The ultrastructure of Drosophila heart cells

Arthropod Structure & Development 41 (2012) 459e474

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The ultrastructure of Drosophila heart cells Christine Lehmacher, Bettina Abeln, Achim Paululat* Department of Biology, Zoology/Developmental Biology, University of Osnabrück, Barbarastraße 11, D-49069 Osnabrück, Germany

a r t i c l e i n f o

a b s t r a c t

Article history: Received 18 October 2011 Received in revised form 21 February 2012 Accepted 23 February 2012

The functionality of the Drosophila heart or dorsal vessel is achieved by contributions from several tissues. The heart tube itself is composed of different types of cardiomyocytes that form an anterior aorta and a posterior heart chamber, inflow tracts and intracardiac valves. Herein we present an in-depth ultrastructural analysis of all cell types present in the Drosophila heart at different developmental stages. We demonstrate that the lumen-forming cardiomyocytes reveal a complex subcellular architecture that changes during development. We show that ostial cells, for which it was previously shown that they are specified during embryogenesis, start to differentiate at the end of embryogenesis displaying opening structures that allow inflow of hemolymph. Furthermore we found, that intracardiac valve cells differentiate during larval development and become enlarged during the 3. instar larval stages by the formation of cellular cytoplasmic free cavities. Moreover we were able to demonstrate, that the alary muscles are not directly connected to the heart tube but by extracellular matrix fibers at any stage of development. Our present work will provide a reference for future investigations on normal heart development and for analyses of mutant phenotypes that are caused by defects on the subcellular level. Ó 2012 Elsevier Ltd. All rights reserved.

Keywords: Drosopohila Heart Dorsal vessel Circulatory system Cardiomyocytes Pericardial cells Alary muscles Ostia Ultrastructure

1. Introduction The embryonic and larval heart of Drosophila have been well proven in the past as a valuable model of cardiogenesis in general. More recently, the heart of the adult fly is increasingly considered as an experimentally treatable system to study clinical relevant cardiac disorders related to human genes with homologous in the fly genome. The development of new technologies, e.g. the noninvasive analysis of heart beating rates or heart malformations under various genetic or physiological conditions, by making use of high-speed video microscopy analysis (Ocorr et al., 2009; Paternostro et al., 2001), GFP-transgenes (Tao et al., 2007; Yi et al., 2006) or OCT (optical coherence tomography) (Choma et al., 2006, 2010), just to mention a few examples, made Drosophila an excellent choice to investigate normal and abnormal heart functionality. Although the availability of new technologies have significantly enhanced the attractiveness of the Drosophila “heart model”, the phenotypic characterization of heart malformations still requires high-resolution imaging techniques like TEM to analyze ultrastructural changes, e.g. in the architecture of the cytoskeleton of organelle organization within cardiomyocytes or associated * Corresponding author. Department of Biology, University of Osnabrück, Barbarastraße 11, D-49069 Osnabrück, Germany. Tel.: þ49 0 541 9692861; fax: þ49 0 541 9692587. E-mail address: [email protected] (A. Paululat). 1467-8039/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.asd.2012.02.002

structures. For example, one of the key proteins related to inheritable forms of Alzheimer disease is Presenilin, a transmembrane protein that is part of the gamma-secretase protease complex. Whereas humans have two presenilin genes (PSEN1 and PSEN2), Drosophila harbors only a single orthologe (dPsn). Overexpression or downregulation of dPsn results in cardiac dysfunction monitored by OCT and is associated with changes in the structure of Z-discs, density of myofibrils, swollen or degenerative mitochondria, appearance of vacuolar structures in cardiomyocytes of adult flies (Li et al., 2011), phenotypes that only can be visualized by TEM. This makes clear that a detailed knowledge of the normal ultrastructure of the cell types that constitute the heart of Drosophila at the different developmental stages, from embryogenesis to adulthood, is quite important for future investigations on the role of conserved genes that are relevant in cardiogenesis and cardiac function in flies and vertebrates. Cardiogenesis in flies begins during embryogenesis with the specification of the cell types that constitute the heart tube and associated tissue, including contractile cardiomyocytes, ostiaforming cardiomyocytes in the posterior heart proper, different types of pericardial cells that adhere to the heart tube and alary muscles that span the heart to epidermal attachment points. The newly hatched larvae possess already a functional heart that pumps the hemolymph from posterior to the anterior region of the animal. During larval growth the number of cardiomyocytes remains constant but each cell elongates dramatically accompanied by

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changes on the ultrastructural level, and the luminal space build by cardiomyocytes increases significantly. During metamorphosis the heart undergoes a remodeling process that finally results in a formation of the adult heart that remains unchanged until death. Comparing the embryonic and the adult heart, several significant differences can be found. First, the ultrastructure of the contractile cardiomyocytes changes. This includes the number of myofilaments, their position and orientation, the size, the number and location of mitochondria, the formation of the sarcoplasmic reticulum and the overall shape of the lumen-forming cells. In addition, a distinct number of developmentally arrested cardiomyocytes start to differentiate. These are the 4 anterior aortal pairs of ostial cells that form the anterior inflow tracts of the adult heart, together with the fifth larval pair that corresponds to the first inflow tract of the larval heart chamber, and the intracardiac valve cells that divide the adult heart into four chambers (Molina and Cripps, 2001; Monier et al., 2005; Sellin et al., 2006; Wasserthal, 2007; Zeitouni et al., 2007). Second, cells associated with the heart tube disappear or newly develop from precursors. From the 120e130 pericardial cells in the embryo only about 40e50 survive into adulthood (Alvarez et al., 2003; Sellin et al., 2006). 8 of them differentiate into the so-called wing hearts (Tögel et al., 2008), the others act as heart associated nephrocytes in the adult (Das et al., 2007, 2008; Weavers et al., 2008). Embryonic pericardial cells are similar in their ultrastructure compared to the adult pericardial cells, indicating, that they adopt full functionality early during differentiation. Furthermore, at the end of the 3. larval stage, longitudinal syncytial muscles, located ventrally to the heart tube, appear. Recently it was shown that these muscles derive from a population of cells originating from the lymph glands (Shah et al., 2011). Thirdly, the syncytial alary muscles, that span the heart tube laterally to the epidermis, change their type of contact to the heart tube (present work). Changes in the overall architecture of the Drosophila heart and changes on the ultrastructural level reflect the adaptation of the circulatory system to the different physiological requirements of the embryonic, larval and adult heart. Our present work aimed at the comparison of the ultrastructure of the different cell types constituting the heart at different developmental stages. It should be noted, that several authors have already analyzed the ultrastructure of Drosophila heart cells, but mainly focusing on certain cell types at certain developmental stages. E.g. pericardial cells were analyzed in adult flies or larvae but not at embryonic stages (Mills and King, 1965) or embryonic and larval stages were not considered (Curtis et al., 1999). In addition, morphologists have been interested in the ultrastructure of heart cells of various insects for a long time e but mostly excluded Drosophila from their investigations. Herein we present an in-depth ultrastructural analysis of all cell types present in the Drosophila heart at different developmental stages, thus our work may provide a reference for future studies on heart anatomy and malformations causing defects on the subcellular level. 2. Material and methods 2.1. Fly stocks w1118 was used as wild-type strain. For visualizing heart cells we used UASmCD8::GFP (Bloomington Stock center) crossed to hand CGal4 (our laboratory). We used hand C-GFP (Sellin et al., 2006) for phalloidin stainings.

