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Sample records for drosophila tendon cells

  1. Singling out Drosophila tendon cells: a dialogue between two distinct cell types.

    PubMed

    Volk, T

    1999-11-01

    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.

  2. Functional Conservation of the Glide/Gcm Regulatory Network Controlling Glia, Hemocyte, and Tendon Cell Differentiation in Drosophila.

    PubMed

    Cattenoz, Pierre B; Popkova, Anna; Southall, Tony D; Aiello, Giuseppe; Brand, Andrea H; Giangrande, Angela

    2016-01-01

    High-throughput screens allow us to understand how transcription factors trigger developmental processes, including cell specification. A major challenge is identification of their binding sites because feedback loops and homeostatic interactions may mask the direct impact of those factors in transcriptome analyses. Moreover, this approach dissects the downstream signaling cascades and facilitates identification of conserved transcriptional programs. Here we show the results and the validation of a DNA adenine methyltransferase identification (DamID) genome-wide screen that identifies the direct targets of Glide/Gcm, a potent transcription factor that controls glia, hemocyte, and tendon cell differentiation in Drosophila. The screen identifies many genes that had not been previously associated with Glide/Gcm and highlights three major signaling pathways interacting with Glide/Gcm: Notch, Hedgehog, and JAK/STAT, which all involve feedback loops. Furthermore, the screen identifies effector molecules that are necessary for cell-cell interactions during late developmental processes and/or in ontogeny. Typically, immunoglobulin (Ig) domain-containing proteins control cell adhesion and axonal navigation. This shows that early and transiently expressed fate determinants not only control other transcription factors that, in turn, implement a specific developmental program but also directly affect late developmental events and cell function. Finally, while the mammalian genome contains two orthologous Gcm genes, their function has been demonstrated in vertebrate-specific tissues, placenta, and parathyroid glands, begging questions on the evolutionary conservation of the Gcm cascade in higher organisms. Here we provide the first evidence for the conservation of Gcm direct targets in humans. In sum, this work uncovers novel aspects of cell specification and sets the basis for further understanding of the role of conserved Gcm gene regulatory cascades.

  3. Functional Conservation of the Glide/Gcm Regulatory Network Controlling Glia, Hemocyte, and Tendon Cell Differentiation in Drosophila

    PubMed Central

    Cattenoz, Pierre B.; Popkova, Anna; Southall, Tony D.; Aiello, Giuseppe; Brand, Andrea H.; Giangrande, Angela

    2016-01-01

    High-throughput screens allow us to understand how transcription factors trigger developmental processes, including cell specification. A major challenge is identification of their binding sites because feedback loops and homeostatic interactions may mask the direct impact of those factors in transcriptome analyses. Moreover, this approach dissects the downstream signaling cascades and facilitates identification of conserved transcriptional programs. Here we show the results and the validation of a DNA adenine methyltransferase identification (DamID) genome-wide screen that identifies the direct targets of Glide/Gcm, a potent transcription factor that controls glia, hemocyte, and tendon cell differentiation in Drosophila. The screen identifies many genes that had not been previously associated with Glide/Gcm and highlights three major signaling pathways interacting with Glide/Gcm: Notch, Hedgehog, and JAK/STAT, which all involve feedback loops. Furthermore, the screen identifies effector molecules that are necessary for cell-cell interactions during late developmental processes and/or in ontogeny. Typically, immunoglobulin (Ig) domain–containing proteins control cell adhesion and axonal navigation. This shows that early and transiently expressed fate determinants not only control other transcription factors that, in turn, implement a specific developmental program but also directly affect late developmental events and cell function. Finally, while the mammalian genome contains two orthologous Gcm genes, their function has been demonstrated in vertebrate-specific tissues, placenta, and parathyroid glands, begging questions on the evolutionary conservation of the Gcm cascade in higher organisms. Here we provide the first evidence for the conservation of Gcm direct targets in humans. In sum, this work uncovers novel aspects of cell specification and sets the basis for further understanding of the role of conserved Gcm gene regulatory cascades. PMID:26567182

  4. Functional Conservation of the Glide/Gcm Regulatory Network Controlling Glia, Hemocyte, and Tendon Cell Differentiation in Drosophila.

    PubMed

    Cattenoz, Pierre B; Popkova, Anna; Southall, Tony D; Aiello, Giuseppe; Brand, Andrea H; Giangrande, Angela

    2016-01-01

    High-throughput screens allow us to understand how transcription factors trigger developmental processes, including cell specification. A major challenge is identification of their binding sites because feedback loops and homeostatic interactions may mask the direct impact of those factors in transcriptome analyses. Moreover, this approach dissects the downstream signaling cascades and facilitates identification of conserved transcriptional programs. Here we show the results and the validation of a DNA adenine methyltransferase identification (DamID) genome-wide screen that identifies the direct targets of Glide/Gcm, a potent transcription factor that controls glia, hemocyte, and tendon cell differentiation in Drosophila. The screen identifies many genes that had not been previously associated with Glide/Gcm and highlights three major signaling pathways interacting with Glide/Gcm: Notch, Hedgehog, and JAK/STAT, which all involve feedback loops. Furthermore, the screen identifies effector molecules that are necessary for cell-cell interactions during late developmental processes and/or in ontogeny. Typically, immunoglobulin (Ig) domain-containing proteins control cell adhesion and axonal navigation. This shows that early and transiently expressed fate determinants not only control other transcription factors that, in turn, implement a specific developmental program but also directly affect late developmental events and cell function. Finally, while the mammalian genome contains two orthologous Gcm genes, their function has been demonstrated in vertebrate-specific tissues, placenta, and parathyroid glands, begging questions on the evolutionary conservation of the Gcm cascade in higher organisms. Here we provide the first evidence for the conservation of Gcm direct targets in humans. In sum, this work uncovers novel aspects of cell specification and sets the basis for further understanding of the role of conserved Gcm gene regulatory cascades. PMID:26567182

  5. A Tendon Cell Specific RNAi Screen Reveals Novel Candidates Essential for Muscle Tendon Interaction.

    PubMed

    Tiwari, Prabhat; Kumar, Arun; Das, Rudra Nayan; Malhotra, Vivek; VijayRaghavan, K

    2015-01-01

    Tendons are fibrous connective tissue which connect muscles to the skeletal elements thus acting as passive transmitters of force during locomotion and provide appropriate body posture. Tendon-derived cues, albeit poorly understood, are necessary for proper muscle guidance and attachment during development. In the present study, we used dorsal longitudinal muscles of Drosophila and their tendon attachment sites to unravel the molecular nature of interactions between muscles and tendons. We performed a genetic screen using RNAi-mediated knockdown in tendon cells to find out molecular players involved in the formation and maintenance of myotendinous junction and found 21 candidates out of 2507 RNAi lines screened. Of these, 19 were novel molecules in context of myotendinous system. Integrin-βPS and Talin, picked as candidates in this screen, are known to play important role in the cell-cell interaction and myotendinous junction formation validating our screen. We have found candidates with enzymatic function, transcription activity, cell adhesion, protein folding and intracellular transport function. Tango1, an ER exit protein involved in collagen secretion was identified as a candidate molecule involved in the formation of myotendinous junction. Tango1 knockdown was found to affect development of muscle attachment sites and formation of myotendinous junction. Tango1 was also found to be involved in secretion of Viking (Collagen type IV) and BM-40 from hemocytes and fat cells. PMID:26488612

  6. Coordinated Development of Muscles and Tendon-Like Structures: Early Interactions in the Drosophila Leg

    PubMed Central

    Soler, Cedric; Laddada, Lilia; Jagla, Krzysztof

    2016-01-01

    The formation of the musculoskeletal system is a remarkable example of tissue assembly. In both vertebrates and invertebrates, precise connectivity between muscles and skeleton (or exoskeleton) via tendons or equivalent structures is fundamental for movement and stability of the body. The molecular and cellular processes underpinning muscle formation are well-established and significant advances have been made in understanding tendon development. However, the mechanisms contributing to proper connection between these two tissues have received less attention. Observations of coordinated development of tendons and muscles suggest these tissues may interact during the different steps in their development. There is growing evidence that, depending on animal model and muscle type, these interactions can take place from progenitor induction to the final step of the formation of the musculoskeletal system. Here, we briefly review and compare the mechanisms behind muscle and tendon interaction throughout the development of vertebrates and Drosophila before going on to discuss our recent findings on the coordinated development of muscles and tendon-like structures in Drosophila leg. By altering apodeme formation (the functional Drosophila equivalent of tendons in vertebrates) during the early steps of leg development, we affect the spatial localization of subsequent myoblasts. These findings provide the first evidence of the developmental impact of early interactions between muscle and tendon-like precursors, and confirm the appendicular Drosophila muscle system as a valuable model for studying these processes. PMID:26869938

  7. Mesenchymal stem cell applications to tendon healing

    PubMed Central

    Chaudhury, Salma

    2012-01-01

    Summary Tendons are often subject to age related degenerative changes that coincide with a diminished regenerative capacity. Torn tendons often heal by forming scar tissue that is structurally weaker than healthy native tendon tissue, predisposing to mechanical failure. There is increasing interest in providing biological stimuli to increase the tendon reparative response. Stem cells in particular are an exciting and promising prospect as they have the potential to provide appropriate cellular signals to encourage neotendon formation during repair rather than scar tissue. Currently, a number of issues need to be investigated further before it can be determined whether stem cells are an effective and safe therapeutic option for encouraging tendon repair. This review explores the in-vitro and invivo evidence assessing the effect of stem cells on tendon healing, as well as the potential clinical applications. PMID:23738300

  8. Amontillado is required for Drosophila Slit processing and for tendon-mediated muscle patterning

    PubMed Central

    Ordan, Elly

    2016-01-01

    ABSTRACT 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. PMID:27628033

  9. The cell biology of suturing tendons.

    PubMed

    Wong, J K F; Alyouha, S; Kadler, K E; Ferguson, M W J; McGrouther, D A

    2010-07-01

    Trauma by suturing tendon form areas devoid of cells termed "acellular zones" in the matrix. This study aimed to characterise the cellular insult of suturing and acellular zone formation in mouse tendon. Acellular zone formation was evaluated using single grasping sutures placed using flexor tendons with time lapse cell viability imaging for a period of 12h. Both tension and injury were required to induce cell death and cell movement in the formation of the acellular zone. DNA fragmentation studies and transmission electron microscopy indicated that cells necrosed. Parallel in vivo studies showed that cell-to-cell contacts were disrupted following grasping by the suture in tensioned tendon. Without tension, cell death was lessened and cell-to-cell contacts remained intact. Quantitative immunohistochemistry and 3D cellular profile mapping of wound healing markers over a one year time course showed that acellular zones arise rapidly and showed no evidence of healing whilst the wound healing response occurred in the surrounding tissues. The acellular zones were also evident in a standard modified "Kessler" clinical repair. In conclusion, the suture repair of injured tendons produces acellular zones, which may potentially cause early tendon failure. PMID:20600895

  10. The Effects of Glucocorticoid on Tendon and Tendon Derived Cells.

    PubMed

    Dean, Benjamin John Floyd; Carr, Andrew Jonathan

    2016-01-01

    Glucocorticoids are generally used to relieve pain and/or inflammation in a wide variety of musculoskeletal disorders including osteoarthritis, inflammatory arthritis, tendinopathy and degenerative spine disease. Glucocorticoids reduce tendon derived cell proliferation in vitro and reduce extracellular matrix synthesis both in vitro and in vivo, in particular type I collagen synthesis. Glucocorticoids also appear to result in acute deleterious changes in healthy in vivo tendon including collagen necrosis, collagen disorganisation and inflammatory cell infiltration; while the overall effect of glucocorticoid administration on the mechanical properties of healthy in vivo tendon are generally negative. Overall the existing in vitro and in vivo evidence suggests that glucocorticoids should be used with caution in treating painful tendinopathy. Certainly a real need exists to follow up the long term clinical effects of glucocorticoid in treating tendinopathy, as there is currently a paucity of evidence in this area. However in this context while the short term benefits are clear, glucocorticoids remain a useful treatment option provided they are used in the right patients in sensible moderation. PMID:27535266

  11. Tendon Stem Cells: Mechanobiology and Development of Tendinopathy.

    PubMed

    Wang, James H-C; Komatsu, Issei

    2016-01-01

    Millions of people suffer from tendon injuries in both occupational and athletic settings. However, the restoration of normal structure and function to injured tendons still remains as one of the greatest challenges in orthopaedics and sports medicine. In recent years, a remarkable advancement in tendon research field has been the discovery of tendon stem/progenitor cells (TSCs). Unlike tenocytes, the predominant resident cell in tendons, TSCs have the ability to self-renew and multi-differentiate. Because of these distinct properties, TSCs may play a critical role in tendon physiology as well as pathology such as tendinopathy, which is a prevalent chronic tendon injury. Additionally, because TSCs are tendon-specific stem cells, they could potentially be used in tendon tissue engineering in vitro, and serve as a promising cell source for cell-based therapy to effectively repair or even regenerate injured tendons in clinical settings. PMID:27535248

  12. In vivo identity of tendon stem cells and the roles of stem cells in tendon healing.

    PubMed

    Tan, Qi; Lui, Pauline Po Yee; Lee, Yuk Wa

    2013-12-01

    We investigated the spatial distribution of stem cells in tendons and the roles of stem cells in early tendon repair. The relationship between tendon-derived stem cells (TDSCs) isolated in vitro and tendon stem cells in vivo was also explored. Iododeoxyuridine (IdU) label-retaining method was used for labeling stem cells in rat patellar tendons with and without injury. Co-localization of label-retaining cells (LRCs) with different markers was done by immunofluorescent staining. TDSCs were isolated from patellar tendon mid-substance after IdU pulsing, and the expression of different markers in fresh and expanded cells was done by immunofluorescent staining. More LRCs were found at the peritenon and tendon-bone junction compared with the mid-substance. Some LRCs at the peritenon were located at the perivascular niche. The LRC number and the expression of proliferative, tendon-related, pluripotency, and pericyte-related markers in LRCs in the window wound increased. Most of the freshly isolated TDSCs expressed IdU, and some TDSCs expressed pericyte-related markers, which were lost during expansion. Both freshly isolated and subcultured TDSCs expressed pluripotency markers, which were absent in LRCs in intact tendons. In conclusion, we identified LRCs at the peritenon, mid-substance, and tendon-bone junction. There were both vascular and non-vascular sources of LRCs at the peritenon, while the source of LRCs at the mid-substance was non-vascular. LRCs participated in tendon repair via migration, proliferation, activation for tenogenesis, and increased pluripotency. Some LRCs in the window wound were pericyte like. Most of the mid-substance TDSCs were LRCs. The pluripotency markers and pericyte-related marker in LRCs might be important for function after injury.

  13. Engineered scaffold-free tendon tissue produced by tendon-derived stem cells.

    PubMed

    Ni, Ming; Rui, Yun Feng; Tan, Qi; Liu, Yang; Xu, Liang Liang; Chan, Kai Ming; Wang, Yan; Li, Gang

    2013-03-01

    Most of the exogenous biomaterials for tendon repair have limitations including lower capacity for inducing cell proliferation and differentiation, poorer biocompatibility and remodeling potentials. To avoid these shortcomings, we intend to construct an engineered tendon by stem cells and growth factors without exogenous scaffolds. In this study, we produced an engineered scaffold-free tendon tissue (ESFTT) in vitro and investigated its potentials for neo-tendon formation and promoting tendon healing in vivo. The ESFTT, produced via tendon-derived stem cells (TDSCs) by treatment of connective tissue growth factor (CTGF) and ascorbic acid in vitro, was characterized by histology, qRT-PCR and immunohistochemistry methods. After ESFTT implanted into the nude mouse, the in vivo fluorescence imaging, histology and immunohistochemistry examinations showed neo-tendon formation. In a rat patellar tendon window injury model, the histology, immunohistochemistry and biomechanical testing data indicated ESFTT could significantly promote tendon healing. In conclusion, this is a proof-of-concept study demonstrating that ESFTT could be a potentially new approach for tendon repair and regeneration.

  14. Informing Stem Cell-Based Tendon Tissue Engineering Approaches with Embryonic Tendon Development.

    PubMed

    Okech, William; Kuo, Catherine K

    2016-01-01

    Adult tendons fail to regenerate normal tissue after injury, and instead form dysfunctional scar tissue with abnormal mechanical properties. Surgical repair with grafts is the current standard to treat injuries, but faces significant limitations including pain and high rates of re-injury. To address this, we aim to regenerate new, normal tendons to replace dysfunctional tendons. A common approach to tendon tissue engineering is to design scaffolds and bioreactors based on adult tendon properties that can direct adult stem cell tenogenesis. Despite significant progress, advances have been limited due, in part, to a need for markers and potent induction cues. Our goal is to develop novel tendon tissue engineering approaches informed by embryonic tendon development. We are characterizing structure-property relationships of embryonic tendon to identify design parameters for three-dimensional scaffolds and bioreactor mechanical loading systems to direct adult stem cell tenogenesis. We will review studies in which we quantified changes in the mechanical and biochemical properties of tendon during embryonic development and elucidated specific mechanisms of functional property elaboration. We then examined the effects of these mechanical and biochemical factors on embryonic tendon cell behavior. Using custom-designed bioreactors, we also examined the effects of dynamic mechanical loading and growth factor treatment on embryonic tendon cells. Our findings have established cues to induce tenogenesis as well as metrics to evaluate differentiation. We finish by discussing how we have evaluated the tenogenic differentiation potential of adult stem cells by comparing their responses to that of embryonic tendon cells in these culture systems.

  15. Famotidine suppresses osteogenic differentiation of tendon cells in vitro and pathological calcification of tendon in vivo.

    PubMed

    Yamamoto, Kenichi; Hojo, Hironori; Koshima, Isao; Chung, Ung-il; Ohba, Shinsuke

    2012-12-01

    Heterotopic ossification or calcification follows any type of musculoskeletal trauma and is known to occur after arthroplasties of hip, knee, shoulder, or elbow; fractures; joint dislocations; or tendon ruptures. Histamine receptor H2 (Hrh2) has been shown to be effective for reducing pain and decreasing calcification in patients with calcifying tendinitis, which suggested that H2 blockers were effective for the treatment of tendon ossification or calcification. However, the detailed mechanisms of its action on tendon remain to be clarified. We investigated the mechanisms underlying H2 blocker-mediated suppression of tendon calcification, with a focus on the direct action of the drug on tendon cells. Famotidine treatment suppressed the mRNA expressions of Col10a1 and osteocalcin, ossification markers, in a tendon-derived cell line TT-D6, as well as a preosteoblastic one MC3T3-E1. Both of the cell lines expressed Hrh2; histamine treatment induced osteocalcin expression in these cells. Famotidine administration suppressed calcification in the Achilles tendon of ttw mice, a mouse model of ectopic ossification. These data suggest that famotidine inhibits osteogenic differentiation of tendon cells in vitro, and this inhibition may underlie the anti-calcification effects of the drug in vivo. This study points to the use of H2 blockers as a promising strategy for treating heterotopic ossification or calcification in tendon, and provides evidence in support of the clinical use of famotidine.

  16. The effect of decellularized matrices on human tendon stem/progenitor cell differentiation and tendon repair.

    PubMed

    Yin, Zi; Chen, Xiao; Zhu, Ting; Hu, Jia-jie; Song, Hai-xin; Shen, Wei-liang; Jiang, Liu-yun; Heng, Boon Chin; Ji, Jun-feng; Ouyang, Hong-Wei

    2013-12-01

    It is reported that decellularized collagen matrices derived from dermal skin and bone have been clinically used for tendon repair. However, the varying biological and physical properties of matrices originating from different tissues may influence the differentiation of tendon stem cells, which has not been systematically evaluated. In this study, the effects of collagenous matrices derived from different tissues (tendon, bone and dermis) on the cell differentiation of human tendon stem/progenitor cells (hTSPCs) were investigated, in the context of tendon repair. It was found that all three matrices supported the adhesion and proliferation of hTSPCs despite differences in topography. Interestingly, tendon-derived decellularized matrix promoted the tendinous phenotype in hTSPCs and inhibited their osteogenesis, even under osteogenic induction conditions, through modulation of the teno- and osteolineage-specific transcription factors Scleraxis and Runx2. Bone-derived decellularized matrix robustly induced osteogenic differentiation of hTSPCs, whereas dermal skin-derived collagen matrix had no apparent effect on hTSPC differentiation. Based on the specific biological function of the tendon-derived decellularized matrix, a tissue-engineered tendon comprising TSPCs and tendon-derived matrix was successfully fabricated for Achilles tendon reconstruction. Implantation of this cell-scaffold construct led to a more mature structure (histology score: 4.08 ± 0.61 vs. 8.51 ± 1.66), larger collagen fibrils (52.2 ± 1.6 nm vs. 47.5 ± 2.8 nm) and stronger mechanical properties (stiffness: 21.68 ± 7.1 Nm m(-1) vs.13.2 ± 5.9 Nm m(-1)) of repaired tendons compared to the control group. The results suggest that stem cells promote the rate of repair of Achilles tendon in the presence of a tendinous matrix. This study thus highlights the potential of decellularized matrix for future tissue engineering applications, as well as developing a practical strategy for functional tendon

  17. Cell therapies for tendons: old cell choice for modern innovation.

    PubMed

    Petrou, Ilias G; Grognuz, Anthony; Hirt-Burri, Nathalie; Raffoul, Wassim; Applegate, Lee Ann

    2014-01-01

    Although tissue engineering and cell therapies are becoming realistic approaches for medical therapeutics, it is likely that musculoskeletal applications will be among the first to benefit on a large scale. Cell sources for tissue engineering and cell therapies for tendon pathologies are reviewed with an emphasis on small defect tendon injuries as seen in the hand which could adapt well to injectable cell administration. Specifically, cell sources including tenocytes, tendon sheath fibroblasts, bone marrow or adipose-derived stem cells, amniotic cells, placenta cells and platelet-derivatives have been proposed to enhance tendon regeneration. The associated advantages and disadvantages for these different strategies will be discussed and evolving regulatory requirements for cellular therapies will also be addressed. Human progenitor tenocytes, along with their clinical cell banking potential, will be presented as an alternative cell source solution. Similar cell banking techniques have already been described with other progenitor cell types in the 1950's for vaccine production, and these "old" cell types incite potentially interesting therapeutic options that could be improved with modern innovation for tendon regeneration and repair.

  18. The effect of tendon surface treatment on cell attachment for potential enhancement of tendon graft healing: an ex vivo model.

    PubMed

    Hashimoto, Takahiro; Sun, Yu-Long; An, Kai-Nan; Amadio, Peter C; Zhao, Chunfeng

    2012-12-01

    For both tendon allografts and autografts, the surface, initially optimized for gliding, may not be ideal to facilitate tissue integration for graft healing to host tendon or bone. As a prelude to studying tendon-bone integration, we investigated the effect of surface treatments with trypsin or mechanical abrasion on cell attachment to the tendon surface in a canine ex vivo intrasynovial tendon tissue culture model. Intrasynovial tendon allograft surfaces were seeded with cells after the following treatments: (1) no treatment, (2) mechanical abrasion, (3) trypsin, and (4) abrasion and trypsin. The area covered by cells was determined using confocal laser microscopy at one and two weeks. Results were compared to untreated extrasynovial tendon. Additional tendons were characterized with scanning electron microscopy. Tendons with trypsin treatment had significantly more surface coverage with cells than the other groups, after both one and two weeks of culture. In terms of the cellular shape and size, cells on tendons with trypsin treatment spread more and were more polygonal in shape, whereas tendons with mechanical abrasion with/without trypsin treatment contained smaller, more spindle-like cells. Surface roughening can affect cell behavior with topographical stimulation. Trypsin surface digestion exposes a mesh-like structure on the tendon surface, which could enhance cell adherence and, possibly, tendon/bone healing.

  19. Drosophila's contribution to stem cell research

    PubMed Central

    Singh, Gyanesh

    2016-01-01

    The discovery of Drosophila stem cells with striking similarities to mammalian stem cells has brought new hope for stem cell research. Recent developments in Drosophila stem cell research is bringing wider opportunities for contemporary stem cell biologists. In this regard, Drosophila germ cells are becoming a popular model of stem cell research. In several cases, genes that controlled Drosophila stem cells were later discovered to have functional homologs in mammalian stem cells. Like mammals, Drosophila germline stem cells (GSCs) are controlled by both intrinsic as well as external signals. Inside the Drosophila testes, germline and somatic stem cells form a cluster of cells (the hub). Hub cells depend on JAK-STAT signaling, and, in absence of this signal, they do not self-renew. In Drosophila, significant changes occur within the stem cell niche that contributes to a decline in stem cell number over time. In case of aging Drosophila, somatic niche cells show reduced DE-cadherin and unpaired (Upd) proteins. Unpaired proteins are known to directly decrease stem cell number within the niches, and, overexpression of upd within niche cells restored GSCs in older males also . Stem cells in the midgut of Drosophila are also very promising. Reduced Notch signaling was found to increase the number of midgut progenitor cells. On the other hand, activation of the Notch pathway decreased proliferation of these cells. Further research in this area should lead to the discovery of additional factors that regulate stem and progenitor cells in Drosophila. PMID:26180635

  20. Drosophila's contribution to stem cell research.

    PubMed

    Singh, Gyanesh

    2015-01-01

    The discovery of Drosophila stem cells with striking similarities to mammalian stem cells has brought new hope for stem cell research. Recent developments in Drosophila stem cell research is bringing wider opportunities for contemporary stem cell biologists. In this regard, Drosophila germ cells are becoming a popular model of stem cell research. In several cases, genes that controlled Drosophila stem cells were later discovered to have functional homologs in mammalian stem cells. Like mammals, Drosophila germline stem cells (GSCs) are controlled by both intrinsic as well as external signals. Inside the Drosophila testes, germline and somatic stem cells form a cluster of cells (the hub). Hub cells depend on JAK-STAT signaling, and, in absence of this signal, they do not self-renew. In Drosophila, significant changes occur within the stem cell niche that contributes to a decline in stem cell number over time. In case of aging Drosophila, somatic niche cells show reduced DE-cadherin and unpaired (Upd) proteins. Unpaired proteins are known to directly decrease stem cell number within the niches, and, overexpression of upd within niche cells restored GSCs in older males also . Stem cells in the midgut of Drosophila are also very promising. Reduced Notch signaling was found to increase the number of midgut progenitor cells. On the other hand, activation of the Notch pathway decreased proliferation of these cells. Further research in this area should lead to the discovery of additional factors that regulate stem and progenitor cells in Drosophila. PMID:26180635

  1. Harnessing endogenous stem/progenitor cells for tendon regeneration

    PubMed Central

    Lee, Chang H.; Lee, Francis Y.; Tarafder, Solaiman; Kao, Kristy; Jun, Yena; Yang, Guodong; Mao, Jeremy J.

    2015-01-01

    Current stem cell–based strategies for tissue regeneration involve ex vivo manipulation of these cells to confer features of the desired progenitor population. Recently, the concept that endogenous stem/progenitor cells could be used for regenerating tissues has emerged as a promising approach that potentially overcomes the obstacles related to cell transplantation. Here we applied this strategy for the regeneration of injured tendons in a rat model. First, we identified a rare fraction of tendon cells that was positive for the known tendon stem cell marker CD146 and exhibited clonogenic capacity, as well as multilineage differentiation ability. These tendon-resident CD146+ stem/progenitor cells were selectively enriched by connective tissue growth factor delivery (CTGF delivery) in the early phase of tendon healing, followed by tenogenic differentiation in the later phase. The time-controlled proliferation and differentiation of CD146+ stem/progenitor cells by CTGF delivery successfully led to tendon regeneration with densely aligned collagen fibers, normal level of cellularity, and functional restoration. Using siRNA knockdown to evaluate factors involved in tendon generation, we demonstrated that the FAK/ERK1/2 signaling pathway regulates CTGF-induced proliferation and differentiation of CD146+ stem/progenitor cells. Together, our findings support the use of endogenous stem/progenitor cells as a strategy for tendon regeneration without cell transplantation and suggest this approach warrants exploration in other tissues. PMID:26053662

  2. Stem Cell Applications in Tendon Disorders: A Clinical Perspective

    PubMed Central

    Young, Mark

    2012-01-01

    Tendon injuries are a common cause of morbidity and a significant health burden on society. Tendons are structural tissues connecting muscle to bone and are prone to tearing and tendinopathy, an overuse or degenerative condition that is characterized by failed healing and cellular depletion. Current treatments, for tendon tear are conservative, surgical repair or surgical scaffold reconstruction. Tendinopathy is treated by exercises, injection therapies, shock wave treatments or surgical tendon debridement. However, tendons usually heal with fibrosis and scar tissue, which has suboptimal tensile strength and is prone to reinjury, resulting in lifestyle changes with activity restriction. Preclinical studies show that cell therapies have the potential to regenerate rather than repair tendon tissue, a process termed tenogenesis. A number of different cell lines, with varying degrees of differentiation, have being evaluated including stem cells, tendon derived cells and dermal fibroblasts. Even though cellular therapies offer some potential in treating tendon disorders, there have been few published clinical trials to determine the ideal cell source, the number of cells to administer, or the optimal bioscaffold for clinical use. PMID:22448174

  3. The promoting effect of pentadecapeptide BPC 157 on tendon healing involves tendon outgrowth, cell survival, and cell migration.

    PubMed

    Chang, Chung-Hsun; Tsai, Wen-Chung; Lin, Miao-Sui; Hsu, Ya-Hui; Pang, Jong-Hwei Su

    2011-03-01

    Pentadecapeptide BPC 157, composed of 15 amino acids, is a partial sequence of body protection compound (BPC) that is discovered in and isolated from human gastric juice. Experimentally it has been demonstrated to accelerate the healing of many different wounds, including transected rat Achilles tendon. This study was designed to investigate the potential mechanism of BPC 157 to enhance healing of injured tendon. The outgrowth of tendon fibroblasts from tendon explants cultured with or without BPC 157 was examined. Results showed that BPC 157 significantly accelerated the outgrowth of tendon explants. Cell proliferation of cultured tendon fibroblasts derived from rat Achilles tendon was not directly affected by BPC 157 as evaluated by MTT assay. However, the survival of BPC 157-treated cells was significantly increased under the H(2)O(2) stress. BPC 157 markedly increased the in vitro migration of tendon fibroblasts in a dose-dependent manner as revealed by transwell filter migration assay. BPC 157 also dose dependently accelerated the spreading of tendon fibroblasts on culture dishes. The F-actin formation as detected by FITC-phalloidin staining was induced in BPC 157-treated fibroblasts. The protein expression and activation of FAK and paxillin were determined by Western blot analysis, and the phosphorylation levels of both FAK and paxillin were dose dependently increased by BPC 157 while the total amounts of protein was unaltered. In conclusion, BPC 157 promotes the ex vivo outgrowth of tendon fibroblasts from tendon explants, cell survival under stress, and the in vitro migration of tendon fibroblasts, which is likely mediated by the activation of the FAK-paxillin pathway. PMID:21030672

  4. Tendon progenitor cells in injured tendons have strong chondrogenic potential: the CD105-negative subpopulation induces chondrogenic degeneration.

    PubMed

    Asai, Shuji; Otsuru, Satoru; Candela, Maria Elena; Cantley, Leslie; Uchibe, Kenta; Hofmann, Ted J; Zhang, Kairui; Wapner, Keith L; Soslowsky, Louis J; Horwitz, Edwin M; Enomoto-Iwamoto, Motomi

    2014-12-01

    To study the cellular mechanism of the tendon repair process, we used a mouse Achilles tendon injury model to focus on the cells recruited to the injured site. The cells isolated from injured tendon 1 week after the surgery and uninjured tendons contained the connective tissue progenitor populations as determined by colony-forming capacity, cell surface markers, and multipotency. When the injured tendon-derived progenitor cells (inTPCs) were transplanted into injured Achilles tendons, they were not only integrated in the regenerating area expressing tenogenic phenotype but also trans-differentiated into chondrogenic cells in the degenerative lesion that underwent ectopic endochondral ossification. Surprisingly, the micromass culture of the inTPCs rapidly underwent chondrogenic differentiation even in the absence of exogenous bone morphogenetic proteins or TGFβs. The cells isolated from human ruptured tendon tissues also showed connective tissue progenitor properties and exhibited stronger chondrogenic ability than bone marrow stromal cells. The mouse inTPCs contained two subpopulations one positive and one negative for CD105, a coreceptor of the TGFβ superfamily. The CD105-negative cells showed superior chondrogenic potential in vitro and induced larger chondroid degenerative lesions in mice as compared to the CD105-positive cells. These findings indicate that tendon progenitor cells are recruited to the injured site of tendons and have a strong chondrogenic potential and that the CD105-negative population of these cells would be the cause for chondroid degeneration in injured tendons. The newly identified cells recruited to the injured tendon may provide novel targets to develop therapeutic strategies to facilitate tendon repair.

  5. Chondrogenic differentiation and lubricin expression of caprine infraspinatus tendon cells.

    PubMed

    Funakoshi, Tadanao; Spector, Myron

    2010-06-01

    Reparative strategies for the treatment of injuries to tendons, including those of the rotator cuff of the shoulder, need to address the formation of the cartilage which serves as the attachment apparatus to bone and which forms at regions undergoing compressive loading. Moreover, recent work indicates that cells employed for rotator cuff repair may need to synthesize a lubricating glycoprotein, lubricin, which has recently been found to play a role in tendon tribology. The objective of the present study was to investigate the chondrogenic differentiation and lubricin expression of caprine infraspinatus tendon cells in monolayer and three-dimensional culture, and to compare the behavior with bone marrow-derived mesenchymal stem cells (MSCs). The results demonstrated that while tendon cells in various media, including chondrogenic medium, expressed lubricin, virtually none of the MSCs synthesized this important lubricating molecule. Also of interest was that the cartilage formation capacity of the tendon cells grown in pellet culture in chondrogenic medium was comparable with MSCs. These data inform the use of tendon cells for rotator cuff repair, including for fibrocartilaginous zones.

  6. An ink surgical marker pen is damaging to tendon cells

    PubMed Central

    Franklin, S. L.; Jayadev, C.; Poulsen, R.; Hulley, P.; Price, A.

    2012-01-01

    Objectives Surgical marking during tendon surgery is often used for technical and teaching purposes. This study investigates the effect of a gentian violet ink marker pen, a common surgical marker, on the viability of the tissue and cells of tendon. Methods In vitro cell and tissue methods were used to test the viability of human hamstring explants and the migrating tenocytes in the presence of the gentian violet ink. Results The outcome of this study was that a constituent of the surgical marker pen causes cell and tissue death in culture, implying the same would occur in vivo. Conclusions This is a cause for concern when marking tendon during surgical procedures, as it may compromise healing and repair and potentially contribute to a poor outcome. The authors suggest that an alternative surgical marking procedure should be found, or that all marker pens should undergo testing on human tendon tissue in vitro prior to use. PMID:23610669

  7. The long head of the biceps tendon is a suitable cell source for tendon tissue regeneration

    PubMed Central

    Pietschmann, Matthias F.; Gülecyüz, Mehmet F.; Ficklscherer, Andreas; Jansson, Volkmar; Müller, Peter E.

    2014-01-01

    Introduction Tendon tissue engineering (TTE) tries to produce tendinous tissue of high quality to replace dysfunctional tissue. One possible application of TTE might be the replacement of ruptured tissue of the rotator cuff. Autologous tenocytes seem to be most suitable as no differentiation in vitro is necessary. Today it is still uncertain if there is a difference between tendon-derived cells (TDC) of different native tissues. Moreover, the search for suitable scaffolds is another important issue in TTE. Material and methods This study compared TDC of the long head of the biceps tendon (LHB), the anterior cruciate ligament (ACL) and the tendon of the musculus semitendinosus (TMS). The TDC were isolated using the cell migration method. Cell morphology was assessed using light microscopy and gene expression was performed using polymerase chain reaction (PCR). Afterwards, cell seeding efficiency and proliferation were tested on a collagen I scaffold using the WST-1 assay. Results were confirmed using H + E staining. Results The TDC of the LHB showed higher expression levels of collagen type I and decorin (p < 0.01) compared to TDC of other origin. Results showed efficient cell seeding and proliferation within the scaffold. Proliferation within the scaffold was not as high as when cells were cultivated without a scaffold. Conclusions The TDC of the LHB seems to be the most suitable cell source. Further research is necessary to find out if the results can be transferred to an in vivo model. The new collagen I scaffold seems to offer an opportunity to combine good biocompatibility and mechanical strength. PMID:25097592

  8. The effect of enrofloxacin on cell proliferation and proteoglycans in horse tendon cells.

    PubMed

    Yoon, J H; Brooks, R L; Khan, A; Pan, H; Bryan, J; Zhang, J; Budsberg, S C; Mueller, P O E; Halper, J

    2004-02-01

    Fluoroquinolone antibiotics have been used widely in humans and domestic animals, including horses, because of their broad-spectrum bactericidal activity, and relative safety. The use of fluoroquinolones, however, is not without risk. Tendonitis and spontaneous tendon rupture have been reported in people during or following therapy with fluoroquinolones. We have studied the effects of enrofloxacin, a fluoroquinolone antibiotic used commonly in domestic animals, on tendon cell cultures established from equine superficial digital flexor tendons. Effects on cell proliferation and morphology were studied using cell counting and scanning electron microscopy. Monosaccharide content and composition was determined by gas chromatography-mass spectrometry analysis. Western and Northern blot analyses were utilized to evaluate the synthesis and expression of two proteoglycans, biglycan and decorin. Our data demonstrate that enrofloxacin inhibits cell proliferation, induces morphological changes, decreases total monosacharide content and alters small proteoglycan synthesis at the glycosylation level in equine tendon cell cultures. These effects are more pronounced in juvenile tendon cells than in adult equine tendon cells. We hypothesize that morphological changes and inhibition of cell proliferation are a result of impaired production of biglycan and decorin, proteoglycans involved in fibrillogenesis of collagen, the most important structural component of the tendon of enrofloxacin-treated tendon cells. Our findings suggest that fluoroquinolones should be used with caution in horses, especially in foals.

  9. Human iPSC-Derived Neural Crest Stem Cells Promote Tendon Repair in a Rat Patellar Tendon Window Defect Model

    PubMed Central

    Xu, Wei; Wang, Yequan; Liu, Erfu; Sun, Yanjun; Luo, Ziwei; Xu, Zhiling; Liu, Wanqian; Zhong, Li; Lv, Yonggang; Wang, Aijun; Tang, Zhenyu; Li, Song

    2013-01-01

    Induced pluripotent stem cells (iPSCs) hold great potential for cell therapy and tissue engineering. Neural crest stem cells (NCSCs) are multipotent that are capable of differentiating into mesenchymal lineages. In this study, we investigated whether iPSC-derived NCSCs (iPSC-NCSCs) have potential for tendon repair. Human iPSC-NCSCs were suspended in fibrin gel and transplanted into a rat patellar tendon window defect. At 4 weeks post-transplantation, macroscopical observation showed that the repair of iPSC-NCSC-treated tendons was superior to that of non-iPSC-NCSC-treated tendons. Histological and mechanical examinations revealed that iPSC-NCSCs treatment significantly enhanced tendon healing as indicated by the improvement in matrix synthesis and mechanical properties. Furthermore, transplanted iPSC-NCSCs produced fetal tendon-related matrix proteins, stem cell recruitment factors, and tenogenic differentiation factors, and accelerated the host endogenous repair process. This study demonstrates a potential strategy of employing iPSC-derived NCSCs for tendon tissue engineering. PMID:23815150

  10. Effects of celecoxib on proliferation and tenocytic differentiation of tendon-derived stem cells

    SciTech Connect

    Zhang, Kairui; Zhang, Sheng; Li, Qianqian; Yang, Jun; Dong, Weiqiang; Wang, Shengnan; Cheng, Yirong; Al-Qwbani, Mohammed; Wang, Qiang; Yu, Bin

    2014-07-18

    Highlights: • Celecoxib has no effects on TDSCs cell proliferation in various concentrations. • Celecoxib reduced mRNAs levels of tendon associated transcription factor. • Celecoxib reduced mRNAs levels of main tendon associated collagen. • Celecoxib reduced mRNAs levels of tendon associated molecules. - Abstract: NSAIDs are often ingested to reduce the pain and improve regeneration of tendon after tendon injury. Although the effects of NSAIDs in tendon healing have been reported, the data and conclusions are not consistent. Recently, tendon-derived stem cells (TDSCs) have been isolated from tendon tissues and has been suggested involved in tendon repair. Our study aims to determine the effects of COX-2 inhibitor (celecoxib) on the proliferation and tenocytic differentiation of TDSCs. TDSCs were isolated from mice Achilles tendon and exposed to celecoxib. Cell proliferation rate was investigated at various concentrations (0.1, 1, 10 and 100 μg/ml) of celecoxib by using hemocytometer. The mRNA expression of tendon associated transcription factors, tendon associated collagens and tendon associated molecules were determined by reverse transcription-polymerase chain reaction. The protein expression of Collagen I, Collagen III, Scleraxis and Tenomodulin were determined by Western blotting. The results showed that celecoxib has no effects on TDSCs cell proliferation in various concentrations (p > 0.05). The levels of most tendon associated transcription factors, tendon associated collagens and tendon associated molecules genes expression were significantly decreased in celecoxib (10 μg/ml) treated group (p < 0.05). Collagen I, Collagen III, Scleraxis and Tenomodulin protein expression were also significantly decreased in celecoxib (10 μg/ml) treated group (p < 0.05). In conclusion, celecoxib inhibits tenocytic differentiation of tendon-derived stem cells but has no effects on cell proliferation.

  11. Tendon cell outgrowth rates and morphology associated with kevlar-49.

    PubMed

    Zimmerman, M; Gordon, K E

    1988-12-01

    A rat tendon cell model was used to evaluate the in vitro biocompatibility of kevlar-49. The cell response to kevlar was compared to carbon AS-4 and nylon sutures. Three trials were run and cell growth rates were statistically similar for all the materials tested. A separate experiment was conducted in which the same fiber materials were placed in the same Petri dish. Again, the rates were similar for each material. Finally, the cells were observed with a scanning electron microscope, and the three classic cell morphologies associated with this tendon cell model were observed. Also, cellular attachment to the fiber and cellular encapsulation of the fiber were identical for the three materials tested. Kevlar-49 proved to be comparable to carbon AS4 and nylon sutures in terms of cellular response and cell outgrowth rates.

  12. Live cell imaging in Drosophila melanogaster.

    PubMed

    Parton, Richard M; Vallés, Ana Maria; Dobbie, Ian M; Davis, Ilan

    2010-04-01

    Although many of the techniques of live cell imaging in Drosophila melanogaster are also used by the greater community of cell biologists working on other model systems, studying living fly tissues presents unique difficulties with regard to keeping the cells alive, introducing fluorescent probes, and imaging through thick, hazy cytoplasm. This article outlines the major tissue types amenable to study by time-lapse cinematography and different methods for keeping the cells alive. It describes various imaging and associated techniques best suited to following changes in the distribution of fluorescently labeled molecules in real time in these tissues. Imaging, in general, is a rapidly developing discipline, and recent advances in imaging technology are able to greatly extend what can be achieved with live cell imaging of Drosophila tissues. As far as possible, this article includes the latest technical developments and discusses likely future developments in imaging methods that could have an impact on research using Drosophila. PMID:20360379

  13. Effects of lubricant and autologous bone marrow stromal cell augmentation on immobilized flexor tendon repairs.

    PubMed

    Zhao, Chunfeng; Ozasa, Yasuhiro; Shimura, Haruhiko; Reisdorf, Ramona L; Thoreson, Andrew R; Jay, Gregory; Moran, Steven L; An, Kai-Nan; Amadio, Peter C

    2016-01-01

    The purpose of the study was to test a novel treatment that carbodiimide-derivatized-hyaluronic acid-lubricin (cd-HA-lubricin) combined cell-based therapy in an immobilized flexor tendon repair in a canine model. Seventy-eight flexor tendons from 39 dogs were transected. One tendon was treated with cd-HA-lubricin plus an interpositional graft of 8 × 10(5) BMSCs and GDF-5. The other tendon was repaired without treatment. After 21 day of immobilization, 19 dogs were sacrificed; the remaining 20 dogs underwent a 21-day rehabilitation protocol before euthanasia. The work of flexion, tendon gliding resistance, and adhesion score in treated tendons were significantly less than the untreated tendons (p < 0.05). The failure strength of the untreated tendons was higher than the treated tendons at 21 and 42 days (p < 0.05). However, there is no significant difference in stiffness between two groups at day 42. Histologic analysis of treated tendons showed a smooth surface and viable transplanted cells 42 days after the repair, whereas untreated tendons showed severe adhesion formation around the repair site. The combination of lubricant and cell treatment resulted in significantly improved digit function, reduced adhesion formation. This novel treatment can address the unmet needs of patients who are unable to commence an early mobilization protocol after flexor tendon repair. PMID:26177854

  14. Therapeutic Roles of Tendon Stem/Progenitor Cells in Tendinopathy

    PubMed Central

    Zhang, Xin; Lin, Yu-cheng; Rui, Yun-feng; Xu, Hong-liang; Chen, Hui; Wang, Chen; Teng, Gao-jun

    2016-01-01

    Tendinopathy is a tendon disorder characterized by activity-related pain, local edema, focal tenderness to palpation, and decreased strength in the affected area. Tendinopathy is prevalent in both athletes and the general population, highlighting the need to elucidate the pathogenesis of this disorder. Current treatments of tendinopathy are both conservative and symptomatic. The discovery of tendon stem/progenitor cells (TSPCs) and erroneous differentiation of TSPCs have provided new insights into the pathogenesis of tendinopathy. In this review, we firstly present the histopathological characteristics of tendinopathy and explore the cellular and molecular cues in the pathogenesis of tendinopathy. Current evidence of the depletion of the stem cell pool and altered TSPCs fate in the pathogenesis of tendinopathy has been presented. The potential regulatory factors for either tenogenic or nontenogenic differentiation of TSPCs are also summarized. The regulation of endogenous TSPCs or supplementation with exogenous TSPCs as therapeutic targets for the treatment of tendinopathy is proposed. Therefore, inhibiting the erroneous differentiation of TSPCs and regulating the differentiation of TSPCs into tendon cells might be important areas of future research and could provide new clinical treatments for tendinopathy. The current evidence suggests that TSPCs are promising therapeutic targets for the management of tendinopathy. PMID:27195010

  15. Progress Towards Drosophila Epithelial Cell Culture

    PubMed Central

    Simcox, Amanda

    2015-01-01

    Drosophila epithelial research is at the forefront of the field; however, there are no well-characterized epithelial cell lines that could provide a complementary in vitro model for studies conducted in vivo. Here, a protocol is described that produces epithelial cell lines. The method uses genetic manipulation of oncogenes or tumor suppressors to induce embryonic primary culture cells to rapidly progress to permanent cell lines. It is, however, a general method and the type of cells that comprise a given line is not controlled experimentally. Indeed, only a small fraction of the lines produced are epithelial in character. For this reason, additional work needs to be done to develop a more robust epithelial cell-specific protocol. It is expected that Drosophila epithelial cell lines will have great utility for in vitro analysis of epithelial biology, particularly high-throughput analyses such as RNAi screens. PMID:23097097

  16. Multiple Giant Cell Tumours of Tendon Sheath: A Rare Occurrence

    PubMed Central

    Pathade, Smita Charandas; Kurpad, Ramkumar; Tauheed, Mohammed

    2014-01-01

    Giant Cell Tumours Of Tendon Sheath (GCTTS) are the second most frequent soft tissue tumours affecting the hand with an overall incidence of 1 in 50,000 individuals. These tumours are usually localized and solitary, with multiple GCTTS occurring rarely. Multi-centric origin is considered unusual and very few cases of multiple GCTTS have been reported till date. Here, we report a rare case of a 26-year-old female who presented with multiple painless swellings on palmar aspect of little finger of right hand since six months. Clinical diagnosis of Dupuytren’s contracture was given. Intraoperative examination revealed multiple separate nodules, firmly attached to the flexor tendon synovial sheath. Histopathology showed features of GCTTS. PMID:24596760

  17. Multiple giant cell tumours of tendon sheath: a rare occurrence.

    PubMed

    Pathade, Smita Charandas; Kurpad, Ramkumar; Tauheed, Mohammed

    2014-01-01

    Giant Cell Tumours Of Tendon Sheath (GCTTS) are the second most frequent soft tissue tumours affecting the hand with an overall incidence of 1 in 50,000 individuals. These tumours are usually localized and solitary, with multiple GCTTS occurring rarely. Multi-centric origin is considered unusual and very few cases of multiple GCTTS have been reported till date. Here, we report a rare case of a 26-year-old female who presented with multiple painless swellings on palmar aspect of little finger of right hand since six months. Clinical diagnosis of Dupuytren's contracture was given. Intraoperative examination revealed multiple separate nodules, firmly attached to the flexor tendon synovial sheath. Histopathology showed features of GCTTS.

  18. Giant Cell Tumor of Tendon Sheath in the Knee

    PubMed Central

    Ghnaimat, Malek; Alodat, Mohannad; Aljazazi, Mohammad; Al-Zaben, Raad; Alshwabkah, Jamal

    2016-01-01

    The giant cell tumor of the tendon sheath (GCTTS) is a benign lesion which arises from the synovium of a joint, bursa or tendon sheath, with 85% of the tumors occurring in the fingers and 12% of the tumors located in large joints such as the knee and ankle. The GCTTS is usually monoarticular, slowly proliferative and rarely locally aggressive. This paper reports three cases of this rare lesion in the knee. Patients presented with painful swelling in the anterior knee, MRI showed localized soft tissue masses which were able to be excised. A follow up of the cases showed no recurrences. This case report emphasizes the importance of considering GCTTS in the differential diagnosis of soft tissue swelling and pain in large joints.

  19. Lovastatin-Mediated Changes in Human Tendon Cells.

    PubMed

    Kuzma-Kuzniarska, Maria; Cornell, Hannah R; Moneke, Michael C; Carr, Andrew J; Hulley, Philippa A

    2015-10-01

    Statins are among the most widely prescribed drugs worldwide. Numerous studies have shown their beneficial effects in prevention of cardiovascular disease through cholesterol-lowering and anti-atherosclerotic properties. Although some statin patients may experience muscle-related symptoms, severe side effects of statin therapy are rare, primarily due to extensive first-pass metabolism in the liver. Skeletal muscles appear to be the main site of side effects; however, recently some statin-related adverse effects have been described in tendon. The mechanism behind these side effects remains unknown. This is the first study that explores tendon-specific effects of statins in human primary tenocytes. The cells were cultured with different concentrations of lovastatin for up to 1 week. No changes in cell viability or morphology were observed in tenocytes incubated with therapeutic doses. Short-term exposure to lovastatin concentrations outside the therapeutic range had no effect on tenocyte viability; however, cell migration was reduced. Simvastatin and atorvastatin, two other drug family members, also reduced the migratory properties of the cells. Prolonged exposure to high concentrations of lovastatin induced changes in cytoskeleton leading to cell rounding and decreased levels of mRNA for matrix proteins, but increased BMP-2 expression. Gap junctional communication was impaired but due to cell shape change and separation rather than direct gap junction inhibition. These effects were accompanied by inhibition of prenylation of Rap1a small GTPase. Collectively, we showed that statins in a dose-dependent manner decrease migration of human tendon cells, alter their expression profile and impair the functional network, but do not inhibit gap junction function.

  20. Giant cell tumour of tendon sheath with simultaneous two tendon involvement of the foot treated with excision of the tumour and reconstruction of the flexor retinaculum using tibialis posterior tendon in a paediatric patient: A rare case report.

    PubMed

    Tiwari, Vivek; Ansari, Tahir; Mittal, Samarth; Sharma, Pankaj; Nalwa, Aasma

    2015-12-01

    Giant cell tumour of tendon sheath is a benign soft tissue tumour arising from the tendon sheath. The involvement of foot and ankle by such tumours is relatively rare. Children are not commonly afflicted by this condition. All such tumours are reported to arise either from a single tendon sheath or one joint. We report a case of giant cell tumour of tendon sheath in a 12-year-old child, arising simultaneously from the tendon sheaths of tibialis posterior and flexor digitorum longus tendons, as well as extending into the ankle joint. It was treated by complete excision of the mass along with the tendon sheaths with reconstruction of the flexor retinaculum. The location of the tumour, age of the patient, diffuse nature of the tumour and novel technique of reconstruction of the flexor retinaculum make this case extremely rare and the first to be reported in literature.

  1. Giant cell tumour of tendon sheath with simultaneous two tendon involvement of the foot treated with excision of the tumour and reconstruction of the flexor retinaculum using tibialis posterior tendon in a paediatric patient: A rare case report.

    PubMed

    Tiwari, Vivek; Ansari, Tahir; Mittal, Samarth; Sharma, Pankaj; Nalwa, Aasma

    2015-12-01

    Giant cell tumour of tendon sheath is a benign soft tissue tumour arising from the tendon sheath. The involvement of foot and ankle by such tumours is relatively rare. Children are not commonly afflicted by this condition. All such tumours are reported to arise either from a single tendon sheath or one joint. We report a case of giant cell tumour of tendon sheath in a 12-year-old child, arising simultaneously from the tendon sheaths of tibialis posterior and flexor digitorum longus tendons, as well as extending into the ankle joint. It was treated by complete excision of the mass along with the tendon sheaths with reconstruction of the flexor retinaculum. The location of the tumour, age of the patient, diffuse nature of the tumour and novel technique of reconstruction of the flexor retinaculum make this case extremely rare and the first to be reported in literature. PMID:26564735

  2. Tendon Reattachment to Bone in an Ovine Tendon Defect Model of Retraction Using Allogenic and Xenogenic Demineralised Bone Matrix Incorporated with Mesenchymal Stem Cells

    PubMed Central

    2016-01-01

    Background Tendon-bone healing following rotator cuff repairs is mainly impaired by poor tissue quality. Demineralised bone matrix promotes healing of the tendon-bone interface but its role in the treatment of tendon tears with retraction has not been investigated. We hypothesized that cortical demineralised bone matrix used with minimally manipulated mesenchymal stem cells will result in improved function and restoration of the tendon-bone interface with no difference between xenogenic and allogenic scaffolds. Materials and Methods In an ovine model, the patellar tendon was detached from the tibial tuberosity and a complete distal tendon transverse defect measuring 1 cm was created. Suture anchors were used to reattach the tendon and xenogenic demineralised bone matrix + minimally manipulated mesenchymal stem cells (n = 5), or allogenic demineralised bone matrix + minimally manipulated mesenchymal stem cells (n = 5) were used to bridge the defect. Graft incorporation into the tendon and its effect on regeneration of the enthesis was assessed using histomorphometry. Force plate analysis was used to assess functional recovery. Results Compared to the xenograft, the allograft was associated with significantly higher functional weight bearing at 6 (P = 0.047), 9 (P = 0.028), and 12 weeks (P = 0.009). In the allogenic group this was accompanied by greater remodeling of the demineralised bone matrix into tendon-like tissue in the region of the defect (p = 0.015), and a more direct type of enthesis characterized by significantly more fibrocartilage (p = 0.039). No failures of tendon-bone healing were noted in either group. Conclusion Demineralised bone matrix used with minimally manipulated mesenchymal stem cells promotes healing of the tendon-bone interface in an ovine model of acute tendon retraction, with superior mechanical and histological results associated with use of an allograft. PMID:27606597

  3. Stem cell technology for tendon regeneration: current status, challenges, and future research directions

    PubMed Central

    Lui, Pauline Po Yee

    2015-01-01

    Tendon injuries are a common cause of physical disability. They present a clinical challenge to orthopedic surgeons because injured tendons respond poorly to current treatments without tissue regeneration and the time required for rehabilitation is long. New treatment options are required. Stem cell-based therapies offer great potential to promote tendon regeneration due to their high proliferative, synthetic, and immunomodulatory activities as well as their potential to differentiate to the target cell types and undergo genetic modification. In this review, I first recapped the challenges of tendon repair by reviewing the anatomy of tendon. Next, I discussed the advantages and limitations of using different types of stem cells compared to terminally differentiated cells for tendon tissue engineering. The safety and efficacy of application of stem cells and their modified counterparts for tendon tissue engineering were then summarized after a systematic literature search in PubMed. The challenges and future research directions to enhance, optimize, and standardize stem cell-based therapies for augmenting tendon repair were then discussed. PMID:26715856

  4. Drosophila neural stem cells in brain development and tumor formation.

    PubMed

    Jiang, Yanrui; Reichert, Heinrich

    2014-01-01

    Neuroblasts, the neural stem cells in Drosophila, generate the complex neural structure of the central nervous system. Significant progress has been made in understanding the mechanisms regulating the self-renewal, proliferation, and differentiation in Drosophila neuroblast lineages. Deregulation of these mechanisms can lead to severe developmental defects and the formation of malignant brain tumors. Here, the authors review the molecular genetics of Drosophila neuroblasts and discuss some recent advances in stem cell and cancer biology using this model system.

  5. Injury induces a change in the functional characteristics of cells recovered from equine tendon.

    PubMed

    Kihara, Rina; Kasashima, Yoshinori; Arai, Katsuhiko; Miyamoto, Yasunori

    2011-01-01

    Injury initiates a repair process characterized by influx of fibroblasts and the rapid formation of fibrous scar tissue and subsequent tissue contraction. The response to injury and behavior of the different tendon fibroblast populations, however, has been poorly characterized. We hypothesized that the fibroblasts recovered from tendon with acute injury would exhibit different cell properties relating to adhesion, migration and tensegrity. To test this hypothesis we evaluated the ability of fibroblasts recovered from normal and injured equine superficial digital flexor tendons (SDFTs). The injured tendon-derived cells showed greater contraction of the collagen gel but poorer adhesion to pepsin-digested collagen, and migration over extracellular matrix proteins compared to normal SDFT-derived fibroblasts. Thus, the cells present within the tendon after injury display different behavior related to wound healing. PMID:24833988

  6. The effects of scaffold architecture and fibrin gel addition on tendon cell phenotype.

    PubMed

    Pawelec, K M; Wardale, R J; Best, S M; Cameron, R E

    2015-01-01

    Development of tissue engineering scaffolds relies on careful selection of pore architecture and chemistry of the cellular environment. Repair of skeletal soft tissue, such as tendon, is particularly challenging, since these tissues have a relatively poor healing response. When removed from their native environment, tendon cells (tenocytes) lose their characteristic morphology and the expression of phenotypic markers. To stimulate tendon cells to recreate a healthy extracellular matrix, both architectural cues and fibrin gels have been used in the past, however, their relative effects have not been studied systematically. Within this study, a combination of collagen scaffold architecture, axial and isotropic, and fibrin gel addition was assessed, using ovine tendon-derived cells to determine the optimal strategy for controlling the proliferation and protein expression. Scaffold architecture and fibrin gel addition influenced tendon cell behavior independently in vitro. Addition of fibrin gel within a scaffold doubled cell number and increased matrix production for all architectures studied. However, scaffold architecture dictated the type of matrix produced by cells, regardless of fibrin addition. Axial scaffolds, mimicking native tendon, promoted a mature matrix, with increased tenomodulin, a marker for mature tendon cells, and decreased scleraxis, an early transcription factor for connective tissue. This study demonstrated that both architectural cues and fibrin gel addition alter cell behavior and that the combination of these signals could improve clinical performance of current tissue engineering constructs.

  7. Drosophila grim induces apoptosis in mammalian cells.

    PubMed Central

    Clavería, C; Albar, J P; Serrano, A; Buesa, J M; Barbero, J L; Martínez-A, C; Torres, M

    1998-01-01

    Genetic studies have shown that grim is a central genetic switch of programmed cell death in Drosophila; however, homologous genes have not been described in other species, nor has its mechanism of action been defined. We show here that grim expression induces apoptosis in mouse fibroblasts. Cell death induced by grim in mammalian cells involves membrane blebbing, cytoplasmic loss and nuclear DNA fragmentation. Grim-induced apoptosis is blocked by both natural and synthetic caspase inhibitors. We found that grim itself shows caspase-dependent proteolytic processing of its C-terminus in vitro. Grim-induced death is antagonized by bcl-2 in a dose-dependent manner, and neither Fas signalling nor p53 are required for grim pro-apoptotic activity. Grim protein localizes both in the cytosol and in the mitochondria of mouse fibroblasts, the latter location becoming predominant as apoptosis progresses. These results show that Drosophila grim induces death in mammalian cells by specifically acting on mitochondrial apoptotic pathways executed by endogenous caspases. These findings advance our knowledge of the mechanism by which grim induces apoptosis and show the conservation through evolution of this crucial programmed cell death pathway. PMID:9857177

  8. Biology and mechano-response of tendon cells: progress overview and perspectives†

    PubMed Central

    Sun, Hui B.; Schaniel, Christoph; Leong, Daniel J.; Wang, James H-C.

