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

INTRODUCTION: 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. RESULTS: 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. CONCLUSIONS: 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.

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.

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.

Roundabout is required in the visceral mesoderm for proper microvillus length in the hindgut epithelium.

INTRODUCTION: In this study we examined Roundabout signaling in the Drosophila embryonic hindgut. RESULTS: Slit and its receptors Roundabout (Robo) and Roundabout 2 (Robo2) localize to discrete regions in the hindgut epithelium and surrounding visceral mesoderm. Loss of robo, robo2 or slit did not disrupt overall hindgut patterning. However, slit and robo mutants showed a decrease in microvillus length on the boundary cells of the hindgut epithelium. Rescue and overexpression analysis revealed that robo is specifically required in the visceral mesoderm for correct microvillus length in the underlying hindgut epithelium. Expression of robo in the visceral mesoderm of robo mutant embryos restored normal microvillus length, while overexpression of robo resulted in an increase in microvillus length. Microvillus length was also increased in robo2 mutants suggesting that robo2 may antagonize robo function in the hindgut. CONCLUSION: Together, these results establish a novel, dose-dependent role for Robo in regulating microvilli growth and provide in vivo evidence for the role of the visceral mesoderm in controlling morphological changes in the underlying intestinal epithelium.

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.

RacGAP50C directs perinuclear gamma-tubulin localization to organize the uniform microtubule array required for Drosophila myotube extension.

The microtubule (MT) cytoskeleton is reorganized during myogenesis as individual myoblasts fuse into multinucleated myotubes. Although this reorganization has long been observed in cell culture, these findings have not been validated during development, and proteins that regulate this process are largely unknown. We have identified a novel postmitotic function for the cytokinesis proteins RacGAP50C (Tumbleweed) and Pavarotti as essential regulators of MT organization during Drosophila myogenesis. We show that the localization of the MT nucleator gamma-tubulin changes from diffuse cytoplasmic staining in mononucleated myoblasts to discrete cytoplasmic puncta at the nuclear periphery in multinucleated myoblasts, and that this change in localization depends on RacGAP50C. RacGAP50C and gamma-tubulin colocalize at perinuclear sites in myotubes, and in RacGAP50C mutants gamma-tubulin remains dispersed throughout the cytoplasm. Furthermore, we show that the mislocalization of RacGAP50C in pavarotti mutants is sufficient to redistribute gamma-tubulin to the muscle fiber ends. Finally, myotubes in RacGAP50C mutants have MTs with non-uniform polarity, resulting in multiple guidance errors. Taken together, these findings provide strong evidence that the reorganization of the MT network that has been observed in vitro plays an important role in myotube extension and muscle patterning in vivo, and also identify two molecules crucial for this process.

Cytoskeletal remodeling during myotube assembly and guidance: coordinating the actin and microtubule networks.

The formation of a multinucleated muscle fiber from individual myoblasts is a complex morphological event that requires dramatic cytoskeletal rearrangements. This multistep process includes myoblast fusion, myotube migration and elongation, myotube target recognition, and finally attachment to form a stable adhesion complex. Many of the studies directed towards understanding the developmental process of muscle morphogenesis at the cellular level have relied on forward genetic screens in model systems such as Drosophila melanogaster for mutations affecting individual stages in myogenesis. Through the analyses of these gene products, proteins that regulate the actin or microtubule cytoskeleton have emerged as important players in each of these steps. We recently demonstrated that RacGAP50C, an essential protein that functions as a cytoskeletal regulator during cell division, also plays an important role in organizing the polarized microtubule network in the elongating myotube. Here we review the current literature regarding Drosophila myogenesis and illustrate several steps of muscle development with respect to the diverse roles that the cytoskeleton plays during this process. Furthermore, we discuss the significance of cytoskeletal coordination during these multiple steps.

Preparation of embryos for electron microscopy of the Drosophila embryonic heart tube.

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

Repulsion by Slit and Roundabout prevents Shotgun/E-cadherin-mediated cell adhesion during Drosophila heart tube lumen formation.

During Drosophila melanogaster heart development, a lumen forms between apical surfaces of contralateral cardioblasts (CBs). We show that Slit and its receptor Roundabout (Robo) are required at CB apical domains for lumen formation. Mislocalization of Slit outside the apical domain causes ectopic lumen formation and the mislocalization of cell junction proteins, E-cadherin (E-Cad) and Enabled, without disrupting overall CB cell polarity. Ectopic lumen formation is suppressed in robo mutants, which indicates robo's requirement for this process. Genetic evidence suggests that Robo and Shotgun (Shg)/E-Cad function together in modulating CB adhesion. robo and shg/E-Cad transheterozygotes have lumen defects. In robo loss-of-function or shg/E-Cad gain-of-function embryos, lumen formation is blocked because of inappropriate CB adhesion and an accumulation of E-Cad at the apical membrane. In contrast, shg/E-Cad loss-of-function or robo gain-of-function blocks lumen formation due to a loss of CB adhesion. Our data show that Slit and Robo pathways function in lumen formation as a repulsive signal to antagonize E-Cad-mediated cell adhesion.

Lateral positioning at the dorsal midline: Slit and Roundabout receptors guide Drosophila heart cell migration.

Heart morphogenesis requires the coordinated regulation of cell movements and cell-cell interactions between distinct populations of cardiac precursor cells. Little is known about the mechanisms that organize cardiac cells into this complex structure. In this study, we analyzed the role of Slit, an extracellular matrix protein and its transmembrane receptors Roundabout (Robo) and Roundabout2 (Robo2) during morphogenesis of the Drosophila heart tube, a process analogous to early heart formation in vertebrates. During heart assembly, two types of progenitor cells align into rows and coordinately migrate to the dorsal midline of the embryo, where they merge to assemble a linear heart tube. Here we show that cardiac-specific expression of Slit is required to maintain adhesion between cells within each row during dorsal migration. Moreover, differential Robo expression determines the relative distance each row is positioned from the dorsal midline. The innermost CBs express only Robo, whereas the flanking pericardial cells express both receptors. Removal of robo2 causes pericardial cells to shift toward the midline, whereas ectopic robo2 in CBs drives them laterally, resulting in an unfused heart tube. We propose a model in which Slit has a dual role during assembly of the linear heart tube, functioning to regulate both cell positioning and adhesive interactions between migrating cardiac precursor cells.