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The fabulous destiny of the Drosophila heart
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The fabulous destiny of the Drosophila heart Caroline Medioni1,2,*, Se´bastien Se´natore1,*, Pierre-Adrien Salmand1, Nathalie Laleve´e1, Laurent Perrin1 and Michel Se´me´riva1 For the last 15 years the fly cardiovascular system has attracted developmental geneticists for its potential as a model system of organogenesis. Heart development in Drosophila indeed provides a remarkable system for elucidating the basic molecular and cellular mechanisms of morphogenesis and, more recently, for understanding the genetic control of cardiac physiology. The success of these studies can in part be attributed to multidisciplinary approaches, the multiplicity of existing genetic tools, and a detailed knowledge of the system.Striking similarities with vertebrate cardiogenesis have long been stressed, in particular concerning the conservation of key molecular regulators of cardiogenesis and the new data presented here confirm Drosophila cardiogenesis as a model not only for organogenesis but also for the study of molecular mechanisms of human cardiac disease. Addresses 1 Institut de Biologie du De´veloppement de Marseille-Luminy (IBDML), UMR 6216 CNRS-Universite´ de la Me´diterrane´e, Campus de Luminy, Case 907, 13288 Marseille Cedex 09, France 2 Present address, Institute de Biologie du Developpement et Cancer (IBDC), UMR 6543 CNRS-Universite´ Nice, Sophia Antipolis, Parc Valrose, 06108 Nice Cedex 2, France Corresponding author: Se´me´riva, Michel ([email protected]) *Equal contributors.
Current Opinion in Genetics & Development 2009, 19:518–525 This review comes from a themed issue on Differentiation and gene regulation Edited by Markus Affolter and Rolf Zeller Available online 28th August 2009 0959-437X/$ – see front matter # 2009 Elsevier Ltd. All rights reserved. DOI 10.1016/j.gde.2009.07.004
In Drosophila, the heart — also named the cardiac tube or dorsal vessel — constitutes the entire cardiovascular system of the organism that operates in an open circulation. Like all organs regarded as ‘hearts’, the fly heart is a muscular pump contracting automatically to distribute blood through the organism. It is constituted by a small number of myocytes, which can be individually identified in vivo, such that analysis can be conducted at the resolution of a single cell. The ultimate fate and emergence of the specific physiological properties associated with genetic diversification of cardiac cells can be followed throughout the whole developmental process. In addition, cardiac activity is dispensable for viability in the Current Opinion in Genetics & Development 2009, 19:518–525
fly, favoring the analysis of genes essential for heart development and function through targeted gene expression or gene inactivation. Numerous studies have recently provided important insights into various aspects of cardiogenesis in fly, including organogenesis, the genetic control of cardiogenesis, and the regulation of cardiac physiology. Here, our aim is not to exhaustively review what is known about Drosophila cardiogenesis, as this has already excellently, and sometimes very recently been done [1,2,3,4,5]. Instead, we will provide the reader with recent insights into elements of morphogenesis, morphology, and physiology that help to understand the fly cardiovascular system as a functional organ and underline the particular attributes that make it a unique system. Secondly, we will argue why it can be anticipated that current research efforts will lead, in a near future, to an integrative view of cardiogenesis through a Systems Biology perspective. Finally, since homology with mammal cardiogenesis has been often invoked to justify the study of the fly heart [6], we will conclude with recent evolutionary considerations of the origin of cardiovascular systems.
Formation of the cardiac tube: a new model of tubulogenesis The embryonic cardiac tube is constituted by two rows of myoendothelial cardiac cells surrounding a lumen, to which are attached excretory and nonmyogenic pericardial cells. Remarkable similarities between Drosophila cardiac tube morphogenesis and the formation of primary axial vessels in vertebrates, including the primary heart tube, have long been stressed [7]. As in vertebrates, the cells constituting the tube originate from bilateral mesodermal precursors that migrate and eventually converge at the midline to form a tube ([8–10] and Figure 1). Three recent studies [11,12,13] have shown how cell morphogenesis, adhesion, and polarity are controlled to trigger Drosophila cardiac tube and lumen formation. These studies identify new molecular and cellular mechanisms of tubulogenesis, significantly distinct from those invoked in well-documented models of tubulogenesis [14]. Formation of the cardiac lumen relies on the dynamic control of cell shape changes and on the establishment of membrane domains conferring polarity to the cardiac cell (Figure 1). Distinct membrane domains in cardiac cells can be identified on the basis of specific cell polarity markers: firstly, www.sciencedirect.com
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Figure 1
Drosophila cardiac tube formation during embryogenesis. (a) Schematic representation of Drosophila embryos showing the cardiac tube with cardiac cells or cardioblasts (CB) in gray and pericardial cells (PC) in blue at stage 13 (only one row is shown); at stage 14, the two contralateral rows of CBs are shown; at stage 16, the tube is closed. (b) Confocal images of embryos at the same stages as in (A) and labeled with cytoplasmic GFP under the control of heart specific enhancer sequences of dHand [41]. (c) Close up of the CB and PC before, during, and after their migration. Before migration, CBs are rather rounded, then when they start migrating (stage 14), they become triangular and form a leading edge in the direction of their migration. Soon after, the dorsal L-domains contact (stage 15) and CBs adopt a crescent-like shape to join together their ventral L-domains, closing the tube and forming a central lumen at stage 16. The J-domains are marked in red, the L-domain in green, and the PA-domain in purple. The identified markers expressed in each of these distinct membrane domains are written in the same colors as the domains. The signaling pathways involved in the establishment of membrane domains are mentioned. The positive regulations required for the specification of the membrane domains are represented with arrows and the negative regulations with closed bars.
the domain facing the lumen and showing basal membrane attributes, the L(umenal)-domain; secondly, the domains forming adherent junctions between cardiac cells of opposite rows at the dorsal and ventral part of the tube, the J(unction)-domain; and thirdly, the domain making contacts between cardiac cells and pericardial cells, the P(ericardial) A(dhesion)-domain. In contrast, apical domains found in most epithelial cells are absent [15]. The specification and maintenance of each of these membrane domains require specific genetic regulators loss of function of which disrupts tubulogenesis and lumen formation. In particular, the Slit-Robo pathway controls cardiac cell shape change and establishment of the respective J/L-domains. This pathway is required for the specification of nonadherent and (or) repulsive L-domains, in part by antagonizing the function of DE-cadherin in establishing adherent junctions www.sciencedirect.com
[13,12,16]. Inactivation of the Slit-Robo pathway leads to expansion of the J-domain at the expense of the L-domain, a phenotype that can also be obtained by overexpressing DE-cadherin. Likewise, signaling by heterotrimeric Go proteins is required for the specification of the PA domain and the localisation in the PA domain of membrane proteins usually found in epithelial septate junction (SJ) [11]. Disrupting the Gao, Gg1 pathway delocalises SJ proteins, leads to nonadherent pericardial cells and affects lumen formation. SJ proteins play a noncanonical function in cardiac cells, as SJs are absent from the fly heart tube [8]. Beyond defining novel mechanisms of lumen formation, these studies are noteworthy because their analyses of cell polarity markers show that the membrane domain organization of cardiac cells is radically different from that of Current Opinion in Genetics & Development 2009, 19:518–525
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epithelial cells, which have been the principal focus of investigations of tubulogenesis to date. Cardiac cell polarity is in fact more reminiscent of that of endothelial cells in which the luminal membrane domain has basal membrane attributes [17] that determine the manner by which endothelial tubulogenesis proceeds. These findings suggest that the Drosophila cardiac tube may provide a model system for the study of endothelial tube and vessel morphogenesis, in particular for large median vessels.
