Cellular mechanisms governing synaptic development in Drosophila melanogaster

The neuromuscular connections of Drosophila are ideally suited for studying synaptic function and development. Hypotheses about cell recognition can be tested in a simple array of pre-and postsynaptic elements. Drosophila muscle fibers are multiply innervated by individually identifiable motoneurons. The neurons express several synaptic cotransmitters, including glutamate, proctolin, and octopamine, and are specialized by their synaptic morphology, neurotransmitters, and connectivity. During larval development the initial motoneuron endings grow extensively over the surface of the muscle fibers, and differentiate synaptic boutons of characteristic morphology. While considerable growth occurs postembryonically, the initial wiring of motoneurons to muscle fibers is accomplished during mid-to-late embryogenesis (stages 15–17). Efferent growth cones sample multiple muscle fibers with rapidly moving filopodia. Upon reaching their target muscle fibers, the growth cones rapidly differentiate into synaptic contacts whose morphology prefigures that of the larval junction. Mismatch experiments show that growth cones recognize specific muscle fibers, and can do so when the surrounding musculature is radically altered. However, when denied their normal targets, motoneurons can establish functional synapses on alternate muscle fibers. Blocking synaptic activity with either injected toxins or ion channel mutants does not derange synaptogenesis, but may influence the number of motor ending processes. The molecular mechanisms governing cellular recognition during synaptogenesis remain to be identified. However, several cell surface glycoproteins known to mediate cellular adhesion events in vitro are expressed by the developing synapses. Furthermore, enhancer detector lines have identified genes with expression restricted to small subsets of muscle fibers and /or motoneurons during the period of synaptogenesis. These observations suggest that in Drosophila a mechanism of target chemoaffinity may be involved in the genesis of stereotypic synaptic wiring. © 1993 John Wiley & Sons, Inc.     

The translation factors of Drosophila melanogaster

Synthesis of polypeptides from mRNA (translation) is a fundamental cellular process that is coordinated and catalyzed by a set of canonical ‘translation factors’. Surprisingly, the translation factors of Drosophila melanogaster have not yet been systematically identified, leading to inconsistencies in their nomenclature and shortcomings in functional (Gene Ontology, GO) annotations. Here, we describe the complete set of translation factors in D. melanogaster, applying nomenclature already in widespread use in other species, and revising their functional annotation. The collection comprises 43 initiation factors, 12 elongation factors, 3 release factors and 6 recycling factors, totaling 64 of which 55 are cytoplasmic and 9 are mitochondrial. We also provide an overview of notable findings and particular insights derived from Drosophila about these factors. This catalog, together with the incorporation of the improved nomenclature and GO annotation into FlyBase, will greatly facilitate access to information about the functional roles of these important proteins.     

Genotoxicity of lapachol evaluated by wing spot test of Drosophila melanogaster

This study investigated the genotoxicity of Lapachol (LAP) evaluated by wing spot test of Drosophila melanogaster in the descendants from standard (ST) and high bioactivation (HB) crosses. This assay detects the loss of heterozygosity of marker genes expressed phenotypically on the fly's wings. Drosophila has extensive genetic homology to mammals, which makes it a suitable model organism for genotoxic investigations. Three-day-old larvae from ST crosses (females flr(3)/TM3, Bd(s) x males mwh/mwh), with basal levels of the cytochrome P450 and larvae of high metabolic bioactivity capacity (HB cross) (females ORR; flr(3)/TM3, Bd(s) x males mwh/mwh), were used. The results showed that LAP is a promutagen, exhibiting genotoxic activity in larvae from the HB cross. In other words, an increase in the frequency of spots is exclusive of individuals with a high level of the cytochrome P450. The results also indicate that recombinogenicity is the main genotoxic event induced by LAP.     

