Chromatin organization in the nucleus represents an important aspect of transcription regulation. Most of the studies so far focused on the chromatin structure in cultured cells or in fixed tissue preparations. Here, we discuss the various approaches for deciphering chromatin 3D organization with an emphasis on the advantages of live imaging approaches.
The three-dimensional organization of chromatin contributes to transcriptional control, but information about native chromatin distribution is limited. Imaging chromatin in live Drosophila larvae, with preserved nuclear volume, revealed that active and repressed chromatin separates from the nuclear interior and forms a peripheral layer underneath the nuclear lamina. This is in contrast to the current view that chromatin distributes throughout the nucleus. Furthermore, peripheral chromatin organization was observed in distinct Drosophila tissues, as well as in live human effector T lymphocytes and neutrophils. Lamin A/C up-regulation resulted in chromatin collapse toward the nuclear center and correlated with a significant reduction in the levels of active chromatin. Physical modeling suggests that binding of lamina-associated domains combined with chromatin self-attractive interactions recapitulate the experimental chromatin distribution profiles. Together, our findings reveal a novel mode of mesoscale organization of peripheral chromatin sensitive to lamina composition, which is evolutionary conserved.
Intact-organism imaging of Drosophila larvae reveals and quantifies chromatin-aqueous phase separation. The chromatin can be organized near the lamina layer of the nuclear envelope, conventionally fill the nucleus, be organized centrally, or as a wetting droplet. These transitions are controlled by changes in nuclear volume and the interaction of chromatin with the lamina (part of the nuclear envelope) at the nuclear periphery. Using a simple polymeric model that includes the key features of chromatin self-attraction and its binding to the lamina, we demonstrate theoretically that it is the competition of these two effects that determines the mode of chromatin distribution. The qualitative trends as well as the composition profiles obtained in our simulations compare well with the observed intact-organism imaging and quantification. Since the simulations contain only a small number of physical variables we can identify the generic mechanisms underlying the changes in the observed phase separations.
DNA endoreplication has been implicated as a cell strategy to grow in size and in tissue injury. Here, we demonstrate that barrier to autointegration factor (BAF), represses endoreplication in Drosophila myofibers. We show that BAF localization at the nuclear envelope was eliminated either in mutants of the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex, in which the LEM-domain protein Otefin was similarly excluded, or after disruption of the nucleus-sarcomere connections. Furthermore, BAF localization at the nuclear envelope required the activity of the BAF kinase VRK1/Ball, and consistently non-phosphorytable BAF-GFP was excluded from the nuclear envelope. Importantly, removal of BAF from the nuclear envelope correlated with increased DNA content in the myonuclei. E2F1, a key regulator of endoreplication was found to overlap BAF localization at the myonuclear envelope, and BAF removal from the nuclear envelope resulted with increased E2F1 levels in the nucleoplasm, and subsequent elevated DNA content. We suggest that LINC-dependent, and phospho-sensitive attachment of BAF to the nuclear envelope, through its binding to Otefin, tethers E2F1 to the nuclear envelope thus inhibiting its accumulation at the nucleoplasm.
Muscle contractions produce reiterated cytoplasmic mechanical variations, which potentially influence nuclear mechanotransduction, however information regarding the dynamics of muscle nuclei (myonuclei) in the course of muscle contraction is still missing. Towards that end, a minimal constraint device was designed in which intact live Drosophilalarva is imaged, while its muscles still contract. The device is placed under spinning disc confocal microscope enabling imaging of fluorescently labeled sarcomeres and nuclei during muscle contraction, without any external stimulation. As a proof of principle we studied myonuclei dynamics in wild-type, as well as inNesprin/klarmutant larvae lacking proper nuclear-cytoskeletal connections. Myonuclei in control larvae exhibited comparable dynamics in the course of multiple contractile events, independent of their position along the muscle fiber. In contrast, myonuclei of mutant larvae displayed differential dynamics at distinct positions along individual myofibers. Moreover, we identified a linear link between myonuclear volume and its acceleration values during muscle contraction which, inNesprin/klarmutants exhibited an opposite tendency relative to control. Estimation of the drag force applied on individual myonuclei revealed that force fluctuations in time, but not the average force, differed significantly between control andNesprin/klarmutant, and were considerably higher in the mutant myonuclei. Taken together these results imply significant alterations in the mechanical dynamics of individual myonuclei in theNesprin/klarmyonuclei relative to control. Such differences provide novel mechanical insight intoNesprinfunction in contractile muscles, and might reveal the mechanical basis underlying Nesprin-related human diseases.
Differentiation of germline stem cells (GSCs) in the Drosophila ovary is induced by somatic escort cells (ECs), which extend membrane protrusions encapsulating the germline cells (GCs). Germline encapsulation requires activated epidermal growth factor receptor (Egfr) signaling within the ECs, following secretion of its ligands from the GCs. We show that the conserved family of irre cell recognition module (IRM) proteins is essential for GC encapsulation by ECs, with a requirement for roughest (rst) and kin of irre (kirre) in the germline and for sticks and stones (sns) and hibris (hbs) in ECs. In the absence of IRM components in their respective cell types, EC extensions are reduced concomitantly with a decrease in Egfr signaling in these cells. Reintroducing either activated Egfr in the ECs, or overexpressing its ligand Spitz (Spi) from the germline, rescued the requirement for IRM proteins in both cell types. These experiments introduce novel essential components, the IRM proteins, into the process of inductive interactions between GCs and ECs, and imply that IRM-mediated activity is required upstream of the Egfr signaling.
The cytoplasm of striated myofibers contains a large number of membrane organelles, including sarcoplasmic reticulum (SR), T-tubules and the nuclear membrane. These organelles maintain a characteristic juxtaposition that appears to be essential for efficient inter-membranous exchange of RNA, proteins and ions. We found that the membrane-associated Muscle-specific alpha 2/delta (Ma2/d) subunit of the Ca2+ channel complex localizes to the SR and T-tubules, and accumulates at the myonuclear surfaces. Furthermore, Ma2/d mutant larval muscles exhibit nuclear positioning defects, disruption of the nuclear-SR juxtapositioning, as well as impaired larval locomotion. Ma2/d localization at the nuclear membrane depends on the proper function of the nesprin ortholog Msp300 and the BAR domain protein Amphiphysin (Amph). Importantly, live imaging of muscle contraction in intact Drosophila larvae indicated altered distribution of Sarco/Endoplamic Reticulum Ca2+-ATPase (SERCA) around the myonuclei of Ma2/d mutant larvae. Co-immunoprecipitation analysis supports association between Ma2/d and Amph, and indirectly with Msp300. We therefore suggest that Ma2/d, in association with Msp300 and Amph, mediates interactions between the SR and the nuclear membrane.
Nuclear mechanotransduction has been implicated in the control of chromatin organization; however, its impact on functional contractile myofibers is unclear. We found that deleting components of the linker of nucleoskeleton and cytoskeleton (LINC) complex in Drosophila melanogaster larval muscles abolishes the controlled and synchronized DNA endoreplication, typical of nuclei across myofibers, resulting in increased and variable DNA content in myonuclei of individual myofibers. Moreover, perturbation of LINC-independent mechanical input after knockdown of beta-Integrin in larval muscles similarly led to increased DNA content in myonuclei. Genome-wide RNA-polymerase II occupancy analysis in myofibers of the LINC mutant klar indicated an altered binding profile, including a significant decrease in the chromatin regulator barrier-to-autointegration factor (BAF) and the contractile regulator Troponin C. Importantly, muscle-specific knockdown of BAF led to increased DNA content in myonuclei, phenocopying the LINC mutant phenotype. We propose that mechanical stimuli transmitted via the LINC complex act via BAF to regulate synchronized cell-cycle progression of myonuclei across single myofibers.
The musculoskeletal and proprioceptive sensory systems exhibit intricate crosstalk between force generation, force sensation and force transmission, all of which are critical for coordinated animal locomotion. Recent developmental studies of the musculoskeletal and proprioceptive units of the invertebrate Drosophila embryo, have revealed several common molecular and structural principles mediating the formation of each of these systems. These common principles, as well as the differences between the developmental programs of the two systems, are discussed. Interestingly, a molecular pathway triggered by the Neuregulin/Vein ligand-dependent activation of the epidermal growth factor receptor (EGFR) pathway, which upregulates the early growth response (EGR)-like transcription factor Stripe, is utilized not only by the Drosophila muscle-tendon and proprioceptive organ-ectoderm attachment, but also by their vertebrate counterparts. An additional theme that has been observed during the development of the musculoskeletal system in both invertebrates and vertebrates is the functional importance of the extracellular matrix and its adhesion receptors. The contribution of mechanical forces to proper junction formation between muscles and tendons and between the sensory cap/ligament cells and their epidermal attachment cells is discussed. The structural and genetic similarities between the musculoskeletal and the proprioceptive systems offer new perspectives as to their common developmental nature.
