Fat & fabulous: Bifunctional lipids in the spotlight

Understanding biological processes at the mechanistic level requires a systematic charting of the physical and functional links between all cellular components. While protein–protein and protein–nucleic acid networks have been subject to many global surveys, other critical cellular components such as membrane lipids have rarely been studied in large-scale interaction screens. Here, we review the development of photoactivatable and clickable lipid analogues–so-called bifunctional lipids–as novel chemical tools that enable a global profiling of lipid–protein interactions in biological membranes. Recent studies indicate that bifunctional lipids hold great promise in systematic efforts to dissect the elaborate crosstalk between proteins and lipids in live cells and organisms. This article is part of a Special Issue entitled Tools to study lipid functions.     

Embryonic origin and differentiation of the Drosophila heart

We have followed the normal development of the different cell types associated with the Drosophila dorsal vessel, i.e. cardioblasts, pericardial cells, alary muscles, lymph gland and ring gland, by using several tissue-specific markers and transmission electron microscopy. Precursors of pericardial cells and cardioblasts split as two longitudinal rows of cells from the lateral mesoderm of segments T2-A7 (cardiogenic region) during stage 12. The lymph gland and dorsal part of the ring gland (corpus allatum) originate from clusters of lateral mesodermal cells located in T3 and T1/dorsal ridge, respectively. Cardioblast precursors are strictly segmentally organized; each of T2-A6 gives rise to six cardioblasts. While moving dorsally during the stages leading up to dorsal closure, cardioblast precursors become flattened, polarized cells aligned in a regular longitudinal row. At dorsal closure, the leading edges of the cardioblast precursors meet their contralateral counterparts. The lumen of the dorsal vessel is formed when the trailing edges of the cardioblast precursors of either side bend around and contact each other. The amnioserosa invaginates during dorsal closure and is transiently attached to the cardioblasts; however, it does not contribute to the cells associated with the dorsal vessel and degenerates during late embryogenesis. We describe ultrastructural characteristics of cardioblast differentiation and discuss similarities between cardioblast development and capillary differentiation in vertebrates. Correspondence to: V. Hartenstein     

The patterns of wingless,decapentaplegic, and tinman position the Drosophila heart

Two secreted signaling molecules, wingless (wg) and decapentaplegic (dpp), are required to specify the heart in Drosophila. wg and dpp are also required to specify other cell types within the mesoderm and in many other regions of the embryo. Because the spatial patterns of wg and dpp are dynamic, different populations of mesodermal cells are exposed to different combinations of wg and/or dpp at different times. To determine whether the patterns of wg and dpp expression provide unique positional information for the specification of heart precursors, we altered these patterns. Our data suggest that wg and dpp contribute progressively to the elaboration of the expression pattern of the mesoderm-specific homeobox-containing gene tinman (tin), and that the overlap of wg and dpp at an early stage (9) as well as at a later stage (11) in the presence of tin-expressing cells directs cardiac-specific differentiation. Furthermore, ectopic tin expression in the ectoderm at wg/dpp intersects (the primordia of the thoracic imaginal disks) also leads to cardiac-specific differentiation, suggesting that tin confers mesoderm-specificity to the wg/dpp response. We conclude that ectopic heart can be generated by altering the patterns of wg and dpp within the tin-expressing mesoderm, or by ectopic induction of tin within the wg- and dpp-expressing ectoderm.     

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.     

The heterotrimeric protein Go is required for the formation of heart epithelium in Drosophila.

The gene encoding the alpha subunit of the Drosophila Go protein is expressed early in embryogenesis in the precursor cells of the heart tube, of the visceral muscles, and of the nervous system. This early expression coincides with the onset of the mesenchymal-epithelial transition to which are subjected the cardial cells and the precursor cells of the visceral musculature. This gene constitutes an appropriate marker to follow this transition. In addition, a detailed analysis of its expression suggests that the cardioblasts originate from two subpopulations of cells in each parasegment of the dorsal mesoderm that might depend on the wingless and hedgehog signaling pathways for both their determination and specification. In the nervous system, the expression of Goalpha shortly precedes the beginning of axonogenesis. Mutants produced in the Goalpha gene harbor abnormalities in the three tissues in which the gene is expressed. In particular, the heart does not form properly and interruptions in the heart epithelium are repeatedly observed, henceforth the brokenheart (bkh) name. Furthermore, in the bkh mutant embryos, the epithelial polarity of cardial cells was not acquired (or maintained) in various places of the cardiac tube. We predict that bkh might be involved in vesicular traffic of membrane proteins that is responsible for the acquisition of polarity.     