CNBP mediates neural crest cell expansion by controlling cell proliferation and cell survival during rostral head development

Striking conservation in various organisms suggests that cellular nucleic acid binding protein (CNBP) plays a fundamental biological role across different species. Recently, it was reported that CNBP is required for forebrain formation during chick and mouse embryogenesis. In this study, we have used the zebrafish model system to expand and contextualize the basic understanding of the molecular mechanisms of CNBP activity during vertebrate head development. We show that zebrafish cnbp is expressed in the anterior CNS in a similar fashion as has been observed in early chick and mouse embryos. Using antisense morpholino oligonucleotide knockdown assays, we show that CNBP depletion causes forebrain truncation while trunk development appears normal. A substantial reduction in cell proliferation and an increase in cell death were observed in the anterior regions of cnbp morphant embryos, mainly within the cnbp expression territory. In situ hybridization assays show that CNBP depletion does not affect CNS patterning while it does cause depletion of neural crest derivatives. Our data suggest an essential role for CNBP in mediating neural crest expansion by controlling proliferation and cell survival rather than via a cell fate switch during rostral head development. This possible role of CNBP may not only explain the craniofacial anomalies observed in zebrafish but also those reported for mice and chicken and, moreover, demonstrates that CNBP plays an essential and conserved role during vertebrate head development.     

Biosynthesis and metabolism of sulfated glycosaminoglycans during Drosophila melanogaster development

We developed a simple methodology for labeling sulfated glycosaminoglycans (GAGs) in adult Drosophila melanogaster and studied some aspects of the biosynthesis and metabolism of these polymers during development. Adult D. melanogaster flies were fed with Na(2)(35)SO(4) for 72 h. During this period, (35)S-sulfate was incorporated into males and females and used to synthesize (35)S-sulfate-heparan sulfate (HS) and (35)S-sulfate-chondroitin sulfate (CS). The incorporation of (35)S-sulfate into HS was higher when compared to CS. In a pulse-chase experiment, we observed that (35)S-sulfate incorporated into adult female was recovered in embryos and used for the synthesis of new (35)S-sulfate-GAGs after 2 h of embryonic development. The synthesis of CS was higher than that of HS, indicating a change in the metabolism of these glycans from adult to embryonic and larval stages. Analysis of the CS in embryonic and larval tissues revealed the occurrence of nonsulfated and 4-sulfated disaccharide units in embryos, L1 and L2. In L3, in addition to these disaccharides, we also detected significant amount of 6-sulfated units that are reported here for the first time. Immunohistochemical analysis indicated that HS and CS were present in nonequivalent structures in adult and larval stages of the fly. Overall, these results indicate that (35)S-sulfate-precursors are transferred from adult to embryonic and larval tissues and used to assemble different morphological structures during development.     

Membrane potential regulates Hedgehog signalling in the Drosophila wing imaginal disc

While the membrane potential of cells has been shown to be patterned in some tissues, specific roles for membrane potential in regulating signalling pathways that function during development are still being established. In the Drosophila wing imaginal disc, Hedgehog (Hh) from posterior cells activates a signalling pathway in anterior cells near the boundary which is necessary for boundary maintenance. Here, we show that membrane potential is patterned in the wing disc. Anterior cells near the boundary, where Hh signalling is most active, are more depolarized than posterior cells across the boundary. Elevated expression of the ENaC channel Ripped Pocket (Rpk), observed in these anterior cells, requires Hh. Antagonizing Rpk reduces depolarization and Hh signal transduction. Using genetic and optogenetic manipulations, in both the wing disc and the salivary gland, we show that membrane depolarization promotes membrane localization of Smoothened and augments Hh signalling, independently of Patched. Thus, membrane depolarization and Hh‐dependent signalling mutually reinforce each other in cells immediately anterior to the compartment boundary.     