Slit cleavage into N-terminal and C-terminal polypeptides is essential for restricting the range of Slit activity. Although the Slit cleavage site has been characterized previously and is evolutionally conserved, the identity of the protease that cleaves Slit remains elusive. Our previous analysis indicated that Slit cleavage is essential to immobilize the active Slit-N at the tendon cell surfaces, mediating the arrest of muscle elongation. In an attempt to identify the protease required for Slit cleavage we performed an RNAi-based assay in the ectoderm and followed the process of elongation of the lateral transverse muscles toward tendon cells. The screen led to the identification of the Drosophila homolog of pheromone convertase 2 (PC2), Amontillado (Amon), as an essential protease for Slit cleavage. Further analysis indicated that Slit mobility on SDS polyacrylamide gel electrophoresis (SDS-PAGE) is slightly up-shifted in amon mutants, and its conventional cleavage into the Slit-N and Slit-C polypeptides is attenuated. Consistent with the requirement for amon to promote Slit cleavage and membrane immobilization of Slit-N, the muscle phenotype of amon mutant embryos was rescued by co-expressing a membrane-bound form of full-length Slit lacking the cleavage site and knocked into the slit locus. The identification of a novel protease component essential for Slit processing may represent an additional regulatory step in the Slit signaling pathway.
Skeletal muscle is highly sensitive to mutations in genes that participate in membrane stability and cellular attachment, which often leads to muscular dystrophy. Here we show that Thrombospondin-4 (Thbs4) regulates skeletal muscle integrity and its susceptibility to muscular dystrophy through organization of membrane attachment complexes. Loss of the Thbs4 gene causes spontaneous dystrophic changes with aging and accelerates disease in 2 mouse models of muscular dystrophy, while overexpression of mouse Thbs4 is protective and mitigates dystrophic disease. In the myofiber, Thbs4 selectively enhances vesicular trafficking of dystrophin-glycoprotein and integrin attachment complexes to stabilize the sarcolemma. In agreement, muscle-specific overexpression of Drosophila Tsp or mouse Thbs4 rescues a Drosophila model of muscular dystrophy with augmented membrane residence of beta P5 integrin. This functional conservation emphasizes the fundamental importance of Thbs' as regulators of cellular attachment and membrane stability and identifies Thbs4 as a potential therapeutic target for muscular dystrophy.
Muscle nuclei are exposed to variable cytoplasmic strain produced by muscle contraction and relaxation, but their morphology remains stable. Still, the mechanism responsible for maintaining myonuclear architecture, and its importance, is currently elusive. Herein, we uncovered a unique myonuclear scaffold in Drosophila melanogaster larval muscles, exhibiting both elastic features contributed by the stretching capacity of MSP300 (nesprin) and rigidity provided by a perinuclear network of microtubules stabilized by Shot (spectraplakin) and EB1. Together, they form a flexible perinuclear shield that protects myonuclei from intrinsic or extrinsic forces. The loss of this scaffold resulted in significantly aberrant nuclear morphology and subsequently reduced levels of essential nuclear factors such as lamin A/C, lamin B, and HP1. Overall, we propose a novel mechanism for maintaining myonuclear morphology and reveal its critical link to correct levels of nuclear factors in differentiated muscle fibers. These findings may shed light on the underlying mechanism of various muscular dystrophies.
The formation of functional musculoskeletal system relies on proper connectivity between muscles and their corresponding tendon cells. In Drosophila, larval muscles are born during early embryonic stages, and elongate toward tendons that are embedded within the ectoderm in later. The Slit/Robo signaling pathway had been implicated in the process of muscle elongation toward tendons. Here we discuss our recent findings regarding the critical contribution of Slit cleavage for immobilization and stabilization of the Slit signal on the tendon cells. Slit cleavage produces 2 polypeptides, the N-terminal Slit-N, which is extremely stable, undergoes oligomerization, and associates with the tendon cell surfaces, and the C-terminal Slit-C, which rapidly degrades. Slit cleavage leads to immobilization of Slit signaling on tendons, leading to a short-range repulsion, which eventually arrest further muscle elongation. Robo2, which is co-expressed with Slit by the tendon cells facilitates Slit cleavage. This activity does not require the cytoplasmic signaling domain of Robo2. We suggest that Robo2-dependent Slit cleavage, and the formation of Slit-N oligomers on the tendon cell surfaces direct muscle elongation, and provide a stop signal for the approaching muscle, through binding to Robo and Robo3 receptors expressed by the muscles.
During organogenesis, secreted signaling proteins direct cell migration towards their target tissue. In Drosophila embryos, developing muscles are guided by signals produced by tendons to promote the proper attachment of muscles to tendons, essential for proper locomotion. Previously, the repulsive protein Slit, secreted by tendon cells, has been proposed to be an attractant for muscle migration. However, our findings demonstrate that through tight control of its distribution, Slit repulsion is used for both directing and arresting muscle migration. We show that Slit cleavage restricts its distribution to tendon cells, allowing it to function as a short-range repellent that directs muscle migration and patterning, and promotes their halt upon reaching the target site. Mechanistically, we show that Slit processing produces a rapidly degraded C-terminal fragment and an active, stable N-terminal polypeptide that is tethered to the tendon cell membrane, which further protects it from degradation. Consistently, the requirement for Slit processing can be bypassed by providing an uncleavable, membrane-bound form of Slit that is stable and is retained on expressing tendon cells. Moreover, muscle elongation appears to be extremely sensitive to Slit levels, as replacing the entire full-length Slit with the stable Slit-N-polypeptide results in excessive repulsion, which leads to a defective muscle pattern. These findings reveal a novel cleavage-dependent regulatory mechanism controlling Slit spatial distribution, which may operate in other Slit-dependent processes.
Nuclear morphology and architecture are maintained by nucleoskeletal elements including, chromatin associated factors and nuclear lamina components both of which are involved in the regulation of chromatin 3D organization [1, 2]. As such, differentiated cells maintain a tissue-specific nuclear distribution of the eu- and heterochromatin and contain steady-state levels of nuclear lamina components and chromatin factors. The mechanical forces applied to the cell nuclei in a given tissue provide a mechano-sensitive signal, which is tightly linked to the levels of the nuclear lamina component lamin A/C . Since lamin A/C associates and regulates the activity of nuclear factors including chromatin regulators and transcription factors it may facilitate the interpretation of the mechanical signal from the cytoplasm into the nucleus. [first paragraph]
Contractile muscle fibers produce enormous intrinsic forces during contraction/relaxation waves. These forces are directly applied to their cytoplasmic organelles including mitochondria, sarcoplasmic reticulum, and multiple nuclei. Data from our analysis of Drosophila larval somatic muscle fibers suggest that an intricate network of organized microtubules (MT) intermingled with Spectrin-Repeat-Containing Proteins (SRCPs) are major structural elements that protect muscle organelles and maintain their structure and position during muscle contraction. Whereas the perinuclear MT network provides structural rigidity to the myonucleus, the SRCPs Nesprin and Spectraplakin form semiflexible filamentous biopolymer networks, providing nuclei with the elasticity required to resist the contractile cytoplasmic forces produced by the muscle. Spectrin repeats are domains found in numerous structural proteins, which are able to unfold under tension and are subject to mechanical stresses in the cell. This unique composite scaffold combines rigidity and resilience in order to neutralize the oscillating cellular forces occurring during muscle contraction/relaxation waves and thereby protect myonuclei. We suggest that the elastic properties of SRCPs are critical for nuclear protection and proper function in muscle fibers.