    2015-01-01

    In this review, we summarize the group discussions on Cell Biology & Mechanics from the 2014 ORS/ISMMS New Frontiers in Tendon Research Conference. The major discussion topics included: 1) the biology of tendon stem/progenitor cells (TSPCs) and the potential of stem cell-based tendon therapy using TSPCs and other types of stem cells, namely, embryonic and/or induced pluripotent stem cells (iPSCs), 2) the biological concept and potential impact of cellular senescence on tendon aging, tendon injury repair and the development of degenerative disease, and 3) the effects of tendon cells’ mechano-response on tendon cell fate and metabolism. For each topic, a brief overview is presented which summarizes the major points discussed by the group participants. The focus of the discussions ranged from current research progress, challenges and opportunities, to future directions on these topics. In the preparation of this manuscript, authors consulted relevant references as a part of their efforts to present an accurate view on the topics discussed. PMID:25728946

  9. Assessment of stem cell carriers for tendon tissue engineering in pre-clinical models

    PubMed Central

    2014-01-01

    Tendon injuries are prevalent and problematic, especially among young and otherwise healthy individuals. The inherently slow innate healing process combined with the inevitable scar tissue formation compromise functional recovery, imposing the need for the development of therapeutic strategies. The limited number of low activity/reparative capacity tendon-resident cells has directed substantial research efforts towards the exploration of the therapeutic potential of various stem cells in tendon injuries and pathophysiologies. Severe injuries require the use of a stem cell carrier to enable cell localisation at the defect site. The present study describes advancements that injectable carriers, tissue grafts, anisotropically orientated biomaterials, and cell-sheets have achieved in preclinical models as stem cell carriers for tendon repair. PMID:25157898

  10. Controlled delivery of mesenchymal stem cells and growth factors using a nanofiber scaffold for tendon repair

    PubMed Central

    Manning, CN; Schwartz, AG; Liu, W; Xie, J; Havlioglu, N; Sakiyama-Elbert, SE; Silva, MJ; Xia, Y; Gelberman, RH; Thomopoulos, S

    2013-01-01

    Outcomes after tendon repair are often unsatisfactory, despite improvements in surgical techniques and rehabilitation methods. Recent studies aimed at enhancing repair have targeted the paucicellular nature of tendon for enhancing repair; however, most approaches for delivering growth factors and cells have not been designed for dense connective tissues such as tendon. Therefore, we developed a scaffold capable of delivering growth factors and cells in a surgically manageable form for tendon repair. The growth factor PDGF-BB along with adipose-derived mesenchymal stem cells (ASCs) was incorporated into a heparin/fibrin-based delivery system (HBDS). This hydrogel was then layered with an electrospun nanofiber poly-lactic-co-glycolic acid (PLGA) backbone. The HBDS allowed for the concurrent delivery of PDGF-BB and ASCs in a controlled manner, while the PLGA backbone provided structural integrity for surgical handling and tendon implantation. In vitro studies verified that the cells remained viable, and that sustained growth factor release was achieved. In vivo studies in a large animal tendon model verified that the approach was clinically relevant, and that the cells remained viable in the tendon repair environment. Only a mild immunoresponse was seen at dissection, histologically, and at the mRNA level; fluorescently-labeled ASCs and the scaffold were found at the repair site 9 days postoperatively; and increased total DNA was observed in ASC-treated tendons. The novel layered scaffold has the potential for improving tendon healing due to its ability to deliver both cells and growth factors simultaneously in a surgically convenient manner. PMID:23416576

  11. Restricted differentiation potential of progenitor cell populations obtained from the equine superficial digital flexor tendon (SDFT).

    PubMed

    Williamson, Kate Ann; Lee, Katie Joanna; Humphreys, William James Edward; Comerford, Eithne Josephine Veronica; Clegg, Peter David; Canty-Laird, Elizabeth Gail

    2015-06-01

    The aim of this study was to characterize stem and progenitor cell populations from the equine superficial digital flexor tendon, an energy-storing tendon with similarities to the human Achilles tendon, which is frequently injured. Using published methods for the isolation of tendon-derived stem/progenitor cells by low-density plating we found that isolated cells possessed clonogenicity but were unable to fully differentiate towards mesenchymal lineages using trilineage differentiation assays. In particular, adipogenic differentiation appeared to be restricted, as assessed by Oil Red O staining of stem/progenitor cells cultured in adipogenic medium. We then assessed whether differential adhesion to fibronectin substrates could be used to isolate a population of cells with broader differentiation potential. However we found little difference in the stem and tenogenic gene expression profile of these cells as compared to tenocytes, although the expression of thrombospondin-4 was significantly reduced in hypoxic conditions. Tendon-derived stem/progenitor cells isolated by differential adhesion to fibronectin had a similar differentiation potential to cells isolated by low density plating, and when grown in either normoxic or hypoxic conditions. In summary, we have found a restricted differentiation potential of cells isolated from the equine superficial digital flexor tendon despite evidence for stem/progenitor-like characteristics. PMID:25877997

  12. Nanostructured Tendon-Derived Scaffolds for Enhanced Bone Regeneration by Human Adipose-Derived Stem Cells.

    PubMed

    Ko, Eunkyung; Alberti, Kyle; Lee, Jong Seung; Yang, Kisuk; Jin, Yoonhee; Shin, Jisoo; Yang, Hee Seok; Xu, Qiaobing; Cho, Seung-Woo

    2016-09-01

    Decellularized matrix-based scaffolds can induce enhanced tissue regeneration due to their biochemical, biophysical, and mechanical similarity to native tissues. In this study, we report a nanostructured decellularized tendon scaffold with aligned, nanofibrous structures to enhance osteogenic differentiation and in vivo bone formation of human adipose-derived stem cells (hADSCs). Using a bioskiving method, we prepared decellularized tendon scaffolds from tissue slices of bovine Achilles and neck tendons with or without fixation, and investigated the effects on physical and mechanical properties of decellularized tendon scaffolds, based on the types and concentrations of cross-linking agents. In general, we found that decellularized tendon scaffolds without fixative treatments were more effective in inducing osteogenic differentiation and mineralization of hADSCs in vitro. When non-cross-linked decellularized tendon scaffolds were applied together with hydroxyapatite for hADSC transplantation in critical-sized bone defects, they promoted bone-specific collagen deposition and mineralized bone formation 4 and 8 weeks after hADSC transplantation, compared to conventional collagen type I scaffolds. Interestingly, stacking of decellularized tendon scaffolds cultured with osteogenically committed hADSCs and those containing human cord blood-derived endothelial progenitor cells (hEPCs) induced vascularized bone regeneration in the defects 8 weeks after transplantation. Our study suggests that biomimetic nanostructured scaffolds made of decellularized tissue matrices can serve as functional tissue-engineering scaffolds for enhanced osteogenesis of stem cells.

  13. Nanostructured Tendon-Derived Scaffolds for Enhanced Bone Regeneration by Human Adipose-Derived Stem Cells.

    PubMed

    Ko, Eunkyung; Alberti, Kyle; Lee, Jong Seung; Yang, Kisuk; Jin, Yoonhee; Shin, Jisoo; Yang, Hee Seok; Xu, Qiaobing; Cho, Seung-Woo

    2016-09-01

    Decellularized matrix-based scaffolds can induce enhanced tissue regeneration due to their biochemical, biophysical, and mechanical similarity to native tissues. In this study, we report a nanostructured decellularized tendon scaffold with aligned, nanofibrous structures to enhance osteogenic differentiation and in vivo bone formation of human adipose-derived stem cells (hADSCs). Using a bioskiving method, we prepared decellularized tendon scaffolds from tissue slices of bovine Achilles and neck tendons with or without fixation, and investigated the effects on physical and mechanical properties of decellularized tendon scaffolds, based on the types and concentrations of cross-linking agents. In general, we found that decellularized tendon scaffolds without fixative treatments were more effective in inducing osteogenic differentiation and mineralization of hADSCs in vitro. When non-cross-linked decellularized tendon scaffolds were applied together with hydroxyapatite for hADSC transplantation in critical-sized bone defects, they promoted bone-specific collagen deposition and mineralized bone formation 4 and 8 weeks after hADSC transplantation, compared to conventional collagen type I scaffolds. Interestingly, stacking of decellularized tendon scaffolds cultured with osteogenically committed hADSCs and those containing human cord blood-derived endothelial progenitor cells (hEPCs) induced vascularized bone regeneration in the defects 8 weeks after transplantation. Our study suggests that biomimetic nanostructured scaffolds made of decellularized tissue matrices can serve as functional tissue-engineering scaffolds for enhanced osteogenesis of stem cells. PMID:27502160

  14. Enhancement of Tenogenic Differentiation of Human Adipose Stem Cells by Tendon-Derived Extracellular Matrix

    PubMed Central

    Yang, Guang; Rothrauff, Benjamin B.; Lin, Hang; Gottardi, Riccardo; Alexander, Peter G.; Tuan, Rocky S.

    2014-01-01

    Mesenchymal stem cells (MSCs) have gained increasing research interest for their potential in improving healing and regeneration of injured tendon tissues. Developing functional three-dimensional (3D) scaffolds to promote MSC proliferation and differentiation is a critical requirement in tendon tissue engineering. Tendon extracellular matrix has been shown to maintain the tenogenic potential of tendon stem cells and stimulate tenogenesis of human adipose stem cells (hASCs) in 2D culture. This study aims at characterizing the biological composition of urea-extracted fraction of tendon ECM (tECM) and its tenogenic effect on hASCs cultured in a 3D collagen scaffold under uniaxial tension. The tECM obtained was cell-free and rich in ECM proteins. hASCs seeded in tECM supplemented scaffold exhibited significantly increased proliferation and tenogenic differentiation. The presence of tECM also greatly suppressed the osteogenic differentiation of hASCs triggered by uniaxial tension. In addition, tECM-supplemented constructs displayed enhanced mechanical strength, accompanied by reduced expression and activity of MMPs in the seeded hASCs, indicating a regulatory activity of tECM in cell-mediated scaffold remodeling. These findings support the utility of tECM in creating bio-functional scaffolds for tendon tissue engineering. PMID:24044998

  15. Enhancement of tenogenic differentiation of human adipose stem cells by tendon-derived extracellular matrix.

    PubMed

    Yang, Guang; Rothrauff, Benjamin B; Lin, Hang; Gottardi, Riccardo; Alexander, Peter G; Tuan, Rocky S

    2013-12-01

    Mesenchymal stem cells (MSCs) have gained increasing research interest for their potential in improving healing and regeneration of injured tendon tissues. Developing functional three-dimensional (3D) scaffolds to promote MSC proliferation and differentiation is a critical requirement in tendon tissue engineering. Tendon extracellular matrix has been shown to maintain the tenogenic potential of tendon stem cells and stimulate tenogenesis of human adipose stem cells (hASCs) in 2D culture. This study aims at characterizing the biological composition of urea-extracted fraction of tendon ECM (tECM) and its tenogenic effect on hASCs cultured in a 3D collagen scaffold under uniaxial tension. The tECM obtained was cell-free and rich in ECM proteins. hASCs seeded in tECM-supplemented scaffold exhibited significantly increased proliferation and tenogenic differentiation. The presence of tECM also greatly suppressed the osteogenic differentiation of hASCs triggered by uniaxial tension. In addition, tECM-supplemented constructs displayed enhanced mechanical strength, accompanied by reduced expression and activity of MMPs in the seeded hASCs, indicating a regulatory activity of tECM in cell-mediated scaffold remodeling. These findings support the utility of tECM in creating bio-functional scaffolds for tendon tissue engineering.

  16. IL-1β Irreversibly Inhibits Tenogenic Differentiation and Alters Metabolism In Injured Tendon-Derived Progenitor Cells In Vitro

    PubMed Central

    Zhang, Kairui; Asai, Shuji; Yu, Bin; Enomoto-Iwamoto, Motomi

    2015-01-01

    Tendon injuries are common, and the damaged tendon often turns into scar tissue and never completely regains the original biomechanical properties. Previous studies have reported that the mRNA levels of inflammatory cytokines such as IL-1β are remarkably up-regulated in injured tendons. To examine how IL-1β impacts tendon repair process, we isolated the injured tendon-derived progenitor cells (inTPCs) from mouse injured Achilles tendons and studied the effects of IL-1β on the inTPCs in vitro. IL-1β treatment strongly reduced expression of tendon cell markers such as scleraxis and tenomodulin, and also down-regulated gene expression of collagen 1, collagen 3, biglycan and fibromodulin in inTPCs. Interestingly, IL-1β stimulated lactate production with increases in hexokinase II and lactate dehydrogenase expression and a decrease in pyruvate dehydrogenase. Inhibition of lactate production restored IL-1β-induced down-regulation of collagen1 and scleraxis expression. Furthermore, IL-1β significantly inhibited adipogenic, chondrogenic and osteogenic differentiation of inTPCs. Interestingly, inhibition of tenogenic and adipogenic differentiation was not recovered after removal of IL-1β while chondrogenic and osteogenic differentiation abilities were not affected. These findings indicate that IL-1β strongly and irreversibly impairs tenogenic potential and alters glucose metabolism in tendon progenitors appearing in injured tendons. Inhibition of IL-1β may be beneficial for maintaining function of tendon progenitor cells during the tendon repair process. PMID:26051275

  17. A review on the use of cell therapy in the treatment of tendon disease and injuries

    PubMed Central

    Sawadkar, Prasad; Mudera, Vivek

    2014-01-01

    Tendon disease and injuries carry significant morbidity worldwide in both athletic and non-athletic populations. It is estimated that tendon injuries account for 30%−50% of all musculoskeletal injuries globally. Current treatments have been inadequate in providing an accelerated process of repair resulting in high relapse rates. Modern concepts in tissue engineering and regenerative medicine have led to increasing interest in the application of cell therapy for the treatment of tendon disease. This review will explore the use of cell therapy, by bringing together up-to-date evidence from in vivo human and animal studies, and discuss the issues surrounding the safety and efficacy of its use in the treatment of tendon disease. PMID:25383170

  18. Study of Bone Marrow Mesenchymal and Tendon-Derived Stem Cells Transplantation on the Regenerating Effect of Achilles Tendon Ruptures in Rats

    PubMed Central

    Al-ani, Mohanad Kh; Xu, Kang; Sun, Yanjun; Pan, Lianhong; Xu, ZhiLing; Yang, Li

    2015-01-01

    Comparative therapeutic significance of tendon-derived stem cells (TDSCs) and bone marrow mesenchymal stem cells (BMSCs) transplantation to treat ruptured Achilles tendon was studied. Three groups of SD rats comprising 24 rats each, designated as TDSCs and BMSCs, and nontreated were studied for regenerative effects through morpho-histological evaluations and ultimate failure load. For possible mechanism in tendon repair/regeneration through TDSCs and BMSCs, we measured Collagen-I (Col-I), Col-III gene expression level by RT-PCR, and Tenascin-C expression via immunofluorescent assay. TDSCs showed higher agility in tendon healing with better appearance density and well-organized longitudinal fibrous structure, though BMSCs also showed positive effects. Initially the ultimate failure load was considerably higher in TDSCs than other two study groups during the weeks 1 and 2, but at week 4 it attained an average or healthy tendon strength of 30.2 N. Similar higher tendency in Col-I/III gene expression level during weeks 1, 2, and 4 was observed in TDSCs treated group with an upregulation of 1.5-fold and 1.1-fold than the other two study groups. Immunofluorescent assay revealed higher expression of Tenascin-C in TDSCs at week 1, while both TDSCs and BMSCs treated groups showed detectable CM-Dil-labelled cells at week 4. Compared with BMSCs, TDSCs showed higher regenerative potential while treating ruptured Achilles tendons in rats. PMID:26339252

  19. Dynamic loading of electrospun yarns guides mesenchymal stem cells towards a tendon lineage

    PubMed Central

    Bosworth, L.A.; Rathbone, S.R.; Bradley, R.S.; Cartmell, S.H.

    2014-01-01

    Alternative strategies are required when autograft tissue is not sufficient or available to reconstruct damaged tendons. Electrospun fibre yarns could provide such an alternative. This study investigates the seeding of human mesenchymal stem cells (hMSC) on electrospun yarns and their response when subjected to dynamic tensile loading. Cell seeded yarns sustained 3600 cycles per day for 21 days. Loaded yarns demonstrated a thickened cell layer around the scaffold׳s exterior compared to statically cultured yarns, which would suggest an increased rate of cell proliferation and/or matrix deposition, whilst maintaining a predominant uniaxial cell orientation. Tensile properties of cell-seeded yarns increased with time compared to acellular yarns. Loaded scaffolds demonstrated an up-regulation in several key tendon genes, including collagen Type I. This study demonstrates the support of hMSCs on electrospun yarns and their differentiation towards a tendon lineage when mechanically stimulated. PMID:25129861

  20. Release of Tensile Strain on Engineered Human Tendon Tissue Disturbs Cell Adhesions, Changes Matrix Architecture, and Induces an Inflammatory Phenotype

    PubMed Central

    Bayer, Monika L.; Schjerling, Peter; Herchenhan, Andreas; Zeltz, Cedric; Heinemeier, Katja M.; Christensen, Lise; Krogsgaard, Michael; Gullberg, Donald; Kjaer, Michael

    2014-01-01

    Mechanical loading of tendon cells results in an upregulation of mechanotransduction signaling pathways, cell-matrix adhesion and collagen synthesis, but whether unloading removes these responses is unclear. We investigated the response to tension release, with regard to matrix proteins, pro-inflammatory mediators and tendon phenotypic specific molecules, in an in vitro model where tendon-like tissue was engineered from human tendon cells. Tissue sampling was performed 1, 2, 4 and 6 days after surgical de-tensioning of the tendon construct. When tensile stimulus was removed, integrin type collagen receptors showed a contrasting response with a clear drop in integrin subunit α11 mRNA and protein expression, and an increase in α2 integrin mRNA and protein levels. Further, specific markers for tendon cell differentiation declined and normal tendon architecture was disturbed, whereas pro-inflammatory molecules were upregulated. Stimulation with the cytokine TGF-β1 had distinct effects on some tendon-related genes in both tensioned and de-tensioned tissue. These findings indicate an important role of mechanical loading for cellular and matrix responses in tendon, including that loss of tension leads to a decrease in phenotypical markers for tendon, while expression of pro-inflammatory mediators is induced. PMID:24465881

  1. Therapeutic potential of mesenchymal stem cells to treat Achilles tendon injuries.

    PubMed

    Vieira, M H C; Oliveira, R J; Eça, L P M; Pereira, I S O; Hermeto, L C; Matuo, R; Fernandes, W S; Silva, R A; Antoniolli, A C M B

    2014-12-12

    Rupture of the Achilles tendon diminishes quality of life. The gold-standard therapy is a surgical suture, but this presents complications, including wound formation and inflammation. These complications spurred evaluation of the therapeutic potential of mesenchymal stem cells (MSCs) from adipose tissue. New Zealand rabbits were divided into 6 groups (three treatments with two time points each) evaluated at either 14 or 28 days after surgery: cross section of the Achilles tendon (CSAT); CSAT + Suture; and CSAT + MSC. A comparison between all groups at both time points showed a statistically significant increase in capillaries and in the structural organization of collagen in the healed tendon in the CSAT + Suture and CSAT + MSC groups at the 14-day assessment. Comparison between the two time points within the same group showed a statistically significant decrease in the inflammatory process and an increase in the structural organization of collagen in the CSAT and CSAT + MSC groups. A study of the genomic integrity of the cells suggested a linear correlation between an increase of injuries and culture time. Thus, MSC transplantation is a good alternative for treatment of Achilles tendon ruptures because it may be conducted without surgery and tendon suture and, therefore, has no risk of adverse effects resulting from the surgical wound or inflammation caused by nonabsorbable sutures. Furthermore, this alternative treatment exhibits a better capacity for wound healing and maintaining the original tendon architecture, depending on the arrangement of the collagen fibers, and has important therapeutic potential.

  2. Novel fiber-based pure chitosan scaffold for tendon augmentation: biomechanical and cell biological evaluation.

    PubMed

    Nowotny, J; Aibibu, D; Farack, J; Nimtschke, U; Hild, M; Gelinsky, M; Kasten, P; Cherif, Ch

    2016-07-01

    One possibility to improve the mechanical properties after tendon ruptures is augmentation with a scaffold. Based on wet spinning technology, chitosan fibres were processed to a novel pure high-grade multifilament yarn with reproducible quality. The fibres were braided to obtain a 3D tendon scaffold. The CS fibres and scaffolds were evaluated biomechanically and compared to human supraspinatus (SSP) tendons. For the cytobiological characterization, in vitro cell culture experiments with human mesenchymal stem cells (hMSC) were performed. Three types of 3D circular braided scaffolds were fabricated. Significantly, higher ultimate stress values were measured for scaffold with larger filament yarn, compared to scaffold with smaller filament yarn. During cultivation over 28 days, the cells showed in dependence of isolation method and/or donor a doubling or tripling of the cell number or even a six-fold increase on the CS scaffold, which was comparable to the control (polystyrene) or in the case of cells obtained from human biceps tendon even higher proliferation rates. After 14 days, the scaffold surface was covered homogeneously with a cell layer. In summary, the present work demonstrates that braided chitosan scaffolds constitute a straightforward approach for designing tendon analogues, maintaining important flexibility in scaffold design and providing favourable mechanical properties of the resulting construct.

  3. Intestinal stem cells in the adult Drosophila midgut

    SciTech Connect

    Jiang, Huaqi; Edgar, Bruce A.

    2011-11-15

    Drosophila has long been an excellent model organism for studying stem cell biology. Notably, studies of Drosophila's germline stem cells have been instrumental in developing the stem cell niche concept. The recent discovery of somatic stem cells in adult Drosophila, particularly the intestinal stem cells (ISCs) of the midgut, has established Drosophila as an exciting model to study stem cell-mediated adult tissue homeostasis and regeneration. Here, we review the major signaling pathways that regulate the self-renewal, proliferation and differentiation of Drosophila ISCs, discussing how this regulation maintains midgut homeostasis and mediates regeneration of the intestinal epithelium after injury. -- Highlights: Black-Right-Pointing-Pointer The homeostasis and regeneration of adult fly midguts are mediated by ISCs. Black-Right-Pointing-Pointer Damaged enterocytes induce the proliferation of intestinal stem cells (ISC). Black-Right-Pointing-Pointer EGFR and Jak/Stat signalings mediate compensatory ISC proliferation. Black-Right-Pointing-Pointer Notch signaling regulates ISC self-renewal and differentiation.

  4. Restricted differentiation potential of progenitor cell populations obtained from the equine superficial digital flexor tendon (SDFT)

    PubMed Central

    Humphreys, William James Edward; Comerford, Eithne Josephine Veronica; Clegg, Peter David; Canty‐Laird, Elizabeth Gail

    2015-01-01

    ABSTRACT The aim of this study was to characterize stem and progenitor cell populations from the equine superficial digital flexor tendon, an energy‐storing tendon with similarities to the human Achilles tendon, which is frequently injured. Using published methods for the isolation of tendon‐derived stem/progenitor cells by low‐density plating we found that isolated cells possessed clonogenicity but were unable to fully differentiate towards mesenchymal lineages using trilineage differentiation assays. In particular, adipogenic differentiation appeared to be restricted, as assessed by Oil Red O staining of stem/progenitor cells cultured in adipogenic medium. We then assessed whether differential adhesion to fibronectin substrates could be used to isolate a population of cells with broader differentiation potential. However we found little difference in the stem and tenogenic gene expression profile of these cells as compared to tenocytes, although the expression of thrombospondin‐4 was significantly reduced in hypoxic conditions. Tendon‐derived stem/progenitor cells isolated by differential adhesion to fibronectin had a similar differentiation potential to cells isolated by low density plating, and when grown in either normoxic or hypoxic conditions. In summary, we have found a restricted differentiation potential of cells isolated from the equine superficial digital flexor tendon despite evidence for stem/progenitor‐like characteristics. © 2015 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 33:849–858, 2015. PMID:25877997

  5. Immunohistological techniques for studying the Drosophila male germline stem cell.

    PubMed

    Singh, Shree Ram; Hou, Steven X

    2008-01-01

    Stem cells are undifferentiated cells that have a remarkable ability to self-renew and produce differentiated cells that support normal development and tissue homeostasis. This unique capacity makes stem cells a powerful tool for future regenerative medicine and gene therapy. Accumulative evidence suggests that stem cell self-renewal or differentiation is controlled by both intrinsic and extrinsic factors, and that deregulation of stem cell behavior results in cancer formation, tissue degeneration, and premature aging. The Drosophila testis provides an excellent in vivo model for studying and understanding the fundamental cellular and molecular mechanisms controlling stem cell behavior and the relationship between niches and stem cells. At the tip of the Drosophila testes, germline stem cells (GSCs) and somatic stem cells (SSCs) contact each other and share common niches (known as a hub) to maintain spermatogenesis. Signaling pathways, such as the Janus kinase (JAK)/signal transducer and activator of transcription (STAT), bone morphogenetic protein (BMP), ras-associated protein-guanine nucleotide exchange factor for small GTPase (Rap-GEF), and epidermal growth factor receptor (EGFR)/mitogen-activated protein kinase (MAPK), are known to regulate self-renewal or differentiation of Drosophila male germline stem cells. We describe the detailed in vivo immunohistological protocols that mark GSCs, SSCs, and their progeny in Drosophila testes. PMID:18370050

  6. Tendon and ligament engineering in the adult organism: mesenchymal stem cells and gene-therapeutic approaches

    PubMed Central

    Hoffmann, Andrea

    2007-01-01

    Tendons and ligaments are elastic collagenous tissues with similar composition and hierarchical structure, contributing to motion. Their strength is related to the number and size of the collagen fibrils. Collagen fibrils increase in size during development and in response to increased physical demands or training. Tendon disorders are commonly seen in clinical practice and give rise to significant morbidity. Treatment is difficult and patients often suffer from the symptoms for quite a long time. Despite remodelling, the biochemical and mechanical properties of healed tendon tissue never match those of intact tendon. The prerequisite for focussed treatment strategies in the future will be an improved understanding of the molecular events both in the embryo and contributing to regeneration in the adult organism. Novel approaches include the local delivery of growth factors, stem- and tendon-cell-derived therapy, the application of mechanical load and gene-therapeutic approaches based on vehicles encoding selected factors, or combinations of these. Important factors are proteins of the extracellular matrix like the metalloproteinases, growth factors like the bone morphogenetic proteins but also intracellular signalling mediator proteins, such as the Smads and transcription factors from the helix–loop–helix and other families. In this review, we focus specifically on such molecular approaches based on mesenchymal stem cells. PMID:17634943

  7. Tendon repair

    MedlinePlus

    Repair of tendon ... Tendon repair can be performed using: Local anesthesia (the immediate area of the surgery is pain-free) ... a cut on the skin over the injured tendon. The damaged or torn ends of the tendon ...

  8. In vitro effects of glutamate and N-methyl-D-aspartate receptor (NMDAR) antagonism on human tendon derived cells.

    PubMed

    Dean, Benjamin John Floyd; Snelling, Sarah J B; Dakin, Stephanie Georgina; Javaid, Muhammad Kassim; Carr, Andrew Jonathan

    2015-10-01

    It is known that extracellular glutamate concentrations are increased in tendinopathy but the effects of glutamate upon human tendon derived cells are unknown. The primary purpose was to investigate the effect of glutamate exposure on human tendon-derived cells in terms of viability, protein, and gene expression. The second purpose was to assess whether NMDAR antagonism would affect the response of tendon-derived cells to glutamate exposure. Human tendon-derived cells were obtained from supraspinatus tendon tissue obtained during rotator cuff repair (tendon tear derived cells) and from healthy hamstring tendon tissue (control cells). The in vitro impact of glutamate exposure and NMDAR antagonism (MK-801) was measured using the Alamar blue cell viability assay, immunocytochemistry, and quantitative real-time PCR. Glutamate reduced cell viability at 24 h in tendon tear derived cells but not in control cells at concentrations of 7.5 mM and above. Cell viability was significantly reduced after 72 h of 1.875 mM glutamate in both cell groups; this deleterious effect was attenuated by NMDAR antagonism with 10 µM MK-801. Both 24 and 72 h of 1.875 mM glutamate exposure reduced Type 1 alpha 1 collagen (COL1A1) and Type 3 alpha 1 collagen (COL3A1) gene expression, but increased Aggrecan gene expression. We propose that these effects of glutamate on tendon derived cells including reduced cell viability and altered matrix gene expression contribute to the pathogenesis of tendinopathy. PMID:26041147

  9. Cyclic strain alters the expression and release of angiogenic factors by human tendon cells.

    PubMed

    Mousavizadeh, Rouhollah; Khosravi, Shahram; Behzad, Hayedeh; McCormack, Robert G; Duronio, Vincent; Scott, Alex

    2014-01-01

    Angiogenesis is associated with the tissue changes underlying chronic overuse tendinopathy. We hypothesized that repetitive, cyclic loading of human tendon cells would lead to increased expression and activity of angiogenic factors. We subjected isolated human tendon cells to overuse tensile loading using an in vitro model (1 Hz, 10% equibiaxial strain). We found that mechanically stimulated human tendon cells released factors that promoted in vitro proliferation and tube formation by human umbilical vein endothelial cells (HUVEC). In response to cyclic strain, there was a transient increase in the expression of several angiogenic genes including ANGPTL4, FGF-2, COX-2, SPHK1, TGF-alpha, VEGF-A and VEGF-C, with no change in anti-angiogenic genes (BAI1, SERPINF1, THBS1 and 2, TIMP1-3). Cyclic strain also resulted in the extracellular release of ANGPTL4 protein by tendon cells. Our study is the first report demonstrating the induction of ANGPTL4 mRNA and release of ANGPTL4 protein in response to cyclic strain. Tenocytes may contribute to the upregulation of angiogenesis during the development of overuse tendinopathy. PMID:24824595

  10. A systems biology approach to defining regulatory mechanisms for cartilage and tendon cell phenotypes

    PubMed Central

    Mueller, A. J.; Tew, S. R.; Vasieva, O.; Clegg, P. D.; Canty-Laird, E. G.

    2016-01-01

    Phenotypic plasticity of adult somatic cells has provided emerging avenues for the development of regenerative therapeutics. In musculoskeletal biology the mechanistic regulatory networks of genes governing the phenotypic plasticity of cartilage and tendon cells has not been considered systematically. Additionally, a lack of strategies to effectively reproduce in vitro functional models of cartilage and tendon is retarding progress in this field. De- and redifferentiation represent phenotypic transitions that may contribute to loss of function in ageing musculoskeletal tissues. Applying a systems biology network analysis approach to global gene expression profiles derived from common in vitro culture systems (monolayer and three-dimensional cultures) this study demonstrates common regulatory mechanisms governing de- and redifferentiation transitions in cartilage and tendon cells. Furthermore, evidence of convergence of gene expression profiles during monolayer expansion of cartilage and tendon cells, and the expression of key developmental markers, challenges the physiological relevance of this culture system. The study also suggests that oxidative stress and PI3K signalling pathways are key modulators of in vitro phenotypes for cells of musculoskeletal origin. PMID:27670352

  11. Carpal tunnel syndrome caused by a giant cell tumour of the flexor tendon sheath.

    PubMed

    Meek, Marcel F; Sheikh, Zahid A; Quinton, David N

    2014-02-01

    A 76-year-old woman developed right carpal tunnel syndrome after being conservatively treated for tenosynovitis of the flexor tendons with associated mild carpal tunnel syndrome. A magnetic resonance imaging scan showed a tumour in the carpal tunnel. Re-exploration showed that the median nerve was being compressed by a giant cell tumour of the flexor tendon sheaths. Appropriate imaging is advised in patients with additional findings (such as swelling) or in patients with secondary carpal tunnel syndrome and incomplete response to conservative treatment, to exclude a space-occupying lesion.

  12. Musculoskeletal diseases—tendon

    PubMed Central

    Sakabe, Tomoya; Sakai, Takao

    2011-01-01

    Introduction Tendons establish specific connections between muscles and the skeleton by transferring contraction forces from skeletal muscle to bone thereby allowing body movement. Tendon physiology and pathology are heavily dependent on mechanical stimuli. Tendon injuries clinically represent a serious and still unresolved problem since damaged tendon tissues heal very slowly and no surgical treatment can restore a damaged tendon to its normal structural integrity and mechanical strength. Understanding how mechanical stimuli regulate tendon tissue homeostasis and regeneration will improve the treatment of adult tendon injuries that still pose a great challenge in today's medicine. Source of data This review summarizes the current status of tendon treatment and discusses new directions from the point of view of cell-based therapy and regenerative medicine approach. We searched the available literature using PubMed for relevant original articles and reviews. Growing points Identification of tendon cell markers has enabled us to study precisely tendon healing and homeostasis. Clinically, tissue engineering for tendon injuries is an emerging technology comprising elements from the fields of cellular source, scaffold materials, growth factors/cytokines and gene delivering systems. Areas timely for developing research The clinical settings to establish appropriate microenvironment for injured tendons with the combination of these novel cellular- and molecular-based scaffolds will be critical for the treatment. PMID:21729872

  13. Biologics for tendon repair☆

    PubMed Central

    Docheva, Denitsa; Müller, Sebastian A.; Majewski, Martin; Evans, Christopher H.

    2015-01-01

    Tendon injuries are common and present a clinical challenge to orthopedic surgery mainly because these injuries often respond poorly to treatment and require prolonged rehabilitation. Therapeutic options used to repair ruptured tendons have consisted of suture, autografts, allografts, and synthetic prostheses. To date, none of these alternatives has provided a successful long-term solution, and often the restored tendons do not recover their complete strength and functionality. Unfortunately, our understanding of tendon biology lags far behind that of other musculoskeletal tissues, thus impeding the development of new treatment options for tendon conditions. Hence, in this review, after introducing the clinical significance of tendon diseases and the present understanding of tendon biology, we describe and critically assess the current strategies for enhancing tendon repair by biological means. These consist mainly of applying growth factors, stem cells, natural biomaterials and genes, alone or in combination, to the site of tendon damage. A deeper understanding of how tendon tissue and cells operate, combined with practical applications of modern molecular and cellular tools could provide the long awaited breakthrough in designing effective tendon-specific therapeutics and overall improvement of tendon disease management. PMID:25446135

  14. Mechanisms of planar cell polarity establishment in Drosophila

    PubMed Central

    Carvajal-Gonzalez, Jose Maria

    2014-01-01

    Correct patterning and polarization of epithelial and mesenchymal cells are essential for morphogenesis and function of all organs and organisms. Epithelial cells are generally polarized in two axes: (a) the ubiquitous apical-basal axis and (b) polarity within the plane of the epithelium. The latter is generally referred to as planar cell polarity (PCP) and also is found in several contexts of mesenchymal cell patterning. In Drosophila, all adult structures display PCP features, and two conserved molecular systems (the Fat [Ft]/Dachsous [Ds] system and the Frizzled [Fz]/PCP pathway) that regulate this process have been identified. Although significant progress has been made in dissecting aspects of PCP signaling within cells, much remains to be discovered about the mechanisms of long-range and local PCP cell-cell interactions. Here, we discuss the current models based on Drosophila studies and incorporate recent insights into this long-standing cell and developmental biology problem. PMID:25580252

  15. Generation of stable Drosophila cell lines using multicistronic vectors.

    PubMed

    González, Monika; Martín-Ruíz, Itziar; Jiménez, Silvia; Pirone, Lucia; Barrio, Rosa; Sutherland, James D

    2011-01-01

    Insect cell culture is becoming increasingly important for applications including recombinant protein production and cell-based screening with chemical or RNAi libraries. While stable mammalian cell lines expressing a protein of interest can be efficiently prepared using IRES-based vectors or viral-based approaches, options for stable insect cell lines are more limited. Here, we describe pAc5-STABLEs, new vectors for use in Drosophila cell culture to facilitate stable transformation. We show that viral-derived 2A-like (or "CHYSEL") peptides function in Drosophila cells and can mediate the multicistronic expression of two or three proteins of interest under control of the Actin5C constitutive promoter. The current vectors allow mCherry and/or GFP fusions to be generated for positive selection by G418 resistance in cells and should serve as a flexible platform for future applications. PMID:22355594

  16. Allogenous tendon stem/progenitor cells in silk scaffold for functional shoulder repair.

    PubMed

    Shen, Weiliang; Chen, Jialin; Yin, Zi; Chen, Xiao; Liu, Huanhuan; Heng, Boon Chin; Chen, Weishan; Ouyang, Hong-Wei

    2012-01-01

    Tendon stem/progenitor cells (TSPCs) were recently identified within tendon tissues. The aim of this study was to investigate TSPC-seeded knitted silk-collagen sponge scaffold for functional shoulder repair. The multidifferentiation potential, proliferation, and immune properties of TSPCs were investigated in vitro, while the efficacy of TSPC-seeded knitted silk-collagen sponge scaffolds in promoting rotator cuff regeneration was evaluated in vivo within a rabbit model. TSPCs, which exhibited universal stem cell characteristics (i.e., clonogenicity, high proliferative capacity, and multidifferentiation potential), nonimmunogenicity, and immunosuppression, proliferated well on our scaffold in vitro. Implantation of allogenous TSPC-seeded scaffolds within a rabbit rotator cuff injury model did not elicit an immunological reaction, but instead increased fibroblastic cell ingrowth and reduced infiltration of lymphocytes within the implantation sites at 4 and 8 weeks postsurgery. After 12 weeks, the allogenous TSPC-treated group exhibited increased collagen deposition and had better structural and biomechanical properties compared to the control group. This study thus demonstrated that the allogenous TSPC-seeded knitted silk-collagen sponge scaffold enhanced the efficacy of rotator cuff tendon regeneration by differentiating into tenocytes, and by secreting anti-inflammatory cytokines that prevent immunological rejection. Hence, allogenous TSPC-seeded knitted silk-collagen sponge scaffolds can be a clinically useful application for tendon tissue engineering.

  17. Moderate Exercise Mitigates the Detrimental Effects of Aging on Tendon Stem Cells.

    PubMed

    Zhang, Jianying; Wang, James H-C

    2015-01-01

    Aging is known to cause tendon degeneration whereas moderate exercise imparts beneficial effects on tendons. Since stem cells play a vital role in maintaining tissue integrity, in this study we aimed to define the effects of aging and moderate exercise on tendon stem/progenitor cells (TSCs) using in vitro and in vivo models. TSCs derived from aging mice (9 and 24 months) proliferated significantly slower than TSCs obtained from young mice (2.5 and 5 months). In addition, expression of the stem cell markers Oct-4, nucleostemin (NS), Sca-1 and SSEA-1 in TSCs decreased in an age-dependent manner. Interestingly, moderate mechanical stretching (4%) of aging TSCs in vitro significantly increased the expression of the stem cell marker, NS, but 8% stretching decreased NS expression. Similarly, 4% mechanical stretching increased the expression of Nanog, another stem cell marker, and the tenocyte-related genes, collagen I and tenomodulin. However, 8% stretching increased expression of the non-tenocyte-related genes, LPL, Sox-9 and Runx-2, while 4% stretching had minimal effects on the expression of these genes. In the in vivo study, moderate treadmill running (MTR) of aging mice (9 months) resulted in the increased proliferation rate of aging TSCs in culture, decreased lipid deposition, proteoglycan accumulation and calcification, and increased the expression of NS in the patellar tendons. These findings indicate that while aging impairs the proliferative ability of TSCs and reduces their stemness, moderate exercise can mitigate the deleterious effects of aging on TSCs and therefore may be responsible for decreased aging-induced tendon degeneration. PMID:26086850

  18. Dissecting Social Cell Biology and Tumors Using Drosophila Genetics

    PubMed Central

    Pastor-Pareja, José Carlos; Xu, Tian

    2014-01-01

    Cancer was seen for a long time as a strictly cell-autonomous process in which oncogenes and tumor-suppressor mutations drive clonal cell expansions. Research in the past decade, however, paints a more integrative picture of communication and interplay between neighboring cells in tissues. It is increasingly clear as well that tumors, far from being homogenous lumps of cells, consist of different cell types that function together as complex tissue-level communities. The repertoire of interactive cell behaviors and the quantity of cellular players involved call for a social cell biology that investigates these interactions. Research into this social cell biology is critical for understanding development of normal and tumoral tissues. Such complex social cell biology interactions can be parsed in Drosophila. Techniques in Drosophila for analysis of gene function and clonal behavior allow us to generate tumors and dissect their complex interactive biology with cellular resolution. Here, we review recent Drosophila research aimed at understanding tissue-level biology and social cell interactions in tumors, highlighting the principles these studies reveal. PMID:23988119

  19. Mesenchymal stromal cells from bone marrow treated with bovine tendon extract acquire the phenotype of mature tenocytes☆

    PubMed Central

    Augusto, Lívia Maria Mendonça; Aguiar, Diego Pinheiro; Bonfim, Danielle Cabral; dos Santos Cavalcanti, Amanda; Casado, Priscila Ladeira; Duarte, Maria Eugênia Leite

    2016-01-01

    Objective This study evaluated in vitro differentiation of mesenchymal stromal cells isolated from bone marrow, in tenocytes after treatment with bovine tendon extract. Methods Bovine tendons were used for preparation of the extract and were stored at −80 °C. Mesenchymal stromal cells from the bone marrow of three donors were used for cytotoxicity tests by means of MTT and cell differentiation by means of qPCR. Results The data showed that mesenchymal stromal cells from bone marrow treated for up to 21 days in the presence of bovine tendon extract diluted at diminishing concentrations (1:10, 1:50 and 1:250) promoted activation of biglycan, collagen type I and fibromodulin expression. Conclusion Our results show that bovine tendon extract is capable of promoting differentiation of bone marrow stromal cells in tenocytes. PMID:26962503

  20. Application of stem cells derived from the periodontal ligament or gingival tissue sources for tendon tissue regeneration.

    PubMed

    Moshaverinia, Alireza; Xu, Xingtian; Chen, Chider; Ansari, Sahar; Zadeh, Homayoun H; Snead, Malcolm L; Shi, Songtao

    2014-03-01

    Tendon injuries are often associated with significant dysfunction and disability due to tendinous tissue's very limited self-repair capacity and propensity for scar formation. Dental-derived mesenchymal stem cells (MSCs) in combination with appropriate scaffold material present an alternative therapeutic option for tendon repair/regeneration that may be advantageous compared to other current treatment modalities. The MSC delivery vehicle is the principal determinant for successful implementation of MSC-mediated regenerative therapies. In the current study, a co-delivery system based on TGF-β3-loaded RGD-coupled alginate microspheres was developed for encapsulating periodontal ligament stem cells (PDLSCs) or gingival mesenchymal stem cells (GMSCs). The capacity of encapsulated dental MSCs to differentiate into tendon tissue was investigated in vitro and in vivo. Encapsulated dental-derived MSCs were transplanted subcutaneously into immunocompromised mice. Our results revealed that after 4 weeks of differentiation in vitro, PDLSCs and GMSCs as well as the positive control human bone marrow mesenchymal stem cells (hBMMSCs) exhibited high levels of mRNA expression for gene markers related to tendon regeneration (Scx, DCn, Tnmd, and Bgy) via qPCR measurement. In a corresponding in vivo animal model, ectopic neo-tendon regeneration was observed in subcutaneous transplanted MSC-alginate constructs, as confirmed by histological and immunohistochemical staining for protein markers specific for tendons. Interestingly, in our quantitative PCR and in vivo histomorphometric analyses, PDLSCs showed significantly greater capacity for tendon regeneration than GMSCs or hBMMSCs (P < 0.05). Altogether, these findings indicate that periodontal ligament and gingival tissues can be considered as suitable stem cell sources for tendon engineering. PDLSCs and GMSCs encapsulated in TGF-β3-loaded RGD-modified alginate microspheres are promising candidates for tendon regeneration.

  1. Live Imaging of Border Cell Migration in Drosophila.

    PubMed

    Dai, Wei; Montell, Denise J

    2016-01-01

    Border cells are a cluster of cells that migrate from the anterior tip of the Drosophila egg chamber to the border of the oocyte in stage 9. They serve as a useful model to study collective cell migration in a native tissue environment. Here we describe a protocol for preparing ex vivo egg chamber cultures from transgenic flies expressing fluorescent proteins in the border cells, and using confocal microscopy to take a multi-positional time-lapse movie. We include an image analysis method for tracking border cell cluster dynamics as well as tracking individual cell movements. PMID:27271901

  2. Live Imaging of Border Cell Migration in Drosophila.

    PubMed

    Dai, Wei; Montell, Denise J

    2016-01-01

    Border cells are a cluster of cells that migrate from the anterior tip of the Drosophila egg chamber to the border of the oocyte in stage 9. They serve as a useful model to study collective cell migration in a native tissue environment. Here we describe a protocol for preparing ex vivo egg chamber cultures from transgenic flies expressing fluorescent proteins in the border cells, and using confocal microscopy to take a multi-positional time-lapse movie. We include an image analysis method for tracking border cell cluster dynamics as well as tracking individual cell movements.

  3. Heterogeneous expression of Drosophila gustatory receptors in enteroendocrine cells.

    PubMed

    Park, Jeong-Ho; Kwon, Jae Young

    2011-01-01

    The gastrointestinal tract is emerging as a major site of chemosensation in mammalian studies. Enteroendocrine cells are chemosensory cells in the gut which produce regulatory peptides in response to luminal contents to regulate gut physiology, food intake, and glucose homeostasis, among other possible functions. Increasing evidence shows that mammalian taste receptors and taste signaling molecules are expressed in enteroendocrine cells in the gut. Invertebrate models such as Drosophila can provide a simple and genetically tractable system to study the chemosensory functions of enteroendocrine cells in vivo. To establish Drosophila enteroendocrine cells as a model for studying gut chemosensation, we used the GAL4/UAS system to examine the expression of all 68 Gustatory receptors (Grs) in the intestine. We find that 12 Gr-GAL4 drivers label subsets of enteroendocrine cells in the midgut, and examine colocalization of these drivers with the regulatory peptides neuropeptide F (NPF), locustatachykinin (LTK), and diuretic hormone 31 (DH31). RT-PCR analysis provides additional evidence for the presence of Gr transcripts in the gut. Our results suggest that the Drosophila Grs have chemosensory roles in the intestine to regulate physiological functions such as food uptake, nutrient absorption, or sugar homeostasis. PMID:22194978

  4. Electrospun fibre diameter, not alignment, affects mesenchymal stem cell differentiation into the tendon/ligament lineage.

    PubMed

    Cardwell, Robyn D; Dahlgren, Linda A; Goldstein, Aaron S

    2014-12-01

    Efforts to develop engineered tendons and ligaments have focused on the use of a biomaterial scaffold and a stem cell source. However, the ideal scaffold microenvironment to promote stem cell differentiation and development of organized extracellular matrix is unknown. Through electrospinning, fibre scaffolds can be designed with tailorable architectures to mimic the intended tissue. In this study, the effects of fibre diameter and orientation were examined by electrospinning thin mats, consisting of small (< 1 µm), medium (1-2 µm) or large (> 2 µm) diameter fibres with either random or aligned fibre orientation. C3H10T1/2 model stem cells were cultured on the six different electrospun mats, as well as smooth spin-coated films, and the morphology, growth and expression of tendon/ligament genes were evaluated. The results demonstrated that fibre diameter affects cellular behaviour more significantly than fibre alignment. Initially, cell density was greater on the small fibre diameter mats, but similar cell densities were found on all mats after an additional week in culture. After 2 weeks, gene expression of collagen 1α1 and decorin was increased on all mats compared to films. Expression of the tendon/ligament transcription factor scleraxis was suppressed on all electrospun mats relative to spin-coated films, but expression on the large-diameter fibre mats was consistently greater than on the medium-diameter fibre mats. These results suggest that larger-diameter fibres (e.g. > 2 µm) may be more suitable for in vitro development of a tendon/ligament tissue.

  5. Stem cell therapy: a promising biological strategy for tendon-bone healing after anterior cruciate ligament reconstruction.

    PubMed

    Hao, Zi-Chen; Wang, Shan-Zheng; Zhang, Xue-Jun; Lu, Jun

    2016-04-01

    Tendon-bone healing after anterior cruciate ligament (ACL) reconstruction is a complex process, impacting significantly on patients' prognosis. Natural tendon-bone healing usually results in fibrous scar tissue, which is of inferior quality compared to native attachment. In addition, the early formed fibrous attachment after surgery is often not reliable to support functional rehabilitation, which may lead to graft failure or unsatisfied function of the knee joint. Thus, strategies to promote tendon-bone healing are crucial for prompt and satisfactory functional recovery. Recently, a variety of biological approaches, including active substances, gene transfer, tissue engineering and stem cells, have been proposed and applied to enhance tendon-bone healing. Among these, stem cell therapy has been shown to have promising prospects and draws increasing attention. From commonly investigated bone marrow-derived mesenchymal stem cells (bMSCs) to emerging ACL-derived CD34+ stem cells, multiple stem cell types have been proven to be effective in accelerating tendon-bone healing. This review describes the current understanding of tendon-bone healing and summarizes the current status of related stem cell therapy. Future limitations and perspectives are also discussed.

  6. The effect of anisotropic collagen-GAG scaffolds and growth factor supplementation on tendon cell recruitment, alignment, and metabolic activity

    PubMed Central

    Caliari, Steven R.; Harley, Brendan A.C.

    2014-01-01

    Current surgical and tissue engineering approaches for treating tendon injuries have shown limited success, suggesting the need for new biomaterial strategies. Here we describe the development of an anisotropic collagen-glycosaminoglycan (CG) scaffold and use of growth factor supplementation strategies to create a 3D platform for tendon tissue engineering. We fabricated cylindrical CG scaffolds with aligned tracks of ellipsoidal pores that mimic the native physiology of tendon by incorporating a directional solidification step into a conventional lyophilization strategy. By modifying the freezing temperature, we created a homologous series of aligned CG scaffolds with constant relative density and degree of anisotropy but a range of pore sizes (55–243 μm). Equine tendon cells showed greater levels of attachment, metabolic activity, and alignment as well as less cell-mediated scaffold contraction, when cultured in anisotropic scaffolds compared to an isotropic CG scaffold control. The anisotropic CG scaffolds also provided critical contact guidance cues for cell alignment. While tendon cells were randomly oriented in the isotropic control scaffold and the transverse (unaligned) plane of the anisotropic scaffolds, significant cell alignment was observed in the direction of the contact guidance cues in the longitudinal plane of the anisotropic scaffolds. Scaffold pore size was found to significantly influence tendon cell viability, proliferation, penetration into the scaffold, and metabolic activity in a manner predicted by cellular solids arguments. Finally, the addition of the growth factors PDGF-BB and IGF-1 to aligned CG scaffolds was found to enhance tendon cell motility, viability, and metabolic activity in dose-dependent manners. This work suggests a composite strategy for developing bioactive, 3D material systems for tendon tissue engineering. PMID:21550653

  7. Tendonitis (image)

    MedlinePlus

    ... tendon. It can occur as a result of injury, overuse, or with aging as the tendon loses elasticity. Any action that places prolonged repetitive strain on the forearm muscles can cause tendonitis. The ...

  8. Spatiotemporal calcium signaling in a Drosophila melanogaster cell line stably expressing a Drosophila muscarinic acetylcholine receptor.

    PubMed

    Cordova, D; Delpech, V Raymond; Sattelle, D B; Rauh, J J

    2003-11-01

    A muscarinic acetylcholine receptor (mAChR), DM1, expressed in the nervous system of Drosophila melanogaster, has been stably expressed in a Drosophila S2 cell line (S2-DM1) and used to investigate spatiotemporal calcium changes following agonist activation. Carbamylcholine (CCh) and oxotremorine are potent agonists, whereas application of the vertebrate M1 mAChR agonist, McN-A-343, results in a weak response. Activation of S2-DM1 receptors using CCh resulted in an increase in intracellular calcium ([Ca(2+)](i)) that was biphasic. Two distinct calcium sources were found to contribute to calcium signaling: (1) internal stores that are sensitive to both thapsigargin and 2-aminoethoxydiphenyl borate and (2) capacitative calcium entry. Spatiotemporal imaging of individual S2-DM1 cells showed that the CCh-induced [Ca(2+)](i) transient resulted from a homogeneous calcium increase throughout the cell, indicative of calcium release from internal stores. In contrast, ionomycin induced the formation of a "calcium ring" at the cell periphery, consistent with external calcium influx. PMID:12827518

  9. Repair of patellar tendon injuries using a cell-collagen composite.

    PubMed

    Awad, Hani A; Boivin, Gregory P; Dressler, Matthew R; Smith, Frost N L; Young, Randell G; Butler, David L

    2003-05-01

    Collagen gels were seeded with rabbit bone marrow-derived mesenchymal stem cells (MSCs) and contracted onto sutures at initial cell densities of 1, 4, and 8 million cells/ml. These MSC-collagen composites were then implanted into full thickness, full length, central defects created in the patellar tendons of the animals providing the cells. These autologous repairs were compared to natural repair of identical defects on the contralateral side. Biomechanical, histological, and morphometric analyses were performed on both repair tissue types at 6, 12, and 26 weeks after surgery. Repair tissues containing the MSC-collagen composites showed significantly higher maximum stresses and moduli than natural repair tissues at 12 and 26 weeks postsurgery. However, no significant differences were observed in any dimensional or mechanical properties of the repair tissues across seeding densities at each evaluation time. By 26 weeks, the repairs grafted with MSC-collagen composites were one-fourth of the maximum stress of the normal central portion of the patellar tendon with bone ends. The modulus and maximum stress of the repair tissues grafted with MSC-collagen composites increased at significantly faster rates than did natural repairs over time. Unexpectedly, 28% of the MSC-collagen grafted tendons formed bone in the regenerating repair site. Except for increased repair tissue volume, no significant differences in cellular organization or histological appearance were observed between the natural repairs and MSC-collagen grafted repairs. Overall, these results show that surgically implanting tissue engineered MSC-collagen composites significantly improves the biomechanical properties of tendon repair tissues, although greater MSC concentrations produced no additional significant histological or biomechanical improvement.

  10. Giant Cell Tumor of the Patella Tendon Sheath Presenting as a Painful Locked Knee

    PubMed Central

    Panagopoulos, Andreas; Tsoumpos, Pantelis; Tatani, Irini; Iliopoulos, Ilias; Papachristou, Dionysios

    2015-01-01

    Patient: Male, 26 Final Diagnosis: Giant cell tumor of the patella tendon seath Symptoms: Efusion • locking knee • pain Medication: None Clinical Procedure: Arthroscopy and open resection of the tumor Specialty: Orthopedics and Traumatology Objective: Rare disease Background: The giant cell tumor of the tendon sheath (GCT-TS) is a benign proliferative synovial tumor manifesting as an intra-articular solitary nodule. When it involves the infrapatellar fat pad it can present acutely as a painful locked knee. Case Report: A 26-year-old white male presented with a 2-week history of painful locking in his right knee. Clinical examination revealed lack of extension by approximately 20°. To help establish the diagnosis, an MRI scan of the right knee was performed, showing a large (5×4×2 cm), oval, well-circumscribed mass with a low-intensity homogenous signal. The size of the mass prohibited the removal by arthroscopy and we therefore proceeded with an open arthrotomy. Histological examination showed a tendosynovial giant cell tumor of the patella tendon sheath. At the latest follow-up, 2 years postoperatively, there was no local tumor recurrence. Conclusions: These rare tumorous lesions should be included in the differential diagnosis of painful locking knee, especially in the absence of definite traumatic history. PMID:26302970

  11. Effect of young extrinsic environment stimulated by hypoxia on the function of aged tendon stem cell.

    PubMed

    Jiang, Dapeng; Jiang, Zhitao; Zhang, Yubo; Wang, Shuai; Yang, Shulong; Xu, Bo; Yang, Mowen; Li, Zhaozhu

    2014-11-01

    Tendon stem cells (TSCs), recently identified as tendon cells, play an important role in maintaining the homeostasis of tendon tissue. Age-related decrease in the function of TSCs has been reported. Recent reports demonstrated that hypoxic condition is advantageous for efficient expansion of TSCs. Moreover, the impaired function of aged stem cells could be modulated by exposing them to a young environment. Therefore, we investigated the effects of hypoxic-conditioned culture medium (HCCM) from young TSCs on the proliferation, migration, senescence, and tenocyte phenotype of aged TSCs. TSCs were isolated, and the conditioned medium was collected. There were 4 groups: young TSCs, aged TSCs, aged TSCs + aged HCCM, and aged TSCs + young HCCM. The proliferative capacity, migration, β-galactosidase activity, and tenogenic differentiation potential of TSCs were assessed. Our results showed that HCCM enhanced the proliferation and migration potential of aged TSCs. Moreover, the senescence-associated β-galactosidase activity of aged TSCs was decreased by young HCCM. After being cultured in the young HCCM, the expressions of tenocyte-related genes in aged TSCs were significantly enhanced. Together, results of this study indicate that HCCM from young TSCs may represent an effective strategy to improve the impaired function of aged TSCs.

  12. Indirect Co-Culture with Tendons or Tenocytes Can Program Amniotic Epithelial Cells towards Stepwise Tenogenic Differentiation

    PubMed Central

    Barboni, Barbara; Curini, Valentina; Russo, Valentina; Mauro, Annunziata; Di Giacinto, Oriana; Marchisio, Marco; Alfonsi, Melissa; Mattioli, Mauro

    2012-01-01

    Background Amniotic epithelial cells (AEC) have potential applications in cell-based therapy. Thus far their ability to differentiate into tenocytes has not been investigated although a cell source providing a large supply of tenocytes remains a priority target of regenerative medicine in order to respond to the poor self-repair capability of adult tendons. Starting from this premise, the present research has been designed firstly to verify whether the co-culture with adult primary tenocytes could be exploited in order to induce tenogenic differentiation in AEC, as previously demonstrated in mesenchymal stem cells. Since the co-culture systems inducing cell differentiation takes advantage of specific soluble paracrine factors released by tenocytes, the research has been then addressed to study whether the co-culture could be improved by making use of the different cell populations present within tendon explants or of the high regenerative properties of fetal derived cell/tissue. Methodology/Principal Findings Freshly isolated AEC, obtained from ovine fetuses at mid-gestation, were co-incubated with explanted tendons or primary tenocytes obtained from fetal or adult calcaneal tendons. The morphological and functional analysis indicated that AEC possessed tenogenic differentiation potential. However, only AEC exposed to fetal-derived cell/tissues developed in vitro tendon-like three dimensional structures with an expression profile of matrix (COL1 and THSB4) and mesenchymal/tendon related genes (TNM, OCN and SCXB) similar to that recorded in native ovine tendons. The tendon-like structures displayed high levels of organization as documented by the cell morphology, the newly deposited matrix enriched in COL1 and widespread expression of gap junction proteins (Connexin 32 and 43). Conclusions/Significance The co-culture system improves its efficiency in promoting AEC differentiation by exploiting the inductive tenogenic soluble factors released by fetal tendon cells or

  13. Superparamagnetic iron oxide nanoparticles as a means to track mesenchymal stem cells in a large animal model of tendon injury.