Morphology and structure of the Drosophila cardiovascular system During late stages of embryogenesis, the cardiac tube, once closed, progressively acquires its functional properties. In particular, muscle striations with Z-bands become apparent. In addition, a cardiovascular valve differentiates at the boundary between the aorta and the heart (Figure 2). During larval stages, the number and identity of cells constituting the cardiovascular system remain unchanged, while growing by 200–500-fold. Likewise, the general morphology of the heart, acquired during embryogenesis, is conserved. Two main domains of the cardiac tube can be distinguished, the heart proper in the posterior abdominal segments and the aorta, itself subdivided into the anterior and posterior aorta, lying respectively in the thoracic and rostral abdominal segments (Figure 2). Several cell types can be identified in the fly heart based on morphology, differentiation properties, and genes expression. In particular, the cardiomyocytes that form the walls of the cardiac tube (Tin-cardiomyocytes) are myoendothelial cells, being both striated mononucleated muscle cells and polarized endothelial cells. The particular arrangement of the muscle fibers, running circularly, mediates wringing of the cardiac cavity and hemolymph expulsion on contraction.
is endowed with automatic and autonomic muscle activity and the sarcomeric apparatus appears very similar to the vertebrate one.
What are the physiological functions of the fly cardiovascular system? The Drosophila heart acts as a myogenic pump to allow the hemolymph circulation through the unique vessel constituted by the cardiovascular system. At a functional level, the cardiac tube is divided into two distinct parts, the aorta and the heart, which parallels its morphology. Only the heart region shows automatic and synchronized beating. The rhythmic and synchronized contraction– relaxation cycles induce a wave from posterior to anterior, suggesting the presence of a dominant pacemaker center in the posterior region. The ostia, the inflow tracts, open and close in phase with the contraction wave [19], leading to hemolymph entry into the cardiac lumen and its subsequent delivery into the hemocoel in the vicinity of the brain lobes by the outflow tract. What might the physiological function of such a cardiovascular system be in an open circulatory system in which oxygenation occurs directly through the tracheal system? The answer to this question remains mysterious, as cardiac activity appears to be dispensable for development and viability: inactivating Calmodulin or L-type Ca2+channels specifically in the cardiac tube, blocks beating of the heart but results in viable and apparently healthy animals (L Perrin, unpublished data). This observation, however, is advantageous when it comes to screening genes for essential heart functions without impairing the general viability of the organism. Thus, a systematic screen for genes regulating cardiac function can be conducted, greatly facilitated by the existence of pangenic RNAi libraries which can be specifically driven in the cardiac tube using the UAS–Gal4 system.
As a whole, the Drosophila heart is structurally very different from the complex chambered vertebrate heart. Perhaps, the most striking difference at the structural level lies in the simplicity of the heart tube composed of a single layer of cardiomyocytes directly in contact with the internal (luminal) and external environments. Thus, Drosophila cardiomyocytes have to fulfill dual functions insured in the mammalian heart by myocardial and endocardial cells. This observation must be taken into account when comparing cardiac function in different organisms.
To further investigate the physiological function of the fly heart, methods have been recently developed to measure cardiac parameters, including beat frequency and arrhythmia, systole–diastole diameters [20], and action potentials by the microelectrode method [21]. With these tools in hand, the analysis of the cardiac function of several gene products, including ion channels with known or unknown function in the vertebrate heart, or with multiple vertebrate homologs, has been initiated [22,23,21]. Likewise, it has been shown that cardiac performance of the Drosophila heart decreases with age, as in humans [24,25].
However, it must be emphasized that, besides the differences in organ structure, Drosophila cardiomyocytes have several hallmarks of cardiac cells, in that they are mononucleated striated muscle cells containing TTubule-like structures (Figure 2) similar to those observed in avian cardiomyocytes [18]. The fly heart
Cardiac muscles of all organisms, including Drosophila, are optimized for periodic and synchronized contraction– relaxation through the entire lifetime of the animal. Mechanistically, this integrates cellular machinery that handles calcium to produce cycles of contraction [26]. The genes encoding the required molecular components
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Figure 2
Structure and morphology of the larval cardiac tube. (a) Schema of the larval cardiovascular system. The Drosophila cardiovascular system is constituted of a unique linear tube made by the juxtaposition of two rows of 52 cardiomyocytes delimiting a central lumen in which the hemolymph flows. The linear tube is made of metameric units, extending from thoracic segment T1 to abdominal segment A7, and is divided into distinct regions along the anterior–posterior axis displaying specific morphology and function: the anterior aorta, the posterior aorta, and the heart. To the cardiac tube are associated the pericardial cells and seven pairs of alary muscles. Heart myocytes expressing Tinman; aorta myocytes expressing Tinman;
ostia cells forming the inflow tracts and expressing Seven-up;
cardiovascular valve;
Seven-up expressing aorta cells;
pericardial cells;
alary muscles. (b–g) Phalloidin staining (F-Actin) of representative views of the different subdomains of the cardiac tube.
Cardiomyocytes contain striated muscles all along the tube. However the number, the size, and the orientation of the muscle fibers are specific to each of the subdivisions. In particular, note the circular/transversal orientation of the fibers in the posterior aorta and the heart (c, d), in contrast to the longitudinal orientation recovered in the anterior aorta (b) and the most posterior segment in A7 (g). (f) Ostia cells are labeled by GFP expression under the control of Seven-up (in green). They lack (or are very poor in) myofibrils. (e) Alary muscles are firmly attached to the cardiomyocytes. (h) A threedimensional Volocity (Improvision Inc., PerkinElmer) reconstitution of a part of the heart stained with phalloidin (see film in Supplementary Material). The insert shows a transversal view in which the lumen (L) is visible. The asterisks show the position of the ostia. The arrow heads point to the cell junctions contracted by the contralateral cardiomyocytes. (i, j) Electron microscopy images of third instar larval heart. Note the highly invaginated luminal (L) and basal membranes of the cardiomyocytes, covered by a thick layer of extracellular matrix. These plasma membrane invaginations are reminiscent to T-Tubules (white arrows), and are in some places in contact with the sarcoplasmic reticulum (white arrow heads). The black arrows point to myofibrils. The dotted line in (j) points to a transversal section of the myofibrils. m, mitochondria.
are present in the Drosophila genome, and, for some of them, they have been shown to be expressed and functional in the fly cardiovascular system [27,28,3]. This conservation also holds for many other ion transporters www.sciencedirect.com
[29]. This commonalty in ion transporters content supports the view that the basic mechanisms of cardiac activity and automaticity might be conserved between fly and human. Current Opinion in Genetics & Development 2009, 19:518–525
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Genetic control of cardiogenesis and Genes Regulatory Networking Cardiogenesis in Drosophila constitutes an example of well-understood organogenesis in which the function of a number of regulatory factors (either Transcription Factors (TFs) or signaling pathways) has been exhaustively studied and described. Conserved TFs are required to progressively drive specification, diversification, and differentiation of cardiomyocytes and the acquisition of physiological function, in collaboration with cell-intrinsically or cell-extrinsically acting signaling pathways. We will not describe in detail the studies which have so far identified and characterized the TFs and signaling pathways involved in Drosophila cardiogenesis, as they have been thoroughly reviewed [1–5]. These data, although still fragmentary, have been source of wealth for Cripps and Olson [1] in tentatively constructing a cardiogenic Gene Regulatory Network (GRN [30]). The next step will undoubtedly be to generate ‘dynamic’ GRNs, integrating time (e.g.: developmental stages) and cell diversification, a seemingly obtainable goal in Drosophila cardiogenesis [4], to ultimately lead to an integrated and overall picture of organogenesis. These studies have also illustrated the concept of maintained function or redeployment of functions throughout various stages of development. For example, two Hox genes, Ubx and AbdA, regulate successive and distinct genetic programs in Drosophila cardiogenesis. They show equivalent function in cell lineage choice for the posterior aorta and the heart, versus anterior aorta, which develops in the absence of Hox activity [31]. Later in development, AbdA and Ubx displays distinct functions, being responsible for differentiation into the functional heart and the posterior aorta, respectively [32,33,34,3]. Finally, Hox function is required for heart remodeling at metamorphosis [35]. At the same line, the highly dynamic expression of Tinman during cardiac development originates from the activity of three developmentally regulated enhancers, tinB, tinD, and tinC [36–38]. Furthermore, depending on the developmental stage, Tinman induces distinct genetic programs [38]. Sequential functions of key regulatory genes appear therefore to be controlled by distinct upstream regulatory inputs.
