TENTH ANNUAL EUROPEAN MEETING ON THE NEUROGENETICS OF DROSOPHILA

Neuchâtel, Switzerland 4–8 September, 2004

REAL-TIME GLIOGENESIS IN THE WHOLE FLY: A NOVEL MODEL TO STUDY MIGRATION AND PROLIFERATION

BENOIT AIGOUY, VÉRONIQUE VAN DE BOR, MARCEL BOEGLIN, and ANGELA GIANGRANDE

Institut de Génétique et Biologie Moléculaire et Cellulaire, 67404 Illkirch, France

Migration and proliferation have been mostly explored in culture systems or fixed preparations. We have developed a simple genetic model, involving chains of glia moving along fly wing nerves, to follow such dynamic processes by time lapse in the whole animal. We show that glia undergo extensive cytoskeleton and mitotic apparatus rearrangements orienting them along underlying axons during division and migration. Single-cell labeling identifies different glia: pioneers with high filopodial (exploratory) activity and less active followers. During migration and proliferation, glia contact axons and other glia. In combination with time-lapse, altering this cellular environment by genetic means or ablation of single cells has allowed to us define the role of specific cell interactions in different aspects of glial development. Neuron-glia interactions control the direction of migration, whereas repulsive interactions between overlapping glia control the extent of movement. Finally, autonomous cues control proliferation, which defines the final glial cell number.

DUAL ROLE FOR CK2β IN THE DROSOPHILA CIRCADIAN OSCILLATOR

BIKEM AKTEN, EIKE JAUCH, GABI PUTZ, MARTIN HEISENBERG, THOMAS RAABE, and F. ROB JACKSON

Department of Neuroscience, Tufts University School of Medicine, Boston, MA 20111, USA

In Drosophila melanogaster, the oscillatory abundance of central clock proteins, such as PERIOD (PER) and TIMELESS (TIM), is maintained by phosphorylation events, which are required for their nuclear entry and cellular stability. These events are sustained by several kinases including Casein Kinase 2 (CK2), which consists of a regulatory β subunit (CK2β) and a catalytic α subunit (CK2α). Andante mutants carry a point mutation within a conserved domain of CK2β known to be important for its interaction with CK2α. Our studies have shown that Andante mutants display a long-period rhythm phenotype similar to CK2α mutants, and the two act together to regulate nuclear translocation of PER and TIM. We have now shown that CK2β can physically and genetically interact with another phosphorylating enzyme called ribosomal kinase S6K2. In mammals, S6K2 is known to be a light- and clock-regulated kinase activated by the MAPK pathway. In Drosophila, S6K2 mRNA oscillates in abundance, suggesting a similar function. We show that S6K2 mutants display a short-period rhythm phenotype, which is rescued by transgenic expression of the gene. Furthermore, S6K2 function is required within clock cells, as overexpression in these cells rescues the rhythm phenotype. Finally, we show that Andante is epistatic to S6K2, indicating that S6K2 regulates CK2β activity. We are currently investigating how this interaction affects the molecular clock to better understand the dual role of CK2β in the Drosophila circadian oscillator.

MECHANICS OF MECHANOSENSORY ADAPTATION IN DROSOPHILA

JOERG T. ALBERT and MARTIN GÖPFERT

Institute of Zoology/Animal Physiology, University of Cologne, 50923 Cologne, Germany

Adaptation is a vital feature of sensation, preserving sensitivity during sustained stimulation. Vertebrate hair cells display an unconventional form of sensory adaptation, which is based on a series of distinct micromechanical events. The gating spring model provides a mechanistic concept of how this adaptation works. The model assumes that the pull of a gating spring opens the mechanotransduction channels. Adaptation then actively adjusts the tension of this spring in either of two ways: by a rapid, calcium-dependent reclosure of the transducer channels (fast adaptation) or by a slower, myosin-dependent displacement of the spring-channel complex itself (slow adaptation). We have identified the mechanical key signatures of hair cell adaptation in the antennal hearing organ of the fly. Mutant analysis shows that both fast and slow adaptation occur in Drosophila and depend on (i) the physiological condition of the animal, (ii) the integrity of the neurons, and (iii) the proper function of the transduction machinery, including the mechanotransduction channels. These findings put forward Drosophila for the study of hair cell adaptation and suggest the gating spring model as a general concept in mechanosensation. Overexpression of the Ip3kinase gene at olfactory receptor neurons modifies olfactory perception in Drosophila melanogaster.

THE CAMP TRANSDUCTION CASCADE IN THE OLFACTORY RECEPTION OF DROSOPHILA MELANOGASTER

ESTHER ALCORTA, CAROLINA GOMEZ-DIAZ, and MARTIN FERNANDO

Department of Functional Biology University of Oviedo, 33.006 Oviedo, Spain

Two main transduction pathways depending on the second messengers IP3 and cAMP, respectively, appear to be involved in olfactory reception. Expression data of intermediate genes of the cAMP transduction cascade, as well as changes in electrophysiological responses to odorants at the olfactory receptor organs of mutants for these genes, suggested a role for this route in olfactory reception. However, analysis of behavioral data should overcome the problem of non-specific gene expression at the receptor level. Oour studies are aimed at directly relating the cAMP transduction pathway at the reception level and the olfactory perception deduced from behavioral experiments. Using the Gal4/UAS system for overexpressing the dnc gene in different olfactory receptor neuron subsets we were able to generate restricted dominant mutations. Abnormal olfactory behavior in response to ethanol, acetone or ethyl acetate was found in 62.5% of the 8 hybrid Gal4/UAS-dnc tested stocks, depending on the affected neuronal subset and according to the correspondent olfactory neuron specificity profile. The high ratio of affected lines suggests that cAMP transduction cascade mediation of olfactory reception is a general process. Finally, these results are compared to the extent of behavioral changes induced by acting on the IP3 route.

A ROLE FOR THE ION CHANNEL NARROW ABDOMEN IN NEURAL OUTPUT OF THE DROSOPHILA CIRCADIAN PACEMAKER

RAVI ALLADA and BRIDGET LEAR

Department of Neurobiology, Northwestern University, Evanston, IL 60208, USA

In flies and mammals, conserved transcriptional feedback loops drive circadian behaviors, yet little is known about how these components regulate neuronal activity relevant to behavior. We have examined loss-of-function mutants of a conserved Drosophila ion channel, narrow abdomen (na). na mutants fail to increase activity in anticipation of light-dark (LD) transitions and exhibit weak locomotor rhythms during constant darkness (DD). Using tissue-specific rescue, we find that na expression within circadian neurons is sufficient to fully rescue both the LD anticipation and DD rhythmicity phenotypes. However, the core clock component, PERIOD, continues to oscillate in the pacemaker neurons of na mutants in LD and DD. The neuropeptide PDF is essential for robust rhythmic behavior and is rhythmically expressed in pacemaker neuron projections. In na mutants, PDF expression is substantially elevated consistent with a reduction in neuropeptide release. We propose that this channel serves as a critical link between transcriptional feedback loops and neural outputs in pacemaker neurons.

NEW SCREENS FOR GENES CONTROLLING GLIAL DEVELOPMENT IN THE EMBRYONIC NERVOUS SYSTEM

BENJAMIN ALTENHEIN

Institute for Genetics, University of Mainz, 55128 Mainz, Germany

Glial cells play an essential role in the correct formation of the CNS. Though many aspects of neuronal development and the proper guidance of their axons has been described in detail, only little is known about the developmental program underlying glial cell differentiation. Two different screens were performed in our lab in order to identify genes involved in different aspects of gliogenesis. An EMS mutagenesis screen was designed which allows an efficient detection and evaluation of mutant phenotypes. The Gal4-UAS system was used to drive a nuclear GFP in glial cells. After mutagenesis the stocks were rebalanced with a newly created Gal80 balancer, thus enabling us to easily identify homozygous mutant embryos. Two thousand and one hundred (2,100) mutant stocks were screened with respect to aberrations in the development and migration of glial cells. Several classes of phenotypes were observed and glial specific ones were further characterized. The second screen is based on the microarray technique and its focus lies on downstream targets of the master gene in gliogenesis, glial cells missing (gcm). This screen comprises for the first time the gcm loss-of-function in comparison to wild-type embryos, as well as ectopic expression of gcm. Both were independently performed in time course experiments through embryogenesis with six to eight different time points.

EVOLUTION OF EYES AND PHOTORECEPTOR CELLS IN BILATERIA

DETLEV ARENDT

European Molecular Biology Laboratory, 69012 Heidelberg, Germany

We investigated brain and eye development at trochophora larval stages in the ragworm Platynereis dumerilii (Polychaeta, Annelida, Lophotrochozoa) to detect similarities and differences with insects and vertebrates, and to learn from this about the evolution of photosensitive-neuroendocrine systems in the Bilateria. Morphological and molecular approaches are combined in a novel Evo-devo approach, the molecular comparison of cell types (“comparative molecular cell biology”). For example, we find that ciliary photoreceptor cells detected in the Platynereis brain, and the ciliary photoreceptor cells of the vertebrate retina, the rods and cones, share a conserved molecular fingerprint – expressing orthologous transcription factors for their specification as well as a conserved, orthologous subtype of the opsin photopigment. The ciliary photoreceptor cell type with ciliary opsin is present in Anopheles but has been lost in Drosophila. The rhabdomeric photoreceptor cells of the Platynereis eyes share their molecular fingerprint with the rhabdomeric insect photoreceptors, and with the ganglion cells of the vertebrate retina. We thus have begun to unravel homologies of cell types across Bilateria that will help reconstruct the evolution of eyes and brains in Bilateria.

IDENTIFICATION OF A CANDIDATE GENE INVOLVED IN AXON GUIDANCE AT THE MIDLINE

HANNA ASLAM and GUY TEAR

Department of Neurobiology, Kings College, London SE1 1UL, UK

During embryonic development, axons on one side of the nervous system must recognize and choose between crossing or not crossing the embryonic midline. Commissural axons rarely re-cross the midline. To secure this structure, the extra-cellular matrix family of Slit proteins mediates a local repulsive function at the midline through the receptor Roundabout (Robo). To enable commissural axons to cross the midline Robo levels are downregulated in these axons and then up-regulated after crossing. This downregulation process involves the transmembrane protein, Commissureless (Comm) which reduces cell surface levels of Robo, thereby allowing axons to cross the midline. However, the exact mechanism behind this downregulation is unknown. Flies were mutagenized and analysed for axon guidance defects. From this screen we have identified two allelic mutants in which axons appear to cross and recross the midline, a phenotype similarly found in robo mutants. These mutations were mapped to the 26D region. We identified a P-element (insert) allele, which led to identification of a candidate gene, CG9535. It encodes a product with UDP-N-acetylglucosamine diphosporylase activity. Glycosylation is a common and complex form of post-translational protein modification and is the most abundant modification seen in nature.

FUNCTIONAL ANALYSIS OF THE DROSOPHILA HOMOLOG OF HUMAN GIT1

SAMI BAHRI, FOOK SION HING, FEN GOH YU, EDWARD MANSER, LOUIS LIM, and XIAOHANG YANG

Institute of Molecular and Cell Biology, 117609 Singapore

GIT1 is a mammalian G-protein-coupled receptor kinase-interacting protein that controls a variety of cellular functions in cell culture assays, including synapse formation, focal adhesions, cell motility and endocytosis. The Drosophila genome contains a single GIT1 homologous gene, dgit, and like its mammalian counterparts, it encodes a putative multidomain protein comprising an ARF-GAP domain in its N-terminus followed by four ankyrin repeats, a SHD domain and a C-terminal GRK-binding region. In the late embryo, dgit RNA is heavily enriched in larval muscles but not CNS. In order to characterize the function of dgit in vivo, we have carried out a P-element mutagenesis screen to isolate dgit mutant alleles and to characterize their phenotypic consequences on animal development. Out of ∼ 200 excisions, we obtained three alleles that delete into the coding region of dgit. The extent of DNA lesions in these alleles were determined by PCR and sequencing. Preliminary characterizations of the dgit mutants show that they are largely homozygous viable, flightless, and exhibit wing-wrinkling defects at 100% penetrance. Homozygous dgit mutant females also show reduced fertility. The dgit mutants will be characterized with various muscle and synaptic markers.

SYNAPTIC STRENGTHENING MEDIATED BY BMP-DEPENDENT RETROGRADE SIGNALING IN THE DROSOPHILA CNS

RICHARD BAINES

Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK

Retrograde signaling is an essential component of synaptic development and physiology. Previous studies show that BMP-dependent retrograde signalling is required for the proper development of the neuromuscular junction (nmj) in Drosophila. These studies, moreover, raised the significant possibility that the development of central motor circuitry might similarly be reliant on such signalling. To test this hypothesis, retrograde signalling between postsynaptic motoneurons and their presynaptic interneurons was examined. Increased postsynaptic cAMP, the result of targeted expression of an adenylate cylase, encoded by rutabaga, is sufficient to strengthen synaptic transmission at these identified central synapses. We have obtained results showing that the underlying mechanism is dependent on BMP retrograde signalling. Thus, presynaptic expression of an activated TGF-beta receptor, Thickvien (Tkv), or postsynaptic expression of a TGF-beta ligand, Glass bottom-boat (Gbb), is sufficient to phenocopy strengthening of synaptic transmission. In the absence of Gbb, endogenous synaptic transmission is significantly weakened; moreover, increased postsynaptic cAMP is unable to potentiate synaptic function. Potentiation of presynaptic neuro-transmitter release, mediated by increased postsynaptic expression of Gbb, is dependent on normal cholinergic activity, indicative that either the secretion of this retrograde signal, or its transduction, is activity-dependent.

GLIAL DEVELOPMENT IN DROSOPHILA: A GENOME-WIDE SCREEN FOR GLIAL GENES

ANGELA BECKER, B. ALTENHEIN, B. BECKMANN, J. HOHEISEL, and GERHARD TECHNAU

Institute for Genetics, University of Mainz, 55128 Mainz, Germany

Expression of the master regulator gene glial cells missing (gcm) induces gliogenesis in the embryonic CNS of Drosophila. Whereas the function of gcm as a binary switch between neuronal and glial fate is well described, little is known about further genes that maintain and regulate glial differentiation. In order to identifying genes acting downstream of gcm, we performed a genome-wide microarray screen. We performed two timecourse experiments throughout embryogenesis. RNA was extracted from up to eight different stages from embryos with ectopic expression of gcm in the entire CNS (thus transforming most CNS cells into glial cells) and from mutant embryos, which lack gcm function (and thus lack all lateral glial cells). Both were compared with wild type on a whole-genome microarray (Flyarray, Heidelberg) comprising all 20,000 predicted genes plus 2,600 novel annoted genes. At least four independent repeat experiments were done for each stage compared. Microarray data were sorted with respect to strength and profile of differential expression, overlap between both experiments, and clustering according to function.

IDENTIFICATION AND CHARACTERIZATION OF GENES INVOLVED IN GLIAL CELL DIFFERENTIATION AND MIGRATION

RUTH MARIE BECKERVORDERSANDFORTH, DIANA CLEPPIEN, OLAF VEF, BENJAMIN ALTENHEIN, and GERHARD TECHNAU

Institute of Genetics, University of Mainz, 55128 Mainz, Germany

To identify genes involved in glial cell differentiation and migration, we designed an EMS mutagenesis screen for the second chromosome. Several genetic tools have been newly combined to make the screening procedure faster and more efficient. Using GFP as an in vivo marker for visualizing the glial cells guaranteed a fast and inexpensive screening procedure and therefore an efficient selection of mutants. We used the GAL4/UAS system to pattern the GFP expression. In several steps DTS chromosomes were used to facilitate the crossings. To identify the mutants in vivo we produced a Gal80 balancer. As Gal80 acts as a repressor of Gal4-driven GFP expression is only detectable in homozygous mutants. About 2,100 lethal mutations were generated and the stocks were screened for their phenotype. Among different interesting phenotypes we chose those lines that show a severe reduction in the number of glial cells and some with clear migration defects for further analysis. Phenotypes of these mutants are being analyzed, as revealed by antibody staining against glial and neuronal proteins and confocal microscopy; and the genetics of these variants is being effected by cytological mapping of the mutations and complementation analysis.

SQUEEZE DETERMINES IDENTITY OF CORAZONIN NEUROSECRETORY CELLS

JONATHAN BENITO, MARTA MAGARIÑOS, PILAR HERRERO PILAR, IMMACULADA CANAL LAURA TORROJA LAURA, and ISABEL MOLINA

Universidad Autónoma de Madrid, 28049 Madrid, Spain

Corazonin is a neuropeptide able that is to act as a neurotransmitter and as neurohormone in all insects in which it has been studied. Based on its expression pattern in Drosophila, corazonin has been postulated to act as a neurohormone in neurosecretory cells from the lateral protocerebrum or as a neurotransmitter in interneurons from the ventral ganglion. In spite of its well-studied expression pattern in a variety of insects, the general biological function carried out by corazonin is not well understood. Previous work has shown that expression of the neuropeptide FMRFamide in Tv cells require the presence of Apterous and Squeeze, two transcription factors involved in determining neuronal identity. Corazonin and Squeeze colocalize in all the expression domain of corazonin. Our results show that squeeze mutants present a decrease in the number of corazonin-expressing cells in the lateral protocerebrum, which innervate the corpora cardiaca. Interneurons expressing corazonin in the ventral ganglia seem not to be affected in squeeze mutants. Corazonin presence is independent of Apterous in both neurosecretory and interneurons cells.

A CRITICAL ROLE FOR CYCLIN E IN GENERATING SEGMENT-SPECIFIC DIFFERENCES IN NEUROBLAST LINEAGES IN DROSOPHILA

CHRISTIAN BERGER, S.K. PALLAVI, P. MOHIT, L.S. SHASHIDHARA, and GERHARD TECHNAU

Institute of Genetics, University of Mainz, 55099 Mainz, Germany; Center for Cellular and Molecular Biology, 500 007 Hyderabad, India

The mechanisms leading to cell diversity in the central nervous system (CNS) represent a major problem in developmental biology. Here we have examined the mechanism by which segment-specific differences are generated within a neuroblast lineage. In thoracic segments, the NB6-4t lineage generates 4 to 6 neurons and 3 glial cells, while in abdominal segments it (NB6-4a) gives rise to only two glial cells (Schmidt et al., Devel. Biol. 189, 186–204, 1997). The first division of NB6-4t is asymmetric, leading to the expression of the pros and gcm genes only in glial precursor cells. NB6-4a undergoes a symmetric division, directly giving rise to two glial cells, both of which express pros and gcm (Akiyama-Oda et al., Development 126, 1967–1674, 1999; Akiyama-Oda et al., Development 127, 3513–3522, 2000; Freeman & Doe, Development 128, 4103–4112, 2001). We show that NB6-4t represents the ground state, which does not require input of any homeotic genes, whereas NB6-4a is specified by homeotic genes abdA and AbdB. They specify the 6-4a lineage by down-regulating the levels of CycE. We also provide evidence that, independent of its role in cell proliferation, CycE plays a direct role in specifying the NB6-4t lineage. Loss of CycE gene function leads to the homeotic transformation of NB6-4t to NB6-4a, whereas ectopic CycE expression in abdominal segments causes reverse transformations. CycE is required and sufficient to down-regulate pros and gcm in the neuronal lineages of 6-4t. Corresponding phenotypes were observed in loss- and gain-of-function genetic backgrounds involving factors that directly interact with CycE function (e.g., dacapo, E2f), but not of genes involved in regulating G1-S or G2-M transition (e.g., Rbf, CycA, cdc2).

CENTROPHOBISM/THIGMOTAXIS BEHAVIOR, A NEW ROLE FOR THE MUSHROOM BODIES IN DROSOPHILA

MORGANE BESSON, CHRISTINE SEMINATORE and JEAN-RENÉ MARTIN

NAMC, CNRS, UMR-8620, Université Paris-Sud, 91405 Orsay, France

Insects like vertebrates exhibit spatially complex locomotor activity pattern when foraging and navigating. Recently, as in mammals, in an open-field situation it has been shown that Drosophila presents avoidance of a center zone and, conversely, a natural proclivity to stay near the perimeters, an effect that can be interpreted as centrophobism/ thigmotaxis. Here, we further characterized this center zone avoidance effect and studied the underlying neural mechanisms that can support such behavior. In Drosophila, mushroom bodies (MBs) are well characterized for their implication in olfactory learning and memory. In an open-field situation in which the fly locomotor activity is recorded by video-tracking, we show that this center avoidance is greatly diminished in hydroxyurea-ablated MBs flies, as well as in mutants known to affect MBs function, suggesting a new role for the MBs. Furthermore, using enhancer-trap P[GAL4] lines specific of the different subgroup of MB lobes, to target the temperature-sensitive allele of the Drosophila dynamin gene shibire and consequently disrupt synaptic transmission, we show that the centrophobism/thigmotaxis is changed when we specifically block the gamma lobes, while blocking the alpha/beta lobes does not lead to such an effect, suggesting a functional specialization of MBs lobes. Thus, Drosophila may serve as a new model system for elucidating the genetic and the neural basis of such complex centrophobism/thigmotaxis phenomena.

ASYMMETRIC LOCALIZATION OF NUMB IS ESSENTIAL FOR DROSOPHILA SENSE-ORGAN DEVELOPMENT

SHEETAL BHALERAO, DANIELA BERDNIK, and JUERGEN KNOBLICH

Institute of Molecular Pathology, 1030 Vienna, Austria

Drosophila external sensory (ES) organs consist of a hair, socket, neuron, and sheath cell, which arise from a single sensory organ precursor (SOP) in two rounds of asymmetric cell divisions. During each of these divisions, Numb protein localizes asymmetrically and segregates into one of the two daughter cells. In numb mutants these divisions become symmetric, leading to the formation of four socket cells. Conversely, overexpression of Numb leads to the formation of four neurons. Although this qualifies Numb as a segregating determinant, it is unclear whether Numb has to localize asymmetrically during mitosis to specify correct cell fates in the ES lineage. In a genetic screen for mutants affecting ES organ development, we identified several new numb alleles. In one of these, Numb is present but fails to localize asymmetrically during mitosis. The mutation maps to a single amino acid in an N-terminal region of Numb that is necessary and sufficient for asymmetric localization. The mutant protein can interact with α-Adaptin and Sanpodo, the known downstream interaction partners of Numb. Overexpression of the mutant protein leads to the characteristic four-neuron phenotype. Although this indicates that the mutant protein is functional, the mutants show a fully penetrant loss-of-function four-socket phenotype. Our results suggest that it is not the presence of Numb but rather its asymmetric distribution between the two daughter cells that is responsible for cell-fate determination.

DIVERSE FUNCTIONS FOR FRUITLESS-M PROTEINS IN THE DEVELOPMENT OF A SEXUALLY DIMORPHIC CNS

JEAN-CHRISTOPHE BILLETER, DONALD A. GAILEY, and STEPHEN F. GOODWIN

Anderson College, University of Glasgow, Glasgow G11 6NU, UK

In Drosophila, most aspects of male reproductive behavior are determined by fruitless (fru). A class of fru transcripts restricted to ca. 2000 pupal and adult CNS neurons is translated in males only. These FruM proteins are BTB-Zinc finger transcription factors. The 3′ends of fru transcripts are spliced to introduce alternative exons (A, B or C) encoding Zn-finger motifs resulting in FruMA, MB and MC isoforms. How do these isoforms establish male reproductive behaviors? To begin to address this question we examined the involvement of FruM isoforms in the development of two male-specific structures: the Muscle of Lawrence, whose development depends on the sex of the motorneuron innervating it, and a cluster of serotonergic neurons innervating the internal reproductive organs. Using a fru-gal4 driver, we expressed each isoform in females and in fru-mutant males. We found that only one is sufficient and necessary for the formation of the MOL in fru males and can ectopically induce MOL formation in females, with a concomitant change in the motorneuron. We observed that each isoform is sufficient to induce differentiation of male-specific serotonergic neurons in females and rescue a subpopulation of the serotonergic neurons in fru mutants. Our data suggest that FruM isoforms act in a context-dependent manner, assuming diverse roles in the differentiation of male-specific neural and neuro-muscular structures involved in reproductive behavior.

TWO DROSOPHILA G-PROTEIN-COUPLED RECEPTORS WITH DIFFERENTIAL TISSUE DISTRIBUTIONS ARE ACTIVATED BY ENDOGENOUS TACHYKININ-RELATED PEPTIDES

RYAN T. BIRSE, ERIK C. JOHNSON, KHADIJEH REZAEI, PAUL H. TAGHERT, and DICK R. NÄSSEL

Department of Zoology, Stockholm University, 10691 Stockholm, Sweden

Neuropeptides related to vertebrate tachykinins have been identified in Drosophila. Two Drosophila G-protein-coupled receptors, designated NKD (CG6515) and DTKR (CG7887), cloned earlier, display sequence similarities to mammalian tachykinin receptors but were until now not characterized with endogenous Drosophila tachykinins (DTKs). We have shown that one of the six DTKs can induce recruitment of barrestin2 via activation of either NKD or DTKR transfected into human embryonic kidney (HEK-293) cells. Furthemore we determined that cells transfected with DTKR display dose-dependent increases in both intracellular calcium and cyclic AMP in response to the different DTKs. However, no responses were seen in NKD-transfected cells. DTKs also induced internalization of NKD- and DTKR-GFP fusion constructs in HEK-293 cells. We generated anti-receptor antisera and showed that the two receptors are distributed in mutually exclusive patterns in the CNS and that only NKD was found in the midgut. Quantitative real-time PCR analysis demonstrated differential levels of mRNA for the two receptors in different tissues. Our findings suggest that NKD and DTKR are the likely DTK receptors. Since the receptors display differential properties in HEK-293 cells and differential distribution in vivo, we propose that Drosophila uses two functionally and spatially separate tachykinin signaling systems.

REGENERATION AFTER CELLULAR DAMAGE IN THE VENTRAL MIDLINE OF THE EMBRYONIC DROSOPHILA CNS

TORSTEN BOSSING and ANDREA BRAND

Wellcome Trust/Cancer Research U.K. Gurdon Institute, Cambridge CB2 1QR, UK

We show that the mechanical damage of undifferentiated midline cells in the Drosophila embryonic CNS causes extra divisions in adjacent midline cells. Cells born by these regenerative divisions replace the damaged cells. Neither neuroblasts, the majority of neural precursors in the CNS, nor their daughter cells (ganglion mother cells) can replace ablated cells. The extra divisions in the midline are not caused by the loss of cell adhesion. Undifferentiated midline siblings are membrane connected and show a high apical accumulation of microtubules. Microtubule depolymerization in undifferentiated midline cells and mechanical damage results in the expression of the mitotic inducer String and abnormal entry into mitosis. With the onset of differentiation the membrane connection, microtubule accumulation, and regenerative capacity are lost. Neither mechanical damage nor microtubule depolymerisation can trigger an abnormal entry into mitosis of differentiated midline cells. Ventral midline cells have the ability to replace damaged siblings by an extra division. Our results indicate that the division might be triggered by a disruption of the microtubule cytoskeleton. The ependymal nature of the ventral midline and its analogy to the vertebrate floorplate and suggests that the ventral midline could serve as a model system to study cellular regeneration in the vertebrate CNS.

DIFFERENTIATION AND CANCER: ATOH1 AND MERKEL CELL CARCINOMA

WOUTER BOSSUYT, PETER MARYNEN PETER, and BASSEM HASSAN

Laboratory of Neurogenetics, Department of Human Genetics, University of Leuven, 3000 Leuven, Belgium

Dedifferentiation is a cardinal process during oncogenesis. We investigate the genetics of a peculiar type of neuronal skin cancer: Merkel cell carcinoma (MCC). Cell lines derived form MCC fall in two groups. One retains the expression of ATOH1, the earliest differentiation marker of Merkel cells; and the other does not. ATOH1 is the human ortholog of the proneural bHLH transcription factor Atonal in Drosophila. Cell lines lacking ATOH1 expression proliferate rapidly with an average duplication time of 20–30 h and show higher activation of the Notch signaling. These cells mimic as such an undifferentiated state. In contrast, cell lines expressing ATOH1 have very little Notch signaling and a duplication rate of more than 90 h. We asked if restoration of ATOH1 expression could inhibit Merkel cell carcinoma. Overexpression of ATOH1 in the aggressive Merkel cell carcinoma cell lines resulted in apoptosis in 90% of the cells. Some cells with resorted ATOH1 expression survived, but these cells proliferated three times slower, thus mimicking those cells with endogenous ATOH1 expression. We suspect that the relation between ATOH1 and Notch is important in this process. Thus, we use Drosophila melanogaster to study the mechanism by which atonal regulates Notch signalling and proliferation.

IDENTIFICATION OF NEW GENETIC ELEMENTS INVOLVED IN OXIDATIVE STRESS-INDUCED NEURODEGENERATION

JOSÉ A BOTELLA, CRISTOPH GRÜNEWALD, and STEPHAN SCHNEUWLY

Institute of Zoology, University of Regensburg, 93051 Regensburg, Germany

Oxidative stress is considered to be a common underlying factor involved in many neurodegenerative diseases. Reactive oxygen species by intracellular reactions to O₂ is the major source of oxidative stress. Aerobic organisms have succeeded in developing cellular mechanisms of defense against this toxicity. Hyperoxia treatment (99.5% O₂) causes a rapid neurodegenerative phenotype in wild-type flies. We have recently reported the isolation of a new gene involved in this process: the Drosophila gene sniffer, which codes for a carbonyl reductase (Botella et al., 2004). sniffer is required for neuronal cell survival, and it is essential to protect flies against the toxic effects of oxygen under normal oxygen conditions. Its overexpression in wild-type flies also induces a strong neuroprotective effect on neurons under hyperoxia conditions. In an effort to identify new genes involved in the process of oxidative stress-induced neurodegeneration, we have performed a screen to identify genomic regions that genetically interact with sniffer. We also carried out a genome-wide expression analysis to identify genes whose expression in brain is altered by hyperoxia treatment. This approach is proving a convenient way to identify new genes involved in the defense against oxidative stress-induced neurodegeneration.

DISSECTING THE ROLE OF SHORT STOP DURING THE DE-NOVO FORMATION OF DENDRITES AND NEUROMUSCULAR TERMINALS

WOLFGANG BOTTENBERG, MICNAEL MENDE MICHAEL, PETER KOLODZIEJ, and ANDREAS PROKOP

School of Biological Sciences, University of Manchester, Manchester M13 9PT, UK; Institute for Genetics, University of Mainz, 55128 Mainz, Germany; Vanderbilt University Medical Center, Nashville, TN 37235, USA

Short stop (Shot) is essential for growth and differentiation of two distinct synaptic compartments of motorneurons, dendrites, and neuromuscular terminals. Shot is a Spectraplakin protein (Röper et al., 2002) composed of Actin-binding, Plakin-like, Spectrin repeat-like, EF-hand and Gas2-like domains. Different isoforms of Shot are expressed differentially and required in diverse developmental processes, which—apart from their requirement at synaptic compartments—include epidermal and tracheal differentiation, along with axonal growth. So far, Shot has been suggested to tether Actin and microtubules (MT) and to regulate MT organization through interaction with MT capping proteins. To address molecular interactions of Shot during the formation of synaptic compartments and regulation of its activity in these contexts, we need to pinpoint the domains/isoforms of Shot required for its localization and/or function at synaptic compartments. To this end, we correlate data from rescue experiments, analysis of different mutant alleles, and stainings with different in-situ probes or antibodies. So far we have clear indications for the requirement of N-terminal Shot domains for the formation of synaptic compartments (see also abstract by Mende et al.).

