Drosophila Dorsal Vessel Research Articles

Enabled/VASP is required to mediate proper sealing of opposing cardioblasts during Drosophila dorsal vessel formation.

The Drosophila dorsal vessel (DV) is comprised of two opposing rows of cardioblasts (CBs) that migrate toward the dorsal midline during development. While approaching the midline, CBs change shape, enabling dorsal and ventral attachments with their contralateral partners to create a linear tube with a central lumen. We previously demonstrated DV closure occurs via a "buttoning" mechanism where specific CBs advance ahead of their lateral neighbors, and attach creating transient holes, which eventually seal. Here, we investigate the role of the actin-regulatory protein enabled (Ena) in DV closure. Loss of Ena results in DV cell shape and alignment defects. Live analysis of DV formation in ena mutants shows a reduction in CB leading edge protrusion length and gaps in the DV between contralateral CB pairs. These gaps occur primarily between a specific genetic subtype of CBs, which express the transcription factor seven-up (Svp) and form the ostia inflow tracts of the heart. In WT embryos these gaps between Svp+ CBs are observed transiently during the final stages of DV closure. Our data suggest that Ena modulates the actin cytoskeleton in order to facilitate the complete sealing of the DV during the final stages of cardiac tube formation.

Gia/Mthl5 is an aorta specific GPCR required for Drosophila heart tube morphology and normal pericardial cell positioning

G-protein signaling is known to be required for cell-cell contacts during the development of the Drosophila dorsal vessel. However, the identity of the G protein-coupled receptor (GPCR) that regulates this signaling pathway activity is unknown. Here we describe the identification of a novel cardiac specific GPCR, called Gia, for “GPCR in aorta”. Gia is the only heart-specific GPCR identified in Drosophila to date and it is specifically expressed in cardioblasts that fuse at the dorsal midline to become the aorta. Gia is the only Drosophila gene so far identified for which expression is entirely restricted to cells of the aorta. Deletion of Gia led to a broken-hearted phenotype, characterized by pericardial cells dissociated from cardioblasts and abnormal distribution of cell junction proteins. Both phenotypes were similar to those observed in mutants of the heterotrimeric cardiac G proteins. Lack of Gia also led to defects in the alignment and fusion of cardioblasts in the aorta. Gia forms a protein complex with G-αo47A, the alpha subunit of the heterotrimeric cardiac G proteins and interacts genetically with G-αo47A during cardiac morphogenesis. Our study identified Gia as an essential aorta-specific GPCR that functions upstream of cardiac heterotrimeric G proteins and is required for morphological integrity of the aorta during heart tube formation. These studies lead to a redefinition of the bro phenotype, to encompass morphological integrity of the heart tube as well as cardioblast-pericardial cell spatial interactions.

Tinman Regulates NetrinB in the Cardioblasts of the Drosophila Dorsal Vessel.

Morphogenesis of the Drosophila dorsal vessel (DV) shares similarities with that of the vertebrate heart. Precursors line up at both sides of the embryo, migrate towards the midline and fuse to form a tubular structure. Guidance receptors and their ligands have been implicated in this process in vertebrates and invertebrates, as have been a series of evolutionarily conserved cardiogenic transcriptional regulators including Tinman, the Drosophila homolog of the transcription factor Nkx-2.5. NetrinB (NetB), a repulsive ligand for the Unc-5 receptor is required to preserve the dorsal vessel hollow. It localizes to the luminal space of the dorsal vessel but its source and its regulation is unknown. Here, using genetics together with in situ hybridization with single cell resolution, we show how tin is required for NetrinB expression in cardioblasts during DV tubulogenesis and sufficient to promote NetB transcription ectopically. We further identify a dorsal vessel-specific NetB enhancer and show that it is also regulated by tin in a similar fashion to NetB.

The Unc-5 Receptor Is Directly Regulated by Tinman in the Developing Drosophila Dorsal Vessel.