    PubMed

    Scharf, Alexandra; Holmes, Shannon; Thoresen, Merrilee; Mumaw, Jennifer; Stumpf, Alaina; Peroni, John

    2015-01-01

    The goal of this study was to establish an SPIO-based cell-tracking method in an ovine model of tendonitis and to determine if this method may be useful for further study of cellular therapies in tendonitis in vivo. Functional assays were performed on labeled and unlabeled cells to ensure that no significant changes were induced by intracellular SPIOs. Following biosafety validation, tendon lesions were mechanically (n = 4) or chemically (n = 4) induced in four sheep and scanned ex vivo at 7 and 14 days to determine the presence and distribution of intralesional cells. Ovine MSCs labeled with 50 µg SPIOs/mL remained viable, proliferate, and undergo tri-lineage differentiation (p < 0.05). Labeled ovine MSCs remained detectable in vitro in concentrated cell numbers as low as 10 000 and in volumetric distributions as low as 100 000 cells/mL. Cells remained detectable by MRI at 7 days, as confirmed by correlative histology for dually labeled SPIO+/GFP+ cells. Histological evidence at 14 days suggested that SPIO particles remained embedded in tissue, providing MRI signal, although cells were no longer present. SPIO labeling has proven to be an effective method for cell tracking for a large animal model of tendon injury for up to 7 days post-injection. The data obtained in this study justify further investigation into the effects of MSC survival and migration on overall tendon healing and tissue regeneration.

  14. Cell Surface Changes Associated with Cellular Immune Reactions in Drosophila

    NASA Astrophysics Data System (ADS)

    Nappi, Anthony J.; Silvers, Michael

    1984-09-01

    In Drosophila melanogaster a temperature-induced change in immune competence accompanies cell surface alterations that cause its blood cells to adhere and to encapsulate a parasite. At 29 degrees C the blood cells of the tumorous-lethal (Tuml) mutant show a high degree of immune competence and encapsulate the eggs of the parasitic wasp Leptopilina heterotoma. At 21 degrees C the blood cells are essentially immune incompetent. High percentages of lectin binding cells were found under conditions which potentiated cellular encapsulation responses. Some immune reactive blood cells did not bind lectin. The low percentages of lectin binding cells in susceptible hosts suggest that developing parasites alter the cell surface of the blood cells of immune reactive hosts.

  15. Cell surface changes associated with cellular immune reactions in Drosophila.

    PubMed

    Nappi, A J; Silvers, M

    1984-09-14

    In Drosophila melanogaster a temperature-induced change in immune competence accompanies cell surface alterations that cause its blood cells to adhere and to encapsulate a parasite. At 29 degrees C the blood cells of the tumorous-lethal (Tuml) mutant show a high degree of immune competence and encapsulate the eggs of the parasitic wasp Leptopilina heterotoma. At 21 degrees C the blood cells are essentially immune incompetent. High percentages of lectin binding cells were found under conditions which potentiated cellular encapsulation responses. Some immune reactive blood cells did not bind lectin. The low percentages of lectin binding cells in susceptible hosts suggest that developing parasites alter the cell surface of the blood cells of immune reactive hosts. PMID:6433482

  16. Assaying Blood Cell Populations of the Drosophila melanogaster Larva

    PubMed Central

    Petraki, Sophia; Alexander, Brandy; Brückner, Katja

    2015-01-01

    In vertebrates, hematopoiesis is regulated by inductive microenvironments (niches). Likewise, in the invertebrate model organism Drosophila melanogaster, inductive microenvironments known as larval Hematopoietic Pockets (HPs) have been identified as anatomical sites for the development and regulation of blood cells (hemocytes), in particular of the self-renewing macrophage lineage. HPs are segmentally repeated pockets between the epidermis and muscle layers of the larva, which also comprise sensory neurons of the peripheral nervous system. In the larva, resident (sessile) hemocytes are exposed to anti-apoptotic, adhesive and proliferative cues from these sensory neurons and potentially other components of the HPs, such as the lining muscle and epithelial layers. During normal development, gradual release of resident hemocytes from the HPs fuels the population of circulating hemocytes, which culminates in the release of most of the resident hemocytes at the beginning of metamorphosis. Immune assaults, physical injury or mechanical disturbance trigger the premature release of resident hemocytes into circulation. The switch of larval hemocytes between resident locations and circulation raises the need for a common standard/procedure to selectively isolate and quantify these two populations of blood cells from single Drosophila larvae. Accordingly, this protocol describes an automated method to release and quantify the resident and circulating hemocytes from single larvae. The method facilitates ex vivo approaches, and may be adapted to serve a variety of developmental stages of Drosophila and other invertebrate organisms. PMID:26650404

  17. Thermal energy enhances cell-mediated contraction of lax rat tail tendon fascicles following exercise

    PubMed Central

    Lavagnino, Michael; Malek, Kirollos; Gardner, Keri L.; Arnoczky, Steven P.

    2015-01-01

    Summary Background: the application of thermal energy (TE) has shown promise in the treatment of tendinopathy. However, the precise mechanism(s) of action of this therapy is unclear. The loss of tendon cell homeostatic tension, due to loading-induced laxity, produces catabolic changes associated with tendinopathy. This catabolic activity can be inhibited through the re-establishment of a normal tensile environment via a cellular contraction mechanism. We hypothesized that application of TE will enhance the contraction rate of lax rat tail tendon fascicles (RTTfs) in an in vitro model. Methods: following loading, 10 lax RTTfs from each mature rat (n=5) were treated once daily for 7 days with TE by replacing the culture media at 37°C (control) with 42°C media. Using calibrated photographs, RTTf lengths were measured daily. Additional RTTfs were utilized to investigate any changes in material (n=12) and/or histological (n=12) properties with TE. Results: TE significantly increased the contraction rate of RTTfs (p>0.001) without altering the material or histological properties. Conclusion: these results demonstrate that TE significantly enhances the contraction rate of previously exercised tendons. The ability to more quickly re-establish a normal mechanobiological environment, thus minimizing any catabolic changes, may explain the beneficial effects reported with applied TE in tendinopathy treatment. PMID:25878989

  18. Extra-Articular Diffuse Giant Cell Tumor of the Tendon Sheath: A Report of 2 Cases.

    PubMed

    Savvidou, Olga D; Mavrogenis, Andreas F; Sakellariou, Vasilios I; Chloros, George D; Sarlikiotis, Thomas; Papagelopoulos, Panayiotis J

    2016-06-01

    Two rare cases of extra-articular diffuse variant giant cell tumor of the tendon sheath are presented, at the elbow of a 68-year-old female and the foot of a 56-year-old male. Both patients presented with a palpable masses and marginal excision was performed; histological sections confirmed the diagnosis of extra-articular giant cell tumor. No adjuvant therapy was administered. At the latest follow-up, minimum 24 months after excision both patients were disease-free. PMID:27517076

  19. Extra-Articular Diffuse Giant Cell Tumor of the Tendon Sheath: A Report of 2 Cases

    PubMed Central

    Savvidou, Olga D.; Mavrogenis, Andreas F.; Sakellariou, Vasilios I.; Chloros, George D.; Sarlikiotis, Thomas; Papagelopoulos, Panayiotis J.

    2016-01-01

    Two rare cases of extra-articular diffuse variant giant cell tumor of the tendon sheath are presented, at the elbow of a 68-year-old female and the foot of a 56-year-old male. Both patients presented with a palpable masses and marginal excision was performed; histological sections confirmed the diagnosis of extra-articular giant cell tumor. No adjuvant therapy was administered. At the latest follow-up, minimum 24 months after excision both patients were disease-free. PMID:27517076

  20. Centrosome and microtubule instability in aging Drosophila cells

    NASA Technical Reports Server (NTRS)

    Schatten, H.; Chakrabarti, A.; Hedrick, J.

    1999-01-01

    Several cytoskeletal changes are associated with aging which includes alterations in muscle structure leading to muscular atrophy, and weakening of the microtubule network which affects cellular secretion and maintenance of cell shape. Weakening of the microtubule network during meiosis in aging oocytes can result in aneuploidy or trisomic zygotes with increasing maternal age. Imbalances of cytoskeletal organization can lead to disease such as Alzheimer's, muscular disorders, and cancer. Because many cytoskeletal diseases are related to age we investigated the effects of aging on microtubule organization in cell cultures of the Drosophila cell model system (Schneider S-1 and Kc23 cell lines). This cell model is increasingly being used as an alternative system to mammalian cell cultures. Drosophila cells are amenable to genetic manipulations and can be used to identify and manipulate genes which are involved in the aging processes. Immunofluorescence, scanning, and transmission electron microscopy were employed for the analysis of microtubule organizing centers (centrosomes) and microtubules at various times after subculturing cells in fresh medium. Our results reveal that centrosomes and the microtubule network becomes significantly affected in aging cells after 5 days of subculture. At 5-14 days of subculture, 1% abnormal out of 3% mitoses were noted which were clearly distinguishable from freshly subcultured control cells in which 3% of cells undergo normal mitosis with bipolar configurations. Microtubules are also affected in the midbody during cell division. The midbody in aging cells becomes up to 10 times longer when compared with midbodies in freshly subcultured cells. During interphase, microtubules are often disrupted and disorganized, which may indicate improper function related to transport of cell organelles along microtubules. These results are likely to help explain some cytoskeletal disorders and diseases related to aging.

  1. Dissecting mitosis with laser microsurgery and RNAi in Drosophila cells.

    PubMed

    Pereira, António J; Matos, Irina; Lince-Faria, Mariana; Maiato, Helder

    2009-01-01

    Progress from our present understanding of the mechanisms behind mitosis has been compromised by the fact that model systems that were ideal for molecular and genetic studies (such as yeasts, C. elegans, or Drosophila) were not suitable for intracellular micromanipulation. Unfortunately, those systems that were appropriate for micromanipulation (such as newt lung cells, PtK1 cells, or insect spermatocytes) are not amenable for molecular studies. We believe that we can significantly broaden this scenario by developing high-resolution live cell microscopy tools in a system where micromanipulation studies could be combined with modern gene-interference techniques. Here we describe a series of methodologies for the functional dissection of mitosis by the use of simultaneous live cell microscopy and state-of-the-art laser microsurgery, combined with RNA interference (RNAi) in Drosophila cell lines stably expressing fluorescent markers. This technological synergism allows the specific targeting and manipulation of several structural components of the mitotic apparatus in different genetic backgrounds, at the highest spatial and temporal resolution. Finally, we demonstrate the successful adaptation of agar overlay flattening techniques to human HeLa cells and discuss the advantages of its use for laser micromanipulation and molecular studies of mitosis in mammals.

  2. In situ cell-matrix mechanics in tendon fascicles and seeded collagen gels: implications for the multiscale design of biomaterials.

    PubMed

    Duncan, Neil A; Bruehlmann, Sabina B; Hunter, Christopher J; Shao, Xinxin; Kelly, Elizabeth J

    2014-01-01

    Designing biomaterials to mimic and function within the complex mechanobiological conditions of connective tissues requires a detailed understanding of the micromechanical environment of the cell. The objective of our study was to measure the in situ cell-matrix strains from applied tension in both tendon fascicles and cell-seeded type I collagen scaffolds using laser scanning confocal microscopy techniques. Tendon fascicles and collagen gels were fluorescently labelled to simultaneously visualise the extracellular matrix and cell nuclei under applied tensile strains of 5%. There were significant differences observed in the micromechanics at the cell-matrix scale suggesting that the type I collagen scaffold did not replicate the pattern of native tendon strains. In particular, although the overall in situ tensile strains in the matrix were quite similar (∼2.5%) between the tendon fascicles and the collagen scaffolds, there were significant differences at the cell-matrix boundary with visible shear across cell nuclei of >1 μm measured in native tendon which was not observed at all in the collagen scaffolds. Similarly, there was significant non-uniformity of intercellular strains with relative sliding observed between cell rows in tendon which again was not observed in the collagen scaffolds where the strain environment was much more uniform. If the native micromechanical environment is not replicated in biomaterial scaffolds, then the cells may receive incorrect or mixed mechanical signals which could affect their biosynthetic response to mechanical load in tissue engineering applications. This study highlights the importance of considering the microscale mechanics in the design of biomaterial scaffolds and the need to incorporate such features in computational models of connective tissues.

  3. Tendon Innervation.

    PubMed

    Ackermann, Paul W; Salo, Paul; Hart, David A

    2016-01-01

    The regulation of tendon metabolism including the responses to loading is far from being well understood. During the last decade, however, accumulating data show that tendon innervation in addition to afferent functions, via efferent pathways has a regulatory role in tendon homeostasis via a wide range of neuromediators, which coordinate metabolic and neuro-inflammatory pathways.Innervation of intact healthy tendons is localized in the surrounding structures, i.e paratenon, endotenon and epitenon, whereas the tendon proper is practically devoid of neuronal supply. This anatomical finding reflects that the tendon metabolism is regulated from the tendon envelope, i.e. interfascicular matrix (see Chap. 1 ).Tendon innervation after injury and during repair, however, is found as extensive nerve ingrowth into the tendon proper, followed by a time-dependent emergence of different neuronal mediators, which amplify and fine-tune inflammatory and metabolic pathways in tendon regeneration. After healing nerve fibers retract to the tendon envelope.In tendinopathy innervation has been identified to consist of excessive and protracted nerve ingrowth in the tendon proper, suggesting pro-inflammatory, nociceptive and hypertrophic (degenerative) tissue responses.In metabolic disorders such as eg. diabetes impaired tendon healing has been established to be related to dysregulation of neuronal growth factors.Targeted approaches to the peripheral nervous system including neuronal mediators and their receptors may prove to be effective therapies for painful, degenerative and traumatic tendon disorders. PMID:27535247

  4. PROGRAMMED CELL DEATH IN EXTRAOCULAR MUSCLE TENDON/SCLERA PRECURSORS

    EPA Science Inventory

    Abstract

    Purpose: This study was designed to examine the occurrence of natural cell death in the periocular mesenchyme of mouse embryos.

    Methods: Vital staining with LysoTracker Red and Nile blue sulphate as well as terminal nick end labeling (TUNEL) were utiliz...

  5. Switching cell fates in the developing Drosophila eye.

    PubMed

    Mavromatakis, Yannis Emmanuel; Tomlinson, Andrew

    2013-11-01

    The developing Drosophila ommatidium is characterized by two distinct waves of pattern formation. In the first wave, a precluster of five cells is formed by a complex cellular interaction mechanism. In the second wave, cells are systematically recruited to the cluster and directed to their fates by developmental cues presented by differentiating precluster cells. These developmental cues are mediated through the receptor tyrosine kinase (RTK) and Notch (N) signaling pathways and their combined activities are crucial in specifying cell type. The transcription factor Lozenge (Lz) is expressed exclusively in second wave cells. Here, we ectopically supply Lz to precluster cells and concomitantly supply the various RTK/N codes that specify each of three second wave cell fates. We thereby reproduce molecular markers of each of the second wave cell types in precluster cells and draw three inferences. First, we confirm that Lz provides key intrinsic information to second wave cells. We can now combine this with the RTK/N signaling to provide a cell fate specification code that entails both extrinsic and intrinsic information. Second, the reproduction of each second wave cell type in the precluster confirms the accuracy of the RTK/N signaling code. Third, RTK/N signaling and Lz need only be presented to the cells for a short period of time in order to specify their fate. PMID:24067351

  6. Multiple Giant Cell Tumors of Tendon Sheath Found within a Single Digit of a 9-Year-Old

    PubMed Central

    Fitzhugh, Valerie A.; Gibson, Peter D.; Didesch, Jacob; Ahmed, Irfan

    2016-01-01

    Giant cell tumor of tendon sheath is one of the most common soft tissue tumors of the hand. These tumors typically occur in the third or fourth decade of life and present as solitary nodules on a single digit. Currently, the greatest reported number of lesions found within a single digit is five. Although uncommon, giant cell tumor of tendon sheath does occur in the pediatric population. Herein we present a report of a rare case of GCTTS in a child in which seven lesions were identified within a single digit—the greatest number of lesions within a single digit reported to date. PMID:27595029

  7. New Twists in Drosophila Cell Signaling.

    PubMed

    Shilo, Ben-Zion

    2016-04-01

    The discovery of a handful of conserved signaling pathways that dictate most aspects of embryonic and post-embryonic development of multicellular organisms has generated a universal view of animal development (Perrimon, N., Pitsouli, C., and Shilo, B. Z. (2012)Cold Spring Harb. Perspect. Biol.4, a005975). Although we have at hand most of the "hardware" elements that mediate cell communication events that dictate cell fate choices, we are still far from a comprehensive mechanistic understanding of these processes. One of the next challenges entails an analysis of developmental signaling pathways from the cell biology perspective. Where in the cell does signaling take place, and how do general cellular machineries and structures contribute to the regulation of developmental signaling? Another challenge is to examine these signaling pathways from a quantitative perspective, rather than as crude on/off switches. This requires more precise measurements, and incorporation of the time element to generate a dynamic sequence instead of frozen snapshots of the process. The quantitative outlook also brings up the issue of precision, and the unknown mechanisms that buffer variability in signaling between embryos, to produce a robust and reproducible output. Although these issues are universal to all multicellular organisms, they can be effectively tackled in theDrosophilamodel, by a combination of genetic manipulations, biochemical analyses, and a variety of imaging techniques. This review will present some of the recent advances that were accomplished by utilizing the versatility of theDrosophilasystem.

  8. The Dynamics in Epithelial Cell Intercalation in Drosophila Morphogenesis

    NASA Astrophysics Data System (ADS)

    Wolf, Fred; Reichl, Lars; Kong, Deqing; Zhang, Yujun; Eule, Stephan; Metzger, Jakob; Großhans, Jörg

    2015-03-01

    Epithelial cell rearrangement is important for many processes in morphogenesis. During germband extension in early gastrulation of Drosophila embryos, exchange of neighbors is achieved by junction remodeling that follows a topological T1 process. Its first step is the constriction of dorsal-ventral junctions and fusion of two 3x vertices into a 4x vertex a process believed to be junction autonomous. We established a high throughput imaging pipeline, by which we recorded, segmented and analysed more than 1000 neighbor exchanges in drosophila embryos. Characterizing the dynamics of junction lengths we find that the constriction of cell contacts follows intriguingly simple quantitative laws. (1) The mean contact length decreases approximately as a square root of time to collapse. (2) The time dependent variance of contact lengths is proportional to the square of the mean. (3) The time dependent probability density of the contact lengths remains close to Gaussian during the entire process. These observations are sufficient to derive a stochastic differential equation for contact length that captures the non-equilibrium statistical mechanics of contact collapse. Supported by the German Research Foundation.

  9. Mechanical Actuation Systems for the Phenotype Commitment of Stem Cell-Based Tendon and Ligament Tissue Substitutes.

    PubMed

    Govoni, Marco; Muscari, Claudio; Lovecchio, Joseph; Guarnieri, Carlo; Giordano, Emanuele

    2016-04-01

    High tensile forces transmitted by tendons and ligaments make them susceptible to tearing or complete rupture. The present standard reparative technique is the surgical implantation of auto- or allografts, which often undergo failure.Currently, different cell types and biomaterials are used to design tissue engineered substitutes. Mechanical stimulation driven by dedicated devices can precondition these constructs to a remarkable degree, mimicking the local in vivo environment. A large number of dynamic culture instruments have been developed and many appealing results collected. Of the cells that have been used, tendon stem cells are the most promising for a reliable stretch-induced tenogenesis, but their reduced availability represents a serious limitation to upscaled production. Biomaterials used for scaffold fabrication include both biological molecules and synthetic polymers, the latter being improved by nanotechnologies which reproduce the architecture of native tendons. In addition to cell type and scaffold material, other variables which must be defined in mechanostimulation protocols are the amplitude, frequency, duration and direction of the applied strain. The ideal conditions seem to be those producing intermittent tension rather than continuous loading. In any case, all physical parameters must be adapted to the specific response of the cells used and the tensile properties of the scaffold. Tendon/ligament grafts in animals usually have the advantage of mechanical preconditioning, especially when uniaxial cyclic forces are applied to cells engineered into natural or decellularized scaffolds. However, due to the scarcity of in vivo research, standard protocols still need to be defined for clinical applications.

  10. Human Adipose Stem Cells Differentiated on Braided Polylactide Scaffolds Is a Potential Approach for Tendon Tissue Engineering.

    PubMed

    Vuornos, Kaisa; Björninen, Miina; Talvitie, Elina; Paakinaho, Kaarlo; Kellomäki, Minna; Huhtala, Heini; Miettinen, Susanna; Seppänen-Kaijansinkko, Riitta; Haimi, Suvi

    2016-03-01

    Growing number of musculoskeletal defects increases the demand for engineered tendon. Our aim was to find an efficient strategy to produce tendon-like matrix in vitro. To allow efficient differentiation of human adipose stem cells (hASCs) toward tendon tissue, we tested different medium compositions, biomaterials, and scaffold structures in preliminary tests. This is the first study to report that medium supplementation with 50 ng/mL of growth and differentiation factor-5 (GDF-5) and 280 μM l-ascorbic acid are essential for tenogenic differentiation of hASCs. Tenogenic medium (TM) was shown to significantly enhance tendon-like matrix production of hASCs compared to other tested media groups. Cell adhesion, proliferation, and tenogenic differentiation of hASCs were supported on braided poly(l/d)lactide (PLA) 96l/4d copolymer filament scaffolds in TM condition compared to foamed poly(l-lactide-co-ɛ-caprolactone) (PLCL) 70L/30CL scaffolds. A uniform cell layer formed on braided PLA 96/4 scaffolds when hASCs were cultured in TM compared to maintenance medium (MM) condition after 14 days of culture. Furthermore, total collagen content and gene expression of tenogenic marker genes were significantly higher in TM condition after 2 weeks of culture. The elastic modulus of PLA 96/4 scaffold was more similar to the elastic modulus reported for native Achilles tendon. Our study showed that the optimized TM is needed for efficient and rapid in vitro tenogenic extracellular matrix production of hASCs. PLA 96/4 scaffolds together with TM significantly stimulated hASCs, thus demonstrating the potential clinical relevance of this novel and emerging approach to tendon injury treatments in the future.

  11. A possible link between loading, inflammation and healing: Immune cell populations during tendon healing in the rat

    PubMed Central

    Blomgran, Parmis; Blomgran, Robert; Ernerudh, Jan; Aspenberg, Per

    2016-01-01

    Loading influences tendon healing, and so does inflammation. We hypothesized that the two are connected. 48 rats underwent Achilles tendon transection. Half of the rats received Botox injections into calf muscles to reduce mechanical loading. Cells from the regenerating tissue were analyzed by flow cytometry. In the loaded group, the regenerating tissue contained 83% leukocytes (CD45+) day 1, and 23% day 10. The M1/M2 macrophage ratio (CCR7/CD206) peaked at day 3, while T helper (CD3+CD4+) and Treg cells (CD25+ Foxp3+) increased over time. With Botox, markers associated with down-regulation of inflammation were more common day 5 (CD163, CD206, CD25, Foxp3), and M1 or M2 macrophages and Treg cells were virtually absent day 10, while still present with full loading. The primary variable, CCR7/CD206 ratio day 5, was higher with full loading (p = 0.001) and the Treg cell fraction was lower (p < 0.001). Free cage activity loading is known to increase size and strength of the tendon in this model compared to Botox. Loading now appeared to delay the switch to an M2 type of inflammation with more Treg cells. It seems a prolonged M1 phase due to loading might make the tendon regenerate bigger. PMID:27405922

  12. Prosomes and heat shock complexes in Drosophila melanogaster cells.

    PubMed Central

    de Sa, C M; Rollet, E; de Sa, M F; Tanguay, R M; Best-Belpomme, M; Scherrer, K

    1989-01-01

    Prosomes and heat shock protein (HSP) complexes isolated from the cytoplasm of Drosophila cells in culture were biochemically and immunologically characterized. The two complexes were found to separate on sucrose gradients, allowing the analysis of their protein constituents by two-dimensional polyacrylamide gel electrophoresis and by reaction with anti-HSP sera and prosome-specific monoclonal antibodies. All of the prosomal proteins were found to be clearly distinct from the HSP; none of the prosomal proteins was synthesized de novo in heat shock. However, an antiprosome (anti-p27K) monoclonal antibody (mouse anti-duck) recognizing the Drosophila p29K prosomal protein allowed immunoprecipitation from a heat-shocked postmitochondrial supernatant of the crude HSP complex, including the low- and the high-molecular-weight components, in particular the 70 x 10(3)-molecular weight HSP. The highly purified small 16S HSP complex still contained this preexistent p29K prosomal protein, which thus also seems to be a metabolically stable constituent of the HSP complex. The significance of this structural and possibly functional relationship between prosomes and HSP, involving the highly ubiquitous and evolutionarily conserved prosomal protein p27/29K, remains to be elucidated. Images PMID:2503709

  13. Strategies to engineer tendon/ligament-to-bone interface: Biomaterials, cells and growth factors.

    PubMed

    Font Tellado, Sonia; Balmayor, Elizabeth R; Van Griensven, Martijn

    2015-11-01

    Integration between tendon/ligament and bone occurs through a specialized tissue interface called enthesis. The complex and heterogeneous structure of the enthesis is essential to ensure smooth mechanical stress transfer between bone and soft tissues. Following injury, the interface is not regenerated, resulting in high rupture recurrence rates. Tissue engineering is a promising strategy for the regeneration of a functional enthesis. However, the complex structural and cellular composition of the native interface makes enthesis tissue engineering particularly challenging. Thus, it is likely that a combination of biomaterials and cells stimulated with appropriate biochemical and mechanical cues will be needed. The objective of this review is to describe the current state-of-the-art, challenges and future directions in the field of enthesis tissue engineering focusing on four key parameters: (1) scaffold and biomaterials, (2) cells, (3) growth factors and (4) mechanical stimuli.

  14. Volumetric Measurements of Amnioserosa Cells in Developing Drosophila

    NASA Astrophysics Data System (ADS)

    Mashburn, David; Jayasinghe, Aroshan; Hutson, Shane

    2013-03-01

    The behavior of cells in tissue in developing Drosophila melanogaster has become increasingly clearer over the past few decades, in large part due to advances in imaging techniques, genetic markers, predictive modeling, and micromanipulation (notably laser microsurgery). We now know apical contractions in amnioserosa cells are a significant factor in large scale processes like germ band retraction and dorsal closure. Also, laser microsurgery induces cellular recoil that strongly mimics a 2D elastic sheet. Still, what we know about these processes comes entirely from the apical surface where the standard fluorescent markers like cadherin are located, but many open questions exist concerning the remaining ``dark'' portion of cells. Does cell volume remain constant during contraction or do cells leak? Also, what shape changes do cells undergo? Do they bulge, wedge, contract prismatically, or something else? By using a marker that labels the entire membrane of amnioserosa cells (Resille, 117) and adapting our watershed segmentation routines for 4D datasets, we have been able to quantify the entire volumetric region of cells in tissue through time and compare changes in apical area and volume. Preliminary results suggest a fairly constant volume over the course of a contraction cycle.

  15. Planar cell polarity: the Dachsous/Fat system contributes differently to the embryonic and larval stages of Drosophila

    PubMed Central

    Saavedra, Pedro; Brittle, Amy; Palacios, Isabel M.; Strutt, David; Casal, José; Lawrence, Peter A.

    2016-01-01

    ABSTRACT The epidermal patterns of all three larval instars (L1–L3) of Drosophila are made by one unchanging set of cells. The seven rows of cuticular denticles of all larval stages are consistently planar polarised, some pointing forwards, others backwards. In L1 all the predenticles originate at the back of the cells but, in L2 and L3, they form at the front or the back of the cell depending on the polarity of the forthcoming denticles. We find that, to polarise all rows, the Dachsous/Fat system is differentially utilised; in L1 it is active in the placement of the actin-based predenticles but is not crucial for the final orientation of the cuticular denticles, in L2 and L3 it is needed for placement and polarity. We find Four-jointed to be strongly expressed in the tendon cells and show how this might explain the orientation of all seven rows. Unexpectedly, we find that L3 that lack Dachsous differ from larvae lacking Fat and we present evidence that this is due to differently mislocalised Dachs. We make some progress in understanding how Dachs contributes to phenotypes of wildtype and mutant larvae and adults. PMID:26935392

  16. A simplified model of ephitelial cell hair orientation in Drosophila

    NASA Astrophysics Data System (ADS)

    Garcia-Vergara, Mauricio; Gomez-Correa, Gilberto; Ramirez-Santiago, Guillermo

    2012-02-01

    Epithelia cells are polarized along an axis perpendicular to the apical-basal axis, --``Planar cell polarization'' (PCP)--. In Drosophila adult cuticle cells are hexagonally packed and the PCP gives rise to the elaboration of an actin-rich hair that develops from one of the hexagon vertex and pointing distally. Genetic analyses have identified a group of proteins whose activities are required to polarize each cell and produce the phenomenon of PCP. To describe the PCP in the epithelia some quantitative models intended to explain this phenomenon by invoking diffusion of several proteins and all their interactions. Here we propose a simpler model consisting of two reaction-diffusion equations that describe the redistribution process of two chemical agents inside a cell. This redistribution occurs as a response to an external gradient of a quimio-attractor. We emulate the collective cell polarization by introducing ``interactions'' between neighboring cells that propagate trough the epithelia. This collective polarization gives rise to an orientational pattern in the actin-rich hairs.

  17. Smad8/BMP2–Engineered Mesenchymal Stem Cells Induce Accelerated Recovery of the Biomechanical Properties of the Achilles Tendon

    PubMed Central

    Pelled, Gadi; Snedeker, Jess G.; Ben-Arav, Ayelet; Rigozzi, Samuela; Zilberman, Yoram; Kimelman-Bleich, Nadav; Gazit, Zulma; Müller, Ralph; Gazit, Dan

    2012-01-01

    Summary Tendon tissue regeneration is an important goal for orthopedic medicine. We hypothesized that implantation of Smad8/BMP2–engineered MSCs in a full-thickness defect of the Achilles tendon (AT) would induce regeneration of tissue with improved biomechanical properties. A 2 mm defect was created in the distal region of murine ATs. The injured tendons were then sutured together or given implants of genetically engineered MSCs (GE group), nonengineered MSCs (CH3 group), or fibrin gel containing no cells (FG group). Three weeks later the mice were killed, and their healing tendons were excised and processed for histological or biomechanical analysis. A biomechanical analysis showed that tendons that received implants of genetically engineered MSCs had the highest effective stiffness (> 70% greater than natural healing, p < 0.001) and elastic modulus. There were no significant differences in either ultimate load or maximum stress among the treatment groups. Histological analysis revealed a tendon-like structure with elongated cells mainly in the GE group. ATs that had been implanted with Smad8/BMP2–engineered stem cells displayed a better material distribution and functional recovery than control groups. While additional study is required to determine long-term effects of GE MSCs on tendon healing, we conclude that genetically engineered MSCs may be a promising therapeutic tool for accelerating short-term functional recovery in the treatment of tendon injuries. PMID:22696396

  18. Slit/Robo signaling regulates cell fate decisions in the intestinal stem cell lineage of Drosophila

    PubMed Central

    Biteau, Benoît; Jasper, Heinrich

    2014-01-01

    To maintain tissue homeostasis, cell fate decisions within stem cell lineages have to respond to the needs of the tissue. This coordination of lineage choices with regenerative demand remains poorly characterized. Here we identify a signal from enteroendocrine cells (EEs) that controls lineage specification in the Drosophila intestine. We find that EEs secrete Slit, a ligand for the Robo2 receptor in stem cells (ISCs) that limits ISC commitment to the endocrine lineage, establishing negative feedback control of EE regeneration. We further show that this lineage decision is made within ISCs and requires induction of the transcription factor Prospero in ISCs. Our work identifies a new function for the conserved Slit/Robo pathway in the regulation of adult stem cells, establishing negative feedback control of ISC lineage specification as a critical strategy to preserve tissue homeostasis. Our results further amend the current understanding of cell fate commitment within the Drosophila ISC lineage. PMID:24931602

  19. Slit/Robo signaling regulates cell fate decisions in the intestinal stem cell lineage of Drosophila.

    PubMed

    Biteau, Benoît; Jasper, Heinrich

    2014-06-26

    In order to maintain tissue homeostasis, cell fate decisions within stem cell lineages have to respond to the needs of the tissue. This coordination of lineage choices with regenerative demand remains poorly characterized. Here, we identify a signal from enteroendocrine cells (EEs) that controls lineage specification in the Drosophila intestine. We find that EEs secrete Slit, a ligand for the Robo2 receptor in intestinal stem cells (ISCs) that limits ISC commitment to the endocrine lineage, establishing negative feedback control of EE regeneration. Furthermore, we show that this lineage decision is made within ISCs and requires induction of the transcription factor Prospero in ISCs. Our work identifies a function for the conserved Slit/Robo pathway in the regulation of adult stem cells, establishing negative feedback control of ISC lineage specification as a critical strategy to preserve tissue homeostasis. Our results further amend the current understanding of cell fate commitment within the Drosophila ISC lineage. PMID:24931602

  20. Slit/Robo signaling regulates cell fate decisions in the intestinal stem cell lineage of Drosophila.

    PubMed

    Biteau, Benoît; Jasper, Heinrich

    2014-06-26

    In order to maintain tissue homeostasis, cell fate decisions within stem cell lineages have to respond to the needs of the tissue. This coordination of lineage choices with regenerative demand remains poorly characterized. Here, we identify a signal from enteroendocrine cells (EEs) that controls lineage specification in the Drosophila intestine. We find that EEs secrete Slit, a ligand for the Robo2 receptor in intestinal stem cells (ISCs) that limits ISC commitment to the endocrine lineage, establishing negative feedback control of EE regeneration. Furthermore, we show that this lineage decision is made within ISCs and requires induction of the transcription factor Prospero in ISCs. Our work identifies a function for the conserved Slit/Robo pathway in the regulation of adult stem cells, establishing negative feedback control of ISC lineage specification as a critical strategy to preserve tissue homeostasis. Our results further amend the current understanding of cell fate commitment within the Drosophila ISC lineage.

  1. Regulation of apoptosis of rbf mutant cells during Drosophila development

    PubMed Central

    Tanaka-Matakatsu, Miho; Xu, Jinhua; Cheng, Leping; Du, Wei

    2008-01-01

    Inactivation of the retinoblastoma gene Rb leads to defects in cell proliferation, differentiation, or apoptosis, depending on specific cell or tissue types. To gain insights into the genes that can modulate the consequences of Rb inactivation, we carried out a genetic screen in Drosophila to identify mutations that affected apoptosis induced by inactivation of the Retinoblastoma-family protein (rbf) and identified a mutation that blocked apoptosis induced by rbf. We found this mutation to be a new allele of head involution defective (hid) and showed that hid expression is deregulated in rbf mutant cells in larval imaginal discs. We identified an enhancer that regulates hid expression in response to developmental cues as well as to radiation and demonstrated that this hid enhancer is directly repressed by RBF through an E2F binding site. These observations indicate that apoptosis of rbf mutant cells is mediated by an upregulation of hid. Finally, we showed that bantam, a miRNA that regulates hid translation, is expressed in the interommatidial cells in the larval eye discs and modulates the survival of rbf mutant cells. PMID:19100727

  2. Cell-selective labelling of proteomes in Drosophila melanogaster

    PubMed Central

    Erdmann, Ines; Marter, Kathrin; Kobler, Oliver; Niehues, Sven; Abele, Julia; Müller, Anke; Bussmann, Julia; Storkebaum, Erik; Ziv, Tamar; Thomas, Ulrich; Dieterich, Daniela C.

    2015-01-01

    The specification and adaptability of cells rely on changes in protein composition. Nonetheless, uncovering proteome dynamics with cell-type-specific resolution remains challenging. Here we introduce a strategy for cell-specific analysis of newly synthesized proteomes by combining targeted expression of a mutated methionyl-tRNA synthetase (MetRS) with bioorthogonal or fluorescent non-canonical amino-acid-tagging techniques (BONCAT or FUNCAT). Substituting leucine by glycine within the MetRS-binding pocket (MetRSLtoG) enables incorporation of the non-canonical amino acid azidonorleucine (ANL) instead of methionine during translation. Newly synthesized proteins can thus be labelled by coupling the azide group of ANL to alkyne-bearing tags through ‘click chemistry'. To test these methods for applicability in vivo, we expressed MetRSLtoG cell specifically in Drosophila. FUNCAT and BONCAT reveal ANL incorporation into proteins selectively in cells expressing the mutated enzyme. Cell-type-specific FUNCAT and BONCAT, thus, constitute eligible techniques to study protein synthesis-dependent processes in complex and behaving organisms. PMID:26138272

  3. Dicer assay in Drosophila S2 cell extract.

    PubMed

    Yang, Baojun; Li, Hongwei

    2011-01-01

    Double-stranded RNA (dsRNA) is the trigger of RNA interference (RNAi)-mediated gene regulation. Dicer processes dsRNAs into short interfering RNAs (siRNAs), which are incorporated into the effector RNA-induced silencing complex (RISC) and direct degradation of homologous target mRNAs. In plants and invertebrates, the RNAi machinery also acts as an antiviral mechanism through production of viral siRNAs by Dicer and silencing of replicating viruses. Viral suppressors of RNAi (VSRs) are encoded by some viruses and serve as a strategy to counteract the RNAi-based antiviral immunity. In this chapter, we describe a Dicer activity assay in extracts prepared from Drosophila melanogaster S2 cells. We also introduce a simple procedure to study VSR activity in the in vitro Dicer assay. PMID:21431688

  4. How Obesity Affects Tendons?

    PubMed

    Abate, Michele; Salini, Vincenzo; Andia, Isabel

    2016-01-01

    Several epidemiological and clinical observations have definitely demonstrated that obesity has harmful effects on tendons. The pathogenesis of tendon damage is multi-factorial. In addition to overload, attributable to the increased body weight, which significantly affects load-bearing tendons, systemic factors play a relevant role. Several bioactive peptides (chemerin, leptin, adiponectin and others) are released by adipocytes, and influence tendon structure by means of negative activities on mesenchymal cells. The ensuing systemic state of chronic, sub-clinic, low-grade inflammation can damage tendon structure. Metabolic disorders (diabetes, impaired glucose tolerance, and dislipidemia), frequently associated with visceral adiposity, are concurrent pathogenetic factors. Indeed, high glucose levels increase the formation of Advanced Glycation End-products, which in turn form stable covalent cross-links within collagen fibers, modifying their structure and functionality.Sport activities, so useful for preventing important cardiovascular complications, may be detrimental for tendons if they are submitted to intense acute or chronic overload. Therefore, two caution rules are mandatory: first, to engage in personalized soft training program, and secondly to follow regular check-up for tendon pathology.

  5. How Obesity Affects Tendons?

    PubMed

    Abate, Michele; Salini, Vincenzo; Andia, Isabel

    2016-01-01

    Several epidemiological and clinical observations have definitely demonstrated that obesity has harmful effects on tendons. The pathogenesis of tendon damage is multi-factorial. In addition to overload, attributable to the increased body weight, which significantly affects load-bearing tendons, systemic factors play a relevant role. Several bioactive peptides (chemerin, leptin, adiponectin and others) are released by adipocytes, and influence tendon structure by means of negative activities on mesenchymal cells. The ensuing systemic state of chronic, sub-clinic, low-grade inflammation can damage tendon structure. Metabolic disorders (diabetes, impaired glucose tolerance, and dislipidemia), frequently associated with visceral adiposity, are concurrent pathogenetic factors. Indeed, high glucose levels increase the formation of Advanced Glycation End-products, which in turn form stable covalent cross-links within collagen fibers, modifying their structure and functionality.Sport activities, so useful for preventing important cardiovascular complications, may be detrimental for tendons if they are submitted to intense acute or chronic overload. Therefore, two caution rules are mandatory: first, to engage in personalized soft training program, and secondly to follow regular check-up for tendon pathology. PMID:27535258

  6. Where do injectable stem cell treatments apply in treatment of muscle, tendon, and ligament injuries?

    PubMed

    Mautner, Kenneth; Blazuk, Joseph

    2015-04-01

    Treatment options for muscle, tendon, and ligament injuries span a constantly evolving spectrum. For years, treatments focused on symptomatic relief. Closer scrutiny of symptomatic treatment suggests that the provision of transient relief of symptoms may have caused more harm than good. Cortisone injections provide a trade-off of short-term relief for poorer long-term outcomes. When conventional treatment failed, patients have faced limited options including surgery, which has increased risk and limited efficacy. Regenerative injections offer a more robust option for soft tissue disease. Basic science and clinical studies show conflicting results to support the use of platelet-rich plasma injections for soft tissue disorders, and even fewer trials have focused on injectable stem cells with limited findings. Additional studies are needed to determine the potential benefits of this regenerative therapy. PMID:25864658

  7. Synovial Mesenchymal Stem Cells Promote Meniscus Regeneration Augmented by an Autologous Achilles Tendon Graft in a Rat Partial Meniscus Defect Model

    PubMed Central

    Ozeki, Nobutake; Muneta, Takeshi; Matsuta, Seiya; Koga, Hideyuki; Nakagawa, Yusuke; Mizuno, Mitsuru; Tsuji, Kunikazu; Mabuchi, Yo; Akazawa, Chihiro; Kobayashi, Eiji; Saito, Tomoyuki; Sekiya, Ichiro

    2015-01-01

    Although meniscus defects and degeneration are strongly correlated with the later development of osteoarthritis, the promise of regenerative medicine strategies is to prevent and/or delay the disease's progression. Meniscal reconstruction has been shown in animal models with tendon grafting and transplantation of mesenchymal stem cells (MSCs); however, these procedures have not shown the same efficacy in clinical studies. Here, our aim was to investigate the ability of tendon grafts pretreated with exogenous synovial-derived MSCs to prevent cartilage degeneration in a rat partial meniscus defect model. We removed the anterior half of the medial meniscus and grafted autologous Achilles tendons with or without a 10-minute pretreatment of the tendon with synovial MSCs. The meniscus and surrounding cartilage were evaluated at 2, 4, and 8 weeks (n = 5). Tendon grafts increased meniscus size irrespective of synovial MSCs. Histological scores for regenerated menisci were better in the tendon + MSC group than in the other two groups at 4 and 8 weeks. Both macroscopic and histological scores for articular cartilage were significantly better in the tendon + MSC group at 8 weeks. Implanted synovial MSCs survived around the grafted tendon and native meniscus integration site by cell tracking assays with luciferase+, LacZ+, DiI+, and/or GFP+ synovial MSCs and/or GFP+ tendons. Flow cytometric analysis showed that transplanted synovial MSCs retained their MSC properties at 7 days and host synovial tissue also contained cells with MSC characteristics. Synovial MSCs promoted meniscus regeneration augmented by autologous Achilles tendon grafts and prevented cartilage degeneration in rats. Stem Cells 2015;33:1927–1938 PMID:25993981

  8. Induction temperature of human heat shock factor is reprogrammed in a Drosophila cell environment

    NASA Astrophysics Data System (ADS)

    Clos, Joachim; Rabindran, Sridhar; Wisniewski, Jan; Wu, Carl

    1993-07-01

    HEAT shock factor (HSF)1,2, the transcriptional activator of eukaryotic heat shock genes, is induced to bind DNA by a monomer to trimer transition involving leucine zipper interactions3,4. Although this mode of regulation is shared among many eukaryotic species, there is variation in the temperature at which HSF binding activity is induced. We investigated the basis of this variation by analysing the response of a human HSF expressed in Drosophila cells and Drosophila HSF expressed in human cells. We report here that the temperature that induces DNA binding and trimerization of human HSF in Drosophila was decreased by ~10 °C to the induction temperature for the host cell, whereas Drosophila HSF expressed in human cells was constitutively active. The results indicate that the activity of HSF in vivo is not a simple function of the absolute environmental temperature.

  9. Glial cell development and function in the Drosophila visual system

    PubMed Central

    CHOTARD, CAROLE; SALECKER, IRIS

    2008-01-01

    In the developing nervous system, building a functional neuronal network relies on coordinating the formation, specification and survival to diverse neuronal and glial cell subtypes. The establishment of neuronal connections further depends on sequential neuron–neuron and neuron–glia interactions that regulate cell-migration patterns and axon guidance. The visual system of Drosophila has a highly regular, retinotopic organization into reiterated interconnected synaptic circuits. It is therefore an excellent invertebrate model to investigate basic cellular strategies and molecular determinants regulating the different developmental processes that lead to network formation. Studies in the visual system have provided important insights into the mechanisms by which photoreceptor axons connect with their synaptic partners within the optic lobe. In this review, we highlight that this system is also well suited for uncovering general principles that underlie glial cell biology. We describe the glial cell subtypes in the visual system and discuss recent findings about their development and migration. Finally, we outline the pivotal roles of glial cells in mediating neural circuit assembly, boundary formation, neural proliferation and survival, as well as synaptic function. PMID:18333286

  10. Tip cell-derived RTK signaling initiates cell movements in the Drosophila stomatogastric nervous system anlage.

    PubMed

    González-Gaitán, M; Jäckle, H

    2000-10-01

    The stomatogastric nervous system (SNS) of Drosophila is a simply organized neural circuitry that innervates the anterior enteric system. Unlike the central and the peripheral nervous systems, the SNS derives from a compact epithelial anlage in which three invagination centers, each giving rise to an invagination fold headed by a tip cell, are generated. Tip cell selection involves lateral inhibition, a process in which Wingless (Wg) activity adjusts the range of Notch signaling. Here we show that RTK signaling mediated by the Drosophila homolog of the epidermal growth factor receptor, DER, plays a key role in two consecutive steps during early SNS development. Like Wg, DER signaling participates in adjusting the range of Notch-dependent lateral inhibition during tip cell selection. Subsequently, tip cells secrete the DER ligand Spitz and trigger local RTK signaling, which initiates morphogenetic movements resulting in the tip cell-directed invaginations within the SNS anlage.

  11. Migration of Drosophila intestinal stem cells across organ boundaries

    PubMed Central

    Takashima, Shigeo; Paul, Manash; Aghajanian, Patrick; Younossi-Hartenstein, Amelia; Hartenstein, Volker

    2013-01-01

    All components of the Drosophila intestinal tract, including the endodermal midgut and ectodermal hindgut/Malpighian tubules, maintain populations of dividing stem cells. In the midgut and hindgut, these stem cells originate from within larger populations of intestinal progenitors that proliferate during the larval stage and form the adult intestine during metamorphosis. The origin of stem cells found in the excretory Malpighian tubules (‘renal stem cells’) has not been established. In this paper, we investigate the migration patterns of intestinal progenitors that take place during metamorphosis. Our data demonstrate that a subset of adult midgut progenitors (AMPs) move posteriorly to form the adult ureters and, consecutively, the renal stem cells. Inhibiting cell migration by AMP-directed expression of a dominant-negative form of Rac1 protein results in the absence of stem cells in the Malpighian tubules. As the majority of the hindgut progenitor cells migrate posteriorly and differentiate into hindgut enterocytes, a group of the progenitor cells, unexpectedly, invades anteriorly into the midgut territory. Consequently, these progenitor cells differentiate into midgut enterocytes. The midgut determinant GATAe is required for the differentiation of midgut enterocytes derived from hindgut progenitors. Wingless signaling acts to balance the proportion of hindgut progenitors that differentiate as midgut versus hindgut enterocytes. Our findings indicate that a stable boundary between midgut and hindgut/Malpighian tubules is not established during early embryonic development; instead, pluripotent progenitor populations cross in between these organs in both directions, and are able to adopt the fate of the organ in which they come to reside. PMID:23571215

  12. Intracellular collagen fibrils: evidence of an intracellular source from experiments with tendon fibroblasts and fibroblastic tumour cells.

    PubMed Central

    Michna, H

    1988-01-01

    This study was designed to substantiate one or both of the two hypotheses for the explanation of intracellular collagen fibrils in collagen-producing cells. The more obvious is the phagocytosis of extracellular collagen fibrils by the cell and the other is a form of autophagocytosis of newly synthesised collagenous products. Information was collected on fibroblasts from murine tendons after exercise and simultaneously stimulating collagen synthesis by treatment with an anabolic steroid hormone. Moreover, in vivo and in vitro fibroblastic tumour cells which demonstrate enhanced protein synthesis were also treated with the anabolic steroid. The findings of intracellular collagen fibrils in tendon fibroblasts and the sarcoma cells after experimentally stimulating collagen synthesis are discussed in the light of the hypothesis that the findings may represent steps of autophagocytosis of newly synthesised collagenous products in the absence of a control mechanism to remove collagenous products which cannot be secreted. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:3225213

  13. Niche Appropriation by Drosophila Intestinal Stem Cell Tumors

    PubMed Central

    Patel, Parthive H.; Dutta, Devanjali; Edgar, Bruce A.

    2015-01-01

    Mutations that inhibit differentiation in stem cell lineages are a common early step in cancer development, but precisely how a loss of differentiation initiates tumorigenesis is unclear. We investigated Drosophila intestinal stem cell (ISC) tumors generated by suppressing Notch (N) signaling, which blocks differentiation. Notch-defective ISCs require stress-induced divisions for tumor initiation and an autocrine EGFR ligand, Spitz, during early tumor growth. Upon achieving a critical mass these tumors displace surrounding enterocytes, competing with them for basement membrane space and causing their detachment, extrusion and apoptosis. This loss of epithelial integrity induces JNK and Yki/YAP activity in enterocytes and, consequently, their expression of stress-dependent cytokines (Upd2, Upd3). These paracrine signals, normally used within the stem cell niche to trigger regeneration, propel tumor growth without the need for secondary mutations in growth signaling pathways. The appropriation of niche signaling by differentiation-defective stem cells may be a common mechanism of early tumorigenesis. PMID:26237646

  14. Tools for Targeted Genome Engineering of Established Drosophila Cell Lines

    PubMed Central

    Cherbas, Lucy; Hackney, Jennifer; Gong, Lei; Salzer, Claire; Mauser, Eric; Zhang, Dayu; Cherbas, Peter

    2015-01-01

    We describe an adaptation of φC31 integrase–mediated targeted cassette exchange for use in Drosophila cell lines. Single copies of an attP-bounded docking platform carrying a GFP-expression marker, with or without insulator elements flanking the attP sites, were inserted by P-element transformation into the Kc167 and Sg4 cell lines; each of the resulting docking-site lines carries a single mapped copy of one of the docking platforms. Vectors for targeted substitution contain a cloning cassette flanked by attB sites. Targeted substitution occurs by integrase-mediated substitution between the attP sites (integrated) and the attB sites (vector). We describe procedures for isolating cells carrying the substitutions and for eliminating the products of secondary off-target events. We demonstrate the technology by integrating a cassette containing a Cu2+-inducible mCherry marker, and we report the expression properties of those lines. When compared with clonal lines made by traditional transformation methods, which lead to the illegitimate insertion of tandem arrays, targeted insertion lines give more uniform expression, lower basal expression, and higher induction ratios. Targeted substitution, though intricate, affords results that should greatly improve comparative expression assays—a major emphasis of cell-based studies. PMID:26450921

  15. Comparative Study on Functional Effects of Allotransplantation of Bone Marrow Stromal Cells and Adipose Derived Stromal Vascular Fraction on Tendon Repair: A Biomechanical Study in Rabbits

    PubMed Central

    Behfar, Mehdi; Javanmardi, Sara; Sarrafzadeh-Rezaei, Farshid

    2014-01-01

    Objective Tendon never returns to its complete biological and mechanical properties after repair. Bone marrow and, recently, adipose tissue have been used as sources of mesenchymal stem cells which have been proven to enhance tendon healing. In the present study, we compared the effects of allotransplantation of bone marrow derived mesenchymal stromal cells (BMSCs) and adipose derived stromal vascular fraction (SVF) on tendon mechanical properties after experimentally induced flexor tendon transection. Materials and Methods In this experimental study, we used 48 adult male New Zealand white rabbits. Twelve of rabbits were used as donors of bone marrow and adipose tissue, the rest were divided into control and treatment groups. The injury model was a unilateral complete transection of the deep digital flexor tendon. Immediately after suture repair, 4×106cells of either fresh SVF from enzymatic digestion of adipose tissue or cultured BMSCs were intratendinously injected into tendon stumps in the treatment groups. Controls received phosphate-buffered saline (PBS). Immobilization with a cast was continued for two weeks after surgery. Animals were sacrificed three and eight weeks after surgery and tendons underwent mechanical evaluations. The differences among the groups were analyzed using the analysis of variance (ANOVA) test followed by Tukey’s multiple comparisons test. Results Stromal cell transplantation resulted in a significant increase in ultimate and yield loads, energy absorption, and stress of repairs compared to the controls. However, there were no statistically significant changes detected in terms of stiffness. In comparison, we observed no significant differences at the third week between SVF and BMSCs treated tendons in terms of all load related properties. However, at the eighth week SVF transplantation resulted in significantly increased energy absorption, stress and stiffness compared to BMSCs. Conclusion The enhanced biomechanical properties of

  16. Haemocytes control stem cell activity in the Drosophila intestine.

    PubMed

    Ayyaz, Arshad; Li, Hongjie; Jasper, Heinrich

    2015-06-01

    Coordination of stem cell activity with inflammatory responses is critical for regeneration and homeostasis of barrier epithelia. The temporal sequence of cell interactions during injury-induced regeneration is only beginning to be understood. Here we show that intestinal stem cells (ISCs) are regulated by macrophage-like haemocytes during the early phase of regenerative responses of the Drosophila intestinal epithelium. On tissue damage, haemocytes are recruited to the intestine and secrete the BMP homologue DPP, inducing ISC proliferation by activating the type I receptor Saxophone and the Smad homologue SMOX. Activated ISCs then switch their response to DPP by inducing expression of Thickveins, a second type I receptor that has previously been shown to re-establish ISC quiescence by activating MAD. The interaction between haemocytes and ISCs promotes infection resistance, but also contributes to the development of intestinal dysplasia in ageing flies. We propose that similar interactions influence pathologies such as inflammatory bowel disease and colorectal cancer in humans. PMID:26005834

  17. Diverse Hormone Response Networks in 41 Independent Drosophila Cell Lines

    DOE PAGESBeta

    Stoiber, Marcus; Celniker, Susan; Cherbas, Lucy; Brown, Ben; Cherbas, Peter

    2016-01-15

    Steroid hormones induce cascades of gene activation and repression with transformative effects on cell fate . Steroid transduction plays a major role in the development and physiology of nearly all metazoan species, and in the progression of the most common forms of cancer. Despite the paramount importance of steroids in developmental and translational biology, a complete map of transcriptional response has not been developed for any hormone . In the case of 20-hydroxyecdysone (ecdysone) in Drosophila melanogaster, these trajectories range from apoptosis to immortalization. We mapped the ecdysone transduction network in a cohort of 41 cell lines, the largest suchmore » atlas yet assembled. We found that the early transcriptional response mirrors the distinctiveness of physiological origins: genes respond in restricted patterns, conditional on the expression levels of dozens of transcription factors. Only a small cohort of genes is constitutively modulated independent of initial cell state. Ecdysone-responsive genes tend to organize into directional same-stranded units, with consecutive genes induced from the same strand. Here, we identify half of the ecdysone receptor heterodimer as the primary rate-limiting step in the response, and find that initial receptor isoform levels modulate the activated cohort of target transcription factors. In conclusion, this atlas of steroid response reveals organizing principles of gene regulation by a model type II nuclear receptor and lays the foundation for comprehensive and predictive understanding of the ecdysone transduction network in the fruit fly.« less

  18. Haemocytes control stem cell activity in the Drosophila intestine.

    PubMed

    Ayyaz, Arshad; Li, Hongjie; Jasper, Heinrich

    2015-06-01

    Coordination of stem cell activity with inflammatory responses is critical for regeneration and homeostasis of barrier epithelia. The temporal sequence of cell interactions during injury-induced regeneration is only beginning to be understood. Here we show that intestinal stem cells (ISCs) are regulated by macrophage-like haemocytes during the early phase of regenerative responses of the Drosophila intestinal epithelium. On tissue damage, haemocytes are recruited to the intestine and secrete the BMP homologue DPP, inducing ISC proliferation by activating the type I receptor Saxophone and the Smad homologue SMOX. Activated ISCs then switch their response to DPP by inducing expression of Thickveins, a second type I receptor that has previously been shown to re-establish ISC quiescence by activating MAD. The interaction between haemocytes and ISCs promotes infection resistance, but also contributes to the development of intestinal dysplasia in ageing flies. We propose that similar interactions influence pathologies such as inflammatory bowel disease and colorectal cancer in humans.

  19. Molecular Evolution of Drosophila Germline Stem Cell and Neural Stem Cell Regulating Genes

    PubMed Central

    Choi, Jae Young; Aquadro, Charles F.

    2015-01-01

    Here, we study the molecular evolution of a near complete set of genes that had functional evidence in the regulation of the Drosophila germline and neural stem cell. Some of these genes have previously been shown to be rapidly evolving by positive selection raising the possibility that stem cell genes as a group have elevated signatures of positive selection. Using recent Drosophila comparative genome sequences and population genomic sequences of Drosophila melanogaster, we have investigated both long- and short-term evolution occurring across these two different stem cell systems, and compared them with a carefully chosen random set of genes to represent the background rate of evolution. Our results showed an excess of genes with evidence of a recent selective sweep in both germline and neural stem cells in D. melanogaster. However compared with their control genes, both stem cell systems had no significant excess of genes with long-term recurrent positive selection in D. melanogaster, or across orthologous sequences from the melanogaster group. The evidence of long-term positive selection was limited to a subset of genes with specific functions in both the germline and neural stem cell system. PMID:26507797

  20. A Model System in S2 Cells to Test the Functional Activities of Drosophila Insulators.

    PubMed

    Tikhonov, M; Gasanov, N B; Georgiev, P; Maksimenko, O

    2015-01-01

    Insulators are a special class of regulatory elements that can regulate interactions between enhancers and promoters in the genome of high eukaryotes. To date, the mechanisms of insulator action remain unknown, which is primarily related to the lack of convenient model systems. We suggested studying a model system which is based on transient expression of a plasmid with an enhancer of the copia transposable element, in Drosophila embryonic cell lines. We demonstrated that during transient transfection of circle plasmids with a well-known Drosophila insulator from the gypsy retrotransposon, the insulator exhibits in an enhancer-blocking assay the same properties as in Drosophila stable transgenic lines. Therefore, the Drosophila cell line is suitable for studying the main activities of insulators, which provides additional opportunities for investigating the functional role of certain insulator proteins.

  1. Scaffolds in Tendon Tissue Engineering

    PubMed Central

    Longo, Umile Giuseppe; Lamberti, Alfredo; Petrillo, Stefano; Maffulli, Nicola; Denaro, Vincenzo

    2012-01-01

    Tissue engineering techniques using novel scaffold materials offer potential alternatives for managing tendon disorders. Tissue engineering strategies to improve tendon repair healing include the use of scaffolds, growth factors, cell seeding, or a combination of these approaches. Scaffolds have been the most common strategy investigated to date. Available scaffolds for tendon repair include both biological scaffolds, obtained from mammalian tissues, and synthetic scaffolds, manufactured from chemical compounds. Preliminary studies support the idea that scaffolds can provide an alternative for tendon augmentation with an enormous therapeutic potential. However, available data are lacking to allow definitive conclusion on the use of scaffolds for tendon augmentation. We review the current basic science and clinical understanding in the field of scaffolds and tissue engineering for tendon repair. PMID:22190961

  2. Mitochondrial fusion is regulated by Reaper to modulate Drosophila programmed cell death

    PubMed Central

    Thomenius, M; Freel, C D; Horn, S; Krieser, R; Abdelwahid, E; Cannon, R; Balasundaram, S; White, K; Kornbluth, S

    2011-01-01

    In most multicellular organisms, the decision to undergo programmed cell death in response to cellular damage or developmental cues is typically transmitted through mitochondria. It has been suggested that an exception is the apoptotic pathway of Drosophila melanogaster, in which the role of mitochondria remains unclear. Although IAP antagonists in Drosophila such as Reaper, Hid and Grim may induce cell death without mitochondrial membrane permeabilization, it is surprising that all three localize to mitochondria. Moreover, induction of Reaper and Hid appears to result in mitochondrial fragmentation during Drosophila cell death. Most importantly, disruption of mitochondrial fission can inhibit Reaper and Hid-induced cell death, suggesting that alterations in mitochondrial dynamics can modulate cell death in fly cells. We report here that Drosophila Reaper can induce mitochondrial fragmentation by binding to and inhibiting the pro-fusion protein MFN2 and its Drosophila counterpart dMFN/Marf. Our in vitro and in vivo analyses reveal that dMFN overexpression can inhibit cell death induced by Reaper or γ-irradiation. In addition, knockdown of dMFN causes a striking loss of adult wing tissue and significant apoptosis in the developing wing discs. Our findings are consistent with a growing body of work describing a role for mitochondrial fission and fusion machinery in the decision of cells to die. PMID:21475305

  3. Antagonistic functions of two stardust isoforms in Drosophila photoreceptor cells.

    PubMed

    Bulgakova, Natalia A; Rentsch, Michaela; Knust, Elisabeth

    2010-11-15

    Membrane-associated guanylate kinases (MAGUKs) are scaffolding proteins that organize supramolecular protein complexes, thereby partitioning the plasma membrane into spatially and functionally distinct subdomains. Their modular organization is ideally suited to organize protein complexes with cell type- or stage-specific composition, or both. Often more than one MAGUK isoform is expressed by one gene in the same cell, yet very little is known about their individual in vivo functions. Here, we show that two isoforms of Drosophila stardust, Sdt-H (formerly called Sdt-B2) and Sdt-D, which differ in their N terminus, are expressed in adult photoreceptors. Both isoforms associate with Crumbs and PATJ, constituents of the conserved Crumbs-Stardust complex. However, they form distinct complexes, localized at the stalk, a restricted region of the apical plasma membrane. Strikingly, Sdt-H and Sdt-D have antagonistic functions. While Sdt-H overexpression increases stalk membrane length and prevents light-dependent retinal degeneration, Sdt-D overexpression reduces stalk length and enhances light-dependent retinal degeneration. These results suggest that a fine-tuned balance of different Crumbs complexes regulates photoreceptor homeostasis.