2 h at 4  C. Embryos were postfixed in 1% osmium tetroxide for 1 h at 4  C, dehydrated in a graded ethanol series and infiltrated in three successive steps. Firstly, embryos were infiltrated in 100% acetone (2  5 min), secondly in a mixture of 100% acetone and Epon 812 (1:1) overnight at room temperature (RT) and thirdly in Epon 812 for 4 h and afterward overnight, each time at RT. Embryos were lastly embedded in Epon 812 and polymerized for 48 h at 60  C. Drosophila larvae, pupae and adults were fixed in a mixture of 1% glutaraldehyde and 4% formaldehyde in PBS overnight, for 4 h at room temperature and then at 4  C. To allow optimal penetration, larvae were prepared in the fixative by opening the ventral body side with a scalpel. Pupae were taped down, afterward the puparium was removed and the cuticle was perforated. After carbon dioxide anesthesia, the ventral and most anterior body side of adult Drosophila was cut off with a razor blade. After fixation larvae, pupae and adults were postfixed in 1% osmium tetroxide in PBS for 1e5 h at room temperature, dehydrated in a graded ethanol series and stored in 100% ethanol overnight at 4  C. After an intermediate step in a mixture of 100% ethanol and propylenoxide (1:1) for 10 min and two successive steps in propylenoxide for 5 min, the specimens were embedded in Epon 812. Polymerization was carried out at 60  C for 48e72 h. For light microscopy, the specimens were serially semi-thin sectioned (0.5e1.0 mm) with a histological diamond knife on a Leica Ultracut and stained with toluidine blue at 70 C for 2 min. Images were acquired using the Axiovision software (Zeiss, Germany) in conjunction with a Zeiss Axiocam MRc 5 digital camera mounted on a Zeiss Axioskop 2 microscope. Ultra-thin sections (70 nm) for transmission electron microscopy were performed with a diamond knife on a Leica Ultracut. Sections were mounted on one slot grids, contrasted with 2% uranyl acetate (30 min) and lead citrate (20 min) using the Nanofilm Surface Analysis. Specimens were investigated with a Zeiss 902 transmission electron microscope (60 kV). 2.3. Phalloidin and antibody staining, confocal microscopy Third instar larvae and adults were dissected in PBS, fixed in 4% Paraformaldehyde for 1 h, washed in PBS several times and stained with Phalloidin-TRITC (100 mg/5 ml EtOH, diluted 1:50 in PBS) for 1 h. Afterward the specimens were embedded in Fluoromount-G (SouthernBiotech). Images were captured with a confocal microscope, Zeiss LSM 5 Pascal (Osnabrueck) and Leica TCSAOBS SP2 at the Scientific-Technical Services, University of Oviedo. AntiPericardin (Hybridoma) was used at a dilution of 1:5. Stainings were performed as described (Albrecht et al., 2006). 2.4. 3-D reconstruction Series of semi-thin sections (0.5 mm) were photographed on a Zeiss Axioskop 2 with a 40  /0.75 Plan-Neofluar objective using the Axiovision software (Zeiss, Germany) and a Zeiss Axiocam MRc 5 digital camera. Images were aligned and processed using the software Amira 5.0.1 (Visage Imaging). For the reconstruction, distinct structures were manually outlined and subsequently rendered into a 3-D surface model using “unconstrained smoothing” option. 3. Results 3.1. Cardiomyocytes

2.2. Histology and electron microscopy Drosophila melanogaster embryos (white1118) were fixed in 2% glutaraldehyde and 1% osmium tetroxide in cacodylate buffer for

The 104 embryonic cardiomyoblasts differentiate into three different cell types, the contractile cardiomyocytes that form the heart lumen, segmentally arranged pairs of ostial cells that serve as

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hemolymph inflow devices and the intracardiac valves that divide the heart into chambers (see Fig. 1). The first two cell types can be distinguished early in the embryo by molecular markers, e.g. by the expression of the COUP-TF homologe Seven-up that is restricted to ostia-forming cells (Lo and Frasch, 2001), whereas all other cardiomyocytes express the NK-transcription factor Tinman (Zaffran et al., 2006). Three pairs of intracardiac valve cells differentiate during metamorphosis in the abdominal segments A2-A4 from Tinman expressing cardiomyocytes under the control of the PDGFVEGF pathway (Monier et al., 2005; Zeitouni et al., 2007). Furthermore, the posterior portion of the heart undergoes remodeling during metamorphosis, resulting in a changed overall number of functional ostia and the formation of a terminal heart chamber (Monier et al., 2005; Sellin et al., 2006).

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First we investigated the ultrastructure of the Tinman expressing contractile cardiac cells, which are 76 cells out of 104 in the embryo and 58 cells out of 84 in the adult fly. We found that these cells e at the late embryo e are not distinguishable from each other and from ostial or potentially existing valve progenitor cells with the exception, that functional ostia (the three pairs in the posterior heart proper) start to differentiate connecting channels between the heart lumen and the body cavity (Fig. 4AeB). At the end of embryogenesis, cardiomyoblasts harbor large numbers of mitochondria evenly distributed within the cell and the single nucleus is always located at the center and takes up more than half of the cell area (Fig. 2A). A typical basement membrane with the occurrence of hemi-adherens zones is found on both the luminal and abluminal side of the cardiomyoblast (Fig. 2C). Opposing cardiomyoblasts that

Fig. 1. (AeB) Schematic representation of heart cells and their differentiation process from embryonic to adult developmental stage. (A) Cardiomyoblasts differentiate into three different cell types: the contractive cardiomyocytes that form the heart lumen, ostial cells forming the inflow tracts and valve forming cardiomyocytes. Ostia-forming cardiomyocytes are characterized by a lateral entrance for an inflow of hemolymph during diastole (red arrows). Valve forming cardiomyocytes are characterized by cellular cavities (in green). (B) Schematic diagram of cardiomyocyte morphology during Drosophila development. During embryogenesis cardiomyoblasts form a heart lumen in between and thus organize a functional hemolymph circulation. Cardiomyoblasts stay in an undifferentiated stage until larval development begins. In 3. larva, muscular components are properly arranged and allow symmetrical contraction. After metamorphosis, mature cardiomyocytes are built up and indicate all characteristics of Drosophila wild-type heart muscle. Abbreviations: a ¼ anterior, A2eA4 ¼ abdominal segment 2e4, AJ ¼ adherens junction, p ¼ posterior. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 2. Differentation process of cardiomyoblasts into larval cardiomyocytes. (AeC) Cross ultra-thin sections through undifferentiated embryonic cardiac cells (cardiomyoblasts, cb) of the heart proper. The cbs are mononucleated cells and enclose the heart lumen. The cbs are dorsally and ventrally connected to each other by spot-adherens junctions (white arrows in A and detail B). Toward the luminal and abluminal cell side a layer of extracellular matrix (basement membrane) surrounds the cbs. Hemi-adherens junctions connect cbs toward the extracellular matrix (detail C). (DeD0 ) Cross ultra-thin sections through cardiomyocytes (cm) of the heart of 1. instar larva. The cms are thin and yet undifferentiated. Note that only a few circularly arranged myofilaments are present. (E) Phalloidin staining of F-Actin in cms demonstrates the myofilament array in the larval heart portion. Note the irregularities in the arrangement of myofilaments, several myofilaments are not orientated in a clear circular manner (white arrows). (F) Semi-thin section through the larval heart. The heart is located next to the tracheae. Cms are fully differentiated and generate contractive activity. (GeH) Cross ultra-thin sections through cms shown in F. (G) Irregularities in the myofilament array are visible. Circularly and irregularly arranged myofilaments alternate randomly within the cms. The cms show discontinuities in Z-disc formation (indicated by black arrows) and surrounding basement membranes toward luminal and abluminal side. (H) Larval cms are closely connected by characteristical junctional complexes. Black arrowheads indicate the firmly adhering myofibrils to the membranes at the junctional complex. Abbreviations: a ¼ anterior, AJ ¼ adherens junction, d ¼ dorsal, cm ¼ cardiomyocyte, hl ¼ heart lumen, m ¼ mitochondria, myo ¼ myofilaments, pc ¼ pericardial cell. Scale bars: A ¼ 2 mm, BeC ¼ 30 nm, D ¼ 2 mm, D0 ¼ 0.5 mm, EeF ¼ 20 mm, GeH ¼ 400 nm.