Coordinated locomotion of an organism relies on the development of proper musculoskeletal connections. In Drosophila, the Slit-Robo signaling pathway guides muscles to tendons. Here, we show that the Slit receptor Roundabout 2 (Robo2) plays a non-cell-autonomous role in directing muscles to their corresponding tendons. Robo2 is expressed by tendons, and its non-signaling activity in these cells promotes Slit cleavage, producing a cleaved Slit N-terminal guidance signal that provides short-range signaling into muscles. Consistently, robo2 mutant embryos exhibited a muscle phenotype similar to that of slit, which could not be rescued by muscle-specific Robo2 expression but rather by ectodermally derived Robo2. Alternatively, this muscle phenotype could be induced by tendon-specific robo2 RNAi. We further show that membrane immobilization of Slit or its Nterminal cleaved form (Slit-N) on tendons bypasses the functional requirement for Robo2 in tendons, verifying that the major role of Robo2 is to promote the association of Slit with the tendon cell membrane. Slit-N tends to oligomerize whereas full-length uncleavable Slit does not. It is therefore proposed that Slit-N oligomers, produced at the tendon membrane by Robo2, signal to the approaching muscle by combined Robo1 and Robo3 activity. These findings establish a Robo2-mediated mechanism, independent of signaling, that is essential to limiting Slit distribution and which might be relevant to the regulation of Slit-mediated shortrange signaling in additional systems. 2015, Published by The Company of Biologists Ltd.
The Drosophila model represents an attractive system in which to study the functional contribution of specific genes to organ development. Within the embryo, the heart tube serves as an informative developmental paradigm to analyze functional aspects of matricellular proteins. Here, we describe two essential extracellular matricellular proteins, Multiplexin (Mp) and Lonely heart (Loh). Each of these proteins contributes to the development and morphogenesis of the heart tube by regulating the activity/localization of essential extracellular proteins. Mp, which is secreted by heart cardioblasts and is specifically distributed in the lumen of the heart tube, binds to the signaling protein Slit, and facilitates its local signaling at the heart's luminal domain. Loh is an ADAMTS-like protein, which serves as an adapter protein to Pericardin (a collagen-like protein), promoting its specific localization at the abluminal domain of the heart tube. We also introduce the Drosophila orthologues of matricellular proteins present in mammals, including Thrombospondin, and SPARC, and discuss a possible role for Teneurins (Ten-A and Ten-M) in the heart. Understanding the role of these proteins provides a novel developmental perspective into the functional contribution of matricellular proteins to organ development (C) 2014 Published by Elsevier B.V.
Post-transcriptional regulation of RNA stability and localization underlies a wide array of developmental processes, such as axon guidance and epithelial morphogenesis. In Drosophila, ectopic expression of the classically Golgi peripheral protein dGRASP at the plasma membrane is achieved through its mRNA targeting at key developmental time-points, in a process critical to follicular epithelium integrity. However, the trans-acting factors that tightly regulate the spatio-temporal dynamics of dgrasp are unknown. Using an in silico approach, we identified two putative HOW Response Elements (HRE1 and HRE2) within the dgrasp open reading frame for binding to Held Out Wings (HOW), a member of the Signal Transduction and Activation of RNA family of RNA-binding proteins. Using RNA immunoprecipitations, we confirmed this by showing that the short cytoplasmic isoform of HOW binds directly to dgrasp HRE1. Furthermore, HOW loss of function in vivo leads to a significant decrease in dgrasp mRNA levels. We demonstrate that HRE1 protects dgrasp mRNA from cytoplasmic degradation, but does not mediate its targeting. We propose that this binding event promotes the formation of ribonucleoprotein particles that ensure dgrasp stability during transport to the basal plasma membrane, thus enabling the local translation of dgrasp for its roles at non-Golgi locations.
The Drosophila heart tube represents a structure that similarly to vertebrates' primary heart tube exhibits a large lumen; the mechanisms promoting heart tube morphology in both Drosophila and vertebrates are poorly understood. We identified Multiplexin (Mp), the Drosophila orthologue of mammalian Collagen-XV/XVIII, and the only structural heart-specific protein described so far in Drosophila, as necessary and sufficient for shaping the heart tube lumen, but not that of the aorta. Mp is expressed specifically at the stage of heart tube closure, in a polarized fashion, uniquely along the cardioblasts luminal membrane, and its absence results in an extremely small heart tube lumen. Importantly, Mp forms a protein complex with Slit, and interacts genetically with both slit and robo in the formation of the heart tube. Overexpression of Mp in cardioblasts promotes a large heart lumen in a Slit-dependent manner. Moreover, Mp alters Slit distribution, and promotes the formation of multiple Slit endocytic vesicles, similarly to the effect of overexpression of Robo in these cells. Our data are consistent with Mp-dependent enhancement of Slit/Robo activity and signaling, presumably by affecting Slit protein stabilization, specifically at the lumen side of the heart tube. This activity results with a Slit-dependent, local reduction of F-actin levels at the heart luminal membrane, necessary for forming the large heart tube lumen. Consequently, lack of Mp results in decreased diastolic capacity, leading to reduced heart contractility, as measured in live fly hearts. In summary, these findings show that the polarized localization of Mp controls the direction, timing, and presumably the extent of Slit/Robo activity and signaling at the luminal membrane of the heart cardioblasts. This regulation is essential for the morphogenetic changes that sculpt the heart tube in Drosophila, and possibly in forming the vertebrates primary heart tube.
Striated muscles contain a tightly ordered cytoplasm in which the shape and size of the nuclei are comparable and nuclear distribution is uniform. These features were recently shown to be essential for muscle function. In an attempt to elucidate mechanisms regulating the position and shape of myonuclei, we analyzed the function of the two KASH proteins that are uniquely present in the Drosophila genome, MSP-300 and Klarsicht, both expressed in striated muscles. We demonstrated that both KASH proteins cooperate to construct a unique ring composed of MSP-300 protein that surrounds and attached to the nuclear envelope. The MSP-300 nuclear ring structure recruits and associates with a network of polarized astral microtubules that enables the dynamic movement and uniform spacing between the nuclei in each muscle fiber.
Striated muscle fibers are characterized by their tightly organized cytoplasm. Here, we show that the Drosophila melanogaster KASH proteins Klarsicht (Klar) and MSP-300 cooperate in promoting even myonuclear spacing by mediating a tight link between a newly discovered MSP-300 nuclear ring and a polarized network of astral microtubules (aMTs). In either klar or msp-300(Delta KASH), or in klar and msp-300 double heterozygous mutants, the MSP-300 nuclear ring and the aMTs retracted from the nuclear envelope, abrogating this even nuclear spacing. Anchoring of the myonuclei to the core acto-myosin fibrillar compartment was mediated exclusively by MSP-300. This protein was also essential for promoting even distribution of the mitochondria and ER within the muscle fiber. Larval locomotion is impaired in both msp-300 and klar mutants, and the klar mutants were rescued by muscle-specific expression of Klar. Thus, our results describe a novel mechanism of nuclear spacing in striated muscles controlled by the cooperative activity of MSP-300, Klar, and astral MTs, and demonstrate its physiological significance.
Drosophila melanogaster Held Out Wings (HOW) is a conserved RNA-binding protein (RBP) belonging to the STAR family, whose closest mammalian ortholog Quaking (QKI) has been implicated in embryonic development and nervous system myelination. The HOW RBP modulates a variety of developmental processes by controlling mRNA levels and the splicing profile of multiple key regulatory genes; however, mechanisms regulating its activity in tissues have yet to be elucidated. Here, we link receptor tyrosine kinase (RTK) signaling to the regulation of QKI subfamily of STAR proteins, by showing that HOW undergoes phosphorylation by MAPK/ERK. Importantly, we show that this modification facilitates HOW dimerization and potentiates its ability to bind RNA and regulate its levels. Employing an antibody that specifically recognizes phosphorylated HOW, we show that HOW is phosphorylated in embryonic muscles and heart cardioblasts in vivo, thus documenting for the first time Serine/Threonine (Ser/Thr) phosphorylation of a STAR protein in the context of an intact organism. We also identify the sallimus/D-titin (s/s) gene as a novel muscle target of HOW-mediated negative regulation and further show that this regulation is phosphorylation-dependent, underscoring the physiological relevance of this modification. Importantly, we demonstrate that HOW Thr phosphorylation is reduced following muscle-specific knock down of Drosophila MAPK rolled and that, correspondingly, Sls is elevated in these muscles, similarly to the HOW RNAi effect. Taken together, our results provide a coherent mechanism of differential HOW activation; MAPK/ERK-dependent phosphorylation of HOW promotes the formation of HOW dimers and thus enhances its activity in controlling mRNA levels of key muscle-specific genes. Hence, our findings bridge between MAPK/ERK signaling and RNA regulation in developing muscles.