  4. Antagonistic Functions of Two Stardust Isoforms in Drosophila Photoreceptor Cells

    PubMed Central

    Bulgakova, Natalia A.; Rentsch, Michaela

    2010-01-01

    Membrane-associated guanylate kinases (MAGUKs) are scaffolding proteins that organize supramolecular protein complexes, thereby partitioning the plasma membrane into spatially and functionally distinct subdomains. Their modular organization is ideally suited to organize protein complexes with cell type- or stage-specific composition, or both. Often more than one MAGUK isoform is expressed by one gene in the same cell, yet very little is known about their individual in vivo functions. Here, we show that two isoforms of Drosophila stardust, Sdt-H (formerly called Sdt-B2) and Sdt-D, which differ in their N terminus, are expressed in adult photoreceptors. Both isoforms associate with Crumbs and PATJ, constituents of the conserved Crumbs–Stardust complex. However, they form distinct complexes, localized at the stalk, a restricted region of the apical plasma membrane. Strikingly, Sdt-H and Sdt-D have antagonistic functions. While Sdt-H overexpression increases stalk membrane length and prevents light-dependent retinal degeneration, Sdt-D overexpression reduces stalk length and enhances light-dependent retinal degeneration. These results suggest that a fine-tuned balance of different Crumbs complexes regulates photoreceptor homeostasis. PMID:20861315

  5. Chromatin regulates origin activity in Drosophila follicle cells.

    PubMed

    Aggarwal, Bhagwan D; Calvi, Brian R

    2004-07-15

    It is widely believed that DNA replication in multicellular animals (metazoa) begins at specific origins to which a pre-replicative complex (pre-RC) binds. Nevertheless, a consensus sequence for origins has yet to be identified in metazoa. Origin identity can change during development, suggesting that there are epigenetic influences. A notable example of developmental specificity occurs in Drosophila, where somatic follicle cells of the ovary transition from genomic replication to exclusive re-replication at origins that control amplification of the eggshell (chorion) protein genes. Here we show that chromatin acetylation is critical for this developmental transition in origin specificity. We find that histones at the active origins are hyperacetylated, coincident with binding of the origin recognition complex (ORC). Mutation of the histone deacetylase (HDAC) Rpd3 induced genome-wide hyperacetylation, genomic replication and a redistribution of the origin-binding protein ORC2 in amplification-stage cells, independent of effects on transcription. Tethering Rpd3 or Polycomb proteins to the origin decreased its activity, whereas tethering the Chameau acetyltransferase increased origin activity. These results suggest that nucleosome acetylation and other epigenetic changes are important modulators of origin activity in metazoa. PMID:15254542

  6. Comparison of Potentials of Stem Cells Isolated from Tendon and Bone Marrow for Musculoskeletal Tissue Engineering

    PubMed Central

    Tan, Qi; Rui, Yun Feng; Wong, Yin Mei

    2012-01-01

    The use of tendon-derived stem cells (TDSCs) as a cell source for musculoskeletal tissue engineering has not been compared with that of bone marrow stromal cells (BMSC). This study compared the mesenchymal stem cell (MSC) and embryonic stem cells (ESC) markers, clonogenicity, proliferative capacity, and multilineage differentiation potential of rat TDSC and BMSC in vitro. The MSC and ESC marker profiles of paired TDSC and BMSC were compared using flow cytometry and quantitative real-time polymerase chain reaction (qRT-PCR), respectively. Their clonogenicity and proliferative capacity were compared using colony-forming and 5-bromo-2′-deoxyuridine assays, respectively. The expression of tenogenic, osteogenic, and chondrogenic markers at basal state were examined using qRT-PCR. Their osteogenic, chondrogenic, and adipogenic differentiation potentials were compared using standard assays. TDSC and BMSC showed similar expression of CD90 and CD73. TDSC expressed higher levels of Oct4 than BMSC. TDSC exhibited higher clonogenicity, proliferated faster, and expressed higher tenomodulin, scleraxis, collagen 1 α 1 (Col1A1), decorin, alkaline phosphatase, Col2A1, and biglycan messenger RNA levels than BMSC. There was higher calcium nodule formation and osteogenic marker expression in TDSC than BMSC upon osteogenic induction. More chondrocyte-like cells and higher glycosaminoglycan deposition and chondrogenic marker expression were observed in TDSC than BMSC upon chondrogenic induction. There were more oil droplets and expression of an adipogenic marker in TDSC than BMSC upon adipogenic induction. TDSC expressed higher Oct4 levels, which was reported to positively regulate mesendodermal lineage differentiation, showed higher clonogenicity and proliferative capacity, and had greater tenogenic, osteogenic, chondrogenic, and adipogenic markers and differentiation potential than BMSC. TDSC might be a better cell source than BMSC for musculoskeletal tissue regeneration. PMID

  7. EXCISION OF GIANT CELL TUMOR OF TENDON SHEATH WITH BONE INVOLVEMENT BY MEANS OF DOUBLE ACCESS APPROACH: CASE REPORT

    PubMed Central

    Alves, Marcelo de Pinho Teixeira

    2015-01-01

    Giant cell tumors of the tendon sheath are common lesions and are the second most frequent tumors in the hand, after synovial cysts. They are diagnosed by means of clinical examination and complementary examinations (simple radiography and magnetic resonance). Erosion and invasion of the phalangeal bone affected may be seen on radiological examination. Magnetic resonance may show a “fluorescent or radiant effect” may be observed, caused by the high quantity of hemosiderin inside the tumor. Surgical treatment is the commonest practice, and complete excision is important for avoiding recurrence of the tumor, especially when bone invasion is observed on imaging examinations, which is generally related to greater tumor recurrence. In this paper, a case of a giant cell tumor of the tendon sheath in the middle phalanx of the third finger of a 45-year-old female patient is presented. This was successfully treated by means of surgery using a double access approach (dorsal and volar). PMID:27026996

  8. A Systematic Cell-Based Analysis of Localization of Predicted Drosophila Peroxisomal Proteins.

    PubMed

    Baron, Matthew N; Klinger, Christen M; Rachubinski, Richard A; Simmonds, Andrew J

    2016-05-01

    Peroxisomes are membrane-bound organelles found in almost all eukaryotic cells. They perform specialized biochemical functions that vary with organism, tissue or cell type. Mutations in human genes required for the assembly of peroxisomes result in a spectrum of diseases called the peroxisome biogenesis disorders. A previous sequence-based comparison of the predicted proteome of Drosophila melanogaster (the fruit fly) to human proteins identified 82 potential homologues of proteins involved in peroxisomal biogenesis, homeostasis or metabolism. However, the subcellular localization of these proteins relative to the peroxisome was not determined. Accordingly, we tested systematically the localization and selected functions of epitope-tagged proteins in Drosophila Schneider 2 cells to determine the subcellular localization of 82 potential Drosophila peroxisomal protein homologues. Excluding the Pex proteins, 34 proteins localized primarily to the peroxisome, 8 showed dual localization to the peroxisome and other structures, and 26 localized exclusively to organelles other than the peroxisome. Drosophila is a well-developed laboratory animal often used for discovery of gene pathways, including those linked to human disease. Our work establishes a basic understanding of peroxisome protein localization in Drosophila. This will facilitate use of Drosophila as a genetically tractable, multicellular model system for studying key aspects of human peroxisome disease. PMID:26865094

  9. Rho1-Wnd signaling regulates loss-of-cell polarity-induced cell invasion in Drosophila.

    PubMed

    Ma, X; Chen, Y; Zhang, S; Xu, W; Shao, Y; Yang, Y; Li, W; Li, M; Xue, L

    2016-02-18

    Both cell polarity and c-Jun N-terminal kinase (JNK) activity are essential to the maintenance of tissue homeostasis, and disruption of either is commonly seen in cancer progression. Despite the established connection between loss-of-cell polarity and JNK activation, much less is known about the molecular mechanism by which aberrant cell polarity induces JNK-mediated cell migration and tumor invasion. Here we show results from a genetic screen using an in vivo invasion model via knocking down cell polarity gene in Drosophila wing discs, and identify Rho1-Wnd signaling as an important molecular link that mediates loss-of-cell polarity-triggered JNK activation and cell invasion. We show that Wallenda (Wnd), a protein kinase of the mitogen-activated protein kinase kinase kinase family, by forming a complex with the GTPase Rho1, is both necessary and sufficient for Rho1-induced JNK-dependent cell invasion, MMP1 activation and epithelial-mesenchymal transition. Furthermore, Wnd promotes cell proliferation and tissue growth through wingless production when apoptosis is inhibited by p35. Finally, Wnd shows oncogenic cooperation with Ras(V12) to trigger tumor growth in eye discs and causes invasion into the ventral nerve cord. Together, our data not only provides a novel mechanistic insight on how cell polarity loss contributes to cell invasion, but also highlights the value of the Drosophila model system to explore human cancer biology.

  10. High-pressure paint-gun injury of the finger simulating giant cell tumor of tendon sheath.

    PubMed

    Stefanato, Catherine M; Turner, Matthew S; Bhawan, Jag

    2005-02-01

    High-pressure paint guns deliver paint at approximately 3000 pounds per square inch. At this pressure, paint will penetrate the skin and spread quickly through fascial planes and tendon sheaths. The present case is that of a lesion from the finger of a 35-year-old white male in whom a history was initially unavailable. Histologic examination revealed diffuse fibrohistiocytic proliferation and giant cells, with numerous darkly pigmented, uniformly small-sized particles throughout the lesion. The initial impression was that of a giant cell tumor of tendon sheath. However, the pigment particles were negative for Perls stain, and polariscopic examination revealed clear refractile fragments. These findings raised the possibility that the lesion was the result of a traumatic event. On further inquiry, it was revealed that the patient had sustained a high-pressure paint-gun injury 1 year earlier. The simulation, histopathologically, of a giant cell tumor of tendon sheath by a high-pressure paint-gun injury has not, to our knowledge, been reported previously, nor has the histologic finding of small, uniformly sized pigment particles and polarizable refractile fragments in this particular type of injury.

  11. Longitudinal Cell Tracking and Simultaneous Monitoring of Tissue Regeneration after Cell Treatment of Natural Tendon Disease by Low-Field Magnetic Resonance Imaging

    PubMed Central

    Berner, Dagmar; Brehm, Walter; Gerlach, Kerstin; Gittel, Claudia; Offhaus, Julia; Paebst, Felicitas; Scharner, Doreen; Burk, Janina

    2016-01-01

    Treatment of tendon disease with multipotent mesenchymal stromal cells (MSC) is a promising option to improve tissue regeneration. To elucidate the mechanisms by which MSC support regeneration, longitudinal tracking of MSC labelled with superparamagnetic iron oxide (SPIO) by magnetic resonance imaging (MRI) could provide important insight. Nine equine patients suffering from tendon disease were treated with SPIO-labelled or nonlabelled allogeneic umbilical cord-derived MSC by local injection. Labelling of MSC was confirmed by microscopy and MRI. All animals were subjected to clinical, ultrasonographical, and low-field MRI examinations before and directly after MSC application as well as 2, 4, and 8 weeks after MSC application. Hypointense artefacts with characteristically low signal intensity were identified at the site of injection of SPIO-MSC in T1- and T2∗-weighted gradient echo MRI sequences. They were visible in all 7 cases treated with SPIO-MSC directly after injection, but not in the control cases treated with nonlabelled MSC. Furthermore, hypointense artefacts remained traceable within the damaged tendon tissue during the whole follow-up period in 5 out of 7 cases. Tendon healing could be monitored at the same time. Clinical and ultrasonographical findings as well as T2-weighted MRI series indicated a gradual improvement of tendon function and structure. PMID:26880932

  12. Analysis of cell identity, morphology, apoptosis and mitotic activity in a primary neural cell culture system in Drosophila

    PubMed Central

    2012-01-01

    In Drosophila, most neurogenetic research is carried out in vivo. Mammalian research demonstrates that primary cell culture techniques provide a powerful model to address cell autonomous and non-autonomous processes outside their endogenous environment. We developed a cell culture system in Drosophila using wildtype and genetically manipulated primary neural tissue for long-term observations. We assessed the molecular identity of distinct neural cell types by immunolabeling and genetically expressed fluorescent cell markers. We monitored mitotic activity of cell cultures derived from wildtype and tumorous larval brains. Our system provides a powerful approach to unveil developmental processes in the nervous system and to complement studies in vivo. PMID:22554060

  13. The cell-mediated immunity of Drosophila melanogaster: hemocyte lineages, immune compartments, microanatomy and regulation.

    PubMed

    Honti, Viktor; Csordás, Gábor; Kurucz, Éva; Márkus, Róbert; Andó, István

    2014-01-01

    In the animal kingdom, innate immunity is the first line of defense against invading pathogens. The dangers of microbial and parasitic attacks are countered by similar mechanisms, involving the prototypes of the cell-mediated immune responses, the phagocytosis and encapsulation. Work on Drosophila has played an important role in promoting an understanding of the basic mechanisms of phylogenetically conserved modules of innate immunity. The aim of this review is to survey the developments in the identification and functional definition of immune cell types and the immunological compartments of Drosophila melanogaster. We focus on the molecular and developmental aspects of the blood cell types and compartments, as well as the dynamics of blood cell development and the immune response. Further advances in the characterization of the innate immune mechanisms in Drosophila will provide basic clues to the understanding of the importance of the evolutionary conserved mechanisms of innate immune defenses in the animal kingdom. PMID:23800719

  14. Lysyl Oxidase Activity Is Required for Ordered Collagen Fibrillogenesis by Tendon Cells.

    PubMed

    Herchenhan, Andreas; Uhlenbrock, Franziska; Eliasson, Pernilla; Weis, MaryAnn; Eyre, David; Kadler, Karl E; Magnusson, S Peter; Kjaer, Michael

    2015-06-26

    Lysyl oxidases (LOXs) are a family of copper-dependent oxido-deaminases that can modify the side chain of lysyl residues in collagen and elastin, thereby leading to the spontaneous formation of non-reducible aldehyde-derived interpolypeptide chain cross-links. The consequences of LOX inhibition in producing lathyrism are well documented, but the consequences on collagen fibril formation are less clear. Here we used β-aminoproprionitrile (BAPN) to inhibit LOX in tendon-like constructs (prepared from human tenocytes), which are an experimental model of cell-mediated collagen fibril formation. The improvement in structure and strength seen with time in control constructs was absent in constructs treated with BAPN. As expected, BAPN inhibited the formation of aldimine-derived cross-links in collagen, and the constructs were mechanically weak. However, an unexpected finding was that BAPN treatment led to structurally abnormal collagen fibrils with irregular profiles and widely dispersed diameters. Of special interest, the abnormal fibril profiles resembled those seen in some Ehlers-Danlos Syndrome phenotypes. Importantly, the total collagen content developed normally, and there was no difference in COL1A1 gene expression. Collagen type V, decorin, fibromodulin, and tenascin-X proteins were unaffected by the cross-link inhibition, suggesting that LOX regulates fibrillogenesis independently of these molecules. Collectively, the data show the importance of LOX for the mechanical development of early collagenous tissues and that LOX is essential for correct collagen fibril shape formation. PMID:25979340

  15. Drosophila Adiponectin Receptor in Insulin Producing Cells Regulates Glucose and Lipid Metabolism by Controlling Insulin Secretion

    PubMed Central

    Kwak, Su-Jin; Hong, Seung-Hyun; Bajracharya, Rijan; Yang, Se-Yeol; Lee, Kyu-Sun; Yu, Kweon

    2013-01-01

    Adipokines secreted from adipose tissue are key regulators of metabolism in animals. Adiponectin, one of the adipokines, modulates pancreatic beta cell function to maintain energy homeostasis. Recently, significant conservation between Drosophila melanogaster and mammalian metabolism has been discovered. Drosophila insulin like peptides (Dilps) regulate energy metabolism similarly to mammalian insulin. However, in Drosophila, the regulatory mechanism of insulin producing cells (IPCs) by adipokine signaling is largely unknown. Here, we describe the discovery of the Drosophila adiponectin receptor and its function in IPCs. Drosophila adiponectin receptor (dAdipoR) has high homology with the human adiponectin receptor 1. The dAdipoR antibody staining revealed that dAdipoR was expressed in IPCs of larval and adult brains. IPC- specific dAdipoR inhibition (Dilp2>dAdipoR-Ri) showed the increased sugar level in the hemolymph and the elevated triglyceride level in whole body. Dilps mRNA levels in the Dilp2>dAdipoR-Ri flies were similar with those of controls. However, in the Dilp2>dAdipoR-Ri flies, Dilp2 protein was accumulated in IPCs, the level of circulating Dilp2 was decreased, and insulin signaling was reduced in the fat body. In ex vivo fly brain culture with the human adiponectin, Dilp2 was secreted from IPCs. These results indicate that adiponectin receptor in insulin producing cells regulates insulin secretion and controls glucose and lipid metabolism in Drosophila melanogaster. This study demonstrates a new adipokine signaling in Drosophila and provides insights for the mammalian adiponectin receptor function in pancreatic beta cells, which could be useful for therapeutic application. PMID:23874700

  16. Drosophila Wnt and STAT Define Apoptosis-Resistant Epithelial Cells for Tissue Regeneration after Irradiation.

    PubMed

    Verghese, Shilpi; Su, Tin Tin

    2016-09-01

    Drosophila melanogaster larvae irradiated with doses of ionizing radiation (IR) that kill about half of the cells in larval imaginal discs still develop into viable adults. How surviving cells compensate for IR-induced cell death to produce organs of normal size and appearance remains an active area of investigation. We have identified a subpopulation of cells within the continuous epithelium of Drosophila larval wing discs that shows intrinsic resistance to IR- and drug-induced apoptosis. These cells reside in domains of high Wingless (Wg, Drosophila Wnt-1) and STAT92E (sole Drosophila signal transducer and activator of transcription [STAT] homolog) activity and would normally form the hinge in the adult fly. Resistance to IR-induced apoptosis requires STAT and Wg and is mediated by transcriptional repression of the pro-apoptotic gene reaper. Lineage tracing experiments show that, following irradiation, apoptosis-resistant cells lose their identity and translocate to areas of the wing disc that suffered abundant cell death. Our findings provide a new paradigm for regeneration in which it is unnecessary to invoke special damage-resistant cell types such as stem cells. Instead, differences in gene expression within a population of genetically identical epithelial cells can create a subpopulation with greater resistance, which, following damage, survive, alter their fate, and help regenerate the tissue. PMID:27584613

  17. A Systematic Analysis of Drosophila Regulatory Peptide Expression in Enteroendocrine Cells

    PubMed Central

    Chen, Ji; Kim, Seol-min; Kwon, Jae Young

    2016-01-01

    The digestive system is gaining interest as a major regulator of various functions including immune defense, nutrient accumulation, and regulation of feeding behavior, aside from its conventional function as a digestive organ. The Drosophila midgut epithelium is completely renewed every 1–2 weeks due to differentiation of pluripotent intestinal stem cells in the midgut. Intestinal stem cells constantly divide and differentiate into enterocytes that secrete digestive enzymes and absorb nutrients, or enteroendocrine cells that secrete regulatory peptides. Regulatory peptides have important roles in development and metabolism, but study has mainly focused on expression and functions in the nervous system, and not much is known about the roles in endocrine functions of enteroendocrine cells. We systemically examined the expression of 45 regulatory peptide genes in the Drosophila midgut, and verified that at least 10 genes are expressed in the midgut enteroendocrine cells through RT-PCR, in situ hybridization, antisera, and 25 regulatory peptide-GAL transgenes. The Drosophila midgut is highly compartmentalized, and individual peptides in enteroendocrine cells were observed to express in specific regions of the midgut. We also confirmed that some peptides expressed in the same region of the midgut are expressed in mutually exclusive enteroendocrine cells. These results indicate that the midgut enteroendocrine cells are functionally differentiated into different subgroups. Through this study, we have established a basis to study regulatory peptide functions in enteroendocrine cells as well as the complex organization of enteroendocrine cells in the Drosophila midgut. PMID:27025390

  18. Drosophila Wnt and STAT Define Apoptosis-Resistant Epithelial Cells for Tissue Regeneration after Irradiation

    PubMed Central

    Su, Tin Tin

    2016-01-01

    Drosophila melanogaster larvae irradiated with doses of ionizing radiation (IR) that kill about half of the cells in larval imaginal discs still develop into viable adults. How surviving cells compensate for IR-induced cell death to produce organs of normal size and appearance remains an active area of investigation. We have identified a subpopulation of cells within the continuous epithelium of Drosophila larval wing discs that shows intrinsic resistance to IR- and drug-induced apoptosis. These cells reside in domains of high Wingless (Wg, Drosophila Wnt-1) and STAT92E (sole Drosophila signal transducer and activator of transcription [STAT] homolog) activity and would normally form the hinge in the adult fly. Resistance to IR-induced apoptosis requires STAT and Wg and is mediated by transcriptional repression of the pro-apoptotic gene reaper. Lineage tracing experiments show that, following irradiation, apoptosis-resistant cells lose their identity and translocate to areas of the wing disc that suffered abundant cell death. Our findings provide a new paradigm for regeneration in which it is unnecessary to invoke special damage-resistant cell types such as stem cells. Instead, differences in gene expression within a population of genetically identical epithelial cells can create a subpopulation with greater resistance, which, following damage, survive, alter their fate, and help regenerate the tissue. PMID:27584613

  19. Lipogems Product Treatment Increases the Proliferation Rate of Human Tendon Stem Cells without Affecting Their Stemness and Differentiation Capability

    PubMed Central

    Randelli, Pietro; Menon, Alessandra; Ragone, Vincenza; Creo, Pasquale; Bergante, Sonia; Randelli, Filippo; De Girolamo, Laura; Alfieri Montrasio, Umberto; Banfi, Giuseppe; Cabitza, Paolo; Tettamanti, Guido; Anastasia, Luigi

    2016-01-01

    Increasing the success rate of rotator cuff healing remains tremendous challenge. Among many approaches, the possibility of activating resident stem cells in situ, without the need to isolate them from biopsies, could represent valuable therapeutic strategy. Along this line, it has been recently demonstrated that lipoaspirate product, Lipogems, contains and produces growth-factors that may activate resident stem cells. In this study, human tendon stem cells (hTSCs) from the rotator cuff were cocultured in a transwell system with the Lipogems lipoaspirate product and compared to control untreated cells in terms of cell proliferation, morphology, stem cell marker and VEGF expression, and differentiation and migration capabilities. Results showed that the Lipogems product significantly increases the proliferation rate of hTSCs without altering their stemness and differentiation capability. Moreover, treated cells increase the expression of VEGF, which is crucial for the neovascularization of the tissue during the healing process. Overall, this study supports that directly activating hTSCs with the Lipogems lipoaspirate could represent a new practical therapeutic approach. In fact, obtaining a lipoaspirate is easier, safer, and more cost-effective than harvesting cells from tendon or bone marrow biopsies, expanding them in GMP facility and then reinjecting them in the patient. PMID:27057170

  20. Lipogems Product Treatment Increases the Proliferation Rate of Human Tendon Stem Cells without Affecting Their Stemness and Differentiation Capability.

    PubMed

    Randelli, Pietro; Menon, Alessandra; Ragone, Vincenza; Creo, Pasquale; Bergante, Sonia; Randelli, Filippo; De Girolamo, Laura; Alfieri Montrasio, Umberto; Banfi, Giuseppe; Cabitza, Paolo; Tettamanti, Guido; Anastasia, Luigi

    2016-01-01

    Increasing the success rate of rotator cuff healing remains tremendous challenge. Among many approaches, the possibility of activating resident stem cells in situ, without the need to isolate them from biopsies, could represent valuable therapeutic strategy. Along this line, it has been recently demonstrated that lipoaspirate product, Lipogems, contains and produces growth-factors that may activate resident stem cells. In this study, human tendon stem cells (hTSCs) from the rotator cuff were cocultured in a transwell system with the Lipogems lipoaspirate product and compared to control untreated cells in terms of cell proliferation, morphology, stem cell marker and VEGF expression, and differentiation and migration capabilities. Results showed that the Lipogems product significantly increases the proliferation rate of hTSCs without altering their stemness and differentiation capability. Moreover, treated cells increase the expression of VEGF, which is crucial for the neovascularization of the tissue during the healing process. Overall, this study supports that directly activating hTSCs with the Lipogems lipoaspirate could represent a new practical therapeutic approach. In fact, obtaining a lipoaspirate is easier, safer, and more cost-effective than harvesting cells from tendon or bone marrow biopsies, expanding them in GMP facility and then reinjecting them in the patient.

  1. Runx2-Modified Adipose-Derived Stem Cells Promote Tendon Graft Integration in Anterior Cruciate Ligament Reconstruction

    PubMed Central

    Zhang, Xin; Ma, Yong; Fu, Xin; Liu, Qiang; Shao, Zhenxing; Dai, Linghui; Pi, Yanbin; Hu, Xiaoqing; Zhang, Jiying; Duan, Xiaoning; Chen, Wenqing; Chen, Ping; Zhou, Chunyan; Ao, Yingfang

    2016-01-01

    Runx2 is a powerful osteo-inductive factor and adipose-derived stem cells (ADSCs) are multipotent. However, it is unknown whether Runx2-overexpressing ADSCs (Runx2-ADSCs) could promote anterior cruciate ligament (ACL) reconstruction. We evaluated the effect of Runx2-ADSCs on ACL reconstruction in vitro and in vivo. mRNA expressions of osteocalcin (OCN), bone sialoprotein (BSP) and collagen I (COLI) increased over time in Runx2-ADSCs. Runx2 overexpression inhibited LPL and PPARγ mRNA expressions. Runx2 induced alkaline phosphatase activity markedly. In nude mice injected with Runx2-ADSCs, promoted bone formation was detected by X-rays 8 weeks after injection. The healing of tendon-to-bone in a rabbit model of ACL reconstruction treated with Runx2-ADSCs, fibrin glue only and an RNAi targeting Runx2, was evaluated with CT 3D reconstruction, histological analysis and biomechanical methods. CT showed a greater degree of new bone formation around the bone tunnel in the group treated with Runx2-ADSCs compared with the fibrin glue group and RNAi Runx2 group. Histology showed that treatment with Runx2-ADSCs led to a rapid and significant increase at the tendon-to-bone compared with the control groups. Biomechanical tests demonstrated higher tendon pullout strength in the Runx2-ADSCs group at early time points. The healing of the attachment in ACL reconstruction was enhanced by Runx2-ADSCs. PMID:26743583

  2. Tie-mediated signal from apoptotic cells protects stem cells in Drosophila melanogaster.

    PubMed

    Xing, Yalan; Su, Tin Tin; Ruohola-Baker, Hannele

    2015-01-01

    Many types of normal and cancer stem cells are resistant to killing by genotoxins, but the mechanism for this resistance is poorly understood. Here we show that adult stem cells in Drosophila melanogaster germline and midgut are resistant to ionizing radiation (IR) or chemically induced apoptosis and dissect the mechanism for this protection. We find that upon IR the receptor tyrosine kinase Tie/Tie-2 is activated, leading to the upregulation of microRNA bantam that represses FOXO-mediated transcription of pro-apoptotic Smac/DIABLO orthologue, Hid in germline stem cells. Knockdown of the IR-induced putative Tie ligand, Pvf1, a functional homologue of human Angiopoietin, in differentiating daughter cells renders germline stem cells sensitive to IR, suggesting that the dying daughters send a survival signal to protect their stem cells for future repopulation of the tissue. If conserved in cancer stem cells, this mechanism may provide therapeutic options for the eradication of cancer. PMID:25959206

  3. Increased centrosome amplification in aged stem cells of the Drosophila midgut

    SciTech Connect

    Park, Joung-Sun; Pyo, Jung-Hoon; Na, Hyun-Jin; Jeon, Ho-Jun; Kim, Young-Shin; Arking, Robert; Yoo, Mi-Ae

    2014-07-25

    Highlights: • Increased centrosome amplification in ISCs of aged Drosophila midguts. • Increased centrosome amplification in ISCs of oxidative stressed Drosophila midguts. • Increased centrosome amplification in ISCs by overexpression of PVR, EGFR, and AKT. • Supernumerary centrosomes can be responsible for abnormal ISC polyploid cells. • Supernumerary centrosomes can be a useful marker for aging stem cells. - Abstract: Age-related changes in long-lived tissue-resident stem cells may be tightly linked to aging and age-related diseases such as cancer. Centrosomes play key roles in cell proliferation, differentiation and migration. Supernumerary centrosomes are known to be an early event in tumorigenesis and senescence. However, the age-related changes of centrosome duplication in tissue-resident stem cells in vivo remain unknown. Here, using anti-γ-tubulin and anti-PH3, we analyzed mitotic intestinal stem cells with supernumerary centrosomes in the adult Drosophila midgut, which may be a versatile model system for stem cell biology. The results showed increased centrosome amplification in intestinal stem cells of aged and oxidatively stressed Drosophila midguts. Increased centrosome amplification was detected by overexpression of PVR, EGFR, and AKT in intestinal stem cells/enteroblasts, known to mimic age-related changes including hyperproliferation of intestinal stem cells and hyperplasia in the midgut. Our data show the first direct evidence for the age-related increase of centrosome amplification in intestinal stem cells and suggest that the Drosophila midgut is an excellent model for studying molecular mechanisms underlying centrosome amplification in aging adult stem cells in vivo.

  4. Cdk4 functions in multiple cell types to control Drosophila intestinal stem cell proliferation and differentiation.

    PubMed

    Adlesic, Mojca; Frei, Christian; Frew, Ian J

    2016-01-01

    The proliferation of intestinal stem cells (ISCs) and differentiation of enteroblasts to form mature enteroendocrine cells and enterocytes in the Drosophila intestinal epithelium must be tightly regulated to maintain homeostasis. We show that genetic modulation of CyclinD/Cdk4 activity or mTOR-dependent signalling cell-autonomously regulates enterocyte growth, which influences ISC proliferation and enteroblast differentiation. Increased enterocyte growth results in higher numbers of ISCs and defective enterocyte growth reduces ISC abundance and proliferation in the midgut. Adult midguts deficient for Cdk4 show severe disruption of intestinal homeostasis characterised by decreased ISC self-renewal, enteroblast differentiation defects and low enteroendocrine cell and enterocyte numbers. The ISC/enteroblast phenotypes result from a combination of cell autonomous and non-autonomous requirements for Cdk4 function. One non-autonomous consequence of Cdk4-dependent deficient enterocyte growth is high expression of Delta in ISCs and Delta retention in enteroblasts. We postulate that aberrant activation of the Delta-Notch pathway is a possible partial cause of lost ISC stemness. These results support the idea that enterocytes contribute to a putative stem cell niche that maintains intestinal homeostasis in the Drosophila anterior midgut. PMID:26879465

  5. Discovery of progenitor cell signatures by time-series synexpression analysis during Drosophila embryonic cell immortalization

    PubMed Central

    Dequéant, Mary-Lee; Fagegaltier, Delphine; Hu, Yanhui; Spirohn, Kerstin; Simcox, Amanda; Hannon, Gregory J.; Perrimon, Norbert

    2015-01-01

    The use of time series profiling to identify groups of functionally related genes (synexpression groups) is a powerful approach for the discovery of gene function. Here we apply this strategy during RasV12 immortalization of Drosophila embryonic cells, a phenomenon not well characterized. Using high-resolution transcriptional time-series datasets, we generated a gene network based on temporal expression profile similarities. This analysis revealed that common immortalized cells are related to adult muscle precursors (AMPs), a stem cell-like population contributing to adult muscles and sharing properties with vertebrate satellite cells. Remarkably, the immortalized cells retained the capacity for myogenic differentiation when treated with the steroid hormone ecdysone. Further, we validated in vivo the transcription factor CG9650, the ortholog of mammalian Bcl11a/b, as a regulator of AMP proliferation predicted by our analysis. Our study demonstrates the power of time series synexpression analysis to characterize Drosophila embryonic progenitor lines and identify stem/progenitor cell regulators. PMID:26438832

  6. Cdk4 functions in multiple cell types to control Drosophila intestinal stem cell proliferation and differentiation

    PubMed Central

    Adlesic, Mojca; Frei, Christian; Frew, Ian J.

    2016-01-01

    ABSTRACT The proliferation of intestinal stem cells (ISCs) and differentiation of enteroblasts to form mature enteroendocrine cells and enterocytes in the Drosophila intestinal epithelium must be tightly regulated to maintain homeostasis. We show that genetic modulation of CyclinD/Cdk4 activity or mTOR-dependent signalling cell-autonomously regulates enterocyte growth, which influences ISC proliferation and enteroblast differentiation. Increased enterocyte growth results in higher numbers of ISCs and defective enterocyte growth reduces ISC abundance and proliferation in the midgut. Adult midguts deficient for Cdk4 show severe disruption of intestinal homeostasis characterised by decreased ISC self-renewal, enteroblast differentiation defects and low enteroendocrine cell and enterocyte numbers. The ISC/enteroblast phenotypes result from a combination of cell autonomous and non-autonomous requirements for Cdk4 function. One non-autonomous consequence of Cdk4-dependent deficient enterocyte growth is high expression of Delta in ISCs and Delta retention in enteroblasts. We postulate that aberrant activation of the Delta–Notch pathway is a possible partial cause of lost ISC stemness. These results support the idea that enterocytes contribute to a putative stem cell niche that maintains intestinal homeostasis in the Drosophila anterior midgut. PMID:26879465

  7. Hyperuricemia in Tendons.

    PubMed

    Andia, Isabel; Abate, Michele

    2016-01-01

    Hyperuricemia, particularly gout, and the immune inflammatory response are highly integrated. Both, long standing hyperuricemia and monosodium urate (MSU) crystal deposition can challenge tendon homeostasis because of their potential to cause inflammation to the host. Knowledge is emerging from clinical imaging research depicting where MSU crystals deposit, including patellar tendon, triceps and quadriceps tendons. Remarkably, subclinical tendon inflammation and damage are also present in asymptomatic hyperuricemia. Monosodium urate crystals act as danger activating molecular patterns (DAMPs), activating the inflammasome and inducing the secretion of IL-1beta, a key mediator of the inflammatory response. The crucial role of IL-1beta in driving the inflammatory events during gout attacks is supported by the clinical efficacy of IL-1beta blockade. Some data implicating IL-1beta as an initiator of tendinopathy exist, but the link between hyperuricemia and the development of tendinopathy remains to be validated. Further knowledge about the interactions of uric acid with both innate immune and tendon cells, and their consequences may help to determine if there is a subclass of hyperuricemic-tendinopathy. PMID:27535254

  8. Nuclear proteins in Drosophila melanogaster cells after heat shock and their binding to homologous DNA.

    PubMed Central

    Falkner, F G; Biessmann, H

    1980-01-01

    After 5 minutes heat shock at 37 degrees C Drosophila melanogaster Kc-cell nuclear proteins were extracted wit 0.4M NaCl and compared by SDS gel electrophoresis with extracts from cells grown at 25 degrees C. Two proteins (39 000 and 46 000) were only found in heat shock nuclei. Reconstitution with total Drosophila DNA or a DNA fragment from the heat inducible locus 87A/C covalently coupled to sepharose was performed. In the presence of calf thymus competitor DNA these proteins and also others of lower molecular weight showed preferential binding to the homologous DNA. Images PMID:6777762

  9. Genotoxicity studies of methyl isocyanate in Salmonella, Drosophila, and cultured Chinese hamster ovary cells

    SciTech Connect

    Mason, J.M.; Zeiger, E.; Haworth, S.; Ivett, J.; Valencia, R.

    1987-01-01

    The genotoxic effects of methyl isocyanate (MIC) were investigated using four short-term tests: the Salmonella reversion assay (Ames test), the Drosophila sex-linked recessive lethal assay, and the sister chromatic exchange (SCE) and chromosomal aberration assays in cultured Chinese hamster ovary (CHO) cells. No evidence was found for the induction of mutations in either Salmonella or Drosophila. MIC did, however, induce SCEs and chromosomal aberrations in CHO cells both in the presence and absence of Aroclor-induced rat liver S-9.

  10. Mesenchymal Stem Cell Therapy Regenerates the Native Bone-Tendon Junction after Surgical Repair in a Degenerative Rat Model

    PubMed Central

    Nourissat, Geoffroy; Diop, Amadou; Maurel, Nathalie; Salvat, Colette; Dumont, Sylvie; Pigenet, Audrey; Gosset, Marjolaine; Houard, Xavier; Berenbaum, Francis

    2010-01-01

    Background The enthesis, which attaches the tendon to the bone, naturally disappears with aging, thus limiting joint mobility. Surgery is frequently needed but the clinical outcome is often poor due to the decreased natural healing capacity of the elderly. This study explored the benefits of a treatment based on injecting chondrocyte and mesenchymal stem cells (MSC) in a new rat model of degenerative enthesis repair. Methodology The Achilles' tendon was cut and the enthesis destroyed. The damage was repaired by classical surgery without cell injection (group G1, n = 52) and with chondrocyte (group G2, n = 51) or MSC injection (group G3, n = 39). The healing rate was determined macroscopically 15, 30 and 45 days later. The production and organization of a new enthesis was assessed by histological scoring of collagen II immunostaining, glycoaminoglycan production and the presence of columnar chondrocytes. The biomechanical load required to rupture the bone-tendon junction was determined. Principal Findings The spontaneous healing rate in the G1 control group was 40%, close to those observed in humans. Cell injection significantly improved healing (69%, p = 0.0028 for G2 and p = 0.006 for G3) and the load-to-failure after 45 days (p<0.05) over controls. A new enthesis was clearly produced in cell-injected G2 and G3 rats, but not in the controls. Only the MSC-injected G3 rats had an organized enthesis with columnar chondrocytes as in a native enthesis 45 days after surgery. Conclusions Cell therapy is an efficient procedure for reconstructing degenerative entheses. MSC treatment produced better organ regeneration than chondrocyte treatment. The morphological and biomechanical properties were similar to those of a native enthesis. PMID:20805884

  11. Histological and Immunohistochemical Evaluation of Autologous Cultured Bone Marrow Mesenchymal Stem Cells and Bone Marrow Mononucleated Cells in Collagenase-Induced Tendinitis of Equine Superficial Digital Flexor Tendon

    PubMed Central

    Crovace, Antonio; Lacitignola, Luca; Rossi, Giacomo; Francioso, Edda

    2010-01-01

    The aim of this study was to compare treatment with cultured bone marrow stromal cells (cBMSCs), bone marrow Mononucleated Cells (BMMNCs), and placebo to repair collagenase-induced tendinitis in horses. In six adult Standardbred horses, 4000 IU of collagenase were injected in the superficial digital flexor tendon (SDFT). Three weeks after collagenase treatment, an average of either 5.5 × 106 cBMSCs or 1.2 × 108 BMMNCs, fibrin glue, and saline solution was injected intralesionally in random order. In cBMSC- and BMMNCS-treated tendons, a high expression of cartilage oligomeric matrix protein (COMP) and type I collagen, but low levels of type III collagen were revealed by immunohistochemistry, with a normal longitudinally oriented fiber pattern. Placebo-treated tendons expressed very low quantities of COMP and type I collagen but large numbers of randomly oriented type III collagen fibers. Both cBMSC and BMMNCS grafts resulted in a qualitatively similar heling improvement of tendon extracellular matrix, in terms of the type I/III collagen ratio, fiber orientation, and COMP expression. PMID:20445779

  12. A non-signaling role of Robo2 in tendons is essential for Slit processing and muscle patterning.

    PubMed

    Ordan, Elly; Volk, Talila

    2015-10-15

    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 N-terminal 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 short-range signaling in additional systems.

  13. Mechanism of osteogenic and adipogenic differentiation of tendon stem cells induced by sirtuin 1.

    PubMed

    Liu, Junpeng; Han, Weifeng; Chen, Lei; Tang, Kanglai

    2016-08-01

    The aim of the present study was to assess the expression of sirtuin (Sirt)1 in tendon stem cells (TSCs) and to elucidate its association with osteogenic and adipogenic differentiation of TSCs. Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) and western blot analyses were performed to detect Sirt1 mRNA and protein levels in TSCs, respectively. TSCs were positive for Sirt1 expression, which was elevated by Sirt1 activator SRT1720 in a time- and concentration- dependent manner, and decreased by Sirt1 inhibitor EX527. TSCs were treated with SRT1720 and EX527 for various time periods and resulting changes in osteogenic and adipogenic protein markers were analyzed using alizarin red and oil red O staining. According to RT-qPCR and western blot analyses, the associated factors β‑catenin, Runt-related transcription factor 2 (Runx2) and bone morphogenetic protein 2 were elevated following increases of Sirt1 levels, while CCAAT/enhancer binding protein (CEBP)α and peroxisome proliferator-activated receptor (PPAR)γ were decreased. These results suggested that osteogenic differentiation capacity was enhanced, while adipogenic differentiation capacity declined. Further mechanistic study revealed that phosphoinositide‑3 kinase (PI3K) and AKT were decreased following activation of Sirt1. In conclusion, the present study suggested that Sirt1 promotes the osteogenic differentiation of TSCs through upregulating β‑catenin and Runx2 and inhibits the adipogenic differentiation of TSCs through the PI3K/AKT pathway with downregulation of CEBPα and PPARγ. PMID:27357961

  14. Effect of adipose-derived stromal cells and BMP12 on intrasynovial tendon repair: A biomechanical, biochemical, and proteomics study.

    PubMed

    Gelberman, Richard H; Shen, Hua; Kormpakis, Ioannis; Rothrauff, Benjamin; Yang, Guang; Tuan, Rocky S; Xia, Younan; Sakiyama-Elbert, Shelly; Silva, Matthew J; Thomopoulos, Stavros

    2016-04-01

    The outcomes of flexor tendon repair are highly variable. As recent efforts to improve healing have demonstrated promise for growth factor- and cell-based therapies, the objective of the current study was to enhance repair via application of autologous adipose derived stromal cells (ASCs) and the tenogenic growth factor bone morphogenetic protein (BMP) 12. Controlled delivery of cells and growth factor was achieved in a clinically relevant canine model using a nanofiber/fibrin-based scaffold. Control groups consisted of repair-only (no scaffold) and acellular scaffold. Repairs were evaluated after 28 days of healing using biomechanical, biochemical, and proteomics analyses. Range of motion was reduced in the groups that received scaffolds compared to normal. There was no effect of ASC + BMP12 treatment for range of motion or tensile properties outcomes versus repair-only. Biochemical assays demonstrated increased DNA, glycosaminoglycans, and crosslink concentration in all repair groups compared to normal, but no effect of ASC + BMP12. Total collagen was significantly decreased in the acellular scaffold group compared to normal and significantly increased in the ASC + BMP12 group compared to the acellular scaffold group. Proteomics analysis comparing healing tendons to uninjured tendons revealed significant increases in proteins associated with inflammation, stress response, and matrix degradation. Treatment with ASC + BMP12 amplified these unfavorable changes. In summary, the treatment approach used in this study induced a negative inflammatory reaction at the repair site leading to poor healing. Future approaches should consider cell and growth factor delivery methods that do not incite negative local reactions.

  15. Updates in biological therapies for knee injuries: tendons.

    PubMed

    Demange, Marco Kawamura; de Almeida, Adriano Marques; Rodeo, Scott A

    2014-09-01

    Tendons are subjected to tendinopathies caused by inflammation, degeneration, and weakening of the tendon, due to overuse and trauma, which may eventually lead to tendon rupture. Recently, there has been increasing interest in biological approaches to augment tissue healing. Tendon healing occurs through a dynamic process with inflammation, cellular proliferation, and tissue remodeling. In this review article, we discuss the more frequently proposed biological therapies for tendon injuries as platelet-rich plasma, mesenchymal stem cells, extracorporeal shockwave, and scaffolds.

  16. In Vivo Evaluation of Adipose-Derived Stromal Cells Delivered with a Nanofiber Scaffold for Tendon-to-Bone Repair.

    PubMed

    Lipner, Justin; Shen, Hua; Cavinatto, Leonardo; Liu, Wenying; Havlioglu, Necat; Xia, Younan; Galatz, Leesa M; Thomopoulos, Stavros

    2015-11-01

    Rotator cuff tears are common and cause a great deal of lost productivity, pain, and disability. Tears are typically repaired by suturing the tendon back to its bony attachment. Unfortunately, the structural (e.g., aligned collagen) and compositional (e.g., a gradient in mineral) elements that produce a robust attachment in the healthy tissue are not regenerated during healing, and the repair is prone to failure. Two features of the failed healing response are deposition of poorly aligned scar tissue and loss of bone at the repair site. Therefore, the objective of the current study was to improve tendon-to-bone healing by promoting aligned collagen deposition and increased bone formation using a biomimetic scaffold seeded with pluripotent cells. An aligned nanofibrous poly(lactic-co-glycolic acid) scaffold with a gradient in mineral content was seeded with adipose-derived stromal cells (ASCs) and implanted at the repair site of a rat rotator cuff model. In one group, cells were transduced with the osteogenic factor bone morphogenetic protein 2 (BMP2). The healing response was examined in four groups (suture only, acellular scaffold, cellular scaffold, and cellular BMP2 scaffold) using histologic, bone morphology, and biomechanical outcomes at 14, 28, and 56 days. Histologically, the healing interface was dominated by a fibrovascular scar response in all groups. The acellular scaffold group showed a delayed healing response compared to the other groups. When examining bone morphology parameters, bone loss was evident in the cellular BMP2 group compared to other groups at 28 days. When examining repair-site mechanical properties, strength and modulus were decreased in the cellular BMP2 groups compared to other groups at 28 and 56 days. These results indicated that tendon-to-bone healing in this animal model was dominated by scar formation, preventing any positive effects of the implanted biomimetic scaffold. Furthermore, cells transduced with the osteogenic factor

  17. Microtubule inhibitors block the morphological changes induced in Drosophila blood cells by a parasitoid wasp factor.

    PubMed

    Rizki, R M; Rizki, T M

    1990-03-15

    The shape change of Drosophila melanogaster blood cells (lamellocytes) from discoidal to bipolar that is caused by a factor from the female parasitoid Leptopilina heterotoma is blocked by the tubulin inhibitors vinblastine and vincristine in vitro. The actin inhibitor, cytochalasin B, causes arborization of Drosophila lamellocytes and acts synergistically with the wasp factor to alter lamellocyte morphology. Lamellocyte aborization induced by cytochalasin B is blocked by simultaneous treatment with vinblastine. These observations indicate that the changes in lamellocyte shape induced by both the wasp factor and cytochalasin B require microtubule assembly. PMID:2311722

  18. Using Fluorescent Reporters to Monitor Autophagy in the Female Germline Cells in Drosophila melanogaster.

    PubMed

    Jacomin, Anne-Claire; Nezis, Ioannis P

    2016-01-01

    Oogenesis is a fundamental biological process for the transmission of genetic information to the next generations. Drosophila has proven to be a valuable model for elucidating the molecular and cellular mechanisms involved in this developmental process. It has been shown that autophagy participates in the maturation of the egg chamber. Here we provide a protocol for monitoring and quantification of the autophagic process in the Drosophila germline cells using the fluorescent reporters mCherry-DmAtg8a and GFP-mCherry-DmAtg8a. PMID:27557573

  19. Extracorporeal Shock Wave Treatment (ESWT) enhances the in vitro-induced differentiation of human tendon-derived stem/progenitor cells (hTSPCs)

    PubMed Central

    Leone, Laura; Raffa, Salvatore; Vetrano, Mario; Ranieri, Danilo; Malisan, Florence; Scrofani, Cristina; Vulpiani, Maria Chiara; Ferretti, Andrea; Torrisi, Maria Rosaria; Visco, Vincenzo

    2016-01-01

    Extracorporeal shock wave therapy (ESWT) is a non-invasive and innovative technology for the management of specific tendinopathies. In order to elucidate the ESWT-mediated clinical benefits, human Tendon-derived Stem/Progenitor cells (hTSPCs) explanted from 5 healthy semitendinosus (ST) and 5 ruptured Achilles (AT) tendons were established. While hTSPCs from the two groups showed similar proliferation rates and stem cell surface marker profiles, we found that the clonogenic potential was maintained only in cells derived from healthy donors. Interestingly, ESWT significantly accelerated hTSPCs differentiation, suggesting that the clinical benefits of ESWT may be ascribed to increased efficiency of tendon repair after injury. PMID:26843618

  20. Expression of the hepatitis B virus surface antigen in Drosophila S2 cells

    PubMed Central

    Santos, Alexandra S.; Spina, Ângela; Pereira, Carlos A.

    2008-01-01

    Drosophila melanogaster S2 cells were transfected with a plasmid vector (pAcHBsAgHy) containing the S gene, coding for the hepatitis B virus surface antigen (HBsAg), under control of the constitutive drosophila actin promoter (pAc), and the hygromycin B (Hy) selection gene. The vector was introduced into Schneider 2 (S2) Drosophila cells by DNA transfection and a cell population (S2AcHBsAgHy) was selected by its resistance to hygromycin B. The pAcHBsAgHy vector integrated in transfected S2 cell genome and approximately 1,000 copies per cell were found in a higher HBsAg producer cell subpopulation. The HBsAg production varied in different subpopulations, but did not when a given subpopulation was cultivated in different culture flasks. Higher HBsAg expression was found in S2AcHBsAgHy cells cultivated in Insect Xpress medium (13.5 μg/1E7 cells) and SFX medium (7 μg/1E7 cells) in comparison to SF900II medium (0.6 μg/1E7 cells). An increase of HBsAg was observed in culture maintained under hygromycin selection pressure. Data presented in the paper show that S2AcHBsAgHy cells produce efficiently the HBsAg which is mainly found in the cell supernatant, suggesting that HBsAg is secreted from the cells. The data also show that our approach using the Drosophila expression system is suitable for the preparation of other viral protein preparation. PMID:19003172

  1. Wnt ligands regulate Tkv expression to constrain Dpp activity in the Drosophila ovarian stem cell niche

    PubMed Central

    Luo, Lichao; Wang, Huashan; Fan, Chao; Liu, Sen

    2015-01-01

    Stem cell self-renewal versus differentiation is regulated by the niche, which provides localized molecules that favor self-renewal. In the Drosophila melanogaster female germline stem cell (GSC) niche, Decapentaplegic (Dpp), a fly transforming growth factor β molecule and well-established long-range morphogen, acts over one cell diameter to maintain the GSCs. Here, we show that Thickveins (Tkv; a type I receptor of Dpp) is highly expressed in stromal cells next to Dpp-producing cells and functions to remove excess Dpp outside the niche, thereby spatially restricting its activity. Interestingly, Tkv expression in these stromal cells is regulated by multiple Wnt ligands that are produced by the niche. Our data demonstrate a self-restraining mechanism by which the Drosophila ovarian GSC niche acts to define its own boundary. PMID:26008746

  2. Induction of aversive learning through thermogenetic activation of Kenyon cell ensembles in Drosophila

    PubMed Central

    Vasmer, David; Pooryasin, Atefeh; Riemensperger, Thomas; Fiala, André

    2014-01-01

    Drosophila represents a model organism to analyze neuronal mechanisms underlying learning and memory. Kenyon cells of the Drosophila mushroom body are required for associative odor learning and memory retrieval. But is the mushroom body sufficient to acquire and retrieve an associative memory? To answer this question we have conceived an experimental approach to bypass olfactory sensory input and to thermogenetically activate sparse and random ensembles of Kenyon cells directly. We found that if the artifical activation of Kenyon cell ensembles coincides with a salient, aversive stimulus learning was induced. The animals adjusted their behavior in a subsequent test situation and actively avoided reactivation of these Kenyon cells. Our results show that Kenyon cell activity in coincidence with a salient aversive stimulus can suffice to form an associative memory. Memory retrieval is characterized by a closed feedback loop between a behavioral action and the reactivation of sparse ensembles of Kenyon cells. PMID:24860455

  3. Bioreactor Design for Tendon/Ligament Engineering

    PubMed Central

    Wang, Tao; Gardiner, Bruce S.; Lin, Zhen; Rubenson, Jonas; Kirk, Thomas B.; Wang, Allan; Xu, Jiake

    2013-01-01

    Tendon and ligament injury is a worldwide health problem, but the treatment options remain limited. Tendon and ligament engineering might provide an alternative tissue source for the surgical replacement of injured tendon. A bioreactor provides a controllable environment enabling the systematic study of specific biological, biochemical, and biomechanical requirements to design and manufacture engineered tendon/ligament tissue. Furthermore, the tendon/ligament bioreactor system can provide a suitable culture environment, which mimics the dynamics of the in vivo environment for tendon/ligament maturation. For clinical settings, bioreactors also have the advantages of less-contamination risk, high reproducibility of cell propagation by minimizing manual operation, and a consistent end product. In this review, we identify the key components, design preferences, and criteria that are required for the development of an ideal bioreactor for engineering tendons and ligaments. PMID:23072472

  4. Vesicular stomatitis virus growth in Drosophila melanogaster cells: G protein deficiency.

    PubMed

    Wyers, F; Richard-Molard, C; Blondel, D; Dezelee, S

    1980-01-01

    In cultured Drosophila melanogaster cells, vesicular stomatitis virus (VSV) established a persistent, noncytopathic infection. No inhibition of host protein synthesis occurred even though all cells were initially infected. No defective interfering particles were detected, which would explain the establishment of the carrier state. In studies of the time course of viral protein synthesis in Drosophila cells, N, NS, and M viral polypeptides were readily detected within 1 h of infection. The yield of G protein and one of its precursors; G1, was very low at any time of the virus cycle; the released viruses always contained four to five times less G than those produced by chicken embryo cells, whatever the VSV strain or serotype used for infection and whatever the Drosophila cell line used as host. Actinomycin D added to the cells before infection enhanced VSV growth up to eight times. G and G1 synthesis increased much more than that of the other viral proteins when the cells were pretreated with the drug; nevertheless, the released viruses exhibited the same deficiency in G protein as the VSV released from untreated cells. Host cell control on both G-protein maturation process and synthesis at traduction level is discussed in relation to G biological properties.

  5. During Drosophila disc regeneration, JAK/STAT coordinates cell proliferation with Dilp8-mediated developmental delay.

    PubMed

    Katsuyama, Tomonori; Comoglio, Federico; Seimiya, Makiko; Cabuy, Erik; Paro, Renato

    2015-05-01

    Regeneration of fragmented Drosophila imaginal discs occurs in an epimorphic manner involving local cell proliferation at the wound site. After disc fragmentation, cells at the wound site activate a restoration program through wound healing, regenerative cell proliferation, and repatterning of the tissue. However, the interplay of signaling cascades driving these early reprogramming steps is not well-understood. Here, we profiled the transcriptome of regenerating cells in the early phase within 24 h after wounding. We found that JAK/STAT signaling becomes activated at the wound site and promotes regenerative cell proliferation in cooperation with Wingless (Wg) signaling. In addition, we showed that the expression of Drosophila insulin-like peptide 8 (dilp8), which encodes a paracrine peptide to delay the onset of pupariation, is controlled by JAK/STAT signaling in early regenerating discs. Our findings suggest that JAK/STAT signaling plays a pivotal role in coordinating regenerative disc growth with organismal developmental timing.

  6. Drosophila as a model for the two myeloid blood cell systems in vertebrates

    PubMed Central

    Gold, Katrina S.; Brückner, Katja

    2016-01-01

    Fish, mice and men rely on two coexisting myeloid blood cell systems. One is sustained by hematopoietic progenitor cells, which reside in specialized microenvironments in hematopoietic organs and give rise to cells of the monocyte lineage. The other system corresponds to the independent lineage of self-renewing tissue macrophages, which colonize organs during embryonic development and are maintained during later life by proliferation in local tissue microenvironments. However, little is known about the nature of these microenvironments and their regulation. Moreover, many vertebrate tissues contain a mix of both tissue-resident and monocyte-derived macrophages, posing a challenge to the study of lineage-specific regulatory mechanisms and function. This review highlights how research in the simple model organism Drosophila melanogaster can address many of these outstanding questions in the field. Drawing parallels between hematopoiesis in Drosophila and vertebrates, we illustrate the evolutionary conservation of the two myeloid systems across animal phyla. Much like vertebrates, Drosophila possesses a lineage of self-renewing tissue-resident macrophages, as well as a ‘definitive’ lineage of macrophages that derive from hematopoiesis in the progenitor-based lymph gland. We summarize key findings from Drosophila hematopoiesis that illustrate how local microenvironments, systemic signals, immune challenges and nervous inputs regulate adaptive responses of tissue-resident macrophages and progenitor-based hematopoiesis to achieve optimal fitness of the animal. PMID:24946019

  7. MRI-Based Assessment of Intralesional Delivery of Bone Marrow-Derived Mesenchymal Stem Cells in a Model of Equine Tendonitis

    PubMed Central

    Scharf, Alexandra; Holmes, Shannon P.; Thoresen, Merrilee; Mumaw, Jennifer; Stumpf, Alaina

    2016-01-01

    Ultrasound-guided intralesional injection of mesenchymal stem cells (MSCs) is held as the benchmark for cell delivery in tendonitis. The primary objective of this study was to investigate the immediate cell distribution following intralesional injection of MSCs. Unilateral superficial digital flexor tendon (SDFT) lesions were created in the forelimb of six horses and injected with 10 × 106 MSCs labeled with superparamagnetic iron oxide nanoparticles (SPIOs) under ultrasound guidance. Assays were performed to confirm that there were no significant changes in cell viability, proliferation, migration, or trilineage differentiation due to the presence of SPIOs. Limbs were imaged on a 1.5-tesla clinical MRI scanner postmortem before and after injection to determine the extent of tendonitis and detect SPIO MSCs. Clusters of labeled cells were visible as signal voids in 6/6 subjects. Coalescing regions of signal void were diffusely present in the peritendinous tissues. Although previous reports have determined that local injury retains cells within a small radius of the site of injection, our study shows greater than expected delocalization and relatively few cells retained within collagenous tendon compared to surrounding fascia. Further work is needed if this is a reality in vivo and to determine if directed intralesional delivery of MSCs is as critical as presently thought. PMID:27746821

  8. Three-dimensional structure of Drosophila testis tip: the spatial relation between dividing cells.

    PubMed

    Lee, Kyung Eun; Han, Sung Sik; Jeon, Hyesung

    2013-08-01

    At the apical tip of Drosophila testis, there is a stem cell niche known as the proliferation center, where the stem cells are maintained by hub cell cluster for the regulation of differentiation and proliferation. Germline stem cells go through mitosis four times from one primary spermatogonial cell to the 16-cell stage before the maturation. The cells derived from the same germline stem cell are located within one cyst, an enclosed system by two cyst cells, and they are connected by the intercellular bridges called ring canals. In this study, the three-dimensional (3D) structure of Drosophila testis tip was reconstructed from serial sections. The size of cells at each stage was compared in volume from the 3D structure. The stages of cells in a cyst could be distinguishable exactly by counting the cells linked with intercellular bridges in 3D-reconstructed structure. The cysts containing the same stage cells appeared in the horizontal plane. Both the germline stem cell directly attached to the hub cell and the spermatogonial cells detached from the hub cell were divided at the almost perpendicular direction to the spermatogonial cell layers. The dividing phase in one cyst was delayed gradually through the cytoplasmic region of intercellular bridge.