Fig. 3. Development of adult heart cells. (A) Cross semi-thin section through pupal cardiomyocytes (cm) in an early pupal stage, just 20 h after puparium formation. The gross morphology is comparable to the embryonic heart with laterally located alary muscles and ECM fibers. (BeC) Cross ultra-thin sections through pupal cms shown in A. (B) The basement membrane at both cell sides is thicker and denser compared to its appearance before metamorphosis. Numerous hemi-adherens junctions are located to both sides. Additionally, several patches of cross-sectioned myofilaments are present with a random arrangement in the cms (indicated by black arrows). Outpockets are completely absent. (C) Spot-adherens junctions connect the cms (both membranes are marked by black arrows). (D) Phalloidin staining of F-Actin shows the regular and circular arrangement of myofilaments within the cms. (E) Cross semi-thin section through adult cms located in abdominal segment 1. The cms form a dominant heart muscle layer with cell nuclei located in protrusions toward the luminal cell side. (FeJ) Cross and sagittal ultra-thin sections through adult cms shown in E. (FeF0 ) The cms are enlarged and more pronounced. Myofilaments are exclusively arranged in circular direction and Z-discs are discontinuous (indicated by black arrows) and regularly located between outpockets filled with numerous mitochondria. Sarcoplasmic reticulum is highly abundant within the I-band, whereas dyads are detectable throughout the sarcomeres. (G) Junctions comparable in structure to intercalated discs connect cms in adult hearts. Here, the cell membranes of two adjacent cms are extensively intertwined and bound together by adherens junctions. Myofibrils in the two interlocking cms are firmly anchored to the membrane at the junctional complex (indicated by black arrowheads). (H) Sagittal section through the t-system of adult cms. Numerous t-tubules are present throughout adult cms, visible as deep invaginations of the sarcolemma. (I) Note the co-formation of hemi-adherens junctions and Z-disc alignment. (J) Dyadic couplings frequently appear in adult cms. Here, the t-tubule is intimately associated with one terminal cisterna/sarcoplasmic reticulum. Abbreviations: a ¼ anterior, AJ ¼ adherens junction, APF ¼ after puparium formation, cm ¼ cardiomyocyte, fc ¼ fat cell, hl ¼ heart lumen, m ¼ mitochondria, pc ¼ pericardial cell, sr ¼ sarcoplasmic reticulum, vlm ¼ ventral longitudinal muscle. Scale bars: A ¼ 20 mm, BeC ¼ 400 nm, D ¼ 10 mm, E ¼ 20 mm, F ¼ 400 nm, F0 ¼ 200 nm, GeH ¼ 400 nm, IeJ ¼ 100 nm.

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Fig. 4. Ostial cells in the embryonic, larval and adult hearts. (AeB) Cross ultra-thin sections through ostial cells of a stage 16/17 embryo. The ostia of the heart proper were identified by counting the nuclei of cardiomyoblasts (cb) in serial sections starting from the posterior end of the heart. Note that ostial cells at this stage are morphologically identical to other cbs with the exception, that channel-like structures between two ostial cells forming one lateral inflow tract become visible (indicated by black arrowheads). (C) Schematic cross

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form the heart lumen in between are sealed at dorsal and ventral membrane domains by adherens junctions that appear in the TEM as attachment spots to the extracellular matrix (ECM) (Fig. 2B). A difference is seen in the extension of the dorsal and ventral adhesive domain between cardiomyoblasts in the aorta (small luminal diameter) and the heart proper (large luminal diameter). Cardiomyoblasts of the aorta exhibit a larger dorsal adhesive zone and a small ventral adhesive zone whereas the cardiomyoblasts of the heart proper display similar sized adhesive membrane domains. This observation is in accordance with a recent study, in which immunofluorescence staining on cryo-sections was used to study the distribution of membrane proteins at the luminal and adhesive membrane domains (Harpaz and Volk, 2012). During larval development, the cardiomyocytes differentiate and the three cell types that constitute the heart become unequivocally distinguishable in the TEM (see also next chapters). In the 1. instar larva, we found an enrichment of circularly arranged myofilaments in distinct areas of the heart cells, most abundantly seen in the dorsal regions of the cells (Fig. 2DeD0 ). Here, less than five parallel arranged myofilaments exist within the cross-sectioned heart muscle layer, resulting in an entire thin-shaped heart wall of the 1. instar larva. Myofilament formation proceeds during the next larval stages, accompanied by cell growth and enlargement of the heart lumen (Fig. 2F). At 3. larval stage all cardiomyocytes including the ostial and intracardiac valve cells, which are now morphologically clearly distinguishable from other cardiomyocytes, exhibit thin and thick filaments oriented in different directions. That means, that these myofilaments are irregularly arranged and alternate within the cardiac cells (Fig. 2E and G). Although the basic array is a circular one, several myofilaments change their well-regulated orientation due to the contraction state of the heart tube. The general organization of sarcomeres in heart cells is similar to what is described for striated muscle cells in insects (Auber, 1967; Crossley, 1968; Elder, 1975; Shafiq, 1963; Smith, 1966; Toselli and Pepe, 1968). The Aband is about 1 mm long; I-bands are present but not always evident in contracted sarcomeres. The Z-discs are not continuous across the sarcomere, and in contracted sarcomeres, thick myofilaments occur in the discontinuities in the Z-disc (Fig. 2G). Well-defined H-zones are not evident and M-lines are not seen. A basement membrane with a thickness of about w90 nm defines the luminal and abluminal cell surface of every cardiomyocyte. Large numbers of mitochondria are concentrated in outpockets of the sarcolemma on both the luminal and abluminal sides of the heart cell and the single nucleus is located in these cellular protrusions, but always in the luminal ones. Between outpockets the sarcolemma invaginates regularly, once per sarcomere. This type of invagination extends to the level of the first myofibril, enabling the cell membrane to make close contact with the outermost Z-disc. The basement membrane follows the invaginating plasma membrane. These characteristics