TIMING OF GENE EXPRESSION DURING DROSOPHILA NEURAL DEVELOPMENT: TEMPORAL AND TERMINAL DETERMINANTS

THOMAS BRODY, ALEXANDER KUZIN, and WARD ODENWALD

Laboratory of Neurochemistry, Neurogentics Unit, National Institutes of Health, Bethesda, MD 20895, USA

During Drosophila neuroblast-lineage development a transcription-factor network, the Hb- > Kr- > Pdm- > Cas- > Gh cascade, regulates temporal transitions in gene expression, resulting in the generation of functionally diverse neural subtypes. We have discovered a number of genes with temporally restricted expression that are potential targets of the temporal network. These factors were found by using a candidate-gene approach through examination of the dynamics of expression of over a hundred known neural determinants. We have assessed the expression of five temporally regulated genes: three of them, Fas2 and Connectin, both axon guidance determinants, and Lin-28, encoding an RNA-binding protein, are expressed in early temporal windows; the other two genes, Tap (the Drosophila NeuroD homolog) and TKR (a BTB domain protein), are expressed late. We have found that Tap is ectopically expressed in Cas mutants. In addition, we have generated Lin-28 mutants by P-element excision. We have also examined the function of the homeodomain transcription factor Prospero in determining gene expression during the terminal divisions of neural precursors. Four gene products—Nerfin-1 (a zinc-finger transcription factor whose mutants exhibit an axon guidance phenotype), Synaptotagmin (a calcium sensor for vesicular fusion), Smooth (an RNA-binding protein), and TKR—are not fully expressed in prospero mutants. This study broadens the known targets and functions of Prospero during CNS development. Finally, we have used bioinformatics to find repeated, clustered, and positionally conserved motifs in putative regulatory sequences (non-coding DNA flanking the ORFs) of genes that encode proteins involved in determination of cell fate and axon guidance of pioneer neurons. We are analyzing a number of motifs shared by these genes that may serve as docking sites for transcriptional regulators acting in common on genes expressed in this early temporal window.

CLARIFYING THE ROLE OF INSULIN PRODUCING CELLS IN THE ADULT DROSOPHILA BRAIN

SUSANNE BUCH, CHRISTPH MELCHER, INGO ZINKE INGO, and MICHAEL PANKRATZ

Institut fu¨r Toxikologie und Genetik, Forschungszentrum Karlsruhe, 76344 Eggenstein-Leopoldshafen, Germany

In Drosophila, seven putative insulin homologs (Dilps) are known; three of them are expressed in a set of neurosecretory cells in the median Drosophila brain called insulin-producing cells (IPCs). Ablation of these cells results in severe changes in larval development. It leads to a developmental delay, growth retardation and larval lethality resembling insulin-receptor mutant phenotype. Flies that succeed in reaching adult stage are smaller and less fertile than the controls. Developmental expression profile of the Dilps showed that mRNA expression levels of dilp2 and 5 do not change during larval development, whereas dilp3 is only weakly expresssed in first instar but increases continuously with each developmental stage. We generated dilp3-promoter-Gal4 lines, crossed them with UAS-rpr to destroy the mNSCs. Analysis showed that there was no ablation of cells in dilp3-Gal4/UAS-rpr larvae, but there was a partial ablation of the mNSCs in the adult fly. The next step was to determine whether this has an effect on the adult. Additionally, another approach was made to disturb the function of the cells by introducing UAS-TeTxLc. This is thought to inhibit neuronal signalling by blocking synaptic vesicle secretion. Microarray analysis of these two crosses revealed that there are different groups of genes affected. Ongoing feeding experiments and phenotypical studies concerning fertility and lifespan might reveal the function of the Drosophila insulin in growth, aging, and reproduction.

THE SEGMENT POLARITY GENE MIDLINE IS REQUIRED FOR CNS FORMATION AND VENTRAL EPIDERMAL PATTERNING

MARITA BUESCHER, PIA SVENDSEN, MURNI TIO, CINDY MISKOLCZI-MCCALLUM, GUY TEAR, WILLIAM BROOK, and WILLIAM CHIA

MRC Centre for Developmental Neurobiology, King's College, London SE1 1UL, UK

We have identified novel alleles of the previously described mutation midline (mid). Mutant alleles of mid were first identified in the classic screen for segmentation genes (mid1, mid2). mid mutant larvae are characterized by patches of naked cuticle in the ventralmost part of the abdominal denticle belts. We identified the mid gene product, a T-box transcription factor (CG6634), which shows an expression pattern characteristic of segment polarity genes. Our studies suggest that mid acts in the formation and specification of neuroblasts and ventral epidermal patterning. mid is expressed from stage 5 onwards in ectodermal stripes from which the neuroblasts of rows 6, 7 and 1/2 delaminate. Loss of mid function leads to the loss of row1/2 neuroblasts, most noteably NB1-1, in alternate segments. The failure of NB1-1 formation results in the absense of the pcc neuron in odd-numbered segments, which in turn causes an axonal phenotype: the longitudinal axon tracts are disrupted in alternate segments. Furthermore, our studies indicate that mid contributes to the specification of neuroblasts by negatively regulating the expression of eagle. The cuticle phenotype of mid larvae suggests that the underlying defect is a gain of Wingless signaling. Our studies show that mid is a repressor of wg transcription. In mid-mutant embryos two wg-expression domains are established in response to Hh-signaling. We conclude that in normal development mid acts to break the symmetry of Hh-dependent activation of wg expression.

ODOR AND TASTE CODING

JOHN CARLSON, ELISSA HALLEM, ANUPAMA DAHANUKAR, WYNAND VAN DER GOES VAN NATERS, and SYLWESTER CHYB

Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06520, USA

We isolated two families of 60 genes each, the Or genes and the Gr genes, which encode odor and gustatory receptors. We developed an in vivo functional expression system to identify the ligand specificities of individual odor receptors. The system uses a mutation that deletes two adjacent Or genes, thereby eliminating the response of a particular neuron. We introduce into the mutant neuron another receptor gene and determine the odor specificity thereby conferred. This approach allowed a systematic investigation of the odor response spectra of the antennal repertoire of odor receptors. Some receptors responded strongly to many of the tested odors, whereas others responded strongly to only one or none. Likewise, some odors strongly activated many receptors, and some odors activated only one. Some individual receptors respond in different ways to different odorants: they are activated by some odorants and inhibited by others. Finally, the work allowed us to deduce which receptors are expressed in which types of neuron in the antenna, thereby establishing a receptor-to-neuron map of the olfactory system. Analysis of the Gr genes revealed that one, Gr5a, maps to a locus associated with taste perception of the disaccharide trehalose. We found that Gr5a is required for both physiological and behavioral response to trehalose, and that expression of Gr5a in cultured cells confers response to this sugar. We conclude that Gr5a encodes a taste receptor for trehalose.

WAVE/SCAR, A MULTIFUNCTIONAL COMPLEX COORDINATING DIFFERENT ASPECTS OF NEURONAL CONNECTIVITY

P. CARRERA, A. SCHENCK, A. QURASHI A, A. GALY, C. DIEBOLD and A. GIANGRANDE

Institut de Génétique et Biologie Moléculaire et Cellulaire & CNRS/INSERM/ULP, 67404 Illkirch, France

Neuronal plasticity requires actin cytoskeleton remodeling and local protein translation in response to extracellular signals. Rho GTPase pathways control actin reorganization, while Fragile-X Mental Retardation Protein (FMRP) regulates the synthesis of specific proteins. Mutations affecting either pathway produce neuronal connectivity defects in model organisms and mental retardation in humans. We previously showed that CYFIP, the fly ortholog of vertebrate FMRP interactors CYFIP1 and CYFIP2, interacts biochemically and genetically with dFMR1 and dRac1. CYFIP mutations affect axons and synapses, much like mutations in dFMR1 (the Drosophila FMR1 ortholog) and in Rho GTPase dRac1. Thus, CYFIP provides a bridge between signal-dependent cytoskeleton remodeling and translation. Like SCAR and Kette, CYFIP belongs to the WAVE/SCAR complex, which transduces Rac1 signaling to trigger Arp2/3 dependent actin nucleation. Our recent data indicate that neuronal defects of SCAR, CYFIP, and Kette mutants are, despite the initially proposed function of CYFIP and Kette as SCAR silencers, indistinguishable and are involved in processes as diverse as ectopic midline crossing and nerve branching, as well as synapse undergrowth at the larval neuromuscular junction. The common phenotypes of single mutants are readily explained by the finding that loss of anyone of the three proteins leads to degradation of its partners. As a consequence, each mutant is unambiguously judged as defective in multiple components of the complex, even though each component affects different signaling pathways. Indeed, SCAR-Arp2/3 signaling is known to control axonogenesis whereas CYFIP signaling to the Fragile-X Mental Retardation Protein fly ortholog contributes to synapse morphology. Thus, the WAVE/SCAR complex constitutes a multifunctional unit orchestrating different pathways and aspects of neuronal connectivity.

SYNAPTIC TRANSMISSION AND MITOCHONDRIA ARE DEFECTIVE IN DROSOPHILA LARVAE OVER-EXPRESSING HUMAN TAU

FRANCIS CHEE, AMRIT MUDHER, MATT CUTTLE, SIMON LOVESTONE, and DAVID SHEPHERD

Cell Sciences, University of Southampton, Southampton SO16 7PX, UK

Selective expression of human Tau protein (0N3R) in Drosophila larval motor neurons caused a significant reduction in the incorporation of FM1-43 during endocytosis in the readily releasable pool at neuromuscular junctions (NMJs). Synaptic transmission was also affected by Tau over-expression. Under normal conditions the evoked synaptic potential produced from the Tau-expressing motor neuron was indistinguishable from wild type. However, when NMJs were depolarized by high frequency tetanic stimuli, NMJs showed significant dysfunction. Excitatory junctional potentials (EJPs) amplitude over time was significantly different to wild-type EJPs. Tau-expressing NMJs showed a significant decrease in mitochondrial membrane potential. Collapsing mitochondrial membrane potential in presynaptic mitochondria of wild-type larvae produced a similar train of EJPs to those seen in the Tau-expressing NMJs. Our results suggest that over-expression of Tau in vivo disrupts the function of mitochondria at the synapse, predisposing the synaptic terminal to vesicle recycling failure and leading to defective synaptic transmission.

BLOCKING G-AXON PRUNING IN DROSOPHILA MUSHROOM BODY IMPAIRS SHORT-TERM MEMORY BUT NOT LONG-TERM MEMORY

CHRISTELLE CLOUET, ELANA TOKMATCHEVA, and JEAN-MAURICE DURA

Institut de Génétique Humaine, CNRS UPR 1142, 34396 Montpellier, France

Drosophila mushroom bodies (MB) are involved in short-term memory (STM) and in long-term memory (LTM), but the relation between STM and LTM has remained enigmatic. STM appears associated with g and ab neurons, whereas LTM requires the integrity of the a' and/or a vertical axons. During metamorphosis, early larval g neurons undergo pruning of larval-specific dendrites and axons followed by outgrowth of adult-specific processes, while a'b’ late larval and ab pupal MB neurons are not remodelled. Here we show that the Hr39 nuclear hormone receptor ectopic/over-expression in MB neurons blocks the remodelling of the g axons leaving apparently unaffected the a'b’ and ab neurons. The blockage of the g neurons remodelling impairs STM at the adult stage. However, LTM of these flies is perfectly normal. This result shows that MB larval pruning is necessary for adult memory and that STM and LTM may be independent memory systems acting in parallel.

TWO FUNCTIONAL TYROSINE DECARBOXYLASE GENES IN DROSOPHILA

SHANNON COLE, GINGER CARNY, BARBARA TAYLOR, and JAY HIRSH

Department of Biology, University of Virginia, Charlottesville, VA 22903, USA

The trace biogenic amine tyramine is present in the nervous systems of animals ranging from C. elegans and Drosophila to mammals. Though little is known about the functional role of tyramine in the CNS, it has been implicated in the etiology and progression of depression plus anxiety disorders, as well as in modulating cocaine responses in Drosophila. In C. elegans, tyramine is synthesized in vivo by the enzyme tyrosine decarboxylase (TDC), but no other animal TDCs have been identified. To further clarify the role of tyramine in the CNS, we have cloned two candidate TDC genes from Drosophila, dTdc1 and dTdc2. We find that both gene products are functional in vivo, although only dTdc2 shows extensive neural expression. Flies with a mutation in dTdc2 lack neural tyramine and octopamine and are female-sterile due to egg retention. This phenotype can be partially rescued by the neural expression of dTdc1, but attempted rescue with ectopically expressed dTdc2 paradoxically results in severe dopamine depletion and early larval death. These intriguing results demonstrate that the precise regulation of TDCs in the nervous system is critical for survival. Preliminary evidence also indicates that this pathway is modulated transcriptionally by exposure to free-base cocaine. We are currently exploring the involvement of dTdc1 and 2 in cocaine responses and hope to more clearly define the neural targets for tyramine and octopamine.

CIRCADIAN RHYTHMS IN PERIOD NULL MUTANTS

BEN COLLINS, STEPHANE DISSEL, EZIO ROSATO EZIO, and CHARALAMBOS P. KYRIACOU

Department of Genetics, University of Leicester, Leicester LE1 7RH, UK

The circadian clock of Drosophila melanogaster is generated by interlocked feedback loops, and null mutations in core clock genes such as period and timeless cause behavioral arrhythmicity. Remarkably, when we combined a period-null mutation with a severe mutation in the gene encoding a circadian photoreceptor, cryptochrome, full circadian rhythmicity was restored in light-dark cycles. This effect was lost in timeless-null mutant backgrounds, and we provide an explanation for these results based on the differential sensitivities of subsets of pacemaker neurons to light. We have therefore uncovered a novel, period-independent role for the timeless gene in the Drosophila circadian pacemaker.

A GLIMPSE OF THE NEUROARCHITECTURE OF THE LARVAL GUSTATORY SYSTEM

JULIEN COLOMB, NICOLA GRILLENZONI, ARIANE RAMAEKERS, and REINHARD STOCKER

Department of Biology, University of Fribourg, 1700 Fribourg, Switzerland

There is increased focus on gustatory coding using Drosophila as a model system. Recent studies provide evidence that the quality of taste and the stimulus site are represented in the anatomical projection pattern in the adult suboesophageal ganglion (SOG). The CNS of the larva is much smaller and simpler in its organization than the adult one, although it must solve the same problems. As in the adult fly, different organs sensing gustatory cues are involved in different behaviors (chemotaxis, control of feeding rate, learning). For these reasons, we assume that the larva will become an important model system for studying gustatory coding. In a first step toward understanding this code, we have begun to investigate the neuroarchitecture of the system. Applying an antibody for visualizing landmarks in the brain, we followed the central projections of different gustatory neurons using the GAL4/UAS system. We are mapping subregions of the SOG involved in the processing of gustatory information, and we plan to look for target interneurons that may receive, modulate or transmit this information.

MINIBRAIN REGULATES CELL CYCLE EXIT DURING POSTEMBRYONIC NEUROGENESIS

JORDI COLONQUES, JULIAN CERON, BARBARA HÄMMERLE, and FRANCISCO J. TEJEDOR

Instituto Neurociencias (UMH-CSIC), 03550 Sant Joan d'Alacant, Spain

minibrain (mnb) mutants exhibit a reduced size of certain adult brain regions, specially the optic lobes. This reduced size is produced by a decrease in the number of neurons generated during the proliferative processes of postembryonic development. Most precisely, this neuronal deficit is originated during the neurogenic divisions of neuroblasts (Nb) at late third instar larvae. At this stage, each Nb divides asymmetrically to yield another Nb and a ganglion mother cell (GMC), which in turn divides into two ganglion cells (GC). We have found that the product of this gene (MNB) is transiently expressed in new-born GCs immediately after the division of the GMC. In mnb mutants, GCs do not stop dividing but go into an additional cell cycle and eventually die. This cell death yields the neuronal deficit. Conversely, ectopic expression of MNB induces cell cycle arrest. We have been compiling data showing the involvement of MNB in the regulation of neuronal cell cycle exit, leading to insights about molecular mechanisms underlying the regulation of postembryonic neurogenesis by MNB.

MODELLING SPORADIC PARKINSON's DISEASE IN DROSOPHILA MELANOGASTER

HÉLÈNE COULOM and SERGE BIRMAN

Laboratoire Génétique et Physiologie du Développement, CNRS, Université de la Méditerranée, 13288 Marseille, France

Parkinson's disease (PD) is the most frequent neurodegenerative movement disorder in humans, characterized by the selective loss of brain nigrostriatal dopaminergic neurons. If major insights into the genes responsible for some rare hereditary cases have arisen, the etiology of sporadic cases remains unknown. Epidemiological studies have suggested an association with environmental toxins, mainly mitochondrial complex-I inhibitors such as the widely used pesticide rotenone. During the last years, Drosophila melanogaster has been used to study several neurodegenerative diseases, including serving as a genetic model of PD. We have studied the neurodegenerative and behavioral effects of a sublethal chronic exposure to rotenone in Drosophila. After several days, the treated flies presented characteristic locomotor impairments that increased with the dose of rotenone. Immunocytochemical analysis demonstrated a dramatic and selective loss of dopaminergic neurons in all brain clusters. The addition of L-dopa into the feeding medium rescued the behavioral deficits but not neuronal death, as is the case in human PD patients. In contrast, the antioxidant melatonin alleviated both symptomatic impairment and neuronal loss, supporting the idea that this agent may be beneficial in the treatment of PD. Therefore, chronic exposure to pesticides recapitulates key aspects of PD in Drosophila and provides a new in vivo model for studying the mechanisms of dopaminergic neuro-degeneration.

LIGHT-DEPENDENT SUBCELLULAR TRANSLOCATION OF GQα IN DROSOPHILA PHOTORECEPTORS IS FACILITATED BY THE MYOSIN III PROTEIN, NINA-C

MICHELLE CRONIN, FENGQIU DIAO, and SUSAN TSUNODA

Department of Biology, Boston University, Boston, MA 02215, USA

An important property of photoreceptors is their ability to maintain a high sensitivity while adapting to different background light intensities. One mechanism by which invertebrates and vertebrates may accomplish this is to regulate the number of available signaling components. We examine the light-dependent translocation of the Gqα-protein in Drosophila photoreceptors between the signaling compartment, the rhabdomere, and the cell body. Gqα is exclusively localized in the rhabdomeres in the dark and translocates to the cell body upon illumination. Higher light intensities increased the quantity of Gqα translocated out of the rhabdomeres from 20 to 75%, consistent with a mechanism for light adaptation. Translocation from the rhabdomere to the cell body is rapid, taking less than five minutes, while translocation in the reverse direction is complete after one hour. Genetic analyses of phototransduction mutants demonstrate that the translocation of Gqα requires the light-activation of rhodopsin, but none of the known downstream components, suggesting that the signaling pathway triggering translocation occurs upstream of Gqα. Finally, we show that ninaC mutants display a significantly reduced rate of Gqα transport from the cell body to the rhabdomere, suggesting that NINA-C may function as a light-dependent, plus-end motor involved in facilitating the transport of Gqα.

EFFECTS OF SEROTONIN AND MDMA IN MODULATING SENSORY-CNS-MOTOR CIRCUIT IN A SEMI-INTACT PREPARATION OF DROSOPHILA LARVAE

SAMEERA DASARI and ROBIN COOPER

Dept. of Biology, University of Kentucky, Lexington, KY 40506, USA

Discrete identifiable motor units that are well defined in anatomic and physiologic function can be recruited selectively and driven depending on the sensory stimulus intensity, duration, and frequency through a sensory-CNS circuit in 3^rd-instar larvae. Monitoring the excitatory postsynaptic potentials on the prominent ventral longitudinal body wall muscles (e.g., m6) provides additional insight into how the selective motor units might be recruited within intact animals. We also introduce the actions of the neuromodulators [serotonin (5HT), octopamine (OA), and dopamine (DA)] on the inducible patterns. 5HT (10 µM), OA (1 µM) and DA (10 µM) all enhanced evoked motor unit activity during sensory stimulation. OA (1 & 10 µM) causes waves on motor-unit activity, which is coordinated. DA and OA depressed synaptic transmission at the NMJ, and 5HT resulted in only a slight depression. We extended this method study by applying 3,4-methylenedioxymethamphetamine (MDMA), a common drug of abuse in humans. It is known that MDMA compels mammalian serotonergic neurons to release serotonin (5HT). We have shown that MDMA works through a non-serotonergic mechanism at the NMJ, since it is insensitive to exogenous 5HT. The sensory-CNS motor circuit is also modulated by MDMA (10 µM) in a similar fashion as for 5HT. We have demonstrated that MDMA's action is presynaptic in enhancing transmitter release. Additional actions of MDMA's activity are being assessed by studies of the heart rate in larval Drosophila.

A ROLE FOR THE ADULT FAT BODY IN MALE COURTSHIP BEHAVIOR

BRIGITTE DAUWALDER, ANNA A. LAZAREVA, WILLIAM W. MATTOX, and PAUL E. HARDIN

Department of Biology and Biochemistry, University of Houston, Houston, TX 77204, USA

The takeout gene is a target of the sex-determination pathway. A mutation in takeout affects male courtship and interacts genetically with mutations in fruitless. takeout (to) is expressed in head fat body specifically in males as well as antennae in both sexes. Feminization of to-expressing tissues drastically reduces male courtship, suggesting that the male identity of cells containing this gene's product is important for normal male courtship. To test whether the fat body, a secretory and non-neuronal tissue, mediates this effect, we feminized this tissue using fat-body-specific promoter sequences from the Lsp-2 gene. One Lsp-2 promoter fragment directs expression only in larval fat body, while another drives expression in both larval and adult fat body. The larval fat body persists through metamorphosis but is replaced within the first few days of adulthood with adult fat-body cells. Feminization of the adult, but not the larval, fat body significantly reduced male courtship, suggesting that the correct sexual identity of the adult fat body is crucial to normal courtship. Given that the fat body is a major secretory tissue, we hypothesize that it interacts with the nervous system via factors that are secreted into the hemolymph. One of these factors may be the to product, Takeout. Indeed, we found by Western analysis that Takeout is present in the male hemolymph, indicating that it may play a sex-specific role as a diffusible factor.

IDENTIFICATION OF SLOWMO INTERACTING PROTEINS

CHIS DEE and KEVIN MOFFAT

Departmemt of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK

We have previously shown that null mutations in slowmo cause a 1^st instar lethal phenotype. Embryos developed normally and hatched but have clear locomotor defects, producing larvae that gave fewer and slower peristaltic wave movements than wild type. The phenotype became more severe until death before the second instar moult. Driving UAS-tetanus-toxin in a neuronal population identified by a P[GAL]4 enhancer trap in the gene gave a similar phenotype, whereby the larvae were unable to correctly perform peristaltic wave movements. Western-blot analysis and GFP tagging have shown the protein to be associated with mitochondria. Drosophila contains two further homologs which we have shown also to be located in the mitochondria. Homologs are found in all multicellular organisms examined; however, their function is unknown. To further our analysis we have performed a yeast two-hybrid screen with Slowmo as the bait, leading to identification of three potential binding partners for Slowmo.

ADENOSINE-TO-INOSINE RNA EDITING AT A CONSERVED PROTEIN KINASE TARGET SITE IN SYNAPSIN PRE-mRNA OF DROSOPHILA

SOEREN DIEGELMANN, URSULA WERNER URSULA, and ERICH BUCHNER

Biozentrum, University of Würzburg, 97074 Würzburg, Germany

Synapsins are abundant synaptic vesicle associated phosphoproteins that are involved in the fine regulation of neurotransmitter release. The Drosophila member of this protein family (Klagges et al., 1996, J. Neurosci. 16, 3154) contains three conserved domains and is expressed in most synaptic terminals. Synapsin knock-out flies show no obvious structural defect but are disturbed in complex behaviour (Godenschwege et al., 2004, Eur. J. Neurosci. 20, 611–622). Here we provide evidence for adenosine-to-inosine (A-to-I) RNA editing at the highly conserved target site P1 of calcium/calmodulin dependent protein kinase I/IV and protein kinase A in the N-terminal A-domain of synapsin. The result of this editing can be observed during all developmental stages and in both isolated heads and bodies. It is also seen in newly collected wild-type strains and thus does not represent an adaptation to laboratory culture conditions. A likely editing site-complementary sequence has been found in a downstream intron, indicating that the synapsin pre-mRNA can form a double-stranded RNA structure that is required for editing by the adenosine deaminase acting on RNA (ADAR) enzyme. We therefore propose that this enzyme is responsible for the mRNA modification we have demonstrated.

GENOME-WIDE STUDY OF SEX-PEPTIDE RESPONSE

ELENA DOMANITSKAYA, HUANFA LIU, and ERIC KUBLI

Institute of Zoology, University of Zürich, 8057 Zürich, Switzerland

In D. melanogaster mating changes physiology and behavior of females. Virgin females readily accept males; after mating, females reject courting males. Moreover egg production and oviposition are induced by copulation. Sex Peptide (SP, 36 aa) is produced in the accessory glands of males and transferred to females during copulation. SP is the major player in the stimulation of egg-laying and rejection behavior. In spite of our extensive knowledge about the nature of SP, the molecular mechanism of the response cascade is unknown. Several attempts have been made to improve our understanding of this pathway. Two binding sites have been found: one is localized in the CNS, the other in the genital tract. The receptor is expressed in a developmentally regulated manner. As the cAMP level is crucial for eliciting the post-mating responses, this molecule is assumed to belong to be of G-protein-coupled receptor type. Based on these facts we carried out a microarray experiment and defined a candidate (GRHR). To identify all transcripts exhibiting SP-dependent expression patterns, we performed the following microarray analysis: analyzed RNA extracted from heads and abdomen of females mated with mutant SP⁰ males and normal SP⁺ males, respectively, 4 hours after copulation. The same analysis was performed for females mated with males carrying a partly deleted ORF [SP^Del(2-7)], allowing us to analyze these process in context of separate functions of the C- versus N-terminal part of the Sex Peptide molecule.

NOVEL BEHAVIORAL DEFECTS ASSOCIATED WITH APTEROUS VISUAL RESPONSES

BELÉN DORADO, PILAR HERRO, ANTONELLA LANNINI, ANTONIO PRADO, and INMACULADA CANAL

Department of Biology, Universidad Autonoma de Madrid, Facultad de Ciencias, 28049 Madrid, Spain; Department of Physiology, Universidad Pablo de Olavide, 41013 Sevilla, Spain

The Drosophila apterous gene encodes a LIM-HD protein that functions in a wide variety of developmental events, including axonal navigation and fasciculation. We have previously described that hypomorphic and null apterous alleles cause severe defects in axon pathfinding and fasciculation in the neuropiles of the visual system. Surprisingly, the severity of the defects does not clearly correlate with the pattern of apterous expression in the optic lobes. The most striking defects are seen at the inner chiasm level. As a consequence, fibbers that form the inner chiasm fail to retinotopically connect the columns of the medulla, lobula and lobula plate properly, and instead choose incorrect pathways. In order to investigate whether this axonal phenotype of apterous flies is related to visual motion and non-motion, behavioral analyses using an apterous enhancer, which mediates rescue of viability, have been carried out. Our results are consistent with the documented role of this retinotopic map in collate and integrate information about directional motion, orientation and visual cues. Furthermore, these results show that apterous structural defects in the optic lobes correlate with the relevant behavioral phenotypes.

PROLIFERATIVE ACTIVITIES OF POSTEMBRYONIC NEUROBLASTS IN THE OPTIC LOBE OF DROSOPHILA MELANOGASTER

BORIS EGGER and ANDREA BRAND

Wellcome Trust/Gurdon Institute, University of Cambridge, Cambridge CB2 1QR, UK

Neural stem cells can divide symmetrically, giving rise to two identical daughter cells; or asymmetrically, produce two daughter cells with different developmental fates. In Drosophila most neural stem cells or neuroblasts divide in an asymmetric mode, producing a smaller ganglion mother cell and self-renewing. Recent work, however, suggests that postembryonic Nbs (pNbs) in the larval optic lobe initially divide symmetrically before switching to an asymmetric division mode. This is of great interest, because vertebrate neural stem cells might undergo similar division patterns in developing neuroepithelia. First, by using clonal labeling techniques combined with time-lapse confocal microscopy, we are describing the division pattern of pNbs in the developing optic lobe. Second, temporal transcription profiles of optic lobe pNbs will reveal changes in gene expression that might be associated with changes in proliferation patterns. We have adapted magnetic cell sorting and single-cell isolation techniques to semi-purity pNbs for subsequent microarray experiments. Currently, we are analyzing regulatory sequences of transcription factors and are screening GAL4 enhancer trap lines, which will enable us to trace and isolate optic-lobe pNbs. This work aims to contribute to the understanding of the cellular mechanisms underlying normal stem cell proliferation, which is a prerequisite to addressing tumorigenesis and cell replacement therapies in neurodegenerative diseases.

PHEROMONE-SPECIFIC ASSOCIATIVE MEMORY

AKI EJIMA and LESLIE GRIFFITH

Deptartment of Biology, Brandeis University, Waltham, MA 02454, USA

Associative memory formation requires that an animal choose a predictor of the experience it needs to remember. We demonstrate that Drosophila males can be trained to discriminate between different types of female pheromones and suppress courtship, specifically to the type of female that was associated with unsuccessful courtship. Trainer-specific learning is mediated by olfactory cues and modifies the male's processing of those cues. Concurrent and sequential presentation of different pheromones demonstrated that the ability to retain memory of pheromonal cues can be modified by the temporal order in which they appear. In addition, olfactory deprivation allowed to males to utilize other sensory pathways to produce associative memory, implying that there is a high degree of flexibility in the types of cues a male can associate with unsuccessful courtship. Flies can also employ artificial odorants, such as benzaldehyde, as associative cues. Based on these observations, together with the behavioral phenotypes of mutant flies, we propose a circuit model controlling males’ responses to females, in which neuronal communication between the antennal lobe and mushroom body consolidates memory modification of the courtship pathway.

OCTOPAMINERGIC MODULATION OF DISTANCE AND FORCE PRODUCTION DURING JUMPING IN WILD-TYPE AND MUTANT DROSOPHILA MELANOGASTER

CHRIS ELLIOTT, NINA ZUMSTEIN, OLVIER FORMAN, UPENDRA NONGTHOMBA, and JOHN SPARROW

Department of Biology, University of York, York YO10 5YW, UK

We have combined physiological, behavioural and genetic approaches to test whether octopamine modulates jumping in Drosophila. We used a sensitive strain-gauge to record the force produced by the main jumping muscle of female flies. In Canton-S and Texas wild-type strains, the force peaks at 101±4.4 µN after 8.2 ms. Wild-type Drosophila (with their wings removed) jump 28.6±0.7 mm (mean±SEM). For a female wild-type Drosophila, jumping 30 mm corresponds to a kinetic energy of 200 nJ on take-off (allowing 20% of energy for air resistance). From this we calculate that the time to take-off is 5.0 ms, and the peak force should be 274 µN (137 µN/leg). We conclude that substantial energy storage is not a feature of Drosophila jumping. We predicted, from the role of octopamine in enhancing muscle tension in several locust muscles, that genetic manipulation of the octopaminergic system would directly affect force production and jumping in Drosophila. The mutants Tbhn^M18 (caused by a null mutation in the gene for an enzyme necessary for octopamine synthesis) and TyrR^hono (caused by a null mutation in the gene for an octopamine receptor) jumped less far (20.7±0.7 and 20.7±0.4 mm) and produced less force (52% and 55% of wild-type values). From the reduced distance and force production in these mutants, we conclude that, in Drosophila, octopamine modulates escape jumping.

PHENOTYPIC ANALYSES OF A MUTATION THAT AFFECTS FATE DETERMINATION OF THE OLFACTORY RECEPTOR NEURONS IN DROSOPHILA

KEITA ENDO, YUKA YODA, KEN-ICHI KIMURA, and CHIHIRO HAMA

Center for Developmental Biology, RIKEN, 650-0047 Kobe, Japan

The olfactory sensory system in Drosophila comprises ∼ 1,300 olfactory-receptor neurons (ORNs), each of which expresses only one of ∼ 60 odorant receptors (ORs). ORNs expressing a given OR project axons to only one or two topographically fixed positions in the antennal lobe and form specific glomeruli with dendrites of the second- order neurons. This clear relationship between ORNs and their projecting glomeruli makes this system an attractive model to reveal mechanisms of the axonal projection in a complex neural circuitry. To approach this issue, we conducted a MARCM-based genetic screen for mutations that affect the axonal projection of the ORNs. Mutant phenotypes were visualized by a Gal4 line, AM29, which labels ∼ 20 pairs of ORNs; one of these neurons projects to a medially located glomerulus, DM6, the other to a dorso-laterally located glomerulus, DL4. One of the mutations we have identified in the screen is K1514, which causes both of the AM29-positive ORNs to project to DM6. In addition, this phenotype was observed only when the mutant clones were generated no later than early pupal stage, when the olfactory progenitor cells are determined in the eye-antennal disc. These data suggest that the cell fate determination is compromised in the olfactory progenitor cells in the mutant clones, which results in the transformation of one of the AM29-positive ORNs to the other. We are currently investigating this idea, while mapping the locus defined by this novel mutation.