  9. Wildtype adult stem cells, unlike tumor cells, are resistant to cellular damages in Drosophila.

    PubMed

    Ma, Meifang; Zhao, Hang; Zhao, Hanfei; Binari, Richard; Perrimon, Norbert; Li, Zhouhua

    2016-03-15

    Adult stem cells or residential progenitor cells are critical to maintain the structure and function of adult tissues (homeostasis) throughout the lifetime of an individual. Mis-regulation of stem cell proliferation and differentiation often leads to diseases including cancer, however, how wildtype adult stem cells and cancer cells respond to cellular damages remains unclear. We find that in the adult Drosophila midgut, intestinal stem cells (ISCs), unlike tumor intestinal cells, are resistant to various cellular damages. Tumor intestinal cells, unlike wildtype ISCs, are easily eliminated by apoptosis. Further, their proliferation is inhibited upon autophagy induction, and autophagy-mediated tumor inhibition is independent of caspase-dependent apoptosis. Interestingly, inhibition of tumorigenesis by autophagy is likely through the sequestration and degradation of mitochondria, as compromising mitochondria activity in these tumor models mimics the induction of autophagy and increasing the production of mitochondria alleviates the tumor-suppression capacity of autophagy. Together, these data demonstrate that wildtype adult stem cells and tumor cells show dramatic differences in sensitivity to cellular damages, thus providing potential therapeutic implications targeting tumorigenesis. PMID:26845534

  10. Active JNK-dependent secretion of Drosophila Tyrosyl-tRNA synthetase by loser cells recruits haemocytes during cell competition.

    PubMed

    Casas-Tintó, Sergio; Lolo, Fidel-Nicolás; Moreno, Eduardo

    2015-12-11

    Cell competition is a process by which the slow dividing cells (losers) are recognized and eliminated from growing tissues. Loser cells are extruded from the epithelium and engulfed by the haemocytes, the Drosophila macrophages. However, how macrophages identify the dying loser cells is unclear. Here we show that apoptotic loser cells secrete Tyrosyl-tRNA synthetase (TyrRS), which is best known as a core component of the translational machinery. Secreted TyrRS is cleaved by matrix metalloproteinases generating MiniTyr and EMAP fragments. EMAP acts as a guiding cue for macrophage migration in the Drosophila larvae, as it attracts the haemocytes to the apoptotic loser cells. JNK signalling and Kish, a component of the secretory pathway, are autonomously required for the active secretion of TyrRS by the loser cells. Altogether, this mechanism guarantees effective removal of unfit cells from the growing tissue.

  11. Active JNK-dependent secretion of Drosophila Tyrosyl-tRNA synthetase by loser cells recruits haemocytes during cell competition.

    PubMed

    Casas-Tintó, Sergio; Lolo, Fidel-Nicolás; Moreno, Eduardo

    2015-01-01

    Cell competition is a process by which the slow dividing cells (losers) are recognized and eliminated from growing tissues. Loser cells are extruded from the epithelium and engulfed by the haemocytes, the Drosophila macrophages. However, how macrophages identify the dying loser cells is unclear. Here we show that apoptotic loser cells secrete Tyrosyl-tRNA synthetase (TyrRS), which is best known as a core component of the translational machinery. Secreted TyrRS is cleaved by matrix metalloproteinases generating MiniTyr and EMAP fragments. EMAP acts as a guiding cue for macrophage migration in the Drosophila larvae, as it attracts the haemocytes to the apoptotic loser cells. JNK signalling and Kish, a component of the secretory pathway, are autonomously required for the active secretion of TyrRS by the loser cells. Altogether, this mechanism guarantees effective removal of unfit cells from the growing tissue. PMID:26658841

  12. A GATA/RUNX cis-regulatory module couples Drosophila blood cell commitment and differentiation into crystal cells.

    PubMed

    Ferjoux, Géraldine; Augé, Benoit; Boyer, Karène; Haenlin, Marc; Waltzer, Lucas

    2007-05-15

    Members of the RUNX and GATA transcription factor families play critical roles during hematopoiesis from Drosophila to mammals. In Drosophila, the formation of the crystal cell hematopoietic lineage depends on the continuous expression of the lineage-specific RUNX factor Lozenge (Lz) and on its interaction with the GATA factor Serpent (Srp). Crystal cells are the main source of prophenoloxidases (proPOs), the enzymes required for melanization. By analyzing the promoter regions of several insect proPOs, we identify a conserved GATA/RUNX cis-regulatory module that ensures the crystal cell-specific expression of the three Drosophila melanogaster proPO. We demonstrate that activation of this module requires the direct binding of both Srp and Lz. Interestingly, a similar GATA/RUNX signature is over-represented in crystal cell differentiation markers, allowing us to identify new Srp/Lz target genes by genome-wide screening of Drosophila promoter regions. Finally, we show that the expression of lz in the crystal cells also relies on Srp/Lz-mediated activation via a similar module, indicating that crystal cell fate choice maintenance and activation of the differentiation program are coupled. Based on our observations, we propose that this GATA/RUNX cis-regulatory module may be reiteratively used during hematopoietic development through evolution.

  13. The Hippo signalling pathway maintains quiescence in Drosophila neural stem cells

    PubMed Central

    Ding, Rouven; Weynans, Kevin; Bossing, Torsten; Barros, Claudia S.; Berger, Christian

    2016-01-01

    Stem cells control their mitotic activity to decide whether to proliferate or to stay in quiescence. Drosophila neural stem cells (NSCs) are quiescent at early larval stages, when they are reactivated in response to metabolic changes. Here we report that cell-contact inhibition of growth through the canonical Hippo signalling pathway maintains NSC quiescence. Loss of the core kinases hippo or warts leads to premature nuclear localization of the transcriptional co-activator Yorkie and initiation of growth and proliferation in NSCs. Yorkie is necessary and sufficient for NSC reactivation, growth and proliferation. The Hippo pathway activity is modulated via inter-cellular transmembrane proteins Crumbs and Echinoid that are both expressed in a nutrient-dependent way in niche glial cells and NSCs. Loss of crumbs or echinoid in the niche only is sufficient to reactivate NSCs. Finally, we provide evidence that the Hippo pathway activity discriminates quiescent from non-quiescent NSCs in the Drosophila nervous system. PMID:26821647

  14. The Hippo signalling pathway maintains quiescence in Drosophila neural stem cells.

    PubMed

    Ding, Rouven; Weynans, Kevin; Bossing, Torsten; Barros, Claudia S; Berger, Christian

    2016-01-29

    Stem cells control their mitotic activity to decide whether to proliferate or to stay in quiescence. Drosophila neural stem cells (NSCs) are quiescent at early larval stages, when they are reactivated in response to metabolic changes. Here we report that cell-contact inhibition of growth through the canonical Hippo signalling pathway maintains NSC quiescence. Loss of the core kinases hippo or warts leads to premature nuclear localization of the transcriptional co-activator Yorkie and initiation of growth and proliferation in NSCs. Yorkie is necessary and sufficient for NSC reactivation, growth and proliferation. The Hippo pathway activity is modulated via inter-cellular transmembrane proteins Crumbs and Echinoid that are both expressed in a nutrient-dependent way in niche glial cells and NSCs. Loss of crumbs or echinoid in the niche only is sufficient to reactivate NSCs. Finally, we provide evidence that the Hippo pathway activity discriminates quiescent from non-quiescent NSCs in the Drosophila nervous system.

  15. Repair of full-thickness tendon injury using connective tissue progenitors efficiently derived from human embryonic stem cells and fetal tissues.

    PubMed

    Cohen, Shahar; Leshansky, Lucy; Zussman, Eyal; Burman, Michael; Srouji, Samer; Livne, Erella; Abramov, Natalie; Itskovitz-Eldor, Joseph

    2010-10-01

    The use of stem cells for tissue engineering (TE) encourages scientists to design new platforms in the field of regenerative and reconstructive medicine. Human embryonic stem cells (hESC) have been proposed to be an important cell source for cell-based TE applications as well as an exciting tool for investigating the fundamentals of human development. Here, we describe the efficient derivation of connective tissue progenitors (CTPs) from hESC lines and fetal tissues. The CTPs were significantly expanded and induced to generate tendon tissues in vitro, with ultrastructural characteristics and biomechanical properties typical of mature tendons. We describe a simple method for engineering tendon grafts that can successfully repair injured Achilles tendons and restore the ankle joint extension movement in mice. We also show the CTP's ability to differentiate into bone, cartilage, and fat both in vitro and in vivo. This study offers evidence for the possibility of using stem cell-derived engineered grafts to replace missing tissues, and sets a basic platform for future cell-based TE applications in the fields of orthopedics and reconstructive surgery.

  16. A Distinct Perisynaptic Glial Cell Type Forms Tripartite Neuromuscular Synapses in the Drosophila Adult

    PubMed Central

    Strauss, Alexandra L.; Kawasaki, Fumiko; Ordway, Richard W.

    2015-01-01

    Previous studies of Drosophila flight muscle neuromuscular synapses have revealed their tripartite architecture and established an attractive experimental model for genetic analysis of glial function in synaptic transmission. Here we extend these findings by defining a new Drosophila glial cell type, designated peripheral perisynaptic glia (PPG), which resides in the periphery and interacts specifically with fine motor axon branches forming neuromuscular synapses. Identification and specific labeling of PPG was achieved through cell type-specific RNAi-mediated knockdown (KD) of a glial marker, Glutamine Synthetase 2 (GS2). In addition, comparison among different Drosophila neuromuscular synapse models from adult and larval developmental stages indicated the presence of tripartite synapses on several different muscle types in the adult. In contrast, PPG appear to be absent from larval body wall neuromuscular synapses, which do not exhibit a tripartite architecture but rather are imbedded in the muscle plasma membrane. Evolutionary conservation of tripartite synapse architecture and peripheral perisynaptic glia in vertebrates and Drosophila suggests ancient and conserved roles for glia-synapse interactions in synaptic transmission. PMID:26053860

  17. Posterior Tibial Tendon Dysfunction

    MedlinePlus

    ... when the posterior tibial tendon becomes inflamed or torn. As a result, the tendon may not be ... repetitive use. Once the tendon becomes inflamed or torn, the arch will slowly fall (collapse) over time. ...

  18. Achilles tendon repair

    MedlinePlus

    Achilles tendon rupture-surgery; Percutaneous Achilles tendon rupture repair ... To fix your torn Achilles tendon, the surgeon will: Make a cut down the back of your heel Make several small cuts rather than one large cut ...

  19. Achilles tendon rupture - aftercare

    MedlinePlus

    Heel cord tear; Calcaneal tendon rupture ... MRI scan to see what type of Achilles tendon tear you have. An MRI is a type ... partial tear means at least some of the tendon is still OK. A full tear means your ...

  20. Tendon latch

    SciTech Connect

    Watkins, B. J.

    1985-01-01

    A latch connects tendons run from a floating platform to a socket in a foundation on the sea floor. The latch includes a latch body having a plurality of dogs disposed within and urgible outward from the latch body. A piston is releasably disposed within the latch body above the dogs and moves downwardly when released to urge the dogs outwardly from the body into latching engagement with the socket. A trigger mechanism in the latch releases the piston when the latch body lands in the socket and contacts a trigger pin projecting upwardly from the bottom of the socket. A series of wedges are disposed exteriorally on the body and inhibit lateral movement of the body relative to the socket when the tendon is subjected to a cycle bending loads.

  1. Evolution of mitochondrial cell death pathway: Proapoptotic role of HtrA2/Omi in Drosophila

    SciTech Connect

    Igaki, Tatsushi; Suzuki, Yasuyuki; Tokushige, Naoko; Aonuma, Hiroka; Takahashi, Ryosuke . E-mail: ryosuket@kuhp.kyoto-u.ac.jp; Miura, Masayuki . E-mail: miura@mol.f.u-tokyo.ac.jp

    2007-05-18

    Despite the essential role of mitochondria in a variety of mammalian cell death processes, the involvement of mitochondrial pathway in Drosophila cell death has remained unclear. To address this, we cloned and characterized DmHtrA2, a Drosophila homolog of a mitochondrial serine protease HtrA2/Omi. We show that DmHtrA2 normally resides in mitochondria and is up-regulated by UV-irradiation. Upon receipt of apoptotic stimuli, DmHtrA2 is translocated to extramitochondrial compartment; however, unlike its mammalian counterpart, the extramitochondrial DmHtrA2 does not diffuse throughout the cytosol but stays near the mitochondria. RNAi-mediated knock-down of DmHtrA2 in larvae or adult flies results in a resistance to stress stimuli. DmHtrA2 specifically cleaves Drosophila inhibitor-of-apoptosis protein 1 (DIAP1), a cellular caspase inhibitor, and induces cell death both in vitro and in vivo as potent as other fly cell death proteins. Our observations suggest that DmHtrA2 promotes cell death through a cleavage of DIAP1 in the vicinity of mitochondria, which may represent a prototype of mitochondrial cell death pathway in evolution.

  2. Non-centrosomal microtubule-organising centres in cold-treated cultured Drosophila cells.

    PubMed

    Cottam, Deborah M; Tucker, John B; Rogers-Bald, Margaret M; Mackie, John B; Macintyre, John; Scarborough, Julie A; Ohkura, Hiroyuki; Milner, Martin J

    2006-02-01

    In this paper we describe a new type of non-centrosomal microtubule-organising centre (MTOC), which is induced by cold treatment of certain cultured Drosophila cells and allows rapid reassembly of microtubule (MT) arrays. Prolonged cooling of two types of cultured Drosophila cells, muscle cells in primary culture and a wing imaginal disc cell line Cl.8+ results in disassembly of MT arrays and induces the formation of clusters of short MTs that have not been described before. Upon rewarming, the clusters are lost and the MT array is re-established within 1 h. In Cl.8+ cells, gamma-tubulin-containing centrosomes are detected, both in cell extensions and in the expected juxtanuclear position, and gamma-tubulin co-localises with the cold-induced MT clusters. The MT plus-end-binding protein, Drosophila EB1, decorates growing tips of MTs extending from clusters. We conclude that the cold-induced MT clusters represent acentrosomal MTOCs, allowing rapid reassembly of MT arrays following exposure to cold.

  3. Cell adhesion and cortex contractility determine cell patterning in the Drosophila retina.

    PubMed

    Käfer, Jos; Hayashi, Takashi; Marée, Athanasius F M; Carthew, Richard W; Graner, François

    2007-11-20

    Because of the resemblance of many epithelial tissues to densely packed soap bubbles, it has been suggested that surface minimization, which drives soap bubble packing, could be governing cell packing as well. We test this by modeling the shape of the cells in a Drosophila retina ommatidium. We use the observed configurations and shapes in wild-type flies, as well as in flies with different numbers of cells per ommatidia, and mutants with cells where E- or N-cadherin is either deleted or misexpressed. We find that surface minimization is insufficient to model the experimentally observed shapes and packing of the cells based on their cadherin expression. We then consider a model in which adhesion leads to a surface increase, balanced by cell cortex contraction. Using the experimentally observed distributions of E- and N-cadherin, we simulate the packing and cell shapes in the wild-type eye. Furthermore, by changing only the corresponding parameters, this model can describe the mutants with different numbers of cells or changes in cadherin expression. PMID:18003929

  4. A simple approach for multicolor immunofluorescence staining in different Drosophila cell types.

    PubMed

    Cipressa, Francesca; Di Giorgio, Maria Laura; Cenci, Giovanni

    2014-06-01

    Multicolor immunostaining analysis is often a desirable tool in cell biology for most researchers. Nonetheless, this is not an easy task and often not affordable by many laboratories as it might require expensive instrumentation and sophisticated analysis software. Here, we describe a simple protocol for performing sequential immunostainings on two different Drosophila specimens. Our strategy relies on an efficient and reproducible method for removal primary antibodies and/or fluorophore-conjugated secondary antibodies that does not affect antigene integrity. We show that alternation of multiple rounds of antibody incubation and removal on the same slide, followed by registration of the same DAPI-stained image, provides a simple framework for the sequential detection of several antigens in the same cell. Given that the sample fixation procedures used for Drosophila tissues are compatible with most specimen processing protocols, we can envisage that the multicolor immunostaining strategy presented here can be also adapted to different samples including mammalian tissues and/or cells.

  5. Functional analysis of Drosophila HSP70 promoter with different HSE numbers in human cells.

    PubMed

    Kust, Nadezda; Rybalkina, Ekaterina; Mertsalov, Ilya; Savchenko, Ekaterina; Revishchin, Alexander; Pavlova, Gali

    2014-01-01

    The activation of genetic constructs including the Drosophila hsp70 promoter with four and eight HSE sequences in the regulatory region has been described in human cells. The promoter was shown to be induced at lower temperatures compared to the human hsp70 promoter. The promoter activity increased after a 60-min heat shock already at 38 °C in human cells. The promoter activation was observed 24 h after heat shock for the constructs with eight HSEs, while those with four HSEs required 48 h. After transplantation of in vitro heat-shocked transfected cells, the promoter activity could be maintained for 3 days with a gradual decline. The promoter activation was confirmed in vivo without preliminary heat shock in mouse ischemic brain foci. Controlled expression of the Gdnf gene under a Drosophila hsp70 promoter was demonstrated. This promoter with four and eight HSE sequences in the regulatory region can be proposed as a regulated promoter in genetic therapeutic systems.

  6. The Drosophila centriole - conversion of doublets into triplets within the stem cell niche.

    PubMed

    Gottardo, Marco; Callaini, Giuliano; Riparbelli, Maria Giovanna

    2015-07-15

    We report, here, that two distinct centriole lineages exist in Drosophila: somatic centrioles usually composed by microtubule doublets and germ line centrioles characterized by triplets. Remarkably, the transition from doublets to triplets in the testis occurs within the stem cell niche with the formation of the C-tubule. We demonstrated that the old mother centriole, which stays in the apical cytoplasm of the male germline stem cells (GSCs), is invariably composed of triplets, whereas its daughter is always built of mixed doublets and triplets. This difference represents the first documentation of a structural asymmetry between mother and daughter centrioles in Drosophila GSCs and might reflect a correlation between the architecture of parent centrioles and their ability to recruit centrosomal proteins. We also found that the old mother centriole is linked to the cell membrane by distinct projections that might play an important role in keeping its apical position during centrosome separation.

  7. Src kinase function controls progenitor cell pools during regeneration and tumor onset in the Drosophila intestine.

    PubMed

    Kohlmaier, A; Fassnacht, C; Jin, Y; Reuter, H; Begum, J; Dutta, D; Edgar, B A

    2015-04-30

    Src non-receptor kinases have been implicated in events late in tumor progression. Here, we study the role of Src kinases in the Drosophila intestinal stem cell (ISC) lineage, during tissue homeostasis and tumor onset. The adult Drosophila intestine contains only two progenitor cell types, division-capable ISCs and their daughters, postmitotic enteroblasts (EBs). We found that Drosophila Src42a and Src64b were required for optimal regenerative ISC division. Conversely, activation of Src42a, Src64b or another non-receptor kinase, Ack, promoted division of quiescent ISCs by coordinately stimulating G1/S and G2/M cell cycle phase progression. Prolonged Src kinase activation caused tissue overgrowth owing to cytokine receptor-independent Stat92E activation. This was not due to increased symmetric division of ISCs, but involved accumulation of weakly specified Notch(+) but division-capable EB-like cells. Src activation triggered expression of a mitogenic module consisting of String/Cdc25 and Cyclin E that was sufficient to elicit division not only of ISCs but also of EBs. A small pool of similarly division-capable transit-amplifying Notch(+) EBs was also identified in the wild type. Expansion of intermediate cell types that do not robustly manifest their transit-amplifying potential in the wild type may also contribute to regenerative growth and tumor development in other tissues in other organisms.

  8. Silver nanoparticles disrupt germline stem cell maintenance in the Drosophila testis

    NASA Astrophysics Data System (ADS)

    Ong, Cynthia; Lee, Qian Ying; Cai, Yu; Liu, Xiaoli; Ding, Jun; Yung, Lin-Yue Lanry; Bay, Boon-Huat; Baeg, Gyeong-Hun

    2016-02-01

    Silver nanoparticles (AgNPs), one of the most popular nanomaterials, are commonly used in consumer products and biomedical devices, despite their potential toxicity. Recently, AgNP exposure was reported to be associated with male reproductive toxicity in mammalian models. However, there is still a limited understanding of the effects of AgNPs on spermatogenesis. The fruit fly Drosophila testis is an excellent in vivo model to elucidate the mechanisms underlying AgNP-induced defects in spermatogenesis, as germ lineages can be easily identified and imaged. In this study, we evaluated AgNP-mediated toxicity on spermatogenesis by feeding Drosophila with AgNPs at various concentrations. We first observed a dose-dependent uptake of AgNPs in vivo. Concomitantly, AgNP exposure caused a significant decrease in the viability and delay in the development of Drosophila in a dose-dependent manner. Furthermore, AgNP-treated male flies showed a reduction in fecundity, and the resulting testes contained a decreased number of germline stem cells (GSCs) compared to controls. Interestingly, testes exposed to AgNPs exhibited a dramatic increase in reactive oxygen species levels and showed precocious GSC differentiation. Taken together, our study suggests that AgNP exposure may increase ROS levels in the Drosophila testis, leading to a reduction of GSC number by promoting premature GSC differentiation.

  9. Silver nanoparticles disrupt germline stem cell maintenance in the Drosophila testis

    PubMed Central

    Ong, Cynthia; Lee, Qian Ying; Cai, Yu; Liu, Xiaoli; Ding, Jun; Yung, Lin-Yue Lanry; Bay, Boon-Huat; Baeg, Gyeong-Hun

    2016-01-01

    Silver nanoparticles (AgNPs), one of the most popular nanomaterials, are commonly used in consumer products and biomedical devices, despite their potential toxicity. Recently, AgNP exposure was reported to be associated with male reproductive toxicity in mammalian models. However, there is still a limited understanding of the effects of AgNPs on spermatogenesis. The fruit fly Drosophila testis is an excellent in vivo model to elucidate the mechanisms underlying AgNP-induced defects in spermatogenesis, as germ lineages can be easily identified and imaged. In this study, we evaluated AgNP-mediated toxicity on spermatogenesis by feeding Drosophila with AgNPs at various concentrations. We first observed a dose-dependent uptake of AgNPs in vivo. Concomitantly, AgNP exposure caused a significant decrease in the viability and delay in the development of Drosophila in a dose-dependent manner. Furthermore, AgNP-treated male flies showed a reduction in fecundity, and the resulting testes contained a decreased number of germline stem cells (GSCs) compared to controls. Interestingly, testes exposed to AgNPs exhibited a dramatic increase in reactive oxygen species levels and showed precocious GSC differentiation. Taken together, our study suggests that AgNP exposure may increase ROS levels in the Drosophila testis, leading to a reduction of GSC number by promoting premature GSC differentiation. PMID:26847594

  10. Phagocytosis genes nonautonomously promote developmental cell death in the Drosophila ovary

    PubMed Central

    Timmons, Allison K.; Mondragon, Albert A.; Schenkel, Claire E.; Yalonetskaya, Alla; Taylor, Jeffrey D.; Moynihan, Katherine E.; Etchegaray, Jon Iker; Meehan, Tracy L.; McCall, Kimberly

    2016-01-01

    Programmed cell death (PCD) is usually considered a cell-autonomous suicide program, synonymous with apoptosis. Recent research has revealed that PCD is complex, with at least a dozen cell death modalities. Here, we demonstrate that the large-scale nonapoptotic developmental PCD in the Drosophila ovary occurs by an alternative cell death program where the surrounding follicle cells nonautonomously promote death of the germ line. The phagocytic machinery of the follicle cells, including Draper, cell death abnormality (Ced)-12, and c-Jun N-terminal kinase (JNK), is essential for the death and removal of germ-line–derived nurse cells during late oogenesis. Cell death events including acidification, nuclear envelope permeabilization, and DNA fragmentation of the nurse cells are impaired when phagocytosis is inhibited. Moreover, elimination of a small subset of follicle cells prevents nurse cell death and cytoplasmic dumping. Developmental PCD in the Drosophila ovary is an intriguing example of nonapoptotic, nonautonomous PCD, providing insight on the diversity of cell death mechanisms. PMID:26884181

  11. Phagocytosis genes nonautonomously promote developmental cell death in the Drosophila ovary.

    PubMed

    Timmons, Allison K; Mondragon, Albert A; Schenkel, Claire E; Yalonetskaya, Alla; Taylor, Jeffrey D; Moynihan, Katherine E; Etchegaray, Jon Iker; Meehan, Tracy L; McCall, Kimberly

    2016-03-01

    Programmed cell death (PCD) is usually considered a cell-autonomous suicide program, synonymous with apoptosis. Recent research has revealed that PCD is complex, with at least a dozen cell death modalities. Here, we demonstrate that the large-scale nonapoptotic developmental PCD in the Drosophila ovary occurs by an alternative cell death program where the surrounding follicle cells nonautonomously promote death of the germ line. The phagocytic machinery of the follicle cells, including Draper, cell death abnormality (Ced)-12, and c-Jun N-terminal kinase (JNK), is essential for the death and removal of germ-line-derived nurse cells during late oogenesis. Cell death events including acidification, nuclear envelope permeabilization, and DNA fragmentation of the nurse cells are impaired when phagocytosis is inhibited. Moreover, elimination of a small subset of follicle cells prevents nurse cell death and cytoplasmic dumping. Developmental PCD in the Drosophila ovary is an intriguing example of nonapoptotic, nonautonomous PCD, providing insight on the diversity of cell death mechanisms. PMID:26884181

  12. Phagocytosis genes nonautonomously promote developmental cell death in the Drosophila ovary.

    PubMed

    Timmons, Allison K; Mondragon, Albert A; Schenkel, Claire E; Yalonetskaya, Alla; Taylor, Jeffrey D; Moynihan, Katherine E; Etchegaray, Jon Iker; Meehan, Tracy L; McCall, Kimberly

    2016-03-01

    Programmed cell death (PCD) is usually considered a cell-autonomous suicide program, synonymous with apoptosis. Recent research has revealed that PCD is complex, with at least a dozen cell death modalities. Here, we demonstrate that the large-scale nonapoptotic developmental PCD in the Drosophila ovary occurs by an alternative cell death program where the surrounding follicle cells nonautonomously promote death of the germ line. The phagocytic machinery of the follicle cells, including Draper, cell death abnormality (Ced)-12, and c-Jun N-terminal kinase (JNK), is essential for the death and removal of germ-line-derived nurse cells during late oogenesis. Cell death events including acidification, nuclear envelope permeabilization, and DNA fragmentation of the nurse cells are impaired when phagocytosis is inhibited. Moreover, elimination of a small subset of follicle cells prevents nurse cell death and cytoplasmic dumping. Developmental PCD in the Drosophila ovary is an intriguing example of nonapoptotic, nonautonomous PCD, providing insight on the diversity of cell death mechanisms.

  13. Insulin-independent role of adiponectin receptor signaling in Drosophila germline stem cell maintenance.

    PubMed

    Laws, Kaitlin M; Sampson, Leesa L; Drummond-Barbosa, Daniela

    2015-03-15

    Adipocytes have key endocrine roles, mediated in large part by secreted protein hormones termed adipokines. The adipokine adiponectin is well known for its role in sensitizing peripheral tissues to insulin, and several lines of evidence suggest that adiponectin might also modulate stem cells/precursors. It remains unclear, however, how adiponectin signaling controls stem cells and whether this role is secondary to its insulin-sensitizing effects or distinct. Drosophila adipocytes also function as an endocrine organ and, although no obvious adiponectin homolog has been identified, Drosophila AdipoR encodes a well-conserved homolog of mammalian adiponectin receptors. Here, we generate a null AdipoR allele and use clonal analysis to demonstrate an intrinsic requirement for AdipoR in germline stem cell (GSC) maintenance in the Drosophila ovary. AdipoR null GSCs are not fully responsive to bone morphogenetic protein ligands from the niche and have a slight reduction in E-cadherin levels at the GSC-niche junction. Conversely, germline-specific overexpression of AdipoR inhibits natural GSC loss, suggesting that reduction in adiponectin signaling might contribute to the normal decline in GSC numbers observed over time in wild-type females. Surprisingly, AdipoR is not required for insulin sensitization of the germline, leading us to speculate that insulin sensitization is a more recently acquired function than stem cell regulation in the evolutionary history of adiponectin signaling. Our findings establish Drosophila female GSCs as a new system for future studies addressing the molecular mechanisms whereby adiponectin receptor signaling modulates stem cell fate.

  14. Fipronil induces apoptosis through caspase-dependent mitochondrial pathways in Drosophila S2 cells.

    PubMed

    Zhang, Baoyan; Xu, Zhiping; Zhang, Yixi; Shao, Xusheng; Xu, Xiaoyong; Cheng, Jiaogao; Li, Zhong

    2015-03-01

    Fipronil is the first phenylpyrazole insecticide widely used in controlling pests, including pyrethroid, organophosphate and carbamate insecticides. It is generally accepted that fipronil elicits neurotoxicity via interactions with GABA and glutamate receptors, although alternative mechanisms have recently been proposed. This study evaluates the genotoxicity of fipronil and its likely mode of action in Drosophila S2 cells, as an in vitro model. Fipronil administrated the concentration- and time-dependent S2 cell proliferation. Intracellular biochemical assays showed that fipronil-induced S2 cell apoptosis coincided with a decrease in the mitochondrial membrane potential and an increase reactive oxygen species generation, a significant decrease of Bcl-2 and DIAP1, and a marked augmentation of Cyt c and caspase-3. Because caspase-3 is the major executioner caspase downstream of caspase-9 in Drosophila, enzyme activity assays were used to determine the activities of caspase-3 and caspase-9. Our results indicated that fipronil effectively induced apoptosis in Drosophila S2 cells through caspase-dependent mitochondrial pathways.

  15. A Drosophila Reporter for the Translational Activation of ATF4 Marks Stressed Cells during Development.

    PubMed

    Kang, Kwonyoon; Ryoo, Hyung Don; Park, Jung-Eun; Yoon, Jee-Hyun; Kang, Min-Ji

    2015-01-01

    Eukaryotic cells have evolved signaling pathways that help to restore cellular homeostasis in response to various physiological or pathological conditions. ATF4 is a transcription factor whose mRNA translation is stimulated in response to stress-activated eIF2alpha kinases. Established conditions that activate eIF2alpha phosphorylation and ATF4 translation include excessive stress in the endoplasmic reticulum (ER) and amino acid deprivation. ATF4 is activated through a unique translational activation mechanism that involves multiple upstream open reading frames (uORFs) in the 5'-untranslated region (UTR), which is conserved from yeast to mammals. Taking advantage of this, we developed a translational activation reporter of ATF4 in Drosophila, in which the dsRed reporter coding sequence was placed downstream of the Drosophila ATF4 5' UTR. This reporter remained inactive in most tissues under normal conditions, but showed dsRed expression when starved, or when challenged with conditions that imposed ER stress. In normally developing flies, a small number of cell types showed reporter expression even without exogenous stress, which included the salivary gland, gut, the male reproductive organ, and the photoreceptor cells, suggestive of inherent stress during the normal development of these cell types. These results establish a new tool to study ATF4-mediated stress response in Drosophila development and disease.

  16. Enhancement of tendon-bone healing for anterior cruciate ligament (ACL) reconstruction using bone marrow-derived mesenchymal stem cells infected with BMP-2.

    PubMed

    Dong, Yu; Zhang, Qingguo; Li, Yunxia; Jiang, Jia; Chen, Shiyi

    2012-10-22

    At present, due to the growing attention focused on the issue of tendon-bone healing, we carried out an animal study of the use of genetic intervention combined with cell transplantation for the promotion of this process. Here, the efficacy of bone marrow stromal cells infected with bone morphogenetic protein-2 (BMP-2) on tendon-bone healing was determined. A eukaryotic expression vector containing the BMP-2 gene was constructed and bone marrow-derived mesenchymal stem cells (bMSCs) were infected with a lentivirus. Next, we examined the viability of the infected cells and the mRNA and protein levels of BMP-2-infected bMSCs. Gastrocnemius tendons, gastrocnemius tendons wrapped by bMSCs infected with the control virus (bMSCs+Lv-Control), and gastrocnemius tendons wrapped by bMSCs infected with the recombinant BMP-2 virus (bMSCs+Lv-BMP-2) were used to reconstruct the anterior cruciate ligament (ACL) in New Zealand white rabbits. Specimens from each group were harvested four and eight weeks postoperatively and evaluated using biomechanical and histological methods. The bMSCs were infected with the lentivirus at an efficiency close to 100%. The BMP-2 mRNA and protein levels in bMSCs were significantly increased after lentiviral infection. The bMSCs and BMP-2-infected bMSCs on the gastrocnemius tendon improved the biomechanical properties of the graft in the bone tunnel; specifically, bMSCs infected with BMP-2 had a positive effect on tendon-bone healing. In the four-week and eight-week groups, bMSCs+Lv-BMP-2 group exhibited significantly higher maximum loads of 29.3 ± 7.4 N and 45.5 ± 11.9 N, respectively, compared with the control group (19.9 ± 6.4 N and 21.9 ± 4.9 N) (P = 0.041 and P = 0.001, respectively). In the eight-week groups, the stiffness of the bMSCs+Lv-BMP-2 group (32.5 ± 7.3) was significantly higher than that of the bMSCs+Lv-Control group (22.8 ± 7.4) or control groups (12.4 ± 6.0) (p = 0.036 and 0.001, respectively). Based on the histological

  17. Functional assessment of gap junctions in monolayer and three-dimensional cultures of human tendon cells using fluorescence recovery after photobleaching

    PubMed Central

    Kuzma-Kuzniarska, Maria; Yapp, Clarence; Pearson-Jones, Thomas W.; Jones, Andrew K.; Hulley, Philippa A.

    2014-01-01

    Abstract. Gap junction-mediated intercellular communication influences a variety of cellular activities. In tendons, gap junctions modulate collagen production, are involved in strain-induced cell death, and are involved in the response to mechanical stimulation. The aim of the present study was to investigate gap junction-mediated intercellular communication in healthy human tendon-derived cells using fluorescence recovery after photobleaching (FRAP). The FRAP is a noninvasive technique that allows quantitative measurement of gap junction function in living cells. It is based on diffusion-dependent redistribution of a gap junction-permeable fluorescent dye. Using FRAP, we showed that human tenocytes form functional gap junctions in monolayer and three-dimensional (3-D) collagen I culture. Fluorescently labeled tenocytes following photobleaching rapidly reacquired the fluorescent dye from neighboring cells, while HeLa cells, which do not communicate by gap junctions, remained bleached. Furthermore, both 18 β-glycyrrhetinic acid and carbenoxolone, standard inhibitors of gap junction activity, impaired fluorescence recovery in tendon cells. In both monolayer and 3-D cultures, intercellular communication in isolated cells was significantly decreased when compared with cells forming many cell-to-cell contacts. In this study, we used FRAP as a tool to quantify and experimentally manipulate the function of gap junctions in human tenocytes in both two-dimensional (2-D) and 3-D cultures. PMID:24390370

  18. Functional assessment of gap junctions in monolayer and three-dimensional cultures of human tendon cells using fluorescence recovery after photobleaching

    NASA Astrophysics Data System (ADS)

    Kuzma-Kuzniarska, Maria; Yapp, Clarence; Pearson-Jones, Thomas W.; Jones, Andrew K.; Hulley, Philippa A.

    2014-01-01

    Gap junction-mediated intercellular communication influences a variety of cellular activities. In tendons, gap junctions modulate collagen production, are involved in strain-induced cell death, and are involved in the response to mechanical stimulation. The aim of the present study was to investigate gap junction-mediated intercellular communication in healthy human tendon-derived cells using fluorescence recovery after photobleaching (FRAP). The FRAP is a noninvasive technique that allows quantitative measurement of gap junction function in living cells. It is based on diffusion-dependent redistribution of a gap junction-permeable fluorescent dye. Using FRAP, we showed that human tenocytes form functional gap junctions in monolayer and three-dimensional (3-D) collagen I culture. Fluorescently labeled tenocytes following photobleaching rapidly reacquired the fluorescent dye from neighboring cells, while HeLa cells, which do not communicate by gap junctions, remained bleached. Furthermore, both 18 β-glycyrrhetinic acid and carbenoxolone, standard inhibitors of gap junction activity, impaired fluorescence recovery in tendon cells. In both monolayer and 3-D cultures, intercellular communication in isolated cells was significantly decreased when compared with cells forming many cell-to-cell contacts. In this study, we used FRAP as a tool to quantify and experimentally manipulate the function of gap junctions in human tenocytes in both two-dimensional (2-D) and 3-D cultures.

  19. Use of double-stranded RNA interference in Drosophila cell lines to dissect signal transduction pathways

    PubMed Central

    Clemens, James C.; Worby, Carolyn A.; Simonson-Leff, Nancy; Muda, Marco; Maehama, Tomohiko; Hemmings, Brian A.; Dixon, Jack E.

    2000-01-01

    We demonstrate the efficacy of double-stranded RNA-mediated interference (RNAi) of gene expression in generating “knock-out” phenotypes for specific proteins in several Drosophila cell lines. We prove the applicability of this technique for studying signaling cascades by dissecting the well-characterized insulin signal transduction pathway. Specifically, we demonstrate that inhibiting the expression of the DSOR1 (mitogen-activated protein kinase kinase, MAPKK) prevents the activation of the downstream ERK-A (MAPK). In contrast, blocking ERK-A expression results in increased activation of DSOR1. We also show that Drosophila AKT (DAKT) activation depends on the insulin receptor substrate, CHICO (IRS1–4). Finally, we demonstrate that blocking the expression of Drosophila PTEN results in the activation of DAKT. In all cases, the interference of the biochemical cascade by RNAi is consistent with the known steps in the pathway. We extend this powerful technique to study two proteins, DSH3PX1 and Drosophila ACK (DACK). DSH3PX1 is an SH3, phox homology domain-containing protein, and DACK is homologous to the mammalian activated Cdc42 tyrosine kinase, ACK. Using RNAi, we demonstrate that DACK is upstream of DSH3PX1 phosphorylation, making DSH3PX1 an identified downstream target/substrate of ACK-like tyrosine kinases. These experiments highlight the usefulness of RNAi in dissecting complex biochemical signaling cascades and provide a highly effective method for determining the function of the identified genes arising from the Drosophila genome sequencing project. PMID:10823906

  20. Trehalase regulates neuroepithelial stem cell maintenance and differentiation in the Drosophila optic lobe.

    PubMed

    Chen, Xi; Quan, Yaru; Wang, Hongbin; Luo, Hong

    2014-01-01

    As one of the major hydrolases in Drosophila, trehalase (Treh) catalyzes the hydrolysis of trehalose into glucose providing energy for flight muscle activity. Treh is highly conserved from bacteria to humans, but little is known about its function during animal development. Here, we analyze the function of Treh in Drosophila optic lobe development. In the optic lobe, neuroepithelial cells (NEs) first divide symmetrically to expand the stem cell pool and then differentiate into neuroblasts, which divide asymmetrically to generate medulla neurons. We find that the knockdown of Treh leads to a loss of the lamina and a smaller medulla. Analyses of Treh RNAi-expressing clones and loss-of-function mutants indicate that the lamina and medulla phenotypes result from neuroepithelial disintegration and premature differentiation into medulla neuroblasts. Although the principal role of Treh is to generate glucose, the Treh loss-of-function phenotype cannot be rescued by exogenous glucose. Thus, our results indicate that in addition to being a hydrolase, Treh plays a role in neuroepithelial stem cell maintenance and differentiation during Drosophila optic lobe development.

  1. Invasive and indigenous microbiota impact intestinal stem cell activity through multiple pathways in Drosophila

    PubMed Central

    Buchon, Nicolas; Broderick, Nichole A.; Chakrabarti, Sveta; Lemaitre, Bruno

    2009-01-01

    Gut homeostasis is controlled by both immune and developmental mechanisms, and its disruption can lead to inflammatory disorders or cancerous lesions of the intestine. While the impact of bacteria on the mucosal immune system is beginning to be precisely understood, little is known about the effects of bacteria on gut epithelium renewal. Here, we addressed how both infectious and indigenous bacteria modulate stem cell activity in Drosophila. We show that the increased epithelium renewal observed upon some bacterial infections is a consequence of the oxidative burst, a major defense of the Drosophila gut. Additionally, we provide evidence that the JAK–STAT (Janus kinase–signal transducers and activators of transcription) and JNK (c-Jun NH2 terminal kinase) pathways are both required for bacteria-induced stem cell proliferation. Similarly, we demonstrate that indigenous gut microbiota activate the same, albeit reduced, program at basal levels. Altered control of gut microbiota in immune-deficient or aged flies correlates with increased epithelium renewal. Finally, we show that epithelium renewal is an essential component of Drosophila defense against oral bacterial infection. Altogether, these results indicate that gut homeostasis is achieved by a complex interregulation of the immune response, gut microbiota, and stem cell activity. PMID:19797770

  2. Time markers for Drosophila morphogenesis based on cell-pattern topology.

    NASA Astrophysics Data System (ADS)

    Zallen, Richard; Zallen, Jennifer A.

    2007-03-01

    Recent work on convergent extension in Drosophila has shown that the accumulation of actin-myosin networks at specific cell interfaces initiates planar polarity and the formation of multicellular rosette structures that contribute to elongation of the body axis [1]. This cell-rearrangement process takes place within a one-cell-thick layer, and the changing two-dimensional cell pattern can be characterized using topological measures such as cell-shape statistics [2]. We find that the timeline for the process contains a well-defined marker corresponding to a sharp increase in the slope of the time dependence of the variance of the cell-shape (number-of-sides) distribution. A rosette in this context is a cluster of cells enclosing high-order vertices at which 4 or 5 or more cells meet. While the cell-shape variance climbs steadily during axis elongation, the frequency of high-order vertices and large rosettes plateaus after 10 and 13 minutes, respectively. These time markers calibrate the conventional timeline descriptors referred to as stages 7 and 8 of embryonic development [3]. [1] J.T. Blankenship et al., Developmental Cell 11, 459 (2006); [2] J.A. Zallen and R. Zallen, J. Phys.: Condensed Matter 16, S5073 (2004); [3] J.A. Campos-Ortega and V. Hartenstein, The embryonic development of Drosophila melanogaster (1985).

  3. Chick tendon fibroblast transcriptome and shape depend on whether the cell has made its own collagen matrix

    PubMed Central

    Yeung, Ching-Yan Chloé; Zeef, Leo A. H.; Lallyett, Chloe; Lu, Yinhui; Canty-Laird, Elizabeth G.; Kadler, Karl E.

    2015-01-01

    Collagen- and fibrin-based gels are extensively used to study cell behaviour. However, 2D–3D and collagen-fibrin comparisons of gene expression, cell shape and mechanotransduction, with an in vivo reference, have not been reported. Here we compared chick tendon fibroblasts (CTFs) at three stages of embryonic development with CTFs cultured in collagen- or fibrin-based tissue engineered constructs (TECs). CTFs synthesised their own collagen matrix in fibrin-based TECs and better recapitulated the gene expression, collagen fibril alignment and cell shape seen in vivo. In contrast, cells in 3D collagen gels exhibited a 2D-like morphology and expressed fewer of the genes expressed in vivo. Analysis of YAP/TAZ target genes showed that collagen gels desensitise mechanotransduction pathways. In conclusion, gene expression and cell shape are similar on plastic and 3D collagen whereas cells in 3D fibrin have a shape and transcriptome better resembling the in vivo situation. Implications for wound healing are discussed. PMID:26337655

  4. Dynamics of cell polarity in tissue morphogenesis: a comparative view from Drosophila and Ciona.

    PubMed

    Veeman, Michael T; McDonald, Jocelyn A

    2016-01-01

    Tissues in developing embryos exhibit complex and dynamic rearrangements that shape forming organs, limbs, and body axes. Directed migration, mediolateral intercalation, lumen formation, and other rearrangements influence the topology and topography of developing tissues. These collective cell behaviors are distinct phenomena but all involve the fine-grained control of cell polarity. Here we review recent findings in the dynamics of polarized cell behavior in both the Drosophila ovarian border cells and the Ciona notochord. These studies reveal the remarkable reorganization of cell polarity during organ formation and underscore conserved mechanisms of developmental cell polarity including the Par/atypical protein kinase C (aPKC) and planar cell polarity pathways. These two very different model systems demonstrate important commonalities but also key differences in how cell polarity is controlled in tissue morphogenesis. Together, these systems raise important, broader questions on how the developmental control of cell polarity contributes to morphogenesis of diverse tissues across the metazoa.

  5. Dynamics of cell polarity in tissue morphogenesis: a comparative view from Drosophila and Ciona

    PubMed Central

    Veeman, Michael T.; McDonald, Jocelyn A.

    2016-01-01

    Tissues in developing embryos exhibit complex and dynamic rearrangements that shape forming organs, limbs, and body axes. Directed migration, mediolateral intercalation, lumen formation, and other rearrangements influence the topology and topography of developing tissues. These collective cell behaviors are distinct phenomena but all involve the fine-grained control of cell polarity. Here we review recent findings in the dynamics of polarized cell behavior in both the Drosophila ovarian border cells and the Ciona notochord. These studies reveal the remarkable reorganization of cell polarity during organ formation and underscore conserved mechanisms of developmental cell polarity including the Par/atypical protein kinase C (aPKC) and planar cell polarity pathways. These two very different model systems demonstrate important commonalities but also key differences in how cell polarity is controlled in tissue morphogenesis. Together, these systems raise important, broader questions on how the developmental control of cell polarity contributes to morphogenesis of diverse tissues across the metazoa. PMID:27303647

  6. The Drosophila selectin furrowed mediates intercellular planar cell polarity interactions via frizzled stabilization.

    PubMed

    Chin, Mei-Ling; Mlodzik, Marek

    2013-09-16

    Establishment of planar cell polarity (PCP) in a tissue requires coordination of directional signals from cell to cell. It is thought that this is mediated by the core PCP factors, which include cell-adhesion molecules. Here, we demonstrate that furrowed, the Drosophila selectin, is required for PCP generation. Disruption of PCP in furrowed-deficient flies results from a primary defect in Fz levels and cell adhesion. Furrowed localizes at or near apical junctions, largely colocalizing with Frizzled and Flamingo (Fmi). It physically interacts with and stabilizes Frizzled, and it mediates intercellular Frizzled-Van Gogh (Vang)/Strabismus interactions, similarly to Fmi. Furrowed does so through a homophilic cell-adhesion role that is distinct from its known carbohydrate-binding function described during vertebrate blood-cell/endothelial cell interactions. Importantly, the carbohydrate function is dispensable for PCP establishment. In vivo studies suggest that Furrowed functions partially redundantly with Fmi, mediating intercellular Frizzled-Vang interactions between neighboring cells.

  7. Multipotent stem cells in the Malpighian tubules of adult Drosophila melanogaster

    PubMed Central

    Singh, Shree Ram; Hou, Steven X.

    2009-01-01

    Summary Excretion is an essential process of an organism's removal of the waste products of metabolism to maintain a constant chemical composition of the body fluids despite changes in the external environment. Excretion is performed by the kidneys in vertebrates and by Malpighian tubules (MTs) in Drosophila. The kidney serves as an excellent model organ to investigate the cellular and molecular mechanisms underlying organogenesis. Mammals and Drosophila share common principles of renal development. Tissue homeostasis, which is accomplished through self-renewal or differentiation of stem cells, is critical for the maintenance of adult tissues throughout the lifetime of an animal. Growing evidence suggests that stem cell self-renewal and differentiation is controlled by both intrinsic and extrinsic factors. Deregulation of stem cell behavior results in cancer formation, tissue degeneration, and premature aging. The mammalian kidney has a low rate of cellular turnover but has a great capacity for tissue regeneration following an ischemic injury. However, there is an ongoing controversy about the source of regenerating cells in the adult kidney that repopulate injured renal tissues. Recently, we identified multipotent stem cells in the MTs of adult Drosophila and found that these stem cells are able to proliferate and differentiate in several types of cells in MTs. Furthermore, we demonstrated that an autocrine JAK-STAT (Janus kinase–signal transducers and activators of transcription) signaling regulates stem cell self-renewal or differentiation of renal stem cells. The Drosophila MTs provide an excellent in vivo system for studying the renal stem cells at cellular and molecular levels. Understanding the molecular mechanisms governing stem cell self-renewal or differentiation in vivo is not only crucial to using stem cells for future regenerative medicine and gene therapy, but it also will increase our understanding of the mechanisms underlying cancer formation

  8. Drosophila cells use nanotube-like structures to transfer dsRNA and RNAi machinery between cells

    PubMed Central

    Karlikow, Margot; Goic, Bertsy; Mongelli, Vanesa; Salles, Audrey; Schmitt, Christine; Bonne, Isabelle; Zurzolo, Chiara; Saleh, Maria-Carla

    2016-01-01

    Tunnelling nanotubes and cytonemes function as highways for the transport of organelles, cytosolic and membrane-bound molecules, and pathogens between cells. During viral infection in the model organism Drosophila melanogaster, a systemic RNAi antiviral response is established presumably through the transport of a silencing signal from one cell to another via an unknown mechanism. Because of their role in cell-cell communication, we investigated whether nanotube-like structures could be a mediator of the silencing signal. Here, we describe for the first time in the context of a viral infection the presence of nanotube-like structures in different Drosophila cell types. These tubules, made of actin and tubulin, were associated with components of the RNAi machinery, including Argonaute 2, double-stranded RNA, and CG4572. Moreover, they were more abundant during viral, but not bacterial, infection. Super resolution structured illumination microscopy showed that Argonaute 2 and tubulin reside inside the tubules. We propose that nanotube-like structures are one of the mechanisms by which Argonaute 2, as part of the antiviral RNAi machinery, is transported between infected and non-infected cells to trigger systemic antiviral immunity in Drosophila. PMID:27255932

  9. Fibrin Gels Exhibit Improved Biological, Structural, and Mechanical Properties Compared with Collagen Gels in Cell-Based Tendon Tissue-Engineered Constructs

    PubMed Central

    Dyment, Nathaniel A.; Lu, Yinhui; Rao, Marepalli; Shearn, Jason T.; Rowe, David W.; Kadler, Karl E.; Butler, David L.

    2015-01-01

    The prevalence of tendon and ligament injuries and inadequacies of current treatments is driving the need for alternative strategies such as tissue engineering. Fibrin and collagen biopolymers have been popular materials for creating tissue-engineered constructs (TECs), as they exhibit advantages of biocompatibility and flexibility in construct design. Unfortunately, a few studies have directly compared these materials for tendon and ligament applications. Therefore, this study aims at determining how collagen versus fibrin hydrogels affect the biological, structural, and mechanical properties of TECs during formation in vitro. Our findings show that tendon and ligament progenitor cells seeded in fibrin constructs exhibit improved tenogenic gene expression patterns compared with their collagen-based counterparts for approximately 14 days in culture. Fibrin-based constructs also exhibit improved cell-derived collagen alignment, increased linear modulus (2.2-fold greater) compared with collagen-based constructs. Cyclic tensile loading, which promotes the maturation of tendon constructs in a previous work, exhibits a material-dependent effect in this study. Fibrin constructs show trending reductions in mechanical, biological, and structural properties, whereas collagen constructs only show improved tenogenic expression in the presence of mechanical stimulation. These findings highlight that components of the mechanical stimulus (e.g., strain amplitude or time of initiation) need to be tailored to the material and cell type. Given the improvements in tenogenic expression, extracellular matrix organization, and material properties during static culture, in vitro findings presented here suggest that fibrin-based constructs may be a more suitable alternative to collagen-based constructs for tissue-engineered tendon/ligament repair. PMID:25266738

  10. Fibrin gels exhibit improved biological, structural, and mechanical properties compared with collagen gels in cell-based tendon tissue-engineered constructs.

    PubMed

    Breidenbach, Andrew P; Dyment, Nathaniel A; Lu, Yinhui; Rao, Marepalli; Shearn, Jason T; Rowe, David W; Kadler, Karl E; Butler, David L

    2015-02-01

    The prevalence of tendon and ligament injuries and inadequacies of current treatments is driving the need for alternative strategies such as tissue engineering. Fibrin and collagen biopolymers have been popular materials for creating tissue-engineered constructs (TECs), as they exhibit advantages of biocompatibility and flexibility in construct design. Unfortunately, a few studies have directly compared these materials for tendon and ligament applications. Therefore, this study aims at determining how collagen versus fibrin hydrogels affect the biological, structural, and mechanical properties of TECs during formation in vitro. Our findings show that tendon and ligament progenitor cells seeded in fibrin constructs exhibit improved tenogenic gene expression patterns compared with their collagen-based counterparts for approximately 14 days in culture. Fibrin-based constructs also exhibit improved cell-derived collagen alignment, increased linear modulus (2.2-fold greater) compared with collagen-based constructs. Cyclic tensile loading, which promotes the maturation of tendon constructs in a previous work, exhibits a material-dependent effect in this study. Fibrin constructs show trending reductions in mechanical, biological, and structural properties, whereas collagen constructs only show improved tenogenic expression in the presence of mechanical stimulation. These findings highlight that components of the mechanical stimulus (e.g., strain amplitude or time of initiation) need to be tailored to the material and cell type. Given the improvements in tenogenic expression, extracellular matrix organization, and material properties during static culture, in vitro findings presented here suggest that fibrin-based constructs may be a more suitable alternative to collagen-based constructs for tissue-engineered tendon/ligament repair.

  11. Drosophila comes of age as a model system for understanding the function of cytoskeletal proteins in cells, tissues, and organisms.

    PubMed

    Rodal, Avital A; Del Signore, Steven J; Martin, Adam C

    2015-05-01

    For the last 100 years, Drosophila melanogaster has been a powerhouse genetic system for understanding mechanisms of inheritance, development, and behavior in animals. In recent years, advances in imaging and genetic tools have led to Drosophila becoming one of the most effective systems for unlocking the subcellular functions of proteins (and particularly cytoskeletal proteins) in complex developmental settings. In this review, written for non-Drosophila experts, we will discuss critical technical advances that have enabled these cell biological insights, highlighting three examples of cytoskeletal discoveries that have arisen as a result: (1) regulation of Arp2/3 complex in myoblast fusion, (2) cooperation of the actin filament nucleators Spire and Cappuccino in establishment of oocyte polarity, and (3) coordination of supracellular myosin cables. These specific examples illustrate the unique power of Drosophila both to uncover new cytoskeletal structures and functions, and to place these discoveries in a broader in vivo context, providing insights that would have been impossible in a cell culture model or in vitro. Many of the cellular structures identified in Drosophila have clear counterparts in mammalian cells and tissues, and therefore elucidating cytoskeletal functions in Drosophila will be broadly applicable to other organisms.

  12. Establishing and maintaining cell polarity with mRNA localization in Drosophila.

    PubMed

    Barr, Justinn; Yakovlev, Konstantin V; Shidlovskii, Yulii; Schedl, Paul

    2016-03-01

    How cell polarity is established and maintained is an important question in diverse biological contexts. Molecular mechanisms used to localize polarity proteins to distinct domains are likely context-dependent and provide a feedback loop in order to maintain polarity. One such mechanism is the localized translation of mRNAs encoding polarity proteins, which will be the focus of this review and may play a more important role in the establishment and maintenance of polarity than is currently known. Localized translation of mRNAs encoding polarity proteins can be used to establish polarity in response to an external signal, and to maintain polarity by local production of polarity determinants. The importance of this mechanism is illustrated by recent findings, including orb2-dependent localized translation of aPKC mRNA at the apical end of elongating spermatid tails in the Drosophila testis, and the apical localization of stardust A mRNA in Drosophila follicle and embryonic epithelia.

  13. Cold-sensing regulates Drosophila growth through insulin-producing cells

    PubMed Central

    Li, Qiaoran; Gong, Zhefeng

    2015-01-01

    Across phyla, body size is linked to climate. For example, rearing fruit flies at lower temperatures results in bigger body sizes than those observed at higher temperatures. The underlying molecular basis of this effect is poorly understood. Here we provide evidence that the temperature-dependent regulation of Drosophila body size depends on a group of cold-sensing neurons and insulin-producing cells (IPCs). Electrically silencing IPCs completely abolishes the body size increase induced by cold temperature. IPCs are directly innervated by cold-sensing neurons. Stimulation of these cold-sensing neurons activates IPCs, promotes synthesis and secretion of Drosophila insulin-like peptides and induces a larger body size, mimicking the effects of rearing the flies in cold temperature. Taken together, these findings reveal a neuronal circuit that mediates the effects of low temperature on fly growth. PMID:26648410

  14. Knockout of Drosophila RNase ZL impairs mitochondrial transcript processing, respiration and cell cycle progression.

    PubMed

    Xie, Xie; Dubrovsky, Edward B

    2015-12-01

    RNase Z(L) is a highly conserved tRNA 3'-end processing endoribonuclease. Similar to its mammalian counterpart, Drosophila RNase Z(L) (dRNaseZ) has a mitochondria targeting signal (MTS) flanked by two methionines at the N-terminus. Alternative translation initiation yields two protein forms: the long one is mitochondrial, and the short one may localize in the nucleus or cytosol. Here, we have generated a mitochondria specific knockout of the dRNaseZ gene. In this in vivo model, cells deprived of dRNaseZ activity display impaired mitochondrial polycistronic transcript processing, increased reactive oxygen species (ROS) and a switch to aerobic glycolysis compensating for cellular ATP. Damaged mitochondria impose a cell cycle delay at the G2 phase disrupting cell proliferation without affecting cell viability. Antioxidants attenuate genotoxic stress and rescue cell proliferation, implying a critical role for ROS. We suggest that under a low-stress condition, ROS activate tumor suppressor p53, which modulates cell cycle progression and promotes cell survival. Transcriptional profiling of p53 targets confirms upregulation of antioxidant and cycB-Cdk1 inhibitor genes without induction of apoptotic genes. This study implicates Drosophila RNase Z(L) in a novel retrograde signaling pathway initiated by the damage in mitochondria and manifested in a cell cycle delay before the mitotic entry.

  15. Knockout of Drosophila RNase ZL impairs mitochondrial transcript processing, respiration and cell cycle progression

    PubMed Central

    Xie, Xie; Dubrovsky, Edward B.

    2015-01-01

    RNase ZL is a highly conserved tRNA 3′-end processing endoribonuclease. Similar to its mammalian counterpart, Drosophila RNase ZL (dRNaseZ) has a mitochondria targeting signal (MTS) flanked by two methionines at the N-terminus. Alternative translation initiation yields two protein forms: the long one is mitochondrial, and the short one may localize in the nucleus or cytosol. Here, we have generated a mitochondria specific knockout of the dRNaseZ gene. In this in vivo model, cells deprived of dRNaseZ activity display impaired mitochondrial polycistronic transcript processing, increased reactive oxygen species (ROS) and a switch to aerobic glycolysis compensating for cellular ATP. Damaged mitochondria impose a cell cycle delay at the G2 phase disrupting cell proliferation without affecting cell viability. Antioxidants attenuate genotoxic stress and rescue cell proliferation, implying a critical role for ROS. We suggest that under a low-stress condition, ROS activate tumor suppressor p53, which modulates cell cycle progression and promotes cell survival. Transcriptional profiling of p53 targets confirms upregulation of antioxidant and cycB-Cdk1 inhibitor genes without induction of apoptotic genes. This study implicates Drosophila RNase ZL in a novel retrograde signaling pathway initiated by the damage in mitochondria and manifested in a cell cycle delay before the mitotic entry. PMID:26553808

  16. Centrosome-dependent asymmetric inheritance of the midbody ring in Drosophila germline stem cell division.

    PubMed

    Salzmann, Viktoria; Chen, Cuie; Chiang, C-Y Ason; Tiyaboonchai, Amita; Mayer, Michael; Yamashita, Yukiko M

    2014-01-01

    Many stem cells, including Drosophila germline stem cells (GSCs), divide asymmetrically, producing one stem cell and one differentiating daughter. Cytokinesis is often asymmetric, in that only one daughter cell inherits the midbody ring (MR) upon completion of abscission even in apparently symmetrically dividing cells. However, whether the asymmetry in cytokinesis correlates with cell fate or has functional relevance has been poorly explored. Here we show that the MR is asymmetrically segregated during GSC divisions in a centrosome age-dependent manner: male GSCs, which inherit the mother centrosome, exclude the MR, whereas female GSCs, which we here show inherit the daughter centrosome, inherit the MR. We further show that stem cell identity correlates with the mode of MR inheritance. Together our data suggest that the MR does not inherently dictate stem cell identity, although its stereotypical inheritance is under the control of stemness and potentially provides a platform for asymmetric segregation of certain factors.