The X+ chronic granulomatous disease as a fabulous model to study the NADPH oxidase complex activation

Chronic granulomatous disease (CGD) is a rare inherited disorder in which phagocytes lack NADPH oxidase activity. Patients with CGD suffer from recurrent bacterial and fungal infections because of the absence of superoxide anions (O2- degrees ) generatingsystem. The NADPH oxidase complex is composed of a membranous cytochrome b558, cytosolic proteins p67phox, p47phox, p40phox and two small GTPases Rac2 and Rap1A. Cytochrome b558 consists of two sub-units gp91phox and p22phox. The most common form of CGD is due to mutations in CYBB gene encoding gp91phox. In some rare cases, the mutated gp91phox is normally expressed but is devoided of oxidase activity. These variants called X+ CGD, have provided interesting informations about oxidase activation mechanisms. However modelization of such variants is necessary to obtain enough biological material for studies at the molecular level. A cellular model (knock-out PLB-985 cells) has been developed for expressing recombinant mutated gp91phox for functional analysis of the oxidase complex. Recent works demonstrated that this cell line genetically deficient in gp91phox is a powerful tool for functional analysis of the NADPH oxidase complex activation.     

Direct influence of serotonin on the larval heart of Drosophila melanogaster

The heart rate (HR) of larval Drosophila is established to be modulated by various neuromodulators. Serotonin (5-HT) showed dose-dependent responses in direct application within semi-intact preparations. At 1 nM, HR decreased by 20% while it increased at 10 nM (10%) and 100 nM (30%). The effects plateaued at 100 nM. The action of 5-HT on the heart was examined with an intact Central Nervous System (CNS) and an ablated CNS. The heart and aorta of dorsal vessel pulsate at different rates at rest and during exposure to 5-HT. Splitting the heart and aorta resulted in a dramatic reduction in pulse rate of both the segments and the addition of 5-HT did not produce regional differences. The split aorta and heart showed a high degree of sensitivity to sham changes of saline but no significant effect to 5-HT. Larvae-fed 5-HT (1 mM) did not show any significant change in HR. Since 3,4-methylenedioxymethamphetamine (MDMA) is known to act as a weak agonist on 5-HT receptors in vertebrates, we tested an exogenous application; however, no significant effect was observed to dosage ranging from 1 nM to 100 μM in larvae with and without an intact CNS. In summary, direct application of 5-HT to the larval heart had significant effects in a dose-dependent manner while MDMA had no effect.     

Matricellular proteins in development: Perspectives from the Drosophila heart

The Drosophila model represents an attractive system in which to study the functional contribution of specific genes to organ development. Within the embryo, the heart tube serves as an informative developmental paradigm to analyze functional aspects of matricellular proteins. Here, we describe two essential extracellular matricellular proteins, Multiplexin (Mp) and Lonely heart (Loh). Each of these proteins contributes to the development and morphogenesis of the heart tube by regulating the activity/localization of essential extracellular proteins. Mp, which is secreted by heart cardioblasts and is specifically distributed in the lumen of the heart tube, binds to the signaling protein Slit, and facilitates its local signaling at the heart's luminal domain. Loh is an ADAMTS-like protein, which serves as an adapter protein to Pericardin (a collagen-like protein), promoting its specific localization at the abluminal domain of the heart tube. We also introduce the Drosophila orthologues of matricellular proteins present in mammals, including Thrombospondin, and SPARC, and discuss a possible role for Teneurins (Ten-A and Ten-M) in the heart. Understanding the role of these proteins provides a novel developmental perspective into the functional contribution of matricellular proteins to organ development.     

The pioneer gene, apontic, is required for morphogenesis and function of the Drosophila heart

In an effort to isolate genes required for heart development and to further our understanding of cardiac specification at the molecular level, we screened PlacZ enhancer trap lines for expression in the Drosophila heart. One of the lines generated in this screen, designated B2-2-15, was particularly interesting because of its early pattern of expression in cardiac precursor cells, which is dependent on the homeobox gene tinman, a key determinant of heart development in Drosophila. We isolated and characterized a gene in the vicinity of B2-2-15 that exhibits an identical expression pattern than the reporter gene of the enhancer trap. The product of his gene, apontic (apt; see also Gellon et al., 1997), does not appear to have any homology with known genes. apt mutant embryos show distinct abnormalities in heart morphology as early as mid-embryonic stages when the heat tube assembles, in that segments of heart cells (those of myocardial and pericardial identity) are often missing. Most strikingly, however, apt mutant embryos or larvae only develop a much reduced heart rate, perhaps because of defects in the assembly of an intact heart tube and/or because of defects in the function or physiological control of the myocardial cells, which normally mediate heart contractions. These cardiac defects may be the cause of death of these mutants during late embryonic or early larval stages.     