EGGHEAD REGULATES PHOTORECEPTOR NEURON PROJECTION IN THE DROSOPHILA LARVAL BRAIN

YUN FAN, MARTIN MÜLLER, SUSANNE FLISTER, BRUNO BELLO, MATTHIAS SOLLER, MARTIN HOLLMANN and HEINRICH REICHERT

Institute of Zoology, Biozentrum/Pharmazentrum, University of Basel, 4056 Basel, Switzerland

In the Drosophila visual system, each class of photoreceptor neurons (R cells) projects to a different synaptic layer in the brain. R1-R6 axons terminate in the lamina, while R7 and R8 axons pass through the lamina and stop in the medulla. The formation of this elaborate projection pattern relies on complex bidirectional interactions between R cell axons and different populations of cells in the target area. Our study shows that egghead (egh) plays a role in target layer selection during larval development. In egh loss-of-function mutants, the targeting of R1-R6 axons is disrupted although the development and migration of lamina neurons, and glial cells are largely normal. In addition, the projections of homozygous egh-null R cell axons into their wild-type target in 3^rd instar larvae, generated by an eye-specific mitotic recombination technique, are largely indistinguishable from wild type. This suggests that egh is required in the target area for the formation of the correct R-cell projection pattern. Further mosaic analysis shows that this requirement is restricted to a specific cell population, a subset of which might act as intermediate target for R-cell growth cones. Since egh encodes a glycosyltransferase, and it is involved in the biosynthesis of glycosphingolipids, we postulate that egh is required for the formation of lipid microdomains in the plasma membrane, which in turn affect R-cell axon targeting by mediate guidance-signal cues.

OPTICAL IMAGING OF ODOR-EVOKED ACTIVITY IN HIGHER BRAIN CENTERS OF DROSOPHILA

ANDRÉ FIALA, THOMAS SPALL, THOMAS RIEMENSPERGER, and ERICH BUCHNER

Department of Genetics and Neurobiology, Biocenter, University of Wu¨rzburg, 97072 Wu¨rzburg, Germany

Drosophila perceives odors by olfactory sensory neurons located on the antennae and maxillary palps. Sensory neurons expressing a certain olfactory receptor and project to the antennal lobe, terminating in defined glomeruli. Thus, a certain odor is represented in the antennal lobe as a specific glomerular activity pattern. Projection neurons (PNs) innervating the glomeruli project to the mushroom-body calyx and the lateral protocerebrum. Expression of the fluorescent calcium sensor cameleon allows to optically record odor-evoked activity in PN terminals in both target neuropils. Using this optical-imaging approach we address the question of how odors are represented in these higher-order brain centers. We show that odors evoke odorant-specific activity patterns in the calyx. In the lateral protocerebrum activity patterns are not resolvable, due to the highly overlapping branching of PN terminals. In both neuropils, an increase in odor concentration leads to an increase in calcium activity. The odorant-specific spatial patterns in the calyx remain constant over several dilution steps. Therefore, odor identity is represented in terms of specific activity patterns in the calyx, whereas odor concentration is represented as intensity of the activity. Current experiments are testing whether repeated stimulation with binary odorant mixtures or pairing of an odorant with a punishing electric shock stimulus modifies those representations.

THE IRRE CELL RECOGNITION MODULE IRM

KARL-FRIEDRICH FISCHBACH, SMITHA VISHNU SMITHA, ALEXANDER HERTENSTEIN, and BERNHARD BONENGEL

Institute of Biology III, Freiburg i. Brsg, 79104 Freiburg, Germany

The fine-tuned expression pattern of many cell-adhesion proteins is essential for the establishment of the well-ordered structure of the optic lobe of Drosophila. Among those are the members of the Irre cell recognition module (IRM): IrreC-rst, Kirre, SNS, and Hbs. Much has been learned by studying the function of the IRM proteins in systems less complex than the brain. They mediate close cellular contacts (e.g., in muscle fusion and in cell sorting within imaginal discs). During optic-lobe development their expression extends from the phase of axonal pathfinding into the period of target recognition and synaptogenesis. We have investigated their exact role in these processes and those of the intracellular interaction partners which we have identified (e.g., D-Mints).

ACTIVITY-DEPENDENT REGULATION OF THE SYNAPTIC LOCALIZATION OF SHAKER- ENCODED POTASSIUM CHANNELS AND NEURO-DEGENERATION

STÉPHANE FRAICHARD, CATALINA RUIZ-CANADA, ELSA ESCAMILLA, BARBARA HÄMMERLE, JOSÉ A. BOTELLA, and FRANCISCO J. TEJEDOR

Instituto Neurociencias, 03550 San Juan Alicante, Spain

Voltage-sensitive K⁺ channels are essential to regulate neuronal excitability. In Drosophila, Shaker potassium channels (Sh Kch) are expressed at the larval NMJ and in the CNS at restricted neuronal populations in the axonal tracks or their synaptic contacts. We have observed that in different hyperexcitable mutants (Sh, eag, Hk, and sei), as well as double-mutant combinations, there is a down-regulation in Sh Kch expression as determined by WB analysis. These changes in expression are accompanied by changes in the repertoire of expressed Sh Kch isoforms. PCR analysis led to the conclusion that these changes do not take place at the transcriptional level. Interestingly, we have found changes in the subcellular localization of Sh Kch in the CNS, as determined by immunocytochemical analysis of mutant brains. Most frequently, these alterations in localization take place by loss of synaptic localization. Thus, we conclude that the synaptic localization of Sh Kch is regulated in an activity-dependent manner, and this leads to changes in the level of such channel proteins. Another rather interesting finding is that all these mutants present neurodegeneration, which could be correlated in a region-specific manner to the mislocalization of Sh Kch. Thus, we propose a model in which this neurodegeneration is caused by Sh Kch delocalization through synaptic disassembling.

SHAGGY, THE HOMOLOG OF GLYCOGEN SYNTHASE KINASE 3, CONTROLS NEUROMUSCULAR JUNCTION GROWTH IN DROSOPHILA

BÉNÉDICTE FRANCO, LAURENT BOGDANIK, YVES BOBINNEC, ALAIN DEBEC, JOËL BOCKAERT; MARIE-LAURE PARMENTIER, and YVES GRAU

Laboratoire de Génomique Fonctionnelle CNRS-UPR 2580, 34094 Montpellier, France

A protein-trap screen using the Drosophila neuromuscular junction (NMJ) as a model synapse was performed in order to identify genes that control synaptic structure or plasticity. We found that Shaggy (Sgg), the Drosophila homolog of mammalian glycogen synthase kinases 3 alpha and beta (GSK-3a and b), two serine/threonine kinases, is concentrated at this synapse. Using various combinations of mutant alleles of shaggy, we found that Shaggy negatively controlled the NMJ growth. In addition, tissue-specific expression of a dominant-negative sgg variant indicated that this kinase is required in the motoneuron, but not in the muscle, to control the NMJ growth. Finally, we show that the Sgg enzyme controls microtubule cytoskeleton dynamics in the motoneuron; and that Futsch, a microtubule associated protein, is required for Sgg function as it influences synaptic growth.

A FLY MODEL FOR RHODOPSINP23H RETINITIS PIGMENTOSA IN HUMANS

ANNE GALY, MICHEL ROUX, JOSÉ SAHEL, THIERRY LEVEILLARD, and ANGELA GIANGRANDE

LPCMR, Hoˆpital St Antoine, 75571 Paris, France

A variety of rhodopsin mutations result in Autosomal Dominant Retinitis Pigmentosa (ADRP). One of the most frequent mutations, substituting Proline 23 with Histidine (RhodopsinP23H), leads to blindness due to severe retinal degeneration. Although cellular and rodent animal models have been developed, the pathogenic mechanism leading to RhoP23H induced cell death is still poorly understood. By using a transgenic Drosophila model we show that a rhodopsin-1 mutation (rhodopsin-1P37H) corresponding to human rhodopsinP23H mutation leads to a dominant photoreceptor degeneration that mimics age-, light-dependent, and progressive ADRP. While most dominant mutations affect rhodopsin trafficking in vertebrates and flies, Rhodopsin-1P37H is properly localized in photoreceptor rhabdomeres and able to transduce the light signal, as demonstrated by electroretinograms. Finally, visual loss and degeneration are accompanied by apoptotic features and prevented by expression of the apoptosis inhibitor p35. The development of a fly model for ADRP makes it possible to devise therapeutic approaches for photoreceptor degeneration in vertebrates.

SHAL IS THE MAJOR A-TYPE CURRENT IN THE DROSOPHILA MUSHROOM-BODY INTRINSIC NEURONS, AND SHAKER SEGREGATES TO A SUBSET OF THESE CELLS

GABRIEL GASQUE, PEDRO LABARCA, ENRIQUE REYNAUD, and ALBERTO DARSZON

Instituto de Biotecnología, Universidad Nacional Autónoma de México, 62210 Cuernavaca, Mor, Mexico

Shaker, which codes for A-type K⁺ currents, is enriched in the locus of olfactory learning, the mushroom bodies (MBs). Mutations at the Shaker locus alter excitability, synaptic function, and olfactory learning. However, a direct link between Shaker channels and MB neuron (MBN) physiology is missing. We employed molecular approaches— combined with biophysical, genetic, and pharmacological scrutiny–to assess the contribution of Shaker polypeptides to the K⁺ current of the MBNs. We found that transcripts for Shab, Shaw, Shaker, and Shal—of which the latter also codes for A-type currents—are present in MBNs. The biophysical data suggest Shaker channel segregation to a subset of MBNs presenting half-inactivation voltages (V1/2) more positive than − 73 mV, whereas most express Shal as the major component. Pharmacological analysis showed that all MBNs were sensitive to 4-aminopyridine, a non-specific A-type current blocker; but a subset of neurons displayed little sensitivity to a Shal-specific toxin, and the toxin-insensitive currents exhibited V1/2 values attributable to Shaker channels. Moreover, the absence of functional Shaker channels slowed down K⁺ current inactivation and decreased current amplitude in only a subset of KCs. Our findings provide the first direct evidence that altered Shaker channel function modifies the physiology of MBNs. Surprisingly, Shaker-dependent neurons segregate to a minor fraction (20–30%) of MBNs, while most have Shal as the major A-type current component.

PERSISTENCE OF COMPLEX LARVAL CHEMOSENSORY ORGANS THROUGH METAMORPHOSIS IN DROSOPHILA

NANAË GENDRE, KARIN LÜER, SANDRINE FRICHE, NICOLA GRILLENZONI, ARIANE RAMAEKERS, GERHARD TECHNAU, and REINHARD STOCKER

Department of Biology, University of Fribourg, 1700 Fribourg, Switzerland; Institute for Genetics, University of Mainz, 55122 Mainz, Germany

The sense organs of adult Drosophila derive essentially from imaginal discs and hence are adult-specific. Experimental evidence presented here, however, suggests a different developmental design for the three largely gustatory sense organs located along the pharynx. In a comprehensive cellular analysis, we show that the posterior-most of the three organs derives directly from a similar larval organ and that the two other organs arise by splitting of a second larval organ. Interestingly, these two larval organs persist despite extensive reorganization of the pharynx. Thus, most of the neurons of the three adult organs are surviving larval neurons. However, the anterior organ includes some sensilla that are generated during pupal stages. Also, we observe apoptosis of the 3rd larval pharyngeal organ. Hence, our experimental data show the integration of complex, fully differentiated larval sense organs into the nervous system of the adult fly and demonstrate the embryonic origin of their neurons. Moreover, they identify metamorphosis of this sensory system as a complex process involving neuronal persistence, generation of additional neurons, and neuronal death. Our conclusions are based on combined analysis of reporter expression from P[GAL4] driver lines, horseradish peroxidase injections into blastoderm stage embryos, cell labeling via heat shock induced FLP-out in the embryo, bromodeoxy-uridine birth dating, and staining for programmed cell death.

SLOWPOKE K⁺ CHANNEL GENE REGULATION MEDIATES RAPID DRUG TOLERANCE

ALFREDO GHEZZI, YAZAN AL-HASAN, and NIGEL ATKINSON

Section of Neurobiology, University of Texas, Austin, TX 78712, USA

Changes in neural activity caused by exposure to drugs may trigger homeostatic mechanisms that attempt to restore normal neural excitability. Many alcohols and solvent anesthetics such as ethanol, benzyl alcohol, toluene, trichloroethylene, and chloroform alter neural excitability. A single sedation with the anesthetic benzyl alcohol changes the expression the of Ca²⁺-activated K⁺ channel gene slowpoke (slo) and induces rapid drug tolerance. We demonstrate linkage between these two phenomena using a mutation and a transgene. A mutation that eliminates slo expression prevents tolerance, while expression from an inducible slo transgene causes tolerance in naive animals. This channel integrates two basic methods of neural signaling: Ca²⁺ and changes in the electrical potential and therefore is a likely contributor in the homeostatic mechanism. The change in slo expression appears to be specific to the nervous system, and development of tolerance is dependant on neurally expressed Slowpoke polypeptide(s) only. The behavioral response to benzyl alcohol can be separated into an initial phase of extreme hyperkinesis and a subsequent phase of sedation. The hyperkinetic phase causes a drop in slo gene expression and makes animals more sensitive to benzyl alcohol. It is the sedative phase that stimulates slo expression and induces tolerance. We demonstrate that the expression level of slowpoke is a predictor of drug sensitivity.

FISHY FLY AND FLYING FISH: WHAT DO THEY HAVE IN COMMON?

ALAIN GHYSEN

Inserm E343, Université de Monpellier II, 34095 Monpellier, France

Answer: their nervous system. The similarities between the nervous systems of insects and of vertebrates are piling up, suggesting that the common ancestor to all living triploblasts (and possibly more) already enjoyed a fairly sophisticated nervous system. This begs another question: why has there been such a narrow bottleneck in evolution, that all present species are derived from a single, neurally complex ancestor species?

OVEREXPRESSION OF THE IP3 KINASE GENE AT OLFACTORY-RECEPTOR NEURONS MODIFIES OLFACTORY PERCEPTION IN DROSOPHILA MELANOGASTER

CAROLINA GOMEZ-DIAZ, FERNANDO MARTIN, and ESTHER ALCORTA

Dept. of Functional Biology, University of Oviedo, 33.006 Oviedo, Spain

The IP3 route is considered one of the two main transduction cascades that mediate olfactory reception in Drosophila melanogaster. Lately, the traditional use of mutants in genes coding for intermediate products of the transduction process to study their effect in olfactory reception is being substituted by the generation of directed dominant mutants using the Gal-4/UAS method (Brand and Perrimon, 1993, Development 118, 401–415.). This approach allows overexpression of the targeted gene in specific cellular subsets and, therefore, functional studies in complete living animals, in which not only intermediate signals but also the final behavioral consequences can be evaluated. In this report we describe the effects on olfactory perception of overexpressing the IP3 K gene (IP3K) at different olfactory receptor neuron subsets. By generating Gal-4/UAS-IP3K doubly-transgene flies, in which the IP3K gene mRNA is overexpressed by more than three times in certain olfactory-receptor neuron subgroups, specific behavioral defects have been detected according to the neuronal olfactory profile. Six strains out of eight displayed abnormal behavioral responses to ethyl acetate, acetone, ethanol, or propionaldehyde, confirming the role of IP3K, and consequently the IP3 transduction cascade, in mediating olfactory information at the reception level.

A COCHLEAR AMPLIFIER IN DROSOPHILA

MARTIN C. GÖPFERT, ANDY D.L. HUMPHRIS, JOERG T. ALBERT, and DANIEL ROBERT

Volkswagen-Foundation Research Group, Institute of Zoology, University of Cologne, 50923 Cologne, Germany

The cochlear amplifier is the dominant unifying concept of how our ears work. The concept assumes that the cochlea is endowed with a biological energy source that feeds back mechanical energy into the vibrations inside the ear, thus amplifying the ear's mechanical input. Motile hair cells provide an energy source, yet the question of whether and how much energy these cells contribute within intact auditory systems has remained uncertain. We have quantified this energy contribution for the motile mechanosensory neurons in the auditory system of Drosophila by analyzing the mechanics of the antennal sound receiver to which these neurons connect. Using dead flies and live mechanosensory mutants (tilB2, btv5P1, nompA2) with defective neurons as a background, we show that the intact, motile neurons exhibit power gain. In wild-type flies, this gain amounts to a mean total energy of 19 zJ (19 × 10⁻²¹ Joules), corresponding to 4.6 times the thermal energy that the neurons add to the receiver's Brownian motion. Larger energy contributions (200 zJ) associate with self-sustained oscillations, suggesting that the neurons adjust their energy expenditure to maintain the ear on the verge of an oscillatory instability, thus maximizing the sensitivity to sound. These findings validate the concept of the cochlear amplifier for Drosophila ears and show that this amplifier resides in motile mechanosensory cells.

ACTIVATION OF THE THERMOSENSITIVE SHIBIRE ALLELE SHI^TS1 IN SUBGROUPS OF CLOCK NEURONS AFFECTS THE MEAN PERIOD LENGTH OF DROSOPHILA MELANOGASTER

EVA GRIESHABER, CORINNA WÜLBECK, and CHARLOTTE HELFRICH-FÖRSTER

Institute of Zoology, University of Regensburg, 93040 Regensburg, Germany

The circadian clock of the fly D. melanogaster is located in the Lateral and Dorsal Neurons (LNs and DNs). These clock neurons are mutually connected and may communicate with each other to generate a common circadian output. The neuropeptide pigment-dispersing factor (PDF) may be involved in the intercellular talk (Peng et al., PLoS Biol. 1, 2003) and in the output pathway (Helfrich-Förster, J. Comp. Physiol A 182, 1998). It is expressed in half of the LNs and released via unknown mechanisms that are independent of synaptobrevin (Kaneko et al., J. Neurobiol 43, 2000). We tried to interfere with the synaptic signalling of the LNs by blocking endocytosis. This was possible with a thermosensitive mutant allele of the Drosophila shibire gene (shi^ts1) (Koenig et al., J. Cell Biol. 96, 1983). It encodes for dynamin, a GTPase essential for clathrin-mediated endocytosis. The mutant protein SHITS1 impairs the constriction of endocytotic vesicles at high temperatures ( > 28°C), but otherwise functions normally. We expressed shi^TS1 (Kitamoto, J. Neurobiol. 47, 2001) in different subsets of the LNs and found a significant increase in the period length of the circadian activity rhythm, if SHITS1 was expressed in the PDF-neurons. This indicates that clathrin-mediated endocytosis is involved in the communication between the clock neurons and a prolonged signaling via PDF lengthens the period of the entire circadian system.

PROSPERO REGULATES LONGITUDINAL GLIA PROLIFERATION THROUGH NEURON INDEPENDENT AND NEURON-DEPENDENT MECHANISMS

RACHEL GRIFFITHS, JANINE FENTON, and ALICIA HIDALGO

NeuroDevelopment Group, School of Biosciences, University of Birmingham, Edgbaston, Birminghanm B15 2TT, UK

During central-nervous system development, glial cells need to be in the correct number and location, at the correct time, to enable axon guidance and neuropile formation. Repair of the injured or diseased central nervous system will require the manipulation of glial precursors to enable axonal tracts to be rebuilt. Unfortunately, the molecular mechanisms controlling glial precursor proliferative potential are unknown. We show here the mechanisms that regulate glial proliferation prior to and during axon guidance. Glial proliferation is regulated by interactions with axons, and prospero (pros) gene function is required to maintain the mitotic potential of glia. During axon guidance, Pros protein promotes cell proliferation. An interacting gene product, Neuronal Vein, activates the MAPKinase signalling pathway in the glia with highest Pros levels, coupling axon extension with glial proliferation. Later on, Pros maintains glial precursors in an undifferentiated state by activating Notch and antagonizing the p27/p21 homolog Dacapo. This enables pros-expressing cells alone to divide further upon elimination of neurons. Thus, Pros plays a key role adjusting glial number to axons during development.

MORNING AND EVENING PEAKS OF LOCOMOTION ARE CONTROLLED BY DIFFERENT CLOCK NEURONS IN THE DROSOPHILA BRAIN

BRIGITTE GRIMA, ELISBETH CHÉLOT, RUOHAN XIA, and FRANÇOIS ROUYER

Institut de Neuobiologie, CNRS, 91198 Gif-sur-Yvette, France

A brain circadian clock controls locomotor activity rhythms in Drosophila. It relies on several groups of clock neurons, among which the PDF-expressing ventral lateral neurons (LNv's) are required for 24-h rhythms in constant darkness. In day-night conditions, adult flies exhibit morning and evening peaks of activity while resting at night and in the middle of the day. How the different neuronal groups control this temporal organization of locomotor activity during a 24-h cycle remains unknown. We have used targeted expression of the period gene to restore clock function to specific subsets of clock neurons in arrhythmic per⁰ mutants. We show that in day-night conditions, Per protein expression restricted to the LNv's restores a morning peak of activity, whereas Per in both the LNv's and PDF-negative dorsal lateral neurons (LNd's) restores the evening peak. In the absence of light cues, LNv and LNd oscillators both contribute to build a unimodal activity distribution, although PDF-expressing LNv's alone are sufficient to drive a sustained 24-h activity rhythm.

INSULIN SIGNALING AND GROWTH CONTROL IN DROSOPHILA

ERNST HAFEN

Institut of Zoology, University of Zürich, 8057 Zürich, Switzerland

The development of a correctly proportioned organism requires an intricate genetic control that coordinates growth of the different tissues and integrates environmental influence such as nutrients, temperature and oxygen. To unravel the genetic control of growth we started different genetic screens to identify genes involved in the regulation of organ size and cell size. These results have show that the insulin and TOR signal pathways play a central, evolutionarily-conserved role in growth control. In Drosophila, the insulin pathway is regulated by insulin-like growth factors that are secreted from different neurosecretory cells in the CNS. Results have been obtained pertaining to the emerging genetic network involved in growth control.

GABA-B RECEPTOR EXPRESSION AND MODULATION OF INTRACELLULAR CALCIUM IN CIRCADIAN CLOCK NEURONS OF DROSOPHILA

YASUTAKA HAMASAKA, CHRISTIAN WEGENER, and DICK R. NÄSSEL

Department of Zoology, Stockholm University, 10691 Stockholm, Sweden

Pacemaker neurons, s-LNv's, of Drosophila's “master” circadian-clock structures are located laterally in each brain hemisphere. These clock neurons are likely to receive multiple inputs that mediate endogenous and exogenous time cues. Little is known about connections between s-LNv's and other neurons and about neurotransmitters acting on the s-LNv's. Here we utilized a s-LNv-specific GAL4 line (w;Pdf-gal4) that directed expression of green fluorescent protein (GFP) to identify clock neurons in dissociated cell culture and showed that s-LNv's respond to gamma-aminobutyric acid (GABA). We monitored intracellular calcium levels in the GFP-labelled s-LNv's loaded with the calcium-sensitive dye Fura-2. Calcium levels decreased when applying GABA and the duration of this decrease were dose-dependent. The calcium response was blocked by the metabotropic GABA-B-receptor (GABA-B-R) antagonist CGP54626 but not by the ionotropic GABA-A-R antagonist picrotoxin. Using an antiserum against one of the two Drosophila GABA-B-R subunits (GABA-B-R2), required for a functional receptor, we showed receptor expression on the s-LNv's. Immunocytochemistry also revealed that GABAergic neurons seem to contact dendrites of the s-LNv's. Our results suggest that these vento-lateral clock neurons are modulated by GABAergic neurons, possibly via intracellular calcium. We also describe the general distribution of GABA-B-R2 immunoreactivity in the Drosophila CNS.

ASSOCIATIVE LEARNING AND MEMORY MEDIATED BY DOPAMINE AND OCTOPAMINE RECEPTORS THAT ARE HIGHLY ENRICHED IN MUSHROOM-BODY NEUROPIL OF THE DROSOPHILA BRAIN

KYUNG-AN HAN, YOUNG-CHO KIM, HYUN-GWAN LEE, and KASEY SOSKA

Department of Biology, Pennsylvania State University, University Park, PA 16802, USA

Biogenic amines exert profound effects on diverse behavioral adaptations in all animals. To elucidate their functions and underlying mechanisms for learning and memory, we identified three receptors—D1 dopamine receptors dDA1 and DAMB and the octopamine receptor OAMB—for their prominent expression in the mushroom bodies (MB), brain structures essential for olfactory conditioning. Notably, each receptor has a distinct expression pattern in MB substructures. In vitro, all three receptors activate cAMP increases, while DAMB and OAMB additionally induce intracellular Ca²⁺ increases. These findings suggest that dDA1, DAMB, and OAMB modulate cAMP and/or Ca²⁺ signaling in the MB that underlies associative learning and memory. Also, their differential distribution in the MB implies that each receptor may play a role in different behavioral plasticity. To test this, we generated mutations in all three receptors and are testing them in three behavioral assays: aversive and appetitive conditionings and courtship conditioning. These paradigms test the fly's ability to associate olfactory inputs (conditioned stimuli) with diverse unconditioned stimuli (US) presented through different sensory modalities. Thus, this study will elucidate whether dDA1, DAMB and OAMB function in processing distinct US information for associative learning and memory. Our preliminary studies, indeed, reveal non-overlapping phenotypes of mutants altered in the genes encoding dDA1, DAMB, and OAMB in these assays. Supported by US NIH grant R01 NS38346.

ACTIN UP: SKELETONS IN THE APP AND FMRP CLOSET

BASSEM HASSAN, SIMON REEVE, MAARTEN LEYSSEN, LAURA BASSETTO, and DERYA AYAZ

VIB, University of Leuven School of Medicine, 3000 Leuven, Belgium

Causes of the loss of neuronal structure and function can be divided into two main categories: developmental and degenerative. In the first, mutations in genes essential for forming a functional nervous system cause a developmental defect. In the second, the nervous system forms and functions normally and then degenerates. Two classic examples are the mental retardation disorder Fragile-X for the first and Alzheimer's disease for the second. In both cases the mechanisms of action of the genes responsible—those encoding FMRP and APP—are essentially unknown. We show that both genes act by regulating the actin cytoskeleton. This regulation is essential for their function in controlling neuronal extension and sprouting. APP initiates a signaling cascade, which requires Abl tyrosine kinase, Rac1, and Profilin; while FMRP controls Profilin levels directly by binding to its mRNA.

SQUEEZE IMPLICATED IN LEUCOKININ EXPRESSION OF THE LHLK NEURONS OF THE DROSOPHILA PROTOCEREBRUM

PILAR HERRERO, MARTA MAGARIÑOS, ISABEL MOLINA, BELEN DORADO, INMACULADA CANAL, and LAURA TORROJA

Departamento le Bioloǵia, Universidad Autónoma de Madrid, 28049 Madrid, Spain

Neuroendocrine cell differentiation involves multiple stages of regulatory gene activity. The LIM Homeodomain proteins form a family of transcriptional regulators involved on that process of cell differentiation. We have already shown that the LIM-HD protein Apterous regulates leucokinin peptide expression in a pair of lateral horn protocerebrum neurons (LHLK cells). Apterous seems not to be necessary for leucokinin determination in other cells. Previously, it was shown that both Apterous and the zinc-finger protein Squeeze are required to specify neuropeptide cell identiy in Tv cells by regulating FMRFamide expression in them. We show here that Squeeze is also necessary in LHLK cells for leucokinin expression. Our results show that 90% of the LHLK cells and 97% of the subesophagic leucokinin neurons are depleted of the neuropeptide in squeeze (sqz) mutant larvae. As expected, in apterous larvae the subesophagic neurons express leucokinin normally. We performed rescue experiments by driving UAS-sqz expression under the control of several Gal4 lines. Leucokinin expression was partially restored in LHLK and subesophagic leucokinin neurons. We also observed ectopic leucokinin expression when both proteins were co-expressed in gain-of-function experiments. From this genetic analysis we suggest that, similar to FMRFamide, leucokinin expression depends on at least two regulator factors: Squeeze and Apterous.

SHAW POTASSIUM (K⁺) CHANNEL GENES IN DROSOPHILA

JAMES J.L. HODGE, DEMIAN PARK, JAMES C. CHOI, MARK MATTALIANO, CAHIR O'KANE, and LESLIE C. GRIFFITH

Department of Biology & Volen Center for Complex Systemss Brandeis University, Waltham, MA 02454, USA

We describe a second member of the Shaw family, Shawl. In-situ hybridization showed that Shaw and Shawl have largely non-overlapping expression patterns in embryos. Shaw is expressed mainly in excitable cells of the CNS and PNS of late embryos. Shawl is expressed in many non-excitable cell types: ubiquitously until the germband extends, then transiently in the developing CNS and PNS, becoming restricted to progressively smaller subsets of the CNS throughout embryogenesis. Ectopic expression of full-length or a truncated form of Shaw within the CNS result in subunits, which are differentially localized and produce flies with an eclosion phenotype that is under control of the CCAP-neuropeptide circuit. More widespread expression of Shaw in the nervous system resulted in a reduction in body mass, ether-induced shaking, and developmental lethality. Motorneuron expression of either transgene caused a reduction in EJP amplitude. Increased stimulation strength in animals expressing full-length Shaw elicited an EJP of almost wild-type amplitude. Increasing stimulation strength in animals expressing the truncated protein failed to rescue the EJP amplitude. Muscle spikes could not be evoked in muscles expressing full-length Shaw, while expression of truncated Shaw had little effect on muscle properties. A large reduction in bouton number accompanied presynaptic expression of the Shaw transgenes, whereas muscle expression had little effect on NMJ morphology.

CONTROL OF INNER OPTIC-CHIASM DEVELOPMENT BY GLIAL EXPRESSION OF DROSOPHILA OPTOMOTOR-BLIND

KERSTIN HOFMEYER, RORIS KRETZSCHMAR, and GERT PFLUGFELDER

Universities of Mainz and Würzburg, 55128 Mainz, Germany; NYU Medical Center, New York, NY 10016; Oregon Health and Sciences University, Portland, OR 97201, USA

The inner optic chiasm (IOC) connects three neighboring neuropil regions of the Drosophila optic lobes. Several types of neurons project through the IOC. Little is known about the development of their projection patterns. Certain regulatory mutations in optomotor-blind (omb), about 100 kb downstream of the promoter, affect the development of the horizontal and vertical system cells. The lack of these cells appears to cause the eponymous optomotor-blind phenotype. Deletion of the optic lobe regulatory region 2 (OLR2), located between 80 and 100 kb downstream of the promoter, causes additional defects in the optic lobes. Among others structures, that of the IOC is perturbed. Fibers that normally seem to project from the edges of the neuropil regions, in the mutant, appear to cut deeply into the lobula. omb is expressed in many cell types of the developing optic lobes. ORL2 deletions cause the selective loss of omb expression from glial cells of the larval IOC. Loss of omb expression does not lead to the elimination of these cells, which continue to express several tested glial markers. DNA fragments from ORL2 were tested for enhancer activity in transgenic flies. A 6.5 kb fragment (omb-C) drives expression in the affected IOC cell population. Expression of UAS-omb under ombC-Gal4 control in an omb mutant background rescued the IOC developmental defect. This demonstrates a necessary and sufficient role of optomotor-blind in the IOC glia for IOC patterning.

CIS-REGULATORY ELEMENTS REQUIRED FOR THE TRANSCRIPTIONAL CONTROL OF THE PRONEURAL GENE AMOS

EIMEAR HOLOHAN and ANDREW JARMAN

School of Biomedical and Clinical Laboratory Sciences, University of Edinburgh, Edinburgh EH8 9XD, UK

Proneural genes (basic-helix-loop-helix transcription factors) are key regulators of neurogenesis, and to a large extent the pattern of neurogenesis is decided by their pattern of expression. In Drosophila, there are a number of different proneural genes, namely the achaete-scute complex, atonal, and amos. The proneural gene amos is required for sense-organ precursors in the olfactory sensilla and a subset of multiple dendritic neurons. We identified enhancer elements by cloning fragments based on the sequence upstream of amos into a transformation vector containing a reporter gene and by assessing the ability of different fragments to drive reporter gene expression in transgenic flies in a pattern resembling amos. Subdivision of the amos upstream region has shown that separate enhancer elements are responsible for amos expression in the antennae, as well as in the head and trunk regions of the embryo. Misexpression analysis indicated the possibility of the existence of an autoregulatory component. The presence of E-boxes (binding sites for proneural proteins) in the upstream region indicated that this autoregulation might be direct. We identified a putative Amos/Da binding site, mutation of which led to an overall reduction of reporter gene expression. Verification of this sequence as an Amos/Da binding site will provide a point of reference to investigate how Amos may specifically regulate gene expression.