During early heart morphogenesis cardiac cells migrate in two bilateral opposing rows, meet at the dorsal midline and fuse to form a hollow tube known as the primary heart field in vertebrates or dorsal vessel (DV) in Drosophila. Guidance receptors are thought to mediate this evolutionarily conserved process. A core of transcription factors from the NK2, GATA and T-box families are also believed to orchestrate this process in both vertebrates and invertebrates. Nevertheless, whether they accomplish their function, at least in part, through direct or indirect transcriptional regulation of guidance receptors is currently unknown. In our work, we demonstrate how Tinman (Tin), the Drosophila homolog of the Nkx-2.5 transcription factor, regulates the Unc-5 receptor during DV tube morphogenesis. We use genetics, expression analysis with single cell mRNA resolution and enhancer-reporter assays in vitro or in vivo to demonstrate that Tin is required for Unc-5 receptor expression specifically in cardioblasts. We show that Tin can bind to evolutionary conserved sites within an Unc-5 DV enhancer and that these sites are required for Tin-dependent transactivation both in vitro and in vivo.

Cdc42 is required in a genetically distinct subset of cardiac cells during Drosophila dorsal vessel closure

The embryonic heart tube is formed by the migration and subsequent midline convergence of two bilateral heart fields. In Drosophila the heart fields are organized into two rows of cardioblasts (CBs). While morphogenesis of the dorsal ectoderm, which lies directly above the Drosophila dorsal vessel (DV), has been extensively characterized, the migration and concomitant fundamental factors facilitating DV formation remain poorly understood. Here we provide evidence that DV closure occurs at multiple independent points along the A–P axis of the embryo in a “buttoning” pattern, divergent from the zippering mechanism observed in the overlying epidermis during dorsal closure. Moreover, we demonstrate that a genetically distinct subset of CBs is programmed to make initial contact with the opposing row. To elucidate the cellular mechanisms underlying this process, we examined the role of Rho GTPases during cardiac migration using inhibitory and overexpression approaches. We found that Cdc42 shows striking cell-type specificity during DV formation. Disruption of Cdc42 function specifically prevents CBs that express the homeobox gene tinman from completing their dorsal migration, resulting in a failure to make connections with their partnering CBs. Conversely, neighboring CBs that express the orphan nuclear receptor, seven-up, are not sensitive to Cdc42 inhibition. Furthermore, this phenotype was specific to Cdc42 and was not observed upon perturbation of Rac or Rho function. Together with the observation that DV closure occurs through the initial contralateral pairing of tinman-expressing CBs, our studies suggest that the distinct buttoning mechanism we propose for DV closure is elaborated through signaling pathways regulating Cdc42 activity in this cell type.

Abstract 172: Notch Signaling Induces Endocardial Hematopoiesis And Endocardial Cushion Formation Downstream Of Nkx2-5

A subset of cardiac progenitors display multipotency to give rise to major cardiovascular lineages during cardiogenesis. In addition, we have recently identified Nkx2-5+ endocardial cells in the outflow and atria that contribute to definitive hematopoietic cells in vivo, which represents phylogenetically conserved pathway of cardio-vasculo-hematopoietic differentiation. Here, we have examined the molecular mechanism underlying the endocardial hematopoiesis. Immunostaining and colony assay suggest that Nkx2-5 mutant mice showed significant decrease in CD31+/41+ hemogenic endocardial cells, while lineage tracing on Nkx2-5-null background revealed that Nkx2-5-derived endocardial cells are present. These data indicate that Nkx2-5 is required not for the formation of the Nkx2-5-derived endocardium but for the hemogenic activity of the endocardium. Notch signaling is also active in cushion endocardium and its mutant embryos show a similar defect with Nkx2-5 mutants as previously reported. Inspired by these similarities between the function of Nkx2-5 and Notch signaling during cardiogenesis, we next investigated the impact of Notch activation on the endocardial hematopoiesis. In vivo forced activation of Notch in the developing heart using Nkx2-5-Cre mice led to massive increase in the endocardial hematopoiesis. As Nkx2-5 binding sites are found in the regulatory region of Notch1, RBPjk, and Jag1 genes, we tested whether Notch regulates endocardial hematopoiesis downstream of Nkx2-5. Surprisingly, Notch activation restored the defective endocardial hematopoiesis as well as endocardial cushion formation in Nkx2-5 knockout mice, suggesting that Notch signaling mediates the Nkx2-5-dependent endocardial hematopoiesis and cushion development. Drosophila dorsal vessel and lymph gland are closely related and regulated by tinman and notch. Therefore, Nkx2-5-Notch regulatory pathway represents phylogenetically conserved fundamental mechanism of cardio-vasculo-hematopoietic differentiation pathway.