The Drosophila heart has become an exciting model for elucidating the molecular basis for cardiac function in higher organisms. To complement the genetic approaches that have recently identified an array of genes essential for cardiac function, we developed a method to obtain optimal semi-thin cross sections of embryonic, larval, and adult fly hearts in a desired orientation. A procedure for fluorescent labeling of these sections with multiple markers has also been developed, allowing the detection of proteins at high subcellular resolution. Sections obtained by our method reveal changes in cell shape between embryonic heart and aorta cardioblasts and elucidate the morphology of the adult heart. Analysis of the adult heart reveals the precise cardiac tube morphology, differential distribution of the extracellular matrix protein Laminin within the cardiac tube, as well as individual hand-positive, and Held Out Wings (HOW)-positive luminal cells that might represent blood cells. In summary, our method enables visualization of cross sections of the embryonic and adult hearts at high resolution while maintaining the ability to co-label the sections with multiple markers, thereby facilitating the analysis of cardiac tube formation and maintenance at different developmental stages. (C) 2011 Elsevier Inc. All rights reserved.
The development of imaging methodologies for detecting blood-brain-barrier (BBB) disruption may help predict stroke patient's propensity to develop hemorrhagic complications following reperfusion. We have developed a delayed contrast extravasation MRI-based methodology enabling real-time depiction of subtle BBB abnormalities in humans with high sensitivity to BBB disruption and high spatial resolution. The increased sensitivity to subtle BBB disruption is obtained by acquiring T1-weighted MRI at relatively long delays (similar to 15 minutes) after contrast injection and subtracting from them images acquired immediately after contrast administration. In addition, the relatively long delays allow for acquisition of high resolution images resulting in high resolution BBB disruption maps. The sensitivity is further increased by image preprocessing with corrections for intensity variations and with whole body (rigid+elastic) registration. Since only two separate time points are required, the time between the two acquisitions can be used for acquiring routine clinical data, keeping the total imaging time to a minimum. A proof of concept study was performed in 34 patients with ischemic stroke and 2 patients with brain metastases undergoing high resolution T1-weighted MRI acquired at 3 time points after contrast injection. The MR images were pre-processed and subtracted to produce BBB disruption maps. BBB maps of patients with brain metastases and ischemic stroke presented different patterns of BBB opening. The significant advantage of the long extravasation time was demonstrated by a dynamic-contrast-enhancement study performed continuously for 18 min. The high sensitivity of our methodology enabled depiction of clear BBB disruption in 27% of the stroke patients who did not have abnormalities on conventional contrast-enhanced MRI. In 36% of the patients, who had abnormalities detectable by conventional MRI, the BBB disruption volumes were significantly larger in the map
The blood brain barrier (BBB) is essential for insulation of the nervous system from the surrounding environment. In Drosophila melanogaster, the BBB is maintained by septate junctions formed between subperineurial glia (SPG) and requires the Moody/G protein coupled receptor (GPCR) signaling pathway. In this study, we describe novel specialized actin-rich structures (ARSs) that dynamically form along the lateral borders of the SPG cells. ARS formation and association with nonmuscle myosin is regulated by Moody/GPCR signaling and requires myosin activation. Consistently, an overlap between ARS localization, elevated Ca(2+) levels, and myosin light chain phosphorylation is detected. Disruption of the ARS by inhibition of the actin regulator Arp2/3 complex leads to abrogation of the BBB. Our results suggest a mechanism by which the Drosophila BBB is maintained by Moody/GPCR-dependent formation of ARSs, which is supported by myosin activation. The localization of the ARSs close to the septate junctions enables efficient sealing of membrane gaps formed during nerve cord growth.
The formation of the musculoskeletal system represents an intricate process of tissue assembly involving heterotypic inductive interactions between tendons, muscles and cartilage. An essential component of all musculoskeletal systems is the anchoring of the force-generating muscles to the solid support of the organism: the skeleton in vertebrates and the exoskeleton in invertebrates. Here, we discuss recent findings that illuminate musculoskeletal assembly in the vertebrate embryo, findings that emphasize the reciprocal interactions between the forming tendons, muscle and cartilage tissues. We also compare these events with those of the corresponding system in the Drosophila embryo, highlighting distinct and common pathways that promote efficient locomotion while preserving the form of the organism.
The formation of complex tissues during embryonic development is often accompanied by directed cellular migration towards a target tissue. Specific mutual recognition between the migrating cell and its target tissue leads to the arrest of the cell migratory behavior and subsequent contact formation between the two interacting cell types. Recent studies implicated a novel family of surface proteins containing a trans-membrane domain and single leucine-rich repeat (LRR) domain in inter-cellular recognition and the arrest of cell migration. Here, we describe the involvement of a novel LRR surface protein, LRT, in targeting migrating muscles towards their corresponding tendon cells in the Drosophila embryo. LRT is specifically expressed by the target tendon cells and is essential for arresting the migratory behavior of the muscle cells. Additional studies in Drosophila S2 cultured cells suggest that LRT forms a protein complex with the Roundabout (Robo) receptor, essential for guiding muscles towards their tendon partners. Genetic analysis supports a model in which LRT performs its activity non-autonomously through its interaction with the Robo receptors expressed on the muscle surfaces. These results suggest a novel mechanism of intercellular recognition through interactions between LRR family members and Robo receptors.
The mechanisms by which germline stem cells (GSCs) in the Drosophila testis undergo asymmetric division to regenerate a stem cell as well as a daughter (gonialblast) that will only undergo a further four mitotic divisions prior to entering premeiotic S phase and differentiating into a cyst of spermatocytes are not fully resolved. Here we demonstrate that the HOW RNA-binding protein is required for maintenance of CycB and therefore mitotic progression in GSCs and gonialblasts as well as determining the timing of the spermatogonial divisions. HOW is normally expressed in a complementary pattern to Barn in the germline and barn mRNA is bound by HOW in vivo. Ectopic expression of the HOW(L) isoform is associated with a delay in accumulation of Barn to the level required for differentiation, resulting in extra mitotic divisions. Spatiotemporal regulation of HOW expression is therefore required to specify the four spermatogonial transit-amplifying divisions.
The formation of complex tissues requires the assembly of distinct cell types that often migrate over long distances in order to interact with each other and establish a functional tissue. The establishment of the contractile tissue in the Drosophila embryo has been used as a model system in which to study how the interplay between distinct cell types results in a complex, functioning tissue. The Drosophila contractile tissue is composed of multi-nucleated muscle cells that are attached to individual specialized ectodermal cells, named tendon cells, at each end. The tendon cells are anchored to the cuticle external skeleton on their apical side and form integrin-dependent myotendinous junctions at their basal end. In order for the complex muscle-pattern to form, muscles must undergo several tightly regulated processes: They need to migrate towards their respective tendon cells, arrest migration upon arrival to the tendon cells and form integrin-mediated muscle-tendon junctions (MTJs) in an accurate manner that would guarantee appropriate muscle function. 1,2 The regulation of many of these events is still poorly understood, driving us to search for novel mechanisms that enable the functionality of the contractile tissue.
The correct assembly of the myotendinous junction (MTJ) is crucial for proper muscle function. In Drosophila, this junction comprises hemi-adherens junctions that are formed upon arrival of muscles at their corresponding tendon cells. The MTJ mainly comprises muscle-specific alpha PS2 beta PS integrin receptors and their tendon-derived extracellular matrix ligand Thrombospondin (Tsp). We report the identification and functional analysis of a novel tendon-derived secreted protein named Slowdown (Slow). Homozygous slow mutant larvae exhibit muscle or tendon rupture, sluggish larval movement, partial lethality, and the surviving adult flies are unable to fly. These defects result from improper assembly of the embryonic MTJ. In slow mutants, Tsp prematurely accumulates at muscle ends, the morphology of the muscle leading edge changes and the MTJ architecture is aberrant. Slow was found to form a protein complex with Tsp. This complex is biologically active and capable of altering the morphology and directionality of muscle ends. Our analysis implicates Slow as an essential component of the MTJ, crucial for ensuring muscle and tendon integrity during larval locomotion.
