The EGFR ligands Spitz and Keren act cooperatively in the Drosophila eye

The EGFR signalling cascade is responsible for coordinating a wide variety of events during Drosophila eye development. It remains something of a mystery how it is that cells are able to interpret the signal so as to choose the appropriate response from the battery of possibilities: division, differentiation, cell shape change and so on. Since the cascade is essentially linear below the receptor, different cellular responses cannot be regulated by alternative signal transduction pathways. The main diversity lies upstream, in the multiple activating ligands. Spitz, Gurken and Vein have been long studied, but little is known about the physiological functions of the fourth ligand, Keren, although various roles have been predicted based on the differences between mutants in the known ligands and those of the receptor. Here, we have isolated a mutant in the keren gene, and demonstrate that Keren does indeed participate in EGFR signalling in the eye, where it acts redundantly with Spitz to control R8 spacing, cell clustering and survival. Thus, specificity cannot be determined by ligand choice, and must instead be a consequence of cell-intrinsic factors, although we speculate that there may be some quantitative differences in signalling elicited by the two ligands.     

Differential requirement of EGFR signaling for the expression of defective proventriculus gene in the Drosophila endoderm and ectoderm

A homeobox gene, defective proventriculus (dve), is expressed in various tissues including the ventral ectoderm and midgut. Here, we show the expression pattern of dve in the ventral ectoderm, in which dve expression is induced by Spitz, a ligand for Drosophila epidermal growth factor receptor (EGFR). In spitz mutants, dve expression is only lost in the ventral ectoderm and overexpression of Spitz induces ectopic dve activation in the ventral ectoderm. Dve expression in the middle midgut depends on Decapentaplegic (Dpp) signaling, while expression of a dominant-negative form of Drosophila EGFR (DER(DN)) also causes a marked decrease in dve expression in the middle midgut. Furthermore, heterozygous mutation of thick veins (tkv), a Dpp receptor, strongly enhances the effect of DER(DN). These results indicate that EGFR signaling is crucial for dve expression in the ventral ectoderm and is required in the middle midgut where it cooperates with Dpp signaling.     

Hedgehog does not guide migrating Drosophila germ cells

In many species, the germ cells, precursors of sperm and egg, migrate during embryogenesis. The signals that regulate this migration are thus essential for fertility. In flies, lipid signals have been shown to affect germ cell guidance. In particular, the synthesis of geranylgeranyl pyrophosphate through the 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (Hmgcr) pathway is critical for attracting germ cells to their target tissue. In a genetic analysis of signaling pathways known to affect cell migration of other migratory cells, we failed to find a role for the Hedgehog (Hh) pathway in germ cell migration. However, previous reports had implicated Hh as a germ cell attractant in flies and suggested that Hh signaling is enhanced through the action of the Hmgcr pathway. We therefore repeated several critical experiments and carried out further experiments to test specifically whether Hh is a germ cell attractant in flies. In contrast to previously reported findings and consistent with findings in zebrafish our data do not support the notion that Hh has a direct role in the guidance of migrating germ cells in flies.     

Blockade of the release of the neuropeptide leucokinin to determine its possible functions in fly behavior: Chemoreception assays

Previous studies have revealed leucokinin (LK) expression in the brain and ventral ganglion of Drosophila CNS. One pair of protocerebrum neurons located in the lateral horn area (LHLK) surrounds the peduncles of the mushroom bodies while two pairs of subesophageal neurons (SELKs) project extended processes to the tritocerebrum and through a cervical connection to the ventral ganglion. There, axons of eight or nine pairs of abdominal (ABLK) neurons leave the CNS through the abdominal nerves and processes connecting each other ipsilaterally and contralaterally. The neural functions of LK remain largely unknown, especially those related to Drosophila behavior. Here, we have studied the role of LK in olfactory and gustatory perception by keeping the LK neurons electrically silent through targeted expression of inward rectifier K⁺ channels. In order to examine the effects of LK failure, we first analyzed the dehydration response, comparing the leucokinin-silent individuals with their parents as a control. Our results showed significant differences that demonstrate the effectiveness of the method. We then tested the olfactory behavioral response to a set of odorants over a range of concentrations in a T-maze paradigm in which flies were allowed to choose between the odorant and solvent compartments. The feeding preference assays were carried out on microplates in which flies were allowed to choose between two colored tastes. Our results show that the blockade of LK release alters both olfactory and gustatory responses, and are therefore evidence that this neuropeptide also modulates chemosensory responses through LHLK and SELK neurons.     