Frazzled/DCC facilitates cardiac cell outgrowth and attachment during Drosophila dorsal vessel formation

Drosophila embryonic dorsal vessel (DV) morphogenesis is a highly stereotyped process that involves the migration and morphogenesis of 52 pairs of cardioblasts (CBs) in order to form a linear tube. This process requires spatiotemporally-regulated localization of signaling and adhesive proteins in order to coordinate the formation of a central lumen while maintaining simultaneous adhesion between CBs. Previous studies have shown that the Slit/Roundabout and Netrin/Unc5 repulsive signaling pathways facilitate site-specific loss of adhesion between contralateral CBs in order to form a luminal space. However, the concomitant mechanism by which attraction initiates CB outgrowth and discrete localization of adhesive proteins remains poorly understood. Here we provide genetic evidence that Netrin signals through DCC (Deleted in Colorectal Carcinoma)/UNC-40/Frazzled (Fra) to mediate CB outgrowth and attachment and that this function occurs prior to and independently of Netrin/UNC-5 signaling. fra mRNA is expressed in the CBs prior to and during DV morphogenesis. Loss-of-fra-function results in significant defects in cell shape and alignment between contralateral CB rows. In addition, CB outgrowth and attachment is impaired in both fra loss- and gain-of-function mutants. Deletion of both Netrin genes (NetA and NetB) results in CB attachment phenotypes similar to fra mutants. Similar defects are also seen when both fra and unc5 are deleted. Finally we show that Fra accumulates at dorsal and ventral leading edges of paired CBs, and this localization is dependent upon Netrin. We propose that while repulsive guidance mechanisms contribute to lumen formation by preventing luminal domains from coming together, site-specific Netrin/Frazzled signaling mediates CB attachment.

Spire, an Actin Nucleation Factor, Regulates Cell Division during Drosophila Heart Development

The Drosophila dorsal vessel is a beneficial model system for studying the regulation of early heart development. Spire (Spir), an actin-nucleation factor, regulates actin dynamics in many developmental processes, such as cell shape determination, intracellular transport, and locomotion. Through protein expression pattern analysis, we demonstrate that the absence of spir function affects cell division in Myocyte enhancer factor 2-, Tinman (Tin)-, Even-skipped- and Seven up (Svp)-positive heart cells. In addition, genetic interaction analysis shows that spir functionally interacts with Dorsocross, tin, and pannier to properly specify the cardiac fate. Furthermore, through visualization of double heterozygous embryos, we determines that spir cooperates with CycA for heart cell specification and division. Finally, when comparing the spir mutant phenotype with that of a CycA mutant, the results suggest that most Svp-positive progenitors in spir mutant embryos cannot undergo full cell division at cell cycle 15, and that Tin-positive progenitors are arrested at cell cycle 16 as double-nucleated cells. We conclude that Spir plays a crucial role in controlling dorsal vessel formation and has a function in cell division during heart tube morphogenesis.

Parallel avenues in the evolution of hearts and pumping organs.

Research in animal models established that tinman, a key gene in Drosophila dorsal vessel development, is an orthologue of Nkx2-5, a key gene in vertebrate cardiac development. Similarities between the arthropod dorsal vessel and vertebrate hearts are interpreted in light of concepts such as homology or convergence. We discuss this controversy in the context of the evolution of animal circulatory pumps and propose the distinction between peristaltic and chambered pumps as a fundamental parameter for evolutionary comparisons between bilaterian pumps. Neither homology nor convergence is satisfactory to explain the origins of hearts and pumping organs. Instead, we propose that animal pumps derive from parallel improvements of an ancestral, peristaltic design represented by a layer of myocytes at the external walls of primitive vessels. This paradigm unifies disparate views, impacts our understanding of bilaterian evolution and may be helpful to interpret similarities between pumping organs of phylogenetically relevant species and emerging models.