Signal transduction and activation of RNA (STAR) family of RNA binding proteins are highly conserved through evolution indicating their core role during development, as well as in adult life. This chapter focuses on two Drosophila STAR proteins: Held Out Wing (HOW), the ortholog of mammalian Quaking (QKI) and Kep1, one of the four orthologs of mammalian Sam 68. I will emphasize the orthologs similarities in splicing pattern, functions and mode of actions of the two proteins relying on recent and earlier findings in the field. I will start with general description of the STAR proteins in Drosophila with an emphasis on their specific expression patterns.
Correct muscle migration towards tendon cells, and the adhesion of these two cell types, form the basis for contractile tissue assembly in the Drosophila embryo. While molecules promoting the attraction of muscles towards tendon cells have been described, signals involved in the arrest of muscle migration following the arrival of myotubes at their corresponding tendon cells have yet to be elucidated. Here, we describe a novel tendon-specific transmembrane protein, which we named LRT due to the presence of a leucine-rich repeat domain (LRR) in its extracellular region. Our analysis suggests that LRT acts non-autonomously to better target the muscle and/or arrest its migration upon arrival at its corresponding tendon cell. Muscles in embryos lacking LRT exhibited continuous formation of membrane extensions despite arrival at their corresponding tendon cells, and a partial failure of muscles to target their correct tendon cells. In addition, overexpression of LRT in tendon cells often stalled muscles located close to the tendon cells. LRT formed a protein complex with Robo, and we detected a functional genetic interaction between Robo and LRT at the level of muscle migration behavior. Taken together, our data suggest a novel mechanism by which muscles are targeted towards tendon cells as a result of LRT-Robo interactions. This mechanism may apply to the Robo-dependent migration of a wide variety of cell types.
Keywords: Developmental Biology
The selective sensitivity of cells to programmed cell death (PCD) depends on the positive and negative death-inducing signals that converge into the apoptotic pathway. In Drosophila, the midline glial (MG) cells undergo selective death during development. Here, we show that the long isoform of the RNA-binding protein Held Out Wing (HOW(L)) is essential for enhancing the sensitivity of the MG cells to PCD. In how mutant embryos, the number of MG cells was elevated. This phenotype could be rescued by midline expression of the HOW(L) repressor isoform. In how mutant embryos, the levels of the caspase inhibitor of apoptosis, Diap1 were elevated, in parallel to reduction in the levels of activated caspase. Similarly, reducing the levels of HOW in S2 cells led to elevation of Diap1, whereas over expression of HOW(L) promoted reduction of Diap1 protein as well as mRNA levels. Importantly, deletion of the two HOW binding sites from diap1 30UTR abrogated HOW-dependent repression of Diap1, suggesting that HOW represses diap1 by binding to its 30UTR. These results suggest that HOW(L) enhances the sensitivity of MG cells to apoptotic signals by reducing the levels of diap1 in these cells in, demonstrating a novel mode of regulation of PCD at the mRNA level. (C) 2008 Elsevier Ireland Ltd. All rights reserved.
The regulation of developmental processes at the RNA level enables selective and rapid modulation of gene expression. Studies in model organisms revealed the essential contribution of the signal transduction and activation of RNA (STAR) family of RNA binding proteins to developmental processes. STAR proteins coordinate the proper timing of developmental events by delaying expression or altering the mRNA or protein levels of essential genes. Recent functional analysis of the Drosophila melanogaster STAR protein, Held Out Wing (HOW), in the context of embryonic development, provided insight into its mode of activity. Here, we describe HOW's activity in the temporal repression or elevation of gene expression that is essential for coordinating the correct timing of instructive signals.
The even spreading of mesoderm cells in the Drosophila embryo is essential for its proper patterning by ectodermally derived signals. In how germline clone embryos, defects in mesoderm spreading lead to a partial loss of dorsal mesoderm derivatives. HOW is an RNA- binding protein that is thought to regulate diverse mRNA targets. To identify direct HOW targets, we implemented a series of selection methods on mRNAs whose levels were elevated in how germline clone embryos during the stage of mesoderm spreading. Four mRNAs were found to be specifically elevated in the mesoderm of how germline clone embryos, and to exhibit specific binding to HOW via their 3' UTRs. Importantly, overexpression of three of these genes phenocopied the mesodermspreading phenotype of how germline clone embryos. Further analysis showed that overexpressing one of these genes, miple ( a Drosophila midkine and pleiotrophin heparin- binding growth factor), in the mesoderm led to abnormal scattered MAPK activation, a phenotype that might explain the abnormal mesoderm spreading. In addition, the number of EVE- positive cells, which are responsive to receptor tyrosine kinase ( RTK) signaling, was increased following Miple overexpression in the mesoderm and appeared to be dependent on Heartless function. In summary, our analysis suggests that HOW downregulates the levels of a number of mRNA species in the mesoderm in order to enable proper mesoderm spreading during early embryogenesis.
Regulation of RNA metabolism plays a major role in controlling gene expression during developmental processes. The Drosophila RNA-binding protein Held out wing ( HOW), regulates an array of developmental processes in embryonic and adult growth. We have characterized the primary sequence and secondary structural requirements for the HOW response element (HRE), and show that this site is necessary and sufficient for HOW binding. Based on this analysis, we have identified the Drosophila TGF beta homolog, dpp, as a novel direct target for HOW negative regulation in the wing imaginal disc. The binding of the repressor isoform HOW( L) to the dpp 3' untranslated region (UTR) leads to a reduction of GFP-dpp3'UTR reporter levels in S-2 cells, in an HRE site-dependent manner. Moreover, co-expression of HOW( L) in the wing imaginal disc with a dpp-GFP fusion construct led to a reduction in DPP-GFP levels in a dpp-3'UTR-dependent manner. Conversely, a reduction of the endogenous levels of HOW by targeted expression of HOW-specific double-stranded RNA led to a corresponding elevation in dpp mRNA level in the wing imaginal disc. Thus, by characterizing the RNA sequences that bind HOW, we demonstrate a novel aspect of regulation, at the mRNA level, of Drosophila DPP.
Organogenesis of the somatic musculature in Drosophila is directed by the precise adhesion between migrating myotubes and their corresponding ectodermally derived tendon cells. Whereas the PS integrins mediate the adhesion between these two cell types, their extracellular matrix (ECM) ligands have been only partially characterized. We show that the ECM protein Thrombospondin (Tsp), produced by tendon cells, is essential for the formation of the integrin-mediated myotendinous junction. Tsp expression is induced by the tendon-specific transcription factor Stripe, and accumulates at the myotendinous junction following the association between the muscle and the tendon cell. In tsp mutant embryos, migrating somatic muscles fail to attach to tendon cells and often form hemiadherens junctions with their neighboring muscle cells, resulting in nonfunctional somatic musculature. Talin accumulation at the cytoplasmic faces of the muscles and tendons is greatly reduced, implicating Tsp as a potential integrin ligand. Consistently, purified Tsp C-terminal domain polypeptide mediates spreading of PS2 integrin-expressing S2 cells in a KGD- and PS2-integrin-dependent manner. We propose a model in which the myotendinous junction is formed by the specific association of Tsp with multiple muscle-specific PS2 integrin receptors and a subsequent consolidation of the junction by enhanced tendon-specific production of Tsp secreted into the junctional space.
Terminal differentiation of single cells selected from a group of equivalent precursors may be random, or may be regulated by external signals. In the Drosophila embryo, maturation of a single tendon cell from a field of competent precursors is triggered by muscle-dependent signaling. The transcription factor Stripe was reported to induce both the precursor cell phenotype, as well as the terminal differentiation of muscle-bound tendons. The mechanism by which Stripe activates these distinct differentiation programs remained unclear. Here, we demonstrate that each differentiation state is associated with a distinct Stripe isoform and that the Stripe isoforms direct different transcriptional outputs. Importantly, the transition to the mature differentiation state is triggered post-transcriptionally by enhanced production of the stripeA splice variant, which is typical of the tendon mature state. This elevation is mediated by the RNA-binding protein How( S), with levels sensitive to muscle-dependent signals. In how mutant embryos the expression of StripeA is significantly reduced, while overexpression of How( S) enhances StripeA protein as well as mRNA levels in embryos. Analysis of the expression of a stripeA minigene in S-2 cells suggests that this elevation may be due to enhanced splicing of stripeA. Consistently, stripeA mRNA is specifically reduced in embryos mutant for the splicing factor Crn, which physically interacts with How( S). Thus, we demonstrate a mechanism by which tendon cell terminal differentiation is maintained and reinforced by the approaching muscle.