larval undifferentiated precursor cells [39], the adult heart is formed during metamorphosis through reprogramming of differentiated and already functional larval cardiomyocytes, without cell proliferation or addition of new cells [35,40]. The formation of the adult heart tissue is autonomously orchestrated by the steroid hormone ecdysone [35]. In vivo cell imaging has enabled visualization of the major events concerning individual cell differentiation during metamorphosis, remodeling of larval aortic myocytes into cardiomyocytes with automatic muscular activity, differentiation of new ostia and valves, and transdifferentiation into the newly innervated terminal chamber [35,41]. Control by Hox genes is critical for cardiac remodeling during metamorphosis, as it is for embryonic cardiogenesis. Of particular interest was the observation that heart remodeling involves an ecdysone-dependent modulation of Ubx expression and AbdA activity. Indeed, the specificity of AbdA switches at metamorphosis to induce a novel genetic program leading to transdifferentiation of larval heart myocytes into adult terminal chamber myocytes. While AbdA expression is maintained in persisting myocytes, the output of its transcriptional activity is dramatically changed. For example, the expression of target genes previously activated by AbdA in the larval heart, such as Ih or Ndae1 [3], is repressed. This example of tissue remodeling thus illustrates how developmental and temporal cues impinge on organogenesis and cell differentiation. In addition, a complete picture of gene expression dynamics in the developing adult heart was drawn from whole genome transcriptome profiling analysis [42]. This has revealed a complex and precise regulatory gene network that coordinates the dynamics of gene functions required for the process. Another important finding is that essential signaling pathways at work at particular stages during this process can be revealed by such analysis.
Evolutionary origin of hearts
The adult heart is formed by remodeling of the larval organ
Striking resemblances between Drosophila and vertebrate cardiogenesis have long been observed and reported [7]. In particular, it has been shown that homologous genes, mainly encoding TFs or components of signaling pathways, were expressed in developing fly and vertebrate hearts and were functionally required for cardiogenesis [5,2,4]. Consequently, Drosophila has been proposed as a model organism for studying cardiogenesis in mammals and for learning about molecular mechanisms of human cardiac pathologies (see e.g. Bier and Bodmer [6]).
The formation of the adult fly heart provides a unique example of organ remodeling under nonpathological conditions, in which a mature organ arises from a pre-existing functional organ. In contrast to most of the structures and organs of the adult fly, which are entirely rebuilt from
However the similarities between vertebrate and invertebrate heart morphogenesis are rather superficial and the postulated homology of organogenesis does not withstand in depth analysis. In addition to the highly divergent
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morphology and physiology of the mature hearts, numerous differences (reported above in the manuscript) exist between fly and vertebrate cardiogenesis and these differences are expected to expand as we learn more about the two systems. Likewise, the presence of homologous genes working in the development of superficially similar structures is not necessarily proof that these structures are homologous [43], and this is a real issue when Drosophila is used as a model for cardiogenesis.
This hypothesis also provides an explanation for the mixed morphology and physiology of the fly dorsal vessel, presenting characteristics of both striated cardiac muscles and smooth muscle/endothelial cells. Interestingly, Nkx2-5 is required for the specification of an endothelial/endocardial fate, in addition to its recognized function in the myocardial lineage [46], reinforcing the view of a common origin of endothelial vessels and myocardium.
Two opposing views as to the origins of the circulatory pumps found in vertebrates and insects have been discussed: homology, which implies the existence of a common ancestor and subsequent evolution, and convergence, according to which these organs, although performing some analogous functions, were in fact independently created [30,44]. Considering it unlikely that the common Bilaterian ancestor had a heart, Erwin and Davidson [30] proposed that the homology lies at the level of the genetic pathways assembled early in evolution to give rise to the basic cell types that underlie organ function. The only homology warranted by the conservation of developmental genes such as tinman in the dorsal vessel and of Nkx2-5 in the vertebrate heart would be at the level of the myocytes that form these organs. Homology would thus reflect differentiation rather than morphogenesis. Accordingly, morphogenesis would have diverged, evolving independently to produce an organ displaying the common function of all hearts, blood circulation ensured by a myogenic pump, a function relying on differentiated conserved cardiomyocytes.
Conclusion
Taking their inspiration from the considerations of the zoologist Ruppert, revisited by Hartenstein and Mandal [45], Xavier-Neto et al. [44] attempted to reconcile homology and convergence. They proposed that animal pumps derived from parallel improvements of an ancestral, peristaltic apparatus constituted by a layer of myocytes at the external walls of primitive vessels and originating from the mesodermally derived cells that surround the coelom, the mesothelium. In all coelomates, parts of the mesothelium form the wall of contractile vessels that contain and propel the blood. In arthropods, the embryonic mesothelium forms epithelially lined vascular spaces. In vertebrates, vascular development proceeds beyond the level of a contractile mesothelium, which then produces several populations of precursor cells that assemble into the various tissue layers of the cardiovascular system, including endothelium/endocardium and myocardium. According to this comparative zoology based hypothesis, the Drosophila heart would be as closely related to the myocardium of the vertebrate heart as it is to the endocardium, or vascular endothelium. And indeed, homologous genes expressed in the Drosophila heart are represented with equal likelihood in all tissues of the vertebrate cardiovascular system [45]. www.sciencedirect.com
It is clear from the above discussion that the genes essential for determining cardiomyocyte cell type identity, differentiation, and function have been conserved over evolution because they originate from common ancestors. Consequently, Drosophila seems an appropriate model system to identify such genes and the genetic networks in which they are included. However the developmental programs responsible for the formation and the acquisition of the function of the organ may have substantially diverged, and are likely to be specific to flies and vertebrates, even though the ‘homologous’ organs have been built with the same basic elements. Indeed, one of the main lessons of developmental genetics has been that the developmental and functional consequences of the activity of orthologous regulatory genes are specifically determined by the organism context (see e.g. [47]). An important step in understanding this evolutionary process would be to determine what makes the output of a given developmental program organism-specific. Such an advance is now envisageable in fly cardiogenesis. For example, understanding how GRNs are sculpted by the environment, not only that intrinsic to the organism, such as the genetic background, developmental constrains, or the mechanical response due to hemodynamics, but also by the ‘external’ physical and social environment, should increase our knowledge on this organism–environment interaction. Likewise, Drosophila cardiogenesis could contribute to determining how physiology is dependent upon environment and how the function of conserved ion transporters is impacted by the organism context. Because of the complexity of such systems and mechanisms, this level of analysis will however be possible only for simple biological models. Drosophila constitutes the simplest genetic model with a fluid pumping organ. In addition one can anticipate that this type of information could greatly contribute to understanding the molecular mechanisms of certain human cardiac pathologies. Despite such caveats when attempting to draw homology between heart development in Drosophila and human, the findings reported above illustrate that cardiogenesis in the fly provides a wonderful system for elucidating the molecular and cellular basic mechanisms underlying morphogenesis, and thus constitutes a unique model for organogenesis. A thorough analysis of the morphogenesis Current Opinion in Genetics & Development 2009, 19:518–525
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of the fly cardiac tube should provide further essential insights into the cellular and molecular mechanisms of tubulogenesis. Likewise, current studies on cardiac physiology are promising and should lead to valuable insights on essential mechanisms of heart rhythm control, mechano-electric feedback, and excitation–contraction coupling in cardiomyocytes. Moreover, the developing Drosophila cardiovascular system is particularly well adapted to the generation and integration of genome-wide qualitative and quantitative data to dissect the GRNs that control organogenesis, taking a Systems Biology perspective. Thus, a comprehensive characterization of the dynamic interactions between the multiple components of the developing cardiovascular system appears now reachable. This should allow modeling of the underlying GRNs and provide broad insight into the downstream events that progressively drive cardiac cell diversification and differentiation. Interestingly, the system offers the potential for the establishment of precise and robust modeling of the transcriptional networks at play in any given cell within the organ at any particular developmental stage. This feasible analysis now would allow reconstruction of cell history in terms of GRNs.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.gde.2009. 07.004.