  17. Visualization of TGN-endosome trafficking in mammalian and Drosophila cells.

    PubMed

    Kametaka, Satoshi; Waguri, Satoshi

    2012-01-01

    Mannose 6-phosphate receptors (MPRs) are known to be shuttled between the trans-Golgi network (TGN) and endosomes, thereby several lysosomal hydrolases are delivered through the endocytic pathway into lysosomes. This interorganellar transport is mediated by transport intermediates, now called transport carriers. Previous studies employing green fluorescent protein (GFP)-based live-cell imaging demonstrated that these transport carriers are pleiomorphic structures composed of tubular and vesicular elements. Introducing a time-axis into light microscopic observations enabled us to identify transport carriers that are derived from or targeted at a distinct organelle. In this study, we describe several methods for the observation of GFP-tagged MPRs. Photobleaching the peripheral region of a cell before a time-lapse observation allows us to monitor TGN-derived transport carriers for longer periods (more than 4min). Events of their targeting into endosomes can be visualized by dual-color imaging of both GFP-MPRs and fluorescently tagged transferrin that is internalized by cells. By using a technique of fluorescence recovery after photobleaching (FRAP), we can analyze overall cycling kinetics of MPRs in a single cell. Transport of MPRs is regulated by several cytosolic factors like clathrin adaptors, AP1, and GGAs. The adaptors on the TGN membranes are exchanging with their cytosolic pool, which can also be analyzed by FRAP. In addition, the relationships of the MPR-containing transport carriers that left the TGN and the adaptors can be visualized by dual-color imaging. A similar system of membrane transport and its regulation is well documented in drosophila cells. As Drosophila melanogaster has only a single MPR (LERP), AP1, or GGA, it is an ideal model system for the understanding of specific functions of each cytosolic factor. To visualize these molecules in drosophila cells, however, we need to consider that multiple Golgi dots exist scattered in the cytoplasm. Thus

  18. Ultrastructural and autoradiographic investigations of cell cultures derived from tendons or ligamentous material from patients with fibromatous disorders.

    PubMed

    Neumüller, J; Tohidast-Akrad, M; Ammer, K; Hakimzadeh, A; Stransky, G; Weis, S; Partsch, G; Eberl, R

    1988-01-01

    Cell cultures were derived from tendons or ligamentous material from patients with carpal tunnel syndrome (CTS), Dupuytren's contracture (DP), tendopathia nodosa (TN) and hallux valgus (HV). The ultrastructure of the operation specimens as well as of the cell monolayers was investigated, using a floating sheet method in order to preserve both cell-to-cell contacts and the orientation of the monolayers. The histologic features of the tissues obtained in the operations were correlated with the ultrastructure of the cells in culture derived from these specimens. In DP, above all in the nodules, an activation of the capillary endothelium in the vicinity of myofibroblasts and mast cells was observed. In CTS the collagen fibrils varied extremely in diameter. In DP and TN biopsies a splicing process of helicoidly arranged fibrils could be seen. A disintegration of elastic fibers in the fibrillar and amorphous components was found in DP nodules, HV and TN tissues. Transitional forms between fibroblasts and myofibroblasts were observed not only in DP but also-though in a smaller percentage--in the cultures derived from the other patients. The cells showed organelles for active protein synthesis and transport. Autophagocytosis and the formation of multilamellated bodies took place in TN and HV cultures. In CTS, DP and TN cultures cells were connected via gap junctions. In some cultures, above all in those derived from CTS, monocilia were found. In CTS cultures the formation of intracellular collagen occurred. Growth parameters were rather low in HV cultures. PLmax (maximal pulse labelling index) values were higher in TN cultures than in DP and HV cultures. Plating efficiency (PE) values were higher in cultures derived from cell-rich and capillarized tissues than in biopsies with few cells. PMID:3229549

  19. Ttk69 acts as a master repressor of enteroendocrine cell specification in Drosophila intestinal stem cell lineages.

    PubMed

    Wang, Chenhui; Guo, Xingting; Dou, Kun; Chen, Hongyan; Xi, Rongwen

    2015-10-01

    In adult Drosophila midgut, intestinal stem cells (ISCs) periodically produce progenitor cells that undergo a binary fate choice determined primarily by the levels of Notch activity that they receive, before terminally differentiating into enterocytes (ECs) or enteroendocrine (EE) cells. Here we identified Ttk69, a BTB domain-containing transcriptional repressor, as a master repressor of EE cell specification in the ISC lineages. Depletion of ttk69 in progenitor cells induced ISC proliferation and caused all committed progenitor cells to adopt EE fate, leading to the production of supernumerary EE cells in the intestinal epithelium. Conversely, forced expression of Ttk69 in progenitor cells was sufficient to prevent EE cell specification. The expression of Ttk69 was not regulated by Notch signaling, and forced activation of Notch, which is sufficient to induce EC specification of normal progenitor cells, failed to prevent EE cell specification of Ttk69-depleted progenitors. Loss of Ttk69 led to derepression of the acheate-scute complex (AS-C) genes scute and asense, which then induced prospero expression to promote EE cell specification. These studies suggest that Ttk69 functions in parallel with Notch signaling and acts as a master repressor of EE cell specification in Drosophila ISC lineages primarily by suppressing AS-C genes.

  20. Chinmo is sufficient to induce male fate in somatic cells of the adult Drosophila ovary.

    PubMed

    Ma, Qing; de Cuevas, Margaret; Matunis, Erika L

    2016-03-01

    Sexual identity is continuously maintained in specific differentiated cell types long after sex determination occurs during development. In the adult Drosophila testis, the putative transcription factor Chronologically inappropriate morphogenesis (Chinmo) acts with the canonical male sex determinant DoublesexM (Dsx(M)) to maintain the male identity of somatic cyst stem cells and their progeny. Here we find that ectopic expression of chinmo is sufficient to induce a male identity in adult ovarian somatic cells, but it acts through a Dsx(M)-independent mechanism. Conversely, the feminization of the testis somatic stem cell lineage caused by loss of chinmo is enhanced by expression of the canonical female sex determinant Dsx(F), indicating that chinmo acts in parallel with the canonical sex determination pathway to maintain the male identity of testis somatic cells. Consistent with this finding, ectopic expression of female sex determinants in the adult testis disrupts tissue morphology. The miRNA let-7 downregulates chinmo in many contexts, and ectopic expression of let-7 in the adult testis is sufficient to recapitulate the chinmo loss-of-function phenotype, but we find no apparent phenotypes upon removal of let-7 in the adult ovary or testis. Our finding that chinmo is necessary and sufficient to promote a male identity in adult gonadal somatic cells suggests that the sexual identity of somatic cells can be reprogrammed in the adult Drosophila ovary as well as in the testis. PMID:26811385

  1. A novel Drosophila Girdin-like protein is involved in Akt pathway control of cell size

    SciTech Connect

    Puseenam, Aekkachai; Yoshioka, Yasuhide; Nagai, Rika; Hashimoto, Reina; Suyari, Osamu; Itoh, Masanobu; Enomoto, Atsushi; Takahashi, Masahide; Yamaguchi, Masamitsu

    2009-11-15

    The Akt signaling pathway is well known to regulate cell proliferation and growth. Girdin, a novel substrate of Akt, plays a crucial role in organization of the actin cytoskeleton and cell motility under the control of Akt. We here identified a novel Girdin-like protein in Drosophila (dGirdin), which has two isoforms, dGirdin PA and dGirdin PB. dGirdin shows high homology with human Girdin in the N-terminal and coiled-coil domains, while diverging at the C-terminal domain. On establishment of transgenic fly lines, featuring knockdown or overexpression of dGirdin in vivo, overexpression in the wing disc cells induced ectopic apoptosis, implying a role in directing apoptosis. Knockdown of dGirdin in the Drosophila wing imaginal disc cells resulted in reduction of cell size. Furthermore, this was enhanced by half reduction of the Akt gene dose, suggesting that Akt positively regulates dGirdin. In the wing disc, cells in which dGirdin was knocked down exhibited disruption of actin filaments. From these in vivo analyses, we conclude that dGirdin is required for actin organization and regulation of appropriate cell size under control of the Akt signaling pathway.

  2. Characterization of Drosophila mini-me, a Gene Required for Cell Proliferation and Survival

    PubMed Central

    Jones, Chonnettia; Reifegerste, Rita; Moses, Kevin

    2006-01-01

    In the developing Drosophila eye, the morphogenetic furrow is a developmental organizing center for patterning and cell proliferation. The furrow acts both to limit eye size and to coordinate the number of cells to the number of facets. Here we report the molecular and functional characterization of Drosophila mini-me (mnm), a potential regulator of cell proliferation and survival in the developing eye. We first identified mnm as a dominant modifier of hedgehog loss-of-function in the developing eye. We report that mnm encodes a conserved protein with zinc knuckle and RING finger domains. We show that mnm is dispensable for patterning of the eye disc, but required in the eye for normal cell proliferation and survival. We also show that mnm null mutant cells exhibit altered cell cycle profiles and contain excess nucleic acid. Moreover, mnm overexpression can induce cells to proliferate and incorporate BrdU. Thus, our data implicate mnm as a regulator of mitotic progression during the proliferative phase of eye development, possibly through the control of nucleic acid metabolism. PMID:16547096

  3. Conserved enhancer and silencer elements responsible for differential Adh transcription in Drosophila cell lines.

    PubMed Central

    Ayer, S; Benyajati, C

    1990-01-01

    The distal promoter of Adh is differentially expressed in Drosophila tissue culture cell lines. After transfection with an exogenous Adh gene, there was a specific increase in distal alcohol dehydrogenase (ADH) transcripts in ADH-expressing (ADH+) cells above the levels observed in transfected ADH-nonexpressing (ADH-) cells. We used deletion mutations and a comparative transient-expression assay to identify the cis-acting elements responsible for enhanced Adh distal transcription in ADH+ cells. DNA sequences controlling high levels of distal transcription were localized to a 15-base-pair (bp) region nearly 500 bp upstream of the distal RNA start site. In addition, a 61-bp negative cis-acting element was found upstream from and adjacent to the enhancer. When this silencer element was deleted, distal transcription increased only in the ADH+ cell line. These distant upstream elements must interact with the promoter elements, the Adf-1-binding site and the TATA box, as they only influenced transcription when at least one of these two positive distal promoter elements was present. Internal deletions targeted to the Adf-1-binding site or the TATA box reduced transcription in both cell types but did not affect the transcription initiation site. Distal transcription in transfected ADH- cells appears to be controlled primarily through these promoter elements and does not involve the upstream regulatory elements. Evolutionary conservation in distantly related Drosophila species suggests the importance of these upstream elements in correct developmental and tissue-specific expression of ADH. Images PMID:1694013

  4. Drosophila Abelson kinase mediates cell invasion and proliferation via two distinct MAPK pathways

    PubMed Central

    Singh, Jaskirat; Aaronson, Stuart A.; Mlodzik, Marek

    2010-01-01

    The Abelson (Abl) family of non-receptor tyrosine kinases plays important role in cell morphogenesis, motility and proliferation. Although the function of Abl has been extensively studied in leukemia, its role in epithelial cell invasion remains obscure. Using the Drosophila wing epithelium as an in-vivo model system, we demonstrate that overexpression (activation) of Drosophila Abl (dAbl) causes loss of epithelial apical/basal cell polarity and secretion of matrix metalloproteinases, resulting in a cellular invasion and apoptosis. Our in vivo data indicate that dAbl acts downstream of the Src kinases, which are known regulators of cell adhesion and invasion. Downstream of dAbl, Rac GTPases activate two distinct MAPK pathways: JNK signaling (required for cell invasion and apoptosis) and ERK signaling (inducing cell proliferation). Activated Abl also increases the activity of Src members through a positive feedback loop leading to signal amplification. Thus targeting Src-Abl, using available dual inhibitors, could be of therapeutic importance in tumor cell metastasis. PMID:20453880

  5. Structure-function relationships in tendons: a review

    PubMed Central

    Benjamin, M; Kaiser, E; Milz, S

    2008-01-01

    The purpose of the current review is to highlight the structure-function relationship of tendons and related structures to provide an overview for readers whose interest in tendons needs to be underpinned by anatomy. Because of the availability of several recent reviews on tendon development and entheses, the focus of the current work is primarily directed towards what can best be described as the ‘tendon proper’ or the ‘mid-substance’ of tendons. The review covers all levels of tendon structure from the molecular to the gross and deals both with the extracellular matrix and with tendon cells. The latter are often called ‘tenocytes’ and are increasingly recognized as a defined cell population that is functionally and phenotypically distinct from other fibroblast-like cells. This is illustrated by their response to different types of mechanical stress. However, it is not only tendon cells, but tendons as a whole that exhibit distinct structure-function relationships geared to the changing mechanical stresses to which they are subject. This aspect of tendon biology is considered in some detail. Attention is briefly directed to the blood and nerve supply of tendons, for this is an important issue that relates to the intrinsic healing capacity of tendons. Structures closely related to tendons (joint capsules, tendon sheaths, pulleys, retinacula, fat pads and bursae) are also covered and the concept of a ‘supertendon’ is introduced to describe a collection of tendons in which the function of the whole complex exceeds that of its individual members. Finally, attention is drawn to the important relationship between tendons and fascia, highlighted by Wood Jones in his concept of an ‘ectoskeleton’ over half a century ago – work that is often forgotten today. PMID:18304204

  6. Contribution of growth and cell cycle checkpoints to radiation survival in Drosophila.

    PubMed

    Jaklevic, Burnley; Uyetake, Lyle; Lemstra, Willy; Chang, Julia; Leary, William; Edwards, Anthony; Vidwans, Smruti; Sibon, Ody; Tin Su, Tin

    2006-12-01

    Cell cycle checkpoints contribute to survival after exposure to ionizing radiation (IR) by arresting the cell cycle and permitting repair. As such, yeast and mammalian cells lacking checkpoints are more sensitive to killing by IR. We reported previously that Drosophila larvae mutant for grp (encoding a homolog of Chk1) survive IR as well as wild type despite being deficient in cell cycle checkpoints. This discrepancy could be due to differences either among species or between unicellular and multicellular systems. Here, we provide evidence that Grapes is needed for survival of Drosophila S2 cells after exposure to similar doses of IR, suggesting that multicellular organisms may utilize checkpoint-independent mechanisms to survive irradiation. The dispensability of checkpoints in multicellular organisms could be due to replacement of damaged cells by regeneration through increased nutritional uptake and compensatory proliferation. In support of this idea, we find that inhibition of nutritional uptake (by starvation or onset of pupariation) or inhibition of growth factor signaling and downstream targets (by mutations in cdk4, chico, or dmyc) reduced the radiation survival of larvae. Further, some of these treatments are more detrimental for grp mutants, suggesting that the need for compensatory proliferation is greater for checkpoint mutants. The difference in survival of grp and wild-type larvae allowed us to screen for small molecules that act as genotype-specific radiation sensitizers in a multicellular context. A pilot screen of a small molecule library from the National Cancer Institute yielded known and approved radio-sensitizing anticancer drugs. Since radiation is a common treatment option for human cancers, we propose that Drosophila may be used as an in vivo screening tool for genotype-specific drugs that enhance the effect of radiation therapy. PMID:17028317

  7. Drosophila as a model to study the role of blood cells in inflammation, innate immunity and cancer

    PubMed Central

    Wang, Lihui; Kounatidis, Ilias; Ligoxygakis, Petros

    2014-01-01

    Drosophila has a primitive yet effective blood system with three types of haemocytes which function throughout different developmental stages and environmental stimuli. Haemocytes play essential roles in tissue modeling during embryogenesis and morphogenesis, and also in innate immunity. The open circulatory system of Drosophila makes haemocytes ideal signal mediators to cells and tissues in response to events such as infection and wounding. The application of recently developed and sophisticated genetic tools to the relatively simple genome of Drosophila has made the fly a popular system for modeling human tumorigensis and metastasis. Drosophila is now used for screening and investigation of genes implicated in human leukemia and also in modeling development of solid tumors. This second line of research offers promising opportunities to determine the seemingly conflicting roles of blood cells in tumor progression and invasion. This review provides an overview of the signaling pathways conserved in Drosophila during haematopoiesis, haemostasis, innate immunity, wound healing and inflammation. We also review the most recent progress in the use of Drosophila as a cancer research model with an emphasis on the roles haemocytes can play in various cancer models and in the links between inflammation and cancer. PMID:24409421

  8. A feedback mechanism converts individual cell features into a supracellular ECM structure in Drosophila trachea.

    PubMed

    Öztürk-Çolak, Arzu; Moussian, Bernard; Araújo, Sofia J; Casanova, Jordi

    2016-02-02

    The extracellular matrix (ECM), a structure contributed to and commonly shared by many cells in an organism, plays an active role during morphogenesis. Here, we used the Drosophila tracheal system to study the complex relationship between the ECM and epithelial cells during development. We show that there is an active feedback mechanism between the apical ECM (aECM) and the apical F-actin in tracheal cells. Furthermore, we reveal that cell-cell junctions are key players in this aECM patterning and organisation and that individual cells contribute autonomously to their aECM. Strikingly, changes in the aECM influence the levels of phosphorylated Src42A (pSrc) at cell junctions. Therefore, we propose that Src42A phosphorylation levels provide a link for the ECM environment to ensure proper cytoskeletal organisation.

  9. A feedback mechanism converts individual cell features into a supracellular ECM structure in Drosophila trachea

    PubMed Central

    Öztürk-Çolak, Arzu; Moussian, Bernard; Araújo, Sofia J; Casanova, Jordi

    2016-01-01

    The extracellular matrix (ECM), a structure contributed to and commonly shared by many cells in an organism, plays an active role during morphogenesis. Here, we used the Drosophila tracheal system to study the complex relationship between the ECM and epithelial cells during development. We show that there is an active feedback mechanism between the apical ECM (aECM) and the apical F-actin in tracheal cells. Furthermore, we reveal that cell-cell junctions are key players in this aECM patterning and organisation and that individual cells contribute autonomously to their aECM. Strikingly, changes in the aECM influence the levels of phosphorylated Src42A (pSrc) at cell junctions. Therefore, we propose that Src42A phosphorylation levels provide a link for the ECM environment to ensure proper cytoskeletal organisation. DOI: http://dx.doi.org/10.7554/eLife.09373.001 PMID:26836303

  10. Enhancer modeling uncovers transcriptional signatures of individual cardiac cell states in Drosophila

    PubMed Central

    Busser, Brian W.; Haimovich, Julian; Huang, Di; Ovcharenko, Ivan; Michelson, Alan M.

    2015-01-01

    Here we used discriminative training methods to uncover the chromatin, transcription factor (TF) binding and sequence features of enhancers underlying gene expression in individual cardiac cells. We used machine learning with TF motifs and ChIP data for a core set of cardiogenic TFs and histone modifications to classify Drosophila cell-type-specific cardiac enhancer activity. We show that the classifier models can be used to predict cardiac cell subtype cis-regulatory activities. Associating the predicted enhancers with an expression atlas of cardiac genes further uncovered clusters of genes with transcription and function limited to individual cardiac cell subtypes. Further, the cell-specific enhancer models revealed chromatin, TF binding and sequence features that distinguish enhancer activities in distinct subsets of heart cells. Collectively, our results show that computational modeling combined with empirical testing provides a powerful platform to uncover the enhancers, TF motifs and gene expression profiles which characterize individual cardiac cell fates. PMID:25609699

  11. Dpp-expressing and non-expressing cells: two different populations of growing cells in Drosophila.

    PubMed

    Arias, Carolina; Fussero, Gimena; Zacharonok, Marcelo; Macías, Ana

    2015-01-01

    There are different models that explain growth during development. One model is based on insect and amphibian regeneration studies. This model proposes that growth is directed by pattern, and growth takes place by intercalation at a growth discontinuity; therefore, proliferation should surround the discontinuity. Currently, this model, apart from regenerative studies on mostly adult patterning, has not found supporting evidence in Drosophila that shows proliferation surrounding a discontinuity. Despite this lack of evidence, the importance of discontinuities has been shown in different experiments, even under wt conditions, more specifically in the formation of the leg joints because of the occurrence of cell death at their boundaries. Here, we show the existence of a sharp discontinuity in Decapentaplegic (Dpp) in the genital discs at the third larvae stage (L3), which determines the upregulation in the Jun-NH2-Terminal-Kinase (JNK) pathway, reaper (rpr), head involution defective (hid) and active caspases from its boundaries. The proliferation and cell death surrounding the discontinuity suggest that growth can proceed by intercalation and competitive death takes place in this area. Finally, we show that the Rpr, Grim and Hid (RGH) products are a few of the factors that define the growth discontinuity because they are negative regulators of growth, a new function that is unique from their known functions in apoptosis.

  12. A dynamic population of stromal cells contributes to the follicle stem cell niche in the Drosophila ovary

    PubMed Central

    Sahai-Hernandez, Pankaj; Nystul, Todd G.

    2013-01-01

    Epithelial stem cells are maintained within niches that promote self-renewal by providing signals that specify the stem cell fate. In the Drosophila ovary, epithelial follicle stem cells (FSCs) reside in niches at the anterior tip of the tissue and support continuous growth of the ovarian follicle epithelium. Here, we demonstrate that a neighboring dynamic population of stromal cells, called escort cells, are FSC niche cells. We show that escort cells produce both Wingless and Hedgehog ligands for the FSC lineage, and that Wingless signaling is specific for the FSC niche whereas Hedgehog signaling is active in both FSCs and daughter cells. In addition, we show that multiple escort cells simultaneously encapsulate germ cell cysts and contact FSCs. Thus, FSCs are maintained in a dynamic niche by a non-dedicated population of niche cells. PMID:24131631

  13. Spen is required for pigment cell survival during pupal development in Drosophila.

    PubMed

    Querenet, Matthieu; Goubard, Valerie; Chatelain, Gilles; Davoust, Nathalie; Mollereau, Bertrand

    2015-06-15

    Apoptosis is required during development to eliminate superfluous cells and sculpt tissues; spatial and timed control of apoptosis ensures that the necessary number of cells is eliminated at a precise time in a given tissue. The elimination of supernumerary pigment or inter-ommatidial cells (IOCs) depends on cell-cell communication and is necessary for the formation of the honeycomb-like structure of the Drosophila eye. However, the mechanisms occurring during pupal development and controlling apoptosis of superfluous IOC in space and time remain unclear. Here, we found that split-ends (spen) is required for IOC survival at the time of removal of superfluous IOCs. Loss of spen function leads to abnormal removal of IOCs by apoptosis. We show that spen is required non-autonomously in cone cells for the survival of IOCs by positively regulating the Spitz/EGFR pathway. We propose that Spen is an important survival factor that ensures spatial control of the apoptotic wave that is necessary for the correct patterning and formation of the Drosophila eye.

  14. Effect of heat shock on poly(ADP-ribose) synthetase and DNA repair in Drosophila cells

    SciTech Connect

    Nolan, N.L.; Kidwell, W.R.

    1982-04-01

    Poly(ADP-ribose) synthetase, a chromatin-bound enzyme which attaches polyanionic chains of ADP-ribose to nuclear proteins, was found to be temperature sensitive in intact Drosophila melanogaster cells. The synthetase was completely inactivated by heat-shocking the cells at 37/sup 0/C for 5 min, a condition which had no appreciable effect on the subsequent growth of Drosophila cells at their physiological temperature. The heat-shock effect on synthetase was reversible; enzyme activity began to reappear about 2 hr post heat shock. During the 2-hr interval when poly(ADP-ribose) synthetase was absent, the cells were competent in repair of ..gamma..-ray-induced DNA strand breaks as shown by DNA sedimentation studies on alkaline sucrose gradients. It is thus concluded that poly(ADP-ribose) synthesis is unnecessary for repair of DNA strand breaks introduced by irradiation. The same conclusion was reached from the fact that two inhibitors of poly(ADP-ribose) synthetase 3-aminobenzamide and 5-methylnicotinamide, failed to block repair of ..gamma..-ray-induced DNA chain breaks even though both inhibitors reduced the amount of poly(ADP-ribose) synthesized in cells by 50-75%. Although it was found that the repair of DNA strand breaks is independent of poly(ADP-ribose) synthesis, irradiation does activate the synthetase in control cells, as shown by radioimmunoassay of poly(ADP-ribose) levels.

  15. GATAe regulates intestinal stem cell maintenance and differentiation in Drosophila adult midgut.

    PubMed

    Okumura, Takashi; Takeda, Koji; Kuchiki, Megumi; Akaishi, Marie; Taniguchi, Kiichiro; Adachi-Yamada, Takashi

    2016-02-01

    Adult intestinal tissues, exposed to the external environment, play important roles including barrier and nutrient-absorption functions. These functions are ensured by adequately controlled rapid-cell metabolism. GATA transcription factors play essential roles in the development and maintenance of adult intestinal tissues both in vertebrates and invertebrates. We investigated the roles of GATAe, the Drosophila intestinal GATA factor, in adult midgut homeostasis with its first-generated knock-out mutant as well as cell type-specific RNAi and overexpression experiments. Our results indicate that GATAe is essential for proliferation and maintenance of intestinal stem cells (ISCs). Also, GATAe is involved in the differentiation of enterocyte (EC) and enteroendocrine (ee) cells in both Notch (N)-dependent and -independent manner. The results also indicate that GATAe has pivotal roles in maintaining normal epithelial homeostasis of the Drosophila adult midgut through interaction of N signaling. Since recent reports showed that mammalian GATA-6 regulates normal and cancer stem cells in the adult intestinal tract, our data also provide information on the evolutionally conserved roles of GATA factors in stem-cell regulation. PMID:26719127

  16. From pathogens to microbiota: How Drosophila intestinal stem cells react to gut microbes.

    PubMed

    Bonfini, Alessandro; Liu, Xi; Buchon, Nicolas

    2016-11-01

    The intestine acts as one of the interfaces between an organism and its external environment. As the primary digestive organ, it is constantly exposed to a multitude of stresses as it processes and absorbs nutrients. Among these is the recurring damage induced by ingested pathogenic and commensal microorganisms. Both the bacterial activity and immune response itself can result in the loss of epithelial cells, which subsequently requires replacement. In the Drosophila midgut, this regenerative role is fulfilled by intestinal stem cells (ISCs). Microbes not only trigger cell loss and replacement, but also modify intestinal and whole organism physiology, thus modulating ISC activity. Regulation of ISCs is integrated through a complex network of signaling pathways initiated by other gut cell populations, including enterocytes, enteroblasts, enteroendocrine and visceral muscles cells. The gut also receives signals from circulating immune cells, the hemocytes, to properly respond against infection. This review summarizes the types of gut microbes found in Drosophila, mechanisms for their elimination, and provides an integrated view of the signaling pathways that regulate tissue renewal in the midgut.

  17. Asrij maintains the stem cell niche and controls differentiation during Drosophila lymph gland hematopoiesis.

    PubMed

    Kulkarni, Vani; Khadilkar, Rohan J; Magadi, Srivathsa S; Srivathsa, M S; Inamdar, Maneesha S

    2011-01-01

    Several signaling pathways control blood cell (hemocyte) development in the Drosophila lymph gland. Mechanisms that modulate and integrate these signals are poorly understood. Here we report that mutation in a conserved endocytic protein Asrij affects signal transmission and causes aberrant lymph gland hematopoiesis. Mammalian Asrij (Ociad1) is expressed in stem cells of the blood vascular system and is implicated in several cancers. We found that Drosophila Asrij is a pan-hemocyte marker and localizes to a subset of endocytic vesicles. Loss of asrij causes hyperproliferation of lymph gland lobes coupled with increased hemocyte differentiation, thereby depleting the pool of quiescent hemocyte precursors. This co-relates with fewer Col+ cells in the hematopoietic stem cell niche of asrij mutants. Asrij null mutants also show excess specification of crystal cells that express the RUNX factor Lozenge (Lz), a target of Notch signaling. Asrij mutant lymph glands show increased N in sorting endosomes suggesting aberrant trafficking. In vitro assays also show impaired traffic of fluorescent probes in asrij null hemocytes. Taken together our data suggest a role for Asrij in causing increased Notch signaling thereby affecting hemocyte differentiation. Thus, conserved endocytic functions may control blood cell progenitor quiescence and differentiation. PMID:22110713

  18. A Systems-Level Interrogation Identifies Regulators of Drosophila Blood Cell Number and Survival

    PubMed Central

    Makhijani, Kalpana; Alexander, Brandy; Perrimon, Norbert; Brückner, Katja

    2015-01-01

    In multicellular organisms, cell number is typically determined by a balance of intracellular signals that positively and negatively regulate cell survival and proliferation. Dissecting these signaling networks facilitates the understanding of normal development and tumorigenesis. Here, we study signaling by the Drosophila PDGF/VEGF Receptor (Pvr) in embryonic blood cells (hemocytes) and in the related cell line Kc as a model for the requirement of PDGF/VEGF receptors in vertebrate cell survival and proliferation. The system allows the investigation of downstream and parallel signaling networks, based on the ability of Pvr to activate Ras/Erk, Akt/TOR, and yet-uncharacterized signaling pathway/s, which redundantly mediate cell survival and contribute to proliferation. Using Kc cells, we performed a genome wide RNAi screen for regulators of cell number in a sensitized, Pvr deficient background. We identified the receptor tyrosine kinase (RTK) Insulin-like receptor (InR) as a major Pvr Enhancer, and the nuclear hormone receptors Ecdysone receptor (EcR) and ultraspiracle (usp), corresponding to mammalian Retinoid X Receptor (RXR), as Pvr Suppressors. In vivo analysis in the Drosophila embryo revealed a previously unrecognized role for EcR to promote apoptotic death of embryonic blood cells, which is balanced with pro-survival signaling by Pvr and InR. Phosphoproteomic analysis demonstrates distinct modes of cell number regulation by EcR and RTK signaling. We define common phosphorylation targets of Pvr and InR that include regulators of cell survival, and unique targets responsible for specialized receptor functions. Interestingly, our analysis reveals that the selection of phosphorylation targets by signaling receptors shows qualitative changes depending on the signaling status of the cell, which may have wide-reaching implications for other cell regulatory systems. PMID:25749252

  19. A histological study of macroscopically normal equine digital flexor tendons.

    PubMed

    Webbon, P M

    1978-10-01

    The normal appearance of the superficial (SFT) and deep (DFT) digital flexor tendons was described and the difference between their histological structures was emphasised. Further differences were recognised between different sites from the same tendon and between tendons in the fore and hind limbs of the same animal. Both of the tendons underwent changes with age but although a number of alterations in the histological appearance were described, a particular change, involving a patchy loss of stainable nuclei, was found at the common site of SFT injuries. While this appearance has been seen in injured tendons and described as tendon necrosis, the author warns that no cause and effect relationship has been established between the acellularity and the clinical lesions. Neither is it certain that the loss of tendon cells results in mechanical weakness of the tendon.

  20. Preparation and characterization of decellularized tendon slices for tendon tissue engineering.

    PubMed

    Ning, Liang-Ju; Zhang, Yi; Chen, Xiao-He; Luo, Jing-Cong; Li, Xiu-Qun; Yang, Zhi-Ming; Qin, Ting-Wu

    2012-06-01

    To develop a naturally derived tendon tissue engineering scaffold with the preservation of the native ultrastructure, tensile strength, and biochemical composition of the tendon extracellular matrix (ECM), decellularized tendon slices (DTSs) were prepared using repetitive freeze/thaw of the intact Achilles tendons, frozen section, and nuclease treatment. The DTSs were characterized in the native ultrastructure, mechanical properties, biochemical composition, and cytocompatibility. Histological examination and DNA quantification analysis confirmed that cells were completely removed from tendon tissue by repetitive freeze/thaw in combination with nuclease treatment 12 h. The intrinsic ultrastructure of tendon tissue was well preserved based on scanning electron microscopy examination. The tensile strength of the DTSs was retained 85.62% of native tendon slice. More than 93% of proteoglycans (fibromodulin, biglycan) and growth factors (TGF-β1, IGF-1, VEGF, and CTGF) inherent in tendon ECM were preserved in the DTSs according to ELISA analysis. Furthermore, the DTSs facilitated attachment and repopulation of NIH-3T3 fibroblasts in vitro. Overall, the DTSs are sheet scaffolds with a combination of elemental mechanical strength and tendon ECM bioactive factors that may have many potential applications in tendon tissue engineering.

  1. Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

    PubMed Central

    Rickert, Christof; Kunz, Thomas; Harris, Kerri-Lee; Whitington, Paul; Technau, Gerhard

    2013-01-01

    In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution. PMID:23486245

  2. An Aminopeptidase in the Drosophila Testicular Niche Acts in Germline Stem Cell Maintenance and Spermatogonial Dedifferentiation.

    PubMed

    Lim, Cindy; Gandhi, Shiv; Biniossek, Martin L; Feng, Lijuan; Schilling, Oliver; Urban, Siniša; Chen, Xin

    2015-10-13

    Extrinsic cues from the niche are known to regulate adult stem cell self-renewal versus differentiation. Here, we report that an aminopeptidase Slamdance (Sda) acts in the Drosophila testicular niche to maintain germline stem cells (GSCs) and regulate progenitor germ cell dedifferentiation. Mutations in sda lead to dramatic testicular niche deterioration and stem cell loss. Recombinant Sda has specific aminopeptidase activity in vitro, and the in vivo function of Sda requires an intact aminopeptidase domain. Sda is required for accumulation of mature DE-cadherin, and overexpression of DE-cadherin rescues most sda mutant phenotypes, suggesting that DE-cadherin is an important target of Sda. Finally, Sda is both necessary and sufficient to promote dedifferentiation during aging and recovery from genetically manipulated depletion of GSCs. Together, our results suggest that a niche factor promotes both stem cell maintenance and progenitor cell dedifferentiation.

  3. An Aminopeptidase in the Drosophila Testicular Niche Acts in Germline Stem Cell Maintenance and Spermatogonial Dedifferentiation.

    PubMed

    Lim, Cindy; Gandhi, Shiv; Biniossek, Martin L; Feng, Lijuan; Schilling, Oliver; Urban, Siniša; Chen, Xin

    2015-10-13

    Extrinsic cues from the niche are known to regulate adult stem cell self-renewal versus differentiation. Here, we report that an aminopeptidase Slamdance (Sda) acts in the Drosophila testicular niche to maintain germline stem cells (GSCs) and regulate progenitor germ cell dedifferentiation. Mutations in sda lead to dramatic testicular niche deterioration and stem cell loss. Recombinant Sda has specific aminopeptidase activity in vitro, and the in vivo function of Sda requires an intact aminopeptidase domain. Sda is required for accumulation of mature DE-cadherin, and overexpression of DE-cadherin rescues most sda mutant phenotypes, suggesting that DE-cadherin is an important target of Sda. Finally, Sda is both necessary and sufficient to promote dedifferentiation during aging and recovery from genetically manipulated depletion of GSCs. Together, our results suggest that a niche factor promotes both stem cell maintenance and progenitor cell dedifferentiation. PMID:26440886

  4. Tendon Vasculature in Health and Disease

    PubMed Central

    Tempfer, Herbert; Traweger, Andreas

    2015-01-01

    Tendons represent a bradytrophic tissue which is poorly vascularized and, compared to bone or skin, heal poorly. Usually, a vascularized connective scar tissue with inferior functional properties forms at the injury site. Whether the increased vascularization is the root cause of tissue impairments such as loss of collagen fiber orientation, ectopic formation of bone, fat or cartilage, or is a consequence of these pathological changes remains unclear. This review provides an overview of the role of tendon vasculature in healthy and chronically diseased tendon tissue as well as its relevance for tendon repair. Further, the nature and the role of perivascular tendon stem/progenitor cells residing in the vascular niche will be discussed and compared to multipotent stromal cells in other tissues. PMID:26635616

  5. Conversion of quiescent niche cells to somatic stem cells causes ectopic niche formation in the Drosophila testis

    PubMed Central

    Hétié, Phylis; de Cuevas, Margaret; Matunis, Erika

    2014-01-01

    Summary Adult stem cells reside in specialized regulatory microenvironments, or niches, where local signals ensure stem cell maintenance. The Drosophila testis contains a well-characterized niche wherein signals from post-mitotic hub cells promote maintenance of adjacent germline stem cells and somatic cyst stem cells (CySCs). Hub cells were considered to be terminally differentiated; here we show that they can give rise to CySCs. Genetic ablation of CySCs triggers hub cells to transiently exit quiescence, delaminate from the hub, and convert into functional CySCs. Ectopic Cyclin D-Cdk4 expression in hub cells is also sufficient to trigger their conversion into CySCs. In both cases, this conversion causes the formation of multiple ectopic niches over time. Therefore, our work provides a model for understanding how oncogenic mutations in quiescent niche cells could promote loss of quiescence, changes in cell fate, and aberrant niche expansion more generally. PMID:24746819

  6. Mononuclear muscle cells in Drosophila ovaries revealed by GFP protein traps

    PubMed Central

    Hudson, Andrew M.; Petrella, Lisa N.; Tanaka, Akemi J.; Cooley, Lynn

    2008-01-01

    Genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into human muscle physiology and disease. Studies in Drosophila have been particularly useful for discovering key genes involved in muscle specification, myoblast fusion, and sarcomere organization. The muscles of the Drosophila female reproductive system have received little attention despite extensive work on oogenesis. We have used newly available GFP protein trap lines to characterize of ovarian muscle morphology and sarcomere organization. The muscle cells surrounding the oviducts are multinuclear with highly organized sarcomeres typical of somatic muscles. In contrast, the two muscle layers of the ovary, which are derived from gonadal mesoderm, have a mesh-like morphology similar to gut visceral muscle. Protein traps in the Fasciclin 3 gene produced Fas3::GFP that localized in dots around the periphery of epithelial sheath cells, the muscle surrounding ovarioles. Surprisingly, the epithelial sheath cells each contain a single nucleus, indicating these cells do not undergo myoblast fusion during development. Consistent with this observation, we were able to use the Flp/FRT system to efficiently generate genetic mosaics in the epithelial sheath, suggesting these cells provide a new opportunity for clonal analysis of adult striated muscle. PMID:18199432

  7. The Molecular Chaperone Hsp90 Is Required for Cell Cycle Exit in Drosophila melanogaster

    PubMed Central

    Bandura, Jennifer L.; Jiang, Huaqi; Nickerson, Derek W.; Edgar, Bruce A.

    2013-01-01

    The coordination of cell proliferation and differentiation is crucial for proper development. In particular, robust mechanisms exist to ensure that cells permanently exit the cell cycle upon terminal differentiation, and these include restraining the activities of both the E2F/DP transcription factor and Cyclin/Cdk kinases. However, the full complement of mechanisms necessary to restrain E2F/DP and Cyclin/Cdk activities in differentiating cells are not known. Here, we have performed a genetic screen in Drosophila melanogaster, designed to identify genes required for cell cycle exit. This screen utilized a PCNA-miniwhite+ reporter that is highly E2F-responsive and results in a darker red eye color when crossed into genetic backgrounds that delay cell cycle exit. Mutation of Hsp83, the Drosophila homolog of mammalian Hsp90, results in increased E2F-dependent transcription and ectopic cell proliferation in pupal tissues at a time when neighboring wild-type cells are postmitotic. Further, these Hsp83 mutant cells have increased Cyclin/Cdk activity and accumulate proteins normally targeted for proteolysis by the anaphase-promoting complex/cyclosome (APC/C), suggesting that APC/C function is inhibited. Indeed, reducing the gene dosage of an inhibitor of Cdh1/Fzr, an activating subunit of the APC/C that is required for timely cell cycle exit, can genetically suppress the Hsp83 cell cycle exit phenotype. Based on these data, we propose that Cdh1/Fzr is a client protein of Hsp83. Our results reveal that Hsp83 plays a heretofore unappreciated role in promoting APC/C function during cell cycle exit and suggest a mechanism by which Hsp90 inhibition could promote genomic instability and carcinogenesis. PMID:24086162

  8. The molecular chaperone Hsp90 is required for cell cycle exit in Drosophila melanogaster.

    PubMed

    Bandura, Jennifer L; Jiang, Huaqi; Nickerson, Derek W; Edgar, Bruce A

    2013-01-01

    The coordination of cell proliferation and differentiation is crucial for proper development. In particular, robust mechanisms exist to ensure that cells permanently exit the cell cycle upon terminal differentiation, and these include restraining the activities of both the E2F/DP transcription factor and Cyclin/Cdk kinases. However, the full complement of mechanisms necessary to restrain E2F/DP and Cyclin/Cdk activities in differentiating cells are not known. Here, we have performed a genetic screen in Drosophila melanogaster, designed to identify genes required for cell cycle exit. This screen utilized a PCNA-miniwhite(+) reporter that is highly E2F-responsive and results in a darker red eye color when crossed into genetic backgrounds that delay cell cycle exit. Mutation of Hsp83, the Drosophila homolog of mammalian Hsp90, results in increased E2F-dependent transcription and ectopic cell proliferation in pupal tissues at a time when neighboring wild-type cells are postmitotic. Further, these Hsp83 mutant cells have increased Cyclin/Cdk activity and accumulate proteins normally targeted for proteolysis by the anaphase-promoting complex/cyclosome (APC/C), suggesting that APC/C function is inhibited. Indeed, reducing the gene dosage of an inhibitor of Cdh1/Fzr, an activating subunit of the APC/C that is required for timely cell cycle exit, can genetically suppress the Hsp83 cell cycle exit phenotype. Based on these data, we propose that Cdh1/Fzr is a client protein of Hsp83. Our results reveal that Hsp83 plays a heretofore unappreciated role in promoting APC/C function during cell cycle exit and suggest a mechanism by which Hsp90 inhibition could promote genomic instability and carcinogenesis. PMID:24086162

  9. Drosophila Condensin II subunit Chromosome-associated protein D3 regulates cell fate determination through non-cell-autonomous signaling

    PubMed Central

    Klebanow, Lindsey R.; Peshel, Emanuela C.; Schuster, Andrew T.; De, Kuntal; Sarvepalli, Kavitha; Lemieux, Madeleine E.; Lenoir, Jessica J.; Moore, Adrian W.; McDonald, Jocelyn A.

    2016-01-01

    The pattern of the Drosophila melanogaster adult wing is heavily influenced by the expression of proteins that dictate cell fate decisions between intervein and vein during development. dSRF (Blistered) expression in specific regions of the larval wing disc promotes intervein cell fate, whereas EGFR activity promotes vein cell fate. Here, we report that the chromatin-organizing protein CAP-D3 acts to dampen dSRF levels at the anterior/posterior boundary in the larval wing disc, promoting differentiation of cells into the anterior crossvein. CAP-D3 represses KNOT expression in cells immediately adjacent to the anterior/posterior boundary, thus blocking KNOT-mediated repression of EGFR activity and preventing cell death. Maintenance of EGFR activity in these cells depresses dSRF levels in the neighboring anterior crossvein progenitor cells, allowing them to differentiate into vein cells. These findings uncover a novel transcriptional regulatory network influencing Drosophila wing vein development, and are the first to identify a Condensin II subunit as an important regulator of EGFR activity and cell fate determination in vivo. PMID:27317808

  10. Cell Competition Drives the Growth of Intestinal Adenomas in Drosophila.

    PubMed

    Suijkerbuijk, Saskia J E; Kolahgar, Golnar; Kucinski, Iwo; Piddini, Eugenia

    2016-02-22

    Tumor-host interactions play an increasingly recognized role in modulating tumor growth. Thus, understanding the nature and impact of this complex bidirectional communication is key to identifying successful anti-cancer strategies. It has been proposed that tumor cells compete with and kill neighboring host tissue to clear space that they can expand into; however, this has not been demonstrated experimentally. Here we use the adult fly intestine to investigate the existence and characterize the role of competitive tumor-host interactions. We show that APC(-/-)-driven intestinal adenomas compete with and kill surrounding cells, causing host tissue attrition. Importantly, we demonstrate that preventing cell competition, by expressing apoptosis inhibitors, restores host tissue growth and contains adenoma expansion, indicating that cell competition is essential for tumor growth. We further show that JNK signaling is activated inside the tumor and in nearby tissue and is required for both tumor growth and cell competition. Lastly, we find that APC(-/-) cells display higher Yorkie (YAP) activity than host cells and that this promotes tumor growth, in part via cell competition. Crucially, we find that relative, rather than absolute, Hippo activity determines adenoma growth. Overall, our data indicate that the intrinsic over-proliferative capacity of APC(-/-) cells is not uncontrolled and can be constrained by host tissues if cell competition is inhibited, suggesting novel possible therapeutic approaches.

  11. Cell Competition Drives the Growth of Intestinal Adenomas in Drosophila

    PubMed Central

    Suijkerbuijk, Saskia J.E.; Kolahgar, Golnar; Kucinski, Iwo; Piddini, Eugenia

    2016-01-01

    Summary Tumor-host interactions play an increasingly recognized role in modulating tumor growth. Thus, understanding the nature and impact of this complex bidirectional communication is key to identifying successful anti-cancer strategies. It has been proposed that tumor cells compete with and kill neighboring host tissue to clear space that they can expand into; however, this has not been demonstrated experimentally. Here we use the adult fly intestine to investigate the existence and characterize the role of competitive tumor-host interactions. We show that APC−/−-driven intestinal adenomas compete with and kill surrounding cells, causing host tissue attrition. Importantly, we demonstrate that preventing cell competition, by expressing apoptosis inhibitors, restores host tissue growth and contains adenoma expansion, indicating that cell competition is essential for tumor growth. We further show that JNK signaling is activated inside the tumor and in nearby tissue and is required for both tumor growth and cell competition. Lastly, we find that APC−/− cells display higher Yorkie (YAP) activity than host cells and that this promotes tumor growth, in part via cell competition. Crucially, we find that relative, rather than absolute, Hippo activity determines adenoma growth. Overall, our data indicate that the intrinsic over-proliferative capacity of APC−/− cells is not uncontrolled and can be constrained by host tissues if cell competition is inhibited, suggesting novel possible therapeutic approaches. PMID:26853366

  12. Morphogenesis of Drosophila melanogaster macrochaetes: cell fate determination for bristle organ.

    PubMed

    Furman, D P; Bukharina, T A

    2012-01-01

    Formation of specialized spatial structures comprising various cell types is most important in the ontogenesis of multicellular organisms. An example is the D. melanogaster bristle organs. Bristles (micro- and macrochaetes) are external sensory organs, elements of the peripheral nervous system, playing the role of mechanoreceptors. Their comparatively simple organization comprising only four specialized cells and a common origin of these cells make macrochaetes a convenient model for studying cell differentiation. The four cells forming bristle organ result from two successive divisions of a single cell, sensory organ precursor (SOP) cell. The number of macrochaetes on drosophila body corresponds to the number of SOP cells. The morphogenesis of macrochaetes comprises three stages, the first two determining a neural fate of the cells. The third stage is cell specialization into components of the bristle organ-neuron, thecogen, tormogen, and trichogen. Development of each bristle commences from segregation of proneural clusters, of 20-30 cells, from the massif of undifferentiated cells of the wing imaginal disc. At this stage, each cluster cell can potentially become a SOP cell. At the second stage, the only SOP cell and its position are determined within each cluster. Finally, two asymmetric divisions of the SOP cell with subsequent differentiation of the daughter cells gives the bristle organ. Several dozens genes are involved in the control of macrochaete morphogenesis. The main component of this system is the proneural genes of achaete-scute complex (AS-C). An increased content of proneural proteins fundamentally distinguished the cells that will follow the neural developmental pathway from the disc epidermal cells. A local AS-C expression, initiated at specified disc sites by specific transcription factors, determines the number and topology of proneural clusters. The expression of AS-C genes, continuing in the cells of the cluster, increases the difference in

  13. Structure-mechanics relationships in mineralized tendons.

    PubMed

    Spiesz, Ewa M; Zysset, Philippe K

    2015-12-01

    In this paper, we review the hierarchical structure and the resulting elastic properties of mineralized tendons as obtained by various multiscale experimental and computational methods spanning from nano- to macroscale. The mechanical properties of mineralized collagen fibres are important to understand the mechanics of hard tissues constituted by complex arrangements of these fibres, like in human lamellar bone. The uniaxial mineralized collagen fibre array naturally occurring in avian tendons is a well studied model tissue for investigating various stages of tissue mineralization and the corresponding elastic properties. Some avian tendons mineralize with maturation, which results in a graded structure containing two zones of distinct morphology, circumferential and interstitial. These zones exhibit different amounts of mineral, collagen, pores and a different mineral distribution between collagen fibrillar and extrafibrillar space that lead to distinct elastic properties. Mineralized tendon cells have two phenotypes: elongated tenocytes placed between fibres in the circumferential zone and cuboidal cells with lower aspect ratios in the interstitial zone. Interestingly some regions of avian tendons seem to be predestined to mineralization, which is exhibited as specific collagen cross-linking patterns as well as distribution of minor tendon constituents (like proteoglycans) and loss of collagen crimp. Results of investigations in naturally mineralizing avian tendons may be useful in understanding the pathological mineralization occurring in some human tendons.

  14. Human hamstring tenocytes survive when seeded into a decellularized porcine Achilles tendon extracellular matrix.

    PubMed

    Lohan, Anke; Stoll, Christiane; Albrecht, Marit; Denner, Andreas; John, Thilo; Krüger, Kay; Ertel, Wolfgang; Schulze-Tanzil, Gundula

    2013-01-01

    Tendon ruptures and defects remain major orthopaedic challenges. Tendon healing is a time-consuming process, which results in scar tissue with an altered biomechanical competence. Using a xenogeneic tendon extracellular matrix (ECM) as a natural scaffold, which can be reseeded with autologous human tenocytes, might be a promising approach to reconstruct damaged tendons. For this purpose, the porcine Achilles (AS) tendons serving as a scaffold were histologically characterized in comparison to human cell donor tendons. AS tendons were decellularized and then reseeded with primary human hamstring tenocytes using cell centrifuging, rotating culture and cell injection techniques. Vitality testing, histology and glycosaminoglycan/DNA quantifications were performed to document the success of tendon reseeding. Porcine AS tendons were characterized by a higher cell and sulfated glycosaminoglycan content than human cell donor tendons. Complete decellularization could be achieved, but led to a wash out of sulfated glycosaminoglycans. Nevertheless, porcine tendon could be recellularized with vital human tenocytes. The recellularization led to a slight increase in cell number compared to the native tendon and some glycosaminoglycan recovery. This study indicates that porcine tendon can be de- and recellularized using adult human tenocytes. Future work should optimize cell distribution within the recellularized tendon ECM and consider tendon- and donor species-dependent differences.

  15. Hedgehog does not guide migrating Drosophila germ cells

    PubMed Central

    Renault, Andrew D.; Ricardo, Sara; Kunwar, Prabhat S.; Santos, Ana; Starz-Gaiano, Michelle; Stein, Jennifer; Lehmann, Ruth

    2009-01-01

    In many species, the germ cells, precursors of sperm and egg, migrate during embryogenesis. The signals that regulate this migration are thus essential for fertility. In flies, lipid signals have been shown to affect germ cell guidance. In particular, the synthesis of geranylgeranyl pyrophosphate through the 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (Hmgcr) pathway is critical for attracting germ cells to their target tissue. In a genetic analysis of signaling pathways known to affect cell migration of other migratory cells, we failed to find a role for the Hedgehog (Hh) pathway in germ cell migration. However, previous reports had implicated Hh as a germ cell attractant in flies and suggested that Hh signaling is enhanced through the action of the Hmgcr pathway. We therefore repeated several critical experiments and carried out further experiments to test specifically whether Hh is a germ cell attractant in flies. In contrast to previously reported findings and consistent with findings in zebrafish our data do not support the notion that Hh has a direct role in the guidance of migrating germ cells in flies. PMID:19389345

  16. Clear cell sarcoma of tendons and aponeuroses of the parapharyngeal space: an unusual localization of a rare tumor (a case report and review of the literature)

    PubMed Central

    Abdellah, Aissa; Soufiane, Berhili; Amine, Bazine; Sanaa, El Majjaoui; Hanan, Elkacemi; Ijlal, Kharbaoui; Rachida, Latib; Basma, El Khannoussi; Tayeb, Kebdani; Noureddine, Benjaafar

    2014-01-01

    The clear cell sarcoma of tendons and aponeuroses (CCSTA) is a rare soft tissue sarcoma in the head and neck region and parapharyngeal space. Over 95% of CCSTAs present in the extremities, with the head and neck region (1.9%) being an unusual site. This study presents an additional case of CCSTA of the head and neck region involving the parapharyngeal space in a 48-year-old men and review of the literature on CCSTA. PMID:25767667

  17. Morphogenesis of the somatic musculature in Drosophila melanogaster

    PubMed Central

    Schulman, Victoria K.; Dobi, Krista C.; Baylies, Mary K.

    2015-01-01

    In Drosophila melanogaster, the somatic muscle system is first formed during embryogenesis, giving rise to the larval musculature. Later during metamorphosis, this system is destroyed and replaced by an entirely new set of muscles in the adult fly. Proper formation of the larval and adult muscles is critical for basic survival functions such as hatching and crawling (in the larva), walking and flying (in the adult), and feeding (at both larval and adult stages). Myogenesis, from mononucleated muscle precursor cells to multinucleated functional muscles, is driven by a number of cellular processes that have begun to be mechanistically defined. Once themesodermal cells destined for themyogenic lineage have been specified, individual myoblasts fuse together iteratively to form syncytial myofibers. Combining cytoplasmic contents demands a level of intracellular reorganization that, most notably, leads to redistribution of the myonuclei to maximize internuclear distance. Signaling from extending myofibers induces terminal tendon cell differentiation in the ectoderm, which results in secure muscle-tendon attachments that are critical formuscle contraction. Simultaneously, muscles become innervated and undergo sarcomerogenesis to establish the contractile apparatus that will facilitate movement. The cellular mechanisms governing these morphogenetic events share numerous parallels to mammalian development, and the basic unit of all muscle, the myofiber, is conserved from flies to mammals. Thus, studies of Drosophila myogenesis and comparisons to muscle development in other systems highlight conserved regulatory programs of biomedical relevance to general muscle biology and studies of muscle disease. PMID:25758712

  18. Transverse Compression of Tendons.

    PubMed

    Salisbury, S T Samuel; Buckley, C Paul; Zavatsky, Amy B

    2016-04-01

    A study was made of the deformation of tendons when compressed transverse to the fiber-aligned axis. Bovine digital extensor tendons were compression tested between flat rigid plates. The methods included: in situ image-based measurement of tendon cross-sectional shapes, after preconditioning but immediately prior to testing; multiple constant-load creep/recovery tests applied to each tendon at increasing loads; and measurements of the resulting tendon displacements in both transverse directions. In these tests, friction resisted axial stretch of the tendon during compression, giving approximately plane-strain conditions. This, together with the assumption of a form of anisotropic hyperelastic constitutive model proposed previously for tendon, justified modeling the isochronal response of tendon as that of an isotropic, slightly compressible, neo-Hookean solid. Inverse analysis, using finite-element (FE) simulations of the experiments and 10 s isochronal creep displacement data, gave values for Young's modulus and Poisson's ratio of this solid of 0.31 MPa and 0.49, respectively, for an idealized tendon shape and averaged data for all the tendons and E = 0.14 and 0.10 MPa for two specific tendons using their actual measured geometry. The compression load versus displacement curves, as measured and as simulated, showed varying degrees of stiffening with increasing load. This can be attributed mostly to geometrical changes in tendon cross section under load, varying according to the initial 3D shape of the tendon. PMID:26833218

  19. Transverse Compression of Tendons.

    PubMed

    Salisbury, S T Samuel; Buckley, C Paul; Zavatsky, Amy B

    2016-04-01

    A study was made of the deformation of tendons when compressed transverse to the fiber-aligned axis. Bovine digital extensor tendons were compression tested between flat rigid plates. The methods included: in situ image-based measurement of tendon cross-sectional shapes, after preconditioning but immediately prior to testing; multiple constant-load creep/recovery tests applied to each tendon at increasing loads; and measurements of the resulting tendon displacements in both transverse directions. In these tests, friction resisted axial stretch of the tendon during compression, giving approximately plane-strain conditions. This, together with the assumption of a form of anisotropic hyperelastic constitutive model proposed previously for tendon, justified modeling the isochronal response of tendon as that of an isotropic, slightly compressible, neo-Hookean solid. Inverse analysis, using finite-element (FE) simulations of the experiments and 10 s isochronal creep displacement data, gave values for Young's modulus and Poisson's ratio of this solid of 0.31 MPa and 0.49, respectively, for an idealized tendon shape and averaged data for all the tendons and E = 0.14 and 0.10 MPa for two specific tendons using their actual measured geometry. The compression load versus displacement curves, as measured and as simulated, showed varying degrees of stiffening with increasing load. This can be attributed mostly to geometrical changes in tendon cross section under load, varying according to the initial 3D shape of the tendon.

  20. Drosophila stem cells share a common requirement for the histone H2B ubiquitin protease scrawny.

    PubMed

    Buszczak, Michael; Paterno, Shelley; Spradling, Allan C

    2009-01-01

    Stem cells within diverse tissues share the need for a chromatin configuration that promotes self-renewal, yet few chromatin proteins are known to regulate multiple types of stem cells. We describe a Drosophila gene, scrawny (scny), encoding a ubiquitin-specific protease, which is required in germline, epithelial, and intestinal stem cells. Like its yeast relative UBP10, Scrawny deubiquitylates histone H2B and functions in gene silencing. Consistent with previous studies of this conserved pathway of chromatin regulation, scny mutant cells have elevated levels of ubiquitinylated H2B and trimethylated H3K4. Our findings suggest that inhibiting H2B ubiquitylation through scny represents a common mechanism within stem cells that is used to repress the premature expression of key differentiation genes, including Notch target genes.

  1. Filopodia-like actin cables position nuclei in association with perinuclear actin in Drosophila nurse cells.

    PubMed

    Huelsmann, Sven; Ylänne, Jari; Brown, Nicholas H

    2013-09-30

    Controlling the position of the nucleus is vital for a number of cellular processes from yeast to humans. In Drosophila nurse cells, nuclear positioning is crucial during dumping, when nurse cells contract and expel their contents into the oocyte. We provide evidence that in nurse cells, continuous filopodia-like actin cables, growing from the plasma membrane and extending to the nucleus, achieve nuclear positioning. These actin cables move nuclei away from ring canals. When nurse cells contract, actin cables associate laterally with the nuclei, in some cases inducing nuclear turning so that actin cables become partially wound around the nuclei. Our data suggest that a perinuclear actin meshwork connects actin cables to nuclei via actin-crosslinking proteins such as the filamin Cheerio. We provide a revised model for how actin structures position nuclei in nurse cells, employing evolutionary conserved machinery.