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also apply for cardiomyocytes in the adult fly, except for the arrangement of myofilaments (Fig. 3DeJ). In cardiomyocytes of the adult, the myofilaments are exclusively grouped in a highly regular and circular manner (Fig. 3D and F). Furthermore, the sarcotubular system is well developed both in the larval and adult cardiomyocytes (Fig. 3F0 and H). The sarcoplasmic reticulum reveals a characteristic arrangement: it envelopes the myofibrils at the Alevels and interweaves through the myofibrils at the I- and Z-levels. The T-tubules are flattened and sac-like, run transversely between the myofibrils and make dyadic couplings with the cisternae of the sarcoplasmic reticulum. These cisternae are filled with electrondense material, i.e. with junctional granules (Fig. 3J), which is in agreement with previous observations (Sommer and Johnson, 1969). Additionally, we found a close association between hemiadherens zones and Z-discs (Fig. 3I). This close physical relationship may constitute the structural basis for the requirement of Integrin for Z-disc formation (Volk et al., 1990). During metamorphosis the cardiomyocytes change their cellular organization. At 20 h after puparium formation (APF), myofilaments are organized in small patches, the luminal as well as the abluminal basement membrane can be well-defined and show a thickness of w150 nm, hemi-adherens junctions are abundant and spotadherens junctions connect cardiomyocytes with one another (Fig. 3AeC). Structures resembling vertebrate intercalated discs are found in the larval and in the adult heart at the regions where contiguous cardiomyocytes contact each other longitudinally as well as transversely. There is a narrow junctional cleft, which is open to the bulk extracellular fluid space (Fig. 2H, Fig. 3G). The intercalated discs are highly convoluted and tortuous in their path, and the disc membranes and adjoining areas are heavily stained in many regions. 3.2. Ostia A distinct subpopulation of cardiomyocytes differentiates into pairs of cells that form the ostia (inflow tracts) of the heart. The embryonic progenitors of the larval ostia are marked by the expression of ostia-specific factors (Svp, Transglutaminase) (Iklé et al., 2008; Molina and Cripps, 2001) but only the 12 posterior most out of 28 ostial cell progenitors differentiate into 3 pairs of functional larval ostia that are located in the posterior heart chamber. During metamorphosis the remaining (4 pairs of) ostial progenitors located within the anterior aorta region differentiate into functional ostial cells and together the first ostial cell pair in the larval heart chamber builds the fifth ostial cell pair of the adult heart. The posterior two pairs of ostia of the larval heart chamber are histolyzed (Fig. 4DeI) (Curtis et al., 1999; Monier et al., 2005; Sellin et al., 2006; Wasserthal, 2007; Zeitouni et al., 2007). As aforementioned, the anterior located embryonic ostial progenitors

view (based on semi-thin sections) through the second pair of larval ostial cells. One lateral ostial inflow tract (indicated by black arrow) is visible and located between anterior and posterior ostial cell. On the opposite site, hemocytes are already visible, but the ostial inflow tract appears more anterior. (C0 ) Cross ultra-thin section through the ostial cell shown in the scheme. Here, the muscular portion of the posterior ostial cell with its elongation toward the heart lumen or so-called posterior ostial lip is shown. (D) 2D-scheme of the adult heart. The scheme shows a dorsal view of the posterior portion of the heart including all differentiated cell types. The five sets of ostia are arranged in a segmental pattern (A1eA5). Cardiomyocytes are colored in white, ostial cells are colored in red and intracardiac valve cells are colored in green. (EeI) Serial, cross semi-thin sections through four of a total of 5 sets of ostial cells. The fifth ostial cell pair is shown in a sagittal overview. Ostia are characterized by invaginations and openings toward the heart lumen allowing an inflow of hemolymph (indicated by black arrows). (J) Cross semi-thin section through the second pair of ostial cells. The ostial lip of the right anterior ostial cell is visible as a protrusion projecting into the heart lumen (indicated by black arrowhead). Note the location of the small nucleus within the ostial lip. (K) Sagittal ultra-thin section through the fifth pair of ostial cells (also shown in I), two of four involved ostial cells are shown here. Anterior as well as posterior ostial lips are visible (indicated by black (anterior) and gray (posterior) arrowheads). Pericardial cells are preferentially seen ventrally to the ostial inflow tracts. (LeM) Sagittal ultra-thin sections through the ostium depicted in K. It is clearly visible that one ostial cell forms one invaginating ostial lip, because the junctional complex is present between two ostial cells in an anterior-posterior direction (indicated by the dashed line). Ultrastructurally, these ostial lips show regularly arranged myofilaments, discontinuous Z-discs and outpockets filled with mitochondria. Numerous hemi-adherens junctions are involved in ostial cell formation. Note also the interaction between Z-disc formation and the arrangement of the hemi-adherens junctions. Both anterior and posterior ostial lip meet each other over protrusion of the sarcolemma located regularly in the middle of each sarcomere (marked by black arrows). Abbreviations: a ¼ anterior, A1-A5 ¼ abdominal segment 1e5, am ¼ alary muscle, AJ ¼ adherens junction, C1-C4 ¼ heart chamber 1e4, fc ¼ fat cell, hl ¼ heart lumen, m ¼ mitochondria, O1eO5 ¼ ostial cell pairs 1e5, p ¼ posterior, pc ¼ pericardial cell, T3 ¼ thoracic segment 3, V1eV3 ¼ valve cell pairs 1e3, vlm ¼ ventral longitudinal muscle. Scale bars: AeB ¼ 2 mm, C ¼ 20 mm, C0 ¼ 2 mm, EeI ¼ 20 mm, J-K ¼ 10 mm, LeM ¼ 0.5 mm.

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Fig. 5. Valve cell formation in larval and adult hearts. (AeB) Phalloidin staining of the larval posterior aorta and heart demonstrates the position of the intracardiac valve located exactly between the two different heart portions. Note the densely packed F-Actin network compared to the surrounding cardiomyocytes. (C) Cross semi-thin section through the larval valve. Two valve cells form one functional valve (cell junctions are indicated by black arrowheads) and have several cellular cavities toward the luminal cell side. Note the peripheral location of the cell nuclei which are separated by the cellular cavities from the basic heart muscle layer. (C0 ) Cross ultra-thin section through the larval valve cells. Note

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are indistinguishable from cardiomyoblasts on the ultrastructural level although already identifiable by the expression of specific marker genes. In contrast, the posterior ostial cells display first morphologically changes at the end of embryogenesis. We found that the cell connection between two ostial cells lying in a row begins to disassociate so that channel-like structures between these cells become visible (Fig. 4AeB). Therefore we conclude that the differentiation of ostia starts at late embryonic stage. Previous work on Drosophila and other insects revealed that the ostia in adults exhibit a specific architecture that allow one way inflow of hemolymph into the heart lumen (Curtis et al., 1999; Glenn et al., 2010; Wasserthal, 2007). We found that the functional ostia of the larvae and of the adults reveal an identical architecture, both on the cellular and the subcellular level, indicating that the specific architecture of ostia is established during larval development (Fig. 4CeC0 ). Respectively two ostial cells form a lockable opening in the heart tube with their ostial lips that extend into the cardiac lumen (Fig. 4JeK). Both anterior as well as posterior ostial lips show an identical muscle pattern with regular sarcomeres limited by discontinuous Z-discs and outpockets to the luminal side (Fig. 4M). However, a basement membrane surrounds the sarcolemma of the ostial lips toward the cell surface. The cell nuclei of ostial cells are always found within the cytoplasma of the lips. These nuclei are smaller compared with those in contractive cardiomyocytes and have a round shape compared with cylindrically formed nuclei in contractive cardiomyocytes. A sagittal section of an ostial cell of the adult heart shows, that the myofilaments of the ostial lips are orientated perpendicular/orthogonally to the myofilaments of the major ostial cell body (Fig. 4L). Upon contraction, this arrangement of myofilaments might be essential to close ostia upon the systolic phase. 3.3. Intracardiac valve Intracardiac valve cells are supposed to be essential for supporting directed hemolymph flow from the posterior heart proper toward the anterior aorta (Zeitouni et al., 2007). The heart of the 3. instar larva harbors one pair of bilaterally located valve cells forming one single valve in abdominal segment A4 which separates the posterior heart chamber and the aorta portion of the heart (Fig. 5AeB). In the adult, three valves, formed by 3 pairs of valve cells, appear in the abdominal segments A2, A3 and A4 respectively and define the four heart chambers. It is currently not known, how the valve cells differentiate, for instance, whether cardiomyoblasts in the embryo are already specified as valve cell progenitors. First we analyzed and compared the larval valve cells with those found in the adult. The valve cells at both stages are much more awkwardly shaped than the neighboring cardiomyocytes due to large cytoplasmic free membrane lined cavities within the cells (Fig. 5CeD and GeI). Another characteristic feature of the valve cells is the appearance of a high number of mitochondria mostly assembled into groups indicating a significant demand for energy (Fig. 5C0 and I). Furthermore valve cells show a characteristic network-like arrangement of myofilaments that make them easily

