Heart development in Drosophila

The Drosophila heart, also called the dorsal vessel, is an organ for hemolymph circulation that resembles the vertebrate heart at its transient linear tube stage. Dorsal vessel morphogenesis shares several similarities with early events of vertebrate heart development and has proven to be an insightful system for the study of cardiogenesis due to its relatively simple structure and the productive use of Drosophila genetic approaches. In this review, we summarize published findings on Drosophila heart development in terms of the regulators and genetic pathways required for cardiac cell specification and differentiation, and organ formation and function. Emerging genome-based strategies should further facilitate the use of Drosophila as an advantageous system in which to identify previously unknown genes and regulatory networks essential for normal cardiac development and function.     

Drosophila models of human neurodegenerative disease

Drosophila has provided a powerful genetic system in which to elucidate fundamental cellular pathways in the context of a developing and functioning nervous system. Recently, Drosophila has been applied toward elucidating mechanisms of human neurodegenerative disease, including Alzheimer's, Parkinson's and Huntington's diseases. Drosophila allows study of the normal function of disease proteins, as well as study of effects of familial mutations upon targeted expression of human mutant forms in the fly. These studies have revealed new insight into the normal functions of such disease proteins, as well as provided models in Drosophila that will allow genetic approaches to be applied toward elucidating ways to prevent or delay toxic effects of such disease proteins. These, and studies to come that follow from the recently completed sequence of the Drosophila genome, underscore the contributions that Drosophila as a model genetic system stands to contribute toward the understanding of human neurodegenerative disease.     

A mighty small heart: the cardiac proteome of adult Drosophila melanogaster

Drosophila melanogaster is emerging as a powerful model system for the study of cardiac disease. Establishing peptide and protein maps of the Drosophila heart is central to implementation of protein network studies that will allow us to assess the hallmarks of Drosophila heart pathogenesis and gauge the degree of conservation with human disease mechanisms on a systems level. Using a gel-LC-MS/MS approach, we identified 1228 protein clusters from 145 dissected adult fly hearts. Contractile, cytostructural and mitochondrial proteins were most abundant consistent with electron micrographs of the Drosophila cardiac tube. Functional/Ontological enrichment analysis further showed that proteins involved in glycolysis, Ca(2+)-binding, redox, and G-protein signaling, among other processes, are also over-represented. Comparison with a mouse heart proteome revealed conservation at the level of molecular function, biological processes and cellular components. The subsisting peptidome encompassed 5169 distinct heart-associated peptides, of which 1293 (25%) had not been identified in a recent Drosophila peptide compendium. PeptideClassifier analysis was further used to map peptides to specific gene-models. 1872 peptides provide valuable information about protein isoform groups whereas a further 3112 uniquely identify specific protein isoforms and may be used as a heart-associated peptide resource for quantitative proteomic approaches based on multiple-reaction monitoring. In summary, identification of excitation-contraction protein landmarks, orthologues of proteins associated with cardiovascular defects, and conservation of protein ontologies, provides testimony to the heart-like character of the Drosophila cardiac tube and to the utility of proteomics as a complement to the power of genetics in this growing model of human heart disease.     

The Hox gene abdominal-A specifies heart cell fate in the Drosophila dorsal vessel

The Drosophila melanogaster dorsal vessel is a linear organ that pumps blood through the body. Blood enters the dorsal vessel in a posterior chamber termed the heart, and is pumped in an anterior direction through a region of the dorsal vessel termed the aorta. Although the genes that specify dorsal vessel cell fate are well understood, there is still much to be learned concerning how cell fate in this linear tube is determined in an anteroposterior manner, either in Drosophila or in any other animal. We demonstrate that the formation of a morphologically and molecularly distinct heart depends crucially upon the homeotic segmentation gene abdominal-A (abd-A). abd-A expression in the dorsal vessel was detected only in the heart, and overexpression of abd-A induced heart fate in the aorta in a cell-autonomous manner. Mutation of abd-A resulted in a loss of heart-specific markers. We also demonstrate that abd-A and sevenup co-expression in cardial cells defined the location of ostia, or inflow tracts. Other genes of the Bithorax Complex do not appear to participate in heart specification, although high level expression of Ultrabithorax is capable of inducing a partial heart fate in the aorta. These findings for the first time demonstrate a specific involvement for Hox genes in patterning the muscular circulatory system, and suggest a mechanism of broad relevance for animal heart patterning.     