  2. Tip cells act as dynamic cellular anchors in the morphogenesis of looped renal tubules in Drosophila.

    PubMed

    Weavers, Helen; Skaer, Helen

    2013-11-11

    Tissue morphogenesis involves both the sculpting of tissue shape and the positioning of tissues relative to one another in the body. Using the renal tubules of Drosophila, we show that a specific distal tubule cell regulates both tissue architecture and position in the body cavity. Focusing on the anterior tubules, we demonstrate that tip cells make transient contacts with alary muscles at abdominal segment boundaries, moving progressively forward as convergent extension movements lengthen the tubule. Tip cell anchorage antagonizes forward-directed, TGF-β-guided tubule elongation, thereby ensuring the looped morphology characteristic of renal tubules from worms to humans. Distinctive tip cell exploratory behavior, adhesion, and basement membrane clearing underlie target recognition and dynamic interactions. Defects in these features obliterate tip cell anchorage, producing misshapen and misplaced tubules with impaired physiological function. PMID:24229645

  3. p53 activity is selectively licensed in the Drosophila stem cell compartment

    PubMed Central

    Wylie, Annika; Lu, Wan-Jin; D’Brot, Alejandro; Buszczak, Michael; Abrams, John M

    2014-01-01

    Oncogenic stress provokes tumor suppression by p53 but the extent to which this regulatory axis is conserved remains unknown. Using a biosensor to visualize p53 action, we find that Drosophila p53 is selectively active in gonadal stem cells after exposure to stressors that destabilize the genome. Similar p53 activity occurred in hyperplastic growths that were triggered either by the RasV12 oncoprotein or by failed differentiation programs. In a model of transient sterility, p53 was required for the recovery of fertility after stress, and entry into the cell cycle was delayed in p53- stem cells. Together, these observations establish that the stem cell compartment of the Drosophila germline is selectively licensed for stress-induced activation of the p53 regulatory network. Furthermore, the findings uncover ancestral links between p53 and aberrant proliferation that are independent of DNA breaks and predate evolution of the ARF/Mdm2 axis. DOI: http://dx.doi.org/10.7554/eLife.01530.001 PMID:24618896

  4. Combinatorial signaling codes for the progressive determination of cell fates in the Drosophila embryonic mesoderm.

    PubMed

    Carmena, A; Gisselbrecht, S; Harrison, J; Jiménez, F; Michelson, A M

    1998-12-15

    Mesodermal progenitors arise in the Drosophila embryo from discrete clusters of lethal of scute (l'sc)-expressing cells. Using both genetic loss-of-function and targeted ectopic expression approaches, we demonstrate here that individual progenitors are specified by the sequential deployment of unique combinations of intercellular signals. Initially, the intersection between the Wingless (Wg) and Decapentaplegic (Dpp) expression domains demarcate an ectodermal prepattern that is imprinted on the adjacent mesoderm in the form of a L'sc precluster. All mesodermal cells within this precluster are competent to respond to a subsequent instructive signal mediated by two receptor tyrosine kinases (RTKs), the Drosophila epidermal growth factor receptor (DER) and the Heartless (Htl) fibroblast growth factor receptor. By monitoring the expression of the diphosphorylated form of mitogen-associated protein kinase (MAPK), we found that these RTKs are activated in small clusters of cells within the original competence domain. Each cluster represents an equivalence group because all members initially resemble progenitors in their expression of both L'sc and mesodermal identity genes. Thus, localized RTK activity induces the formation of mesodermal equivalence groups. The RTKs remain active in the single progenitor that emerges from each cluster under the subsequent inhibitory influence of the neurogenic genes. Moreover, DER and Htl are differentially involved in the specification of particular progenitors. We conclude that distinct cellular identity codes are generated by the combinatorial activities of Wg, Dpp, EGF, and FGF signals in the progressive determination of embryonic mesodermal cells.

  5. The cell adhesion molecule Fasciclin2 regulates brush border length and organization in Drosophila renal tubules.

    PubMed

    Halberg, Kenneth A; Rainey, Stephanie M; Veland, Iben R; Neuert, Helen; Dornan, Anthony J; Klämbt, Christian; Davies, Shireen-Anne; Dow, Julian A T

    2016-01-01

    Multicellular organisms rely on cell adhesion molecules to coordinate cell-cell interactions, and to provide navigational cues during tissue formation. In Drosophila, Fasciclin 2 (Fas2) has been intensively studied due to its role in nervous system development and maintenance; yet, Fas2 is most abundantly expressed in the adult renal (Malpighian) tubule rather than in neuronal tissues. The role Fas2 serves in this epithelium is unknown. Here we show that Fas2 is essential to brush border maintenance in renal tubules of Drosophila. Fas2 is dynamically expressed during tubule morphogenesis, localizing to the brush border whenever the tissue is transport competent. Genetic manipulations of Fas2 expression levels impact on both microvilli length and organization, which in turn dramatically affect stimulated rates of fluid secretion by the tissue. Consequently, we demonstrate a radically different role for this well-known cell adhesion molecule, and propose that Fas2-mediated intermicrovillar homophilic adhesion complexes help stabilize the brush border. PMID:27072072

  6. Screens for piwi suppressors in Drosophila identify dosage-dependent regulators of germline stem cell division.

    PubMed Central

    Smulders-Srinivasan, Tora K; Lin, Haifan

    2003-01-01

    The Drosophila piwi gene is the founding member of the only known family of genes whose function in stem cell maintenance is highly conserved in both animal and plant kingdoms. piwi mutants fail to maintain germline stem cells in both male and female gonads. The identification of piwi-interacting genes is essential for understanding how stem cell divisions are regulated by piwi-mediated mechanisms. To search for such genes, we screened the Drosophila third chromosome ( approximately 36% of the euchromatic genome) for suppressor mutations of piwi2 and identified six strong and three weak piwi suppressor genes/sequences. These genes/sequences interact negatively with piwi in a dosage-sensitive manner. Two of the strong suppressors represent known genes--serendipity-delta and similar, both encoding transcription factors. These findings reveal that the genetic regulation of germline stem cell division involves dosage-sensitive mechanisms and that such mechanisms exist at the transcriptional level. In addition, we identified three other types of piwi interactors. The first type consists of deficiencies that dominantly interact with piwi2 to cause male sterility, implying that dosage-sensitive regulation also exists in the male germline. The other two types are deficiencies that cause lethality and female-specific lethality in a piwi2 mutant background, revealing the zygotic function of piwi in somatic development. PMID:14704180

  7. Effects of lamellolysin from a parasitoid wasp on Drosophila blood cells in vitro.

    PubMed

    Rizki, R M; Rizki, T M

    1991-02-01

    Female parasitoid Leptopilina heterotoma inject a factor, lamellolysin, along with their eggs into the host hemocoel to destroy selectively host hemocytes that encapsulate foreign objects. In parasitized Drosophila melanogaster larvae, these hemocytes (lamellocytes) change from discoidal cells to bipolar cells that no longer adhere to each other to form capsules. To study the effects of lamellolysin on Drosophila lamellocytes in vitro, a giant strain of D. melanogaster was constructed to yield hemolymph with an abundance of lamellocytes. The effect of lamellolysin on the adhesivity of lamellocytes in vitro was demonstrated when the cells were gently rotated in the culture medium. Under these conditions, the bipolar shape of the affected lamellocytes resembled that of lamellocytes in parasitized hosts. When lamellocytes were exposed to lamellolysin in stationary culture medium, the elongation of the bipolar cells continued until they became threadlike. Lamellocytes fragmented in both stationary and rotating culture medium in the presence of lamellolysin, although loss of cellular material was more pronounced in the latter. This study demonstrates that lamellolysin acts directly and destructively on lamellocytes. PMID:1899269

  8. The transcriptional diversity of 25 Drosophila cell lines

    SciTech Connect

    Cherbas, Lucy; Willingham, Aarron; Zhang, Dayu; Yang, Li; Zou, Yi; Eads, Brian D.; Carlson, Joseph W.; Landolin, Jane M.; Kapranov, Philipp; Dumais, Jacqueline; Samsonova, Anastasia; Choi, Jeong-Hyeon; Roberts, Johnny; Davis, Carrie A.; Tang, Haixu; van Baren, Marijke J.; Ghosh, Srinka; Dobin, Alexander; Bell, Kim; Lin, Wei; Langton, Laura; Duff, Michael O.; Tenney, Aaron E.; Zaleski, Chris; Brent, Michael R.; Hoskins, Roger A.; Kaufman, Thomas C.; Andrews, Justen; Graveley, Brenton R.; Perrimon, Norbert; Celniker, Susan E.; Gingeras, Thomas R.; Cherbas, Peter

    2010-12-22

    Drosophila melanogaster cell lines are important resources for cell biologists. In this article, we catalog the expression of exons, genes, and unannotated transcriptional signals for 25 lines. Unannotated transcription is substantial (typically 19% of euchromatic signal). Conservatively, we identify 1405 novel transcribed regions; 684 of these appear to be new exons of neighboring, often distant, genes. Sixty-four percent of genes are expressed detectably in at least one line, but only 21% are detected in all lines. Each cell line expresses, on average, 5885 genes, including a common set of 3109. Expression levels vary over several orders of magnitude. Major signaling pathways are well represented: most differentiation pathways are ‘‘off’’ and survival/growth pathways ‘‘on.’’ Roughly 50% of the genes expressed by each line are not part of the common set, and these show considerable individuality. Thirty-one percent are expressed at a higher level in at least one cell line than in any single developmental stage, suggesting that each line is enriched for genes characteristic of small sets of cells. Most remarkable is that imaginal disc-derived lines can generally be assigned, on the basis of expression, to small territories within developing discs. These mappings reveal unexpected stability of even fine-grained spatial determination. No two cell lines show identical transcription factor expression. We conclude that each line has retained features of an individual founder cell superimposed on a common ‘‘cell line‘‘ gene expression pattern. We report the transcriptional profiles of 25 Drosophila melanogaster cell lines, principally by whole-genome tiling microarray analysis of total RNA, carried out as part of the modENCODE project. The data produced in this study add to our knowledge of the cell lines and of the Drosophila transcriptome in several ways. We summarize the expression of previously annotated genes in each of the 25

  9. Regulatory Networks that Direct the Development of Specialized Cell Types in the Drosophila Heart

    PubMed Central

    Lovato, TyAnna L.; Cripps, Richard M.

    2016-01-01

    The Drosophila cardiac tube was once thought to be a simple linear structure, however research over the past 15 years has revealed significant cellular and molecular complexity to this organ. Prior reviews have focused upon the gene regulatory networks responsible for the specification of the cardiac field and the activation of cardiac muscle structural genes. Here we focus upon highlighting the existence, function, and development of unique cell types within the dorsal vessel, and discuss their correspondence to analogous structures in the vertebrate heart. PMID:27695700

  10. Achilles tendon: US examination

    SciTech Connect

    Fornage, B.D.

    1986-06-01

    Real-time ultrasonography (US) using linear-array probes and a stand-off pad as a ''waterpath'' was performed to evaluate the Achilles tendon in 67 patients (including 24 athletes) believed to have acute or chronic traumatic or inflammatory pathologic conditions. Tendons in 23 patients appeared normal on US scans. The 44 abnormal tendons comprised five complete and four partial ruptures, seven instances of postoperative change, and 28 cases of tendonitis. US depiction of the inner structure of the tendon resulted in the diagnosis of focal abnormalities, including partial ruptures, nodules, and calcifications. Tendonitis was characterized by enlargement and decreased echogenicity of the tendon. The normal US appearance of the Achilles tendon is described.

  11. The Levels of the bancal Product, a Drosophila Homologue of Vertebrate hnRNP K Protein, Affect Cell Proliferation and Apoptosis in Imaginal Disc Cells

    PubMed Central

    Charroux, Bernard; Angelats, Corinne; Fasano, Laurent; Kerridge, Stephen; Vola, Christine

    1999-01-01

    We have characterized the Drosophila bancal gene, which encodes a Drosophila homologue of the vertebrate hnRNP K protein. The bancal gene is essential for the correct size of adult appendages. Reduction of appendage size in bancal mutant flies appears to be due mainly to a reduction in the number of cell divisions in the imaginal discs. Transgenes expressing Drosophila or human hnRNP K are able to rescue weak bancal phenotype, showing the functional similarity of these proteins in vivo. High levels of either human or Drosophila hnRNP K protein in imaginal discs induces programmed cell death. Expression of the antiapoptotic P35 protein suppresses this phenotype in the eye, suggesting that apoptosis is the major cellular defect caused by overexpression of K protein. Finally, the human K protein acts as a negative regulator of bancal gene expression. We propose that negative autoregulation limits the level of Bancal protein produced in vivo. PMID:10523673

  12. Jak-STAT regulation of cyst stem cell development in the Drosophila testis

    PubMed Central

    Sinden, D.; Badgett, M.; Fry, J.; Jones, T.; Palmen, R.; Sheng, X.; Simmons, A.; Matunis, E.; Wawersik, M.

    2012-01-01

    Establishment and maintenance of functional stem cells is critical for organ development and tissue homeostasis. Little is known about the mechanisms underlying stem establishment during organogenesis. Drosophila testes are among the most thoroughly characterized systems for studying stem cell behavior, with germline stem cells (GSCs) and somatic cyst stem cells (CySCs) cohabiting a discrete stem cell niche at the testis apex. GSCs and CySCs are arrayed around hub cells that also comprise the niche and communication between hub cells, GSCs, and CySCs regulates the balance between stem cell maintenance and differentiation. Recent data has shown that functional, asymmetrically dividing GSCs are first established at ~23 hrs after egg laying during Drosophila testis morphogenesis (Sheng et al., 2009). This process correlates with coalescence of the hub, but development of CySCs from somatic gonadal precursors (SGPs) was not examined. Here, we show that functional CySCs are present at the time of GSC establishment, and that Jak-STAT signaling is necessary and sufficient for CySC maintenance shortly thereafter. Furthermore, hyper-activation of Jak in CySCs promotes expansion of the GSC population, while ectopic Jak activation in the germline induces GSC gene expression in GSC daughter cells but does not prevent spermatogenic differentiation. Together, these observations indicate that, similar to adult testes, Jak-STAT signaling from the hub acts on both GSCs and CySC to regulate their development and differentiation, and that additional signaling from CySCs to the GSCs play a dominant role in controlling GSC maintenance during niche formation. PMID:23010510

  13. Functional Significance of the Crystal Cells in the Larva of Drosophila melanogaster

    PubMed Central

    Rizki, M. T. M.; Rizki, Rose M.

    1959-01-01

    Cytoplasmic crystalline inclusions are found in some larval haemocytes of Drosophila melanogaster. Blackening can be experimentally induced in these cells, and previously it was suggested that either the substrate or enzyme for the tyrosine-tyrosinase system leading to melanin production in Drosophila larvae is found in these inclusions in the crystal cells. The present report is an attempt to further localize the enzyme and substrate. Larvae have been fed on food containing α-C14-tyrosine and autoradiographs of the blood cells taken from these larvae subsequently prepared. The C14 activity in the crystal cells is restricted to the crystal inclusions in the cells and is significantly higher than that found in the other type of haemocytes, the plasmatocytes. When samples of the blood cells are incubated in DOPA solution, the extra-crystalline cytoplasm becomes blackened while the crystals themselves remain colorless. These observations are consistent with the hypothesis that the substrate is localized in the crystal inclusions whereas enzyme is found in the surrounding cytoplasm. An in vivo structural isolation would serve to separate enzyme and substrate rather than an inhibition by dehydrogenases as postulated by previous authors. In vitro examination with the phase microscope has shown that the crystal cells rupture easily and the crystals dissolve in the haemolymph. Therefore any treatment which tends to disrupt the structural integrity of the cell will allow the enzyme and substrate to come together. Humoral factors preceding metamorphosis might account for the in vivo release of the enzymatic reaction by initially altering the permeability of the cell. PMID:13654442

  14. Biomechanics of Tendon Transfers.

    PubMed

    Livermore, Andrew; Tueting, Jonathan L

    2016-08-01

    The transfer of tendons in the upper extremity is a powerful technique to restore function to a partially paralyzed hand. The biomechanical principles of muscle tension and tendon excursion dictate motor function both in the native as well as transferred states. Appropriately tensioning transferred tendons to maximize the function of the associated muscle remains an area of focused research. Newer methods of tendon coaptation have proven similar in strength to the standard Pulvertaft weave, affording more options to the surgeon. PMID:27387073

  15. p38 MAPK Signaling in Postnatal Tendon Growth and Remodeling

    PubMed Central

    Schwartz, Andrew J.; Sarver, Dylan C.; Sugg, Kristoffer B.; Dzierzawski, Justin T.; Gumucio, Jonathan P.; Mendias, Christopher L.

    2015-01-01

    Tendon is a dynamic tissue whose structure and function is influenced by mechanical loading, but little is known about the fundamental mechanisms that regulate tendon growth and remodeling in vivo. Data from cultured tendon fibroblasts indicated that the p38 MAPK pathway plays an important role in tendon fibroblast proliferation and collagen synthesis in vitro. To gain greater insight into the mechanisms of tendon growth, and explore the role of p38 MAPK signaling in this process, we tested the hypotheses that inducing plantaris tendon growth through the ablation of the synergist Achilles tendon would result in rapid expansion of a neotendon matrix surrounding the original tendon, and that treatment with the p38 MAPK inhibitor SB203580 would prevent this growth. Rats were treated with vehicle or SB203580, and subjected to synergist ablation by bilateral tenectomy of the Achilles tendon. Changes in histological and biochemical properties of plantaris tendons were analyzed 3, 7, or 28 days after overload, and comparisons were made to non-overloaded animals. By 28 days after overload, tendon mass had increased by 30% compared to non-overloaded samples, and cross-sectional area (CSA) increased by around 50%, with most of the change occurring in the neotendon. The expansion in CSA initially occurred through the synthesis of a hyaluronic acid rich matrix that was progressively replaced with mature collagen. Pericytes were present in areas of active tendon growth, but never in the original tendon ECM. Inhibition of p38 MAPK resulted in a profound decrease in IL6 expression, and had a modest effect on the expression of other ECM and cell proliferation genes, but had a negligible impact on overall tendon growth. The combined results from this study provided novel insights into tendon mechanobiology, and suggest that p38 MAPK signaling does not appear to be necessary for tendon growth in vivo. PMID:25768932

  16. The Hedgehog Signalling Pathway in Cell Migration and Guidance: What We Have Learned from Drosophila melanogaster

    PubMed Central

    Araújo, Sofia J.

    2015-01-01

    Cell migration and guidance are complex processes required for morphogenesis, the formation of tumor metastases, and the progression of human cancer. During migration, guidance molecules induce cell directionality and movement through complex intracellular mechanisms. Expression of these molecules has to be tightly regulated and their signals properly interpreted by the receiving cells so as to ensure correct navigation. This molecular control is fundamental for both normal morphogenesis and human disease. The Hedgehog (Hh) signaling pathway is evolutionarily conserved and known to be crucial for normal cellular growth and differentiation throughout the animal kingdom. The relevance of Hh signaling for human disease is emphasized by its activation in many cancers. Here, I review the current knowledge regarding the involvement of the Hh pathway in cell migration and guidance during Drosophila development and discuss its implications for human cancer origin and progression. PMID:26445062

  17. Antioxidant Role for Lipid Droplets in a Stem Cell Niche of Drosophila

    PubMed Central

    Bailey, Andrew P.; Koster, Grielof; Guillermier, Christelle; Hirst, Elizabeth M.A.; MacRae, James I.; Lechene, Claude P.; Postle, Anthony D.; Gould, Alex P.

    2015-01-01

    Summary Stem cells reside in specialized microenvironments known as niches. During Drosophila development, glial cells provide a niche that sustains the proliferation of neural stem cells (neuroblasts) during starvation. We now find that the glial cell niche also preserves neuroblast proliferation under conditions of hypoxia and oxidative stress. Lipid droplets that form in niche glia during oxidative stress limit the levels of reactive oxygen species (ROS) and inhibit the oxidation of polyunsaturated fatty acids (PUFAs). These droplets protect glia and also neuroblasts from peroxidation chain reactions that can damage many types of macromolecules. The underlying antioxidant mechanism involves diverting PUFAs, including diet-derived linoleic acid, away from membranes to the core of lipid droplets, where they are less vulnerable to peroxidation. This study reveals an antioxidant role for lipid droplets that could be relevant in many different biological contexts. PMID:26451484

  18. Drosophila PI4KIIIalpha is required in follicle cells for oocyte polarization and Hippo signaling

    PubMed Central

    Yan, Yan; Denef, Natalie; Tang, Charm; Schüpbach, Trudi

    2011-01-01

    In a genetic screen we isolated mutations in CG10260, which encodes a phosphatidylinositol 4-kinase (PI4KIIIalpha), and found that PI4KIIIalpha is required for Hippo signaling in Drosophila ovarian follicle cells. PI4KIIIalpha mutations in the posterior follicle cells lead to oocyte polarization defects similar to those caused by mutations in the Hippo signaling pathway. PI4KIIIalpha mutations also cause misexpression of well-established Hippo signaling targets. The Merlin-Expanded-Kibra complex is required at the apical membrane for Hippo activity. In PI4KIIIalpha mutant follicle cells, Merlin fails to localize to the apical domain. Our analysis of PI4KIIIalpha mutants provides a new link in Hippo signal transduction from the cell membrane to its core kinase cascade. PMID:21429988

  19. Three subclasses of a Drosophila insulator show distinct and cell type-specific genomic distributions

    PubMed Central

    Bushey, Ashley M.; Ramos, Edward; Corces, Victor G.

    2009-01-01

    Insulators are protein-bound DNA elements that are thought to play a role in chromatin organization and the regulation of gene expression by mediating intra- and interchromosomal interactions. Suppressor of Hair-wing [Su(Hw)] and Drosophila CTCF (dCTCF) insulators are found at distinct loci throughout the Drosophila melanogaster genome and function by recruiting an additional protein, Centrosomal Protein 190 (CP190). We performed chromatin immunoprecipitation (ChIP) and microarray analysis (ChIP–chip) experiments with whole-genome tiling arrays to compare Su(Hw), dCTCF, boundary element-associated factor (BEAF), and CP190 localization on DNA in two different cell lines and found evidence that BEAF is a third subclass of CP190-containing insulators. The DNA-binding proteins Su(Hw), dCTCF, and BEAF show unique distribution patterns with respect to the location and expression level of genes, suggesting diverse roles for these three subclasses of insulators in genome organization. Notably, cell line-specific localization sites for all three DNA-binding proteins as well as CP190 indicate multiple levels at which insulators can be regulated to affect gene expression. These findings suggest a model in which insulator subclasses may have distinct functions that together organize the genome in a cell type-specific manner, resulting in differential regulation of gene expression. PMID:19443682

  20. Plant Defense Inhibitors Affect the Structures of Midgut Cells in Drosophila melanogaster and Callosobruchus maculatus.

    PubMed

    Li-Byarlay, Hongmei; Pittendrigh, Barry R; Murdock, Larry L

    2016-01-01

    Plants produce proteins such as protease inhibitors and lectins as defenses against herbivorous insects and pathogens. However, no systematic studies have explored the structural responses in the midguts of insects when challenged with plant defensive proteins and lectins across different species. In this study, we fed two kinds of protease inhibitors and lectins to the fruit fly Drosophila melanogaster and alpha-amylase inhibitors and lectins to the cowpea bruchid Callosobruchus maculatus. We assessed the changes in midgut cell structures by comparing them with such structures in insects receiving normal diets or subjected to food deprivation. Using light and transmission electron microscopy in both species, we observed structural changes in the midgut peritrophic matrix as well as shortened microvilli on the surfaces of midgut epithelial cells in D. melanogaster. Dietary inhibitors and lectins caused similar lesions in the epithelial cells but not much change in the peritrophic matrix in both species. We also noted structural damages in the Drosophila midgut after six hours of starvation and changes were still present after 12 hours. Our study provided the first evidence of key structural changes of midguts using a comparative approach between a dipteran and a coleopteran. Our particular observation and discussion on plant-insect interaction and dietary stress are relevant for future mode of action studies of plant defensive protein in insect physiology. PMID:27594789

  1. Functional dissection of the Hox protein Abdominal-B in Drosophila cell culture

    SciTech Connect

    Zhai, Zongzhao; Yang, Xingke; Lohmann, Ingrid

    2011-11-04

    Highlights: Black-Right-Pointing-Pointer ct340 CRM was identified to be the posterior spiracle enhancer of gene cut. Black-Right-Pointing-Pointer ct340 is under the direct transcriptional control of Hox protein Abd-B. Black-Right-Pointing-Pointer An efficient cloning system was developed to assay protein-DNA interaction. Black-Right-Pointing-Pointer New features of Abd-B dependent target gene regulation were detected. -- Abstract: Hox transcription factors regulate the morphogenesis along the anterior-posterior (A/P) body axis through the interaction with small cis-regulatory modules (CRMs) of their target gene, however so far very few Hox CRMs are known and have been analyzed in detail. In this study we have identified a new Hox CRM, ct340, which guides the expression of the cell type specification gene cut (ct) in the posterior spiracle under the direct control of the Hox protein Abdominal-B (Abd-B). Using the ct340 enhancer activity as readout, an efficient cloning system to generate VP16 activation domain fusion protein was developed to unambiguously test protein-DNA interaction in Drosophila cell culture. By functionally dissecting the Abd-B protein, new features of Abd-B dependent target gene regulation were detected. Due to its easy adaptability, this system can be generally used to map functional domains within sequence-specific transcriptional factors in Drosophila cell culture, and thus provide preliminary knowledge of the protein functional domain structure for further in vivo analysis.

  2. Tension, cell shape and triple-junction angle anisotropy in the Drosophila germband

    NASA Astrophysics Data System (ADS)

    Lacy, Monica; Hutson, M. Shane; Meyer, Christian; McDonald, Xena

    In the field of tissue mechanics, the embryonic development of Drosophila melanogaster offers many opportunities for study. One of Drosophila's most crucial morphogenetic stages is the retraction of an epithelial tissue called the germband. During retraction, the segments of the retracting germband, as well as the individual germband cells, elongate in response to forces from a connected tissue, the amnioserosa. Modeling of this elongation, based on tissue responses to laser wounding, has plotted the internal germband tension against the external amnioserosa stress, creating a phase space to determine points and regions corresponding to stable elongation. Although the resulting fits indicate a necessary opposition of internal and external forces, they are inconclusive regarding the exact balance. We will present results testing the model predictions by measuring cell shapes and the correlations between cell-edge directions and triple-junction angles. These measures resolve the ambiguity in pinpointing the internal-external force balance for each germband segment. Research was supported by NIH Grant Numbers 1R01GM099107 and 1R21AR068933.

  3. Plant Defense Inhibitors Affect the Structures of Midgut Cells in Drosophila melanogaster and Callosobruchus maculatus

    PubMed Central

    Li-Byarlay, Hongmei; Pittendrigh, Barry R.; Murdock, Larry L.

    2016-01-01

    Plants produce proteins such as protease inhibitors and lectins as defenses against herbivorous insects and pathogens. However, no systematic studies have explored the structural responses in the midguts of insects when challenged with plant defensive proteins and lectins across different species. In this study, we fed two kinds of protease inhibitors and lectins to the fruit fly Drosophila melanogaster and alpha-amylase inhibitors and lectins to the cowpea bruchid Callosobruchus maculatus. We assessed the changes in midgut cell structures by comparing them with such structures in insects receiving normal diets or subjected to food deprivation. Using light and transmission electron microscopy in both species, we observed structural changes in the midgut peritrophic matrix as well as shortened microvilli on the surfaces of midgut epithelial cells in D. melanogaster. Dietary inhibitors and lectins caused similar lesions in the epithelial cells but not much change in the peritrophic matrix in both species. We also noted structural damages in the Drosophila midgut after six hours of starvation and changes were still present after 12 hours. Our study provided the first evidence of key structural changes of midguts using a comparative approach between a dipteran and a coleopteran. Our particular observation and discussion on plant–insect interaction and dietary stress are relevant for future mode of action studies of plant defensive protein in insect physiology. PMID:27594789

  4. Positive feedback and mutual antagonism combine to polarize Crumbs in the Drosophila follicle cell epithelium.

    PubMed

    Fletcher, Georgina C; Lucas, Eliana P; Brain, Ruth; Tournier, Alexander; Thompson, Barry J

    2012-06-19

    Epithelial tissues are composed of polarized cells with distinct apical and basolateral membrane domains. In the Drosophila ovarian follicle cell epithelium, apical membranes are specified by Crumbs (Crb), Stardust (Sdt), and the aPKC-Par6-cdc42 complex. Basolateral membranes are specified by Lethal giant larvae (Lgl), Discs large (Dlg), and Scribble (Scrib). Apical and basolateral determinants are known to act in a mutually antagonistic fashion, but it remains unclear how this interaction generates polarity. We have built a computer model of apicobasal polarity that suggests that the combination of positive feedback among apical determinants plus mutual antagonism between apical and basal determinants is essential for polarization. In agreement with this model, in vivo experiments define a positive feedback loop in which Crb self-recruits via Crb-Crb extracellular domain interactions, recruitment of Sdt-aPKC-Par6-cdc42, aPKC phosphorylation of Crb, and recruitment of Expanded (Ex) and Kibra (Kib) to prevent endocytic removal of Crb from the plasma membrane. Lgl antagonizes the operation of this feedback loop, explaining why apical determinants do not normally spread into the basolateral domain. Once Crb is removed from the plasma membrane, it undergoes recycling via Rab11 endosomes. Our results provide a dynamic model for understanding how epithelial polarity is maintained in Drosophila follicle cells.

  5. Plant Defense Inhibitors Affect the Structures of Midgut Cells in Drosophila melanogaster and Callosobruchus maculatus

    PubMed Central

    Li-Byarlay, Hongmei; Pittendrigh, Barry R.; Murdock, Larry L.

    2016-01-01

    Plants produce proteins such as protease inhibitors and lectins as defenses against herbivorous insects and pathogens. However, no systematic studies have explored the structural responses in the midguts of insects when challenged with plant defensive proteins and lectins across different species. In this study, we fed two kinds of protease inhibitors and lectins to the fruit fly Drosophila melanogaster and alpha-amylase inhibitors and lectins to the cowpea bruchid Callosobruchus maculatus. We assessed the changes in midgut cell structures by comparing them with such structures in insects receiving normal diets or subjected to food deprivation. Using light and transmission electron microscopy in both species, we observed structural changes in the midgut peritrophic matrix as well as shortened microvilli on the surfaces of midgut epithelial cells in D. melanogaster. Dietary inhibitors and lectins caused similar lesions in the epithelial cells but not much change in the peritrophic matrix in both species. We also noted structural damages in the Drosophila midgut after six hours of starvation and changes were still present after 12 hours. Our study provided the first evidence of key structural changes of midguts using a comparative approach between a dipteran and a coleopteran. Our particular observation and discussion on plant–insect interaction and dietary stress are relevant for future mode of action studies of plant defensive protein in insect physiology.

  6. The cell adhesion molecule Fasciclin2 regulates brush border length and organization in Drosophila renal tubules

    PubMed Central

    Halberg, Kenneth A.; Rainey, Stephanie M.; Veland, Iben R.; Neuert, Helen; Dornan, Anthony J.; Klämbt, Christian; Davies, Shireen-Anne; Dow, Julian A. T.

    2016-01-01

    Multicellular organisms rely on cell adhesion molecules to coordinate cell–cell interactions, and to provide navigational cues during tissue formation. In Drosophila, Fasciclin 2 (Fas2) has been intensively studied due to its role in nervous system development and maintenance; yet, Fas2 is most abundantly expressed in the adult renal (Malpighian) tubule rather than in neuronal tissues. The role Fas2 serves in this epithelium is unknown. Here we show that Fas2 is essential to brush border maintenance in renal tubules of Drosophila. Fas2 is dynamically expressed during tubule morphogenesis, localizing to the brush border whenever the tissue is transport competent. Genetic manipulations of Fas2 expression levels impact on both microvilli length and organization, which in turn dramatically affect stimulated rates of fluid secretion by the tissue. Consequently, we demonstrate a radically different role for this well-known cell adhesion molecule, and propose that Fas2-mediated intermicrovillar homophilic adhesion complexes help stabilize the brush border. PMID:27072072

  7. Transposon Dysregulation Modulates dWnt4 Signaling to Control Germline Stem Cell Differentiation in Drosophila

    PubMed Central

    Upadhyay, Maitreyi; Martino Cortez, Yesenia; Wong-Deyrup, SiuWah; Tavares, Leticia; Schowalter, Sean; Flora, Pooja; Hill, Corinne; Nasrallah, Mohamad Ali; Chittur, Sridar; Rangan, Prashanth

    2016-01-01

    Germline stem cell (GSC) self-renewal and differentiation are required for the sustained production of gametes. GSC differentiation in Drosophila oogenesis requires expression of the histone methyltransferase dSETDB1 by the somatic niche, however its function in this process is unknown. Here, we show that dSETDB1 is required for the expression of a Wnt ligand, Drosophila Wingless type mouse mammary virus integration site number 4 (dWnt4) in the somatic niche. dWnt4 signaling acts on the somatic niche cells to facilitate their encapsulation of the GSC daughter, which serves as a differentiation cue. dSETDB1 is known to repress transposable elements (TEs) to maintain genome integrity. Unexpectedly, we found that independent upregulation of TEs also downregulated dWnt4, leading to GSC differentiation defects. This suggests that dWnt4 expression is sensitive to the presence of TEs. Together our results reveal a chromatin-transposon-Wnt signaling axis that regulates stem cell fate. PMID:27019121

  8. Capping protein beta is required for actin cytoskeleton organisation and cell migration during Drosophila oogenesis.

    PubMed

    Ogienko, Anna A; Karagodin, Dmitry A; Lashina, Valentina V; Baiborodin, Sergey I; Omelina, Eugeniya S; Baricheva, Elina M

    2013-02-01

    Capping protein (CP) is a well-characterised actin-binding protein important for regulation of actin filament (AF) assembly. CP caps the barbed end of AFs, inhibiting the addition and loss of actin monomers. In Drosophila melanogaster, the gene encoding CP β-subunit is named capping protein beta (cpb; see Hopmann et al. [1996] J Cell Biol 133: 1293-305). The cpb level is reduced in the Drosophila bristle actin cytoskeleton and becomes disorganised with abnormal morphology. A reduced level of the CP protein in ovary results in disruption of oocyte determination, and disturbance of nurse cell (NC) cortical integrity and dumping. We describe novel defects appearing in cpb mutants during oogenesis, in which cpb plays an important role in border and centripetal follicle cell migration, ring canal development and cytoplasmic AF formation. The number of long cytoplasmic AFs was dramatically reduced in cpb hypomorphs and abnormal actin aggregates was seen on the inner side of NC membranes. A hypothesis to explain the formation of abnormal short-cut cytoplasmic AFs and actin aggregates in the cpb mutant NCs was proffered, along with a discussion of the reasons for 'dumpless' phenotype formation in the mutants.

  9. Posterior midgut epithelial cells differ in their organization of the membrane skeleton from other drosophila epithelia.

    PubMed

    Baumann, O

    2001-11-01

    In epithelial cells, the various components of the membrane skeleton are segregated within specialized subregions of the plasma membrane, thus contributing to the development and stabilization of cell surface polarity. It has previously been shown that, in various Drosophila epithelia, the membrane skeleton components ankyrin and alphabeta-spectrin reside at the lateral surface, whereas alphabeta(H)-spectrin is restricted to the apical domain. By use of confocal immunofluorescence microscopy, the present study characterizes the membrane skeleton of epithelial cells in the posterior midgut, leading to a number of unexpected results. First, ankyrin and alphabeta-spectrin are not detected on the entire lateral surface but appear to be restricted to the apicolateral area, codistributing with fasciclin III at smooth septate junctions. The presumptive ankyrin-binding proteins neuroglian and Na(+),K(+)-ATPase, however, do not colocalize with ankyrin. Second, alphabeta(H)-spectrin is enriched at the apical domain but is also present in lower amounts on the entire lateral surface, colocalizing apicolaterally with ankyrin/alphabeta-spectrin. Finally, despite the absence of zonulae adherentes, F-actin, beta(H)-spectrin, and nonmuscle myosin-II are enriched in the midlateral region. Thus, the model established for the organization of the membrane skeleton in Drosophila epithelia does not hold for the posterior midgut, and there is quite some variability between the different epithelia with respect to the organization of the membrane skeleton.

  10. Using EGFP fusions to monitor the functional expression of GPCRs in the Drosophila Schneider 2 cells

    PubMed Central

    Brillet, Karl; Perret, Bénédicte G.; Klein, Valérie; Pattus, Franc

    2008-01-01

    In combining fluorescence measurements with ligand binding assays, the versatility of the EGFP C-terminally fused to the human mu opioid receptor (EGFP-hMOR) has been exploited to notably improve the expression level of functional G protein-coupled receptors in Drosophila S2 cells. A selected array of efficient optimization approaches is presented herein, ranging from a cell-sorting method, allowing for a substantial enrichment in EGFP-hMOR expressing cells, to the addition of chemical and pharmacological chaperones, significantly enhancing the yield and the activity of the expressed receptors. Consistent with previous studies, significant discrepancies were observed between the total amounts of fluorescent receptors over a limited subpopulation capable of ligand binding, even after expression optimization. Subsequently, membrane isopycnic centrifugation experiments allowed to separate the ligand binding active from the non-active membrane fraction, the latter most probably containing misfolded receptors. Taken together, these results illustrate a coherent set of advantageous productive and preparative methods for the production of GPCRs in the highly valuable Drosophila S2 expression system. PMID:19003178

  11. Piwi maintains germline stem cells and oogenesis in Drosophila through negative regulation of Polycomb group proteins.

    PubMed

    Peng, Jamy C; Valouev, Anton; Liu, Na; Lin, Haifan

    2016-03-01

    The Drosophila melanogaster Piwi protein regulates both niche and intrinsic mechanisms to maintain germline stem cells, but its underlying mechanism remains unclear. Here we report that Piwi interacts with Polycomb group complexes PRC1 and PRC2 in niche and germline cells to regulate ovarian germline stem cells and oogenesis. Piwi physically interacts with the PRC2 subunits Su(z)12 and Esc in the ovary and in vitro. Chromatin coimmunoprecipitation of Piwi, the PRC2 enzymatic subunit E(z), histone H3 trimethylated at lysine 27 (H3K27me3) and RNA polymerase II in wild-type and piwi mutant ovaries demonstrates that Piwi binds a conserved DNA motif at ∼ 72 genomic sites and inhibits PRC2 binding to many non-Piwi-binding genomic targets and H3K27 trimethylation. Moreover, Piwi influences RNA polymerase II activities in Drosophila ovaries, likely via inhibiting PRC2. We hypothesize that Piwi negatively regulates PRC2 binding by sequestering PRC2 in the nucleoplasm, thus reducing PRC2 binding to many targets and influencing transcription during oogenesis. PMID:26780607

  12. Bioinformatic analyses of sense and antisense expression from terminal inverted repeat transposons in Drosophila somatic cells.

    PubMed

    Harrington, Andrew W; Steiniger, Mindy

    2016-01-01

    Understanding regulation of transposon movement in somatic cells is important as mobile elements can cause detrimental genomic rearrangements. Generally, transposons move via one of 2 mechanisms; retrotransposons utilize an RNA intermediate, therefore copying themselves and amplifying throughout the genome, while terminal inverted repeat transposons (TIR Tns) excise DNA sequences from the genome and integrate into a new location. Our recently published work indicates that retrotransposons in Drosophila tissue culture cells are actively transcribed in the antisense direction. Our data support a model in which convergent transcription of retrotransposons from intra element transcription start sites results in complementary RNAs that hybridize to form substrates for Dicer-2, the endogenous small interfering (esi)RNA generating enzyme. Here, we extend our previous analysis to TIR Tns. In contrast to retrotransposons, our data show that antisense TIR Tn RNAs result from transcription of intronic TIR Tns oriented antisense to their host genes. Also, disproportionately less esiRNAs are generated from TIR transcripts than from retrotransposons and transcription of very few individual TIR Tns could be confirmed. Collectively, these data support a model in which TIR Tns are regulated at the level of Transposase production while retrotransposons are regulated with esiRNA post-transcriptional mechanisms in Drosophila somatic cells. PMID:26986720

  13. Piwi maintains germline stem cells and oogenesis in Drosophila through negative regulation of Polycomb Group proteins

    PubMed Central

    Peng, Jamy C.; Valouev, Anton; Liu, Na; Lin, Haifan

    2015-01-01

    The Drosophila Piwi protein regulates both niche and intrinsic mechanisms to maintain germline stem cells, but its underlying mechanism remains unclear. Here we report that Piwi cooperates with Polycomb Group complexes PRC1 and PRC2 in niche and germline cells to regulate ovarian germline stem cells and oogenesis. Piwi physically interacts with PRC2 subunits Su(z)12 and Esc in the ovary and in vitro. Chromatin co-immunoprecipitation of Piwi, the PRC2 enzymatic subunit E(z), lysine-27-tri-methylated histone 3 (H3K27m3), and RNA polymerase II in wild-type and piwi mutant ovaries reveals that Piwi binds a conserved DNA motif at ~72 genomic sites, and inhibits PRC2 binding to many non-Piwi-binding genomic targets and H3K27 tri-methylation. Moreover, Piwi influences RNA Polymerase II activities in Drosophila ovaries likely via inhibiting PRC2. We hypothesize that Piwi negatively regulates PRC2 binding by sequestering PRC2 in the nucleoplasm, thus reducing PRC2 binding to many targets and influences transcription during oogenesis. PMID:26780607

  14. Wingless signaling and the control of cell shape in Drosophila wing imaginal discs.

    PubMed

    Widmann, Thomas J; Dahmann, Christian

    2009-10-01

    The control of cell morphology is important for shaping animals during development. Here we address the role of the Wnt/Wingless signal transduction pathway and two of its target genes, vestigial and shotgun (encoding E-cadherin), in controlling the columnar shape of Drosophila wing disc cells. We show that clones of cells mutant for arrow (encoding an essential component of the Wingless signal transduction pathway), vestigial or shotgun undergo profound cell shape changes and are extruded towards the basal side of the epithelium. Compartment-wide expression of a dominant-negative form of the Wingless transducer T-cell factor (TCF/Pangolin), or double-stranded RNA targeting vestigial or shotgun, leads to abnormally short cells throughout this region, indicating that these genes act cell autonomously to maintain normal columnar cell shape. Conversely, overexpression of Wingless, a constitutively-active form of the Wingless transducer beta-catenin/Armadillo, or Vestigial, results in precocious cell elongation. Co-expression of Vestigial partially suppresses the abnormal cell shape induced by dominant-negative TCF. We conclude that Wingless signal transduction plays a cell-autonomous role in promoting and maintaining the columnar shape of wing disc cells. Furthermore, our data suggest that Wingless controls cell shape, in part, through maintaining vestigial expression. PMID:19627985

  15. Asymmetric distribution of Spalt in Drosophila wing squamous and columnar epithelia ensures correct cell morphogenesis

    PubMed Central

    Tang, Wenqian; Wang, Dan; Shen, Jie

    2016-01-01

    The Drosophila wing imaginal disc is a sac-like structure that is composed of two opposing cell layers: peripodial epithelium (PE, also known as squamous epithelia) and disc proper (DP, also known as pseudostratified columnar epithelia). The molecular mechanism of cell morphogenesis has been well studied in the DP but not in the PE. Although proper Dpp signalling activity is required for proper PE formation, the detailed regulation mechanism is poorly understood. Here, we found that the Dpp target gene sal is only expressed in DP cells, not in PE cells, although pMad is present in the PE. Increasing Dpp signalling activity cannot activate Sal in PE cells. The absence of Sal in the PE is essential for PE formation. The ectopic expression of sal in PE cells is sufficient to increase the PE cell height. Down-regulation of sal in the DP reduced DP cell height. We further demonstrated that the known PE cell height regulator Lines, which can convert PE into a DP cell fate, is mediated by sal mis-activation in PE because sal-RNAi and lines co-expression largely restores PE cell morphology. By revealing the microtubule distribution, we demonstrated that Lines- and Sal-heightened PE cells are morphologically similar to the intermediate cell with cuboidal morphology. PMID:27452716

  16. Wingless signaling and the control of cell shape in Drosophila wing imaginal discs.

    PubMed

    Widmann, Thomas J; Dahmann, Christian

    2009-10-01

    The control of cell morphology is important for shaping animals during development. Here we address the role of the Wnt/Wingless signal transduction pathway and two of its target genes, vestigial and shotgun (encoding E-cadherin), in controlling the columnar shape of Drosophila wing disc cells. We show that clones of cells mutant for arrow (encoding an essential component of the Wingless signal transduction pathway), vestigial or shotgun undergo profound cell shape changes and are extruded towards the basal side of the epithelium. Compartment-wide expression of a dominant-negative form of the Wingless transducer T-cell factor (TCF/Pangolin), or double-stranded RNA targeting vestigial or shotgun, leads to abnormally short cells throughout this region, indicating that these genes act cell autonomously to maintain normal columnar cell shape. Conversely, overexpression of Wingless, a constitutively-active form of the Wingless transducer beta-catenin/Armadillo, or Vestigial, results in precocious cell elongation. Co-expression of Vestigial partially suppresses the abnormal cell shape induced by dominant-negative TCF. We conclude that Wingless signal transduction plays a cell-autonomous role in promoting and maintaining the columnar shape of wing disc cells. Furthermore, our data suggest that Wingless controls cell shape, in part, through maintaining vestigial expression.

  17. Gprk2 adjusts Fog signaling to organize cell movements in Drosophila gastrulation.

    PubMed

    Fuse, Naoyuki; Yu, Fengwei; Hirose, Susumu

    2013-10-01

    Gastrulation of Drosophila melanogaster proceeds through sequential cell movements: ventral mesodermal (VM) cells are induced by secreted Fog protein to constrict their apical surfaces to form the ventral furrow, and subsequently lateral mesodermal (LM) cells involute toward the furrow. How these cell movements are organized remains elusive. Here, we observed that LM cells extended apical protrusions and then underwent accelerated involution movement, confirming that VM and LM cells display distinct cell morphologies and movements. In a mutant for the GPCR kinase Gprk2, apical constriction was expanded to all mesodermal cells and the involution movement was abolished. In addition, the mesodermal cells halted apical constriction prematurely in accordance with the aberrant accumulation of Myosin II. Epistasis analyses revealed that the Gprk2 mutant phenotypes were dependent on the fog gene. Overexpression of Gprk2 suppressed the effects of excess Cta, a downstream component of Fog signaling. Based on these findings, we propose that Gprk2 attenuates and tunes Fog-Cta signaling to prevent apical constriction in LM cells and to support appropriate apical constriction in VM cells. Thus, the two distinct cell movements in mesoderm invagination are not predetermined, but rather are organized by the adjustment of cell signaling.

  18. Augmentation of tendon-to-bone healing.

    PubMed

    Atesok, Kivanc; Fu, Freddie H; Wolf, Megan R; Ochi, Mitsuo; Jazrawi, Laith M; Doral, M Nedim; Lubowitz, James H; Rodeo, Scott A

    2014-03-19

    Tendon-to-bone healing is vital to the ultimate success of the various surgical procedures performed to repair injured tendons. Achieving tendon-to-bone healing that is functionally and biologically similar to native anatomy can be challenging because of the limited regeneration capacity of the tendon-bone interface. Orthopaedic basic-science research strategies aiming to augment tendon-to-bone healing include the use of osteoinductive growth factors, platelet-rich plasma, gene therapy, enveloping the grafts with periosteum, osteoconductive materials, cell-based therapies, biodegradable scaffolds, and biomimetic patches. Low-intensity pulsed ultrasound and extracorporeal shockwave treatment may affect tendon-to-bone healing by means of mechanical forces that stimulate biological cascades at the insertion site. Application of various loading methods and immobilization times influence the stress forces acting on the recently repaired tendon-to-bone attachment, which eventually may change the biological dynamics of the interface. Other approaches, such as the use of coated sutures and interference screws, aim to deliver biological factors while achieving mechanical stability by means of various fixators. Controlled Level-I human trials are required to confirm the promising results from in vitro or animal research studies elucidating the mechanisms underlying tendon-to-bone healing and to translate these results into clinical practice.

  19. Tendon development and diseases.

    PubMed

    Gaut, Ludovic; Duprez, Delphine

    2016-01-01

    Tendon is a uniaxial connective tissue component of the musculoskeletal system. Tendon is involved in force transmission between muscle and bone. Tendon injury is very common and debilitating but tendon repair remains a clinical challenge for orthopedic medicine. In vertebrates, tendon is mainly composed of type I collagen fibrils, displaying a parallel organization along the tendon axis. The tendon-specific spatial organization of type I collagen provides the mechanical properties for tendon function. In contrast to other components of the musculoskeletal system, tendon biology is poorly understood. An important goal in tendon biology is to understand the mechanisms involved in the production and assembly of type I collagen fibrils during development, postnatal formation, and healing processes in order to design new therapies for tendon repair. In this review we highlight the current understanding of the molecular and mechanical signals known to be involved in tenogenesis during development, and how development provides insights into tendon healing processes. WIREs Dev Biol 2016, 5:5-23. doi: 10.1002/wdev.201 For further resources related to this article, please visit the WIREs website.

  20. Pressurized gas filled tendons

    SciTech Connect

    Silcox, W. H.

    1985-06-04

    Pressurized gas filled tubular tendons provide a means for detecting leaks therein. Filling the tendon with a gaseous fluid provides increased buoyancy and reduces the weight supported by the buoyant structure. The use of a corrosion inhibiting gaseous fluid reduces the corrosion of the interior tendon wall.

  1. Extreme divergence of Wolbachia tropism for the stem-cell-niche in the Drosophila testis.

    PubMed

    Toomey, Michelle E; Frydman, Horacio M

    2014-12-01

    Microbial tropism, the infection of specific cells and tissues by a microorganism, is a fundamental aspect of host-microbe interactions. The intracellular bacteria Wolbachia have a peculiar tropism for the stem cell niches in the Drosophila ovary, the microenvironments that support the cells producing the eggs. The molecular underpinnings of Wolbachia stem cell niche tropism are unknown. We have previously shown that the patterns of tropism in the ovary show a high degree of conservation across the Wolbachia lineage, with closely related Wolbachia strains usually displaying the same pattern of stem cell niche tropism. It has also been shown that tropism to these structures in the ovary facilitates both vertical and horizontal transmission, providing a strong selective pressure towards evolutionary conservation of tropism. Here we show great disparity in the evolutionary conservation and underlying mechanisms of stem cell niche tropism between male and female gonads. In contrast to females, niche tropism in the male testis is not pervasive, present in only 45% of niches analyzed. The patterns of niche tropism in the testis are not evolutionarily maintained across the Wolbachia lineage, unlike what was shown in the females. Furthermore, hub tropism does not correlate with cytoplasmic incompatibility, a Wolbachia-driven phenotype imprinted during spermatogenesis. Towards identifying the molecular mechanism of hub tropism, we performed hybrid analyses of Wolbachia strains in non-native hosts. These results indicate that both Wolbachia and host derived factors play a role in the targeting of the stem cell niche in the testis. Surprisingly, even closely related Wolbachia strains in Drosophila melanogaster, derived from a single ancestor only 8,000 years ago, have significantly different tropisms to the hub, highlighting that stem cell niche tropism is rapidly diverging in males. These findings provide a powerful system to investigate the mechanisms and evolution of

  2. Extreme Divergence of Wolbachia Tropism for the Stem-Cell-Niche in the Drosophila Testis

    PubMed Central

    Toomey, Michelle E.; Frydman, Horacio M.

    2014-01-01

    Microbial tropism, the infection of specific cells and tissues by a microorganism, is a fundamental aspect of host-microbe interactions. The intracellular bacteria Wolbachia have a peculiar tropism for the stem cell niches in the Drosophila ovary, the microenvironments that support the cells producing the eggs. The molecular underpinnings of Wolbachia stem cell niche tropism are unknown. We have previously shown that the patterns of tropism in the ovary show a high degree of conservation across the Wolbachia lineage, with closely related Wolbachia strains usually displaying the same pattern of stem cell niche tropism. It has also been shown that tropism to these structures in the ovary facilitates both vertical and horizontal transmission, providing a strong selective pressure towards evolutionary conservation of tropism. Here we show great disparity in the evolutionary conservation and underlying mechanisms of stem cell niche tropism between male and female gonads. In contrast to females, niche tropism in the male testis is not pervasive, present in only 45% of niches analyzed. The patterns of niche tropism in the testis are not evolutionarily maintained across the Wolbachia lineage, unlike what was shown in the females. Furthermore, hub tropism does not correlate with cytoplasmic incompatibility, a Wolbachia-driven phenotype imprinted during spermatogenesis. Towards identifying the molecular mechanism of hub tropism, we performed hybrid analyses of Wolbachia strains in non-native hosts. These results indicate that both Wolbachia and host derived factors play a role in the targeting of the stem cell niche in the testis. Surprisingly, even closely related Wolbachia strains in Drosophila melanogaster, derived from a single ancestor only 8,000 years ago, have significantly different tropisms to the hub, highlighting that stem cell niche tropism is rapidly diverging in males. These findings provide a powerful system to investigate the mechanisms and evolution of

  3. Steroid hormone signaling is essential to regulate innate immune cells and fight bacterial infection in Drosophila.

    PubMed

    Regan, Jennifer C; Brandão, Ana S; Leitão, Alexandre B; Mantas Dias, Angela Raquel; Sucena, Elio; Jacinto, António; Zaidman-Rémy, Anna

    2013-10-01

    Coupling immunity and development is essential to ensure survival despite changing internal conditions in the organism. Drosophila metamorphosis represents a striking example of drastic and systemic physiological changes that need to be integrated with the innate immune system. However, nothing is known about the mechanisms that coordinate development and immune cell activity in the transition from larva to adult. Here, we reveal that regulation of macrophage-like cells (hemocytes) by the steroid hormone ecdysone is essential for an effective innate immune response over metamorphosis. Although it is generally accepted that steroid hormones impact immunity in mammals, their action on monocytes (e.g. macrophages and neutrophils) is still not well understood. Here in a simpler model system, we used an approach that allows in vivo, cell autonomous analysis of hormonal regulation of innate immune cells, by combining genetic manipulation with flow cytometry, high-resolution time-lapse imaging and tissue-specific transcriptomic analysis. We show that in response to ecdysone, hemocytes rapidly upregulate actin dynamics, motility and phagocytosis of apoptotic corpses, and acquire the ability to chemotax to damaged epithelia. Most importantly, individuals lacking ecdysone-activated hemocytes are defective in bacterial phagocytosis and are fatally susceptible to infection by bacteria ingested at larval stages, despite the normal systemic and local production of antimicrobial peptides. This decrease in survival is comparable to the one observed in pupae lacking immune cells altogether, indicating that ecdysone-regulation is essential for hemocyte immune functions and survival after infection. Microarray analysis of hemocytes revealed a large set of genes regulated at metamorphosis by EcR signaling, among which many are known to function in cell motility, cell shape or phagocytosis. This study demonstrates an important role for steroid hormone regulation of immunity in vivo in

  4. Tendon Differentiation on Decellularized Extracellular Matrix Under Cyclic Loading.

    PubMed

    Youngstrom, Daniel W; Barrett, Jennifer G

    2016-01-01

    Tendon bioreactors combine cells, scaffold, and mechanical stimulation to drive tissue neogenesis ex vivo. Faithful recapitulation of the native tendon microenvironment is essential for stimulating graft maturation or modeling tendon biology. As the mediator between cells and mechanical stimulation, the properties of a scaffold constitute perhaps the most essential elements in a bioreactor system. One method of achieving native scaffold properties is to process tendon allograft in a manner that removes cells without modifying structure and function: "decellularization." This chapter describes (1) production of tendon scaffolds derived from native extracellular matrix, (2) preparation of cell-laden scaffolds prior to bioreactor culture, and (3) tissue processing post-harvest for gene expression analysis. These methods may be applied for a variety of applications including graft production, cell priming prior to transplantation and basic investigations of tendon cell biology. PMID:27062597

  5. Tendon Differentiation on Decellularized Extracellular Matrix Under Cyclic Loading.

    PubMed

    Youngstrom, Daniel W; Barrett, Jennifer G

    2016-01-01

    Tendon bioreactors combine cells, scaffold, and mechanical stimulation to drive tissue neogenesis ex vivo. Faithful recapitulation of the native tendon microenvironment is essential for stimulating graft maturation or modeling tendon biology. As the mediator between cells and mechanical stimulation, the properties of a scaffold constitute perhaps the most essential elements in a bioreactor system. One method of achieving native scaffold properties is to process tendon allograft in a manner that removes cells without modifying structure and function: "decellularization." This chapter describes (1) production of tendon scaffolds derived from native extracellular matrix, (2) preparation of cell-laden scaffolds prior to bioreactor culture, and (3) tissue processing post-harvest for gene expression analysis. These methods may be applied for a variety of applications including graft production, cell priming prior to transplantation and basic investigations of tendon cell biology.

  6. Artificial finger joint replacement due to a giant cell tumor of the tendon sheath with bone destruction: A case report

    PubMed Central

    LU, HUI; SHEN, HUI; CHEN, QIANG; SHEN, XIANG-QIAN; WU, SHOU-CHENG

    2015-01-01

    The current study presents the case of a 25-year-old male who developed tumor recurrence of the proximal phalange of the ring finger on the right hand 4 years after partial tumor resection surgery. An X-ray of the right hand showed that the distal bone of the proximal phalange on the ring finger was destroyed. An artificial finger joint replacement was performed using a silicone joint for this unusual tumor recurrence. The pathological findings were indicative of a giant cell tumor of the tendon sheath. As a result of surgery, the patient's proximal interphalangeal point motion recovered to the pre-operative level. The pre-operative and post-operative disabilities of the arm, at shoulder and hand and total activity measurement values were 1.67 and 3.33, and 255 and 243°, respectively. Complications such as tumor recurrence, joint dislocation and the requirement for prosthetic training were not observed during the 5-year follow-up period. PMID:26788157

  7. The role of iron in the proliferation of Drosophila l(2)mbn cells

    SciTech Connect

    Metzendorf, Christoph; Lind, Maria I.

    2010-09-24

    Research highlights: {yields} Establishment of a model system to study the role of iron during proliferation. {yields} Iron deprivation of insect tumorous cell line inhibits cell proliferation. {yields} Iron deprivation causes a reversible cell cycle arrest in G1/S-phase. {yields} Iron deprivation promotes decreased gene expression of cycE. -- Abstract: Iron is essential for life and is needed for cell proliferation and cell cycle progression. Iron deprivation results first in cell cycle arrest and then in apoptosis. The Drosophila tumorous larval hemocyte cell line l(2)mbn was used to study the sensitivity and cellular response to iron deprivation through the chelator desferrioxamine (DFO). At a concentration of 10 {mu}M DFO or more the proliferation was inhibited reversibly, while the amount of dead cells did not increase. FACS analysis showed that the cell cycle was arrested in G1/S-phase and the transcript level of cycE was decreased to less than 50% of control cells. These results show that iron chelation in this insect tumorous cell line causes a specific and coordinated cell cycle arrest.

  8. The migrations of Drosophila muscle founders and primordial germ cells are interdependent.

    PubMed

    Stepanik, Vincent; Dunipace, Leslie; Bae, Young-Kyung; Macabenta, Frank; Sun, Jingjing; Trisnadi, Nathanie; Stathopoulos, Angelike

    2016-09-01

    Caudal visceral mesoderm (CVM) cells migrate from posterior to anterior of the Drosophila embryo as two bilateral streams of cells to support the specification of longitudinal muscles along the midgut. To accomplish this long-distance migration, CVM cells receive input from their environment, but little is known about how this collective cell migration is regulated. In a screen we found that wunen mutants exhibit CVM cell migration defects. Wunens are lipid phosphate phosphatases known to regulate the directional migration of primordial germ cells (PGCs). PGC and CVM cell types interact while PGCs are en route to the somatic gonadal mesoderm, and previous studies have shown that CVM impacts PGC migration. In turn, we found here that CVM cells exhibit an affinity for PGCs, localizing to the position of PGCs whether mislocalized or trapped in the endoderm. In the absence of PGCs, CVM cells exhibit subtle changes, including more cohesive movement of the migrating collective, and an increased number of longitudinal muscles is found at anterior sections of the larval midgut. These data demonstrate that PGC and CVM cell migrations are interdependent and suggest that distinct migrating cell types can coordinately influence each other to promote effective cell migration during development. PMID:27578182

  9. Drosophila spermiogenesis

    PubMed Central

    Fabian, Lacramioara; Brill, Julie A.

