Fluorescent Labeling of Drosophila Heart Structures

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells that align along each side of the heart wall, supportive alary muscles and, in adults, a layer of ventral longitudinal muscle cells. The contractile fibers house conserved constituents of the muscle cytoarchitecture including densely packed bundles of myofibrils and cytoskeletal/submembranous protein complexes, which interact with homologous components of the extracellular matrix. Here we describe a protocol for the fixation and the fluorescent labeling of particular myocardial elements from the hearts of dissected larvae and semi-intact adult Drosophila. Specifically, we demonstrate the labeling of sarcomeric F-actin and of α-actinin in larval hearts. Additionally, we perform labeling of F-actin and α-actinin in myosin-GFP expressing adult flies and of α-actinin and pericardin, a type IV extracellular matrix collagen, in wild type adult hearts. Particular attention is given to a mounting strategy for semi-intact adult hearts that minimizes handling and optimizes the opportunity for maintaining the integrity of the cardiac tubes and the associated tissues. These preparations are suitable for imaging via fluorescent and confocal microscopy. Overall, this procedure allows for careful and detailed analysis of the structural characteristics of the heart from a powerful genetically tractable model system.Download video file.^{(130M, mp4)}     

Visual circuit development in Drosophila

Fly visual circuits are organized into lattice-like arrays and layers. Recent genetic studies have provided insights into how these reiterated structures are assembled through stepwise processes and how precise connections are established during development. Afferent-derived morphogens, such as Hedgehog, play a key role in organizing the overall structure by inducing and recruiting target neurons and glia. In turn, the target-derived ligand DWnt4 guides Frizzled2-expressing photoreceptor afferents to their proper destination. Photoreceptor afferents select specific synaptic targets by forming adhesive interactions and regulating actin cytoskeleton in growth cones. Target specificity is probably achieved by restricting the expression of adhesive molecules, such as Capricious, to appropriate presynaptic and postsynaptic partners, and by differentially regulating the function of broadly expressed adhesive molecules such as N-cadherin.     

Surviving Drosophila eye development

During eye development, cell death interplays dynamically with events of differentiation to achieve the remarkably patterned structure of the fly compound eye. Mutations in genes that affect the normal developmental process can lead to excessive death of progenitor cells, or, alternatively, to the differentiation of supernumerary neurons, pigment and cone cells due to survival of cells that would normally be eliminated. These data reveal that eye development contains cell selection processes: only certain cells are selected to undergo differentiation, and supernumerary cells are actively eliminated by cell death pathways to achieve the highly ordered lattice of the eye. The final number of cells that comprise the eye is controlled through a balance of cell proliferation with proper cell differentiation and removal by cell death.     

Compartments and appendage development in Drosophila

The appendages of Drosophila develop from the imaginal discs. During the extensive growth of these discs cell lineages are for the most part unfixed, suggesting a strong role for cell-cell interactions in controlling the final pattern of differentiation. However, during early and middle stages of development, discs are subdivided by strict lineage restrictions into a small number of spatially distinct compartments. These compartments appear to be maintained by stably inheriting states of gene expression; the compartmentspecific expression of two such ‘selector’ - like genes, engrailed and apterous, are critical for anterior-posterior and dorso-ventral compartmentalization, respectively. Recent work suggests that one purpose of compartmentalization is to establish regions of specialized cells near compartment boundaries via intercompartmental induction, using molecules like the hedgehog protein. Thus, compartments can act as organizing centers for patterning within compartments. Evidence for non-compartmental patterning mechanisms will also be discussed.     

Salivary gland development in Drosophila melanogaster

The Drosophila salivary gland is proving to be an excellent experimental system for understanding how cells commit to specific developmental programs and, once committed, how cells implement such decisions. Through genetic studies, the factors that determine where salivary glands will form, the number of cells committed to a salivary gland fate, and the distinction between the two major cell types (secretory cells and duct cells) have been discovered. Within the next few years, we will learn the molecular details of the interactions among the salivary gland regulators and salivary gland target genes. We will also learn how the early-expressed salivary gland genes coordinate their activities to mediate the morphogenetic movements required to form the salivary gland and the changes in cell physiology required for high secretory activity.     

Neuronal development in the Drosophila retina

Nervous systems of higher organisms are comprised of a variety of cell types which are interconnected in a precise manner. The molecular mechanisms that lead to the specification of neuronal cell types are not well understood. The compound eye of the fruit fly Drosophila is an attractive experimental system to understand these mechanism. The compound eye is a reiterated neural pattern with several hundred unit structures and is amenable to both classical and molecular genetic methods. During the development of the compound eye cell–cell interactions and positional information play a critical role in the determination of cell fate. Recent genetic and molecular studies have provided important clues regarding the nature of the molecules involved in cellular signalling and neuronal differentiation. © 1993 John Wiley & Sons, Inc.     

Autophagy promotes synapse development in Drosophila

Autophagy, a lysosome-dependent degradation mechanism, mediates many biological processes, including cellular stress responses and neuroprotection. In this study, we demonstrate that autophagy positively regulates development of the Drosophila melanogaster larval neuromuscular junction (NMJ). Autophagy induces an NMJ overgrowth phenotype closely resembling that of highwire (hiw), an E3 ubiquitin ligase mutant. Moreover, like hiw, autophagy-induced NMJ overgrowth is suppressed by wallenda (wnd) and by a dominant-negative c-Jun NH₂-terminal kinase (bsk^DN). We show that autophagy promotes NMJ growth by reducing Hiw levels. Thus, autophagy and the ubiquitin–proteasome system converge in regulating synaptic development. Because autophagy is triggered in response to many environmental cues, our findings suggest that it is perfectly positioned to link environmental conditions with synaptic growth and plasticity.     