The NK homeodomain transcription factor Tinman is a direct activator of seven-up in the Drosophila dorsal vessel

A complex regulatory cascade is required for normal cardiac development, and many aspects of this network are conserved from Drosophila to mammals. In Drosophila, the seven-up ( svp) gene, an ortholog of the vertebrate chick ovalbumin upstream promoter transcription factors ( COUP-TFI and II), is initially activated in the cardiac mesoderm and is subsequently restricted to cells forming the cardiac inflow tracts. Here, we investigate svp regulation in the developing cardiac tube. Using bioinformatics, we identify a 1007-bp enhancer of svp which recapitulates its entire expression in the embryonic heart and other mesodermal derivatives, and we show that this enhancer is initially activated by the NK homeodomain factor Tinman (Tin) via two conserved Tin binding sites. Mutation of the Tin binding sites significantly reduces enhancer activity both during normal development and in response to ectopic Tin. This is the first identification of an enhancer for the complex svp gene, demonstrating the effectiveness of bioinformatics tools in assisting in unraveling transcriptional regulatory networks. Our studies define a critical component of the svp regulatory cascade and place gene regulatory events in direct apposition to the formation of critical cardiac structures.

Role of svp in Drosophila Pericardial Cell Growth

The Drosophila dorsal vessel is a segmentally repeated linear organ, in which seven-up (svp) is expressed in two pairs of cardioblasts and two pairs of pericardial cells in each segment. Under the control of hedgehog ( hh) signaling from the dorsal ectoderm, svp participates in diversifying cardioblast identities within each segment. In this experiment, the homozygous embryos of svp mutants exhibited an increase in cell size of Eve positive pericardial cells (EPCs) and a disarranged expression pattern, while the cardioblasts pattern of svp-lacZ expression was normal. In the meantime, the DA1 muscle founders were absent in some segments in svp mutant embryos, and the dorsal somatic muscle patterning was also severely damaged in the late stage mutant embryos, suggesting that svp is required for the differentiation of Eve-positive pericardial cells and DA1 muscle founders and may have a role in EPC cell growth.

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

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

Cell fate decisions in the Drosophila dorsal vessel depend on the multiadapter protein inscuteable

The Drosophila dorsal vessel consists of two cell types, contractile cardiomyoblasts that form a linear tube-like structure, and the loosely associated pericardial cells. All heart cells originate during embryogenesis from the early dorsal mesoderm under the influence of external and internal signals. Recently, it was shown that a subset of heart cells arise throughout asymmetric cell division, dependent on the function of Notch, Sanpodo, and Numb. Here, we show that Inscuteable, a multiadapter protein required for asymmetric cell division, participates in the formation of specific heart cells to distinguish between a myogenic (cardiomyoblast) and a nonmyogenic (pericardial cell) fate.

Homeotic Genes Autonomously Specify the Anteroposterior Subdivision of the Drosophila Dorsal Vessel into Aorta and Heart

The embryonic dorsal vessel in Drosophila possesses anteroposterior polarity and is subdivided into two chamber-like portions, the aorta in the anterior and the heart in the posterior. The heart portion features a wider bore as compared with the aorta and develops inflow valves (ostia) that allow the pumping of hemolymph from posterior toward the anterior. Here, we demonstrate that homeotic selector genes provide positional information that determines the anteroposterior subdivision of the dorsal vessel. Antennapedia ( Antp ), Ultrabithorax ( Ubx ), abdominal-A ( abd-A ), and Abdominal-B ( Abd-B ) are expressed in distinct domains along the anteroposterior axis within the dorsal vessel, and, in particular, the domain of abd-A expression in cardioblasts and pericardial cells coincides with the heart portion. We provide evidence that loss of abd-A function causes a transformation of the heart into aorta, whereas ectopic expression of abd-A in more anterior cardioblasts causes the aorta to assume heart-like features. These observations suggest that the spatially restricted expression and activity of abd-A determine heart identities in cells of the posterior portion of the dorsal vessel. We also show that Abd-B , which at earlier stages is expressed posteriorly to the cardiogenic mesoderm, represses cardiogenesis. In light of the developmental and morphological similarities between the Drosophila dorsal vessel and the primitive heart tube in early vertebrate embryos, these data suggest that Hox genes may also provide important anteroposterior cues during chamber specification in the developing vertebrate heart.