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distinguishable from other cardiomyocytes (Fig. 5A, B, E and F). The valve cells in the larvae and in the adults share these features, indicating that the functional architecture is the same. We also analyzed the heart of late stage embryos for the presence of cells with valve cell characteristics. Due to the constant number of cardiac cells that constitute the heart tube in the embryo and the larva, it is reasonable to argue that the two valve cells differentiate from existing cardiomyoblasts already present in the embryo. Therefore we used serial sections through the relevant heart regions to verify, if cells with valve characteristics are present such early. This is not the case and therefore we conclude that valve cell differentiation starts postembryonically. 3.4. Pericardial cells At the end of embryogenesis three types of pericardial cells (pccells) associate with the dorsal vessel (Fig. 6AeB): a) dorsally located Even-skipped/Tinman expressing cells (2 per abdominal hemisegment), b) ventrally located Tinman expressing cells (4 per abdominal hemisegment) and c) laterally located odd-skipped expressing cells (4 per abdominal hemisegment) (Ward and Skeath, 2000). The last group persists into larval and adult stages and was recently characterized as nephrocytes, a function which was already assumed from previous studies in various insects (Crossley, 1972, 1985; Das et al., 2007, 2008; Fife et al., 1987; Mills and King, 1965; Weavers et al., 2008; Wigglesworth, 1970). The majority of all other embryonic pericardial cells has a so far unknown function and disappears after embryogenesis with the exception of 8 even-skipped pc-cells that give rise to the wing hearts (Lehmacher et al., 2009; Tögel et al., 2008). Therefore we asked, whether the different embryonic pericardial cell subpopulations, which can be identified in TEM-sections due to their position, differ in their ultrastructural features. The second question we aimed to answer was, whether embryonic, larval and adult pericardial cells are differentially organized on the ultrastructural level. We found that from 1. instar larva onwards all pericardial cells share the same principal subcellular organization (Fig. 6FeJ0 ). The organelles are arranged in concentric zones e starting with the outer cortex and ending with a single nucleus in the center of the cell. The cortex is composed of labyrinthine channels and associated organelles. A basement membrane separates the labyrinthine channels from the hemolymph, but does not line the channels. Apposed plasma membranes at the entrance of the channels are bonded/sealed by a desmosome-like structure called surface junctional complex (Fig. 6F and J). This complex appears to control the inflow of substances, of certain molecules into the labyrinthine channels. The channels are extensive in this area and between them clusters of membranous tubules (with diameters about 320 nm) can be seen. Three types of vacuoles, labeled a, b and g can be recognized on the ultrastructural level (a vacuoles are confined to the cortex of the cell, Fig. 6GeI). Vesicular invaginations (coated pits/vesicles) of the plasma membrane occur along the labyrinthine channels (Fig. 6J0 ). Adjacent to the cortex zone, we found the transitional zone (Fig. 6G, visible in larval cells). Knots of tortuous

the enrichment of mitochondria within the cell and the distribution of myofilaments between cellular cavities. (D) 3-D reconstruction of the larval valve based on serial sections. Each valve cell builds up one central cellular cavity surrounded by smaller ones. Cellular cavities are colored in yellow (right cell) and in green (left cell), cell nuclei are colored in blue (right cell) and in red (left cell). (EeF) Phalloidin staining of the adult heart demonstrates the position of both valve cells of the third valve. The staining reveals an increased overall volume of these cells compared with other cardiomyocytes. (GeG0 ) Cross ultra-thin sections through the third adult valve. Both valve cells contain several cellular cavities toward the luminal cell side. Myofilaments are present in patches between the cellular cavities. Cell nuclei are located to the heart lumen. (H) Sagittal semi-thin section through the third adult valve. Two valve cells forming one functional valve are consecutively arranged in the anterior-posterior direction. The cell bodies extend into the heart lumen and lead to an increasing cell volume. Several cellular cavities inside the cells are visible. (I) Sagittal ultra-thin section through posterior valve cell shown in H. Patches with numerous mitochondria are visible throughout the valve cell. Cell nuclei are located on the luminal side and myofilaments stretch inside the whole cell body. Abbreviations: a ¼ anterior, hl ¼ heart lumen, m ¼ mitochondria, myo ¼ myofilaments, n ¼ cell nucleus, pc ¼ pericardial cell, V3 ¼ valve cell pair 3. Scale bars: A ¼ 50 mm, B ¼ 10 mm, C ¼ 10 mm, C0 ¼ 2 mm, E ¼ 50 mm, F-G ¼ 10 mm, G0 ¼ 2 mm, H ¼ 20 mm, I ¼ 1 mm.

Fig. 6. The pericardial cells in Drosophila melanogaster. (AeB) Cross ultra-thin sections through the embryonic heart (stage 17). Undifferentiated cardiomyoblasts form the heart lumen and are flanked by three types of mononucleated pericardial cells (pc), also visible in the accordant schemes. Even-skipped expressing pcs are located dorsally (A), Odd-skipped expressing pcs are located laterally and Tinman expressing pcs are located ventrally to the cardiomyoblasts (B). (CeE) Cross ultra-thin sections through embryonic pcs. Despite their different expression pattern all embryonic types of pcs are morphologically identical with one cell nucleus located in the center that occupies most of the cell volume. Additionally, patches of rough endoplasmic reticulum encircle the nucleus and several mitochondria are randomly distributed throughout the cell body. (FeJ0 ) Cross ultra-thin sections through larval (FeG), pupal (H) and adult (IeJ0 ) pcs. In the 3. larvae, pcs appear to be fully differentiated, visible in their ultrastructure, and maintain their morphology until death. The cortex of pcs harbors labyrinthine channels formed by infoldings of the plasma membrane, between which lie a-vacuoles (J). Note the thick basement membrane as well as numerous coated pits and vesicles forming on labyrinthine channels (F, J and J0 ). Passing inwards in survey, a transitional zone containing mitochondria and tortuous tubules (G and H) merges into a zone of larger vacuoles (b- and g-vacuoles, I). The central part or perinuclear zone of the cell is occupied by endoplasmic reticulum (F0 ) with granules believed to be glycogen. Abbreviations: av ¼ alpha vacuole, am ¼ alary muscle, APF ¼ after puparium formation, bm ¼ basement membrane, cb ¼ cardiomyoblast, cv ¼ coated vesicle, eve ¼ Even-skipped, hl ¼ heart lumen, jc ¼ junctional complex, lc ¼ labyrinthine channel, m ¼ mitochondria, odd ¼ Odd-skipped, pc ¼ pericardial cell, pm ¼ plasma membrane, rER ¼ rough endoplasmic reticulum, te ¼ tubular element, tin ¼ Tinman, tt ¼ tortuous tubule. Scale bars: AeB ¼ 5 mm, CeE ¼ 600 nm, F ¼ 0.5 mm, F0 ¼ 1 mm, GeH ¼ 1 mm, I ¼ 2 mm, JeJ0 ¼ 400 nm.