AN INCREASED RECEPTIVE FIELD OF OLFACTORY RECEPTOR OR43A IN THE ANTENNAL LOBE OF DROSOPHILA REDUCES BENZALDEHYDE-DRIVEN AVOIDANCE BEHAVIOR

BERNHARD HOVEMANN, RAFFAEL KETTLER, SVEN FISCHER, and KLEMENS STÖRTKUHL

Faculty of Chemistry, Ruhr-University Bochum, 44780 Bochum, Germany

Most animals orient themselves in their environment through the perception of olfactory cues. In order to gain insight into the principles of olfactory processing in Drosophila, we misexpressed olfactory receptor Or43a in additional olfactory-receptor neurons of the third antennal segment using enhancer trap line GH320. Application of benzaldehyde, an identified ligand of Or43a, resulted in stimulation of a number of glomeruli in addition to glomerulus DA4, which is the regular target of Or43a-expressing neurons. The behavioral response of GH320/UAS-or43a flies was changed upon benzaldehyde application. Using the T-maze assay, misexpressing flies performed a reduced avoidance reaction to benzaldehyde compared with wild type. This reduction of avoidance could be mimicked in wild-type flies by exposing them to a mixture of benzaldehyde and ethyl acetate. Our results demonstrate the relevance of specific olfactory-sensory input and subsequent processing in the antennal lobe for Drosophila behavior.

DSCAM SIGNALING IN THE REGULATION OF ACTIN DYNAMICS AND NEURITE MORPHOGENESIS

MICHAEL HUGHES and DIETMAR SCHMUCKER

Harvard Medical School, Boston, MA 02115, USA

Drosophila Dscam is a single pass transmembrane protein implicated in axon guidance that is alternately spliced to generate approximately 38,000 different isoforms. These isoforms possess identical molecular architectures but variable amino acid sequences underlying the extracellular and transmembrane domains. Interestingly, all Dscam isoforms contain an identical cytoplasmic domain, suggesting that the core constituents of Dscam's signaling complex are constant. Although Dock and Pak, two signaling molecules involved in axon guidance, physically interact with Dscam, little is known about the mechanism by which Dscam regulates axonal and dendritic growth. We are taking an unbiased, biochemical approach to identify components of Dscam's receptor proximal complex involved in the regulation of neurite morphogenesis.

PHYLOGENETIC SHADOWING OF A DROSOPHILA HISTAMINE-GATED CHLORIDE CHANNEL SUBUNIT

MLADEN IOVCHEV, ALEXANDER BOUTANAEV, DMITRY NURMINSKY, ADRIAN WOLSTENHOLME, ROGER HARDIE, and EUGENE SEMENOV

Institute of Molecular Biology, Bulgarian Academy of Sciences, 1040 Sofia, Bulgaria; Tufts University School of Medicine, Boston, MA 20111, USA; Department of Biology & Biochemistry, University of Bath, Bath BA2, UK; Department of Anatomy, University of Cambridge, Cambridge CB3 2DY, UK

Known mutations in gene hclA (synonym: ort), which encodes for a histamine receptor subunit in D. melanogaster, alter histaminergic transmission and lead to defects in the visual system: neurologic disorders and changed responsiveness to neurotoxins. To determine the extent of evolutionary changes in HCLA (phylogenetic shadowing), we identified HCLAs from two other Drosophila species—D. simulans and D. virilis—and compared them and HCLA from D. pseudoobscura in pairwise sequence alignments to Dm-HCLA. Clustered substitutions of amino acids were found at the N-terminus of the protein, in the putative extracellular domain, and in the C-terminal third of HCLA. The transmembrane domains (TMs) 1-3 do not carry any interspecific variations, and only single substitutions were observed in TM 4. Evolutionary changes are especially frequent in the intracellular loop. Conserved motifs for enzymatic modifications (glycosylation and phosphorylation) are mapped along the HCLA sequence. hclA mRNA is found more abundant in heads than in bodies of adults D. melanogaster, D. simulans, and D. virilis.

THE PROCTOLIN PREPROHORMONE (PROCT) GENE OF DROSOPHILA MELANOGASTER

ELWYN ISAAC, CHRISTINE TAYLOR, ALAN SHIRRAS, and DICK R. NÄSSEL

Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK

The Drosophila Proctolin gene (CG7105, Proct) encodes a precursor protein containing the neuropeptide proctolin (RYLPT), which is expressed in neurons of the CNS in embryos, larvae and adult insects. The preproprotein contains a secretion signal peptide followed by a single copy of RYLPT, flanked by a C-terminal 94 amino acid peptide containing a furin-like processing site. The proprotein structure is conserved (86% sequence identity) in D. pseudoobscura preproproctolin, suggesting that selective pressure has maintained not only the proctolin peptide, but also the C-terminal peptide sequence and that its structure is functionally important. Ectopic expression of Proct in the CNS and midgut confirmed that the predicted preproproctolin can be processed to generate immunoreactive proctolin pentapeptide. Pupae over-expressing Proct displayed a 14% increase in heart rate, providing evidence in support of a cardioacceleratory endocrine function for proctolin. The distribution of proctolin suggests roles as a neuromodulator in motoneurons and interneurons and as a neurohormone that could be released from brain neurosecretory cells with terminations in the ring gland. Proct knock-down flies have been generated and are being compared with wild-type flies in behavioral studies.

POLYAMINE-DEPENDENT CK2BETA FUNCTION IN MUSHROOM-BODY DEVELOPMENT

EIKE JAUCH and THOMAS RAABE

Institut für Medizinische Strahlenkunde, University of Würzburg, 97078 Würzburg, Germany

Analysis of CK2beta mutations in Drosophila melanogaster uncovered a critical function of this regulator of divers Ser/Thr-kinases in mushroom body development and the circadian clock. Our phenotypic analysis of both a hypomorphic and a loss of function allele of CK2beta indicate that impairment of normal CK2beta (protein) function results in a loss of neurons generated by the proliferative activity of the mushroom-body neuroblasts. Previous biochemical experiments underline the importance of a polyamine-binding site located in the acidic loop of CK2beta for its regulatory properties. To look into the assumed regulation of CK2beta signaling by polyamines, we investigated mushroom-body (MB) development under conditions in which normal CK2beta signalling in the MB neuroblasts was unbalanced either by overexpression of a CK2beta protein mutant for its acidic loop or by overexpression of SamDC, a key enzyme of the polyamine biosynthetic pathway. We observe in both experiments a reduced number of mushroom body neurons and therefore suggest that CK2beta signalling is modified by polyamines in vivo.

THE STANDARD BRAIN PROTOCOL: TOWARD A GENETIC ATLAS OF THE BRAIN

ARNIM JENETT, JOHANNES SCHINDELIN, and MARTIN HEISENBERG

Biocenter, University of Würzburg, 97074 Würzburg, Germany

Gene expression patterns in wholemounted fly brains are scanned with the confocal microscope and visualized. These datasets can now be compared and evaluated as aligned standard brains using the Virtual Insect Brain (VIB) protocol. The computation reveals and quantifies inter-individual variability of the expression patterns. Typically, crosses of Gal4 driver and UAS reporter are standardized and compared to expression patterns of other crosses. A databank has been developed to collect and host the standardised expression patterns for usage via the www (http://www.neurofly.de). In the two-component Gal4 enhancer-trap system of Brand & Perrimon (Development 118, 401–415; 1993), tissue specificity is thought to be provided by the expression pattern of Gal4 in the driver line, whereas the reporter construct is usually assumed to have no influence on the pattern. We have compared the expression patterns of Gal4-driver/UAS-reporter combinations differing only in the genomic location of the reporter insert. The expression patterns are highly dependent on the genomic positions of the reporter constructs applied. Using Gal4 enhancer-trap strains as well as promoter strains we found substantial differences in the visualized patterns. While the patterns agreed in several basic features, they differed in many details, which could not be explained assuming differences in general expression levels. Variability among flies of the same genotype was much smaller.

SCREENING FOR GENES THAT INTERACT WITH GLUED DURING SYNAPTOGENESIS IN THE GIANT-FIBER SYSTEM OF DROSOPHILA

LOUISE JOHNS, LISHA MA, and MARCUS ALLEN

Dept. of Biosciences, University of Kent, Canterbury, Canterbury CT2 7NJ, UK

The giant-fiber system is a well-characterized neural circuit in Drosophila. Our research is focused upon the role of the retrograde transport machinery during synaptogenesis between the giant-fiber (GF) and the tergotrochanteral motor neuron (TTMn), both major neurons in the circuit. The Glued gene encodes the p150 component of dynactin, which complexes with the retrograde motor dynein, facilitating retrograde transport along axonal microtubules. The dominant negative mutation Glued¹ results in a C-terminal-truncated protein that disables dynein-dynactin. Selective expression of a mutant C-terminal-truncated Glued protein (Gl^Δ) in the GF disrupts synaptogenesis between the GF and the TTMn. We have performed a screen for dominant enhancers and suppressors of mutant Glued phenotypes to identify other molecules that interact genetically with the retrograde motor during synapse formation. Over 30,000 flies were screened, and 11–15 interesting enhancers or suppressors have been identified. Mapping of mutant etiologies and analyses of mutant phenotypes are in progress.

FUNCTIONAL REQUIREMENT FOR THE RECEPTOR COMPONENT PROTEIN BY A DROSOPHILA DIURETIC HORMONE RECEPTOR

ERIK JOHNSON, ORIE SHAFER, JULIAN DOW, DAVID SCHOOLEY, JENNIFER TRIGG, and PAUL H. TAGHERT

School of Medicine, Washington University, St. Louis, MO 63112, USA

The Drosophila G protein-coupled receptor encoded by CG17415 is related to members of the calcitonin-like receptor family. We report here the identification of a candidate neuropeptide ligand, Diuretic Hormone 31 (DHII). Functional expression of this receptor in a mammalian cell line conferred specificity to this ligand as measured by dose-dependent increases in cAMP levels but notably was dependent upon the co-expression of the Drosophila receptor component protein (dRCP), encoded by the CG4875 gene. A systematic analysis addressed the influence of coexpressing mammalian RAMPs and RCPs, and co-expression of different RAMP subtypes and/or RCP did not alter signaling elicited via CG17415. We conclude that these factors serve redundant functions for this Drosophila receptor in HEK cells. An antibody directed against the receptor revealed expression in a limited number of neurons in the dorsal brain, correlating well with DH31 peptide expression, and furthermore suggests novel roles for DH31 peptidergic signaling. Consistent with predictions concerning expression patterns of a candidate DH31 receptor, CG17415 expressed within the principal cells of Malphigian tubules. The identification of a candidate receptor in Drosophila has two major implications: first, it will allow for a detailed analysis of the mechanisms maintaining osmotic balance in this organism; and second, it will permit a detailed genetic analysis of calcitonin-like receptor signaling.

COMPREHENSIVE SENSORY MAP OF DROSOPHILA AUDITORY NEURONS

AZUSA KAMIKOUCHI, TAKASHI SHIMADA, and KEI ITO

Institute of Molecular and Cellular Biosciences, University of Tokyo, 113-0032 Tokyo, Japan

To clarify mechanisms as to how animals process acoustic information, we initiated a comprehensive mapping of auditory neural network in the Drosophila brain. As a first step, we analyzed the innervation patterns of the neurons of the Johnston's organ (JONs), the mechanoreceptive auditory organ located in the second antennal segment. By screening GAL4 enhancer-trap strains that exclusively label JONs in the antennae, we revealed that JONs and olfactory-receptor neurons form segregated bundles in the antennal nerve, and that the terminals of JONs as a whole spread over broad area of the antennal mechanosensory and motor center (AMMC) rather than being confined within its subarea. We also found a few sub-branches that project to the suboesophageal ganglion or ventrolateral protocerebrum. Because these areas receive input not only from JONs, but also of other sensory modalities, some cross-modal bindings might occur at this level. Using the heat-shock FLP-out system, we found that each single JON innervates specific subregions of the AMMC. Comparing their projection patterns, we identified eleven subregions in the AMMC and classified JONs into four types according to the combination of subregions they innervate. Interestingly, somata of each type of JONs were located in characteristic zones of the JON array in a partially overlapping manner. These results suggest that JONs comprise four subgroups, each of which might participate in different aspects of auditory reception.

CHARACTERIZATION OF HOMEOBRAIN, A HOMEOBOX GENE OF DROSOPHILA MELANOGASTER

PETRA KASPAR, MICHAEL FAUST, RICHARD J. DAVIS, and UWE WALLDORF

University of Saarland, 66421 Homburg, Germany; Department of Pathology, Baylor College of Medicine, Houston, TX 77030, USA

The homeobrain (hbn) gene belongs to the paired-like class of homeobox genes and forms a complex with the genes orthopedia (otp) and drx in region 57B on the right arm of the 2nd chromosome of Drosophila melanogaster. Hbn protein is expressed in the embryonic brain and the ventral nerve chord. This expression pattern suggests that the transcription factor Hbn is involved in the development of brain and nervous system. In larvae the protein is expressed in the optic lobes of the larval brain and in the wing disc. In an EMS-mutagenesis experiment of region 57B we isolated 38 lethal mutations, representing 15 complementation groups. Among these, one fly stock with an embryonic lethal mutation of hbn was identified. An EMS-induced stop codon results in a shortened protein of 86 amino acids (aa's), missing the homeodomain compared with the 409-aa wild-type form. Analysis of the gene function during later stages of development will be possible if non-lethal mutations are available. Such mutations often affect regulatory elements of a gene. We identified a P-element insertion into the 5' regulatory region of hbn that causes a deformed meso-thorax. This deformation also results in a disability of the flies to unfold their wings. This characteristic phenotype may contribute to understand the functions of hbn during Drosophila development.

EXPERIMENTAL EVOLUTION OF IMPROVED LEARNING AND MEMORY IN DROSOPHILA

TADEUSZ KAWECKI and FREDERIC MERY

Dept. of Biology, University of Fribourg, 1700 Fribourg, Switzerland

The process of learning and memory formation in flies is thought to consist of five distinct stages: learning acquisition and four forms (phases) of memory. Which stages of the learning process change if evolution favors an improved learning response, and how readily does this happen? To address this question we exposed outbred laboratory lines to conditions that favored associative aversion learning in the context of oviposition substrate choice. Within 20 generations these selected lines evolved markedly improved learning response, indicating that improved learning can readily evolve under an ecologically relevant regime. The improved response of the selected lines generalized to other olfactory-aversive and appetitive-learning paradigms, (i.e., the response was not specific to the task and stimuli under which the lines were selected). Using an olfactory-mechanical learning paradigm we showed that the selected lines show faster learning acquisition, better anesthesia-resistant memory, and better long-term memory (compared with unselected control lines). However, the saturation level of the learning curve, short- and middle-term memory and olfactory ability did not change. This indicates that some stages in the process of learning and memory formation can evolve independently of the others. Furthermore, analysis of individual replicate lines indicates antagonism between anesthesia-resistant and long-term memory, in agreement with a recent model of memory consolidation.

THE CNS MIDLINE CELLS ARE REQUIRED FOR CORRECT IDENTITY DETERMINATION, PROPER GENERATION OF THE NEUROBLASTS, AND AXON-PATHWAY FORMATION IN THE DROSOPHILA BRAIN

HEE KIM, JIYOUNG SONG, and WAN SEOP CHOI

Department of Chemistry, Konkuk University, 143-701 Seoul, Korea

The CNS midline cells are essential for the proper neural development of the Drosophila ventral nerve cord. It is not well known whether the CNS midline cells are also involved in proper formation of NBs and axon pathway in the Drosophila brain. The roles of the CNS midline cells were investigated by examining the expression of several neuronal, glial and axon pathway markers in the brain of single-minded (sim) mutant embryos. This study revealed that the CNS midline cells are required for the proper expression of early brain NB markers—products of the genes lethal of scute, deadpan, scratch, and seven-up—indicating that the CNS midline cells are essential for brain formation from the initial brain neurogenesis. They are also required for the proper expression of late brain NB markers, castor- and dachsund-encoded proteins, and of a ganglion mother cell marker generated by prospero. Reduced expression of brain neuronal markers in sim embryos is in part caused by defect in cell division of NBs in the Drosophila brain. It was shown that the longitudinal axon pathway in the sim mutant is disconnected between the brain and ventral nerve cord. The defects in the longitudinal axon pathway are not by identity change or by absence of segmental neurons, but by malformation of axonal connection between the brain and ventral nerve cord. This study indicates that the CNS midline cells play critical roles in the formation and differentiation of the Drosophila brain and ventral nerve cord, which could help understand mammalian brain development.

DISTINCT LIGHT-INPUT PATHWAYS INTO CIRCADIAN-CLOCK NEURONS OF THE DROSOPHILA LARVAL BRAIN

ANDRÉ KLARSFELD, MARIE PICOT, CHRISTINE MICHARD-VANHÉE, SÉBASTIEN MALPEL, ELISABETH CHÉLOT, and FRANÇOIS ROUYER

CNRS, 91198 Gif-sur-Yvette, France

The brain circadian clock of adult Drosophila consists of several tens of neurons per hemisphere and may perceive light via as many as four anatomically or molecularly distinct pathways. In contrast, only nine clock neurons have been described in larval brain hemispheres, and the relatively simple larval visual organ [projecting onto one class of clock neurons which express the pigment-dispersing factor (PDF) neuropeptide] and blue-light-sensitive cryptochrome (CRY) seem to provide their only light inputs (Kaneko et al., J. Biol. Rhythms, 2000; Malpel et al., J. Biol. Rhythms, 2004). We have shown that CRY is specifically expressed in only six of the nine larval clock neurons and that its forced expression was able to change the phase of molecular oscillations in some of the normally CRY-deficient cells, the so-called DN2 neurons (Klarsfeld et al., J. Neurosci, 2004). Further analyses of the respective roles of CRY and the visual system in light entrainment, as well as in molecular rhythmicity in constant light, reveal a surprising specificity of input for each clock neuronal group of the larval brain. Notably, proper synchronization of the larval DN2s appears to require the PDF neuropeptide.

THE DROSOPHILA MELANOGASTER HOMOLOG OF THE HUMAN OAZ TRANSCRIPTION FACTOR PLAYS A CRUCIAL ROLE IN FLY DEVELOPMENT

ANNE KRATTINGER, ARIANE RAMAEKERS, NICOLA GRILLENZONI, and REINHARD F. STOCKER

Department of Biology, University of Fribourg, 1700 Fribourg, Switzerland

The GAL4-line GH146, isolated in our laboratory, has been intensively used both for behavioral and developmental studies. We investigated the gene reflecting the particular expression pattern of GH146. By in situ hybridization, we determined the putative gene CG17390 as the best candidate. By homology comparison, we suggest that this gene could be the Drosophila melanogaster homolog of OAZ. Therefore, we call this gene dmoaz (Drosophila melanogaster oaz homolog). The members of this recently identified OAZ family encodes C2H2 zinc-finger transcription factors that play multiple roles during development. To investigate the function of dmoaz, we generated about 200 mutant lines by imprecise excision of the GH146 P-element insertion. After screening, we kept certain lines (93, 151, and 172) for further study. Based on complementation tests we were able to conclude that the line 151 was a weaker mutant line than 93 and 172. These three mutant lines are homozygous lethal. The hatching rate seems to be normal, but development stops at the 1st larval instar. Further investigations are required to clearly analyze the phenotype of these mutants.

GLIAL AND NEURONAL EXPRESSION OF POLYGLUTAMINE PROTEINS INDUCE BEHAVIORAL CHANGES AND INCLUSION FORMATION IN THE DROSOPHILA BRAIN

DORIS KRETZSCHMAR. JAKOB TSCHÄPE, ALEXANDRE BETTENCOURT DA CRUZ, ESTHER ASAN, BURKHARD POECK, ROLAND STRAUSS, and GERT PFLUGFELDER

Universities of Mainz (55128) and Würzburg (97074), Germany; Oregon Health and Sciences University, Portland, OR 97201, USA

Surprisingly little is known about the early role of glia in the familial human CAG expansion diseases. Given the intimate association between neurons and glia, it is unlikely that glia are unaffected bystanders during pathogenesis. Since most CAG expansion genes appear to be expressed fairly ubiquitously in the CNS we investigated, in the Drosophila adult brain, whether glia are refractory to the toxic action of expanded polyglutamine proteins. Glial-specific expression of a truncated and glutamine-expanded (Qn = 78) version of human ataxin 3 (obtained from Nancy Bonini) led to nuclear inclusion formation and cell death (vacuolation). Similar inclusions were observed when the CAG-expanded constructs were expressed in neurons. Glial inclusions had a fibrillary substructure indistinguishable from the aggregates found in neurons. Behavioral changes were observed prior to glial cell death. Changes in fly behavior can therefore be used to monitor early effects of polyglutamine toxicity. Pan-neuronal expression of the same construct resulted in behavioral changes similar to those resulting from glial expression, without any sign of neurodegeneration. This is in agreement with the large body of evidence that neurodegeneration is not a prerequisite for the appearance of neurological symptoms.

CHARACTERIZATION AND MANIPULATION OF A GENE INVOLVED IN PHEROMONAL DISCRIMINATION

EMELIE LANNELONGUE and JEAN-FRANÇOIS FERVEUR

CNRS-UMR5548, Université de Bourgogne, 21 000 Dijon, France

D. melanogaster males show a polymorphism for their production of cuticular pheromones: in strains (such as Tai) collected in African-tropical regions, males produce a high amount of 7-pentacosene (7-P), whereas males of other strains (like Canton-S, CS) produce a low amount of 7-P. There is also a polymorphism for male response toward 7-P, because Tai-like males are inhibited, whereas CS-like males are stimulated- by 7-P. The production of 7-P and the response to 7-P are two characters that have co-evolved, because their variation segregates with different autosomes. One factor that changes male response toward 7-P was mapped on chromosome 3: it matches the CG 8793 sequence that corresponds to a gene without any known function in flies and in human brain. This gene harbors a TPR-like domain that was also found in croocked-neck, a gene required for the specification of embryonic nervous system. CG 8793 codes for a long transcript (3,710 bp) present in both sexes and for a short male-specific transcript (1,720 bp). The short transcript was detected (by RT-PCR) in male legs of Tai-like strains but not in male legs of CS-like strains. A UAS-transgene containing the short transcript was injected in a strain with the CS genetic background. Its ubiquitous expression specifically reduced the behavioral response of transgenic males to Tai males. This indicates that the short transcript contributes to the polymorphism of male responsiveness.

THE MOLECULAR ARCHITECTURE OF AXO-GLIAL JUNCTIONS: GENETIC ANALYSES IN DROSOPHILA

MONIQUE LAVAL, SWATI BANERJEE, MANZOOR A. BHAT, JEAN ANTOINE GIRAULT, and CATHERINE FAIVRE-SARRAILH

UMR 6184 CNRS, 13916 Marseille, France

Ensheathment and myelination share important features in common in Drosophila and vertebrates, including the formation of septate junctions (SJs) that isolate the nerve fibers from the extracellular fluid in Drosophila and are involved in the attachment of myelin loops to axonal surface in vertebrates. In vertebrates, the cell adhesion molecules Caspr/paranodin, F3/contactin and Neurofascin-155 are key components of the paranodal junctions. Neurexin IV (NRX IV) is the fly homolog of Caspr/paranodin, and it plays a crucial role in the formation of glial SJs as well as axonal insulation in the PNS of Drosophila embryos. We report here the genetic, molecular, and biochemical characterization of Drosophila Contactin (DCONT). We show that DCONT, NRG and NRX IV are interdependent for their SJs localization and these proteins form a tripartite complex. Ultrastructural and dye-exclusion analyses of dcont-null embryos show that DCONT is required for organization of SJs and paracellular barrier function. Our studies provide evidence that an interplay between highly conserved cell adhesion molecules mediates the organization of SJs in different species. We are using the available results of a yeast two-hybrid screen that allowed identification of proteins interacting with the intracellular domain of Drosophila NRX IV to characterize new downstream effectors for the DCONT/NRXIV/NRG complex, which could be also operative during axo-glial interactions in vertebrates.

VIDEOTAPING AROUSAL: AUTOMATED TRACKING OF DROSOPHILA FOR HIGH-THROUGHPUT BEHAVIORAL ANALYSES

TIM LEBESTKY, LIHI ZELNIK, PIETRO PERONA, and DAVID ANDERSON

Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA

To assess the molecular nature of arousal, we have developed simple automated behavioral assays to present freely moving animals with different mechanical stimuli of varied frequency and intensity. One such assay follows the startle effects in response to a series of air-puffs, delivered at regular intervals. We observe a rapid escalation of locomotor activity as a function of time and puff number. Furthermore, the stable elevation in locomotor pattern is only achieved by multiple puff events: a single puff stimulus is insufficient to generate a stable, highly aroused state. By initially screening through mutants and collections of flies that are conditionally silenced at specific circuits (Gal4;UAS-shibire^TS), we hope to delineate the molecular mechanisms and neural circuits involved in the generation and modulation of such arousal responses. This automated quantification relies on Fly-Tracker, visual tracking software that we have developed for high spatial and temporal quantification of locomotor patterns. Beyond the current applications, the software is highly versatile and should allow accurate measurement of populations or individual flies in many behavioral contexts. Possible examples would involve neurodegenerative locomotor assays, place-preference assays, assessing social interactions, and courtship behaviors.

HISTOBLAST DEVELOPMENT: A STUDY OF FORMATION OF THE ADULT-FLY ABDOMINAL EPIDERMIS

CLAUDIA LEE, GUY TEAR, and WILLIAM CHIA

MRC Centre for Developmental Neurobiology, King's College, London SE1 1UL, UK

Histoblasts are imaginal cells that give rise to the adult abdominal epidermis. Formed in embryogenesis, they remain quiescent throughout larval stages. During pupariation, ecdysone signalling triggers the histoblasts to divide and migrate, resulting in the expansion and fusion of the nests. The larval epidermal cells located between the nests are removed by autophagy of cells in contact with the expanding histoblast nests. After histoblast fusion is complete, histoblast cells differentiate to produce adult epidermal structures, including cuticle and sensory organs. In comparison to other imaginal systems, histoblast development has not been extensively studied. Potentially, histoblast development can provide a good system to study cell migration, programmed cell death, ecdysone signalling, compartmentalization, and differentiation. We used time-lapse, live-imaging confocal microscopy—using specific drivers and protein trap lines to mark histoblast nests with GFP—in order to characterize the development and especially the migration of histoblasts in larval and pupal stages. Analysis of these movies enables the observation of the direction and domains of migration. We have also examined a collection of pupal-lethal EMS mutants to look for genes involved in abdomen development.

UNRAVELLING AMYLOID PRECURSOR PROTEIN SIGNALING IN DROSOPHILA NEURONS

MAARTEN LEYSSEN, DERYA AYAZ, SIMON REEVE, JIEKUN YAN, BART DE STROOPER, and BASSEM HASSAN

Laboratory of Neurogenetics, Department of Human Genetics, VIB & University of Leuven, 3000 Leuven Belgium

Alzheimer's disease (AD) is the most common neurodegenerative disorder in the western world. Familial forms of the disease have been linked to mutations in the gene encoding Amyloid Precursor Protein (APP). We use Drosophila melanogaster to study neuronal APP signalling in vivo. We specifically express human APP in the rhythm-related Lateral Neurons Ventral (LNv's). APP is transported to the axonal processes and leads to a drastic and dosage dependent increase in the extent of axonal arborisation. We found the membrane-tethered C-terminus to be crucial for this phenotype. Since axonal extension depends critically on the modification of the actin cytoskeleton, we used genetic interaction studies to identify a signaling cascade leading from APP to actin. We investigated whether neuronal upregulation of APPL expression could be also a physiologically relevant paradigm and found high expression of APPL in fly neurons after traumatic brain injury. Since axonal extension and outgrowth under these circumstances is crucial for functional rewiring of the brain, we hypothesize that APP may be important in the functional repair after brain injury.

OTD AND EMS IN ADULT NERVOUS-SYSTEM DEVELOPMENT OF DROSOPHILA MELANOGASTER

ROBERT LICHTNECKERT, BRUNO BELLO, and HEINRICH REICHERT

Biozentrum/Pharmazentrum, University of Basel, 4056 Basel, Switzerland

The homeobox transcription factors Otd and Ems have crucial roles in early embryonic CNS development of Drosophila and mouse. In the otd homozygous mutant fly brain, the anterior-most part of the brain fails to form during embryonic stages. Another brain gap phenotype in a more posterior part of the supraoesophageal ganglion is seen in emshomozygous mutants. Both severe phenotypes result from the absence of a specific subgroup of neurons. CNS development in the holometabolous insect Drosophila melanogaster is characterized by two phases of neurogenesis and neurodifferentiation, giving rise to a functional larval nervous system over the course of only 22 hours and a highly complex adult nervous system during larval and pupal stages. Given the striking functions of otd/ems during embryonic brain development in Drosophila, we are interested in the expression and function of these two head gap genes during adult nervous system development. Expression analysis in the CNS at different larval stages has revealed the continuous expression of both genes in postembryonically differentiating neural cell lineages. Strong correlation with the embryonic expression pattern points towards the maintenance of fate determination of quiescent neuroblasts characterized by specific expression of otd or ems. Since both mutations result in embryonic lethality we are currently performing a func-tional analysis of otd and ems in larval and adult stages by generating homozygous mutant clones.

Out Cold: A DROSOPHILA MODEL OF HUMAN SEIZURE DISORDERS?

HELEN LINDSAY, HOWRY JACOBS, RHONA MILNE, and KEVIN O'DELL

Division of Molecular Genetics, University of Glasgow, Glasgow 611 GNU, UK

We are studying the Out cold (Ocd) mutant with a view to developing a Drosophila model of human seizure disorders, particularly those involved in mitochondrial defects. Ocd is a dominant cold-sensitive paralytic X-linked mutant. The primary objective of this work is to identify the Ocd gene and determine how mutations in it result in its complex phenotype. Previous work suggests that Ocd may be involved in mitochondrial energy metabolism. However, sequence analysis of mitochondrially expressed candidate genes has revealed no coding mutations. To narrow down the Ocd critical region we first used a SNP based mapping approach, which localised Ocd to a 200-kb region encompassing 30 genes. At present we are refining the search further, performing high resolution mapping with molecularly defined P-element insertions. To date, we have successfully narrowed down the interval to six genes, the coding regions of which are being sequenced. Additionally, work is being carried out to further characterize the Ocd phenotype. Preliminary studies suggest sound-induced paralysis of mutants, developmental delay, and semi-viability. We have also performed whole-fly proteome analysis. Ten proteins were identified which show statistically significant differences between wild-type and Ocd flies. Six of these are mitochondrial. Once the Ocd gene has been cloned and characterized we should be able to elucidate how Ocd affects mitochondrial metabolism and impairs neurological activity.

DISCOVERY AND CHARACTERIZATION OF GENES INVOLVED IN DE-NOVO FORMATION OF NEUROMUSCULAR JUNCTIONS OF DROSOPHILA

MOHIDDIN LONE, ZOLTAN ASZTALOS, RICHARD AUBURN, CAHIR O'KANE, ANDREAS PROKOP, JOÃO ROCHA, SEAN SWEENEY, and MINGYAO YANG

School of Biological Sciences, University of Manchester, Manchester M13 9PT, UK

Whereas principles of synaptic function are well understood, there is very little insight into the mechanisms underlying the formation of synapses and synaptic terminals. To identify novel genes involved in these processes we focussed on neuromuscular junctions (NMJ) and screened a collection of strains carrying lethal P-element insertions on the 3rd chromosome (Salzberg et al., 1997, Genetics 147, 1723 ff.). Initial criteria were impaired movement at the late embryonic/early larval stage in the absence of obvious muscle or CNS patterning defects. Remaining candidates were crudely analyzed with synaptic markers, and 13 were selected for closer analysis. Inverse PCR, sequencing, and genetic complementation tests were used to identify the P-element insertion sites, and the respective candidate genes were determined in silico. Following detailed light microscopic analyses of these mutants, two were prioritized for detailed molecular and genetic analysis. In the first mutant, NMJs display increased branching, increased number of boutons and active zones. The second mutation causes fused boutons. We are currently proceeding with the molecular analysis of the respective genes. In-situ hybridization shows one of the genes to be expressed in the embryonic CNS from mid-embryonic stages onwards. Taken together, we uncovered promising candidate genes that may further unravel the mechanisms underlying de-novo formation of neuromuscular junctions in the embryo.