Acknowledgements We thank R Kelly and M Gettings for critical reading and helpful discussions and M Astier for her help in electron microscopy analysis. Work is supported by grants from Association de la Recherche contre le Cancer, Association Franc¸aise contre les Myopathies, Agence Nationale de la Recherche and la Fondation pour la Recherche Me´dicale.
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10. Fremion F, Astier M, Zaffran S, Guillen A, Homburger V, Semeriva M: The heterotrimeric protein Go is required for the formation of heart epithelium in Drosophila. J Cell Biol 1999, 145:1063-1076. 11. Yi P, Johnson AN, Han Z, Wu J, Olson EN: Heterotrimeric G proteins regulate a noncanonical function of septate junction proteins to maintain cardiac integrity in Drosophila. Dev Cell 2008, 15:704-713. Olson and colleagues implicate G protein signaling pathway containing Gg1, b13F and G-oa47A in the control of Drosophila cardiac tube formation. They show that the establishment of the PA domain (pericardial-cardiac cells adhesion) depends on septate junction (SJ) proteins Neurexin-IV, Sinuous, Coracle and Nervana-2 and on their modulation by Gg1. Mutants for these G-proteins or SJ molecules, or combination of both result in the loss of cardiac tube integrity and function: the two rows of cardiac cells are not aligned properly (broken heart phenotype) and the heart rate is strongly reduced. 12. Santiago-Martinez E, Soplop NH, Patel R, Kramer SG: Repulsion by Slit and Roundabout prevents Shotgun/E-cadherinmediated cell adhesion during Drosophila heart tube lumen formation. J Cell Biol 2008, 182:241-248. This study points to the antagonistic interaction between Cadherin and Slit-Robo pathways. They show that robo genetically interacts with DEcadherin/shotgun to prevent junction formation in the luminal domain avoiding the cardiac cells of the two opposite rows to stick. robo loss of function phenocopies shotgun overexpression and leads to the formation of a continuous junction domain between cardiac cells instead of having clear dorsal and ventral junction domains separated by a luminal domain. 13. Medioni C, Astier M, Zmojdzian M, Jagla K, Semeriva M: Genetic control of cell morphogenesis during Drosophila melanogaster cardiac tube formation. J Cell Biol 2008, 182:249-261. The authors demonstrate that cardiac cell shape changes and J/L domains establishment (cell polarity) are controlled by a new genetic pathway including slit and robo, held out wings (how) and dystroglycan (dg), coding axon guidance regulators, RNA binding protein involved in muscle function and a receptor for ECM components, respectively. The loss of function of one of these genes results in the absence of lumen or defects in lumen formation. 14. Lubarsky B, Krasnow MA: Tube morphogenesis: making and shaping biological tubes. Cell 2003, 112:19-28. 15. Bryant DM, Mostov KE: From cells to organs: building polarized tissue. Nat Rev Mol Cell Biol 2008, 9:887-901. 16. Helenius IT, Beitel GJ: The first ‘‘Slit’’ is the deepest: the secret to a hollow heart. J Cell Biol 2008, 182:221-223. A comprehensive comment on articles [12,13]. 17. Iruela-Arispe ML, Davis GE: Cellular and molecular mechanisms of vascular lumen formation. Dev Cell 2009, 16:222-231. 18. Protasi F, Sun XH, Franzini-Armstrong C: Formation and maturation of the calcium release apparatus in developing and adult avian myocardium. Dev Biol 1996, 173:265-278. 19. Wu M, Sato TN: On the mechanics of cardiac function of Drosophila embryo. PLoS ONE 2008, 3:e4045. 20. Fink M, Callol-Massot C, Chu A, Ruiz-Lozano P, Belmonte JC, Giles W, Bodmer R, Ocorr K: A new method for detection and quantification of heartbeat parameters in Drosophila, zebrafish, and embryonic mouse hearts. Biotechniques 2009, 46:101-113. The authors combine high-speed optical recording of beating hearts with a semi-automated analysis to accurately detect and quantify heart rate, diastolic and systolic intervals and diameters, percent fractional shortening, contraction wave velocity, and cardiac arrhythmicity. www.sciencedirect.com
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21. Lalevee N, Monier B, Senatore S, Perrin L, Semeriva M: Control of cardiac rhythm by ORK1, a Drosophila two-pore domain potassium channel. Curr Biol 2006, 16:1502-1508. By a combined genetic and electrophysiological approaches, the authors show that Ork1, a Drosophila K+ channel of the K2P family, controls cardiac rate by modulating the slow predepolarisation phase of the action potential. 22. Ocorr K, Reeves NL, Wessells RJ, Fink M, Chen HS, Akasaka T, Yasuda S, Metzger JM, Giles W, Posakony JW et al.: KCNQ potassium channel mutations cause cardiac arrhythmias in Drosophila that mimic the effects of aging. Proc Natl Acad Sci U S A 2007, 104:3943-3948. K+ currents mediated by the fly homolog of KCNQ, involved in human in Long QT syndrome, contribute to the repolarization, ensuring normal excitation–contraction coupling and rhythmical contraction. 23. Akasaka T, Klinedinst S, Ocorr K, Bustamante EL, Kim SK, Bodmer R: The ATP-sensitive potassium (KATP) channelencoded dSUR gene is required for Drosophila heart function and is regulated by tinman. Proc Natl Acad Sci U S A 2006, 103:11999-12004. These authors show that dSUR, a Sulfonylurea Receptor encoding an ATP-sensitive potassium channel, plays a protective role against hypoxia. 24. Paternostro G, Vignola C, Bartsch DU, Omens JH, McCulloch AD, Reed JC: Age-associated cardiac dysfunction in Drosophila melanogaster. Circ Res 2001, 88:1053-1058. 25. Wessells RJ, Fitzgerald E, Cypser JR, Tatar M, Bodmer R: Insulin regulation of heart function in aging fruit flies. Nat Genet 2004, 36:1275-1281. Interfering with Insulin Receptor signaling exclusively in the heart prevents the decline in cardiac performance with age. Thus, Insulin-IGF signaling influences age-dependent organ physiology and senescence directly and autonomously, in addition to its known systemic effect on lifespan. 26. Bers DM: Cardiac excitation-contraction coupling. Nature 2002, 415:198-205. 27. Sullivan KM, Scott K, Zuker CS, Rubin GM: The ryanodine receptor is essential for larval development in Drosophila melanogaster. Proc Natl Acad Sci U S A 2000, 97:5942-5947. The role of the Ryanodine Receptor has been investigated in Drosophila development by using pharmacological and genetic approaches. The results suggest that the Ryanodine Receptor is required for proper muscle function and may be essential for excitation-contraction coupling in larval muscles, including heart muscles. 28. Sanyal S, Jennings T, Dowse H, Ramaswami M: Conditional mutations in SERCA, the Sarco-endoplasmic reticulum Ca2+ATPase, alter heart rate and rhythmicity in Drosophila. J Comp Physiol [B] 2006, 176:253-263. 29. Littleton JT, Ganetzky B: Ion channels and synaptic organization: analysis of the Drosophila genome. Neuron 2000, 26:35-43. 30. Erwin DH, Davidson EH: The last common bilaterian ancestor. Development 2002, 129:3021-3032. These authors point to the fact there is no convincing evidence that it is specific morphogenetic pathways per se that are conserved, rather than cell-type specification and differentiation processes. 31. Perrin L, Monier B, Ponzielli R, Astier M, Semeriva M: Drosophila cardiac tube organogenesis requires multiple phases of Hox activity. Dev Biol 2004, 272:419-431. Along with Refs. [32,33,34] this paper shows that the activity of two Hox genes, AbdA and Ubx are required for AP axis differentiation of the cardiac tube. 32. Ponzielli R, Astier M, Chartier A, Gallet A, Therond P, Semeriva M: Heart tube patterning in Drosophila requires integration of axial and segmental information provided by the Bithorax Complex genes and hedgehog signaling. Development 2002, 129:4509-4521. See annotation to Ref. [31].