In both vertebrates and invertebrates, glial cells wrap axonal processes to ensure electrical conductance. Here we report that Crooked neck (Crn), the Drosophila homolog of the yeast Clf1p splicing factor, is directing peripheral glial cell maturation. We show that crooked neck is expressed and required in glial cells to control migration and axonal wrapping. Within the cytoplasm, Crn interacts with the RNA-binding protein HOW and then translocates to the nucleus where the Crn/HOW complex controls glial differentiation by facilitating splicing of specific target genes. By using a GFP-exon trap approach, we identified some of the in vivo target genes that encode proteins localized in autocellular septate junctions. In conclusion, here we show that glial cell differentiation is controlled by a cytoplasmic assembly of splicing components, which upon translocation to the nucleus promote the splicing of genes involved in the assembly of cellular junctions.
Background: Cell-cycle progression is tightly regulated during embryonic development. In the Drosophila early embryo, the levels of String/Cdc25 define the precise timing and sites of cell divisions. However, cell-cycle progression is arrested in the mesoderm of gastrulating embryos despite a positive transcriptional string/cdc25 activation provided by the mesoderm-specific action of Twist. Whereas String/Cdc25 is negatively regulated by Tribbles in the mesoderm at these embryonic stages, the factor(s) controlling string/cdc25 mRNA levels has yet to be elucidated. Results: Here, we show that the repressor isoform of the Drosophila RNA binding protein Held Out Wing [HOW(L)] is required to inhibit mesodermal cell division during gastrulation. Embryos mutant for how exhibited an excess of cell divisions, leading to delayed mesoderm invagination. The levels of the mitotic activator string/ cdc25 mRNA in these embryos were significantly elevated. Protein-RNA precipitation experiments show that HOW(L) binds string/cdc25 mRNA. Overexpression of HOW(L) in Schneider cells reduces specifically the steady-state mRNA levels of a gfp reporter fused to string/ cdc25 untranslated region (3'UTR). Conclusions: Our results suggest that in wild-type embryos, string/cdc25 mRNA levels are downregulaled by the repressor isoform HOW(L), which binds directly to string/cdc25 mRNA and regulates its degradation. Thus, we are proposing a novel posttranscriptional mechanism controlling cell-cycle progression in the Drosophila embryo.
Crosstalk between signaling pathways is crucial for the generation of complex and varied transcriptional networks. Antagonism between the EGF-receptor (EGFR) and Notch pathways in particular is well documented, although the underlying mechanism is poorly understood. The global corepressor Groucho (Gro) and its transducin-like Enhancer-of-split (TLE) mammalian homologs mediate repression by a myriad of repressors, including effectors of the Notch, Wnt (Wg) and TGF-beta (Dpp) signaling cascades(1-8). Given that there are genetic interactions between gro and components of the EGFR pathway(9) (ref. 9 and P. H. et al., unpublished results), we tested whether Gro is at a crossroad between this and other pathways. Here we show that phosphorylation of Gro in response to MAPK activation weakens its repressor capacity, attenuating Gro-dependent transcriptional silencing by the Enhancer-of-split proteins, effectors of the Notch cascade. Thus, Gro is a new junction between signaling pathways, enabling EGFR signaling to antagonize transcriptional output by Notch and potentially other Gro-dependent pathways.
Locomotion relies on stable attachment of muscle fibres to their target sites, a process that allows for muscle contraction to generate movement. Here, we show that glide/gcm and glide2/gcm2, the fly glial cell determinants, are expressed in a subpopulation of embryonic tendon cells and required for their terminal differentiation. By using loss-of-function approaches, we show that in the absence of both genes, muscle attachment to tendon cells is altered, even though the molecular cascade induced by stripe, the tendon cell determinant, is normal. Moreover, we show that glide/gcm activates a new tendon cell gene independently of stripe. Finally, we show that segment polarity genes control the epidermal expression of glide/gcm and determine, within the segment, whether it induces glial or tendon cell-specific markers. Thus, under the control of positional cues, glide/gcm triggers a new molecular pathway involved in terminal tendon cell differentiation, which allows the establishment of functional muscle attachment sites and locomotion.
Drosophila proprioceptors (chordotonal organs) are structured as a linear array of four lineage-related cells: a neuron, a glial cell, and two accessory cells, called cap and ligament, between which the neuron is stretched. To function properly as stretch receptors, chordotonal organs must be stably anchored at both edges. The cap cells are anchored to the cuticle through specialized lineage-related attachment cells. However, the mechanism by which the ligament cells at the other edge of the organ attach is not known. Here, we report the identification of specialized attachment cells that anchor the ligament cells of pentascolopidial chordotonal organs (lch5) to the cuticle. The ligament attachment cells are recruited by the approaching ligament cells upon reaching their attachment site, through an EGFR-dependent mechanism. Molecular characterization of lch5 attachment cells demonstrated that they share significant properties with Drosophila tendon cells and with mammalian proprioceptive organs.
Epithelial tissue functions depend largely on a polarized organization of the individual cells. We examined the roles of the Drosophila PDGF/VEGF receptor (PVR) in polarized epithelial cells, with specific emphasis on the wing disc epithelium. Although the receptor is broadly distributed in this tissue, two of its ligands, PVF1 and PVF3 are specifically deposited within the apical extracellular space, implying that polarized apical activation of the receptor takes place. The apical localization of the ligands involves a specialized secretion pathway. Clones for null alleles of Pvr or expression of RNAi constructs showed no phenotypes in the wing disc or pupal wing, suggesting that Pvr plays a redundant role in this tissue. However, when uniform expression of a constitutively dimerizing receptor was induced, loss of epithelial polarity, formation of multiple adherens and septate junctions, and tumorous growth were observed in the wing disc. Elevation of the level of full-length PVR also gave rise to prominent phenotypes, characterized by higher levels of actin microfilaments at the basolateral. areas of the cells and irregular folding of the tissue. Together, these results suggest that polarized PVR activation is necessary for the proper organization of the wing disc epithelium, by regulating the apical assembly of the actin cytoskeleton.
Background: Shot (previously named Kakapo), is a Drosophila Plakin family member containing both Actin binding and microtubule binding domains. In Drosophila, it is required for a wide range of processes, including axon extension, dendrite formation, axonal terminal arborization at the neuromuscular junction, tendon cell development, and adhesion of wing epithelium. Results: To address how Shot exerts its activity at the molecular level, we investigated the molecular interactions of Shot with candidate proteins in mature larval tendon cells. We show that Shot colocalizes with EB1/APC1 and with a compact microtubule array extending between the muscle-tendon junction and the cuticle. Shot forms a protein complex with EB1 via its C-terminal EF-hands and GAS2-containing domains. In tendon cells with reduced Shot activity, EB1/APC1 dissociate from the muscle-tendon junction, and the microtubule array elongates. The resulting tendon cell, although associated with the muscle and the cuticle ends, loses its stress resistance and elongates. Conclusions: Our results suggest that Shot mediates tendon stress resistance by the organization of a compact microtubule network at the muscle-tendon junction. This is achieved by Shot association with the cytoplasmic faces of the basal hemiadherens junction and with the EB1/APC1 complex.
The Drosophila tracheal system is an interconnected tubular respiratory network, which is formed by directed stereotypic migration and fusion of branches. Cell migration and specification are determined by combinatorial signaling of several morphogens secreted from the ectoderm. We report the discovery of a group of ectodermal cells, marked by Stripe (Sr) expression, that coordinates tracheal cell migration in the dorsoventral axis. Sr, an EGR family transcription factor, is known to regulate muscle migration. In this study, we show that Sr ectodermal cells also provide signals that are utilized for tracheal migration. These cues are separated in the time course of embryonic development. Initially, tendon-precursor cells are in close proximity to the tracheal cells, and later, when tracheal migration is complete, the muscles displace the trachea and attach to the tendon cells. sr-mutant embryos exhibit defects in migration of all tracheal branches. Although the FGF ligand Branchless (Bnl) is expressed in a subset of tendon-precursor cells independently of Sr, Bnl functions cooperatively with proteins induced by Sr in attraction of tracheal branches. (C) 2002 Elsevier Science (USA).