AmphiNk2-tin,an amphioxus homeobox gene expressed in myocardial progenitors: insights into evolution of the vertebrate heart

We isolated a full-length cDNA clone of amphioxus AmphiNk2-tin, an NK2 gene similar in sequence to vertebrate NK2 cardiac genes, suggesting a potentially similar function to Drosophila tinman and to vertebrate NK2 cardiac genes during heart development. During the neurula stage of amphioxus, AmphiNk2-tin is expressed first within the foregut endoderm, then transiently in muscle precursor cells in the somites, and finally in some mesoderm cells of the visceral peritoneum arranged in an approximately midventral row running beneath the midgut and hindgut. The peritoneal cells that express AmphiNk2-tin are evidently precursors of the myocardium of the heart, which subsequently becomes morphologically detectable ventral to the gut. The amphioxus heart is a rostrocaudally extended tube consisting entirely of myocardial cells (at both the larval and adult stages); there are no chambers, valves, endocardium, epicardium, or other differentiated features of vertebrate hearts. Phylogenetic analysis of the AmphiNk2-tin sequence documents its close relationship to vertebrate NK2 class cardiac genes, and ancillary evidence suggests a relationship with the Drosophila NK2 gene tinman. Apparently, an amphioxus-like heart, and the developmental program directing its development, was the foundation upon which the vertebrate heart evolved by progressive modular innovations at the genetic and morphological levels of organization.     

Acoustic communication in Drosophila

Acoustic communication during courtship has been extensively studied in many Drosophila species. Here we summarise approaches that have been applied to the study of both song production and hearing. These approaches harnessed a variety of genetic tools available in Drosophila, such as isolation of song or hearing mutants, QTL mapping and transgenesis as well as electrophysiology and behavioural analysis. We also provide a short guide for the methodology used in acoustic studies in Drosophila and discuss prospects and new tools that would benefit future research.     

利用果蝇模型研究人类心脏早期发育的分子机理(英文)

近年来 ,果蝇心脏特化的遗传机制已初步研究清楚 ,但控制人类心脏早期发育的基因尚待鉴定。因为调控果蝇和脊椎动物早期心脏细胞命运定型的途径具有保守性 ,果蝇是一种探讨人类心脏早期发育的分子机理的理想动物模式。为此目的 ,我们采用P转座子和EMS诱变技术建立了约 3 0 0 0个隐性致死基因平衡系。通过心脏前体细胞特异性抗体免疫组化筛选 ,我们检出 2 0 0余个表现心脏突变表型的平衡致死系。我们进一步利用RNAi技术对一些基因的功能进行了初步的研究 ,证明这些基因表现RNAi的突变表型 ,该类突变表型与基因突变时表现的表型相似 ,即心管呈缺陷型或无心脏前体细胞形成。利用果蝇和人类基因组计划获得的成果 ,我们从果蝇心脏侯选基因中初步克隆和鉴定了 5 0个人类同源基因 ,其中 2 0个是新基因。Northen印迹分析表明 ,一部分人类基因在心脏组织中有表达 ,从而为研究这些基因在人类心脏早期发育中的作用提供了信息。目前 ,我们正在建立转基因果蝇 ,以此为模型研究这些基因是否对心肌细胞发生或心肌功能起调控作用。产生心肌细胞突变类型的基因如果类似于人类心脏病综合症 ,则可以作为人类心脏疾病侯选基因作进一步的分析。     

Drosophila with postponed aging as a model for aging research.

Species of the genus Drosophila, commonly known as "fruitflies," are good model systems for research in aging. Drosophila are extremely well-known genetically, developmentally, and otherwise. They are also genetically analogous to mammalian species in most important respects. Previous work with Drosophila has been hampered by inbreeding depression, but more recent work using selection has created Drosophila with postponed aging that is inherited normally. Genetic transformation has also increased Drosophila life spans in some cases. Several biologic approaches have been applied to the analysis of genetically postponed aging in Drosophila: quantitative genetics, organismal physiology, and protein electrophoresis. Ultimately, these different approaches will be integrated into an overall analysis of aging in Drosophila, one that could be valuable for research with other taxa as well.     