HEMICHORDATE NERVE NET PATTERNING: A MIX OF FLIES AND FROGS

CHRISTOPHER LOWE, MARC KIRSCHNER, and JOHN GERHART

Department of Organismal Biology and Anatomy; Systems Biology, Harvard Medical School, Boston, MA 02115, USA; Molecular and Cellular Biology, University of California, Berkeley, CA 94720, USA

The early evolutionary history of deuterostomes has been the subject of an unresolved debate for over 150 years. Developmental genetic data shows great promise to help advance our understanding of this problematic group. We have begun a comprehensive developmental genetic study of a direct-developing enteropneust hemichordate Saccoglossus kowalevskii. By directed cloning and an extensive EST project, we have cloned and examined expression by in-situ hybridization a large number hemichordate orthologs of developmentally important patterning genes that have conserved roles in patterning the CNS of arthropods and chordates. The expression of these orthologs during the development of the diffuse, intra-epidermal nerve net of hemichordates exhibits strikingly similar relative domains in hemichordates in the A/P axis to those in arthropods and chordates, despite fundamental differences in their nervous system organization. However expression is not restricted dorsally or ventrally, but rather is circumferentially uniform. In the dorsoventral dimension, conserved expression domains resemble many of those from Drosophila. The results are discussed in the context of metazoan nervous system evolution and the use of developmental gene-expression data for testing hypotheses of morphological homology.

FUNCTIONAL ANALYSIS OF SYNAPTOTAGMIN'S C2B POLYLYSINE MOTIF

CARIN LOEWEN and NOREEN REIST

Department of Biomedical Sciences, Colorado State University, Fort Collins, CO 80523, USA

Synaptotagmin (Syt) is a synaptic vesicle protein that is required for efficient synaptic transmission. However, its exact roles in the synaptic vesicle cycle remain unclear. While there is strong evidence that Syt is the major Ca²⁺ sensor for synaptic transmission, it has also been implicated in synaptic vesicle docking and recycling. Consistent with a role in recycling, Syt is a high-affinity receptor for the clathrin adaptor-protein complex, AP-2. In vitro biochemical experiments have mapped the AP-2 binding site to the polylysine motif in Syt's C2B domain, and mutations of this motif in Drosophila (Syt K379,380,384Q) result in a ∼ 40% decrease in synaptic transmission. However, the C2B polylysine motif also interacts with several other possible effecter molecules, including PIP₂ and Ca²⁺ channels, consistent with a role in either recycling or docking. To test whether the impaired function of the polylysine motif mutants is due to a recycling deficit, we utilized the activity-dependent dye FM1-43 to measure the kinetics of vesicle membrane internalization. We found no difference in the rate of internalization between mutants and controls, indicating that Syt's C2B polylysine motif is not required for synaptic-vesicle recycling. To further test the function of the C2B polylysine motif, we measured evoked junctional potentials during high frequency stimulation to examine the mutants’ rate of depletion and their subsequent rate of recovery.

DEVELOPMENT OF WIRING SPECIFICITY OF THE DROSOPHILA OLFACTORY CIRCUIT

LIQUN LUO

Department of Biological Sciences, Stanford University, Stanford, CA 94305, USA

As in mammals, Drosophila olfactory receptor neurons (ORNs) that express a given receptor converge their axons onto a common glomerulus in the antennal lobe (AL), creating an odor map in this first olfactory center. AL projection neurons (PNs) send their dendrites into glomeruli and axons to higher brain centers including the mushroom body and the lateral horn. Using MARCM-based systematic clonal analysis, we found that PNs are prespecified by lineage and birth order to send dendrites to specific glomeruli and thereby carry specific olfactory information (Jefferis et al., Nature 414, 2001). Further, we demonstrated that according to glomerular class, PNs have stereotyped axon branching patterns and terminal fields in the lateral horn (Marin et al., Cell 109, 2002; also see Wong et al., Cell 109, 2002). Thus, during the construction of the fly olfactory system a given ORN must target its axons to one of ∼ 50 glomeruli, while a given PN must also target its dendrites to one of ∼ 50 glomeruli; furthermore the PN must coordinate its dendritic target choice with its axon terminal arborization pattern in higher olfactory centers. Our developmental and genetic analyses have begun to shed light on how wiring specificity in the AL is achieved. Surprisingly, PN dendrites and ORN axons appear to have substantial self-organizing properties. Various lines of experimental evidence have been obtained to support these findings and have prompted further efforts involving candidate-gene and forward-genetic approaches to identify genes that direct the establishment of wiring specificity of the olfactory system.

DROSOPHILA HOMOLOG OF RECEPTOR TYROSINE KINASE SUBSTRATE EPS15 IS A KEY COMPONENT OF THE SYNAPTIC VESICLE-RECYCLING MACHINERY

AMITABHA MAJUMDAR, RICHA RIKHY, SHOBHA RAMAGIRI, and K. S KRISHNAN

Tata Institute of Fundamental Research, 400 005 Mumbai, India

The mammalian Eps15 is phosphorylated by EGF receptor tyrosine kinase and has been shown to interact with several components of the endocytic machinery. Genetic studies in C. elegans have implicated Eps-15 in synaptic vesicle recycling. We have identified an Eps-15 mutant in Drosophila which shows reversible paralysis and an altered electroretinogram when exposed to restrictive temperatures. In addition, the temperature-sensitive paralytic defect of shibire^TS is enhanced by this mutant. Eps15 is enriched in the neuromuscular junction in endocytic hot spots in a pattern similar to the expression of dynamin. Vesicle recycling is compromised, as indicated by a reduction in AM1-43 dye uptake. Electrophysiological recordings at the larval neuromuscular junction show an increased quantal size, and electron microscopic sections of synapses show abnormally oversized vesicles in these mutants. Genetic and biochemical studies of interactions with components of endocytic machinery suggest that the Eps-15 gane has a formative role at endocytic hot spots and regulates recruitment of endocytic proteins to vesicular components destined for retrieval.

GENETIC DISSECTION OF THE CIRCUITRY UNDERLYING DROSOPHILA COURTSHIP BEHAVIOR

DEVANAND S. MANOLI, GEOFFREY W. MEISSNER, ROBIN J. STEVENS, EMILY A. OCHOA, WENDY WOO WENDY, JOSÉ CHAVEZ, and BRUCE S. BAKER

Department of Biological Sciences, Stanford University, Stanford CA 94305, USA

Male courtship in Drosophila is regulated by male-specific transcription factors (Fru^M) encoded by the fruitless (fru) locus. Mutant fru phenotypes show that this gene is essential for all aspects of courtship. Fru^M is expressed in ∼ 2% of CNS neurons. Most expression is seen in ∼ 20 clusters of neurons throughout the brain and VNC. In order to dissect the flow of information and circuitry underlying courtship, we are determining the role of individual clusters of neurons for courtship behaviors. A GAL4 enhancer-trap, P52-GAL4, whose expression overlaps with a single, bilaterally-symmetric pair of clusters of Fru^M-expressing neurons in the sub-oesophageal ganglion, was used to eliminate Fru^M expression in these median bundle neurons, via P52-Gal4-directed expression of an RNAi transgene targeting male-specific Fru isoforms ( fru^MIR). Such males display striking, specific defects in courtship (Manoli and Baker, 2004, Nature 430, 564–569), suggesting that these neurons function to coordinate the behaviors that comprise courtship. We have further used targeted expression of the fru^MIR transgene to eliminate Fru^M expression in many other specific regions of the CNS and assayed the effects on courtship behavior. Screening a collection of more than 1,000 Gal4 lines and assaying for intermale chaining behavior, aberrant male-male interactions, defects in courtship, and decreases in fertility, has identified distinct subsets of Fru^M-expressing neurons that function to regulate specific components of courtship.

DEVELOPMENTALLY PROGRAMMED REMODELING OF THE DROSOPHILA OLFACTORY SYSTEM

ELISABETH MARIN and LIQUN LUO

Department of Biological Sciences, Stanford University, Stanford, CA 94305, USA

In the adult fly, olfactory-receptor neurons (ORNs) expressing a given olfactory receptor target a single glomerulus in the antennal lobe (AL). Projection neurons (PNs) relay activity from the AL to higher olfactory centers, the mushroom body calyx and the lateral horn. Here we show that embryonic-born PNs contribute to both the larval and adult olfactory circuits. In the larva, these neurons generally innervate a single glomerulus in the antennal lobe and one or two glomerulus-like substructures in the mushroom-body calyx. Most if not all of these PNs persist to participate in the adult olfactory circuit as well and are pre-specified by birth order to innervate a distinct subset of glomeruli from those of later-born PNs. Developmental studies indicate that these neurons undergo stereotyped pruning of their dendrites and axon branches during early metamorphosis. This pruning requires cell-autonomous func-tion of Ultraspiracle, a co-receptor for the steroid hormone ecdysone, as well as the TGFβ/Activin Type I receptor Baboon.

OLFACTORY LEARNING INDEPENDENT OF THE CAMP SIGNALING PATHWAY

PAVEL MASEK and MARTIN HEISENBERG

Biozentrum, University of Würzburg, 97074 Würzburg, Germany

Drosophila can associate different odors with electroshocks. Several genes of the cAMP-signaling pathway are involved in olfactory short-term memory. For example, memory scores of mutant rutabaga (rut) and dunce (dnc) are about 50% of wild type. Flies can also learn to discriminate between different concentrations of the same odorant. We provided a choice between two concentrations of isoamylacetate (IAA) at a ratio of 1:6. The learning score for 3-min memory is PI = 0.2 for wild-type Canton-S flies. The same scores were obtained for rut²⁰⁸⁰ and dnc^M11 mutant flies. Our data suggest that the 3-min memory for this type of learning does not depend on rut or dnc. In tests involving large concentration differences, short-term memory scores equal those obtained in two-odor learning for wild-type flies as well as for rutabaga. Generalization experiments show that increasing concentration differences lead to behavioral responses similar to differences in odor quality (no generalization). In contrast, at low concentrations two odor qualities can be generalized. Here, rut performs as well as wild type. For two very dist-inct odorants that are not generalized at all, rut gives 50% wild-type learning scores over the whole concentration range. We propose two additive components of short-term memory, one that is rutabaga-dependent and requires different odor qualities in the memory test, and one that is rut-independent and can operate even with small concentration differences in the test.

THE DROSOPHILA SODIUM-CHANNEL GENE PARA: A ROLE IN THE TRANSLATIONAL CONTROL OF NEURONAL EXCITABILITY

CHRISTOPHER MEE and RICHARD BAINES

Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK

Our current study focuses on the Drosophila voltage-gated sodium channel encoded by paralytic (para) and its role in the regulation of membrane excitability. Previous studies have shown that in the absence of cholinergic excitation, sodium currents (INa) in identified motoneurons are significantly elevated, while increased excitation results in a significant reduction in INa. The present study examines changes in para mRNA levels, measured using real time RT-PCR, in activity mutants. We show that para mRNA is significantly increased in the absence of synaptic-vesicle release. By comparison, para mRNA is significantly reduced in genetic backgrounds that increase synaptic excitation. These changes in mRNA are likely due to activity-dependent translational repression. We show that the known translational repressor encoded by pumilio (pum) is both necessary and sufficient for the activity-dependent changes in para mRNA. Bioinformatic analysis of the para transcript shows it to contain sequence specific binding motifs, or Nanos Response Elements (NRE), in the 5′-UTR. These motifs have been shown necessary for Pum protein binding to hunchback (hb) RNA. Binding of Pum to hb leads to translational repression. The presence of these motifs in para indicates a possible direct interaction between the Pum protein and para mRNA, resulting in suppression of the para transcript.

DAILY RHYTHMS IN MORPHOGENESIS OF DROSOPHILA MOTOR TERMINALS

KERSTIN I. MEHNERT, FAHAD ELGHAZALI, PAOLO NEGRO, CHARALAMBOS P. KYRIACOU, and RAFAEL CANTERA

Zoology Department, Stockholm University, 0691 Stockholm, Sweden

In Drosophila melanogaster, locomotor activity follows a circadian rhythm of activity/rest during the light/dark cycle. If locomotor activity influences neuronal morphology, a daily morphological change might be predicted in the neuronal endings on muscles used for locomotion. Here we observe that the morphology of nerve terminals on thoracic flight muscles, changes in complexity between day and night. This striking morphogenetic rhythm is abolished in arrhythmic flies carrying null mutations in the clock genes period (per) or timeless (tim). Surprisingly, whereas the per mutation reduces complexity to a nighttime pattern, tim-null mutants show a dramatic enhancement of the branching pattern of the nerve terminal, far beyond that normally seen in daytime. We do not observe any correlated differences in circadian locomotor behavior between per and tim mutants. Yet, we discovered strikingly opposite effects on the male's ultradian love song cycle, whose expression is mediated by the thoracic nervous system. We propose that, in addition to their function as core clock molecules, per and tim play a novel regulatory role in neuronal morphogenesis.

ACTIVATION OF CAMKII IN THE ADULT DROSOPHILA CNS ALTERS THE SALIENCE OF PHEROMONAL CUES

JENNIFER E. MEHREN and LESLIE C. GRIFFITH

Department of Biology, Brandeis University, Waltham, MA 02454, USA

Upon activation, calcium/calmodulin-dependent protein kinase II (CaMKII) has the ability to phosphorylate itself, becoming Ca²⁺-independent, making it an intriguing player in cellular and behavioral plasticity. We used a tripartite transgenic system to manipulate levels of Ca²⁺-independent CaMKII activity in Drosophila melanogaster, in space and time. Using the Gal4/UAS system combined with the tetracycline-transactivator system, over-expression of constitutively active CaMKII (T287D) was kept off through pupation, followed by incuding its expression in specific areas of the adult nervous system. Memory of flies over-expressing Ca²⁺-independent CaMKII during adulthood was tested in courtship conditioning. One Gal4 line, 30Y, enhanced the response to the mated female during training when driving activated CaMKII. No significant differences in the immediate memory test were seen. A similar but more intermediate effect was seen with MJ85b-Gal4. Two Gal4 lines expressing in the mushroom bodies (MB), 201Y and 29BD, showed training and memory similar to controls. Gal4 lines preferentially expressing in the antennal lobes (AL), or in both AL and MB, showed varied responses. We documented the Gal4 expression patterns in the male antenna, foreleg, and proboscis and found no correlation between sensory expression and the training enhancement. We conclude that this is a central-processing phenomenon, prompting further manipulations aimed at delineating the crucial cells and neurotransmitters involved.

CHARACTERIZATION OF CANDIDATE GUSTATORY INTERNEURONS MODULATING FEEDING BEHAVIOR IN DROSOPHILA

CHRISTOPH MELCHER and MICHAEL J. PANKRATZ

Institut für Toxikologie und Genetik, Forschungszentrum Karlsruhe, 76344 Eggenstein-Leopoldshefen, Germany

Feeding as a fundamental activity of all animals can be regulated by internal energy status or external sensory signals. We characterize a zinc-finger transcription factor, encoded by klumpfuss (klu), which is required for food intake in Drosophila. As 1st-instar larvae, klu mutants stop feeding and display a wandering behavior reminiscent of 3rd-instar wild-type larvae prior to pupariation. Microarray analysis of klu mutants indicates that expression level of the neuropeptide gene hugin (hug) is altered in klu mutants and that hug itself is regulated by food signals. Neuroanatomical analysis demonstrates that hug-mutant neurons project axons to pharyngeal muscles, to the ring gland, and to the mushroom body region whereas hug dendrites are innervated by external gustatory receptor neurons as well as by internal pharyngeal chemosensory organs. Use of tetanus toxin to block synaptic transmission in the hug neurons resulted in specific alterations in the initiation of food intake. Our results provide evidence that hug neurons function as interneurons, that modulate taste mediated feeding behavior in Drosophila. Ongoing analysis of hug-mediated gustatory signalling circuits by calcium imaging and related optical methods should increase our understanding of how taste sensory input modulates feeding behavior.

DPAXILLIN DIRECTLY INTERACTS WITH THE SPECTRAPLAKIN PROTEIN SHORT STOP AND ANTAGONISES ITS GROWTH-PROMOTING ACTIVITY DURING NMJ FORMATION

MICHAEL MENDE, TOBIAS BÖCKERS, HISATAKA SABE, ARUL SUBRAMANIAN, TALIA VOLK TALILA, RYOHEI YAGI, and ANDREAS PROKOP

Institute for Genetics, University of Mainz, 55128 Mainz, Germany

We investigate the function of the cytoskeletal interacting Spectraplakin protein Short Stop (Shot) for growth and differentiation of presynaptic neuromuscular terminals. Here we show a new molecular link explaining how Shot activity can be regulated in this context. When expressing different tagged domains of Shot in motorneurons, especially the N-terminus of Shot is targeted to synapses, potentially interacting with proteins in that location. To uncover respective interactors, we used N-terminal domains as baits in yeast two-hybrid screens. One of the obtained candidates is the small LIM-domain protein Paxillin (DPxn). Interaction of DPxn with Shot is supported by co-IP studies and co-localization with Shot in vivo in SR + cells, tendon cells, and neuromuscular junctions (NMJs). A function of DPxn at the NMJ is revealed upon removal of DPxn function via different genetic deletions or RNA interference, causing irregular overgrowth of motorneuronal terminals. This phenotype depends on the presence of functional Shot protein, suggesting DPxn to act through Shot. Since we observed that the DPxn-Shot interaction is phosphorylation-dependent, and we found highly predicted and conserved PKA phosphorylation sites in DPxn, we could test this possibility. Targeted expression of activated or inhibitory PKA lead to mimics shot- or DPxn-like mutant phenotypes, respectively. We conclude that DPxn acts as a direct negative regulator of growth-promoting Shot activity at the NMJ.

COMPLEX EXPRESSION PATTERN AND NEUROGENIC FUNCTIONS OF DLG GENE

CAROLINA MENDOZA and JIMENA SIERRALTA

Department of Physiology and Biophysics, Universidad de Chile, 6530499 Santiago, Chile

The Drosophila dlg gene is required for processes as different as control of cell proliferation, formation of septate junctions in epithelia, and synaptic junctions in neuromuscular synapses, as well as the maintenance of the polarity in neuroblasts. Recently, we have described dlg splice variants that are expressed in neurons and muscles but are not expressed in epithelia. These products of splice variants have an amino-terminal domain called L27, which is not present in DlgA, the main dlg-encoded isoform previously described. Experiments using RNAi directed against the L27 domain indicate that DLG proteins containing this domain are necessary for proper embryonic neuronal development. Here we show that this knockdown is associated also with a loss of prospero-encoded immunoreactivity in clusters of PNS neuronal precursors and with some loss of repo-encoded immunoreactivity in the CNS, beside the defects in routing of axons. The abnormalities observed in these RNAi experiments apparently do not originate from incorrect neuronal specification before stage 13, because the S97N-containing isoforms are strongly expressed only from stage 13 onwards, and they are not expressed in neuroblasts. The normal expression pattern of 22C10 and FasII are affected in S97N-RNAi embryos, but overexpression of the S97N domain and DlgA and DlgS97 variants in neurons do not affect the expression pattern of these markers in CNS. Finally, from the results of RT-PCR experiments performed on staged embryos, we conclude that the dlg variants with S97N (L27) domain exhibit a transcriptional-regulation mechanism that differs in space and time from that associated with dlg epithelial variants.

SCREENING FOR FACTORS INVOLVED IN DROSOPHILA SYNAPSE FORMATION

CARLOS MERINO, SARA MERTEL, and STEPHAN J. SIGRIST

Max Planck Research Group Neuroplasticity, European Neuroscience Institute, 37075 Göttingen, Germany

Glutamate receptors localized in postsynaptic receptor fields mediate a major fraction of excitatory transmission in our brains. Recent evidence suggests that glutamate receptors can control synapse efficacy over shorter time periods and that long-term changes of glutamatergic synapses can underlie learning and memory processes (Riedel et al., 2004). However, the molecular and cellular basis for forming and modifying glutamatergic synapses remains enigmatic. The glutamatergic neuromuscular synapses of Drosophila larvae (NMJ) are in ultrastructural and functional terms similar to mammalian glutamatergic synapses and are subject to activity dependent long-term plasticity (Sigrist et al., 2000, 2002). We are genetically screening for loci involved in the formation of Drosophila neuromuscular synapses. Transgenic expression of GFP-tagged glutamate receptor DGluRIIA is used to visualize glutamate receptor fields. To visualize active zones, larvae are stainined with the Nc82 antibody (Heimbeck et al., 1999). From screening a collection of P-elements on the X chromosome (Peter et al., 2002), six lines so far show a consistent NMJ phenotypic defect. These lines are being characterized in more detail, including electrophysiologically and by electron microscopy.

FUNCTIONAL CHARACTERIZATION OF BASIGIN, AN IG-CAM WITH POSTSYNAPTIC EXPRESSION AT NEUROMUSCULAR SYNAPSES

SARA MERTEL, FLORENCE BESSE, TOBIAS RASSE, ANNE EPHRUSSI, and STEPHAN SIGRIST

European Neuroscience Institute, Max Planck Society, 37075 Göttingen, Germany; European Molecular Biology Laboratory, 69012 Heidelberg, Germany

Synapses are specialized cell-cell junctions established and maintained by the targeting and localization of specific sets of proteins. Here we use the Drosophila neuromuscular junction (NMJ) to address the cell-biological basis of synapse assembly and maturation. To identify proteins specifically accumulating at the NMJ, we undertook a protein-trap screen to generate GFP-fusion proteins expressed from their endogenous promoters. Using a piggyBac transposable element, which has been shown to preferentially insert downstream of the translation initiation codon, we generated a collection of 350 GFP + lines. Insertions into seven different genes exhibit strong GFP expression at the NMJ. We have focused our work on a gene encoding a polypeptide homologous to the mammalian Basigin/EMMPRIN protein. Using markers for either pre- or post-synaptic compartments, we were able to show that d-Basigin-GFP fusion proteins accumulate in the postsynaptic compartment. We are now combining different approaches (P-element insertions, inducible RNAi constructs) to characterize the function of d-Basigin. Preliminary data suggest that it might regulate NMJ growth and could be essential for muscle integrity.

ANTAGONISTIC REGULATION OF SEVEN UP AND PROSPERO LEADS TO THE CORRECT SPATIOTEMPORAL EXPRESSION OF THE TEMPORAL-SPECIFICATION GENE HUNCHBACK IN NEUROBLASTS OF DROSOPHILA

ULRIKE METTLER, GEORG VOGLER, and JOACHIM URBAN

Institute for Genetics, University of Mainz, 55099 Mainz, Germany

One important principle during neurogenesis in vertebrates as well as invertebrates is the fact that neural progenitors generate different cell types in a reproducible temporal order. In Drosophila this change of temporal identity of the neuroblasts (NBs) is linked to a sequential expression of the transcription factors Hunchback (HB), Krueppel (KR), PDM, and Castor (CAS) (Kambadur et al., 1998; Isshiki et al., 2001; Novotny et al., 2002). These genes are consecutively expressed in a given NB during a certain time window. At the end of each time window, the expression is switched off in the NB but stays on in the GMC and its progeny. Thus, the temporal specification of NB progeny depends strongly on the correct timing of the on and off-switch of these genes within the NB and the GMCs. We were also able to show that the dynamic regulation of hb is mainly brought about by an antagonistic regulation of seven up (svp) and prospero (pros). Svp protein is expressed in the NBs just before the division, which leads to the downregulation of hb. This expression confers competence to both the NB and GMC for mediating a so far unknown mitotic signal for switching-off the hb gene. The activity of Pros, which asymmetrically segregates into the GMC during cell division, blocks this signal within the GMC, leading to hb maintenance. These results provide a first principal insight about how the dynamic lineage-specific regulation of temporal-specification genes might occur.

SEGMENT-SPECIFIC PREVENTION OF PIONEER NEURON APOPTOSIS BY CELL-AUTONOMOUS, POSTMITOTIC HOX GENE ACTIVITY

IRENE MIGUEL-ALIAGA and STEFAN THOR

Division of Biology-IFM, Linkoping University, 58183 Linkoping, Sweden

In vertebrates, neurons often undergo apoptosis after differentiating and extending their axons. By contrast, in the developing nervous system of invertebrate embryos apoptosis typically occurs soon after cells are generated. Here, we show that the Drosophila dMP2 and MP1 pioneer neurons undergo segment-specific apoptosis at late embryonic stages, long after they have extended their axons and have performed their pioneering role to guide follower axons. This segmental specificity is achieved by differential expression of the Hox gene Abdominal B, which in posterior segments prevents pioneer neuron death postmitotically and cell-autonomously, by repressing the RHG-motif cell death activators encoded by reaper and grim. Our results identify the first clear case of a postmitotic cell-autonomous and anti-apoptotic role for a Hox gene in vivo. In addition, they provide a novel mechanism linking Hox-controlled positional information to differences in neuronal architecture along the A/P axis by selective elimination of certain mature neurons.

LOSS OF TORP4A, THE EARLY-ONSET TORSION DYSTONIA HOMOLOG, RESULTS IN RETINAL DEGENERATION IN DROSOPHILA

NARA MURARO and KEVIN MOFFAT

Deptartment of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK

Early-onset torsion dystonia is a human motor disorder resulting from mutation of DYT1, encoding the TorsinA protein. The role of this protein family is unknown. Torp4a immunocytochemistry shows, as in the case pf TorsinA, association with the ER and nuclear envelope. Torp4a is expressed in the ring-gland, imaginal discs, and in the brain and midline glia of 3rd-instar larvae. We have made UAS-transgenes of wild-type and torp4a RNAi constructs. Tissue-specific expression of these gave a rough-eyed phenotype. Sections demonstrate a clear difference in young flies. Over-expression of torp4a causes loss of pigment granules; loss of torp4a function causes an increase in pigment granules in secondary and tertiary pigment cells. Section analysis of older flies (4 and 7 weeks) demonstrates that, in flies lacking detectable Torp4a, dramatic retinal neurodegeneration is seen. Conversely over-expression of Torp4a protects against normal levels of retinal degeneration. We have performed a genetic screen for enhancers of the rough-eye phenotype and identified a number of loci, including several eye pigmentation mutants some of which identify the AP3 complex. (e.g., garnet) We are currently examining potential physical interactions of the identified components by yeast two-hybrid analysis. These are the first reported results from a torsin loss-of-function analysis. From our results so far we propose that the level of Torp4a is crucial to regulate transport to lysosomal-related organelles.

SEARCH FOR FACTORS WITH POLARIZED DISTRIBUTION WITHIN MUSHROOM-BODY NEURONS USING A PROTEIN-TRAP STRATEGY

LUKA NEIDHART, FLORENCE BESSE, and ANNE EPHRUSSI

Developmental Biology Programme, European Molecular Biology Laboratory, 69012 Heidelberg, Germany

Although most mRNAs of mature mammalian neurons have been detected only in the cell body, a few mRNAs have been shown to localize specifically in dendritic processes. Strikingly, recent discoveries provide evidence for the existence of mRNA targeting factors conserved between cell types and organisms. For example, the mammalian homolog of Staufen protein accumulates in dendrites of rat hippocampal cells and is required for the localization of specific mRNAs in this system. In order to gain insights into the precise mechanisms and the biological function in vivo of mRNA targeting in neural cells, we are using the model of larval Drosophila mushroom-body neurons, applying a protein-trap strategy to identify factors with asymmetric distribution within neurons. As part of a consortium (with Christian Klämbt et al.), we are using a piggyBac transposable element to generate new lines and are analyzing the expression pattern of GFP-fusions in the larval brain. Interesting lines have resulted from this screening. In the course of a pilot screen, we fished out several lines showing high expression levels in the mushroom bodies. Even if in this system the lines do not show any polarized expression, our interest in them was maintained, as they all represent insertions into the shaggy gene. Its mammalian homolog, encoding the GSK-3 kinase, has been shown to be implicated in axonal remodelling. Our preliminary data suggest that one function of Shaggy in Drosophila may regulate mushroom-body axonal branching.

A NOVEL POSTSYNAPTIC ROLE FOR DYSTROPHIN IN REGULATING MORPHOLOGY AND FUNCTION OF THE DROSOPHILA NEURO-MUSCULAR JUNCTION

JASPRIEN NOORDERMEER, MARISKA VAN DER PLAS, GONNEKE PILGRAM, ANJA DE JONG, MONIQUE RADJKOEMAR-BANSRAJ, JAAP PLOMP, and LEE FRADKIN

Deptartment of Molecular and Cell Biology, Medical Center, Leiden University, 2333AL Leiden, Netherlands

Mutations in the human dystrophin-encoding gene cause Duchenne and Becker muscular dystrophies. Dystrophin provides both a structural link between cytoskeleton and the extracellular matrix and a scaffold to localize signaling molecules. To further our understanding of dystrophin's roles at the neuromuscular junction, we have determined protein-expression domains of the Drosophila dystrophin isoforms and generated dystrophin mutants. The large isoforms are expressed post-synaptically in the musculature and a smaller isoform is expressed in the CNS. We have characterized mutants lacking all isoforms and mutants that lack the muscle-specific large dystrophin isoforms. While the absence of dystrophin in the muscle does not result in discernable alterations in muscle ultrastructure or resting membrane potential, both synapse morphology and electrophysiology of the neuromuscular junction are abnormal. Mutant synaptic boutons have an increased number of T-bars per active zone. Electrophysiological recordings reveal that, although the amplitudes of spontaneous miniature excitatory junction potentials (mEJPs) are normal, evoked responses (EJPs) are significantly elevated in the mutant, resulting in increased quantal content. Our results suggest that postsynaptically localized dystrophin plays a role in a retrograde signaling pathway, facilitating the homeostatic regulation of the NMJ.

FEMALE SEXUAL RECEPTIVITY IN DROSOPHILA: THE ICEBOX MUTATION MAPS TO THE L1-TYPE CELL ADHESION MOLECULE NEUROGLIAN

KEVIN O'DELL, AHMET CARHAN, FIONA ALLEN, and STEPHEN F. GOODWIN

IBLS Division of Molecular Genetics, University of Glasgow, Glasgow G11 6NU, UK

Relatively little is known about the genes and brain structures that enable virgin female Drosophila to make the decision to mate or not. Classical genetic approaches have identified several mutant females that have a reluctance-to-mate phenotype, but most of these have additional behavioral defects. However, the icebox (ibx) mutation was previously reported to lower the sexual receptivity of females without apparently affecting any other aspect of female behavior. We have shown that the ibx mutation maps to the 7F region of Drosophila's X chromosome to form a complex complementation group with both lethal and viable alleles of neuroglian (nrg). The L1-type cell adhesion molecule encoded by nrg consists of six immunoglobulin-like domains, 5 fibronectin-like domains, one transmembrane domain, and one alternatively spliced intracellular domain. The ibx strain has a missense mutation causing a G92R change in the first immunoglobulin domain of Neuroglian protein. Defects in the central brain of ibx mutants are similar to those observed in another nrg mutant, ceb1. However, both ceb1 homozygous females and ceb1/ibx heterozygous females are receptive. Tissue-specific expression of the non-neural isoform of nrg targeted to the central brain rescues both the mating-receptivity and brain-structure phenotypes of ibx females.

SEARCHING FOR SUPPRESSORS OF TECHNICAL KNOCKOUT, A DROSOPHILA MODEL OF MITOCHONDRIAL DEAFNESS

KEVIN O'DELL, ESKO KEMPPAINEN, DANIEL FERNANDEZ-AYALA, JAMME TOIVONEN, and HOWY JACOBS

IBLS Division of Molecular Genetics, University of Glasgow, Glasgow G11 6NU, UK

Mitochondrial dysfunction results in a wide range of clinical phenotypes including deafness, seizures, short stature and antibiotic sensitivity. However, the mechanism by which mitochondrial mutations lead to this syndrome of phenotypes is poorly understood. To address this, we are characterizing the Drosophila technical knockout (tko) gene, which encodes mitoribosomal protein S12. Flies carrying the tko^25t (L85H) mutation have impaired mitoribosome assembly or stability that leads to a quantitative deficit of mitochondrial translational capacity and consequent respiratory chain deficiency. The resulting mutant phenotypes are remarkably similar to the array of clinical abnormalities. With a view to developing a strategy alleviating symptoms of the condition, we have been searching for mutations that suppress the tko^25t mutant phenotype. Having generated clean, out-bred isogenized tko^25t mutant strains, we are actively selecting against the mutant phenotype. After 30 generations all six selected lines show a strong response and manifest phenotypes intermediate between wild-type and those caused by the tko^25t mutation. A full genome microarray analysis of these lines may identify gene-expression changes that mediate the suppressor response. Curiously, one selected line has almost entirely reverted to wild type in a manner that suggests a single-gene suppressor is involved. This is neither a revertant, nor is it a second-site mutation in tko, indicating that it will be interesting to identify the etiology of this tko suppressor.