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33. Lo PC, Skeath JB, Gajewski K, Schulz RA, Frasch M: Homeotic genes autonomously specify the anteroposterior subdivision of the Drosophila dorsal vessel into aorta and heart. Dev Biol 2002, 251:307-319. See annotation to Ref. [31]. 34. Lovato TL, Nguyen TP, Molina MR, Cripps RM: The Hox gene abdominal-A specifies heart cell fate in the Drosophila dorsal vessel. Development 2002, 129:5019-5027. See annotation to Ref. [31]. 35. Monier B, Astier M, Semeriva M, Perrin L: Steroid-dependent modification of Hox function drives myocyte reprogramming in the Drosophila heart. Development 2005, 132:5283-5293. This article reports cell imaging recording of cardiac tube remodeling during metamorphosis and demonstrates that remodeling is under the control of ecdysone dependent Hox activity. 36. Yin Z, Xu XL, Frasch M: Regulation of the twist target gene tinman by modular cis-regulatory elements during early mesoderm development. Development 1997, 124:4971-4982. 37. Xu X, Yin Z, Hudson JB, Ferguson EL, Frasch M: Smad proteins act in combination with synergistic and antagonistic regulators to target Dpp responses to the Drosophila mesoderm. Genes Dev 1998, 12:2354-2370. 38. Zaffran S, Reim I, Qian L, Lo PC, Bodmer R, Frasch M: Cardioblast-intrinsic Tinman activity controls proper diversification and differentiation of myocardial cells in Drosophila. Development 2006, 133:4073-4083. 39. Thummel CS: Files on steroids–Drosophila metamorphosis and the mechanisms of steroid hormone action. Trends Genet 1996, 12:306-310. 40. Molina MR, Cripps RM: Ostia, the inflow tracts of the Drosophila heart, develop from a genetically distinct subset of cardial cells. Mech Dev 2001, 109:51-59. 41. Sellin J, Albrecht S, Kolsch V, Paululat A: Dynamics of heart differentiation, visualized utilizing heart enhancer elements of the Drosophila melanogaster bHLH transcription factor Hand. Gene Expr Patterns 2006, 6:360-375. 42. Zeitouni B, Senatore S, Severac D, Aknin C, Semeriva M, Perrin L: Signalling pathways involved in adult heart formation revealed by gene expression profiling in Drosophila. PLoS Genet 2007, 3:1907-1921. Major components of five signaling pathways (FGF, VEGF–PDGF, Wnt, Notch, and Toll) show differential expression during cardiac remodeling, suggesting that these particular pathways are functionally involved in the process. Among these 5 pathways, four have been demonstrated by reverse genetic approach to be actually required in particular aspects of the remodeling process. 43. Wagner GP: The developmental genetics of homology. Nat Rev Genet 2007, 8:473-479. 44. Xavier-Neto J, Castro RA, Sampaio AC, Azambuja AP, Castillo HA, Cravo RM, Simoes-Costa MS: Parallel avenues in the evolution of hearts and pumping organs. Cell Mol Life Sci 2007, 64:719-734. This is a fascinating account of heart and pumping organs evolution. 45. Hartenstein V, Mandal L: The blood/vascular system in a phylogenetic perspective. Bioessays 2006, 28:1203-1210. 46. Ferdous A, Caprioli A, Iacovino M, Martin CM, Morris J, Richardson JA, Latif S, Hammer RE, Harvey RP, Olson EN et al.: Nkx2-5 transactivates the Ets-related protein 71 gene and specifies an endothelial/endocardial fate in the developing embryo. Proc Natl Acad Sci U S A 2009, 106:814-819. 47. Gehring WJ, Ikeo K: Pax 6: mastering eye morphogenesis and eye evolution. Trends Genet 1999, 15:371-377. Pax6 and eyeless are orthologous master genes for eye morphogenesis in mouse and Drosophila, respectively. Pax3 when ectopically transferred to the fly is indeed capable of developing a functional eye, although a fly eye and not a mouse one.
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The fabulous destiny of the Drosophila heart Caroline Medioni1,2,*, Se´bastien Se´natore1,*, Pierre-Adrien Salmand1, Nathalie Laleve´e1, Laurent Perrin1 and Michel Se´me´riva1 For the last 15 years the fly cardiovascular system has attracted developmental geneticists for its potential as a model system of organogenesis. Heart development in Drosophila indeed provides a remarkable system for elucidating the basic molecular and cellular mechanisms of morphogenesis and, more recently, for understanding the genetic control of cardiac physiology. The success of these studies can in part be attributed to multidisciplinary approaches, the multiplicity of existing genetic tools, and a detailed knowledge of the system.Striking similarities with vertebrate cardiogenesis have long been stressed, in particular concerning the conservation of key molecular regulators of cardiogenesis and the new data presented here confirm Drosophila cardiogenesis as a model not only for organogenesis but also for the study of molecular mechanisms of human cardiac disease. Addresses 1 Institut de Biologie du De´veloppement de Marseille-Luminy (IBDML), UMR 6216 CNRS-Universite´ de la Me´diterrane´e, Campus de Luminy, Case 907, 13288 Marseille Cedex 09, France 2 Present address, Institute de Biologie du Developpement et Cancer (IBDC), UMR 6543 CNRS-Universite´ Nice, Sophia Antipolis, Parc Valrose, 06108 Nice Cedex 2, France Corresponding author: Se´me´riva, Michel ([email protected]) *Equal contributors.
