利用果蝇模型研究人类心脏早期发育的分子机理

近年来,果蝇心脏转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型。为此目的,我们采用P转座子和EMS诱变技术建立了约3000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,我们选出200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50个人类同源基因,其中20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。     

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。     

果蝇心脏发育研究三十年进展

果蝇(Drosophila melanogaster)作为最早用于研究心脏发育基因调控的模式生物,已经走过三十年的历程。果蝇心脏发育过程经历了胚胎期、幼虫期和成虫期三大阶段。在胚胎早期, Tinman、Dorsocross和Pannier等基因是关键的调控因子。Tinman参与最早的心脏前体细胞分化和心脏细胞形成,而Dorsocross和Pannier则影响心脏前体细胞的定向分化和心脏管腔的形成。进入胚胎晚期和幼虫期,果蝇的心管经历进一步的发展和重塑,该过程主要受到转录因子Hand、Mef2以及Hox基因家族的调控。在成虫期, Hox基因家族和Tinman依旧发挥重要作用。虽然果蝇心脏与脊椎动物成熟心脏存在形态上的差异,但两者心脏的早期发育过程以及调控基因和信号通路都有保守性。本文综述了果蝇心脏发育基因调控研究的三十年进展以及利用果蝇模型研究人类心脏相关疾病的潜在希望。     

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。     

果蝇心脏发育基因调控的研究进展

果蝇心脏的发育是一个受到一系列基因共同调控的复杂过程,这些基因在脊椎动物和无脊椎动物果蝇中具有惊人的相似性,对于它们功能的研究将有助于揭示人类心脏发育的过程及分子控制机理.通过将果蝇作为一种重要的模式动物,对心脏发育基因调控的研究进展作一综述.     

果蝇新基因CG7609的克隆与初步功能研究

通过生物信息学方法和分子克隆技术克隆了一个果蝇新基因CG7609．该基因属于WD40家族,具有7个典型的WD40重复结构域．与人类WDR24基因的同源度很高．胚胎原位杂交和RT-PCR显示其在胚胎早期的中胚层有弱表达,在胚胎晚期主要在肠中表达．通过心脏特异抗体检测CG7609突变体的表型,发现其心脏前体细胞的分化不受影响．但通过果蝇心力衰竭模型分析该基因在成体心脏中的功能,发现CG7609基因缺失后心力衰竭发生率明显高于野生型,而且在与pannier基因配对后心力衰竭率发生较大的变化,这个初步发现推测该基因可能在戍体心脏中具有功能．     

果蝇昼夜节律的调控机制

40多年前的遗传筛选鉴定了第一个果蝇生物钟基因period,开启了果蝇生物钟调控机制的研究。随着更多生物钟基因被发现,一个由转录水平的调控及转录后水平的修饰组成的负反馈环路模型逐步形成,被认为是调控昼夜节律的核心分子机制。生物钟驱动果蝇脑内约150个神经元的活动,这些神经元在不同的环境条件下通过不同的方式互作,共同调控果蝇的行为节律。昼夜环境变化中最显著的是明暗变化。蓝光受体cryptochrome在光对昼夜节律的调控中起重要作用。     

缺失Df(3R)Espl3/TM6C基因片段影响黑腹果蝇的睡眠时间

【目的】果蝇的睡眠活动具有生物节律性, 可受到基因的调控。为了寻找影响果蝇睡眠时间的基因, 本研究对与果蝇睡眠时间相关的基因型进行了筛选。【方法】选择黑腹果蝇Drosophila melanogaster基因缺失系5601, 8904, 7061, 7146, 27327, 669, 8103, 691, 9697, 24416, 26525, 5411, 3096, 5877和7682的7日龄成虫和野生CS品系7日龄成虫为研究对象, 利用果蝇活动监测器系统（Drosophila Activity Monitoring System, DAMS）, 记录果蝇的睡眠时间, 累计计算24 h内果蝇睡眠时间, 将测得的各品系果蝇睡眠时间进行对比分析。【结果】与野生型CS品系7日龄成虫相比, 缺失Df(3R)Espl3/TM6C基因片段的 5601品系7日龄成虫睡眠时间明显缩短（P<0.001）。【结论】缺失Df(3R)Espl3/TM6C基因片段与果蝇睡眠有关。本研究结果为揭示影响果蝇睡眠时间的基因提供数据支持, 进而为研究人类睡眠提供线索。     