IDENTIFICATION OF VISUAL PROJECTION NEURONS THAT CONNECT THE LOBULA AND THE CENTRAL BRAIN

HIDEO OTSUNA and KEI ITO

Institute of Molecular and Cellular Biology, University of Tokyo, 11-0032 Tokyo, Japan

Compared with the neurons that connect different neuropils within the optic lobe (OL), relatively few is known about those that connect the OL and the central brain (termed here visual projection neurons: VPNs). By screening about 4,000 GAL4 enhancer-trap strains, we identified 22 types of VPNs arising from the lobula, among which only seven had been known. Many of them extend their branches in overlapping layers of the lobula, suggesting strong interaction between different VPN types. Of the 22 types, nine consist of relatively large number of neurons (between 13 and ∼ 150 neurons per pathway), each of which has a columnar arborization covering a small visual field. The remaining 13 have tangential or tree-like arborization that covers a large visual area. In the central brain, 11 VPNs project to the ventrolateral protocerebrum (VLPR). Among them six are columnar. Labeling with neuronal synaptobrevin-GFP (fusion protein) indicates pre-synapses locating mainly in the VLPR (10 types). Only two types have their pre-synapses in the OL. Among the seven VPNs that project to the protocerebral regions other than the VLPR, six are tangential/tree-like VPNs whose pre-synapses are located in the OL. Thus, the VLPR is specialized for receiving visual signals; whereas other protocerebra might send centrifugal control signal to the OL, which should affect the whole visual field simultaneously rather than its particular subareas. The resulting effect would then be sent back to the VLPR.

THE DROSOPHILA MELANOGASTER HOMOLOG OF THE NOVEL HUMAN NEUROTROPHIC FACTOR MANF

MARI PALGI, JOHAN PERÄNEN, MART SAARMA, and TAPIO HEINO

Institute of Biotechnology, Helsinki University, 14 Helsini, Finland

Recently a novel mammalian neurotrophic factor MANF (Mesencephalic Astrocyte derived Neurotrophic Factor) has been discovered (J. Molec. Neurosci. 20, 173-187, 2003). MANF is the first neurotrophic factor whose protein sequence is well conserved through evolution from C. elegans to human. We describe the Drosophila melanogaster MANF (DmMANF), the first mammalian neurotrophic factor homolog found in higher invertebrates. We have raised the working antibody towards DmMANF and studied the expression patterns both at mRNA and protein levels during the fly development. Applying P-element excision methods we created DmMANF null alleles. These mutants were larval lethal, and the lethality was rescued by the ectopic overexpression of DmMANF. The mutant embryos, however, showed no clearly abnormal phenotypes. According to Northern blot, RT-PCR, and immunostainings, the DmMANF gene product was maternally contributed. Abolishment of the maternal contribution using the germline mosaic technique revealed dramatic embryonic phenotypes in the PNS and CNS. In the case of abolished maternal contribution of DmMANF, the lethality was observed during the embryogenesis, before hatching. The severity of the phenotype varied from twisted early embryos to wild- type-appearing stage 12-13 embryos, which later on showed misrouted segmental nerves and overcondensed ventral nerve cord. The lack of maternal DmMANF could be paternally rescued by paternal contribution of the normal allele of this gene.

DIVERGENT EVOLUTION IN METABOTROPIC GLUTAMATE RECEPTORS: A NEW RECEPTOR ACTIVATED BY AN ENDOGENOUS LIGAND DIFFERENT FROM GLUTAMATE IN INSECTS

MARIE-LAURE PARMENTIER, CHRISTIAN MITRI, JEAN-PHILIPPE PIN, JOEL BOCKAERT, and YVES GRAU

CNRS, UPR2580, 34094 Montpellier, France

The metabotropic glutamate receptors (mGluRs) are G-protein-coupled receptors (GPCRs) involved in the regulation of glutamatergic synapses. Surprisingly, the evolutionary distant Drosophila mGluR (DmGluAR) shares a very similar pharmacological profile with its mammalian orthologs (mGlu2R and mGlu3R). Such conservation in ligand recognition indicates a strong selective pressure during evolution to maintain the ligand recognition selectivity of mGluRs and suggests that structural constraints within the ligand-binding pocket (LBP) would hinder divergent evolution. Here we report the identification of a new receptor homologous to mGluRs found in Anopheles gambiae, Apis mellifera, and Drosophila melanogaster genomes, which we named AmXR, HBmXR, and DmXR, respectively. 3D modeling of the LBP revealed that the residues contacting the amino-acid moiety of glutamate were conserved in mXRs, whereas the residues interacting with the g-carboxylic group were not. This suggests that the mXRs evolved to recognize an amino acid different from glutamate. The Drosophila cDNA encoding DmXR was isolated and found to be insensitive to glutamate or to any other standard amino acid. However, we found that the DmX receptor was activated by a ligand containing an amino group, which was extracted from Drosophila heads. No ortholog of mXR could be detected in C. elegans or human genomes. These data indicate that the LBP of the mGluRs has diverged in insects to recognize a new ligand.

ATLAS OF THE THIRD-INSTAR LARVAL BRAIN: APPLICATIONS AS A GENE REPOSITORY DATABASE

WAYNE PEREANU and VOLKER HARTENSTEIN

Department of Biology, UCLA, Los Angeles, CA 90024, USA

The Drosophila brain is formed by primary neurons, generated in the embryo, and secondary neurons, born in the larva. The late larval brain is studded with approximately 90 lineages, each one located at an invariant position and represented by an axon bundle that forms an invariant pattern in the neuropile. We have generated a digital model that contains all secondary lineages and relates them to landmarks, most importantly the neuropile compartments, including the mushroom body. This model will be used as a repository for gene-expression data. Given the high numbers and small sizes of cells in the brain, the level of resolution at which expression patterns can be managed meaningfully will, initially, almost certainly not be single cells. However, lineages appear at a level of resolution that may be appropriate. Each lineage occupies a roughly conical or cylindrical volume measuring approximately 20 µm in diameter and 40 µm in height. In a pilot study the expression pattern of engrailed was “warped” into our atlas model. Warping was performed with the Amira software package, using only rigid transformations and uniform scaling. By comparing the position of the volume-rendered engrailed lineage with each of the lineages consecutively, it was determined that the DALv3 lineage expresses this gene. We consider it to be realistic to expect that other expression patterns coinciding with other discrete lineages can be deposited into the larval-brain model in the same manner.

LARVAL DEVELOPMENT OF GLIAL CELLS IN THE DROSOPHILA BRAIN

WAYNE PEREANU and VOLKER HARTENSTEIN

Department of Biology, UCLA, Los Angeles, CA 90024, USA

The CNS glia of Drosophila is divided into midline glia, derived from mesectoderm, and lateral glia, derived from the neuroectoderm. Lateral glia in the CNS are divided into three sub-groups: (1) surface glia, that extend sheath-like processes to wrap around the entire brain; (2) cortex glia which, in the adult, encapsulate each individual neuron by a glial sheath; and (3) neuropile glia, located both around the neuropile as well as between compartments, that wrap the neuropile compartments and major tracts of neurites. Here we generate GFP-labeled clones to illustrate the three types of lateral glia in the larval brain. In addition, analysis of labeled clones provides two strong pieces of evidence that glia in the late larva originate from neuroblasts: (1) the elav-gal4 line used in generating clones is only expressed in neurons and neuroblasts; and (2) with few exceptions, labeled surface and cortex glia are directly adjacent to a labeled neuroblast.

LARVAL MOTOR IMPAIRMENT INDUCED BY RNAI KNOCKDOWN OF D. MELANOGASTER'S ZASP HOMOLOG

SAMANTHA PERON, CLARA BENNA, GIULIANA PERINI, ARAM MEGIGHIAN, MAURO ZORDAN, CARLO REGGIANI, and RODOLFO COSTA

Department of Human Anatomy and Physiology, University of Padova, 35131 Padova, Italy

ZASP is a recently identified PDZ-LIM domain protein localized in the Z-line of vertebrate striated muscle. The ZASP PDZ domain binds to α-actinin, a protein which cross-links actin filaments from opposite sarcomeres to the Z-line and therefore has a major mechanical function in keeping the sarcomeres together. PDZ-LIM proteins are regarded as mediators between cytoskeletal structures and signaling cascades. ZASP is evolutionary conserved, and a bioinformatics analysis of the Drosophila genome revealed an homolog of the mammalian Zasp gene. By using RNA-interference techniques we functionally knocked down Zasp in three Drosophila melanogaster transgenic lines, which were subsequently analyzed using behavioral, morphological, and electrophysiological techniques. The “interfered” flies mainly died during metamorphosis and did not reach the adult stage. Behavioral tests on 3rd-instar larvae revealed a severe motor impairment, which is correlated with electrophysiological alterations. Morphological (including morphometrical) analyses of larval muscles failed to show any modification compared with wild-type controls. These results suggest that the impaired motor behavior could be due to alterations in neuro-muscular junction activity and/or in the mechanical properties of larval muscle fibers.

MASS SPECTROMETRIC ANALYSIS OF IDENTIFIED PEPTIDERGIC NEURONS FROM DROSOPHILA: DOES IT WORK?

REINHARD PREDEL, SUSANNE NEUPERT, and CHRISTIAN WEGENER

Sächsische Akademie der Wissenschaften Leipzig, AG Jena, 7743 Jena, Germany

Mass spectrometric analysis of single cells, performed on giant peptidergic neurons of molluscs, has led to important findings about cellular mechanisms of peptide synthesis and transport. Insects like Drosophila, although containing a number of identifiable neurons in the CNS, seem to be less suitable for such attempts. In comparison with giant molluscan neurons, every Drosophila neuron is a dwarf, which causes numerous problems in dissection and analysis. We here describe approaches to study differential neuropeptide expression or post-translational modifications in morphologically defined parts or single neurons of the nervous system. Our approach has been developed using the larger and more easily accessible homologous neurons of the American cockroach, focussing on FMRFamide related peptides and capa-gene products. We show that despite their small size, neurosecretory D. melanogaster neurons accumulate sufficient material in their somata to be successfully analyzed by mass spectrometric techniques (MALDI-TOF MS). Thus, MALDI-TOF MS appears to be well suited to study differential peptide expression under different physiological and developmental conditions in wild-type and mutant flies. In addition, GAL4-directed expression of GFP offers a unique possibility to identify isolated peptidergic neurons. In perspective, mass spectrometric profiling and Fas2-based morphological charting of peptidergic neurons seems to be a promising combination to establish an atlas of peptidergic fly neurons.

INVESTIGATING THE IMPORTANCE OF THE MOLECULAR DIVERSITY AND ALTERNATIVE SPLICING OF DSCAM

ROLAND PUETTMANN-HOLGADO and DIETMAR SCHMUCKER

Dana Farber Cancer Institute, Boston, MA 20115, USA

Dscam is a member of the immunoglobulin superfamily and orthologous to the human Down Syndrome Cell Adhesion Molecule. In Drosophila tandem arrays of alternative exons 4, 6, 9, and 17 allow the generation of some 38,000 isoforms through alternative splicing. However, it is unclear how the isoforms are expressed and whether the isoform diversity is functionally required. We developed a DNA microarray that allows us to discriminate between all the different variable exons. Using these arrays for expression profiling we have detected tissue and cell-type-specific splicing patterns. We are now analyzing Dscam isoform expression at single-neuron resolution. In order to test whether the molecular diversity is evolutionarily conserved, we used a combination of BLAST searches and cloning and were able to identify Dscam orthologs across four major orders of insects. Strikingly the alternative splicing of exons corresponding to D. melanogaster exons 4, 6, 9 and 17 is highly conserved among insect species (Diptera, Hymenoptera, Lepidoptera, and Coleoptera), which are separated by 300 million years of evolution. To address the issue of specificity functionally we are testing gain- and loss-of-function phenotypes associated with single Dscam isoforms. Preliminary experiments suggest that reduced diversity of Dscam causes defects in synaptic targeting. The evolutionary conservation, together with the functional requirement, strongly suggest an ancient functional role for the molecular diversity generated by the Dscam-encoding gene.

OF NATURE AND NURTURE: DETERMINANTS OF ELECTRICAL PROPERTIES IN EMBRYONIC NEURONS

EDWARD PYM, MIKI FUJIOKA MIKI, JAMES JAYNES, and RICHARD BAINES

Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK

The development of embryonic neurons from birth to maturity has been extensively studied. Whereas developmental processes such as axonal pathfinding and synapse formation have been characterized in some detail, relatively little is known about how neurons develop their functional characteristics (i.e., synaptic integration and action-potential firing). This study investigates the role of both extrinsic and intrinsic factors in the development of functional characteristics in embryonic motor neurons. Each segment of the 1st-instar larvae contains 30 muscles innervated by ∼ 40 motor neurons. The precise pattern of muscle innervation has been shown to arise from differential expression of the transcription factors Even-skipped (Eve), dHb9, Lim3, and Islet. In 1st-instar larvae we have characterized the wild-type functional properties of three identified Eve-expressing neurons: two motoneurons, termed aCC and RP2, and an interneuron termed pCC. Here we show, using whole-cell voltage and current clamp, that alterations in intrinsic transcription-factor expression results in subtle alterations of electrical properties. Manipulation of extrinsic synaptic input, by comparison, has a more pronounced effect. Thus, it appears that extrinsic factors may play a larger role in the determination of functional properties of neurons, whereas intrinsic transcription factors fine-tune a neuron to its role in the embryonic nervous system.

STUDY OF SIGNALING AND REGULATORY PATHWAYS INVOLVED IN NEUROGENESIS

XIAO-JIANG QUAN, SVEN VILAIN, CHINGMAN CHOI, JIEKUN YAN, and BASSEM HASSAN

Labortory of Neurogenetics, Department of Human Genetics, VIB & University of Leuven, 3000 Leuven, Belgium

Studies on transcription factors which regulate early neurogenesis has made it clear that the genetic and molecular mechanisms by which ATONAL (ATO) and NEUROGENIN (NGN) proneural proteins select neural precursor cells (NPCs) are different in the peripheral nervous systems (PNSs) of vertebrates and invertebrates (Quan et al., 2004). This divergence in proneural activity is encoded by three residues in the basic domain of proneural proteins. Differential interactions with different types of zinc (Zn)-finger proteins—Senseless (SENS) in Drosophila (Nolo et al., 2000) and Myelin transcription factor 1 (MyT1) in Xenopus (Bellefroid et al., 1996)—mediate the divergence of ATO and NGN activities. However, no evidence for direct physical binding between bHLH proneural protein and Zn-finger protein has been shown. Thus we are still far from fully understanding the mechanisms of how proneural proteins define and regulate neural lineage development. In order to dissect the molecular basis of this functional divergence, we asked which motif mediates the function of ATO. The chimeric proteins, in which different motifs between ATO and NGN1 were swapped, have been overexpressed to test if they can mimic and rescue the various aspects of the ATO mutant phenotype in Drosophila. Our results indicate that a motif containing three amino acids in the basic domain is necessary and sufficient for proneural function and cell identity specification in the embryo and the eye. However, proper patterning of the eye field requires a second motif in the Helix 2 of ATO. The results of misexpressing ATO, NGN1, and all these chimeric proteins in Drosophila eye further support this conclusion.

IDENTIFICATION OF MUSHROOM-BODY-MINIATURE, A ZINC-FINGER PROTEIN IMPLICATED IN DEVELOPMENT OF THE CENTRAL NERVOUS SYSTEM

THOMAS RAABE, SUSANNE CLEMENS-RICHTER, THOMAS TWARDZIK, ANSELM EBERT, GERTRUD GRAMLICH, and MARTIN HEISENBERG

Institut für Medizinische Strahlenkunde & Biozentrum, University of Würzburg, 97078 Würzburg, Germany

The mushroom bodies (MBs) are bilaterally arranged structures in the protocerebrum of Drosophila. Mutants with altered mushroom-body structure have not only been instrumental to establish their role in distinct behavioral functions but also in the identification of the molecular pathways that control mushroom body development. We present the identification of the mushroom body miniature (mbm) gene. The MBs of mbm females develop normally until the beginning of the 3rd larval instar when the intrinsic neurons of the MBs, the Kenyon cells, start to loose their axonal projections inappropriately. Kenyon-cell perikarya apparently survive, but no regeneration of the axons is seen during metamorphosis, leading to a grossly reduced MB structure in the adult. The most prominent structural feature of the Mbm protein is a pair of zinc fingers, which indicates a function of the protein in binding nucleic acids. Expression of the Mbm protein is not restricted to the mushroom bodies, suggesting a function of Mbm in other aspects of brain development.

OLFACTORY MAPS IN THE LARVAL BRAIN

ARIANE RAMAEKERS, EDWIGE MAGNENAT, NANAË GENDRE, and REINHARD F. STOCKER

Department of Biology, University of Fribourg, 1700 Fribourg, Switzerland

The question of how sensory information is encoded in the brain is one of the major issues in current neuroscience. Spatial maps are a common feature underlying such processes, concerning many sensory modalities and model systems. Spatial maps transform the information perceived by sensory neurons into a spatial pattern of activation of target neurons in the brain. In particular, the representation of olfactory information in the adult brain of Drosophila relies on spatial maps, both in the antennal lobe (AL), and in higher brain centers: the mushroom body calyx and the lateral horn. Compared with the adult system, the larval olfactory system of Drosophila looks rudimentary: 21 olfactory receptor- neurons (ORNs) and 20–25 projection neurons (PNs) appear to be sufficient for a task that requires 1,300 ORNs and 150 PNs in the adult. However, we previously showed that the overall organization of the olfactory system in the larval brain is similar as the adult one. Here we describe the connectivity of individually labeled ORNs and PNs. Our results suggest that, as in the adult brain, afferent terminals of ORNs form a spatial, glomerular-type map in the larval AL. Spatial organization seems to be conserved even in the calyx, as shown by the stereotyped PN projections both at their input and output levels. However, unlike in adults, the larval system may be devoid of redundancy, displaying a proportion of 1 ORN : 1 “glomerulus” in the AL: 1 PN : 1 “glomerulus” in the calyx.

TURNOVER OF SYNAPTIC PROTEINS DURING IN VIVO SYNAPSE FORMATION AND MATURATION

TOBIAS RASSE, WEHNER FOUQUET, ANDREAS SCHMID, ASJA GUZMAN, ROBERT KITTEL, CAROLA SIGRIST CAROLA, CARLO MERINO, MIRIAM RICHTER, and STEPHAN SIGRIST

European Neuroscience Institute, Max Planck-Society, 37073 Göttingen, Germany

In terms of ultrastructure and molecular composition, Drosophila glutamatergic synapses are similar to the glutamatergic synapses important for mammalian information processing. We studied these synapses for the first time under full in-vivo conditions over extended periods. Confocal microscopy on transgenic lines expressing GFP-labeled glutamate receptors revealed that new receptor fields form exclusively de novo and mature in less than a day. In-vivo photobleaching and photoactivation experiments demonstrate that the rapid growth of new receptor fields depends on glutamate receptor entry from diffuse extrasynaptic pools. Mature receptor fields are stable due to both glutamate receptor entry and exit being low. We further apply this assay to explore the molecular dynamics of postsynaptic assembly (e.g., by studying the cytosolic component “pak.”) This pak kinase has a much faster turnover compared to the receptors; thus we can draw mechanistic conclusions, which are relevant for a general understanding of the cellular basis of learning and memory. Furthermore, this work establishes a new assay that allows us to study the dynamics of synaptic proteins at the level of individual synapses in the context of an intact organism.

LABELING OF SINGLE INTERNEURONS IN THE LATE EMBRYONIC CNS OF DROSOPHILA

CHRISTOF RICKERT, KERRI-LEE HARRIS, THOMAS KUNZ, PAUL WHITINGTON, and GERHARD TECHNAU

Institute of Genetics, University of Mainz, 55099 Mainz, Germany; Department of Anatomy and Cell Biology, University of Melbourne, Victoria 3010, Australia

In the Drosophila embryonic ventral nervous system, each hemi-neuromere derives from about 30 neuroblasts and comprises about 350 neurons and 30 glial cells. The entire lineage of each neuroblast is known. However, while most of the glial cells and all motoneurons can be identified individually, the vast majority of the more than 300 interneurons are not described at the single-cell level. Therefore questions remain open: How many interneurons develop an axon during embryogenesis? To what extent exists variability in a neuron's cell body position or axonal/dendritic morphology? Additionally, a detailed knowledge of the morphology of individual cells is a prerequisite for a mutant analysis, for the identification of neuronal circuits and for the clarification of the relationship between developmental and functional units. Here we present an attempt to generate a catalog of all embryonic interneurons. Single cells are labelled in early stage 17 flat preparations. This stage guarantees an elaborate axon morphology but still allows linkage of the respective cell to its lineage. Additionally, labelings can be performed in strains expressing GFP in specific cells in order to add landmark axon bundles or to demonstrate co-labelling with known cell specific markers. 3D-reconstructions of labeled cells allow locating overlapping projection areas between neurons in silico.

RHODOPSIN 6-EXPRESSING PHOTORECEPTOR CELLS APPEAR TO HAVE DIRECT CONTACT TO THE CIRCADIAN PACEMAKER NEURONS OF DROSOPHILA MELANOGASTER

DIRK RIEGER and CHARLOTTE HELFRICH-FÖRSTER

Institute of Zoology, University of Regensburg, 93051 Regensburg, Germany

The compound eyes and the extraretinal H-B eyelets contribute to entrainment of the circadian clock in the fruit fly (Helfrich-Förster et al., 2002, J. Neurosci. 22, 9255–9266; Rieger et al., 2003, J. Biol. Rhythms 18, 377–391). Preliminary anatomical data suggest that photoreceptor cells 7 and 8 (R7 and R8) of the compound eyes and the four photoreceptor cells of the H-B eyelets have direct contacts to circadian pacemaker neurons (the Lateral Neurons or LNs). We are interested to unravel these contacts on an anatomical and physiological basis. First, we looked for synapses between photoreceptor cells and LNs. For this purpose we expressed the synaptic protein synaptobrevin (coupled to GFP) in all photoreceptor cells (or only in Rhodopsin 6-expressing cells) and double-stained these speciments with anti-PDF (pigment-dispersing factor), which is expressed in the ventro-lateral LNs. We found synaptobrevin-GFP labeling in close vicinity to LN fibers, indicating that the R7, R8 cells and the H-B eyelet cells have anatomical contact with LNs. Second, we investigated a possible functional connectivity between photoreceptor cells and LNs by expressing the lectin Wheat Germ Agglutinin (WGA) in the photoreceptor cells or in the LN. WGA can be transferred via active synapses to postsynaptic cells (Tabuchi et al., 2000, J. Neurosci. Res. 59, 94–99). We found WGA labeling in R8 after expressing it in LNs, suggesting functional connectivity between both cell types.

OPTICAL CALCIUM IMAGING OF ACTIVITY IN DOPAMINERGIC NEURONS OF DROSOPHILA IN VIVO

THOMAS RIEMENSPERGER, ERICH BUCHNER, and ANDRÉ FIALA

Biozentrum, University of Würzburg, 97074 Würzburg, Germany

Olfactory learning and memory formation in Drosophila melanogaster is studied extensively using an odor stimulus paired with an electroshock as an aversive reinforcer. Whereas appetitive olfactory learning is dependent on octopamine, the dopaminergic system is necessary for aversive olfactory memory formation. This suggests that dopaminergic neurons might mediate the punishing reinforcer during aversive training. We tested this hypothesis using the fluorescent calcium sensor cameleon to optically record neuronal activity in vivo. Cameleon is driven selectively in dopaminergic neurons using the Gal4-line Th-gal4, which expresses Gal4 under the control of the regulatory sequence of the Tyrosine hydroxylase gene, which generates an enzyme involved in dopamine synthesis. We confirm the high specificity of the Th-gal4 line by double stainings of cameleon, driven by the Gal4 line, and anti-tyrosine hydroxylase or anti-dopamine immunhistochemistry. Several parts of the mushroom bodies, brain centers involved in olfactory learning, are densely innervated. In first experiments we show that somata of single dopaminergic neurons respond to electric-shock stimulation. Current experiments address the question whether all dopaminergic neurons or only a fraction of them are responsive to electric shock. The second question under investigation is whether pairing of odorants with electric shock, thereby mimicking an olfactory training procedure, modifies the response of those neurons.

PHYSIOLOGICAL REQUIREMENT FOR THE GLUTAMATE TRANSPORTER DEAAT1 AT THE ADULT DROSOPHILA NEUROMUSCULAR JUNCTION

THOMAS RIVAL, LAURENT SOUSTELLE, DANIEL CATTAERT, COLETTE STRAMBI, MAGALI ICHÉ, and SERGE BIRMAN

CNRS, Université de la Méditerranée, 13288 Marseille, France

Glutamate is the major excitatory neurotransmitter in the mammalian brain. Specific proteins, the Na⁺/K⁺-dependent high-affinity excitatory amino-acid transporters (EAAT) are involved in the clearance of glutamate from the synaptic cleft and recycling of this transmitter. We recently reported that dEAAT1, the only high-affinity glutamate transporter in Drosophila, plays a crucial role in the prevention of glutamate neurotoxicity in the adult brain. In Drosophila, glutamate is the excitatory neurotransmitter at the neuromuscular junction (NMJ), but the localization and function of glutamate transporters at the NMJ is unknown. Using a specific antibody and transgenic strains, we observed that dEAAT1 is present at adult but, surprisingly, not at larval NMJs, suggesting a physiological maturation of the junction during metamorphosis. We found that dEAAT1 is not expressed in motor neurons but localized in glial extensions that closely follow motor axons to the NMJ. We inactivated the dEAAT1 gene by RNA interference, generating viable adult flies that were able to walk but were flight-defective. Electrophysiological recordings of the thoracic dorso-lateral NMJ showed normal responses to single motor-nerve stimulation. In contrast, the duration of the individual responses was significantly increased during trains of stimulations. These results suggest that the glutamate transporter is required for activity of the NMJ only in adult flies and under conditions of strong stimulations.

INVESTIGATIONS INTO APOPTOSIS IN THE DEVELOPING EMBRYONIC NERVOUS SYSTEM OF DROSOPHILA MELANOGASTER

ANA ROGULJA-ORTMANN, KARIN LÜER, JANINA SEIBERT, and GERHARD TECHNAU

Institute for Genetics, University of Mainz, 55128 Mainz, Germany

One of the key features of animal development is the selective removal of cells through apoptosis. The CNS of Drosophila melanogaster is no exception: a significant number of its cells die during embryonic development. However, very little is known about the pattern of CNS apoptosis and identity of dying cells. We have undertaken three approaches to obtain insights into this process: (1) Analysis of distribution and amounts of dying cells in the CNS throughout development. (2) DiI labeling in an apoptosis-deficient mutant to determine the clonal origin and development of additional cells. The current results suggest that some clones in apoptosis-deficient mutants show no difference compared to wild-type ones, but some do show a difference in cell number. Our current efforts are focused on obtaining data for every neuroblast lineage in the embryonic CNS, and analyzing this data in more detail. (3) Analysis of occurrence of apoptosis in cells expressing various markers. For most of the markers used, there are at least a few dying cells in each stage examined. The combined information obtained from these three approaches will give us an overview of the occurrence of apoptosis during the development of the embryonic CNS and allow us to use the identified cells as models for investigations into the mechanisms controlling developmentally regulated cell death.

EXPLORING THE FORMATION OF NEURO-NEURONAL SYNAPSES IN THE DROSOPHILA EMBRYO

NATALIA SANCHEZ-SORIANO, BARBARA KUEPPERS-MUNTHER, ROBERT LÖHR, WOLFGANG BOTTENBERG, MATTHIAS LANDGRAF, RIKKI HAESSLER, and ANDREAS PROKOP

School of Biological Sciences, University of Manchester, Manchester M13 9PT, UK

The use of NMJs as model systems restricts investigations to one type of glutamatergic junctions, the postsynaptic part of which is not of neuronal but muscular nature. Here we present new strategies and cellular models for the study of synaptic compartments of neuro-neuronal junctions of the Drosophila embryo. Thus, we developed conditions for Drosophila primary cell cultures under which neurons adapt structural and functional properties widely reminiscent of those known from studies in the embryonic CNS (Küppers-Munther et al., Devel. Biol. 269, 459 ff ). In these cultures, synaptic compartments can be visualized and experimentally addressed. In parallel we approach neurons in the developing CNS, where they wire into stereotypically arranged neuronal networks. To this end, we apply mapping strategies based on Fasciclin2 expression, by which projections of individual neurons can be charted precisely and three-dimensionally in the neuropile (Landgraf et al., Devel. Biol. 260, 207 ff ). To characterize the synaptic nature of these neurites, we developed and use different forms of genetic mosaic strategies, which enabled us to demonstrate the existence of reproducible pre- and post-synaptic compartments, which are now amenable to developmental studies (Löhr et al., J. Neurosci. 22, 10357 ff ).

DENDRITES IN ARTHROPODS? INSIGHTS FROM DROSOPHILA MOTORNEURONS

NATALIA SANCHEZ-SORIANO, ROBERT LÖHR, WOLFGANG BOTTENBERG, AFRODITI KERASSOVITI, ELISABETH KNUST, ANDRÉ FIALA, and ANDREAS PROKOP

School of Biological Sciences, University of Manchester, Manchester M13 9PT, UK

Typical neuronal somata in the vertebrate CNS lie in the synaptic core area and give rise to two types of neurites, dendrites and axons (hetero-multipolar). In contrast, neuronal somata of the arthropod CNS lie in the rind, each displaying just one primary neurite (unipolar), which branches thereafter into neurites of unknown synaptic nature. Are these neurites classifiable as axons and dendrites? Here, we demonstrate that side branches of Drosophila motorneurons in the CNS (sprouting off their primary neurites) are true dendrites as defined by their postsynaptic nature, cytoskeletal features, localization of Par-complex gene gene products, their developmental time line, and calcium signaling properties. In addition to these similarities, motorneuronal dendrites can be shifted experimentally from the primary neurite to the soma, an arrangement typical of vertebrate neurons. Furthermore, our cell-culture experiments suggest that a hetero-multi/bipolar shape is the default condition of Drosophila motorneurons. Therefore, the localization of dendrites on the primary neurite during normal CNS development seems to be induced by position-specific signals in the neuropile. We propose that the very different organizations of vertebrate and arthropod CNSs have developed from a common ancestor through cell body shifts, which did not require a major reorganization of the synaptic area.

STUDYING PIONEER NEURONS DURING THE GROWTH OF A DROSOPHILA MOTOR NERVE: INVOLVEMENT OF THE N-CAM HOMOLOG FASCICLIN2

NATALIA SANCHEZ-SORIANO and ANDREAS PROKOP

School of Biological Sciences, University of Manchester, Manchester M13 9PT, UK

The phenomenon of pioneer neurons has been known for almost a century, but so far we know little about mechanisms and molecules involved. We study the formation of the Drosophila ISN nerve in this context. We show that aCC/RP2 and U motorneurons grow together at the leading front of the ISN. Nevertheless, aCC/RP2 neurons are the pioneers and U neurons the followers. First, ablation of aCC/RP2 has a stronger impact on ISN growth than U ablation. Second, strong growth-instructive capabilities of aCC/RP2 are revealed with a stalling approach we used: when stalling aCC/RP2 motoraxons, the whole ISN (including the Us) co-arrests, demonstrating that aCC/RP2 instruct the U growth cones to stop. In contrast, stalled U neurons do not have the same instructive influence on other ISN motorneurons. We also show that instruction of ISN growth requires Fasciclin2: targeted expression of this molecule in Us increases their influence on the ISN, whereas Fasciclin2 loss-of-function variants reduces ISN co-arrest with stalled aCC/RP2 axons. The qualitative differences of both neuron groups are confirmed through our findings that aCC/RP2 growth cones are wider and more complex than those of Us. However, U growth cones adopt aCC/RP2-like wider shapes in a Fasciclin2 loss-of-function genetic background. Therefore, Fasciclin2 is to a degree required and sufficient for pioneer-follower interactions, but its mode of action cannot simply be explained through an equally bidirectional homophilic interaction.