Circadian regulation of egg-laying behavior in fruit flies Drosophila melanogaster

Significant progress has been made in our understanding of the neurogenetics of circadian clocks in fruit flies Drosophila melanogaster. Several pacemaker neurons and clock genes have now been identified and their roles in the cellular and molecular clockwork established. Some recent findings suggest that the basic architecture of the clock is multi-oscillatory; the clock mechanisms in the ventral lateral neurons (LN(v)s) of the fly brain govern locomotor activity and adult emergence rhythms, while the peripheral oscillators located in antennal cells regulate olfactory rhythm. Among circadian phenomena exhibited by Drosophila, the egg-laying rhythm is unique in many ways: (i) this rhythm persists under constant light (LL), while locomotor activity and adult emergence become arrhythmic, (ii) its circadian periodicity is much longer than 24h, and (iii) while egg-laying is rhythmic under constant darkness, the expression of two core clock genes period (per) and timeless (tim), is non-oscillatory in the ovaries. In this paper, we review our current knowledge of the circadian regulation of egg-laying behavior in Drosophila, and provide some possible explanations for its self-sustained nature. We conclude by discussing the existing limitations in our understanding of the regulatory mechanisms and propose few approaches to address them.     

A new elongase selectively expressed in Drosophila male reproductive system

We have identified an elongase gene, elo68alpha, which is specifically transcribed in males. We have characterized the elo68alpha open reading frame, expressed it in fasDelta elo1Delta yeast and showed that it could elongate myristoleic and palmitoleic acids, therefore sharing an Elo1 specificity. This elongase was found to be exclusively expressed in male genital system (testis and ejaculatory bulb). Northern blot analysis showed that the elo68alpha gene was inducible at low temperatures. One P-strain mutant for elo68alpha and three excision lines for this P-element were subsequently studied. The excision line with only 1% elo68alpha expression showed decreased levels of vaccenyl acetate, a male pheromone produced in the ejaculatory bulb. The induction of elo68alpha expression at 21 degrees C was also paralleled with higher vaccenyl acetate production. These results strongly suggest that elo68alpha is involved in the elongation of short unsaturated fatty acids in males and might play a role in vaccenyl acetate biosynthesis.     

Lack of correlation between body mass and metabolic rate in Drosophila melanogaster

We examined the association between body mass and metabolic rate in Drosophila melanogaster under a variety of conditions. These included comparisons of body mass and metabolic rate in flies from different laboratory lines measured at different ages, over different metabolic sampling periods, and comparisons using wet versus dry mass data. In addition, the relationship between body mass and metabolic rate was determined for flies recently collected from wild populations. In no case was there a significant correlation between body mass and metabolic rate. These results indicate that care must be taken when attempting to account for the effects of body mass on metabolic rate. Expressing such data in mass-specific units may be an inappropriate method of attempting to control for the effects of differences in body mass.     