Differential RNA metabolism regulates a wide array of developmental processes. Here, we describe a mechanism that controls the transition from premature Drosophila tendon precursors into mature muscle-bound tendon cells. This mechanism is based on the opposing activities of two isoforms of the RNA binding protein How. While the isoform How(L) is a negative regulator of Stripe, the key modulator of tendon cell differentiation, How(S) isoform elevates Stripe levels, thereby releasing the differentiation arrest induced by How(L). The opposing activities of the How isoforms are manifested by differential rates of mRNA degradation of the target stripe mRNA. This mechanism is conserved, as the mammalian RNA binding Quaking proteins may similarly affect the levels of Krox20, a regulator of Schwann cell maturation.
The gene which is defective in Duchenne muscular dystrophy (DMD) is the largest known gene containing at least 79 introns, some of which are extremely large. The product of the gene in muscle, dystrophin, is a 427 kDa protein. The same gene encodes at least two additional non-muscle full length dystrophin isoforms transcribed from different promoters located in the 5'-end region of the gene, and four smaller proteins transcribed from internal promoters located further downstream, and lack important domains of dystrophin. Several other genes, encoding evolutionarily related proteins, have been identified. To study the evolution of the DMD gene and the significance of its various products, we have searched for genes encoding dystrophin-like proteins in sea urchin and in Drosophila. We previously reported on the characterization of a sea urchin gene encoding a protein which is an evolutionary homologue of Dp116, one of the small products of the mammalian DMD gene, and on the partial sequencing of a large product of the same gene. Here we describe the full-length product which shows strong structural similarity and sequence identity to human dystrophin and utrophin. We also describe a Drosophila gene closely related to the human dystrophin gene. Like the human gene, the Drosophila gene encodes at least three isoforms of full length dystrophin-like proteins (dmDLP1, dmDLP2 and dmDLP3,), regulated by different promoters located at the 5' end of the gene, and a smaller product regulated by an internal promoter (dmDp186). As in mammals, dmDp186 and the dmDLPs share the same C-terminal and cysteine-rich domains which are very similar to the corresponding domains in human dystrophin and utrophin. In addition, dmDp186 contains four of the spectrin-like repeats of the dmDLPs and a unique N-terminal region of 512 amino acids encoded by a single exon. The full length products and the small product have distinct patterns of expression. Thus, the complex structure of the dystrop
The precise match between somatic muscles and their epidermal attachment cells is achieved through a continuous dialogue between these two cell types. Whereas tendon cells direct myotube migration and final patterning, the muscles are essential for the maintenance of the fate of tendon cells. The Drosophila neuregulin-like ligand, Vein, and its receptor, the epidermal growth factor receptor (Egfr), are critical components in the inductive signaling process that takes place between muscles and tendon cells. Additional gene products that relay the Vein-Egfr effect in Drosophila are conserved in the vertebrate neuregulin-mediated cascade. This review describes genetic and molecular aspects of the muscle-tendon inductive processes in Drosophila, and compares them with the relevant mechanisms in the vertebrate embryo.
In Drosophila, a tendon cell is selected from a group of equipotent precursors following its interaction with a muscle cell. This interaction results in elevated levels of the transcription factor Stripe in the future tendon cells. Here we show that the balance between two distinct forms of the RNA-binding protein How maintains low levels of Stripe at the precursor stage and high levels in the mature tendon. The long, nuclear-specific protein How(L) downregulates Stripe protein levels at the precursor stage by binding stripe mRNA and inhibiting its nuclear export. This inhibition is likely to be counteracted by the short How(S) protein, present in both nucleus and cytoplasm, which is upregulated in the muscle-bound tendon cell following EGF receptor activation.
The presence of a single EGF receptor in Drosophila is contrasted by multiple ligands activating it. This work explores the role of two ligands, Spitz and Vein, in the embryonic ventral ectoderm. Spitz is a potent ligand, whereas Vein is an intrinsically weak activating Ligand. We show that secreted Spitz emanating from the midline, triggers expression of vein in the ventral-most cell rows, by inducing expression of the ETS domain transcription factor Pointed P1. In the absence of Vein, lateral cell fates are not induced when Spitz levels are compromised. The positive feedback loop of Vein generates a robust mechanism for patterning the ventral ectoderm.
Activation of the Drosophila EGF-receptor (DER) is spatially and temporally controlled by the release of its various ligands. DER and its ligand Spitz mediate the formation of specific somatic muscle precursors. We show that a second DER ligand, Vein, complements the activity of Spitz in the development of various somatic muscle precursors. In vn mutant embryos, the DER-dependent muscle precursors do not form in some of the segments. This phenotype is significantly enhanced in embryos carrying only one copy of wild type spitz. Our analysis suggests that Vein activation of DER differs qualitatively from that of Spitz in that it does not lead to the expression of the inhibitory protein Argos, possibly leading to a continuous activation of the DER signaling pathway. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved.
In the Drosophila embryo, the correct association of muscles with their specific tendon cells is achieved through reciprocal interactions between these two distinct cell types. Tendon cell differentiation is initiated by activation of the EGF-receptor signaling pathway within these cells by Vein, a neuregulin-like factor secreted by the approaching myotube. Here, we describe the cloning and the molecular and genetic analyses of kakapo, a Drosophila gene, expressed in the tendons, that is essential for muscle-dependent tendon cell differentiation. Kakapo is a large intracellular protein and contains structural domains also found in cytoskeletal-related vertebrate proteins (including plakin, dystrophin, and Gas2 family members). kakapo mutant embryos exhibit abnormal muscle-dependent tendon cell differentiation. A major defect in the kakapo mutant tendon cells is the failure of Vein to be localized at the muscle-tendon junctional site; instead, Vein is dispersed and its levels are reduced. This may lead to aberrant differentiation of tendon cells and consequently to the kakapo mutant deranged somatic muscle phenotype.
Inductive interactions between cells of distinct fates underlie the basis for morphogenesis and organogenesis across species. In the Drosophila embryo, somatic myotubes form specific interactions with their epidermal muscle attachment (EMA) cells. The establishment of these interactions is a first step toward further differentiation of the EMA cells into elongated tendon cells containing an organized array of microtubules and microfilaments. Here we show that the molecular signal for terminal differentiation of tendon cells is the secreted Drosophila neuregulin-like growth factor Vein produced by the myotubes. Although vein mRNA is produced by all of the myotubes, Vein protein is secreted and accumulates specifically at the muscle-tendon cell junctional site. In loss-of-function vein mutant embryos, muscle-dependent differentiation of tendon cells, measured by the level of expression of specific markers (Delilah and beta 1 tubulin) is blocked. When Vein is expressed in ectopic ectodermal cells, it induces the ectopic expression of these genes. Our results favor the possibility that the Drosophila EGF receptor DER/Egfr expressed by the EMA cells functions as a receptor for Vein. We show that Vein/Egfr binding activates the Ras pathway in the EMA cells leading to the transcription of the tendon-specific genes, stripe, delilah, and beta 1 tubulin. In Egfr(1F26) mutant embryos that lack functional Egfr expression, the levels of Delilah and beta 1 tubulin are very low. In addition, the ability of ectopic Vein to induce the expression of Delilah and beta 1 tubulin depends on the presence of functional Egfrs. finally, activation of the Egfr signaling pathway by either ectopically secreted Spitz, or activated Ras, leads to the ectopic expression of Delilah. These results suggest that inductive interactions between myotubes and their epidermal muscle attachment cells are initiated by the binding of Vein, to the Egfr on the surface of EMA cells.