FUNCTION AND MOLECULAR DIVERSITY OF THE HYPERVARIABLE RECEPTOR DSCAM

DIETMAR SCHMUCKER

Department of Neurobiology, Harvard Medical School, Boston, MA 02115, USA

I and my co-workers have identified a neuronal receptor termed Dscam which is required for synaptic targeting. Dscam is the record holder for alternative splicing and a highly complex gene. Tandem arrays of alternative exons 4, 6, 9, and 17 allow the generation of some 38,000 isoforms through alternative splicing. It is, however, unclear whether this molecular diversity is indeed functionally required. Our studies attempt to determine whether the large molecular diversity of Dscam contributes directly to the synaptic specificity. We propose that different nerve cells express different Dscam isoforms and that this specifies a molecular recognition code to provide individual neurons with instructions to connect with the correct target neurons. To address the functional requirement of Dscam, we are using the MARCM system and single cell labeling to analyze pathfinding, targeting, and synaptogenesis of mechanoreceptor neurons (ms-neurons) in the somatosensory system. We have obtained evidence demonstrating that Dscam is essential for targeting of ms-neurons. In addition, we have generated flies that lack a substantial amount of the Dscam receptor diversity but maintain normal amounts of Dscam protein. Remarkably, animals that express a reduced diversity display distinct defects in synaptic targeting. This provides the first evidence for a functional importance of the Dscam receptor diversity.

BIOGENIC AMINES IN THE ANTENNA OF DROSOPHILA MELANOGASTER

SABINE SCHWARZ, ZAINULABEUDDIN SYED, and MARIEN DE BRUYNE

Institute of Neurobiology, Freie Universität Berlin, 14195 Berlin, Germany

As animals need to adapt to physiological and environmental changes, the nervous system has to be plastic. Flexibility in odor coding could involve plasticity in the olfactory receptor neurons themselves. Biogenic amines might play a role here as their receptors have been found in olfactory tissues in different species (tyramine receptor in Drosophila, octopamine receptor in Bombyx and Heliothis), and they have been described to have an effect on the properties of sensory neurons. For example, in silk moths octopamine increases the amplitude of pheromone-receptor potentials and nerve-impulse frequencies. We have studied the expression of biogenic amine receptors in the antenna of Drosophila. RT-PCR experiments confirmed the presence of the tyramine receptor and demonstrated expression of dopamine, serotonin, and octopamine receptors. Several mutants that show defects in biogenic amine synthesis or signaling have been tested in electrophysiological experiments to study their effects on odor responses. Tyramine-receptor mutants showed significantly stronger EAG responses to ethyl acetate but not to other odorants tested. In single-unit recordings we observed a shift in the action potential/sensillum potential relationship in one sensillum class but not in others.

HANGOVER FUNCTION IS REQUIRED FOR PROPER BOUTON FORMATION AT THE NEUROMUSCULAR JUNCTION

ISABELL SCHWENKERT, MIRJAM FRANZ, NATALJA FUNK, and HENRIKE SCHOLZ

Biozentrum, University of Würzburg, 97074 Würzburg, Germany

Previously we identified the Drosophila hangover mutant. This mutant is defective for ethanol tolerance as well as other cellular stress responses such as heat and oxidative stress. hangover encodes for a putative zinc-finger transcription factor that shares high homology to a human zinc-finger protein which has not been characterized yet. In the adult fly, hangover is mainly expressed in neuronal cells. In adult hangover-mutant brains, no obvious structural defects can be detected. To understand the cellular function of hangover, we investigated the larval neuromuscular junction of hangover mutants, an excellent model to study neuronal function at the cellular level. Interestingly hangover mutants display an increase in numbers of synaptic boutons and an increase in numbers of active zones, suggesting that the mutant neurons do not function as well as wild-type ones. The observed phenotype is similar to those of mutants with altered cAMP levels. We are currently investigating possible genetic interactions between hangover and mutants affecting cAMP signaling.

MOLECULAR CLOCKS IN DROSOPHILA'S PACEMAKER NEURONS

ORIE T. SHAFER and PAUL H. TAGHERT

Washington University School of Medicine, St. Louis, MO 63110, USA

The molecular-genetic analysis of Drosophila's circadian clock has resulted in a mechanistic model of clock function that accounts for much of this fly's circadian behavior. At the heart of the model lies two interconnected transcriptional feedback loops. Thus, our present understanding of the clock mechanism in Drosophila accounts only for biological timekeeping within cells, leaving a vast conceptual gap between cellular pacemakers and rhythmic behavioral output. For this reason we have sought to determine with high resolution the dynamics of clock-gene expression within identified subsets of clock neurons and have found that the program of PER and TIM expression differs between subsets of pacemaker neurons. Furthermore, mutations in photoreceptors and genes encoding neuropeptides differentially affect the molecular clock in different subgroups of pacemaker neurons. This in-situ approach to neuronal clocks has made possible a glimpse at how groups of neuronal pacemakers interact within the brain to create a light-sensitive circadian clock. We have gained insights about how photoreceptors and neuropeptides cooperate to set the phase of locomotor rhythms and their underlying neuronal clocks.

COUNTING THE BRAIN CELLS: AUTOMATIC DETECTION OF CELL-BODY POSITIONS USING AN EXCLUDED-VOLUME-EMBEDDING ALGORITHM

TAKASHI SHIMADA, KANTARO KATO, AZUSA KAMIKOUCHI, and KEI ITO

Institute of Molecular and Cellular Biology, University of Tokyo, 112-0003 Tokyo, Japan

Quantitative information about the brain is a prerequisite for understanding its circuit structure. Recent advances in computer-aided image analysis provided useful tools for measuring its size and volume. The number of brain cells, however, still remained essentially in the level of estimation. Counting more than one thousand cells is a daunting task for human eyes. The available automatic cell-counting programs are prone to get affected by noise and signal level variance. To overcome this problem, we developed the new Excluded-Volume-Embedding (EVE) algorithm for detecting the positions and total number of cell bodies automatically from 2D and 3D images. For each pixel/voxel, we first calculate the embedding score, which represents the likelihood of regarding the voxel as the center of a labelled cell. Those voxels that have locally maximum scores are chosen as candidate centres. Then, objects each of which has an exclusive volume (typically, disks in 2D and balls in 3D) are embedded on the candidate centers in a descending order of those scores. This process continues until the score becomes no more than the noise level. The EVE algorithm successfully resolved cells, even when they are tightly packed or partially obscured by noise. Because the process is automated, quantitative comparison between many individuals is now feasible. This enabled us to begin estimating differences of whole- brain cell numbers between animals of separate ages and sex.

THE EVOLUTION OF VERTEBRATE SENSORY SYSTEMS

SEBASTIAN SHIMELD, ANTHONY GRAHAM, and FRANÇOISE MAZET

School of Animal & Microbial Sciences, University of Reading, Reading RG6 6AJ, UK

In vertebrate embryos specialized areas of the cranial ectoderm called placodes contribute to the specialized sense organs and associated ganglia of the vertebrate head. As such, placodes are a key component of what makes a vertebrate different from its closest surviving relatives, amphioxus and tunicates, and may have facilitated the evolution of a predatory lifestyle by primitive vertebrates. In vertebrates, multiple molecular markers have been identified that mark the development of all sensory placodes, of subsets of placodes and of individual placode-derived cell types. We have turned to the tunicate Ciona intestinalis as a model for studying the evolutionary origin of placodes. Using a panel of molecular markers, we have found two areas of the Ciona embryo that bear molecular, morphological and positional similarities to specific vertebrate placodes. Our data indicate that placodes evolved before the emergence of the vertebrates and suggest the evolutionary origin of vertebrate sensory specialization via heterochrony.

LIVE IMAGING OF GLUTAMATE RECEPTOR TRAFFICKING IN SYNAPSE FORMATION OF INTACT DROSOPHILA

STEPHAN J. SIGRIST, TOBIAS RASSE, ANDREAS SCHMID, and WERNHER FOUQET

Max Planck Society, European Neuroscience Institute, 37073 Göttingen, Germany

Both the development as the experience-dependent rewiring of synaptic circuitry depends on the formation of additional synaptic sites. While glutamatergic synapses are the predominant excitatory synapses of our brains, little is known about how they form in vivo. We here for the frist time follow glutamate receptor fields of individual neuromuscular synapses over days—using confocal microscopy on developing Drosophila larvae—which express DGluRIIA-GFP, a functionally GFP-tagged glutamate receptor subunit. We find that, throughout development, new small receptor fields form de novo, which then grow to a mature size at which they remain stable over days. We find functional and structural assembly of the presynaptic site to be tightly coordinated spatiotemporally with the growth of the postsynaptic receptor fields (RFs). We measure receptor entry and exit at developing RFs by in vivo photobleaching and photoactivation experiments. While de-novo forming receptor fields grow by newly synthesized receptors entering at high density, at maturity, RFs density of glutamate receptor entry and exit are low. This study opens the way for analyzing glutamatergic synapse formation in native settings together with using the efficient functional genetics of Drosophila.

IDENTIFICATION OF BRAIN REGIONS CRITICAL FOR COORDINATED MOTOR BEHAVIOR

JULIE SIMPSON and BARRY GANETZKY

Genetics Department, University of Wisconsin, Madison, WI 53706, USA

Using reversible tools to increase and decrease neural activity in distinct groups of neurons, we have identified several brain regions essential for walking and climbing behavior in adult Drosophila. We have used GAL4 lines expressed in defined cell types (glia, muscles, and neurons using particular neurotransmitters) to assess the roles of these cells in motor coordination. Unsurprisingly, inactivation of motor and sensory neurons can impair motor function and result in paralysis and loss of coordination, respectively. Blocking neural activity specifically in cholinergic, GABAergic, and glutamatergic neurons also prevents normal motor function; however serotonergic and dopaminergic neurons do not seem to be acutely required. GAL4 and GAL80 enhancer-trap screens identified restricted expression patterns in the adult brain and thoracic ganglion, within which altered neural activity causes paralysis and seizures. The connectivity of these groups of neurons is being investigated using fluorescent markers targeted to different regions of the neurons and candidate trans-synaptic tracers. We are particularly interested in the roles of the inhibitory neurons in coordinating motor behavior. We are also comparing different ways to alter neural activity. Genetic access to a defined neural circuit with a behavioral output is critical for exploring the role of ion channels and second messengers in processing information. We are developing the tools to identify and manipulate such a circuit.

A GENETIC MOSAIC SCREEN TO IDENTIFY NOVEL GENES INVOLVED IN NEUROBLAST ASYMMETRIC DIVISION

CATHY SLACK, PAUL OVERTON, and WILLIAM CHIA

MRC Centre for Developmental Neurobiology, King's College, London SE1 1UL, UK

Asymmetric cell division is a fundamental mechanism for generating cell-fate diversity during development. Drosophila neuroblasts undergo a series of asymmetric divisions along the apical-basal axis, each of which generates a larger neuroblast and a smaller ganglion mother cell (GMC) into which neural fate determinants are specifically segregated. An apically localized protein complex, containing Inscuteable, Bazooka, Par-6, and atypical Protein Kinase C, as well as Partner of Insc and Gai, is retained by the neuroblast after division. This complex is required for the appropriate orientation of the mitotic spindle as well as for the correct localization of the basal determinants Numb and Prospero via their adapter proteins Partner-of-Numb and Miranda. After segregation of the basal determinants into the GMC, Pros enters the nucleus and directs these cells to undergo a single terminal division. We have conducted a screen of 2,200 EMS-mutagenized 3rd chromosomes to identify novel proteins that function during asymmetric division. To circumvent the limitation of embryonic screens arising from the maternal contribution of many proteins, we have employed the MARCM system to generate clones in 3rd-instar larval neuroblasts. We have identified several novel complementation groups that affect the basal localization of Miranda or the orientation of the mitotic spindle during neuroblast division. Preliminary analyses of these mutant lines have been performed.

ALTERNATIVE SPLICING REGULATES TRANSCRIPTIONAL ACTIVATOR FUNCTION OF EWG

MATTHIAS SOLLER, IRMGARD HAUSSMANN, and KALPANA WHITE

Department of Biology, Brandeis University, Waltham, MA 02454, USA

The erect wing gene (ewg) encodes a transcription factor essential for nervous-system function. Phenotypic analysis indicates that ewg affects synaptic growth at 3rd-instar neuromuscular junctions as well as physiological and metabolic aspects of neurons. ewg RNA is broadly expressed and alternatively spliced in two regions of the gene. From the RNAs of the four major splice forms, however, only one major EWG protein (SC3 ORF) is detected. Nervous-system-specific expression of EWG protein is achieved through inhibition of polyadenylation in the last ewg intron by ELAV, resulting in splicing of this intron and expression of the major EWG isoform. Although the SC3 isoform is the most prevalent and provides full rescue of lethality, analysis of epitope-tagged genomic rescue constructs indicates that other isoforms are also made. Unexpectedly, one of the isoforms runs at the same size on SDS-gels as the SC3 ORF and might have been underestimated. To elucidate the function of these isoforms and the role of post-transcriptional regulation of EWG expression, we tested the rescue capacity of the other three isoforms. Surprisingly, all isoforms can rescue lethality, although with reduced efficiency. Finally, we used RNA expression profiling to determine the transcriptional activity of different EWG isoforms. Our analysis indicates that transcriptional repression is the default function of EWG isoforms and that ELAV-mediated regulation of ewg expression adds an activator function.

CHARACTERIZATION OF NOVEL PROTEINS INVOLVED IN CELLULAR ASYMMETRY IN THE DROSOPHILA CNS

JOHN SOLLY and ANDREA BRAND

WT/CR UK Gurdon Institute, Cambridge CB2 1QR, UK

We are interested in understanding how cellular diversity is generated by asymmetric stem-cell division in the Drosophila CNS. One way asymmetric cell division can occur is through the asymmetric partitioning of cell fate determinants within the mother cell before mitosis. Proteins that contain a PDZ domain are known to be involved in establishing and maintaining cell polarity in species ranging from C. elegans to humans. The Drosophila genome encodes 68 putative PDZ proteins, many of which have not yet been functionally characterized. We are concentrating primarily on uncharacterized PDZ proteins and have cloned 37 cDNAs using Invitrogens GatewayTM cloning technology. This enables us to generate rapidly many different constructs for tagging with, for example, GFP, His, Flag, or myc. We have identified a subset of PDZ genes that is transcribed in the embryonic CNS. These have been separately CFP-tagged at the C terminus and YFP-tagged at the N terminus for the creation of transgenic Drosophila to assay protein localization in vivo. Many of the proteins involved in asymmetric cell division in the CNS are restricted apically or basally in the mother cell prior to mitosis. In addition, all known PDZ proteins are localized asymmetrically. We anticipate, therefore, that several PDZ proteins have as yet uncharacterized functions in asymmetric cell division in the embryonic CNS.

SCREEN FOR MUTATIONS ON 3R FOR DEFECTS IN THE ASYMMETRIC DIVISION OF LARVAL NEUROBLASTS

RITA SOUSA-NUNES, GREG SOMERS, and WILLIAM CHIA

MRC Centre for Developmental Neurobiology, London SE1 1UL, UK

Asymmetric cell division is a fundamental mechanism for generating cell fate diversity during development. In Drosophila embryonic neuroblasts (NBs), the highly conserved polarity-controlling complex Bazooka (Par-3), Par-6, and atypical Protein Kinase C is linked, by Inscuteable, to a second polarity complex containing Partner of Inscuteable (Pins) and GaI. These complexes localize to the apical cortex and are required for the correct orientation of the mitotic spindle and localization of the adaptor protein Miranda (Mir) and the cell-fate determinants Numb and Prospero to the basal cortex. To help understand the mechanisms concerned, we aim to identify novel components involved in asymmetric cell division. The zygotic loss-of-function screens that have brought great insight into the molecules and mechanisms concerning NB asymmetric division are approaching saturation and are hampered by the fact that maternally contributed proteins could mask a mutant phenotype. We have therefore adopted the MARCM system to screen through mutant NB clones in the 3rd-instar larval brain. We are in the process of screening 1,900 EMS-mutagenized FRT82B lines and have identified a number of potentially informative phenotypes. We are characterizing a line that results in Mir mislocalization to the NB cytoplasm. Other phenotypes include clones that lack an obviously large NB, clones where the NB lacks Mir, and also a number of lines where Mir is mislocalized to the centrosomes. [Also see abstract by Slack et al.]

HOX GENE CROSS-REGULATORY INTERACTIONS IN THE EMBRYONIC BRAIN OF DROSOPHILA

SIMON SPRECHER, MARTIN MÜLLER, LARS KAMMERMEIER, DAVID MILLER, THOMAS KAUFMAN HEINRICH REICHERT, and FRANK HIRTH

Zoological Institute, University of Basel, 4056 Basel, Switzerland

During embryonic development of the Drosophila brain, the Hox gene labial is required for the regionalized specification of the tritocerebral neuromere. In order to gain further insight into the mechanisms of Hox gene action in the CNS, we have studied the molecular and genetic basis of cross-regulatory interactions between labial and other more posterior Hox genes using the GAL4-UAS system. Misexpression of posterior Hox genes in the embryonic neuroectoderm results in a labial loss-of function phenotype and a corresponding lack of Labial protein expression in the tritocerebrum. This is due to repression of labial gene transcription in the embryonic brain. Enhancer analysis suggests that this transcriptional repression operates on a 3.65-kb brain-specific labial-enhancer element. A functional analysis of ANTP and UBX protein domains shows that the transcriptional repression of labial requires homeodomain-DNA interactions but is not dependent on a functional hexapeptide. The repressive activity of a Hox protein on labial expression in the tritocerebrum can, however, be abolished by concomitant misexpression of a Hox protein along with the cofactors Homothorax and nuclear-targeted Extradenticle. Taken together, these results provide novel and detailed insight into the cross-regulatory interactions of Hox genes in embryonic brain development and suggest that specification of tritocerebral neuronal identity requires equilibrated levels of a Hox protein plus Hth and n-Exd cofactors.

CONTROL OF AXON EXTENSION IN THE DEVELOPING DROSOPHILA BRAIN BY THE CROSS-TALK OF SIGNAL TRANSDUCTION PATHWAYS

MOHAMMED SRAHNA, MAARTEN LEYSSEN, CHING-MAN CHOI, LEE FRADKIN, JASPRIEN NOORDERMEER, and BASSEM HASSAN

Department of Human Genetics, School of Medicine, University of Leuven, 3000 Leuven, Belgium

Understanding how neurons extend axons for long distances and, consequently, finding ways of regenerating this capacity after injury is a fundamental goal of basic and medical brain research. Although extension, retraction, pathfinding, branching, and synaptogenesis are likely under separate genetic control, little is known about the mechanisms of extension and retraction. We investigated this question in the developing adult brain of Drosophila and find that it is regulated by crosstalk between WNT, FGFR, and JNK signaling. The Rac1 GTPase is an integration point relaying the axon-stabilizing WNT signal and the axon-retracting FGFR signal to modulate the extension-promoting JNK signaling pathway. Activation of JNK signaling is necessary and sufficient for axon extension, whereas the antagonistic WNT and FGFR signals act as a redundant system to minimize variability. The interaction of these different pathways produces a stereotyped connectivity pattern.

SENSORY MAP FORMATION THE ADULT VISUAL AND OLFACTORY SYSTEM

GEORG STEFFES, ARIANE ZIERAU MILAN PETROVIC, MARC LATTEMANN, BRITTA KUHLMANN, and THOMAS HUMMEL

Institut für Neuro- und Verhaltensbiologie, Universität Münster, 48149 Münster, Germany

Our research centers on the molecular mechanism that direct the formation of sensory maps in the Drosophila visual and olfactory system. Photoreceptor cells (R cells) and olfactory receptor neurons (ORNs) project to distinct central target regions, the optic and the antennal lobe, respectively. The visual map is strictly topographic: neighboring photoreceptor cells in the retina project to neighboring target neurons in the brain, such that the spatial relationships between projecting and target populations are maintained. By contrast, ORNs that are randomly distributed in the olfactory epithelium converge onto specific glomeruli, the functional units in the antennal lobes, in a manner that reflects their pattern of olfactory receptor expression. To identify gene functions that underly the different mapping strategies employed by the fly visual and olfactory system we are performing a large-scale mosaic screen. Expression of FLPase under ey-promotor control induces large mutant clones in the eye-antennal disc, affecting both R-cell and ORN precursors. The combination of R-cell and ORN subclass specific GFP-tagged marker lines in the mosaic background allows us to directly compare the role of the affected genes in visual versus olfactory system connectivity. This approach presents a unique opportunity to gain better understanding of how regional specificity of synaptic wiring is achieved.

SCREENING CLIMBING FRUIT FLIES: VISUAL GAP SIZE ESTIMATION DETERMINES THEIR MOTIVATION TO CLIMB

ROLAND STRAUSS and SIMON PICK

Biozentrum, University of Würzburg, 97074 Würzburg, Germany

Drosophila flies are not only good aviators, but also fast walkers and extraordinary climbers with a highly adaptive multi-stage climbing behavior. The objective of this study is to unravel the higher motor control by the brain. Freely walking flies regularly overcome gaps 140% wider than their body size. The decision to initiate climbing is based on visual gap-width estimation, as a comparison of visual mutants and wild-type flies revealed. At narrow gaps, flies persistently attempt to climb and successfully surmount the gap, whereas they avoid vain attempts at large gaps. Width information is conveyed exclusively by the photoreceptor system R1-R6, feeding also into “motion vision.” Binocular vision is not required. No evidence for peering head movements has been found. Instead our data suggest that en-route evaluation of parallax motion generated mainly by vertical edges on the frontal opposite surface is used for width estimation. Gap crossing in Drosophila provides an excellent paradigm for studying behavioral-plasticity and motor control. Among 230 locomotor mutant lines screened for climbing deficits, 42 showed sub-normal motivation or performance. The central complex and the mushroom bodies are involved in different control aspects of gap crossing. Last but not least, we beneficially transfer the flies’ time-tested strategies of autonomous climbing to an experimental walking machine to achieve adaptive and highly maneuverable robots.

AN INNATE AVOIDANCE BEHAVIOR IS DICTATED BY A SPECIFIC OLFACTORY CIRCUIT IN DROSOPHILA

GREG SUH, ALLAN WONG, ANNE HERGARDEN, SEYMOUR BENZER, RICHARD AXEL, and DAVID ANDERSON

Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA

We have developed a novel behavioral paradigm for an innate avoidance response in Drosophila. This paradigm involves avoidance of a putative “alarm” substance (AS) emitted by flies subjected to stress. Responder flies were given a choice in a T-maze between a fresh tube and a conditioned tube in which a set of emitter flies was previously traumatized. Most responders choose the fresh tube: Performance Index values typically fall between 95 and 80 under optimal conditions. Gas-chromatograph/mass-spectrometry and respirometer analyses indicated that CO₂ is a component of the AS; and flies exhibit avoidance response to CO₂ in a dosage-dependent manner. We next sought to functionally map olfactory circuits mediating avoidance response to CO₂. Flies in which a genetically encoded, calcium-responsive indicator (GCaMP) was expressed throughout the antenna lobe revealed that a single pair of the ventralmost glomeruli, known as V, are activated by CO₂. Moreover, functional inactivation of the GR21D1 + sensory neurons, which project to the V glomerulus, using UAS-shibire^TS (see below), was sufficient to abolish the avoidance response to CO₂. We have independently mapped groups of neurons essential for the avoidance response to the AS by carrying out an unbiased neuronal inactivation screen using the Gal4/UAS-shibire^TS system. Flies bearing a Gal4 enhancer trap along with a UAS-shi^TS transgene were assayed in the behavioral paradigm at the non-permissive temperature (30^oC) and were compared to their performance at the permissive temperature (22^oC).

A ROLE FOR EBONY IN THE CIRCADIAN REGULATION OF LOCOMOTOR BEHAVIOR

JOOWON SUH and F. ROB JACKSON

Department of Neuroscience, Tufts University, Boston, MA 02111, USA

ebony mutants exhibit morphological and behavioral phenotypes, including abnormally dark body color and defects in the circadian regulation of locomotor activity. The ebony gene encodes N-β-alanyl-biogenic amine synthase (BAS), which conjugates β-alanine with various kinds of biogenic amines, forming N-β-alanyl-biogenic amine. Consistent with a deficit in BAS activity, ebony mutants have elevated levels of β-alanine and dopamine. We have examined expression patterns of ebony in the CNS to gain insight into how this gene contributes to circadian regulation. We confirm that ebony RNA shows circadian changes in abundance, with a peak near the beginning of the day, a time that correlates with the initiation of locomotor activity. We also show that EBONY protein abundance is higher during the day than at night in both head and brain extracts, indicative of a circadian regulation of BAS activity in the CNS. Immunostaining studies show that EBONY protein is localized in a subpopulation of CNS glial cells rather than neurons. Many of the EBONY-containing glia are close to dopaminergic or serotonergic neurons, suggesting they might modulate aminergic functions, and a small percentage exhibits nuclear staining for the PER and TIM clock proteins. Consistent with a role for ebony-expressing glia in the circadian modulation of activity, flies with defective glial development show altered locomotor activity rhythms. Our results support the idea that glial cells participate in the orchestration of rhythmic behavior.

COMMON EXCITATORY MOTONEURONS IN DROSOPHILA LARVA

EIJI TAKIZAWA, AKIRA KOMATSU, and HIDENOBU TSUJIMURA

Tokyo University of Agriculture and Technology, 183-8509 Tokya, Japan

Insects have several types of motoneurons, which innervate the same muscle and together control its contraction. They have motoneurons, which innervate multiple muscles (common motoneurons), as well as motoneurons innervating a single muscle (specific motoneurons). A well-known example of the common motoneurons is the common inhibitory motoneuron in locust. In Drosophila, however, the presence and properties of this type of motoneurons have not been defined. We identified two common motoneurons in the ventral abdominal ganglia of the 3rd-instar larva: a dorsal common motoneuron and a ventral common motoneuron, using a GAL4 line which drives expression in a small number of neurons. The dorsal common motoneuron was a larval form of embryonic RP2, innervating eight dorsal body-wall muscles with type-Is synaptic terminals. The ventral common motoneuron was an RP like motoneuron, innervating eight ventral body-wall muscles with type-Is terminals. Recording of spontaneous junctional potentials showed that these motoneurons gave a large excitatory potential in muscles, and this piece of physiology occurred simultaneously in different muscles. These common motoneurons generally fired in a burst of large potentials of specific excitatory motoneurons and gave extra large compound potentials. The bursts caused the contraction of the larval body. These results indicate a novel type of common motoneurons and its important role in the control of muscle contraction in insect behavior.

AXON-GLIA ASSOCIATION IN THE DROSOPHILA CNS IS MEDIATED BY TWO CELL ADHESION MOLECULES CONNECTIN AND NEUROGLIAN

KAZUNAGA TAKIZAWA and YASUSHI HIROMI

Center for Developmental Biology, RIKEN CDB, 650-0047 Kobe, Japan

During CNS development, glial cells guide axon pathfinding, whereas axons provide glial migratory pathway. Thus axon-glia interaction is critical for the patterning of the CNS architecture. In the Drosophila embryonic CNS a subtype of glial cells called longitudinal glia (LG) undergo cell migration and associate with longitudinal axon bundles. The initial contact of LG to axons occur when longitudinal pioneer axons start axonogenesis; and LG aligns on the longitudinal axon tract, eventually ensheathing it. Proper glia-axon association requires a glia-specific homeodomain protein REPO; in the repo mutant, LG fails to adhere to the axon bundle but instead is misplaced laterally. Two homophilic cell adhesion molecules Connectin and Neuroglian are expressed on both LG and axons, raising the possibility of their involvement in axon-glia association. Although either single mutant had only minor defects in axon-glia association, a connectin neuroglian double mutant exhibited a synergistic phenotype, resulting in mislocation of LG. In the repo mutant, expression of both Connectin and Neuroglian was severely reduced in glial cells, while their expression on axons remained unaltered. Forced expression of connectin or neuroglian in glial cells was sufficient to rescue the axon-glia association phenotype of repo, implying that these two adhesion molecules are the major players that mediate axon-glia association downstream of REPO.

NATURAL POLYMORPHISM IN CRYPTOCHROME AND LIGHT SENSITIVITY OF THE CIRCADIAN CLOCK

ERAN TAUBER, RODOLFO COSTA, EZIO ROSATO, and CHARALAMBOS P. KYRIACOU

Department of Genetics, University of Leicester, Leicester LE1 7RH, UK

For most organisms light is the main cue for entrainment of the circadian clock. As light conditions vary along latitude, populations from different geographical zones are expected to have different molecular adaptations. Natural polymorphism in the Drosophila period and timeless genes has previously been identified. Since cryptochrome (cry) encodes a dedicated circadian photoreceptor in Drosophila, we have initiated a screen to identify natural cry alleles. We sequenced the complete coding region of 12 natural cry alleles from African, Mediterranean, and European populations. Analysis of the sequences revealed high level of polymorphism consisting of 29 single nucleotide polymorphic sites (SNPs), including seven amino-acid replacements. The L232H replacement involved a significant change in the amino-acid properties. The pattern of SNPs (many intermediate frequency mutations) and strong linkage disequilibrium suggest that this polymorphism is maintained by natural selection and thus is functionally important. We carried light-pulse experiments using different homozygous natural lines and found a significant difference in light sensitivity. A multiple regression model indicated that the L232H replacement is the main factor behind this variation. Analysis of cry sequences from different organisms suggests that this site has undergone rapid evolution.

PATTERNS OF GLIOGENESIS IN THE LARVAL OPTIC LOBES OF DROSOPHILA AND ITS RELATION TO GLIDE/GCM AND GLIDE2

FRANCISCO J. TEJEDOR, JORDI COLONQUES, JULIAN CERON, and ANGELA GIANGRANDE

Instituto de Neurociencias-CSIC, Universidad Miguel Hernandez, Campus de San Juan, 03550 Alicante, Spain

Gliogenesis has been extensively studied in the embryonic CNS of Drosophila where most glial progenitor cells produce both glia and neurons, and few produce only glia. It is also well known that the transcription factor Glide/Gcm is specifically expressed in all glial progenitors cells and is necessary and sufficient to promote gliogenesis. In contrast, little is known about postembryonic gliogenesis and the genetic basis of glial specification in the adult brain. We have studied gliogenesis in the larval optic lobe and have generated a spatio-temporal map with the distribution of glial progenitor anlagen. In parallel, we have studied the expression pattern of glide/gcm and glide2 and their role in the specification of glia in the larval brain. Several major general conclusions can be outlined: (1) Glial progenitors are mostly well segregated from neuroblasts in the larval brain. (2) In contrast to the embryonic CNS, glide/gcm and glide2 present quite complementary expression patterns in the larval brain. (3) Also in contrast, glide/gcm and glide2 expressions in the larval brain are not restricted to glial progenitors, since we have also found expression in some neuroblasts, postmitotic glia, and even in certain neurons. (4) Ectopic expression of glide/gcm is sufficient to produce glial cells, but this is regionally restricted. Altogether, our results suggest a clear diversification of the functional roles played by glide/gcm and glide2 in postembryonic CNS development.

CHARACTERIZATION AND FUNCTION OF THE DROSOPHILA GUSTATORY RECEPTOR GENES IN THE PERCEPTION OF SWEET, BITTER AND PHEROMONE COMPOUNDS

NATASHA THORNE, STEVE BRAY, CAROLINE CHROMEY, and HUBERT AMREIN

Department of Molecular Genetics and Microbiology, Duke University, Durham, NC 27710, USA

In Drosophila, a family of gustatory receptors (GRs) expressed in taste neurons is thought to mediate the recognition of sugars, toxic compounds, and pheromones, thereby controlling feeding and mating behaviors. We have characterized in detail the expression of eight Gr genes in the labial palps, the fly's main taste organ. These genes fall into two distinct groups: seven of them, including Gr66a, are expressed in 22 or fewer taste neurons in each labial palp. Additional experiments show that many of these genes are co-expressed in partially overlapping sets of neurons. In contrast, Gr5a, which encodes a receptor for trehalose, is expressed in a distinct and larger set of taste neurons associated with most chemosensory sensilla, including taste pegs. Mapping the axonal targets of cells expressing Gr66a and Gr5a reveals distinct projection patterns for these two groups of neurons in the brain. Moreover, tetanus-toxin-mediated inactivation of Gr66a- or Gr5a-expressing cells show that these two sets of neurons mediate distinct taste modalities, the perception of bitter (caffeine) and sweet (trehalose) taste, respectively. Earlier investigations have led to the identification of GR68a, a receptor required for male courtship. Thus, the Gr genes appear to accommodate many different kinds of non-volatile chemosensory signals that involve not only feeding or avoidance of harmful substances, but also the recognition of pheromone cues that elicit complex behaviors.

