as in wild-type D(2)R, suggesting that certain cellular processes other than receptor dephosphorylation occurring within the endocytic vesicle are responsible for the resensitization of D(2)R. When dissociation between D(2)R and beta-arrestin was inhibited or when the expression of cellular beta-arrestins was decreased, agonist-induced desensitization of D(2)R did not occur, suggesting that dissociation from beta-arrestin is the main cellular process required for resensitization of D(2)R and is achieved through agonist-induced internalization. These results indicate that, in the regulation of some GPCRs, phosphorylation-independent association with beta-arrestin plays a major role in agonist-induced desensitization.

The Bax/Bak ortholog in Drosophila, Debcl, exerts limited control over programmed cell death

Bcl-2 family members are pivotal regulators of programmed cell death (PCD). In mammals, pro-apoptotic Bcl-2 family members initiate early apoptotic signals by causing the release of cytochrome c from the mitochondria, a step necessary for the initiation of the caspase cascade. Worms and flies do not show a requirement for cytochrome c during apoptosis, but both model systems express pro- and anti-apoptotic Bcl-2 family members. Drosophila encodes two Bcl-2 family members, Debcl (pro-apoptotic) and Buffy (anti-apoptotic). To understand the role of Debcl in Drosophila apoptosis, we produced authentic null alleles at this locus. Although gross development and lifespans were unaffected, we found that Debcl was required for pruning cells in the developing central nervous system. debcl genetically interacted with the ced-4/Apaf1 counterpart dark, but was not required for killing by RHG (Reaper, Hid, Grim) proteins. We found that debclKO mutants were unaffected for mitochondrial density or volume but, surprisingly, in a model of caspase-independent cell death, heterologous killing by murine Bax required debcl to exert its pro-apoptotic activity. Therefore, although debcl functions as a limited effector of PCD during normal Drosophila development, it can be effectively recruited for killing by mammalian members of the Bcl-2 gene family.