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Fig. 7. Morphology of alary muscles and their organization regarding heart formation. (AeC) Schematic diagrams of the heart and its associated tissues from embryogenesis to adulthood based on cross ultra-thin sections. Muscles tissue is colored in red, pericardial cells are colored in gray and tracheae are marked in green. In the embryonic as well as in the larval heart alary muscles (am) stretch laterally toward the lateral epidermis. ECM fibers surround pericardial cells (pc) in a network-like manner and attach finally to the heart cells (A and B). In the adult heart ams stretch dorsally toward the dorsal epidermis and ECM fibers extend between the alary and the cardiac muscles. Here, pericardial cells are located in the open body cavity, are excluded from the muscle-heart-complex and are loosely associated with the heart. (DeD0 ) 3-D reconstruction of the most posterior embryonic alary muscles (based on w50 cross ultra-thin sections). The reconstruction verifies the syncytial organization of the ams and shows clearly the interaction zones between ams, the ECM fiber-network and pcs (D0 ). Ams and nuclei are colored in red, ECM fibers are colored in yellow and pcs are colored in blue. (E) Cross ultra-thin section through embryonic am also used for 3-D reconstruction. Note the syncytial formation of ams with three cell nuclei without separated membranes in between. Myofilaments within the am are not differentiated. (F) Cross ultra-thin section through the ECM fiber-network and the adjacent pc. Close to the ventral pc the ECM fiber-network splits into different directions and encircles the pc. (G) Phalloidin and Pericardin staining of the larval heart revealed that the ECM fibers encircle the pcs and connect both muscle tissues in a network-like manner. Direct muscle to muscle connections between alary and heart muscle are absent. (H) Cross ultra-thin section through one larval am. Adherens zones are formed by the am where the muscle itself ends and the ECM fiber stretches toward the heart muscle. The am is completely differentiated, myofilaments are longitudinally arranged according to the lateral formation of the muscle and Z-discs are discontinuous. (I) The cross ultra-thin section through one larval ECM fiber shows several elastic components visible as electron-dense material. (JeJ0 ) 3-D reconstruction of the third pair of adult ams. Note the different formation of adult ams regarding the overall architecture. The ams suspend between ventral longitudinal muscle and dorsal epidermis and the ECM fibers suspend between am and cardiomyocyte. Consequently, the ams surround the heart muscle toward its lateral surface. Muscle tissues are colored in red, ECM fibers are colored in rose. (K) Cross ultra-thin section through one am of the adult fly. Adherens zones are formed where the muscular portion is terminated. (L) Ultrastructurally, the adult fiber structure is identical to the larval one. Abbreviations: a ¼ anterior, am ¼ alary muscle, cb ¼ cardiomyoblast, eve ¼ even-skipped, hl ¼ heart lumen, m ¼ mitochondria, n1e3 ¼ nucleus 1e3, pc ¼ pericardial cell, t ¼ trachea. Scale bars: AeC ¼ 10 mm, E ¼ 3 mm, F ¼ 1 mm, G ¼ 5 mm, H ¼ 1 mm, I ¼ 150 nm, K ¼ 1 mm, L ¼ 150 nm.

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tubules and mitochondria appear in this transitional zone between labyrinthine channels (exterior surface) and endoplasmic re-ticulum occupying the center of the cell. Tortuous tubules (with diameters of about 530 nm) are elongated, membrane bound, cisternae containing material with affinity for heavy metal salts. Passing in survey from the transitional zone toward the nucleus, a region of heterogeneous vacuoles (e.g. b and g-vacuoles) is entered (Fig. 6I, visible in adult cells). The following perinuclear zone is relatively free from membrane bound vacuoles, but contains stacked cisternae of ribosome-studded endoplasmic reticulum in abundance (Fig. 6F0, visible in larval cells). Prior to that, in embryonic pericardial cells the nuclei take up most of the cellular volume and are associated with several mitochondria and rough endoplasmic reticulum (Fig. 6CeE). We found no morphological differences between the three types of embryonic pc-cells, apart from their defined position toward the cardiomyoblasts (Fig. 6C (dorsal), D (lateral) and E (ventral)). During their differentiation process pericardial cell specific organelles develop and characterize these podocyte-like cells in the following developmental stages from larva onwards. From this point of time, we found no morphological differences throughout development with an identical zonation in every stage from the cortex to the perinuclear zone. 3.5. Alary muscles and their 3-D-fiber-network 7 pairs of alary muscles attach to the dorsal vessel of the embryo and larvae (Bate, 1993; Curtis et al., 1999; Dulcis and Levine, 2003; LaBeau et al., 2009; Rizki, 1978). 3 of them connect to the posterior heart proper and 4 to the anterior larval aorta in a segmental arrangement. In the adult, at least 4 pairs of alary muscles are visible. 2 smaller were found in the third and fourth abdominal segment and two larger in the fifth segment. The most terminal part of the heart is attached toward the epidermis presumably by non-cellular structures. Our TEM analyses of 3. instar larvae revealed that two structural components are essential to form the connection between the heart and the body wall e on the one hand, the alary muscle itself and on the other hand, the ECM fibernetwork (Fig. 7B). The muscular component stretches bilaterally from the body wall (myotendinuous junctions are present, but data are not shown) toward the dorsal midline, but does not adhere directly to cardiomyocytes. Such direct cellecell contact zones were not found at any stage of differentiation from embryo to adulthood. Instead, the non-cellular fibers (ECM) form the connection between the alary and the heart muscle cell by surrounding and enveloping the pericardial cells (Fig. 7G). Alary muscles and cardiomyocytes form adherens zones where the terminal myofilaments end in (in the case of muscle cells) and the heterogeneous collagen fibernetwork starts (Fig. 7H). Additionally, we found elastic fibers (visible as black bands) within the ECM fiber (Fig. 7I). Together, we suggest that the ECM fiber-network represent an indirect cell-to-

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cell contact between two contractive muscle layers in order to make the muscular interaction flexible and elastic. Like skeletal muscle cells the alary muscle cells are multinucleated with at least 3 nuclei per cell and coated by a basement membrane (already visible in the embryo; Fig. 7DeF). The overall architecture of alary muscles in the embryo is identical to the larval ones with the exception, that muscle components are so far not differentiated. After differentiation most of the muscle cell volume is taken up by numerous myofilaments and abundant mitochondria, which typically acculumate, often pairwise, in outpockets. All myofilaments are longitudinally arranged according to their longitudinal elongation and grouped in regular sarcomeres including discontinuous Z-discs. A tubular system (t-system) formed by invaginations of the sarcolemma and a sarcoplasmic reticulum is found, but both structures are less developed compared to the organization in cardiomyocytes. Next we investigated the ultrastructure of alary muscles in adult flies. We found that the inner organization as well as the inner fiber structure is similar to the larval ones (Fig. 7KeL). Differences are seen in the overall architecture. The alary muscles in the adult connect the dorsal vessel toward the dorsal epidermis instead of projecting laterally as seen in larvae. Pericardial cells are located separately from the muscular complex in the adult (Fig. 7C). To demonstrate the interaction between two muscle tissues and the ECM fiber-system we made a 3-D reconstruction through one adult abdominal segment (Fig. 7JeJ0 ). The ECM fibers of the adults are not organized as a network system, but rather function as suspending strands between heart and alary muscle. As previously shown by Miller (1950) and Wasserthal (2007), the anterior portion of the adult heart is completely separated from the open body cavity by muscle cells whereas the posterior part is only incompletely separated. Our analysis showed that two different syncytial muscle types contribute to the formation of sinus-like structures: 1) on the one hand, the alary muscle cells and 2) on the other hand, the ventral longitudinal muscle bundles (see following section). 3.6. Ventral longitudinal muscles During metamorphosis a single layer of longitudinal striated muscles appears at the ventral surface of the heart that corresponds to the dorsal diaphragm described, e.g. by Miller (1950) and others . Recent studies showed that these muscles originate from a subset of larval lymph gland-like cells (Shah et al., 2011). Our TEM analysis of cross-sectioned adult flies revealed, that ten to twelve parallel arranged longitudinal muscles run along the ventral side of the dorsal vessel (Fig. 8AeC0 ). In anterior to posterior direction, we found that at least two muscle fibers in a row stretch along the ventral side of the abdominal heart (“data not shown”). The myofilaments show a striated pattern in longitudinal direction according to the extension of the muscle. Within each fiber