基于RNA-Seq数据识别果蝇剪接位点和可变剪接事件

完整基因结构的预测是当前生命科学研究的一个重要基础课题,其中一个关键环节是剪接位点和各种可变剪接事件的精确识别.基于转录组测序（RNA-seq）数据,识别剪接位点和可变剪接事件是近几年随着新一代测序技术发展起来的新技术策略和方法.本工作基于黑腹果蝇睾丸RNA-seq数据,使用TopHat软件成功识别出39718个果蝇剪接位点,其中有10584个新剪接位点.同时,基于剪接位点的不同组合,针对各类型可变剪接特征开发出计算识别算法,成功识别了8477个可变剪接事件（其中新识别的可变剪接事件3922个）,包括可变供体位点、可变受体位点、内含子保留和外显子缺失4种类型.RT-PCR实验验证了2个果蝇基因上新识别的可变剪接事件,发现了全新的剪接异构体.进一步表明,RNA-seq数据可有效应用于识别剪接位点和可变剪接事件,为深入揭示剪接机制及可变剪接生物学功能提供新思路和新手段.     

Identification of methylated sequences in genomic DNA of adult Drosophila melanogaster

The genome of Drosophila melanogaster contains methylated cytosines. Recent studies indicate that DNA methylation in the fruit fly depends on one DNA methyltransferase, dDNMT2. No obvious phenotype is associated with the downregulation of this DNA methyltransferase. Thus, identifying the target sequences methylated by dDNMT2 may constitute the first step towards understanding the biological functions of this enzyme. We used anti-5-methylcytosine antibodies as affinity column to identify the methylated sequences in the genome of adult flies. Our analysis demonstrates that components of retrotransposons and repetitive DNA sequences are putative substrates for dDNMT2. The methylation status of DNA encoding Gag, a protein involved in delivering the transposition template to its DNA target, was confirmed by sodium bisulfite sequencing.     

Identification of new immune induced molecules in the haemolymph of Drosophila melanogaster by 2D-nanoLC MS/MS

Antimicrobial peptides (AMPs) play an important role in the innate immunity of insects. In Drosophila 17 additional immune induced molecules (DIMs) were found in the haemolymph of adult flies upon septic injury. Previous studies using MALDI mass spectrometry combined with Edman degradation, detected AMPs and DIMs of a predominantly large size. By means of 2D-nanoLC ESI MS/MS, 43 DIMs were identified in this study from the haemolymph of Drosophila third instar larvae 12h after challenge with a mixture of Micrococcus luteus and Escherichia coli. Most peptides were derived from known AMP or DIM precursors, but only four peptides were purified and identified before. The majority of the peptides that we detected were smaller in size. Interestingly, two previously unknown peptide precursors were found and hereby related to immune defense. These include CG7738 and CG32185. Many of the identified peptides are post-translationally modified by an N-terminal pyroglutamic acid and/or a C-terminal amide. Haemolymph of control larvae was treated in the same way and revealed only one peptide.     

Antagonistic relationship between Dpp and EGFR signaling in Drosophila head patterning

The Drosophila eye field that gives rise to the visual system and dorsal head epidermis forms an unpaired anlage located in the dorsal head ectoderm. The eye field expresses and requires both Dpp and EGFR signaling for its development. As shown in previous studies, EGFR is required for cell maintenance in the developing visual system. Dpp initially switches on the early eye genes so and eya in the eye field. Consecutively, high levels of Dpp in the dorsal midline inhibit these genes and promote development of head epidermis. We show that Dpp negatively regulates EGFR signaling, thereby increasing the amount of cell death in the dorsal midline. By this mechanism, Dpp controls the formation of a bilateral visual system and indirectly modulates cell death, which is essential for normal head morphogenesis. Loss of either Dpp or its downstream target, Zen, abolishes head epidermis fate and leads to the misexpression of dp-ERK in the dorsal midline. The resulting morphological phenotype consists of cyclopia, reduction of cell death, and failure of head involution. Ectopic expression of activated EGFR inhibits the Dpp target race and thereby causes cyclopia and defective head involution. We discuss possible mechanisms of Dpp and EGFR interaction in the embryo.     