Profiling catalase gene expression in Drosophila melanogaster during development and aging

Catalase represents one of the key antioxidant enzymes (AOE) in the metabolism of oxygen free radicals. A comprehensive analysis was brought to bear on establishing catalase gene expression profiles during development and aging, with the underlying objective being to identify potential regulatory factors. Expression of the catalase gene exhibits substantial variations during development and aging in a stage- and tissue-specific manner. At the temporal level, previous observations of the coincidence of ecdysteroid pulses with peaks in catalase expression during developmental stages were largely corroborated. In adults, a small but significant decline in catalase expression was noted in adults as a function of age. Spatially, it was ascertained that catalase expression is mostly confined to tissues related to intermediary metabolism, digestive and adipose systems as well as oenocytes. By combining histochemical analysis of reporter gene expression with immunostaining of the endogenous product, it was possible to identify putative positive and negative regulatory elements that control catalase expression. Finally, when adult flies were subjected to various environmental insults, such as heat, paraquat, hyperoxia and H(2)O(2), no significant responses were observed, suggesting that catalase gene expression is largely governed by intrinsic genetic programs.     

The dimensionality of genetic variation for wing shape in Drosophila melanogaster

Absolute constraints are limitations on genetic variation that preclude evolutionary change in some aspect of the phenotype. Absolute constraints may reflect complete absence of variation, lack of genetic variation that extends the range of phenotypes beyond some limit, or lack of additive genetic variation. This last type of absolute constraint is bidirectional, because the mean cannot evolve to be larger or smaller. Most traits do possess genetic variation, so bidirectional absolute constraints are most likely to be detected in a multivariate context, where they would reflect combinations of traits, or dimensions in phenotype space that cannot evolve. A bidirectional absolute constraint will cause the additive genetic covariance matrix (G) to have a rank less than the number of traits studied. In this study, we estimate the rank of the G-matrix for 20 aspects of wing shape in Drosophila melanogaster. Our best estimates of matrix rank are 20 in both sexes. Lower 95% confidence intervals of rank are 17 for females and 18 for males. We therefore find little evidence of bidirectional absolute constraints. We discuss the importance of this result for resolving the relative roles of selection and drift processes versus constraints in the evolution of wing shape in Drosophila.     

Sexual conflict in wing size and shape in Drosophila melanogaster

Intralocus sexual conflict occurs when opposing selection pressures operate on loci expressed in both sexes, constraining the evolution of sexual dimorphism and displacing one or both sexes from their optimum. We eliminated intralocus conflict in Drosophila melanogaster by limiting transmission of all major chromosomes to males, thereby allowing them to win the intersexual tug‐of‐war. Here, we show that this male‐limited (ML) evolution treatment led to the evolution (in both sexes) of masculinized wing morphology, body size, growth rate, wing loading, and allometry. In addition to more male‐like size and shape, ML evolution resulted in an increase in developmental stability for males. However, females expressing ML chromosomes were less developmentally stable, suggesting that being ontogenetically more male‐like was disruptive to development. We suggest that sexual selection over size and shape of the imago may therefore explain the persistence of substantial genetic variation in these characters and the ontogenetic processes underlying them.     

产气荚膜梭菌促进黑腹果蝇的生长和发育

【目的】黑腹果蝇Drosophila melanogaster肠道中栖生着众多微生物,通过分离和研究其内共生菌,以研究肠道菌群的多态性和作用。【方法】利用Hungate滚管技术从黑腹果蝇成虫肠道分离厌氧细菌;通过记录果蝇的发育历期和生长速率,检测该细菌对果蝇发育和生长的影响。【结果】首次从黑腹果蝇肠道内分离到一株产气荚膜梭菌Clostridium perfringens。该菌能够有效地定植到果蝇肠道内,是果蝇肠道共生菌。产气荚膜梭菌显著地缩短无菌果蝇的发育历期,将无菌果蝇成蛹天数由20 d缩短到8.1 d,羽化天数由30 d缩短到12.7 d。该菌还可以提高果蝇生长速率。【结论】本研究揭示了产气荚膜梭菌是果蝇的内共生菌,可以通过提高生长速率而有效地促进果蝇的生长和发育。     

Stage-specific regulation of actin genes in Drosophila wing cells

Extreme and rapid changes in the synthesis of messenger RNAs and proteins accompany differentiation in wing tissues of Drosophila. Of the six actin genes, at least three are expressed in wing cells, some during the most extreme changes in cell shape. However, different messages of the set appear, decay, and reappear on a regulated temporal program. These results show that actin expression is stage-specific in a single cell type.     