Fig. 8. Ventral longitudinal muscles are formed during metamorphosis. (AeA0 ) Cross semi-thin sections through the fourth abdominal segment (A4). Twelve bundles of ventral longitudinal muscles (vlm) are located ventrally toward the heart muscle (indicated by black arrows). (B) 3-D reconstruction based on a total of w80 cross semi-thin sections illustrates the arrangement of the vlms, the adult heart and its associated structures in A4. Also twelve cross-sectioned vlms are located ventrally to the heart tube and are arranged in regular parallel muscle fibers (colored in red). Note that the heart cells are delimited to the body cavity by these vlms and by the adjacent alary muscles (colored in pink) which laterally surround the heart. (CeC0 ) Phalloidin staining of vlm demonstrates their formation pattern ventral to the heart muscle (ventral view). (D) Sagittal ultra-thin section through an individual vlm. Myofilaments are longitudinally arranged, sarcomeres are regularly organized with discontinuous Z-discs and several mitochondria are randomly distributed within the muscle bundles (outpockets are completely absent, although the muscle is fully contracted). (E) Cross ultra-thin section through one nerve bundle, innervating the vlms. The innervation of vlms is a direct one, indicated by the presence of neurohaemal axons in the nerve endings (white arrowheads). (FeH) Cross ultra-thin sections through the most centrally located vlms along the dorsal midline. The multinucleated organization of the vlms is distinguishable by the presence of two nuclei located to the non-cardiac side within one cross-sectioned vlm. Additionally, vlms show identical left-right as well as dorsal-ventral dimensions indicating that all vlms are equally in size. The extracellular matrix (ECM) is moderately enriched within the interspaces between vlms and cardiac muscle and both muscle tissues are connected to the ECM by hemi-adherens junctions (indicated by black arrowheads). (I) Sagittal ultra-thin section through an individual vlm located at the periphery. The section verifies the multinucleated organization of every vlm and demonstrates the apical location of cell nuclei within the vlm. Abbreviations: a ¼ anterior, AJ ¼ adherens junction, am ¼ alary muscle, d ¼ dorsal, fc ¼ fat cell, hl ¼ heart lumen, m ¼ mitochondria, vlm ¼ ventral longitudinal muscle. Scale bars: A ¼ 25 mm, A0 ¼ 10 mm, C ¼ 50 mm, C0 ¼ 10 mm, D ¼ 2 mm, E ¼ 1.5 mm, F ¼ 1 mm, G ¼ 0.5 mm, H ¼ 300 nm, I ¼ 0.5 mm.

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continuous sarcomeres are present, outpocketing is absent, mitochondria are randomly distributed and nuclei are located peripherally in swellings toward the body cavity (Fig. 8D and F). The main features of typical sarcomeres are apparent: namely, A- and Ibands, discontinuous Z-discs, a system of lateral tubules arising as invaginations of the plasma membrane portion of the sarcolemma and dyads formed by the T-system and elements of the sarcoplasmic reticulum. The syncytial organization of the longitudinal muscles is depicted in cross as well as in sagittal sections (Fig. 8G and I). Here, the nuclei lie side by side without being separated by sarcolemma. In both transverse and longitudinal directions, the syncytial organization is clearly visible, indicating cell fusion events of more than three precursor cells. Additionally, the extracellular matrix is more densely packed between the heart and the longitudinal muscles, appearing to separate both muscle layers (Fig. 8H). Small nerve bundles are frequently found near the ventral longitudinal muscle cells and neurohaemal vesicles are often abundant in the nerve endings (Fig. 8E). 4. Discussion In this study we used transmission electron microscopy, fluorescence microscopy and 3-D reconstructions based on serial sections to characterize ultrastructural changes of Drosophila heart cells during development. The Drosophila heart tube is build up by cardiomyocytes with common but also three different major functionalities. All heart cells contribute to lumen formation, a process that takes place during embryogenesis and which is driven by the establishment of specific membrane compartments at the later luminal side of the early cardiomyoblast. Adhesive junctional membrane domains at the dorsal and ventral contact zone seal the luminal space between two opposing cardiomyocytes. The cell membranes facing the lumen are defined by the accumulation of repellents and attractants such as Slit, Robo, Unc5 and Netrin (Albrecht et al., 2011; MacMullin and Jacobs, 2006; Medioni et al., 2008; Qian et al., 2005; Santiago-Martínez et al., 2008). How these proteins are sorted and directed to certain membrane compartments of heart cells is not yet understood. Our present data as well as work from many others showed, that all cardiomyocytes display circularly arranged myofilaments that allow contraction and thereby hemolymph transport through the heart lumen. Comparing embryonic, larval and adult stages it becomes clear, that the number and density of myofilaments increases but not their principal overall arrangement and orientation (Figs. 2and 3). Circular myofilament architecture in heart cells is also described for closely related dipteran species including Protophormia terraenovae (Angioy et al., 1999) and Calliphora erythrocephala (Wasserthal, 1999), whereas helically oriented muscles were found in the phylogenetically more distant located mosquito Anopheles gambiae (Glenn et al., 2010). Such helical orientation of myofilaments is also seen in the Drosophila larval heart (Fig. 2) but we interpret these irregularities as being partly dependent on the contraction and elongation state of the heart (see results). Nevertheless, the circular arrangement of the myofilaments in the larvae is not as regular as in adult heart cells. Both phalloidin stainings and ultrastructural analyses verify these structural differences. Studies by Shah et al. also show a more “irregular” myofilament array in larval heart cells than in adult cardiomyocytes (Shah et al., 2011). Distinct functionalities of subsets of heart cells are reflected by a specific shape and ultrastructure. 28 embryonic cardiac cells out of 104 are known to represent progenitors of ostial cells from which the most posterior 12, those that belong to the heart chamber portion of the heart, differentiate into 3 pairs of functional ostia providing the hemolymph inflow tracts of the larvae (Curtis et al.,