DMMNF: A NEW TRANSCRIPTION FACTOR OF THE FORK HEAD FAMILY IN DROSOPHILA MELANOGASTER

CASAS TINTÓ and GRANADINO GOENECHEA

CIB (CSIC), 28040 Madrid, Spain

DmMNF is a transcription factor of the Fork-head family; nine exons code for two different mRNA originated by alternative splicing. These mRNAs give rise to two isoforms of 654 and 739 aa. The Fork-head domain of these proteins shows an homology of 70% with the Fork-head domain of MNF and ILF factors in vertebrates. In electrophoretic mobility shift assays, we showed that the protein binds the consensus sequences recognized by all the fork head proteins (T/C AAACA). Transfection experiments in Schneider cells showed that both isoforms activate transcription and are located within the nucleus, as expected for a transcription factor. In-situ hybridization experiments showed that DmMNF mRNA localizes in the endoderm of the parasegments 3 and 7 and in the ventral nerve cord. A polyclonal antiserum against DmMNF protein revealed that the expression profile of the protein during embryonic development is coincident with the mRNA expression. The CNS development depends on the spatially restricted expression of homeotic genes that regulate the expression of signaling molecules, such as Dpp and Wg. Flies that over express dpp showed an expansion of DmMNF. In adition, we found consensus sequences in DmMNF promoter for Mad proteins (which function in the Dpp signaling pathway), data supporting our hypothesis that DmMNF could respond to the signaling pathway of Dpp. Mutants for this gene do not develop a CNS and die before embryonic stage 15.

ELAV MULTIMERIZATION DOMAIN

GAKUTA TOBA and KALPANA WHITE

Department of Biology, Brandeis University, Waltham, MA 02454, USA

ELAV is a panneurally expressed RNA-binding protein in Drosophila that is required for both development and maintenance of neurons. It has three RNA recognition motifs (RRM1, 2 and 3) with a hinge region between RRM2 and RRM3. ELAV regulates alternative splicing of several target genes to produce neuron-specific isoforms. We found that ELAV interacts with itself as well as with other Drosophila ELAV-family proteins Rbp9 and Fne in the yeast two-hybrid system. However, structural basis of the multimer formation is not understood. To map the multimerization domain in ELAV, we made a series of deletion mutants and tested their interaction in the yeast two-hybrid system. We found that a fragment (H3) that consists of the hinge region and RRM3 was able to interact with the full length ELAV as well as with the H3 fragment itself. Since RRM1 or RRM2 did not show interaction activity when fused to the hinge, amino acids that are unique to RRM3 are likely to be responsible for the interaction. Sequence comparison of RRMs among ELAV-family proteins revealed some uniquely conserved sequences in RRM3. We mutated the H3 construct by replacing the conserved sequences with the corresponding sequences of RRM2 and tested the interaction by the two-hybrid assay. Three short sequences were found to cause loss of interaction when mutated, indicating importance of those sequences for the interaction.

DOES SHE CHOOSE? A STATISTICAL APPROACH

ENRIQUE TURIÉGANO, SANTIAGO SÁNCHEZ-PAGÉS, PABLO REGUEIRA, JONATHAN BENITO, RUTH MATESANZ, LAURA TORROJA, and INMACULADA CANAL

Departamento de Biologia, Universidad Autonoma de Madrid, 28049 Madrid, Spain

According to evolutionary theories, the best male must be the copulating one. This implies that the mating of a female with a male when there are two candidates must be different from random mating. This deviation from random mating can be caused by two different factors: female discrimination or different male efficacy (mating propensity). In Drosophila it is usually assumed that the male efficacy (calculated as copulation latency, CL) decides whether mating will occur. This assumption would imply that there is no female choice. Therefore, the mating event (when a female is exposed to two males) would occur with the male that has higher efficacy. We have analyzed 3,000 copulations and recorded certain male-associated characteristics of them. We have correlated these characteristics with the CL values of the males. With this correlation we have constructed a model that allows us to obtain theoretic efficiencies of two competing males. Assuming that the most efficient male is the copulating one, the comparison of the former predicted data with its actual competition results will allow us to discriminate between the two hypotheses.

FORMINS IN THE DEVELOPING EMBRYONIC CNS

RICHARD TUXWORTH and WILLIAM CHIA

MRC Centre for Developmental Neurobiology, Kings College, London SE1 1UL, UK

The formins are a family of cytoskeleton proteins able to bind to and stimulate actin polymerisation directly, forming linear actin filaments. Formins have important roles in various aspects of cell behavior that require modulation of the actin cytoskeleton, including cell polarization, migration, adhesion, and cytokinesis. There are several formins in Drosophila, the best characterized being Diaphanous and Cappuccino, which are required for cytokinesis and cell polarity. Here, we describe three novel Drosophila formins that are expressed in the developing embryonic CNS. The first novel formin conforms to the domain structure of the Diaphanous subfamily and is expressed in a pan-neural pattern from embryonic stage 9 onwards. Two others are expressed in the midline glia. Of these, one is a founder member of a new subfamily of formins that lack the profilin-binding FH1 domain (formin-homology 1 domain), which is located immediately N-terminal of the FH2 domain in most formin proteins. This formin is expressed specifically in the midline glia from stage 12 onwards; and mutations disrupt the glia, leading to collapse of commissural axon tracts. Interestingly, the well-characterized enhancer trap AA142 is inserted within this locus. We are performing RNAi experiments to target the neural-specific formins and analyze the resulting mutant phenotypes.

EMBRYONIC DEVELOPMENT OF THE DROSOPHILA BRAIN: A MOLECULAR CHARACTERIZATION OF A PROCEPHALIC TERRITORY SHARING SIMILIARITIES WITH THE VERTEBRATE MIDBRAIN/HINDBRAIN BOUNDARY REGION

ROLF URBACH and GERHARD TECHNAU

Institute for Genetics, University of Mainz, 55122 Mainz, Germany

In vertebrates, the primordium of the brain is subdivided into three basic regions by the expression of Otx/Emx genes (forebrain/anterior midbrain), Hox genes (posterior hindbrain), and Pax2/5/8 genes (intermediate region). Still unclear is whether this tripartite ground pattern, including the midbrain/hindbrain boundary (MHB) in its intermediate region, is an innovation of vertebrates or has evolved earlier in evolution. In order to determine whether a tripartite ground pattern is conserved in early neurogenesis of the Drosophila brain, we examined the expression of homologous genes in the procephalic neuroectoderm and in identified neuroblasts (NBs) during developmental stages 9–11. We show that, based on otd and lab expression, the early brain principally exhibits a tripartite pattern, although Pax2 genes are not expressed in the neuroectoderm/brain NBs of an intermediate zone (IZ). We identified the IZ to encompass a fraction of deutocerebral NBs. Furthermore, otd and unpg are complementarily expressed in NBs, and their domains exhibit a small common interface at the anterior border of the IZ. We describe the early expression of other genes, the vertebrate homologs of which are involved in MHB formation, as well as of DV patterning genes, which specifically exhibit a discontinuous expression at the level of the IZ. Altogether, our results suggest the existence of an ancestral territory within the primordium of the deutocerebrum, which might be evolutionary equivalent to the region of vertebrate MHB.

ENHANCER-PROMOTER COMMUNICATION AND CHROMATIN REMODELING DURING DROSOPHILA NEURAL DEVELOPMENT

LUC VANOLST, CATHERINE FROMENTAL-RAMAIN, and PHILIPPE RAMAIN

Institut de Génétique et Biologie Moléculaire et Cellulaire & CNRS/INSERM/ULP, 67404 Illkirch, France

The GATA factor Pannier (Pnr) activates proneural expression through binding to a remote enhancer of achaete (ac) and scute (sc). Chip bridges Pnr with the (Ac/Sc)-Daughterless heterodimer associated with the promoters of ac and sc and facilitate therefore enhancer-promoter communication during Drosophila neural development. Chromatin is a highly dynamic structure at promoters and plays a crucial role during control of eukaryotic gene expression. Thus, we have previously shown that enhancer-promoter communication is repressed by the DrosophilaBrahma (Brm) complex, homologous to the yeast SWI/SNF. The Brm complex is recruited through dimerization of its Osa subunit with Pnr and Chip. Recent studies have identified a novel chromatin remodeling complex, encompassing Toutatis and the ISWI ATPase and involved in the positive regulation of enhancer-promoter communication. In vitro studies have established the role of such factors as chromatin- remodeling machines. Our studies provide in-vivo links between transcription factors, chromatin-remodeling machines, promoter sequences, and regulation of gene expression.

TOUTATIS, A CHROMATIN REMODELING PROTEIN, POSITIVELY REGULATES ENHANCER-PROMOTER COMMUNICATION DURING DROSOPHILA NEURAL DEVELOPMENT

LUC VANOLST, CATHERINE FROMENTAL-RAMAIN, and PHILIPPE RAMAIN

Institut de Génétique et Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, 67404 Illkirch, France

Enhancer-promoter communication plays a crucial role during Pannier (Pnr)-driven neural development. Thus, Pnr activates proneural achaete/scute (ac/sc) expression through binding to a remote enhancer of the ac/sc complex. Moreover, Chip physically interacts both with Pnr and the (Ac/Sc)–Daughterless heterodimer associated with the promoters of ac and sc to give a proneural complex which facilitates enhancer/promoter communication. Using a two-hybrid screening in yeast, we have identified Toutatis (Tou), a protein which shares functional domains with chromatin remodeling factors including Acf1, a subunit of both ACF (ATP-utilizing Chromatin assembly and remodeling Factor) and CHRAC (CHRomatin Accessibility Complex) and TIP5 (Termination factor TTF1-Interacting Protein 5) of NoRC (Nucleolar Remodeling Complex). Loss-of-function and gain-of-function experiments reveal that Tou functionally cooperate with Pnr and Chip during neural development and positively regulates enhancer-promoter communication during proneural expression, probably via chromatin remodeling. Furthermore, Tou physically interacts with the ISWI ATPase, which is also shown to be necessary during Drosophila development. Thus, Tou, and ISWI may define a novel chromatin-remodeling complex involved in positive regulation of enhancer-promoter communication. These studies will help us to elucidate the complete molecular nature of the remodeling complex as well as its function during Drosophila neural development.

A GENETIC SCREEN TO IDENTIFY GENES INVOLVED IN NEUROGENESIS

SVEN VILAIN, XIAO-JIANG QUAN, SHU HU, and BASSEM HASSAN

Laboratory of Neurogenetics, Department of Human Genetics, University of Leuven, 3000 Leuven, Belgium

During neural development, progenitor cells participate in regulatory networks that influence cell fate decisions and tissue patterning. In our previous work (Quan et al., 2004) we have shown that three amino acids in the basic domain of ATO are necessary and sufficient for ATO proneural activity. In order to find out the modifiers for this motif and to dissect the genetic cascades regulating neurogenesis, we performed a screen in a sensitized background using a chimeric protein that consists of NGN with the swapped basic motif of ATO. A test screen showed that Daughterless and Notch pathway genes were easily picked out, suggesting that the screen is robust and sensitive. We have thus far screened the 2nd and 3rd chromosomes in a primary round. We found 16 positive regions on the 3rd chromosome and 21 positive regions on the 2nd chromosome. A secondary screening will rule out false positives and discriminate between ATO-specific and general proneural-interacting genes, by comparing the modification of scute, ngn, and atonal gene-expression paterns.

TRANSGENIC DISSECTION OF CNS REGIONS IN WHICH THE FRUITLESS GENE IS EXPRESSED TO INFLUENCE DISCRETE COMPONENTS OF DROSOPHILA COURTSHIP

ADRIANA VILLELLA and JEFFREY C. HALL

Department of Biology, Brandeis University, Waltham MA 02454, USA

A gal4-containing enhancer-trap (C309) was previously shown to cause subnormal courtship of males toward females and dramatic interactions among males when driving neural expression of a conditional disrupter of synaptic transmission (shi^TS). We have now analyzed this C309-gal4/UAS-shi^TS combination for effects on more distinct features of male-specific behavior, notably courtship song. This phenotype was mostly eliminated by driving shi^TS at restrictive temperature. Song defects and homosexual behavior are among the more salient effects of fruitless ( fru) mutations. Against a background of other CNS-expressed gal4 drivers that cause males to court other males when united with a transgene encoding the female form (F) of the transformer gene product, the C309/UAS-traF combination was found to induce high levels of intermale courtship; yet they exhibited no defects in singing. Inasmuch as the fru gene acts downstream of tra in Drosophila sex-determination hierarchy, we hypothesized that a fru-Inhibitory RNA (IR) transgene would mimic the effects of traF when driven by C309; this was not the case. To connect the behavioral effects mediated by C309 with CNS expression patterns of male-specific FRUITLESS protein (FRU^M), double-labeling of neurons marked by gal4 driving of GFP revealed several brain and VNC neurons that co-express both the protein and the marker. However, only certain interpretable subsets of the 20 FRU^M-containing clusters in the adult CNS were double-labeled.

THE ADAPTOR PROTEIN D-MINT-1 IS AN INTRACELLULAR INTERACTION PARTNER OF THE NEURONAL CELL ADHESION MOLECULE IRREC-RST

SMITHA VISHNU, RODERICK BRAUN, ALEXANDER HERTENSTEIN, GERT DE COUET GERT, and KARL-FRIEDRICH FISCHBACH

Institute of Biology III, University of Freiburg/Brsg., 79115 Freiburg, Germany

IrreC-rst belongs to the immunoglobulin superfamily of cell-adhesion proteins. It has a multifaced role in Drosophila development, such as spacing of ommatidia in the compound eye, axonal guidance in the optic lobe, development of the somatic musculature in the embryo, and spacing of sensory organs. To date, there is not much known about the molecular mechanisms and biochemical pathways recruited by IrreC-rst. The effects of the C-terminal-deleted form of IrreC-rst protein in rst -CT mutants are highly suggestive of involvement of the molecule in signal transduc-tion. In order to identify possible protein- interaction partners for IrreC-rst and its paralog Kirre, yeast two-hybrid screens were performed using the intracellular domains. Drosophila Mint-1 was positive in both the screens. Based on its protein-protein interaction domains Mint-1 was considered to be an interaction partner for IrreC-rst and Kirre. We identified a second mint gene in Drosophila in the reported sequences from the genome project and named this mint-2. Genetic and biochemical experiments confirmed that Mint-1 interacts with IrreC-rst. Overlapping expression patterns of Mint-1 and IrreC-rst, as well as the similar overexpression phenotypes of Mint-1 compared to that of an extracellularly truncated version of IrreC-rst, indicate that both proteins are likely to be involved in the same pathway.

ANALYSIS OF ZINC-FINGER HOMEODOMAIN PROTEIN 1 AND -2 FUNCTION IN THE DEVELOPING CNS OF DROSOPHILA MELANOGASTER

GEORG VOGLER and JOACHIM URBAN

Institute for Genetics, University of Mainz, 55099 Mainz, Germany

The transcription factors Zfh-1 and fh-2 are both expressed during embryonic Drosophila CNS development. They are conserved among species and thought to act as transcriptional repressors, the zfh1 homolog in vertebrates being involved in TGFbeta signaling. We analyzed the expression patterns of the zfh-1 and zfh-2 genes in the embryonic CNS of Drosophila with regard to the temporal expression within neuroblast lineages and their distribution in glial cells. We could show that the temporal expression patterns of both genes are confined to an early (zfh1) and later part (zfh2) of neuroblast lineages. Additionally, there is a differential distribution in glial cells: zfh-1 in peripheral and subperineurial glia, zfh-2 in subperineurial, cell body and interface glia. In addition, we showed that zfh-2 is alternatively spliced, giving rise to at least six isoforms with different compositions of zinc fingers and alterations in the number of homeodomains. We cloned the 10.5 kb cDNA of zfh2 to design gain-of-function experiments using the Gal4/UAS system. To reveal the function of zfh-1 in the CNS, we analyzed mutant embryos and performed overexpression studies of zfh-1 during embryonic and larval stages. There, we could show that ZFH-1 plays a role in the regulation of specific neuropeptides. Further investigations will include putative connections pf these factors with to TGFbeta signaling, as has been found in vertebrates.

THE DROSOPHILA NC82 ANTIGEN: GENE STRUCTURE, EXPRESSION ANALYSIS, AND LOCALIZATION AT PRESYNAPTIC ACTIVE ZONES

DHANANJAY WAGH, TOBIAS RASSE, ALOIS HOFBAUER, ISABELL SHWENKERT, MARIE-CHRISTIAN DABAUVALLE, STEFAN SIGRIST, SIGRID BUCHNER, HEIKE DÜRRBECK, and ERICH BUCHNER

Department of Genetics and Neurobiology, Biozentrum, University of Würzburg, 97074 Würzburg, Germany

The monoclonal antibody (MAB) nc82 was identified in the screen of a large hybridoma library against Drosophila head homogenate for antibodies binding selectively to synaptic neuropil of the brain. Confocal immunohistochemistry localizes the protein at the active zones of presumably all synaptic terminals. The fact that in each synaptic terminal only the small spots of the active zones are labeled makes this antibody especially useful for confocal immunofluorescence microscopy of Drosophila brain wholemounts, because the resulting neuropil stacks are more transparent compared to stainings with antibodies that bind, for example, to synaptic vesicles. On Western blots MAB nc82 recognizes two protein isoforms of about 190 and 180 kDa. We have identified the gene coding for this active zone protein employing 2D-gel electrophoresis and MALDI-TOF mass spectrometry. RT-PCR sequencing using RNA from larvae and adults—in comparison with cDNA fragments and genomic DNA from Drosophila melanogaster, D. pseudoobscura, and Anopheles—identifies at least two alternatively spliced transcripts and reveals a complex gene structure. Northern Blots of head mRNA identifies transcripts at 10, 6, and 4 kb. In-situ hybridization and antibody staining reveal expression of the gene in presumably all neurons of late embryonic, larval and adult stages. Further analyses of these factors will involve homologies with vertebrate proteins and mutational strategies to reveal the function of the encoded proteins.

FUNCTIONAL CHARACTERIZATION OF THE DROSOPHILA RETICULON LOCUS

SARAH WAKEFIELD and GUY TEAR

MRC Centre For Developmental Neurobiology, King's College, London SE1 1UL, UK

reticulon-like-1 (rtnl1) is the only functional reticulon gene in Drosophila and encodes a protein whose C-terminal domain shares 50% identity to mammalian reticulons, such as Nogo (RTN4), a proposed inhibitory molecule present in myelin that may prevent axon regeneration following spinal-cord injury. Analysis of the GFP-protein trap line G9, which contains an insertion in rtnl1, reveals that, although expression of this gene is widespread, it becomes upregulated in the axons of late stage embryos. rtnl1 produces seven transcripts encoding five polypeptides, and in-situ hybridization shows that these are expressed in different tissue specific patterns. In, particular, expression of the largest transcript becomes confined to the CNS during late embryogenesis. In order to further understand the cellular function of reticulon proteins, two different approaches are being pursued: First, to analyze the loss of function of rtnl1, mutations have been generated by male recombination that sequentially remove different transcripts of the gene; this shows that flies lacking 5 of the 7 transcripts develop normally and have no defects in the CNS in the absence of the CNS specific isoform. Second, a UAS enhancer P-element line (GS9781) inserted in rtnl1 is being used to drive ectopic expression of rtnl1 in the developing eye disc and nervous system. We have characterized the gain-of-function phenotype associated with overexpression of rtnl1 in these tissues.

IDENTIFICATION AND CHARACTERIZATION OF A NEW DROSOPHILA RETINAL HOMEOBOX (DRX) MUTATION

RUTH-JESSICA WILD, MICHAEL FAUST, RICHARD DAVIS, and UWE WALLDORF

University of Saarland, 66421 Homburg/Saar, Germany; Department of Pathology, Baylor College of Medicine, Houston, TX 77303, USA

Retinal homeobox (Rx) genes are highly conserved between vertebrates and Drosophila melanogaster. In contrast to vertebrate Rx, an essential regulator of early eye and brain formation, Drosophila Rx (DRx) has probably no function in the development of the visual system. DRx is expressed during embryonic development in the procephalic region, as well as in the clypeolabrum and, later, in the brain and CNS. Larval expression takes place in the optic lobe and partly in the mushroom bodies. DRx is localized in the 57B cluster of homeobox genes on the right arm of the 2nd chromosome, together with orthopedia (otp) and homeobrain (hbn), and additionally with Act57B. A DRx deletion (drx^ex8) affects DRx and the upstream region of the neighboring Act57B gene. This mutant is missing a clypeus, a component of the feeding apparatus, and shows an abnormal ellipsoid body structure. In an EMS-mutagenesis screen we have found a mutation (drx¹⁰¹⁵⁵), which affects only the DRx gene. This point mutation causes a truncation after the octapeptide and leads to a shortened DRx protein of 230 aa compared with the 902-aa wild-type form. The drx¹⁰¹⁵⁵ mutation is a near embryonic lethal—with a few survivors showing the clypeus phenotype similar to effects of the drx^ex8 deletion. Therefore, we possess a specific tool to analyze and characterize the role of DRx during the Drosophila brain and nervous system development.

THE NEUROPEPTIDES TACHYKININS AFFECT BEHAVIORAL RESPONSES TO ODORS

ÅSA WINTHER and ALBERTO FERRÚS

Department of Zoology, Stockholm University, 106 91 Stockholm, Sweden; Instituto Cajal, 28002 Madrid, Spain

The invertebrate neuropeptide family of tachykinins (Tks) consists of structurally related peptides. They are believed to be evolutionary related to the vertebrate tachykinins. Insect Tks have been found to have multiple in vitro actions, however, the in-vivo actions of the Tks are still unclear. In Drosophila the Tk encoding gene and its five different peptide isoforms (Tk 1–5) have been identified. In an attempt to better understand the in vivo actions of Tks, we have generated Tk-deficient flies using RNAi interference (RNAi). We created transgenic lines expressing a Tk-RNAi construct that was predicted to form double-stranded RNA following splicing. The genomic-cDNA construct is under the control of UAS sequences. We have shown that in flies expressing this construct, the endogenous Tk mRNA and peptide expression is reduced by more than 90%, as determined by real-time quantitative PCR and immunocytochemistry—thus demonstrating that the genomic-cDNA construct is capable of inducing RNAi. In the brain of Drosophila Tks are expressed in interneurons with varicose processes in the antennal lobe neuropil. Their distribution suggests roles in olfactory processing. For this reason we have tested the Tk deficient flies for olfactory behavior using an olfactory choice assay. Dose-response tests of the Tk-RNAi expressing flies suggest that these flies have defective responses to odors.

THE CA²⁺-DEPENDENT VOLTAGE-ACTIVATED K⁺ CHANNEL, SLOWPOKE, CONTRIBUTES TO DROSOPHILA PHOTORECEPTOR RESPONSES

VERENA WOLFRAM and MIKKO JUUSOLA

Physiology Department, University of Cambridge, Cambridge CB2 3EG, UK

In Drosophila the Ca²⁺-dependent voltage-activated K⁺ channel, Slowpoke (SLO), is widely expressed in muscle and neuronal tissue and plays a critical role in regulating neuronal firing patterns and neurotransmitter release. Evidence is accumulating that Slowpoke is expressed in the retina and lamina. Furthermore, the SLO-binding protein, SLOB, which specifically binds to SLO and EAG, is expressed in photoreceptor cells. However, no physiological role has been assigned to slowpoke in the visual system. In R1-6 photoreceptors three voltage-activated K⁺ channels have been identified, a slow and a fast delayed rectifier and an A-type current, Shaker. None of these channels is Ca²⁺-dependent. Our working hypothesis was that SLO might contribute to early visual processing. Using electroretinogram (ERG) recordings we show that slowpoke null mutants, slo² and slo⁴, have altered responses to light. To link these alterations to photoreceptor responses we have recorded intracellularly from wild-type and mutant photoreceptors. Light-induced voltage responses of the mutants have significantly reduced signal-to-noise ratio, owing to reduced amplification and speed. Although many secondary effects are likely to influence these events, it is feasible that slowpoke contributes to the enhancement of transients necessary for normal synaptic signal transfer. To test this hypothesis we are currently doing intracellular recordings from post-synaptic neurons (Large Monopolar Cells).

ATTENUATION OF SENSITIVITY TO TREHALOSE BY EXPRESSION OF AN IP3-ABSORBENT “SPONGE” IN A SUBSET OF GUSTATORY-RECEPTOR CELLS IN DROSOPHILA

DAISUKE YAMAMOTO, K. MATSUMOTO, S. KOHATSU, K. ISONO, H. MATSUBAYASHI, M.-T. YAMAMOTO, K. TAKAHASHI, R. UEDA, K. MIKOSHIBA, and K. USUI-AOKI

Waseda University, 202–0021 Nishi-tokyo, Japan

The “IP3 sponge” was developed as a recombinant hyper-affinity IP3 absorbent, which was constructed on the basis of the ligand-binding site of the mouse type-1 IP3 receptor. We used this IP3 sponge to investigate the possible involvement of IP3 signaling in gustatory transduction in Drosophila. The Tre gene encodes a gustatory-receptor protein that is primarily responsible for trehalose sensitivity. We carried out the feeding preference assay, in which adult flies starved for 20 hours were given a choice to take either 2 mM sucrose or 10 mM trehalose. In another series of experiments, the flies were tested for their ability to distinguish solutions containing two different concentrations of trehalose in a similar two-way choice paradigm. The two solutions used for the assay were colored red or blue, and the relative amount of two solutions consumed by a fly was estimated by the coloration of the abdominal part of the fly. When the IP3 sponge was expressed by means of the Gal4-UAS system with the Tre-promoter-driven Gal4 transgene, acuity in trehalose sensitivity was significantly decreased in both types of assays mentioned above. Similar results were obtained when the IP3 receptor function was inhibited by means of RNAi or hypomorphic mutations. These observations are in keeping with the hypothesis that activation of the Tre-encoded receptor initiates IP3 signaling.

SPECIFICATION OF LONGITUDINAL GLIA

YOSHIHIRO YUASA and YASUSHI HIROMI

National Institute of Genetics, 411-8540 Mishima Shizuoka, Japan

Spatial and temporal regulation of cell fate determination in the nervous system requires integration of various input signals. The longitudinal glia are composed of a heterogeneous population that is subdivided into two glial cell types owing to the Notch signal. A homeodomain transcription factor Prospero (PROS) is expressed in a subset of longitudinal glia and is required for generating the scaffold of the longitudinal tract and neuronal survival. We are analyzing how PROS expression is regulated in the longitudinal glial cells. While PROS expression in longitudinal glia was dependent on the “ets” transcription factor Pointed (PNT), PNT was not sufficient for inducing PROS expression in the embryonic epidermis. To identify other factors involved in pros regulation, we focused on the homeodomain protein REPO and AT-rich binding protein Dead Ringer/Retain (DRI), two transcription factors that are specifically expressed in longitudinal glia. PROS expression was reduced in embryos that are mutant for either repo or dri. When PNT, REPO, and DRI were misexpressed simultaneously, there was a dramatic increase in the number of PROS-positive cells in the epidermis. These three transcription factors also activated expression of a pros enhancer reporter gene. These results suggest that pros integrates inputs from multiple transcription factors to achieve longitudinal glial expression.

AN IMPROVED ELECTROPHYSIO-LOGICAL AND BEHAVIORAL PARADIGM USING TETHERED FLIES DEMONSTRATES OPPOSITE EFFECTS OF DROSOPHILA PKA-RI AND PKA-RII MUTATIONS ON HABITUATION AND DISHABITUATION

XIAOTIAN ZHONG and CHUN-FANG WU

Department of Biological Sciences, University of Iowa, Iowa City, IA 52242, USA

We previously established an electrical stimulation-recording paradigm that demonstrates in tethered flies a physiological analog of habituation in the giant fiber escape circuit (Engel & Wu, 1996, 1998). Altered habituation rates were found in memory mutants rut and dnc, defective in the synthesis and degradation of cAMP, respectively. Here we show an improved paradigm that allows for behavioral performance in tethered flies for physiological and behavioral assessments of dishabituation as well as habituation. We examined mutants of Type I and II regulatory subunits of PKA, two potential downstream mediators of rut and dnc mutations. These mutants, RI^7I5 and RII^EP2162, displayed contrasting vs. phenotypes, in parallel to those of rut vs dnc. Both rut and RII^EP2162 slowed habituation and enhanced dishabituation, while dnc and RI^7I5 accelerated habituation and decreased dishabituation. Biochemically, the RII^EP2162 mutation significantly reduces cAMP-activated PKA activity, resembling the effects of decreased cAMP levels in rut. The RI^7I5 mutation reduces RI expression, causing constitutive activation of PKA and resembling the effects of increased cAMP levels in dnc. Our results demonstrate that cAMP plays a critical role in, and that PKA is the major downstream mediator for, regulation of both habituation and dishabituation.

MUTATIONS AFFECTING SYNAPTIC SPECIFICITY IN THE DROSOPHILA OLFACTORY SYSTEM

ARIANE ZIERAU, GEORG STEFFES, BRITTA KUHLMANN, MARC LATTEMANN, MILAN PETROVIC, and THOMAS HUMMEL

Institut für Neuro- und Verhaltensbiologie, Universität Münster, 41149 Münster, Germany

A striking feature of olfactory system organization is the evolutionarily conserved arrangement of olfactory receptor neuron (ORN) terminals into an odortopic map. To uncover the molecular basis of wiring specificity in the Drosophila olfactory system, we have initiated both forward genetic screens and candidate gene approaches to identify ORN targeting mutants. During pupal development, functionally identical ORN subclasses project their axons to only one of 40–50 distinct glomeruli in the antennal lobe (AL), the primary olfactory center in the brain. We previously demonstrated that the neuronal receptors Dscam and N-Cadherin play crucial roles in determining target specificity for a subclass of ORNs. Dscam mutant ORNs frequently converge onto ectopic glomeruli throughout the AL. In contrast, N-Cadherin mutant ORNs project to the vicinity of their postsynaptic glomerulus, but fail to converge onto their cognate glomerulus supporting the notion that AL targeting and glomerulus formation are distinct steps in constructing an olfactory sensory map. In a subsequently performed large-scale mutagenesis screen we discovered additional mutations that lead to Dscam and Ncad-like ORN targeting defects. Initial phenotypic characterization have been performed, and possible ORN/ORN and ORN/target interactions underlying synaptic specificity in the olfactory system have been inferred accordingly.

FUNCTIONAL CHARACTERIZATION OF A dsRNAi KNOCKDOWN OF THE DROSOPHILA MELANOGASTER HOMOLOG OF HUMAN SURF1

MAURO ZORDAN, ALBERTO PICCIN, PAOLO CISOTTO, CLARA BENNA, MIRKO PEGORARO, GERTRUUS TEKRONNIE, ALESSANDRO AGOSTINO, MASSIMO ZEVIANI, SAMANTHA PERON, CARLO REGGIANI, and RODOLFO COSTA

Department of Biology, University of Padova, 35131 Padova, Italy

In humans, mtDNA mutations are associated with pathologicies involving tissues highly dependent on mitochondrial (mt) energy, such as brain, muscle, and sensorineural epithelia. It appears that all cases of human hereditary motor and sensory neuropathy with optic atrophy are associated with mt alterations. In Drosophila mutants for genes encoding mt proteins show neuromuscular defects, suggesting that the neuromuscular system is sensitive to perturbations in mt function. We are interested in studying the consequences of mt defects on neuromuscular function in Drosophila melanogaster. Loss-of-function mutations of Surf1 in humans cause Leigh Syndrome, which is one of the most common disorders of the mt respiratory chain. We generated three transgenic lines to produce dsRNAi knockdown (KD) of the Drosophila Surf1 homolog. KD individuals die during the late larval period. Confocal analysis shows that muscle fibers are smaller than in wild type. The NMJ shows immaturity. KD larvae show an altered light response. elav-gal4-driven dsRNA-KD Surf1 individuals show an altered optomotor response and ERG. In muscle fiber 6, evoked intracellular EJP, resting membrane potential, and frequency plus amplitude of MEPPS do not show significant variations. Three-day primary cultures of larval CNS neurons show smaller cell body size and reduced number of dendrites.