Measuring passive myocardial stiffness in Drosophila melanogaster to investigate diastolic dysfunction

Aging is marked by a decline in LV diastolic function, which encompasses abnormalities in diastolic relaxation, chamber filling and/or passive myocardial stiffness. Genetic tractability and short life span make Drosophila melanogaster an ideal organism to study the effects of aging on heart function, including senescent-associated changes in gene expression and in passive myocardial stiffness. However, use of the Drosophila heart tube to probe deterioration of diastolic performance is subject to at least two challenges: the extent of genetic homology to mammals and the ability to resolve mechanical properties of the bilayered fly heart, which consists of a ventral muscle layer that covers the contractile cardiomyocytes. Here, we argue for widespread use of Drosophila as a novel myocardial aging model by (1) describing diastolic dysfunction in flies, (2) discussing how critical pathways involved in dysfunction are conserved across species and (3) demonstrating the advantage of an atomic force microscopy-based analysis method to measure stiffness of the multilayered Drosophila heart tube versus isolated myocytes from other model systems. By using powerful Drosophila genetic tools, we aim to efficiently alter changes observed in factors that contribute to diastolic dysfunction to understand how one might improve diastolic performance at advanced ages in humans.     

利用果蝇心力衰竭模型筛选果蝇第2号染色体缺失系

果蝇心脏一直以来都是研究心血管发育的极好模型,许多控制心脏分化和特化的调控基因和信号途径从果蝇到哺乳动物都是保守的.由于近年心力衰竭的发病率不断升高,我们最近又建立了果蝇心力衰竭模型用于大规模筛选和鉴定心力衰竭的相关基因.在这个模型中,适龄的成体果蝇被整齐排列在导电的载玻片上,通过电极短暂刺激30s,使果蝇的心跳频率由正常的3Hz增加到6Hz,停止后检测果蝇心率恢复情况,不能恢复正常心跳频率或出现纤维性震颤的果蝇视为心力衰竭.该模型可以在短期内大规模筛选到与心力衰竭相关的基因.利用此心力衰竭模型,我们筛选了164个果蝇2号染色体缺失系,获得33个候选缺失系.这些候选缺失系的心衰率要么与野生型品系相比差异显著,要么与tinman或panier突变系相比差异显著,提示这些缺失系中可能含有与心力衰竭相关的调控基因.     

利用RNAi技术研究果蝇心脏发育基因的功能

RNAi是近两年发展起来的一种阻抑基因表达的新方法。它通过导入一段与内源基因同源的双链RNA序列（dsRNA）,使内源mRNA降解,从而达到阻抑基因表达的目的。目前已在线虫、果蝇、臭虫、真菌及植物等生物中建立RNAi技术,用于研究某些特定基因或已知基因在特定发育时期的功能。对于难于获得突变体的基因或生物体,RNAi技术尤其有效。虽然果蝇心脏发育基因wingless和tinman在果蝇心脏发育的早期功能已经清楚,它们都与果蝇心脏前体细胞的形成有关,但它们在果蝇心脏发育的后期功能仍有待进一步研究。实验运用RNAi技术,分别将tinman和wingless的dsRNA注入果蝇的早期胚胎,得到了这两个基因的dsRNA干扰表型,与两个基因的突变体表型非常相似,都表现为果蝇心脏前体细胞不能形成或心脏管缺失。尤其是tinman基因的dsRNA,还引起了肠中胚胎层缺失和体壁肌肉组织的紊乱,而wingless基因的dsRNA却只影响心脏的形成,而不影响肠中胚层,说明dsRNA干扰具有非常强的特异性,因而不失为研究果蝇心脏发育基因功能的有效方法。     

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     

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。     

On the Mechanics of Cardiac Function of Drosophila Embryo

The heart is a vital organ that provides essential circulation throughout the body. Malfunction of cardiac pumping, thus, leads to serious and most of the times, to fatal diseases. Mechanics of cardiac pumping is a complex process, and many experimental and theoretical approaches have been undertaken to understand this process. We have taken advantage of the simplicity of the embryonic heart of an invertebrate, Drosophila melanogaster, to understand the fundamental mechanics of the beating heart. We applied a live imaging technique to the beating embryonic heart combined with analytical imaging tools to study the dynamic mechanics of the pumping. Furthermore, we have identified one mutant line that exhibits aberrant pumping mechanics. The Drosophila embryonic heart consists of only 104 cardiac cells forming a simple straight tube that can be easily accessed for real-time imaging. Therefore, combined with the wealth of available genetic tools, the embryonic Drosophila heart may serve as a powerful model system for studies of human heart diseases, such as arrhythmia and congenital heart diseases. We, furthermore, believe our mechanistic data provides important information that is useful for our further understanding of the design of biological structure and function and for engineering the pumps for medical uses.     