高脂饮食诱导果蝇心衰模型的建立

目的建立高脂饮食诱导的果蝇心衰模型。方法选择野生型果蝇w1118果蝇作为实验对象,采用含不同浓度椰油(0%,7.5%,15%,30%)食物喂食果蝇5 d,检测果蝇体内的三酰甘油含量。后续实验以非高脂喂食组(0%椰油)作为对照,30%椰油高脂喂食组作为实验组。负向趋地性实验检测果蝇运动能力。果蝇成体心脏功能分析平台,拍摄果蝇在半解剖条件下的心脏跳动情况(30 s),软件分析获得成体心脏功能指标,包括心跳周期,收缩和舒张直径,心脏收缩力指数等。实时定量PCR检测高脂喂食果蝇心脏的RNA表达变化。结果与非高脂喂食果蝇相比,不同比例高脂喂食果蝇的三酰甘油含量随椰油浓度呈剂量递增(P0.05);高脂饮食显著影响果蝇活动力(P0.001);高脂喂食果蝇的心跳周期缩短(P0.01),心脏收缩力减弱(P0.001);实时定量PCR发现高脂喂食果蝇中,参与脂代谢相关的一些基因fa2h,CG6277,CG3699,CG9914,lip2的表达明显下调(P0.05)。结论高脂饮食严重影响果蝇运动能力和成体心脏功能,成功建立了高脂饮食诱导的果蝇心衰模型。     

Deletion of gene fragment Df(3R)Espl3/TM6C affects sleep duration in Drosophila melanogaster

[目的]果蝇的睡眠活动具有生物节律性,可受到基因的调控.为了寻找影响果蝇睡眠时间的基因,本研究对与果蝇睡眠时间相关的基因型进行了筛选.[方法]选择黑腹果蝇Drosophila melanogaster基因缺失系5601,8904,7061,7146,27327,669,8103,691,9697,24416,26525,5411,3096,5877和7682的7日龄成虫和野生CS品系7日龄成虫为研究对象,利用果蝇活动监测器系统(Drosophila Activity Monitoring System,DAMS),记录果蝇的睡眠时间,累计计算24h内果蝇睡眠时间,将测得的各品系果蝇睡眠时间进行对比分析.[结果]与野生型CS品系7日龄成虫相比,缺失Df(3R)Espl3/TM6C基因片段的5601品系7日龄成虫睡眠时间明显缩短(P＜0.001).[结论]缺失Df(3R)Espl3/TM6C基因片段与果蝇睡眠有关.本研究结果为揭示影响果蝇睡眠时间的基因提供数据支持,进而为研究人类睡眠提供线索.     

钾离子通道蛋白Shaker对果蝇心脏衰老的保护作用

钾离子通道在心肌细胞动作电位复极过程中起着重要作用。钾离子通道蛋白种类繁多,已知钾离子通道蛋白KCNQ和HERG/eag参与心脏动作电位的形成,调节心脏收缩节律。钾离子通道蛋白Shaker是果蝇(Drosophila)体内发现的第一个电压门控钾离子通道,维持神经元和肌肉细胞的电兴奋性,但是目前其在成人心脏功能中的作用仍不清楚。本研究以果蝇为模型,高频电刺激模拟心脏应激状态,观察钾离子通道蛋白shaker基因突变体的心衰发生率。同时,利用心脏特异性启动子hand4.2Gal4特异性敲低钾离子通道蛋白Shaker的表达;果蝇成体心脏生理学功能分析系统分析了1、3、5周龄特异性敲低钾离子通道蛋白Shaker的心脏表型。结果表明,shaker基因突变将严重影响果蝇心脏抗应激能力,表现在高频电刺激后的心力衰竭发生率显著性升高;心脏特异性敲低shaker基因导致5周龄果蝇心律失常发生率显著性增加;心脏特异性敲低HDAC3将显著降低果蝇寿命。综上所述,本研究推测钾离子通道蛋白Shaker在衰老过程中维护果蝇正常的心脏功能。     

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.     