Genetic variation affecting heart rate in Drosophila melanogaster

Heart rate in pre-pupae of Drosophila melanogaster is shown to vary over a wide range from 2.5 to 3.7 beats per second. Quantitative genetic analysis of a sample of 11 highly inbred lines indicates that approaching one-quarter of the total variance in natural populations can be attributed to genetic differences between flies. A hypomorphic allele of the potassium channel gene ether-a-gogo, which is homologous to a human long-QT syndrome susceptibility gene (HERG), has a heart rate at the low end of the wild-type range, but this effect can be suppressed in certain wild-type genetic backgrounds. This study provides a baseline for investigation of pharmacological and other physiological influences on heart rate in the model organism, and implies that quantitative genetic dissection will provide insight into the molecular basis for variation in normal and arrhythmic heart function.     

Screening assays for heart function mutants in Drosophila

The rapid life cycle and genetic tractability of Drosophila make it an ideal organism for large-scale genetic screens. Here we describe a novel assay for pupal heart rate and rhythmicity as well as techniques to measure adult cardiac stress response. These assays can be powerfully combined to concurrently screen for both mutations affecting cardiac function and mutations affecting the age-dependent decline in adult cardiac stress response. Mutations identified in such screens have the potential to contribute greatly to the understanding of both congenital heart disease and the regulation of age-dependent decline in cardiac function in the human population.     

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.     

The ADAM metalloprotease Kuzbanian is crucial for proper heart formation in Drosophila melanogaster

We have screened a collection of EMS mutagenized fly lines in order to identify genes involved in cardiogenesis. In the present work, we have studied a group of alleles exhibiting a hypertrophic heart. Our analysis revealed that the ADAM protein (A Disintegrin And Metalloprotease) Kuzbanian, which is the functional homologue of the vertebrate ADAM10, is crucial for proper heart formation. ADAMs are a family of transmembrane proteins that play a critical role during the proteolytic conversion (shedding) of membrane bound proteins to soluble forms. Enzymes harboring a sheddase function recently became candidates for causing several congenital diseases, like distinct forms of the Alzheimer disease. ADAMs play also a pivotal role during heart formation and vascularisation in vertebrates, therefore mutations in ADAM genes potentially could cause congenital heart defects in humans. In Drosophila, the zygotic loss of an active form of the Kuzbanian protein results in a dramatic excess of cardiomyocytes, accompanied by a loss of pericardial cells. Our data presented herein suggest that Kuzbanian acts during lateral inhibition within the cardiac primordium. Furthermore we discuss a second function of Kuzbanian in heart cell morphogenesis.     

Steroid-dependent modification of Hox function drives myocyte reprogramming in the Drosophila heart

In the Drosophila larval cardiac tube, aorta and heart differentiation are controlled by the Hox genes Ultrabithorax (Ubx) and abdominal A (abdA), respectively. There is evidence that the cardiac tube undergoes extensive morphological and functional changes during metamorphosis to form the adult organ, but both the origin of adult cardiac tube myocytes and the underlying genetic control have not been established. Using in vivo time-lapse analysis, we show that the adult fruit fly cardiac tube is formed during metamorphosis by the reprogramming of differentiated and already functional larval cardiomyocytes, without cell proliferation. We characterise the genetic control of the process, which is cell autonomously ensured by the modulation of Ubx expression and AbdA activity. Larval aorta myocytes are remodelled to differentiate into the functional adult heart, in a process that requires the regulation of Ubx expression. Conversely, the shape, polarity, function and molecular characteristics of the surviving larval contractile heart myocytes are profoundly transformed as these cells are reprogrammed to form the adult terminal chamber. This process is mediated by the regulation of AbdA protein function, which is successively required within these persisting myocytes for the acquisition of both larval and adult differentiated states. Importantly, AbdA specificity is switched at metamorphosis to induce a novel genetic program that leads to differentiation of the terminal chamber. Finally, the steroid hormone ecdysone controls cardiac tube remodelling by impinging on both the regulation of Ubx expression and the modification of AbdA function. Our results shed light on the genetic control of one in vivo occurring remodelling process, which involves a steroid-dependent modification of Hox expression and function.     

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.