Directed intercellular interactions between distinct cell. types underlie the basis for organogenesis during embryonic development, This paper focuses on the establishment of the final somatic muscle pattern in Drosophila, and on the possible cross-talk between the myotubes and the epidermal muscle attachment cells, occurring while both cell types undergo distinct developmental programs. Our findings suggest that the stripe gene is necessary and sufficient to initiate the developmental program of epidermal muscle attachment cells, In stripe mutant embryos, these cells do not differentiate correctly. Ectopic expression of Stripe in various epidermal cells transforms these cells into muscle-attachment cells expressing an array of epidermal muscle attachment cell-specific markers, Moreover, these ectopic epidermal muscle attachment cells are capable of attracting somatic myotubes from a limited distance, providing that the myotube has not yet been attached to or been influenced by a closer wild-type attachment cell. Analysis of the relationships between muscle binding and differentiation of the epidermal muscle attachment cell was performed in mutant embryos in which loss of muscles, or ectopic muscles were induced, This analysis indicated that, although the initial expression of epidermal muscle-attachment cell-specific genes including stripe and groovin is muscle independent, their continuous expression is maintained only in epidermal muscle attachment cells that are connected to muscles, These results suggest that the binding of a somatic muscle to an epidermal muscle attachment cell triggers a signal affecting gene expression in the attachment cell. Taken together, our results suggest the presence of a reciprocal signaling mechanism between the approaching muscles and the epidermal muscle attachment cells, First the epidermal muscle attachment cells signal the myotubes and induce myotube attraction and adhesion to their target cells, Following this binding, the
A Drosophila FGF receptor homolog (DFGF-R2/DFRI) termed Heartless (Htl) is expressed in the embryonic mesoderm. The phenotypes of null mutant embryos demonstrated that Htl is a central player that is required for the development of several mesodermal lineages. No abnormalities in the primary specification of the mesoderm were observed. The first defects were seen as irregular migration and spreading of the mesoderm over the ectoderm. Subsequently, cell fates were not induced in several lineages including the visceral mesoderm, heart, and the dorsal somatic muscles. The defects in the induction of cell fates are likely to result from failure of the mesoderm to spread over the ectoderm and receive patterning signals. The defective spreading could be circumvented in htl mutant embryos by providing an ectopic Dpp patterning signal, leading to the formation of heart and dorsal muscle cells. Htl appears to be required also subsequently during the migration and morphogenesis of the different lineages. Expression of a dominant-negative htl construct after the initial induction of eel fates gave rise to aberrant migration and organization of the visceral mesoderm, heart, and somatic muscles. Thus, a common role for Htl in cell migration and tissue organization may account for the pleiotropic defects of the htl mutation.
Proteins from the spectrin superfamily contribute to cell polarity and shape during the morphogenetic movements that accompany embryogenesis. Drosophila MSP-300, a member of the spectrin superfamily, is expressed in somatic, visceral and heart embryonic muscles. Cloning and sequence analysis of various spliced forms of MSP-300 reveals functional and structural similarities between MSP-300 and vertebrate Dystrophin, the product of the Duchenne Muscular Dystrophy gene. The identification of a strain mutant for the MSP-300 gene is described. Analysis of the somatic muscle phenotype in MSP-300 mutant embryos suggests that the protein contributes to the integrity of the somatic and visceral muscle tissue during periods of significant morphogenetic change. Functional synergism between MSP-300 and laminin is demonstrated by the analysis of the phenotype of embryos mutant for both genes. The enhancement of aberrant muscle phenotype in the double mutants suggests a link between MSP-300 and laminin function in mediating proper extension of the myotube towards the epidermal muscle attachment site. In addition, both genes function to establish gut integrity. In view of the functional and structural similarities between MSP-300 and Dystrophin, it is postulated that Dystrophin is not only required for proper muscle function in adult life but also contributes to muscle morphogenesis during the development of the vertebrate embryo.
We have cloned and molecularly characterized the Drosophila gene stripe (sr) required for muscle-pattern formation in the embryo. Through differential splicing, sr encodes two nuclear protein variants which contain a zinc finger DNA-binding domain in common with the early growth response (egr) family of vertebrate transcription factors. The sr transcripts and their protein products are exclusively expressed in the epidermal muscle attachment cells and their ectodermal precursors, but not in muscles or muscle precursors. The results suggest that sr activity induces a subset of ectodermal cells to develop into muscle attachment sites and to provide spatial cues necessary to orient myotubes along the basal surface of the epidermis to their targeted attachment cells.
Laminin, an extracellular matrix glycoprotein, has been implicated in a wide array of biological activities. Its specific roles during development, however, remain to be elucidated. In this report we describe the specific contribution of laminin to histogenesis and organogenesis during Drosophila embryogenesis. In particular, the function of laminin during mesoderm development and morphogenesis was dissected by analyzing embryos deficient in laminin A chain alone or in both laminin A and B2 chains. We show that laminin function is not required for the early phases of mesoderm patterning and morphogenesis in embryos at the extended germ band phase (stage 11). However, laminin function is required for the proper morphogenesis of the heart, somatic mesoderm, and the gut during later stages of embryonic development. In laminin A-deficient embryos, the heart is ''broken'' as a result of dissociation of the pericardial cells. The somatic myotubes are defective and do not extend properly, and the ventral oblique muscles never reach their proper attachment sites. Finally, during midgut formation, the endoderm fails to undergo the initial columnar polarization in the absence of functional laminin. These studies indicate that the primary function of laminin is to provide structural support for the various cell types, thereby enabling their proper organization into the various organs. (C) 1995 Academic Press, Inc.
The correct patterning of muscles in the Drosophila embryo depends on the migration of developing muscles over the ectoderm and on the attachment of these muscles to specific attachment sites. We investigate the mechanisms that are involved in this process and describe experiments that allow a genetic dissection of the role of the ectoderm in muscle migration and attachment. We show that cells along the segmental border in the ectoderm are used by the developing muscles to reach their attachment sites. These segment border cells are recognized by dissociated myotubes in single suspensions in culture. Thus, developing muscles have properties that allow the specific recognition of the segment border cells and migrate to attach to these cells. The segment border cells are absent in the mutant wingless and naked. In these mutants, the muscles are severely disorganized. We show that this is not a mere consequence of disruption of the epidermis, since, in the mutant patched, where segmental patterning is affected, the segment border cells are present near their normal position; the muscles in this mutant are relatively organized. Similarly, in the mutant lines where ectopic segment border cells are present, the observed muscle derangement correlates well with the ectopic attachment sites that are present. Finally, we have analyzed mutants at the stripe locus and have shown that lethal alleles disrupt muscle organization during embryogenesis. Enhancer-trap alleles of stripe that we have analyzed show reporter gene expression in the segment border cells. Our results indicate a role for the segment border cells in guidance of migrating muscle fibers to their attachment sites.
Myotube migration and the formation of muscle attachments are crucial events for the proper development of muscle patterning in the Drosophila embryo. This paper describes the identification of a new embryonic muscle-specific protein, MSP-300, in Drosophila. This protein is initially expressed by muscle precursors at muscle-ectoderm and muscle-muscle attachment sites. As development continues, MSP-300 becomes associated with muscle myofibrillar network. Studies of the subcellular localization of this muscle-specific protein in primary embryonic cultured myotubes show that MSP-300 decorates actin filaments, and that it is specifically enriched in sites where actin microfilaments are linked to the plasma membrane. Migrating myotubes exhibit high levels of this protein at their leading edge while, in myotubes that have already developed sarcomeric architecture, the protein is localized mainly at the Z-discs. Sequence of a partial 3.9 kb cDNA clone and molecular analysis of the predicted protein sequence of this protein indicates that it encodes a high relative molecular mass protein (approximately 300 X 10(3)), which exhibits at least five spectrin-like repeats. Several properties are shared by MSP-300 and members of the spectrin superfamily: it is associated with actin microfilaments, its sequence exhibits spectrin-like repeats and it is localized at sites where actin is linked to the plasma membrane. This protein could have a developmental role in the formation of muscle-ectoderm attachments and may be involved in myotube migration on the ectoderm.
We report here on a new 135-kd membrane protein which is specifically associated with intercellular adherens-type junctions. This surface component was identified by a monoclonal antibody, ID-7.2.3, raised against detergent-extracted components of membranes of chicken cardiac muscle rich in intercalated discs. The antibodies stain extensively adherens junctions in intact cardiac muscle and in lens, as well as in cultured cells derived from these tissues. In living cultured cells only very little immunolabelling was obtained with ID-7.2.3 antibodies, probably due to the limited accessibility of the antibodies to the intercellular gap. However, upon the removal of extracellular Ca2+ ions a dissociation of the junction occurred, leading to the rapid exposure of the 135-kd protein. Immunoelectron microscopic labelling of EGTA-treated, or detergent-permeabilized cells indicated that the antigen is found along the plasma membrane and highly enriched in contact areas. Double immunolabelling for both the 135-kd protein and vinculin pointed to the close association of the two in intercellular junctions and to the apparent absence of the former protein from the vinculin-rich focal contacts of cultured cells and from dense plaque of smooth muscle. Immunoblotting indicated that the 135-kd protein is present in many tissues but is particularly enriched in heart, lens and brain.