Proteomic analysis of the wing imaginal discs of Drosophila melanogaster

We have combined high-resolution two-dimensional (2-D) gel electrophoresis and mass spectrometry with the aim of identifying proteins represented in the 2-D gel database of the wing imaginal discs of Drosophila melanogaster. First, we obtained a high-resolution 2-D gel pattern of [35S]methionine + [35S]cysteine-labeled polypeptides of Schneider cells, a permanent cell line of Drosophila embryonic origin, and compared it with the standard pattern of polypeptides of the wing imaginal disc. These studies reveal qualitative and quantitative differences between the two samples, but have more than 600 polypeptides in common. Second, we carried out preparative 2-D polyacrylamide gel electrophoresis using Schneider cells mixed with radioactively labeled wing imaginal discs in order to isolate some of the shared polypeptides and characterize them by matrix-assisted laser desorption/ionization-time of flight MALDI-TOF analysis. Using this strategy we identified 100 shared proteins represented in the database, and in each case confirmed their identity by MALDI-TOF/TOF analysis.     

A mutation affecting larval muscle development in Drosophila melanogaster

The phenotypic analysis of a new spontaneous recessive lethal mutation of Drosophila melanogaster is described. The lethal(2)thin mutation maps at 85.6 on chromosome 2 and produces a characteristic long, thin puparium due to an inability to shorten the larval form prior to pupariation. Histological examination of larval muscles and behavioural studies support the hypothesis that the mutation affects the striated structure of the larval muscles in late larval stages. Lethality largely occurs due to an inability to perform the movements necessary for pupation, although there is evidence for larval and possibly embryonic lethal phases.     

Genes required for embryonic muscle development in Drosophila melanogaster A survey of the X chromosome

We have begun a genetic analysis to dissect the process of myogenesis by surveying the X chromosome of Drosophila melanogaster for mutations that affect embryonic muscle development. Using polarised light microscopy and antibody staining techniques we analysed embryos hemizygous for a series of 67 deletion mutations that together cover an estimated 85% of the X chromosome, or 16.5% of the genome. Whereas the mature wild type embryo has a regular array of contractile muscles that insert into the epidermis, 31 of the deletion mutants have defects in muscle pattern, contractility or both, that cannot be attributed simply to epidermal defects and identify functions required for wild type muscle development. We have defined mutant pattern phenotypes that can be described in terms of muscle absences, incomplete myoblast fusion, failure of attachment of the muscle to the epidermis or mispositioning of attachment sites. Thus muscle development can be mutationally disrupted in characteristic and interpretable ways. The areas of overlap of the 31 deletions define 19 regions of the X chromosome that include genes whose products are essential for various aspects of myogenesis. We conclude that our screen can usefully identify loci coding for gene products essential in muscle development.     

异色瓢虫捕食胁迫对黑腹果蝇繁殖与后代发育的影响水

以模式昆虫黑腹果蝇(Drosophila melanogaster)及捕食性天敌异色瓢虫(Harmonia axyridis)为研究系统,通过把异色瓢虫接入到有黑腹果蝇的指形管中,研究了黑腹果蝇雌、雄成虫单独或共同被异色瓢虫捕食胁迫后,其自身寿命、子代的生长发育、繁殖和适合度的变化。结果表明:当雄性黑腹果蝇受到异色瓢虫成虫胁迫时,其寿命明显延长,但胁迫与否对雌性黑腹果蝇的寿命无明显影响;当黑腹果蝇的雌雄成虫同时受到胁迫时,其子代幼虫的发育历期延长,雄性后代寿命也增加,而雌性或雄性黑腹果蝇单独受到胁迫,其子代幼虫发育进度没有明显改变;当初孵黑腹果蝇幼虫受到异色瓢虫成虫直接或间接捕食胁迫后,其后续幼虫的发育历期都会受到影响,其中1龄期受到的间接捕食胁迫作用的影响大于直接捕食胁迫作用,直接捕食胁迫1龄幼虫比间接捕食胁迫更能延长其将来雌性成虫的寿命。