影响果蝇心脏发育的基因突变

最近的研究表明,果蝇与脊椎动物及人的心脏早期发育具有极为相似的基因控制机理,果蝇已成为研究人体心脏早期发育基因控制的理想模式动物。利用化学诱变剂甲磺酸乙酯大规模地诱变影响果蝇心脏发育的基因,利用心脏特异性抗体染色进行筛选,获得了112个有心脏突变表型的致死系,其中32个致死系的心脏畸变表型有别于目前已知心脏发育基因的突变表型。细胞遗传学定位研究表明在多线染色体的13个带纹区的某些隐性致死突变基因是目前未知的,其功能可能与发育有关的基因。     

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained and restrained whole larvae. For direct control of the environment around the heart another approach utilizes the dissected larvae and removal of the internal organs in order to bathe the heart in desired compounds. The exposed heart also allows membrane potentials to be monitored which can give insight of the ionic currents generated by the myocytes and for electrical conduction along the heart tube. These approaches have various advantages and disadvantages for future experiments that are discussed. The larval heart preparation provides an additional model besides the Drosophila skeletal NMJ to investigate the role of intracellular calcium regulation on cellular function. Learning more about the underlying ionic currents that shape the action potentials in myocytes in various species, one can hope to get a handle on the known ionic dysfunctions associated to specific genes responsible for various diseases in mammals.     

果蝇心脏发育起调控作用的候选基因的筛选

果蝇的早期心脏发育与脊椎动物的早期发育模式具有惊人的相似,所以果蝇成为研究脊椎动物心脏发育的模式动物,通过对其心脏发育基因的研究,可加速揭示人体心脏的发育机理。为进一步筛选并克隆出新的心脏发育基因,本实验采用经化学诱变的平衡致死系的果蝇,进行心脏特异性抗体染色,观察到10个致死系表现出心脏突变表型,并将已确定遗传学部位的6个品系缩小到更小区域。 Screening of the Genes in Controlling HeartDevelopment of Drosophila YANG Yue-jun,WU Xiu-shan,LI Min College of life sciences,Hunan Normal University,Changsha 410081,China Abstract:It is becoming increasingly evident that remarkable similaries of heart development are revealed in Drosophila and vertebrate,Therefore Drosophila can be used as a prototype to explore the vertebrate.This can in accelerate to revealing of the machanisms of human heart development.In order to screen and clone new genes that control the heart development,we have established the balanced-lethal lines by chemical mutagen and performed the heart-specific antibody.Ten of lines showed mutant phenotype,of which 6 were determined the smaller genetic sites for gene location. Key words：Drosophila; heart develop; genes     

Chitinase activity during Drosophila development

Before both larval moults in Drosophila melanogaster, the chitin in the cuticle is digested to a significant degree by the moulting fluid. A spurt of chitinase activity appears just before each ecdysis, drops sharply after the first ecdysis, and begins to rise again just about the time that chitin degradation becomes evident. The level of enzyme activity/mg of soluble protein reached just before the second ecdysis is about twice that reached before the first, and this declines gradually after the ecdysis until puparium formation. Chitinase activity is measured with a viscometric assay on a chitosan substrate.The enzyme activity is stable, with no loosely bound cofactor. Data also exist supporting the presence of more than one enzyme fraction in Drosophila with chitinase activity.     

Nitric oxide and Drosophila development

Mechanisms controlling the transition of precursor cells from proliferation to differentiation during organism development determine the distinct anatomical features of tissues and organs. NO may mediate such a transition since it can suppress DNA synthesis and cell proliferation. Inhibition of NOS activity in the imaginal discs of Drosophila larvae results in hypertrophy of tissues and organs of the adult fly, whereas ectopic overexpression of NOS has the reciprocal, hypotrophic, effect. Furthermore, NO production is crucial for the establishment of ordered neuronal connections in the visual system of the fly, indicating that NO affects the acquisition of the differentiated phenotype by the neural tissue. Increasing evidence points to a broad role that NO may play in animal development by acting as an essential negative regulator of precursor cell proliferation during tissue and organ morphogenesis.     

Heparan sulfate proteoglycans in Drosophila neuromuscular development

Heparan sulfate proteoglycans (HSPGs) are glycoconjugates bearing heparan sulfate (HS) chains covalently attached to core proteins, which are ubiquitously distributed on the cell surface and in the extracellular matrix. HSPGs interact with a number of molecules mainly through HS chains, which play critical roles in diverse physiological and disease processes. Among these, recent vertebrate studies showed that HSPGs are closely involved in synapse development and function. However, the detailed molecular mechanisms remain elusive. Genetic studies from fruit flies, Drosophila melanogaster, have begun to reveal the molecular mechanisms by which HSPGs regulate synapse formation at neuromuscular junctions (NMJs). In this review, we introduce Drosophila studies showing how HSPGs regulate various signaling pathways in developing NMJs. This article is part of a Special Issue entitled Neuro-glycoscience, edited by Kenji Kadomatsu and Hiroshi Kitagawa.