Wolbachia effects in Drosophila melanogaster: in search of fitness benefits

Insect endosymbionts often influence host nutrition but these effects have not been comprehensively investigated in Wolbachia endosymbionts that are widespread in insects. Using strains of Drosophila melanogaster with the wMel Wolbachia infection, we showed that Wolbachia did not influence adult starvation resistance. Wolbachia also had no effect on larval development time or the size of emerging adults from a low nutrition medium. While Wolbachia may influence the expression of heat shock proteins, we found that there was no effect on adult heat resistance when tested in terms of survival or virility following heat stress. The absence of nutrition or stress effects suggests that other processes maintain wMel frequencies in natural populations of Drosophila melanogaster.     

Oxygen-sensitive guanylyl cyclases in insects and their potential roles in oxygen detection and in feeding behaviors

Responses to hypoxia and hyperoxia depend critically on the ability of the animal to detect changes in O2 levels. However, it has only been recently that an O2-sensing system has been identified in invertebrates. Evidence is accumulating that this molecular O2 sensor is, surprisingly, a class of soluble guanylyl cyclase (sGC) known as atypical sGCs. It has long been known that the conventional sGC alpha and beta subunits form heterodimeric enzymes that are potently activated by NO, but do not bind O2. By contrast, the Drosophila melanogaster atypical sGC subunits, Gyc-88E, Gyc-89Da and Gyc-89Db, are only slightly sensitive to NO, but are potently activated under hypoxic conditions. Here we review evidence that suggests that the atypical sGCs can function as molecular O2 sensors mediating behavioral responses to hypoxia. Sequence comparisons of other predicted O2-sensitive sGCs suggest that most, if not all, insects express two heterodimeric sGCs; an NO-sensitive isoform and a separate O2-sensitive isoform. Expression data and recent experiments that block the function of cells that express the atypical sGCs and experiments that reduce the cGMP levels in these cells also suggest a role in behavioral responses to sweet tastants.     

Deoxyribonucleoside kinases belonging to the thymidine kinase 2 (TK2)-like group vary significantly in substrate specificity, kinetics and feed-back regulation.

In eukaryotic cells deoxyribonucleoside kinases belonging to three phylogenetic sub-families have been found: (i) thymidine kinase 1 (TK1)-like enzymes, which are strictly pyrimidine deoxyribonucleoside-specific kinases; (ii) TK2-like enzymes, which include pyrimidine deoxyribonucleoside kinases and a single multisubstrate kinase from Drosophila melanogaster (Dm-dNK); and (iii) deoxycytidine/deoxyguanosine kinase (dCK/dGK)-like enzymes, which are deoxycytidine and/or purine deoxyribonucleoside-specific kinases. We cloned and characterized two new deoxyribonucleoside kinases belonging to the TK2-like group from the insect Bombyx mori and the amphibian Xenopus laevis. The deoxyribonucleoside kinase from B. mori (Bm-dNK) turned out to be a multisubstrate kinase like Dm-dNK. But uniquely for a deoxyribonucleoside kinase, Bm-dNK displayed positive cooperativity with all four natural deoxyribonucleoside substrates. The deoxyribonucleoside kinase from X. laevis (Xen-PyK) resembled closely the human and mouse TK2 enzymes displaying their characteristic Michaelis-Menten kinetic with deoxycytidine and negative cooperativity with its second natural substrate thymidine. Bm-dNK, Dm-dNK and Xen-PyK were shown to be homodimers. Significant differences in the feedback inhibition by deoxyribonucleoside triphosphates between these three enzymes were found. The insect multisubstrate deoxyribonucleoside kinases Bm-dNK and Dm-dNK were only inhibited by thymidine triphosphate, while Xen-PyK was inhibited by thymidine and deoxycytidine triphosphate in a complex pattern depending on the deoxyribonucleoside substrate. The broad substrate specificity and different feedback regulation of the multisubstrate insect deoxyribonucleoside kinases may indicate that these enzymes have a different functional role than the other members of the TK2-like group.