Current Opinion in Genetics & Development 2009, 19:518–525 This review comes from a themed issue on Differentiation and gene regulation Edited by Markus Affolter and Rolf Zeller Available online 28th August 2009 0959-437X/$ – see front matter # 2009 Elsevier Ltd. All rights reserved. DOI 10.1016/j.gde.2009.07.004
In Drosophila, the heart — also named the cardiac tube or dorsal vessel — constitutes the entire cardiovascular system of the organism that operates in an open circulation. Like all organs regarded as ‘hearts’, the fly heart is a muscular pump contracting automatically to distribute blood through the organism. It is constituted by a small number of myocytes, which can be individually identified in vivo, such that analysis can be conducted at the resolution of a single cell. The ultimate fate and emergence of the specific physiological properties associated with genetic diversification of cardiac cells can be followed throughout the whole developmental process. In addition, cardiac activity is dispensable for viability in the Current Opinion in Genetics & Development 2009, 19:518–525
fly, favoring the analysis of genes essential for heart development and function through targeted gene expression or gene inactivation. Numerous studies have recently provided important insights into various aspects of cardiogenesis in fly, including organogenesis, the genetic control of cardiogenesis, and the regulation of cardiac physiology. Here, our aim is not to exhaustively review what is known about Drosophila cardiogenesis, as this has already excellently, and sometimes very recently been done [1,2,3,4,5]. Instead, we will provide the reader with recent insights into elements of morphogenesis, morphology, and physiology that help to understand the fly cardiovascular system as a functional organ and underline the particular attributes that make it a unique system. Secondly, we will argue why it can be anticipated that current research efforts will lead, in a near future, to an integrative view of cardiogenesis through a Systems Biology perspective. Finally, since homology with mammal cardiogenesis has been often invoked to justify the study of the fly heart [6], we will conclude with recent evolutionary considerations of the origin of cardiovascular systems.
Formation of the cardiac tube: a new model of tubulogenesis The embryonic cardiac tube is constituted by two rows of myoendothelial cardiac cells surrounding a lumen, to which are attached excretory and nonmyogenic pericardial cells. Remarkable similarities between Drosophila cardiac tube morphogenesis and the formation of primary axial vessels in vertebrates, including the primary heart tube, have long been stressed [7]. As in vertebrates, the cells constituting the tube originate from bilateral mesodermal precursors that migrate and eventually converge at the midline to form a tube ([8–10] and Figure 1). Three recent studies [11,12,13] have shown how cell morphogenesis, adhesion, and polarity are controlled to trigger Drosophila cardiac tube and lumen formation. These studies identify new molecular and cellular mechanisms of tubulogenesis, significantly distinct from those invoked in well-documented models of tubulogenesis [14]. Formation of the cardiac lumen relies on the dynamic control of cell shape changes and on the establishment of membrane domains conferring polarity to the cardiac cell (Figure 1). Distinct membrane domains in cardiac cells can be identified on the basis of specific cell polarity markers: firstly, www.sciencedirect.com
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Figure 1
Drosophila cardiac tube formation during embryogenesis. (a) Schematic representation of Drosophila embryos showing the cardiac tube with cardiac cells or cardioblasts (CB) in gray and pericardial cells (PC) in blue at stage 13 (only one row is shown); at stage 14, the two contralateral rows of CBs are shown; at stage 16, the tube is closed. (b) Confocal images of embryos at the same stages as in (A) and labeled with cytoplasmic GFP under the control of heart specific enhancer sequences of dHand [41]. (c) Close up of the CB and PC before, during, and after their migration. Before migration, CBs are rather rounded, then when they start migrating (stage 14), they become triangular and form a leading edge in the direction of their migration. Soon after, the dorsal L-domains contact (stage 15) and CBs adopt a crescent-like shape to join together their ventral L-domains, closing the tube and forming a central lumen at stage 16. The J-domains are marked in red, the L-domain in green, and the PA-domain in purple. The identified markers expressed in each of these distinct membrane domains are written in the same colors as the domains. The signaling pathways involved in the establishment of membrane domains are mentioned. The positive regulations required for the specification of the membrane domains are represented with arrows and the negative regulations with closed bars.
the domain facing the lumen and showing basal membrane attributes, the L(umenal)-domain; secondly, the domains forming adherent junctions between cardiac cells of opposite rows at the dorsal and ventral part of the tube, the J(unction)-domain; and thirdly, the domain making contacts between cardiac cells and pericardial cells, the P(ericardial) A(dhesion)-domain. In contrast, apical domains found in most epithelial cells are absent [15]. The specification and maintenance of each of these membrane domains require specific genetic regulators loss of function of which disrupts tubulogenesis and lumen formation. In particular, the Slit-Robo pathway controls cardiac cell shape change and establishment of the respective J/L-domains. This pathway is required for the specification of nonadherent and (or) repulsive L-domains, in part by antagonizing the function of DE-cadherin in establishing adherent junctions www.sciencedirect.com
[13,12,16]. Inactivation of the Slit-Robo pathway leads to expansion of the J-domain at the expense of the L-domain, a phenotype that can also be obtained by overexpressing DE-cadherin. Likewise, signaling by heterotrimeric Go proteins is required for the specification of the PA domain and the localisation in the PA domain of membrane proteins usually found in epithelial septate junction (SJ) [11]. Disrupting the Gao, Gg1 pathway delocalises SJ proteins, leads to nonadherent pericardial cells and affects lumen formation. SJ proteins play a noncanonical function in cardiac cells, as SJs are absent from the fly heart tube [8]. Beyond defining novel mechanisms of lumen formation, these studies are noteworthy because their analyses of cell polarity markers show that the membrane domain organization of cardiac cells is radically different from that of Current Opinion in Genetics & Development 2009, 19:518–525
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epithelial cells, which have been the principal focus of investigations of tubulogenesis to date. Cardiac cell polarity is in fact more reminiscent of that of endothelial cells in which the luminal membrane domain has basal membrane attributes [17] that determine the manner by which endothelial tubulogenesis proceeds. These findings suggest that the Drosophila cardiac tube may provide a model system for the study of endothelial tube and vessel morphogenesis, in particular for large median vessels.
Morphology and structure of the Drosophila cardiovascular system During late stages of embryogenesis, the cardiac tube, once closed, progressively acquires its functional properties. In particular, muscle striations with Z-bands become apparent. In addition, a cardiovascular valve differentiates at the boundary between the aorta and the heart (Figure 2). During larval stages, the number and identity of cells constituting the cardiovascular system remain unchanged, while growing by 200–500-fold. Likewise, the general morphology of the heart, acquired during embryogenesis, is conserved. Two main domains of the cardiac tube can be distinguished, the heart proper in the posterior abdominal segments and the aorta, itself subdivided into the anterior and posterior aorta, lying respectively in the thoracic and rostral abdominal segments (Figure 2). Several cell types can be identified in the fly heart based on morphology, differentiation properties, and genes expression. In particular, the cardiomyocytes that form the walls of the cardiac tube (Tin-cardiomyocytes) are myoendothelial cells, being both striated mononucleated muscle cells and polarized endothelial cells. The particular arrangement of the muscle fibers, running circularly, mediates wringing of the cardiac cavity and hemolymph expulsion on contraction.
is endowed with automatic and autonomic muscle activity and the sarcomeric apparatus appears very similar to the vertebrate one.