Dystrophin deficiency in Drosophila reduces lifespan and causes a dilated cardiomyopathy phenotype

A number of studies have been conducted recently on the model organism Drosophila to determine the function of genes involved in human disease, including those implicated in neurological disorders, cancer and metabolic and cardiovascular diseases. The simple structure and physiology of the Drosophila heart tube together with the available genetics provide a suitable in vivo assay system for studying cardiac gene functions. In our study, we focus on analysis of the role of dystrophin (Dys) in heart physiology. As in humans, the Drosophila dys gene encodes multiple isoforms, of which the large isoforms ( DLPs ) and a truncated form ( Dp117 ) are expressed in the adult heart. Here, we show that the loss of dys function in the heart leads to an age-dependent disruption of the myofibrillar organization within the myocardium as well as to alterations in cardiac performance. dys RNAi-mediated knockdown in the mesoderm also shortens lifespan. Knockdown of all or deletion of the large isoforms increases the heart rate by shortening the diastolic intervals (relaxation phase) of the cardiac cycle. Morphologically, loss of the large DLPs isoforms causes a widening of the cardiac tube and a lower fractional shortening, a phenotype reminiscent of dilated cardiomyopathy. The dilated dys mutant phenotype was reversed by expressing a truncated mammalian form of dys ( Dp116 ). Our results illustrate the utility of Drosophila as a model system to study dilated cardiomyopathy and other muscular-dystrophy-associated phenotypes.     

Homeotic selector genes control the patterning of seven-up expressing cells in the Drosophila dorsal vessel

The linear cardiac tube of Drosophila, the dorsal vessel, is an important model organ for the study of cardiac specification and patterning in vertebrates. In Drosophila, the Hox segmentation gene abdominal-A (abd-A) is required for the specification of a functionally distinct heart region at the posterior of the dorsal vessel, from which blood is pumped anteriorly through a tube termed the aorta. Since we have previously shown that the posterior part of the aorta is specified during embryogenesis to form the adult heart during metamorphosis, we determined if the embryonic aorta is also patterned by the function of Hox segmentation genes. Using gain- and loss-of-function experiments, we demonstrate that the three Hox genes expressed in the posterior aorta and heart are sufficient to confer heart or posterior aorta fate throughout the dorsal vessel. Additionally, we demonstrate that Ultrabithorax and abd-A, but not Antennapedia, function to control cell number in the dorsal vessel. These studies add robustness to the model that homeotic selector genes pattern the Drosophila dorsal vessel, and further extend our understanding of how the cardiac tube is patterned in animal models.     

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.     

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.     

Cardiac remodeling in Drosophila arises from changes in actin gene expression and from a contribution of lymph gland-like cells to the heart musculature

Understanding the basis of normal heart remodeling can provide insight into the plasticity of the cardiac state, and into the potential for treating diseased tissue. In Drosophila, the adult heart arises during metamorphosis from a series of events, that include the remodeling of an existing cardiac tube, the elaboration of new inflow tracts, and the addition of a layer of longitudinal muscle fibers. We have identified genes active in all these three processes, and studied their expression in order to characterize in greater detail normal cardiac remodeling. Using a Transglutaminase-lacZ transgenic line, that is expressed in the inflow tracts of the larval and adult heart, we confirm the existence of five inflow tracts in the adult structure. In addition, expression of the Actin87E actin gene is initiated in the remodeling cardiac tube, but not in the longitudinal fibers, and we have identified an Act87E promoter fragment that recapitulates this switch in expression. We also establish that the longitudinal fibers are multinucleated, characterizing these cells as specialized skeletal muscles. Furthermore, we have defined the origin of the longitudinal fibers, as a subset of lymph gland cells associated with the larval dorsal vessel. These studies underline the myriad contributors to the formation of the adult Drosophila heart, and provide new molecular insights into the development of this complex organ.