Reduction of XNkx2-10 expression leads to anterior defects and malformation of the embryonic heart

Normal vertebrate heart development depends upon the expression of homeodomain containing proteins related to the Drosophila gene, tinman. In Xenopus laevis, three such genes have been identified in regions that will eventually give rise to the heart, XNkx2-3, XNkx2-5 and XNkx2-10. Although the expression domains of all three overlap in early development, distinctive differences have been noted. By the time the heart tube forms, there is little XNkx2-10 mRNA detected by in situ analysis in the embryonic heart while both XNkx2-3 and XNkx2-5 are clearly present. In addition, unlike XNkx2-3 and XNkx2-5, injection of XNkx2-10 mRNA does not increase the size of the embryonic heart. We have reexamined the expression and potential role of XNkx2-10 in development via oligonucleotide-mediated reduction of XNkx2-10 protein expression. We find that a decrease in XNkx2-10 leads to a broad spectrum of developmental abnormalities including a reduction in heart size. We conclude that XNkx2-10, like XNkx2-3 and XNkx2-5, is necessary for normal Xenopus heart development.     

Analysis of the expression pattern of Mysidium columbiae wingless provides evidence for conserved mesodermal and retinal patterning processes among insects and crustaceans

The Wnt family includes a number of genes, such as wingless ( wg), which encode secreted glycoproteins that function in numerous developmental patterning processes. In order to gain a better understanding of crustacean pattern formation, a wg orthologue was cloned from the malacostracan crustacean Mysidium columbiae(mysid), and the expression pattern of this gene was compared with that of Drosophila wg. Although Drosophila wg is expressed in many developing tissues, such as the ventral neuroectoderm, M. columbiae wg (mcowg)expression is detected within only a subset of these tissues. mcowg is expressed in the dorsal part of each developing segment and within the developing eye, but not within the ventral neuroectoderm. Dorsal wg expression in Drosophila is required for heart and muscle development, and conservation of this dorsal wgexpression pattern suggests that mcowgmay function to pattern these tissues in mysids. Consistent with this, expression of Even-skipped (Eve) protein in heart precursor and muscle cells, which is dependent on Wg signaling in Drosophila, is also conserved in mysids. Within the developing mysid eye, mcowg is expressed in a pattern that is similar to the expression pattern of Drosophila wg in the fly eye disc. In Drosophila,Wg inhibits neural differentiation at the anterior margin of the eye disc and patterns the dorsal/ventral axis of the eye. These data indicate that mcowg may function similarly during mysid eye development. Analysis of mcowgexpression provides molecular evidence suggesting that the processes of heart, muscle, and eye patterning are likely to be conserved among insects and crustaceans.     

A novel method for obtaining semi-thin cross sections of the Drosophila heart and their labeling with multiple antibodies

The Drosophila heart has become an exciting model for elucidating the molecular basis for cardiac function in higher organisms. To complement the genetic approaches that have recently identified an array of genes essential for cardiac function, we developed a method to obtain optimal semi-thin cross sections of embryonic, larval, and adult fly hearts in a desired orientation. A procedure for fluorescent labeling of these sections with multiple markers has also been developed, allowing the detection of proteins at high subcellular resolution. Sections obtained by our method reveal changes in cell shape between embryonic heart and aorta cardioblasts and elucidate the morphology of the adult heart. Analysis of the adult heart reveals the precise cardiac tube morphology, differential distribution of the extracellular matrix protein Laminin within the cardiac tube, as well as individual hand-positive, and Held Out Wings (HOW)-positive luminal cells that might represent blood cells. In summary, our method enables visualization of cross sections of the embryonic and adult hearts at high resolution while maintaining the ability to co-label the sections with multiple markers, thereby facilitating the analysis of cardiac tube formation and maintenance at different developmental stages.