1999; Iklé et al., 2008; Molina and Cripps, 2001; Monier et al., 2005; Sellin et al., 2006; Wasserthal, 2007; Zeitouni et al., 2007). Due to remodeling processes during metamorphosis, the adult heart contains 5 pairs of functional ostia. We found that larval ostia differentiation starts at the end of embryogenesis as seen by the occurrence of channels between two ostial cells that connect the inner heart lumen with the body cavity. The architecture, which is characterized by the appearance of ostial lips that extend toward the cardiac lumen, is identical in larval and adult ostia and also basically identical in other insects (Curtis et al., 1999; Glenn et al., 2010; Wasserthal, 2007). Thus our data confirm and extend previous observations. A third type of functionality comes from intracardiac valve cells that were previously identified in Drosophila (Miller, 1950; Rizki, 1978; Zeitouni et al., 2007). Intracardiac valves are known from several other hexapod species as being important to regulate hemolymph flow inside the heart, e.g. two such valves with flap like extensions located in the thoracic and abdominal portion of the heart in the Diplurn Campodea augens channel the hemolymph flow (Gereben-Krenn and Pass, 2000). Cushion-like cell evaginations projecting toward the lumen were also found in Calliphora, where they probably play a role in regulation hemolymph passage (Wasserthal, 1999). Other structures, such as cellular pads between ostia, might have similar functions (Pass et al., 2006). Because of the lack of detailed knowledge on the ultrastructure and their developmental origin it is impossible to compare the intracardiac valve cells found in Drosophila with the situation in other species. Nevertheless, we found, that the valve cells in Drosophila exhibit large cytoplasmic free cellular cavities that differentiate in a so far unknown process during larval development and remain present during adulthood. Pericardial cells, that are aligned along the heart tube, were recently characterized as podocyte-like cells with a filtration slit apparatus (Weavers et al., 2008). Although three types of pericardial cells originate during embryogenesis and are characterized by the expression of different molecular markers, our analysis showed that they are indistinguishable on the ultrastructural level. Furthermore we found, that the ultrastructure of larval and adult pericardial cells is the same, with an extensive labyrinth channel system and other cellular characteristics typically found in endocytic active cells. These observations indicate, that the function of pericardial cells as nephrocytes starts during embryogenesis and is maintained to adulthood. During metamorphosis neither morphological nor functional changes affect pericardial cells in Drosophila. Two types of muscles contribute to the architecture of the adult heart, which are the alary muscles present already in the embryo and longitudinal muscles that line the ventral abdominal portion of the heart tube only in the adult fly. Our results confirm previous observations on the syncytial nature of both muscle types (LaBeau et al., 2009). The alary muscles arise from the fusion of at least three myoblasts and attach to the heart tube by fibrillar strands, similarly to that what is found in other insects (Sanger and McCann, 1968; Wasserthal and Wasserthal, 1977). The longitudinal myofilament arrangement in alary muscles antagonizes with the circular arranged myofilaments in cardiomyocytes and may provide tension during heart contraction to support the diastolic phase of heart wall movement. Because ostia are located near the center of each basket formed by the alary muscles and the connective strands, it seems reasonable to follow the hypothesis by Glenn and others, that alary muscles may help to control opening and closing of ostia as well (Glenn et al., 2010). The fact that alary muscles are one of the first muscles which differentiate during embryogenesis (Rugendorff et al., 1994), may indicate that they are also crucial for the establishment of the overall heart architecture.

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Ventral longitudinal muscles below the heart tube in Drosophila definitely contain more than three nuclei per longitudinal fiber. Therefore, many fusion events have to occur during metamorphosis and represent an excellent model system to investigate fusion mechanisms of adult musculature. Precursors of this skeletal-like muscle type originate from several lymph gland-like cells (Shah et al., 2011). Thus, the transformation from non-muscle cells into syncytial muscle fibers makes this system also so interesting. Because of the importance of Drosophila as a genetically treatable model system for heart development and function we hope that our present work might help to analyze and interpret future studies on heart malformations that have an impact on the cellular ultrastructure. Acknowledgments We thank K. Etzold for excellent technical assistance and Esther Maria Alcorta (University of Oviedo, Spain) for allowing B.A. to learn cryo-immunostainings in her lab and Marta Alonso Guervós from the Scientific-Technical Services in Oviedo. This work was supported by grants from the DFG to A.P. (PA517/10-1) and the EUNetwork of Excellence MYORES. References Albrecht, S., Altenhein, B., Paululat, A., 2011. The transmembrane receptor Uncoordinated5 (Unc5) is essential for heart lumen formation in Drosophila melanogaster. Developmental Biology 350, 89e100. Albrecht, S., Wang, S., Holz, A., Bergter, A., Paululat, A., 2006. The ADAM metalloprotease Kuzbanian is crucial for proper heart formation in Drosophila melanogaster. Mechanisms of Development 123, 372e387. Alvarez, A.D., Shi, W., Wilson, B.A., Skeath, J.B., 2003. pannier and pointedP2 act sequentially to regulate Drosophila heart development. Development 130, 3015e3026. Angioy, A.M., Boassa, D., Dulcis, D., 1999. Functional morphology of the dorsal vessel in the adult fly Protophormia terraenovae (Diptera, Calliphoridae). Journal of Morphology 240, 15e31. Auber, J., 1967. Distribution of two kinds of myofilaments in insect muscles. American Zoologist 7, 451e456. Bate, M., 1993. The mesoderm and its derivatives. In: Bate, M., Martinez Arias, A. (Eds.), The Development of Drosophila melanogaster. Cold Spring Harbor Laboratory Press, Plainview, N.Y, pp. 1013e1090. Choma, M.A., Izatt, S.D., Wessells, R.J., Bodmer, R., Izatt, J.A., 2006. Images in cardiovascular medicine: in vivo imaging of the adult Drosophila melanogaster heart with real-time optical coherence tomography. Circulation 114, e35e36. Choma, M.A., Suter, M.J., Vakoc, B.J., Bouma, B.E., Tearney, G.J., 2010. Heart wall velocimetry and exogenous contrast-based cardiac flow imaging in Drosophila melanogaster using Doppler optical coherence tomography. Journal of Biomedical Optics 15, 056020. Crossley, A.C., 1968. The fine structure and mechanism of breakdown of larval intersegmental muscles in the blowfly Calliphora erythrocephala. Journal of Insect Physiology 14, 1389e1407. Crossley, A.C., 1972. Ultrastructure and function of pericardial cells and other nephrocytes in an insect e Calliphora erythrocephala. Tissue & Cell 4, 529e560. Crossley, A.C., 1985. Nephrocytes and pericardial cells. In: Kerkut, G.A., Gilbert, L.I. (Eds.), Comprehensive Insect Physiology, Biochemistry, and Pharmacology. Pergamon Press, Oxford, pp. 487e516. Curtis, N.J., Ringo, J.M., Dowse, H.B., 1999. Morphology of the pupal heart, adult heart, and associated tissues in the fruit fly, Drosophila melanogaster. Journal of Morphology 240, 225e235. Das, D., Aradhya, R., Ashoka, D., Inamdar, M., 2007. Post-embryonic pericardial cells of Drosophila are required for overcoming toxic stress but not for cardiac function or adult development. Cell & Tissue Research 331, 565e570. Das, D., Aradhya, R., Ashoka, D., Inamdar, M., 2008. Macromolecular uptake in Drosophila pericardial cells requires rudhira function. Experimental Cell Research 314, 1804e1810. Dulcis, D., Levine, R.B., 2003. Innervation of the heart of the adult fruit fly, Drosophila melanogaster. Journal of Comparative Neurology 465, 560e578. Elder, H.Y., 1975. Muscle structure. In: Usherwood, P.N.R. (Ed.), Insect Muscle. Academic Press, London, New York, San Francisco, pp. 1e74. Fife, H.G., Reddy Palli, S., Locke, M., 1987. A function for pericardial cells in an insect. Insect Biochemistry 17, 829e840. Gereben-Krenn, B.-A., Pass, G., 2000. Circulatory organs of abdominal appendages in primitive insects (Hexapoda: Archaeognatha, Zygentoma and Ephemeroptera). Acta Zoologica 81, 285e292.

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