What are the physiological functions of the fly cardiovascular system? The Drosophila heart acts as a myogenic pump to allow the hemolymph circulation through the unique vessel constituted by the cardiovascular system. At a functional level, the cardiac tube is divided into two distinct parts, the aorta and the heart, which parallels its morphology. Only the heart region shows automatic and synchronized beating. The rhythmic and synchronized contraction– relaxation cycles induce a wave from posterior to anterior, suggesting the presence of a dominant pacemaker center in the posterior region. The ostia, the inflow tracts, open and close in phase with the contraction wave [19], leading to hemolymph entry into the cardiac lumen and its subsequent delivery into the hemocoel in the vicinity of the brain lobes by the outflow tract. What might the physiological function of such a cardiovascular system be in an open circulatory system in which oxygenation occurs directly through the tracheal system? The answer to this question remains mysterious, as cardiac activity appears to be dispensable for development and viability: inactivating Calmodulin or L-type Ca2+channels specifically in the cardiac tube, blocks beating of the heart but results in viable and apparently healthy animals (L Perrin, unpublished data). This observation, however, is advantageous when it comes to screening genes for essential heart functions without impairing the general viability of the organism. Thus, a systematic screen for genes regulating cardiac function can be conducted, greatly facilitated by the existence of pangenic RNAi libraries which can be specifically driven in the cardiac tube using the UAS–Gal4 system.
As a whole, the Drosophila heart is structurally very different from the complex chambered vertebrate heart. Perhaps, the most striking difference at the structural level lies in the simplicity of the heart tube composed of a single layer of cardiomyocytes directly in contact with the internal (luminal) and external environments. Thus, Drosophila cardiomyocytes have to fulfill dual functions insured in the mammalian heart by myocardial and endocardial cells. This observation must be taken into account when comparing cardiac function in different organisms.
To further investigate the physiological function of the fly heart, methods have been recently developed to measure cardiac parameters, including beat frequency and arrhythmia, systole–diastole diameters [20], and action potentials by the microelectrode method [21]. With these tools in hand, the analysis of the cardiac function of several gene products, including ion channels with known or unknown function in the vertebrate heart, or with multiple vertebrate homologs, has been initiated [22,23,21]. Likewise, it has been shown that cardiac performance of the Drosophila heart decreases with age, as in humans [24,25].
However, it must be emphasized that, besides the differences in organ structure, Drosophila cardiomyocytes have several hallmarks of cardiac cells, in that they are mononucleated striated muscle cells containing TTubule-like structures (Figure 2) similar to those observed in avian cardiomyocytes [18]. The fly heart
Cardiac muscles of all organisms, including Drosophila, are optimized for periodic and synchronized contraction– relaxation through the entire lifetime of the animal. Mechanistically, this integrates cellular machinery that handles calcium to produce cycles of contraction [26]. The genes encoding the required molecular components
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Figure 2
Structure and morphology of the larval cardiac tube. (a) Schema of the larval cardiovascular system. The Drosophila cardiovascular system is constituted of a unique linear tube made by the juxtaposition of two rows of 52 cardiomyocytes delimiting a central lumen in which the hemolymph flows. The linear tube is made of metameric units, extending from thoracic segment T1 to abdominal segment A7, and is divided into distinct regions along the anterior–posterior axis displaying specific morphology and function: the anterior aorta, the posterior aorta, and the heart. To the cardiac tube are associated the pericardial cells and seven pairs of alary muscles. Heart myocytes expressing Tinman; aorta myocytes expressing Tinman;
ostia cells forming the inflow tracts and expressing Seven-up;
cardiovascular valve;
Seven-up expressing aorta cells;
pericardial cells;
alary muscles. (b–g) Phalloidin staining (F-Actin) of representative views of the different subdomains of the cardiac tube.
Cardiomyocytes contain striated muscles all along the tube. However the number, the size, and the orientation of the muscle fibers are specific to each of the subdivisions. In particular, note the circular/transversal orientation of the fibers in the posterior aorta and the heart (c, d), in contrast to the longitudinal orientation recovered in the anterior aorta (b) and the most posterior segment in A7 (g). (f) Ostia cells are labeled by GFP expression under the control of Seven-up (in green). They lack (or are very poor in) myofibrils. (e) Alary muscles are firmly attached to the cardiomyocytes. (h) A threedimensional Volocity (Improvision Inc., PerkinElmer) reconstitution of a part of the heart stained with phalloidin (see film in Supplementary Material). The insert shows a transversal view in which the lumen (L) is visible. The asterisks show the position of the ostia. The arrow heads point to the cell junctions contracted by the contralateral cardiomyocytes. (i, j) Electron microscopy images of third instar larval heart. Note the highly invaginated luminal (L) and basal membranes of the cardiomyocytes, covered by a thick layer of extracellular matrix. These plasma membrane invaginations are reminiscent to T-Tubules (white arrows), and are in some places in contact with the sarcoplasmic reticulum (white arrow heads). The black arrows point to myofibrils. The dotted line in (j) points to a transversal section of the myofibrils. m, mitochondria.
are present in the Drosophila genome, and, for some of them, they have been shown to be expressed and functional in the fly cardiovascular system [27,28,3]. This conservation also holds for many other ion transporters www.sciencedirect.com
[29]. This commonalty in ion transporters content supports the view that the basic mechanisms of cardiac activity and automaticity might be conserved between fly and human. Current Opinion in Genetics & Development 2009, 19:518–525
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Genetic control of cardiogenesis and Genes Regulatory Networking Cardiogenesis in Drosophila constitutes an example of well-understood organogenesis in which the function of a number of regulatory factors (either Transcription Factors (TFs) or signaling pathways) has been exhaustively studied and described. Conserved TFs are required to progressively drive specification, diversification, and differentiation of cardiomyocytes and the acquisition of physiological function, in collaboration with cell-intrinsically or cell-extrinsically acting signaling pathways. We will not describe in detail the studies which have so far identified and characterized the TFs and signaling pathways involved in Drosophila cardiogenesis, as they have been thoroughly reviewed [1–5]. These data, although still fragmentary, have been source of wealth for Cripps and Olson [1] in tentatively constructing a cardiogenic Gene Regulatory Network (GRN [30]). The next step will undoubtedly be to generate ‘dynamic’ GRNs, integrating time (e.g.: developmental stages) and cell diversification, a seemingly obtainable goal in Drosophila cardiogenesis [4], to ultimately lead to an integrated and overall picture of organogenesis. These studies have also illustrated the concept of maintained function or redeployment of functions throughout various stages of development. For example, two Hox genes, Ubx and AbdA, regulate successive and distinct genetic programs in Drosophila cardiogenesis. They show equivalent function in cell lineage choice for the posterior aorta and the heart, versus anterior aorta, which develops in the absence of Hox activity [31]. Later in development, AbdA and Ubx displays distinct functions, being responsible for differentiation into the functional heart and the posterior aorta, respectively [32,33,34,3]. Finally, Hox function is required for heart remodeling at metamorphosis [35]. At the same line, the highly dynamic expression of Tinman during cardiac development originates from the activity of three developmentally regulated enhancers, tinB, tinD, and tinC [36–38]. Furthermore, depending on the developmental stage, Tinman induces distinct genetic programs [38]. Sequential functions of key regulatory genes appear therefore to be controlled by distinct upstream regulatory inputs.
larval undifferentiated precursor cells [39], the adult heart is formed during metamorphosis through reprogramming of differentiated and already functional larval cardiomyocytes, without cell proliferation or addition of new cells [35,40]. The formation of the adult heart tissue is autonomously orchestrated by the steroid hormone ecdysone [35]. In vivo cell imaging has enabled visualization of the major events concerning individual cell differentiation during metamorphosis, remodeling of larval aortic myocytes into cardiomyocytes with automatic muscular activity, differentiation of new ostia and valves, and transdifferentiation into the newly innervated terminal chamber [35,41]. Control by Hox genes is critical for cardiac remodeling during metamorphosis, as it is for embryonic cardiogenesis. Of particular interest was the observation that heart remodeling involves an ecdysone-dependent modulation of Ubx expression and AbdA activity. Indeed, the specificity of AbdA switches at metamorphosis to induce a novel genetic program leading to transdifferentiation of larval heart myocytes into adult terminal chamber myocytes. While AbdA expression is maintained in persisting myocytes, the output of its transcriptional activity is dramatically changed. For example, the expression of target genes previously activated by AbdA in the larval heart, such as Ih or Ndae1 [3], is repressed. This example of tissue remodeling thus illustrates how developmental and temporal cues impinge on organogenesis and cell differentiation. In addition, a complete picture of gene expression dynamics in the developing adult heart was drawn from whole genome transcriptome profiling analysis [42]. This has revealed a complex and precise regulatory gene network that coordinates the dynamics of gene functions required for the process. Another important finding is that essential signaling pathways at work at particular stages during this process can be revealed by such analysis.