Homeotic Genes Autonomously Specify the Anteroposterior Subdivision of the Drosophila Dorsal Vessel into Aorta and Heart

The embryonic dorsal vessel in Drosophila possesses anteroposterior polarity and is subdivided into two chamber-like portions, the aorta in the anterior and the heart in the posterior. The heart portion features a wider bore as compared with the aorta and develops inflow valves (ostia) that allow the pumping of hemolymph from posterior toward the anterior. Here, we demonstrate that homeotic selector genes provide positional information that determines the anteroposterior subdivision of the dorsal vessel. Antennapedia ( Antp), Ultrabithorax ( Ubx), abdominal-A ( abd-A), and Abdominal-B ( Abd-B) are expressed in distinct domains along the anteroposterior axis within the dorsal vessel, and, in particular, the domain of abd-A expression in cardioblasts and pericardial cells coincides with the heart portion. We provide evidence that loss of abd-A function causes a transformation of the heart into aorta, whereas ectopic expression of abd-A in more anterior cardioblasts causes the aorta to assume heart-like features. These observations suggest that the spatially restricted expression and activity of abd-A determine heart identities in cells of the posterior portion of the dorsal vessel. We also show that Abd-B, which at earlier stages is expressed posteriorly to the cardiogenic mesoderm, represses cardiogenesis. In light of the developmental and morphological similarities between the Drosophila dorsal vessel and the primitive heart tube in early vertebrate embryos, these data suggest that Hox genes may also provide important anteroposterior cues during chamber specification in the developing vertebrate heart.

Genetically distinct cardial cells within the Drosophila heart.

Although often viewed as a simple pulsating tube, the Drosophila dorsal vessel is intricate in terms of its structure, cell types, and patterns of gene expression. Two nonidentical groups of cardial cells are observed in segments of the heart based on the differential expression of transcriptional regulators. These include sets of four cell pairs that express the homeodomain protein Tinman (Tin), alternating with groups of two cell pairs that express the orphan steroid hormone receptor Seven Up (Svp). Here we show that these myocardial cell populations are distinct in terms of their formation and gene expression profiles. The Svp-expressing cells are generated by asymmetric cell divisions of precursor cells based on decreases or increases in their numbers in numb or sanpodo mutant embryos. In contrast, the numbers of Tin-expressing cardial cells are unchanged in these genetic backgrounds, suggesting they arise from symmetric cell divisions. One function for Svp in the two pairs of cardial cells is to repress the expression of the tin gene and at least one of its targets, the beta3 tubulin gene. Further differences in the cells are substantiated by the identification of separable enhancers for D-mef2 gene transcription in the distinct cardioblast sets. Taken together, these results demonstrate a greater cellular and genetic complexity of the Drosophila heart than previously appreciated.

A Novel Sulfonylurea Receptor Family Member Expressed in the Embryonic Drosophila Dorsal Vessel and Tracheal System

Sulfonylurea receptors (SURx) are required subunits of the ATP-sensitive potassium channel. SURx alone is electrophysiologically inert. However, when SURx is combined with an inward rectifier Kir6.2 subunit, ATP-sensitive potassium channel activity is generated. We report the identification, characterization, and localization of Dsur, a novel Drosophila gene that is highly related to the vertebrate SUR family. The Dsur coding sequence contains structural features characteristic of the ABC transporter family and, in addition, harbors 1.7 kilobases of a distinctive sequence that does not share homology with any known gene. When Dsur alone is expressed in Xenopus oocytes glibenclamide-sensitive potassium channel activity occurs. During Drosophila embryogenesis, the Dsur gene is specifically expressed in the developing tracheal system and dorsal vessel. Studies of the Drosophila genome support that only a single Dsur gene is present. Our data reveal conservation of glibenclamide-sensitive potassium channels in Drosophila and suggest that Dsur may play an important role during Drosophila embryogenesis. The lack of gene duplication in the Drosophila system provides a unique opportunity for functional studies of SUR using a genetic approach.

Embryonic origin and differentiation of the Drosophila heart.

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