Evolutionary origin of hearts
The adult heart is formed by remodeling of the larval organ
Striking resemblances between Drosophila and vertebrate cardiogenesis have long been observed and reported [7]. In particular, it has been shown that homologous genes, mainly encoding TFs or components of signaling pathways, were expressed in developing fly and vertebrate hearts and were functionally required for cardiogenesis [5,2,4]. Consequently, Drosophila has been proposed as a model organism for studying cardiogenesis in mammals and for learning about molecular mechanisms of human cardiac pathologies (see e.g. Bier and Bodmer [6]).
The formation of the adult fly heart provides a unique example of organ remodeling under nonpathological conditions, in which a mature organ arises from a pre-existing functional organ. In contrast to most of the structures and organs of the adult fly, which are entirely rebuilt from
However the similarities between vertebrate and invertebrate heart morphogenesis are rather superficial and the postulated homology of organogenesis does not withstand in depth analysis. In addition to the highly divergent
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morphology and physiology of the mature hearts, numerous differences (reported above in the manuscript) exist between fly and vertebrate cardiogenesis and these differences are expected to expand as we learn more about the two systems. Likewise, the presence of homologous genes working in the development of superficially similar structures is not necessarily proof that these structures are homologous [43], and this is a real issue when Drosophila is used as a model for cardiogenesis.
This hypothesis also provides an explanation for the mixed morphology and physiology of the fly dorsal vessel, presenting characteristics of both striated cardiac muscles and smooth muscle/endothelial cells. Interestingly, Nkx2-5 is required for the specification of an endothelial/endocardial fate, in addition to its recognized function in the myocardial lineage [46], reinforcing the view of a common origin of endothelial vessels and myocardium.
Two opposing views as to the origins of the circulatory pumps found in vertebrates and insects have been discussed: homology, which implies the existence of a common ancestor and subsequent evolution, and convergence, according to which these organs, although performing some analogous functions, were in fact independently created [30,44]. Considering it unlikely that the common Bilaterian ancestor had a heart, Erwin and Davidson [30] proposed that the homology lies at the level of the genetic pathways assembled early in evolution to give rise to the basic cell types that underlie organ function. The only homology warranted by the conservation of developmental genes such as tinman in the dorsal vessel and of Nkx2-5 in the vertebrate heart would be at the level of the myocytes that form these organs. Homology would thus reflect differentiation rather than morphogenesis. Accordingly, morphogenesis would have diverged, evolving independently to produce an organ displaying the common function of all hearts, blood circulation ensured by a myogenic pump, a function relying on differentiated conserved cardiomyocytes.
Conclusion
Taking their inspiration from the considerations of the zoologist Ruppert, revisited by Hartenstein and Mandal [45], Xavier-Neto et al. [44] attempted to reconcile homology and convergence. They proposed that animal pumps derived from parallel improvements of an ancestral, peristaltic apparatus constituted by a layer of myocytes at the external walls of primitive vessels and originating from the mesodermally derived cells that surround the coelom, the mesothelium. In all coelomates, parts of the mesothelium form the wall of contractile vessels that contain and propel the blood. In arthropods, the embryonic mesothelium forms epithelially lined vascular spaces. In vertebrates, vascular development proceeds beyond the level of a contractile mesothelium, which then produces several populations of precursor cells that assemble into the various tissue layers of the cardiovascular system, including endothelium/endocardium and myocardium. According to this comparative zoology based hypothesis, the Drosophila heart would be as closely related to the myocardium of the vertebrate heart as it is to the endocardium, or vascular endothelium. And indeed, homologous genes expressed in the Drosophila heart are represented with equal likelihood in all tissues of the vertebrate cardiovascular system [45]. www.sciencedirect.com
It is clear from the above discussion that the genes essential for determining cardiomyocyte cell type identity, differentiation, and function have been conserved over evolution because they originate from common ancestors. Consequently, Drosophila seems an appropriate model system to identify such genes and the genetic networks in which they are included. However the developmental programs responsible for the formation and the acquisition of the function of the organ may have substantially diverged, and are likely to be specific to flies and vertebrates, even though the ‘homologous’ organs have been built with the same basic elements. Indeed, one of the main lessons of developmental genetics has been that the developmental and functional consequences of the activity of orthologous regulatory genes are specifically determined by the organism context (see e.g. [47]). An important step in understanding this evolutionary process would be to determine what makes the output of a given developmental program organism-specific. Such an advance is now envisageable in fly cardiogenesis. For example, understanding how GRNs are sculpted by the environment, not only that intrinsic to the organism, such as the genetic background, developmental constrains, or the mechanical response due to hemodynamics, but also by the ‘external’ physical and social environment, should increase our knowledge on this organism–environment interaction. Likewise, Drosophila cardiogenesis could contribute to determining how physiology is dependent upon environment and how the function of conserved ion transporters is impacted by the organism context. Because of the complexity of such systems and mechanisms, this level of analysis will however be possible only for simple biological models. Drosophila constitutes the simplest genetic model with a fluid pumping organ. In addition one can anticipate that this type of information could greatly contribute to understanding the molecular mechanisms of certain human cardiac pathologies. Despite such caveats when attempting to draw homology between heart development in Drosophila and human, the findings reported above illustrate that cardiogenesis in the fly provides a wonderful system for elucidating the molecular and cellular basic mechanisms underlying morphogenesis, and thus constitutes a unique model for organogenesis. A thorough analysis of the morphogenesis Current Opinion in Genetics & Development 2009, 19:518–525
524 Differentiation and gene regulation
of the fly cardiac tube should provide further essential insights into the cellular and molecular mechanisms of tubulogenesis. Likewise, current studies on cardiac physiology are promising and should lead to valuable insights on essential mechanisms of heart rhythm control, mechano-electric feedback, and excitation–contraction coupling in cardiomyocytes. Moreover, the developing Drosophila cardiovascular system is particularly well adapted to the generation and integration of genome-wide qualitative and quantitative data to dissect the GRNs that control organogenesis, taking a Systems Biology perspective. Thus, a comprehensive characterization of the dynamic interactions between the multiple components of the developing cardiovascular system appears now reachable. This should allow modeling of the underlying GRNs and provide broad insight into the downstream events that progressively drive cardiac cell diversification and differentiation. Interestingly, the system offers the potential for the establishment of precise and robust modeling of the transcriptional networks at play in any given cell within the organ at any particular developmental stage. This feasible analysis now would allow reconstruction of cell history in terms of GRNs.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.gde.2009. 07.004.
Acknowledgements We thank R Kelly and M Gettings for critical reading and helpful discussions and M Astier for her help in electron microscopy analysis. Work is supported by grants from Association de la Recherche contre le Cancer, Association Franc¸aise contre les Myopathies, Agence Nationale de la Recherche and la Fondation pour la Recherche Me´dicale.
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