    2012-01-01

    Drosophila melanogaster spermatids undergo dramatic morphological changes as they differentiate from small round cells approximately 12 μm in diameter into highly polarized, 1.8 mm long, motile sperm capable of participating in fertilization. During spermiogenesis, syncytial cysts of 64 haploid spermatids undergo synchronous differentiation. Numerous changes occur at a subcellular level, including remodeling of existing organelles (mitochondria, nuclei), formation of new organelles (flagellar axonemes, acrosomes), polarization of elongating cysts and plasma membrane addition. At the end of spermatid morphogenesis, organelles, mitochondrial DNA and cytoplasmic components not needed in mature sperm are stripped away in a caspase-dependent process called individualization that results in formation of individual sperm. Here, we review the stages of Drosophila spermiogenesis and examine our current understanding of the cellular and molecular mechanisms involved in shaping male germ cell-specific organelles and forming mature, fertile sperm. PMID:23087837

  10. Insulin delays the progression of Drosophila cells through G2/M by activating the dTOR/dRaptor complex

    PubMed Central

    Wu, Mary Y W; Cully, Megan; Andersen, Ditte; Leevers, Sally J

    2007-01-01

    In Drosophila and mammals, insulin signalling can increase growth, progression through G1/S, cell size and tissue size. Here, we analyse the way insulin affects cell size and cell-cycle progression in two haemocyte-derived Drosophila cell lines. Surprisingly, we find that although insulin increases cell size, it slows the rate at which these cells increase in number. By using BrdU pulse-chase to label S-phase cells and follow their progression through the cell cycle, we show that insulin delays progression through G2/M, thereby slowing cell division. The ability of insulin to slow progression through G2/M is independent of its ability to stimulate progression through G1/S, so is not a consequence of feedback by the cell-cycle machinery to maintain cell-cycle length. Insulin's effects on progression through G2/M are mediated by dTOR/dRaptor signalling. Partially inhibiting dTOR/dRaptor signalling by dsRNAi or mild rapamycin treatment can increase cell number in cultured haemocytes and the Drosophila wing, respectively. Thus, insulin signalling can influence cell number depending on a balance between its ability to accelerate progression through G1/S and delay progression through G2/M. PMID:17183368

  11. Tissue engineering in flexor tendon surgery: current state and future advances.

    PubMed

    Galvez, M G; Crowe, C; Farnebo, S; Chang, J

    2014-01-01

    Tissue engineering of flexor tendons addresses a challenge often faced by hand surgeons: the restoration of function and improvement of healing with a limited supply of donor tendons. Creating an engineered tendon construct is dependent upon understanding the normal healing mechanisms of the tendon and tendon sheath. The production of a tendon construct includes: creating a three-dimensional scaffold; seeding cells within the scaffold; encouraging cellular growth within the scaffold while maintaining a gliding surface; and finally ensuring mechanical strength. An effective construct incorporates these factors in its design, with the ultimate goal of creating tendon substitutes that are readily available to the reconstructive hand surgeon.

  12. Socs36E attenuates STAT signaling to optimize motile cell specification in the Drosophila ovary.

    PubMed

    Monahan, Amanda J; Starz-Gaiano, Michelle

    2013-07-15

    The Janus kinase/Signal transducers and activators of transcription (JAK/STAT) pathway determines cell fates by regulating gene expression. One example is the specification of the motile cells called border cells during Drosophila oogenesis. It has been established that too much or too little STAT activity disrupts follicle cell identity and cell motility, which suggests the signaling must be precisely regulated. Here, we find that Suppressor of cytokine signaling at 36E (Socs36E) is a necessary negative regulator of JAK/STAT signaling during border cell specification. We find when STAT signaling is too low to induce migration in the presumptive border cell population, nearby follicle cells uncharacteristically become invasive to enable efficient migration of the cluster. We generated a genetic null allele that reveals Socs36E is required in the anterior follicle cells to limit invasive behavior to an optimal number of cells. We further show Socs36E genetically interacts with the required STAT feedback inhibitor apontic (apt) and APT's downstream target, mir-279, and provide evidence that suggests APT directly regulates Socs36E transcriptionally. Our work shows Socs36E plays a critical role in a genetic circuit that establishes a boundary between the motile border cell cluster and its non-invasive epithelial neighbors through STAT attenuation. PMID:23583584

  13. Induction of focal adhesions and motility in Drosophila S2 cells.

    PubMed

    Ribeiro, Susana A; D'Ambrosio, Michael V; Vale, Ronald D

    2014-12-01

    Focal adhesions are dynamic structures that interact with the extracellular matrix on the cell exterior and actin filaments on the cell interior, enabling cells to adhere and crawl along surfaces. We describe a system for inducing the formation of focal adhesions in normally non-ECM-adherent, nonmotile Drosophila S2 cells. These focal adhesions contain the expected molecular markers such as talin, vinculin, and p130Cas, and they require talin for their formation. The S2 cells with induced focal adhesions also display a nonpolarized form of motility on vitronectin-coated substrates. Consistent with findings in mammalian cells, the degree of motility can be tuned by changing the stiffness of the substrate and was increased after the depletion of PAK3, a p21-activated kinase. A subset of nonmotile, nonpolarized cells also exhibited focal adhesions that rapidly assembled and disassembled around the cell perimeter. Such cooperative and dynamic fluctuations of focal adhesions were decreased by RNA interference (RNAi) depletion of myosin II and focal adhesion kinase, suggesting that this behavior requires force and focal adhesion maturation. These results demonstrate that S2 cells, a cell line that is well studied for cytoskeletal dynamics and readily amenable to protein manipulation by RNAi, can be used to study the assembly and dynamics of focal adhesions and mechanosensitive cell motility.

  14. The tiptop/teashirt genes regulate cell differentiation and renal physiology in Drosophila

    PubMed Central

    Denholm, Barry; Hu, Nan; Fauquier, Teddy; Caubit, Xavier; Fasano, Laurent; Skaer, Helen

    2013-01-01

    The physiological activities of organs are underpinned by an interplay between the distinct cell types they contain. However, little is known about the genetic control of patterned cell differentiation during organ development. We show that the conserved Teashirt transcription factors are decisive for the differentiation of a subset of secretory cells, stellate cells, in Drosophila melanogaster renal tubules. Teashirt controls the expression of the water channel Drip, the chloride conductance channel CLC-a and the Leukokinin receptor (LKR), all of which characterise differentiated stellate cells and are required for primary urine production and responsiveness to diuretic stimuli. Teashirt also controls a dramatic transformation in cell morphology, from cuboidal to the eponymous stellate shape, during metamorphosis. teashirt interacts with cut, which encodes a transcription factor that underlies the differentiation of the primary, principal secretory cells, establishing a reciprocal negative-feedback loop that ensures the full differentiation of both cell types. Loss of teashirt leads to ineffective urine production, failure of homeostasis and premature lethality. Stellate cell-specific expression of the teashirt paralogue tiptop, which is not normally expressed in larval or adult stellate cells, almost completely rescues teashirt loss of expression from stellate cells. We demonstrate conservation in the expression of the family of tiptop/teashirt genes in lower insects and establish conservation in the targets of Teashirt transcription factors in mouse embryonic kidney. PMID:23404107

  15. Tre1, a G Protein-Coupled Receptor, Directs Transepithelial Migration of Drosophila Germ Cells

    PubMed Central

    2003-01-01

    In most organisms, germ cells are formed distant from the somatic part of the gonad and thus have to migrate along and through a variety of tissues to reach the gonad. Transepithelial migration through the posterior midgut (PMG) is the first active step during Drosophila germ cell migration. Here we report the identification of a novel G protein-coupled receptor (GPCR), Tre1, that is essential for this migration step. Maternal tre1 RNA is localized to germ cells, and tre1 is required cell autonomously in germ cells. In tre1 mutant embryos, most germ cells do not exit the PMG. The few germ cells that do leave the midgut early migrate normally to the gonad, suggesting that this gene is specifically required for transepithelial migration and that mutant germ cells are still able to recognize other guidance cues. Additionally, inhibiting small Rho GTPases in germ cells affects transepithelial migration, suggesting that Tre1 signals through Rho1. We propose that Tre1 acts in a manner similar to chemokine receptors required during transepithelial migration of leukocytes, implying an evolutionarily conserved mechanism of transepithelial migration. Recently, the chemokine receptor CXCR4 was shown to direct migration in vertebrate germ cells. Thus, germ cells may more generally use GPCR signaling to navigate the embryo toward their target. PMID:14691551

  16. Lin-28 promotes symmetric stem cell division and drives adaptive growth in the adult Drosophila intestine.

    PubMed

    Chen, Ching-Huan; Luhur, Arthur; Sokol, Nicholas

    2015-10-15

    Stem cells switch between asymmetric and symmetric division to expand in number as tissues grow during development and in response to environmental changes. The stem cell intrinsic proteins controlling this switch are largely unknown, but one candidate is the Lin-28 pluripotency factor. A conserved RNA-binding protein that is downregulated in most animals as they develop from embryos to adults, Lin-28 persists in populations of adult stem cells. Its function in these cells has not been previously characterized. Here, we report that Lin-28 is highly enriched in adult intestinal stem cells in the Drosophila intestine. lin-28 null mutants are homozygous viable but display defects in this population of cells, which fail to undergo a characteristic food-triggered expansion in number and have reduced rates of symmetric division as well as reduced insulin signaling. Immunoprecipitation of Lin-28-bound mRNAs identified Insulin-like Receptor (InR), forced expression of which completely rescues lin-28-associated defects in intestinal stem cell number and division pattern. Furthermore, this stem cell activity of lin-28 is independent of one well-known lin-28 target, the microRNA let-7, which has limited expression in the intestinal epithelium. These results identify Lin-28 as a stem cell intrinsic factor that boosts insulin signaling in intestinal progenitor cells and promotes their symmetric division in response to nutrients, defining a mechanism through which Lin-28 controls the adult stem cell division patterns that underlie tissue homeostasis and regeneration.

  17. Lin-28 promotes symmetric stem cell division and drives adaptive growth in the adult Drosophila intestine

    PubMed Central

    Chen, Ching-Huan; Luhur, Arthur; Sokol, Nicholas

    2015-01-01

    Stem cells switch between asymmetric and symmetric division to expand in number as tissues grow during development and in response to environmental changes. The stem cell intrinsic proteins controlling this switch are largely unknown, but one candidate is the Lin-28 pluripotency factor. A conserved RNA-binding protein that is downregulated in most animals as they develop from embryos to adults, Lin-28 persists in populations of adult stem cells. Its function in these cells has not been previously characterized. Here, we report that Lin-28 is highly enriched in adult intestinal stem cells in the Drosophila intestine. lin-28 null mutants are homozygous viable but display defects in this population of cells, which fail to undergo a characteristic food-triggered expansion in number and have reduced rates of symmetric division as well as reduced insulin signaling. Immunoprecipitation of Lin-28-bound mRNAs identified Insulin-like Receptor (InR), forced expression of which completely rescues lin-28-associated defects in intestinal stem cell number and division pattern. Furthermore, this stem cell activity of lin-28 is independent of one well-known lin-28 target, the microRNA let-7, which has limited expression in the intestinal epithelium. These results identify Lin-28 as a stem cell intrinsic factor that boosts insulin signaling in intestinal progenitor cells and promotes their symmetric division in response to nutrients, defining a mechanism through which Lin-28 controls the adult stem cell division patterns that underlie tissue homeostasis and regeneration. PMID:26487778

  18. Increased longevity mediated by yeast NDI1 expression in Drosophila intestinal stem and progenitor cells

    PubMed Central

    Hur, Jae H.; Bahadorani, Sepehr; Graniel, Jacqueline; Koehler, Christopher L.; Ulgherait, Matthew; Rera, Michael; Jones, D. Leanne; Walker, David W.

    2013-01-01

    A functional decline in tissue stem cells and mitochondrial dysfunction have each been linked to aging and multiple aging-associated pathologies. However, the interplay between energy homeostasis, stem cells, and organismal aging remains poorly understood. Here, we report that expression of the single-subunit yeast alternative NADH dehydrogenase, ndi1, in Drosophila intestinal stem and progenitor cells delays the onset of multiple markers of intestinal aging and extends lifespan. In addition, expression of ndi1 in the intestine increases feeding behavior and results in organismal weight gain. Consistent with increased nutrient uptake, flies expressing ndi1 in the digestive tract display a systemic reduction in the activity of AMP-activated protein kinase (AMPK), a key cellular energy sensor. Together, these results demonstrate that ndi1 expression in the intestinal epithelium is an effective strategy to delay tissue and organismal aging. PMID:24038661

  19. BMP signaling and the maintenance of primordial germ cell identity in Drosophila embryos.

    PubMed

    Deshpande, Girish; Willis, Elinor; Chatterjee, Sandip; Fernandez, Robert; Dias, Kristen; Schedl, Paul

    2014-01-01

    The specification of primordial germ cells (PGCs) and subsequent maintenance of germ-line identity in Drosophila embryos has long been thought to occur solely under the control of cell-autonomous factors deposited in the posterior pole plasm during oogenesis. However, here we document a novel role for somatic BMP signaling in the maintenance of PGC fate during the period leading up to embryonic gonad coalescence. We find that PGCs fail to maintain their germline identity when BMP signaling is compromised. They initiate but are unable to properly assemble the germline stem cell-specific organelle, the spectrosome, and they lose expression of the germline-specific gene Vasa. BMP signaling must, however, be finely tuned as there are deleterious consequences to PGCs when the pathway is excessively active. We show that one mechanism used to calibrate the effects of BMP signals is dependent on the Ubc9 homolog Lesswright (Lwr).

  20. Drosophila Mitochondrial Genetics: Evolution of Heteroplasmy through Germ Line Cell Divisions

    PubMed Central

    Solignac, Michel; Génermont, Jean; Monnerot, Monique; Mounolou, Jean-Claude

    1987-01-01

    The mitochondrial genotype of all F1 female offspring (426 individuals) of a single Drosophila mauritiana female, heteroplasmic for two types of mtDNA (a short and a long genome), was established. All descendants were heteroplasmic. The earliest eggs laid by this female show the cytoplasmic genetic structure of ovariole stem cells at the end of development. Cohorts of females from the eggs laid day after day by this female, throughout the 31 days of its life, provide information on the evolution of the mitochondrial genotypes in the course of successive divisions of stem cells. An increase of the percentage of long DNA in offspring was observed as the female aged. Moreover, the variance of the genotypes increases as rounds of stem cell division progress. These results are supported by observations based on the adults issued from the early and late eggs, for three additional heteroplasmic females. PMID:17246410

  1. Stem-cell-specific endocytic degradation defects lead to intestinal dysplasia in Drosophila

    PubMed Central

    Nagy, Péter; Kovács, Laura; Sándor, Gyöngyvér O.

    2016-01-01

    ABSTRACT UV radiation resistance-associated gene (UVRAG) is a tumor suppressor involved in autophagy, endocytosis and DNA damage repair, but how its loss contributes to colorectal cancer is poorly understood. Here, we show that UVRAG deficiency in Drosophila intestinal stem cells leads to uncontrolled proliferation and impaired differentiation without preventing autophagy. As a result, affected animals suffer from gut dysfunction and short lifespan. Dysplasia upon loss of UVRAG is characterized by the accumulation of endocytosed ligands and sustained activation of STAT and JNK signaling, and attenuation of these pathways suppresses stem cell hyperproliferation. Importantly, the inhibition of early (dynamin-dependent) or late (Rab7-dependent) steps of endocytosis in intestinal stem cells also induces hyperproliferation and dysplasia. Our data raise the possibility that endocytic, but not autophagic, defects contribute to UVRAG-deficient colorectal cancer development in humans. PMID:26921396

  2. STED super-resolution microscopy in Drosophila tissue and in mammalian cells

    NASA Astrophysics Data System (ADS)

    Lau, Lana; Lee, Yin Loon; Matis, Maja; Axelrod, Jeff; Stearns, Tim; Moerner, W. E.

    2011-03-01

    Far-field super-resolution microscopy is a rapidly emerging method that is opening up opportunities for biological imaging beyond the optical diffraction limit. We have implemented a Stimulated Emission Depletion (STED) microscope to image single dye, cell, and tissue samples with 50-80 nm resolution. First, we compare the STED performance imaging single molecules of several common dyes and report a novel STED dye. Then we apply STED to image planar cell polarity protein complexes in intact fixed Drosophila tissue for the first time. Finally, we present a preliminary study of the centrosomal protein Cep164 in mammalian cells. Our images suggest that Cep164 is arranged in a nine-fold symmetric pattern around the centriole, consistent with findings suggested by cryoelectron tomography. Our work demonstrates that STED microscopy can be used for superresolution imaging in intact tissue and provides ultrastructural information in biological samples as an alternative to immuno-electron microscopy.

  3. BMP Signaling and the Maintenance of Primordial Germ Cell Identity in Drosophila Embryos

    PubMed Central

    Deshpande, Girish; Willis, Elinor; Chatterjee, Sandip; Fernandez, Robert; Dias, Kristen; Schedl, Paul

    2014-01-01

    The specification of primordial germ cells (PGCs) and subsequent maintenance of germ-line identity in Drosophila embryos has long been thought to occur solely under the control of cell-autonomous factors deposited in the posterior pole plasm during oogenesis. However, here we document a novel role for somatic BMP signaling in the maintenance of PGC fate during the period leading up to embryonic gonad coalescence. We find that PGCs fail to maintain their germline identity when BMP signaling is compromised. They initiate but are unable to properly assemble the germline stem cell-specific organelle, the spectrosome, and they lose expression of the germline-specific gene Vasa. BMP signaling must, however, be finely tuned as there are deleterious consequences to PGCs when the pathway is excessively active. We show that one mechanism used to calibrate the effects of BMP signals is dependent on the Ubc9 homolog Lesswright (Lwr). PMID:24551179

  4. The Drosophila melanogaster cinnabar gene is a cell autonomous genetic marker in Aedes aegypti (Diptera: Culicidae).

    PubMed

    Sethuraman, Nagaraja; O'Brochta, David A

    2005-07-01

    The cinnabar gene of Drosophila melanogaster (Meigen) encodes for kynurenine hydroxylase, an enzyme involved in ommochrome biosynthesis. This gene is commonly included as a visible genetic marker in gene vectors used to create transgenic Aedes aegypti (L.) that are homozygous for the khw allele, the mosquito homolog of cinnabar. Unexpectedly, the phenotype of cells expressing kynurenine hydroxylase in transgenic Ae. aegypti is cell autonomous as demonstrated by the recovery of insects heterozygous for the kynurenine hydroxylase transgene with mosaic eye color patterns. In addition, a transgenic gynandromorph was recovered in which one-half of the insect was expressing the kynurenine hydroxylase transgene, including one eye with red pigmentation, whereas the other half of the insect was homozygous khw and included a white eye. The cell autonomous behavior of cinnabar in transgenic Ae. aegypti is unexpected and increases the utility of this genetic marker. PMID:16119567

  5. EGFR/Ras Signaling Controls Drosophila Intestinal Stem Cell Proliferation via Capicua-Regulated Genes.

    PubMed

    Jin, Yinhua; Ha, Nati; Forés, Marta; Xiang, Jinyi; Gläßer, Christine; Maldera, Julieta; Jiménez, Gerardo; Edgar, Bruce A

    2015-12-01

    Epithelial renewal in the Drosophila intestine is orchestrated by Intestinal Stem Cells (ISCs). Following damage or stress the intestinal epithelium produces ligands that activate the epidermal growth factor receptor (EGFR) in ISCs. This promotes their growth and division and, thereby, epithelial regeneration. Here we demonstrate that the HMG-box transcriptional repressor, Capicua (Cic), mediates these functions of EGFR signaling. Depleting Cic in ISCs activated them for division, whereas overexpressed Cic inhibited ISC proliferation and midgut regeneration. Epistasis tests showed that Cic acted as an essential downstream effector of EGFR/Ras signaling, and immunofluorescence showed that Cic's nuclear localization was regulated by EGFR signaling. ISC-specific mRNA expression profiling and DNA binding mapping using DamID indicated that Cic represses cell proliferation via direct targets including string (Cdc25), Cyclin E, and the ETS domain transcription factors Ets21C and Pointed (pnt). pnt was required for ISC over-proliferation following Cic depletion, and ectopic pnt restored ISC proliferation even in the presence of overexpressed dominant-active Cic. These studies identify Cic, Pnt, and Ets21C as critical downstream effectors of EGFR signaling in Drosophila ISCs.

  6. Drosophila Sld5 is essential for normal cell cycle progression and maintenance of genomic integrity

    SciTech Connect

    Gouge, Catherine A.; Christensen, Tim W.

    2010-09-10

    Research highlights: {yields} Drosophila Sld5 interacts with Psf1, PPsf2, and Mcm10. {yields} Haploinsufficiency of Sld5 leads to M-phase delay and genomic instability. {yields} Sld5 is also required for normal S phase progression. -- Abstract: Essential for the normal functioning of a cell is the maintenance of genomic integrity. Failure in this process is often catastrophic for the organism, leading to cell death or mis-proliferation. Central to genomic integrity is the faithful replication of DNA during S phase. The GINS complex has recently come to light as a critical player in DNA replication through stabilization of MCM2-7 and Cdc45 as a member of the CMG complex which is likely responsible for the processivity of helicase activity during S phase. The GINS complex is made up of 4 members in a 1:1:1:1 ratio: Psf1, Psf2, Psf3, And Sld5. Here we present the first analysis of the function of the Sld5 subunit in a multicellular organism. We show that Drosophila Sld5 interacts with Psf1, Psf2, and Mcm10 and that mutations in Sld5 lead to M and S phase delays with chromosomes exhibiting hallmarks of genomic instability.

  7. Transition zone assembly and its contribution to axoneme formation in Drosophila male germ cells.

    PubMed

    Vieillard, Jennifer; Paschaki, Marie; Duteyrat, Jean-Luc; Augière, Céline; Cortier, Elisabeth; Lapart, Jean-André; Thomas, Joëlle; Durand, Bénédicte

    2016-09-26

    The ciliary transition zone (TZ) is a complex structure found at the cilia base. Defects in TZ assembly are associated with human ciliopathies. In most eukaryotes, three protein complexes (CEP290, NPHP, and MKS) cooperate to build the TZ. We show that in Drosophila melanogaster, mild TZ defects are observed in the absence of MKS components. In contrast, Cby and Azi1 cooperate to build the TZ by acting upstream of Cep290 and MKS components. Without Cby and Azi1, centrioles fail to form the TZ, precluding sensory cilia assembly, and no ciliary membrane cap associated with sperm ciliogenesis is made. This ciliary cap is critical to recruit the tubulin-depolymerizing kinesin Klp59D, required for regulation of axonemal growth. Our results show that Drosophila TZ assembly in sensory neurons and male germ cells involves cooperative actions of Cby and Dila. They further reveal that temporal control of membrane cap assembly by TZ components and microtubule elongation by kinesin-13 is required for axoneme formation in male germ cells. PMID:27646273

  8. Repression of the Drosophila proliferating-cell nuclear antigen gene promoter by zerknuellt protein

    SciTech Connect

    Yamaguchi, Masamitsu; Hirose, Fumiko; Nishida, Yasuyoshi; Matsukage, Akio )

    1991-10-01

    A 631-bp fragment containing the 5{prime}-flanking region of the Drosophila melanogaster proliferating-cell nuclear antigen (PCNA) gene was placed upstream of the chloramphenicol acetyltransferase (CAT) gene of a CAT vector. A transient expression assay of CAT activity in Drosophila Kc cells transfected with this plasmid and a set of 5{prime}-deletion derivatives revealed that the promoter function resided within a 192-bp region. Cotransfection with a zerknuellt (zen)-expressing plasmid specifically repressed CAT expression. However, cotransfection with expression plasmids for a nonfunctional zen mutation, even skipped, or bicoid showed no significant effect on CAT expression. RNase protection analysis revealed that the repression by zen was at the transcription step. The target sequence of zen was mapped within the 34-bp region of the PCNA gene promoter, even though it lacked zen protein-binding sites. Transgenic flies carrying the PCNA gene regulatory region fused with lacZ were established. These results indicate that zen indirectly represses PCNA gene expression, probably by regulating the expression of some transcription factor(s) that binds to the PCNA gene promoter.

  9. Radial Nerve Tendon Transfers.

    PubMed

    Cheah, Andre Eu-Jin; Etcheson, Jennifer; Yao, Jeffrey

    2016-08-01

    Radial nerve palsy typically occurs as a result of trauma or iatrogenic injury and leads to the loss of wrist extension, finger extension, thumb extension, and a reduction in grip strength. In the absence of nerve recovery, reconstruction of motor function involves tendon transfer surgery. The most common donor tendons include the pronator teres, wrist flexors, and finger flexors. The type of tendon transfer is classified based on the donor for the extensor digitorum communis. Good outcomes have been reported for most methods of radial nerve tendon transfers as is typical for positional tendon transfers not requiring significant power. PMID:27387076

  10. Two Independent Functions of Collier/Early B Cell Factor in the Control of Drosophila Blood Cell Homeostasis

    PubMed Central

    Krzemień, Joanna; Morin-Poulard, Ismaël; Vincent, Alain; Crozatier, Michèle

    2016-01-01

    Blood cell production in the Drosophila hematopoietic organ, the lymph gland, is controlled by intrinsic factors and extrinsic signals. Initial analysis of Collier/Early B Cell Factor function in the lymph gland revealed the role of the Posterior Signaling Center (PSC) in mounting a dedicated cellular immune response to wasp parasitism. Further, premature blood cell differentiation when PSC specification or signaling was impaired, led to assigning the PSC a role equivalent to the vertebrate hematopoietic niche. We report here that Collier is expressed in a core population of lymph gland progenitors and cell autonomously maintains this population. The PSC contributes to lymph gland homeostasis by regulating blood cell differentiation, rather than by maintaining core progenitors. In addition to PSC signaling, switching off Collier expression in progenitors is required for efficient immune response to parasitism. Our data show that two independent sites of Collier/Early B Cell Factor expression, hematopoietic progenitors and the PSC, achieve control of hematopoiesis. PMID:26866694

  11. Luciferase reporter assay in Drosophila and mammalian tissue culture cells

    PubMed Central

    Yun, Chi

    2014-01-01

    Luciferase reporter gene assays are one of the most common methods for monitoring gene activity. Because of their sensitivity, dynamic range, and lack of endogenous activity, luciferase assays have been particularly useful for functional genomics in cell-based assays, such as RNAi screening. This unit describes delivery of two luciferase reporters with other nucleic acids (siRNA /dsRNA), measurement of the dual luciferase activities, and analysis of data generated. The systematic query of gene function (RNAi) combined with the advances in luminescent technology have made it possible to design powerful whole genome screens to address diverse and significant biological questions. PMID:24652620

  12. Drosophila Photoreceptor Cells Exploited for the Production of Eukaryotic Membrane Proteins: Receptors, Transporters and Channels

    PubMed Central

    Panneels, Valérie; Kock, Ines; Krijnse-Locker, Jacomine; Rezgaoui, Meriem; Sinning, Irmgard

    2011-01-01

    Background Membrane proteins (MPs) play key roles in signal transduction. However, understanding their function at a molecular level is mostly hampered by the lack of protein in suitable amount and quality. Despite impressive developments in the expression of prokaryotic MPs, eukaryotic MP production has lagged behind and there is a need for new expression strategies. In a pilot study, we produced a Drosophila glutamate receptor specifically in the eyes of transgenic flies, exploiting the naturally abundant membrane stacks in the photoreceptor cells (PRCs). Now we address the question whether the PRCs also process different classes of medically relevant target MPs which were so far notoriously difficult to handle with conventional expression strategies. Principal Findings We describe the homologous and heterologous expression of 10 different targets from the three major MP classes - G protein-coupled receptors (GPCRs), transporters and channels in Drosophila eyes. PRCs offered an extraordinary capacity to produce, fold and accommodate massive amounts of MPs. The expression of some MPs reached similar levels as the endogenous rhodopsin, indicating that the PRC membranes were almost unsaturable. Expression of endogenous rhodopsin was not affected by the target MPs and both could coexist in the membrane stacks. Heterologous expression levels reached about 270 to 500 pmol/mg total MP, resulting in 0.2–0.4 mg purified target MP from 1 g of fly heads. The metabotropic glutamate receptor and human serotonin transporter - both involved in synaptic transmission - showed native pharmacological characteristics and could be purified to homogeneity as a prerequisite for further studies. Significance We demonstrate expression in Drosophila PRCs as an efficient and inexpensive tool for the large scale production of functional eukaryotic MPs. The fly eye system offers a number of advantages over conventional expression systems and paves the way for in-depth analyses of

  13. Different cell size and cell number contribution in two newly established and one ancient body size cline of Drosophila subobscura.

    PubMed

    Calboli, Federico C F; Gilchrist, George W; Partridge, Linda

    2003-03-01

    Latitudinal genetic clines in body size occur in many ectotherms including Drosophila species. In the wing of D. melanogaster, these clines are generally based on latitudinal variation in cell number. In contrast, differences in wing area that evolve by thermal selection in the laboratory are in general based on cell size. To investigate possible reasons for the different cellular bases of these two types of evolutionary response, we compared the newly established North and South American wing size clines of Drosophila subobscura. The new clines are based on latitudinal variation in cell area in North America and cell number in South America. The ancestral European cline is also based on latitudinal variation in cell number. The difference in the cellular basis of wing size variation in the American clines, which are roughly the same age, together with the similar cellular basis of the new South American cline and the ancient European one, suggest that the antiquity of a cline does not explain its cellular basis. Furthermore, the results indicate that wing size as a whole, rather than its cellular basis, is under selection. The different cellular bases of different size clines are most likely explained either entirely by chance or by different patterns of genetic variance--or its expression--in founding populations.

  14. Regions within a single epidermal cell of Drosophila can be planar polarised independently

    PubMed Central

    Rovira, Miguel; Saavedra, Pedro; Casal, José; Lawrence, Peter A

    2015-01-01

    Planar cell polarity (PCP), the coordinated and consistent orientation of cells in the plane of epithelial sheets, is a fundamental and conserved property of animals and plants. Up to now, the smallest unit expressing PCP has been considered to be an entire single cell. We report that, in the larval epidermis of Drosophila, different subdomains of one cell can have opposite polarities. In larvae, PCP is driven by the Dachsous/Fat system; we show that the polarity of a subdomain within one cell is its response to levels of Dachsous/Fat in the membranes of contacting cells. During larval development, cells rearrange (Saavedra et al., 2014) and when two subdomains of a single cell have different types of neighbouring cells, then these subdomains can become polarised in opposite directions. We conclude that polarisation depends on a local comparison of the amounts of Dachsous and Fat within opposing regions of a cell's membrane. DOI: http://dx.doi.org/10.7554/eLife.06303.001 PMID:25671242

  15. Toll pathway modulates TNF-induced JNK-dependent cell death in Drosophila.

    PubMed

    Wu, Chenxi; Chen, Changyan; Dai, Jianli; Zhang, Fan; Chen, Yujun; Li, Wenzhe; Pastor-Pareja, José Carlos; Xue, Lei

    2015-07-01

    Signalling networks that control the life or death of a cell are of central interest in modern biology. While the defined roles of the c-Jun N-terminal kinase (JNK) pathway in regulating cell death have been well-established, additional factors that modulate JNK-mediated cell death have yet to be fully elucidated. To identify novel regulators of JNK-dependent cell death, we performed a dominant-modifier screen in Drosophila and found that the Toll pathway participates in JNK-mediated cell death. Loss of Toll signalling suppresses ectopically and physiologically activated JNK signalling-induced cell death. Our epistasis analysis suggests that the Toll pathway acts as a downstream modulator for JNK-dependent cell death. In addition, gain of JNK signalling results in Toll pathway activation, revealed by stimulated transcription of Drosomycin (Drs) and increased cytoplasm-to-nucleus translocation of Dorsal. Furthermore, the Spätzle (Spz) family ligands for the Toll receptor are transcriptionally upregulated by activated JNK signalling in a non-cell-autonomous manner, providing a molecular mechanism for JNK-induced Toll pathway activation. Finally, gain of Toll signalling exacerbates JNK-mediated cell death and promotes cell death independent of caspases. Thus, we have identified another important function for the evolutionarily conserved Toll pathway, in addition to its well-studied roles in embryonic dorso-ventral patterning and innate immunity.

  16. The canonical Wg signaling modulates Bsk-mediated cell death in Drosophila

    PubMed Central

    Zhang, S; Chen, C; Wu, C; Yang, Y; Li, W; Xue, L

    2015-01-01

    Cell death is an essential regulatory mechanism for removing unneeded cells in animal development and tissue homeostasis. The c-Jun N-terminal kinase (JNK) pathway has pivotal roles in the regulation of cell death in response to various intrinsic and extrinsic stress signals. The canonical Wingless (Wg) signaling has been implicated in cell proliferation and cell fate decisions, whereas its role in cell death remains largely elusive. Here, we report that activated Bsk (the Drosophila JNK homolog) induced cell death is mediated by the canonical Wg signaling. First, loss of Wg signaling abrogates Bsk-mediated caspase-independent cell death. Second, activation of Wg signaling promotes cell death in a caspase-independent manner. Third, activation of Bsk signaling results in upregulated transcription of wingless (wg) gene. Finally, Wg pathway participates in the physiological function of Bsk signaling in development. These findings not only reveal a previously undiscovered role of Wg signaling in Bsk-mediated cell death, but also provide a novel mechanism for the interplay between the two important signaling pathways in development. PMID:25855961

  17. Functional Analysis of the Drosophila Embryonic Germ Cell Transcriptome by RNA Interference

    PubMed Central

    Bujna, Ágnes; Vilmos, Péter; Spirohn, Kerstin; Boutros, Michael; Erdélyi, Miklós

    2014-01-01

    In Drosophila melanogaster, primordial germ cells are specified at the posterior pole of the very early embryo. This process is regulated by the posterior localized germ plasm that contains a large number of RNAs of maternal origin. Transcription in the primordial germ cells is actively down-regulated until germ cell fate is established. Bulk expression of the zygotic genes commences concomitantly with the degradation of the maternal transcripts. Thus, during embryogenesis, maternally provided and zygotically transcribed mRNAs determine germ cell development collectively. In an effort to identify novel genes involved in the regulation of germ cell behavior, we carried out a large-scale RNAi screen targeting both maternal and zygotic components of the embryonic germ line transcriptome. We identified 48 genes necessary for distinct stages in germ cell development. We found pebble and fascetto to be essential for germ cell migration and germ cell division, respectively. Our data uncover a previously unanticipated role of mei-P26 in maintenance of embryonic germ cell fate. We also performed systematic co-RNAi experiments, through which we found a low rate of functional redundancy among homologous gene pairs. As our data indicate a high degree of evolutionary conservation in genetic regulation of germ cell development, they are likely to provide valuable insights into the biology of the germ line in general. PMID:24896584

  18. Fibrillins in Tendon

    PubMed Central

    Giusti, Betti; Pepe, Guglielmina

    2016-01-01

    Tendons among connective tissue, mainly collagen, contain also elastic fibers (EF) made of fibrillin 1, fibrillin 2 and elastin that are broadly distributed in tendons and represent 1–2% of the dried mass of the tendon. Only in the last years, studies on structure and function of EF in tendons have been performed. Aim of this review is to revise data on the organization of EF in tendons, in particular fibrillin structure and function, and on the clinical manifestations associated to alterations of EF in tendons. Indeed, microfibrils may contribute to tendon mechanics; therefore, their alterations may cause joint hypermobility and contractures which have been found to be clinical features in patients with Marfan syndrome (MFS) and Beals syndrome. The two diseases are caused by mutations in genes FBN1 and FBN2 encoding fibrillin 1 and fibrillin 2, respectively. PMID:27812333

  19. Tendon Structure and Composition.

    PubMed

    Thorpe, Chavaunne T; Screen, Hazel R C

    2016-01-01

    Tendons are soft, fibrous tissues that connect muscle to bone. Their main function is to transfer muscle generated force to the bony skeleton, facilitating movement around a joint, and as such they are relatively passive, inelastic structures, able to resist high forces. Tendons are predominantly composed of collagen, which is arranged in a hierarchical manner parallel to the long axis of the tendon, resulting in high tensile strength. Tendon also contains a range of non-collagenous proteins, present in low amounts, which nevertheless have important functional roles. In this chapter, we describe general tendon composition and structure, and discuss how variations in composition and structure at different levels of the tendon hierarchy confer specific mechanical properties, which are related to tendon function. PMID:27535244

  20. Ibuprofen upregulates expressions of matrix metalloproteinase-1, -8, -9, and -13 without affecting expressions of types I and III collagen in tendon cells.

    PubMed

    Tsai, Wen-Chung; Hsu, Chih-Chin; Chang, Hsiang-Ning; Lin, Yu-Chun; Lin, Miao-Sui; Pang, Jong-Hwei S

    2010-04-01

    Nonsteroidal antiinflammatory drugs are widely used to treat sports-related tendon injuries or tendinopathy. This study was designed to investigate the effect of ibuprofen on expressions of types I and III collagen, as well as collagen-degrading enzymes including matrix metalloproteinase (MMP)-1, -2, -8, -9, and -13. Rat Achilles tendon cells were treated with ibuprofen and then underwent MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Reverse transcription-polymerase chain reaction was used to evaluate mRNA expressions of types I and III collagen, MMP-1, -2, -8, -9, and -13. Protein expressions of types I and III collagen, MMP-1, -8, and -13 were determined by Western blot analysis. Gelatin zymography was used to evaluate the enzymatic activities of MMP-2 and MMP-9. The results revealed that ibuprofen upregulated expressions of MMP-1, -8, -9, and -13, both at mRNA and protein levels. There was no effect of ibuprofen on mRNA and protein expressions of types I and III collagen. Gelatin zymography revealed that the enzymatic activity of MMP-9 was upregulated after ibuprofen treatment. In conclusion, ibuprofen upregulates the expressions of collagenases including MMP-1, -8, -9, and -13 without affecting the expressions of types I and III collagen. These findings suggest a molecular mechanism potentially accounting for the inhibition of tendon healing by ibuprofen.

  1. In vitro investigation of a tissue-engineered cell-tendon complex mimicking the transitional architecture at the ligament-bone interface.

    PubMed

    Wang, Zhibing; Zhang, Yuan; Zhu, Jie; Dong, Shiwu; Jiang, Tao; Zhou, Yue; Zhang, Xia

    2015-03-01

    Restoration of the transitional ligament-bone interface is critical for graft-bone integration. We postulated that an allogenic scaffold mimicking the fibrogenic, chondrogenic, and osteogenic transition gradients could physiologically promote ligament-bone incorporation. The aim of this study was to construct and characterize a composite tendon scaffold with a continuous and heterogeneous transition region mimicking a native ligament insertion site. Genetically modified heterogeneous cell populations were seeded within specific regions of decellularized rabbit Achilles tendons to fabricate a stratified scaffold containing three biofunctional regions supporting fibrogenesis, chondrogenesis, and osteogenesis. The observed morphology, architecture, cytocompatibility, and biomechanics of the scaffolds demonstrated their improved bio-physico-chemical properties. The formation of the transitional regions was augmented via enhanced delivery of two transcription factors, sex determining region Y-box 9 and runt-related transcription factor 2, which also triggered early up-regulated expression of cartilage- and bone-relevant markers, according to quantitative PCR and immunoblot analyses. Gradient tissue-specific matrix formation was also confirmed within the predesignated regions via histological staining and immunofluorescence assays. These results suggest that a transitional interface could be replicated on an engineered tendon through stratified tissue integration. The scaffold offers the advantages of a multitissue transition involving controlled cellular interactions and matrix heterogeneity, which can be applied for the regeneration of the ligament-bone interface.

  2. Somatic-cell mutation induced by short exposures to cigarette smoke in urate-null, oxidative stress-sensitive Drosophila.

    PubMed

    Uchiyama, Tomoyo; Koike, Ryota; Yuma, Yoko; Okamoto, Keinosuke; Arimoto-Kobayashi, Sakae; Suzuki, Toshinori; Negishi, Tomoe

    2016-01-01

    We previously reported that a urate-null strain of Drosophila is hypersensitive to cigarette smoke (CS), and we suggested that CS induces oxidative stress in Drosophila because uric acid is a potent antioxidant. Although the carcinogenic risk of CS exposure is widely recognized; documentation of in vivo genotoxic activity of environmental CS, especially gaseous-phase CS, remains inconclusive. To date, somatic-cell mutations in Drosophila resulting from exposure to CS have not been detected via the somatic mutation and recombination test (wing spot test) with wild-type flies, a widely used Drosophila assay for the detection of somatic-cell mutation; moreover, genotoxicity has not been documented via a DNA repair test that involves DNA repair-deficient Drosophila. In this study, we used a new Drosophila strain (y v ma-l; mwh) to examine the mutagenicity induced by gaseous-phase CS; these flies are urate-null due to a mutation in ma-l, and they are heterozygous for multiple wing hair (mwh), a mutation that functions as a marker for somatic-cell mutation. In an assay with this newly developed strain, a superoxide anion-producing weed-killer, paraquat, exhibited significant mutagenicity; in contrast, paraquat was hardly mutagenic with a wild-type strain. Drosophila larvae were exposed to CS for 2, 4 or 6h, and then kept at 25°C on instant medium until adulthood. After eclosion, mutant spots, which consisted of mutant hairs on wings, were scored. The number of mutant spots increased significantly in an exposure time-dependent manner in the urate-null females (ma-l (-/-)), but not in the urate-positive females (ma-l (+/-)). In this study, we showed that short-term exposure to CS was mutagenic in this in vivo system. In addition, we obtained suggestive data regarding reactive oxygen species production in larva after CS exposure using the fluorescence probe H2DCFDA. These results suggest that oxidative damage, which might be countered by uric acid, was partly responsible

  3. Biological Augmentation of Rotator Cuff Tendon Repair

    PubMed Central

    Kovacevic, David

    2008-01-01

    A histologically normal insertion site does not regenerate following rotator cuff tendon-to-bone repair, which is likely due to abnormal or insufficient gene expression and/or cell differentiation at the repair site. Techniques to manipulate the biologic events following tendon repair may improve healing. We used a sheep infraspinatus repair model to evaluate the effect of osteoinductive growth factors and BMP-12 on tendon-to-bone healing. Magnetic resonance imaging and histology showed increased formation of new bone and fibrocartilage at the healing tendon attachment site in the treated animals, and biomechanical testing showed improved load-to-failure. Other techniques with potential to augment repair site biology include use of platelets isolated from autologous blood to deliver growth factors to a tendon repair site. Modalities that improve local vascularity, such as pulsed ultrasound, have the potential to augment rotator cuff healing. Important information about the biology of tendon healing can also be gained from studies of substances that inhibit healing, such as nicotine and antiinflammatory medications. Future approaches may include the use of stem cells and transcription factors to induce formation of the native tendon-bone insertion site after rotator cuff repair surgery. PMID:18264850

  4. Factors Influencing the Production of MFSV Full-Length Clone: Maize Fine Streak Virus Proteins in Drosophila S2 Cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Maize fine streak virus (MFSV) is negative-sense RNA virus member of the genus Nucleorhabdovirus. Our goal is to determine whether Drosophila S2 cells can support the production of a full-length clone of MFSV. We have previously demonstrated that the full-length MFSV nucleoprotein (N) and phosphopro...

  5. Nanotubes mediate niche-stem-cell signalling in the Drosophila testis.

    PubMed

    Inaba, Mayu; Buszczak, Michael; Yamashita, Yukiko M

    2015-07-16

    Stem cell niches provide resident stem cells with signals that specify their identity. Niche signals act over a short range such that only stem cells but not their differentiating progeny receive the self-renewing signals. However, the cellular mechanisms that limit niche signalling to stem cells remain poorly understood. Here we show that the Drosophila male germline stem cells form previously unrecognized structures, microtubule-based nanotubes, which extend into the hub, a major niche component. Microtubule-based nanotubes are observed specifically within germline stem cell populations, and require intraflagellar transport proteins for their formation. The bone morphogenetic protein (BMP) receptor Tkv localizes to microtubule-based nanotubes. Perturbation of microtubule-based nanotubes compromises activation of Dpp signalling within germline stem cells, leading to germline stem cell loss. Moreover, Dpp ligand and Tkv receptor interaction is necessary and sufficient for microtubule-based nanotube formation. We propose that microtubule-based nanotubes provide a novel mechanism for selective receptor-ligand interaction, contributing to the short-range nature of niche-stem-cell signalling.

  6. Different cell cycle modifications repress apoptosis at different steps independent of developmental signaling in Drosophila

    PubMed Central

    Qi, Suozhi; Calvi, Brian R.

    2016-01-01

    Apoptotic cell death is important for the normal development of a variety of organisms. Apoptosis is also a response to DNA damage and an important barrier to oncogenesis. The apoptotic response to DNA damage is dampened in specific cell types during development. Developmental signaling pathways can repress apoptosis, and reduced cell proliferation also correlates with a lower apoptotic response. However, because developmental signaling regulates both cell proliferation and apoptosis, the relative contribution of cell division to the apoptotic response has been hard to discern in vivo. Here we use Drosophila oogenesis as an in vivo model system to determine the extent to which cell proliferation influences the apoptotic response to DNA damage. We find that different types of cell cycle modifications are sufficient to repress the apoptotic response to ionizing radiation independent of developmental signaling. The step(s) at which the apoptosis pathway was repressed depended on the type of cell cycle modification—either upstream or downstream of expression of the p53-regulated proapoptotic genes. Our findings have important implications for understanding the coordination of cell proliferation with the apoptotic response in development and disease, including cancer and the tissue-specific responses to radiation therapy. PMID:27075174

  7. Cytoskeletal turnover and Myosin contractility drive cell autonomous oscillations in a model of Drosophila Dorsal Closure

    NASA Astrophysics Data System (ADS)

    Machado, P. F.; Blanchard, G. B.; Duque, J.; Gorfinkiel, N.

    2014-06-01

    Oscillatory behaviour in force-generating systems is a pervasive phenomenon in cell biology. In this work, we investigate how oscillations in the actomyosin cytoskeleton drive cell shape changes during the process of Dorsal Closure (DC), a morphogenetic event in Drosophila embryo development whereby epidermal continuity is generated through the pulsatile apical area reduction of cells constituting the amnioserosa (AS) tissue. We present a theoretical model of AS cell dynamics by which the oscillatory behaviour arises due to a coupling between active myosin-driven forces, actin turnover and cell deformation. Oscillations in our model are cell-autonomous and are modulated by neighbour coupling, and our model accurately reproduces the oscillatory dynamics of AS cells and their amplitude and frequency evolution. A key prediction arising from our model is that the rate of actin turnover and Myosin contractile force must increase during DC in order to reproduce the decrease in amplitude and period of cell area oscillations observed in vivo. This prediction opens up new ways to think about the molecular underpinnings of AS cell oscillations and their link to net tissue contraction and suggests the form of future experimental measurements.

  8. Transforming Growth Factor β/activin signalling induces epithelial cell flattening during Drosophila oogenesis

    PubMed Central

    Brigaud, Isabelle; Duteyrat, Jean-Luc; Chlasta, Julien; Le Bail, Sandrine; Couderc, Jean-Louis; Grammont, Muriel

    2015-01-01

    ABSTRACT Although the regulation of epithelial morphogenesis is essential for the formation of tissues and organs in multicellular organisms, little is known about how signalling pathways control cell shape changes in space and time. In the Drosophila ovarian epithelium, the transition from a cuboidal to a squamous shape is accompanied by a wave of cell flattening and by the ordered remodelling of E-cadherin-based adherens junctions. We show that activation of the TGFβ pathway is crucial to determine the timing, the degree and the dynamic of cell flattening. Within these cells, TGFβ signalling controls cell-autonomously the formation of Actin filament and the localisation of activated Myosin II, indicating that internal forces are generated and used to remodel AJ and to promote cytoskeleton rearrangement. Our results also reveal that TGFβ signalling controls Notch activity and that its functions are partly executed through Notch. Thus, we demonstrate that the cells that undergo the cuboidal-to-squamous transition produce active cell-shaping mechanisms, rather than passively flattening in response to a global force generated by the growth of the underlying cells. Thus, our work on TGFβ signalling provides new insights into the mechanisms through which signal transduction cascades orchestrate cell shape changes to generate proper organ structure. PMID:25681395

  9. Cyclin E-dependent protein kinase activity regulates niche retention of Drosophila ovarian follicle stem cells

    PubMed Central

    Wang, Zhu A.; Kalderon, Daniel

    2009-01-01

    Whether stem cells have unique cell cycle machineries and how they integrate with niche interactions remains largely unknown. We identified a hypomorphic cyclin E allele WX that strongly impairs the maintenance of follicle stem cells (FSCs) in the Drosophila ovary but does not reduce follicle cell proliferation or germline stem cell maintenance. CycEWX protein can still bind to the cyclin-dependent kinase catalytic subunit Cdk2, but forms complexes with reduced protein kinase activity measured in vitro. By creating additional CycE variants with different degrees of kinase dysfunction and expressing these and CycEWX at different levels, we found that higher CycE-Cdk2 kinase activity is required for FSC maintenance than to support follicle cell proliferation. Surprisingly, cycEWX FSCs were lost from their niches rather than arresting proliferation. Furthermore, FSC function was substantially restored by expressing either excess DE-cadherin or excess E2F1/DP, the transcription factor normally activated by CycE-Cdk2 phosphorylation of retinoblastoma proteins. These results suggest that FSC maintenance through niche adhesion is regulated by inputs that normally control S phase entry, possibly as a quality control mechanism to ensure adequate stem cell proliferation. We speculate that a positive connection between central regulators of the cell cycle and niche retention may be a common feature of highly proliferative stem cells. PMID:19966222

  10. Activation of EphA4 and EphB2 Reverse Signaling Restores the Age-Associated Reduction of Self-Renewal, Migration, and Actin Turnover in Human Tendon Stem/Progenitor Cells

    PubMed Central

    Popov, Cvetan; Kohler, Julia; Docheva, Denitsa

    2016-01-01

    Tendon tissues, due to their composition and function, are prone to suffer age-related degeneration and diseases as well as to respond poorly to current repair strategies. It has been suggested that local stem cells, named tendon stem/progenitor cells (TSPCs), play essential roles in tendon maintenance and healing. Recently, we have shown that TSPC exhibit a distinct age-related phenotype involving transcriptomal shift, poor self-renewal, and elevated senescence coupled with reduced cell migration and actin dynamics. Here, we report for the first time the significant downregulation of the ephrin receptors EphA4, EphB2 and B4 and ligands EFNB1 in aged-TSPC (A-TSPC). Rescue experiments, by delivery of target-specific clustered proteins, revealed that activation of EphA4- or EphB2-dependent reverse signaling could restore the migratory ability and normalize the actin turnover of A-TSPC. However, only EphA4-Fc stimulation improved A-TSPC cell proliferation to levels comparable to young-TSPC (Y-TSPC). Hence, our novel data suggests that decreased expression of ephrin receptors during tendon aging and degeneration limits the establishment of appropriate cell-cell interactions between TSPC and significantly diminished their proliferation, motility, and actin turnover. Taken together, we could propose that this mechanism might be contributing to the inferior and delayed tendon healing common for aged individuals. PMID:26779014

  11. dNF-YB plays dual roles in cell death and cell differentiation during Drosophila eye development.

    PubMed

    Ly, Luong Linh; Suyari, Osamu; Yoshioka, Yasuhide; Tue, Nguyen Trong; Yoshida, Hideki; Yamaguchi, Masamitsu

    2013-05-15

    Nuclear transcription factor Y (NF-Y) is well characterized in eukaryotes. It consists of three different subunits, NF-YA, NF-YB and NF-YC, all of which are required for formation of the NF-Y complex and DNA-binding. There is a high homology in NF-YB among Drosophila species with 75% identity and 95% similarity overall, especially in the histone-fold motif (HFM) (95% identity and 100% similarity). In the present study, specific knockdown of Drosophila NF-YB (dNF-YB) in eye imaginal discs induced a rough eye phenotype in adults and this phenotype was the result of induction of caspase-dependent apoptosis followed by apoptosis-induced proliferation. Furthermore, knockdown specifically inhibited R7 photoreceptor cell differentiation, independent of the apoptotic function. dNF-YB and dNF-YA indeed form complexes in vivo where they impair R7 photoreceptor cell differentiation by down regulating the mitogen-activated protein kinase (MAPK) pathway. Expression of the sev gene, or the D-raf gene, a downstream component of the MAPK cascade, could rescue the rough eye phenotype and the loss of R7 signals in dNF-YB knockdown flies. The death executioner Bcl-2 (debcl) is the homolog of Bcl-2 in Drosophila melanogaster and its promoter contains four dNF-Y-binding consensus sequences which play positive roles in promoter activity. In chromatin immunoprecipitation assays with anti-dNF-YB antibody and S2 cells, the debcl gene promoter region containing the NF-Y consensus was effectively amplified in immunoprecipitates by polymerase chain reaction. Taken together, these results indicate that dNF-Y regulates debcl gene expression.

  12. Epidermal cells are the primary phagocytes in the fragmentation and clearance of degenerating dendrites in Drosophila

    PubMed Central

    Xiao, Hui; Wang, Denan; Franc, Nathalie C.; Jan, Lily Yeh; Jan, Yuh-Nung

    2014-01-01

    SUMMARY During developmental remodeling, neurites destined for pruning often degenerate on-site. Physical injury also induces degeneration of neurites distal to the injury site. Prompt clearance of degenerating neurites is important for maintaining tissue homeostasis and preventing inflammatory responses. Here we show that in both dendrite pruning and dendrite injury of Drosophila sensory neurons, epidermal cells rather than hemocytes are the primary phagocytes in clearing degenerating dendrites. Epidermal cells act via Draper-mediated recognition to facilitate dendrite degeneration and to engulf and degrade degenerating dendrites. Using multiple dendritic membrane markers to trace phagocytosis, we show that two members of the CD36 family, croquemort (crq) and debris buster (dsb), act at distinct stages of phagosome maturation for dendrite clearance. Our finding reveals the physiological importance of coordination between neurons and their surrounding epidermis, for both dendrite fragmentation and clearance. PMID:24412417

  13. An essential cell cycle regulation gene causes hybrid inviability in Drosophila.

    PubMed

    Phadnis, Nitin; Baker, EmilyClare P; Cooper, Jacob C; Frizzell, Kimberly A; Hsieh, Emily; de la Cruz, Aida Flor A; Shendure, Jay; Kitzman, Jacob O; Malik, Harmit S

    2015-12-18

    Speciation, the process by which new biological species arise, involves the evolution of reproductive barriers, such as hybrid sterility or inviability between populations. However, identifying hybrid incompatibility genes remains a key obstacle in understanding the molecular basis of reproductive isolation. We devised a genomic screen, which identified a cell cycle-regulation gene as the cause of male inviability in hybrids resulting from a cross between Drosophila melanogaster and D. simulans. Ablation of the D. simulans allele of this gene is sufficient to rescue the adult viability of hybrid males. This dominantly acting cell cycle regulator causes mitotic arrest and, thereby, inviability of male hybrid larvae. Our genomic method provides a facile means to accelerate the identification of hybrid incompatibility genes in other model and nonmodel systems.

  14. An essential cell cycle regulation gene causes hybrid inviability in Drosophila.

    PubMed

    Phadnis, Nitin; Baker, EmilyClare P; Cooper, Jacob C; Frizzell, Kimberly A; Hsieh, Emily; de la Cruz, Aida Flor A; Shendure, Jay; Kitzman, Jacob O; Malik, Harmit S

    2015-12-18

    Speciation, the process by which new biological species arise, involves the evolution of reproductive barriers, such as hybrid sterility or inviability between populations. However, identifying hybrid incompatibility genes remains a key obstacle in understanding the molecular basis of reproductive isolation. We devised a genomic screen, which identified a cell cycle-regulation gene as the cause of male inviability in hybrids resulting from a cross between Drosophila melanogaster and D. simulans. Ablation of the D. simulans allele of this gene is sufficient to rescue the adult viability of hybrid males. This dominantly acting cell cycle regulator causes mitotic arrest and, thereby, inviability of male hybrid larvae. Our genomic method provides a facile means to accelerate the identification of hybrid incompatibility genes in other model and nonmodel systems. PMID:26680200

  15. An essential cell cycle regulation gene causes hybrid inviability in Drosophila

    PubMed Central

    Phadnis, Nitin; Baker, EmilyClare P.; Cooper, Jacob C.; Frizzell, Kimberly A.; Hsieh, Emily; de la Cruz, Aida Flor A.; Shendure, Jay; Kitzman, Jacob O.; Malik, Harmit S.

    2015-01-01

    Speciation, the process by which new biological species arise, involves the evolution of reproductive barriers such as hybrid sterility or inviability between populations. However, identifying hybrid incompatibility genes remains a key obstacle in understanding the molecular basis of reproductive isolation. We devised a genomic screen, which identified a cell cycle regulation gene as the cause of male inviability in hybrids between Drosophila melanogaster and D. simulans. Ablation of the D. simulans allele of this gene is sufficient to rescue the adult viability of hybrid males. This dominantly acting cell cycle regulator causes mitotic arrest and, thereby, inviability of male hybrid larvae. Our genomic method provides a facile means to accelerate the identification of hybrid incompatibility genes in other model and non-model systems. PMID:26680200

  16. Vesicular stomatitis virus in Drosophila melanogaster cells: regulation of viral transcription and replication.

    PubMed Central

    Blondel, D; Petitjean, A M; Dezélée, S; Wyers, F

    1988-01-01

    Vesicular stomatitis virus RNA synthesis was investigated during the establishment of persistent infection in Drosophila melanogaster cells. The transcription rate declined as early as 5 h after infection and was strongly inhibited after 7 h, leading to a decrease in viral mRNA levels and in viral protein synthesis rates. Full-length plus-strand antigenomes and minus-strand genomes were detected after a 3-h lag time and accumulated until 15 h after infection. Short encapsidated plus-strand molecules were also generated corresponding to the 5' end of viral defective antigenomes. Assembly and release of virions were not restricted, but their infectivity was extremely reduced. In persistently infected cells, an equilibrium was reached where the level of intracellular genomes maintained was constant and maximal even after the rate of all viral syntheses had decreased. These results are discussed with regard to the establishment of persistent infection. Images PMID:2824851

  17. Dpp signaling determines regional stem cell identity in the regenerating adult Drosophila gastrointestinal tract.

    PubMed

    Li, Hongjie; Qi, Yanyan; Jasper, Heinrich

    2013-07-11

    The gastrointestinal tract is lined by a series of epithelia that share functional requirements but also have distinct, highly specialized roles. Distinct populations of somatic stem cells (SCs) regenerate these epithelia, yet the mechanisms that maintain regional identities of these SCs are not well understood. Here, we identify a role for the BMP-like Dpp signaling pathway in diversifying regenerative processes in the adult gastrointestinal tract of Drosophila. Dpp secreted from enterocytes at the boundary between the posterior midgut and the middle midgut (MM) sets up a morphogen gradient that selectively directs copper cell (CC) regeneration from gastric SCs in the MM and thus determines the size of the CC region. In vertebrates, deregulation of BMP signaling has been associated with Barrett's metaplasia, wherein the squamous esophageal epithelium is replaced by a columnar epithelium, suggesting that the maintenance of regional SC identities by BMP is conserved.

  18. Evaluation of mutagenic, recombinogenic and carcinogenic potential of (+)-usnic acid in somatic cells of Drosophila melanogaster.

    PubMed

    Machado, Nayane Moreira; de Rezende, Alexandre Azenha Alves; Nepomuceno, Júlio César; Tavares, Denise Crispim; Cunha, Wilson Roberto; Spanó, Mário Antônio

    2016-10-01

    The main of this study was to evaluate the mutagenic and carcinogenic potential of (+) - usnic acid (UA), using Somatic Mutation and Recombination Test (SMART) and the test for detecting epithelial tumor clones (wts) in Drosophila melanogaster. Larvae from 72 ± 4 h from Drosophila were fed with UA (5.0, 10.0 or 20.0 mM); urethane (10.0 mM) (positive control); and solvent (Milli-Q water, 1% Tween-80 and 3% ethanol) (negative control). ST cross produced increase in total mutant spots in the individuals treated with 5.0, 10.0 or 20.0 mM of UA. HB cross produced spot frequencies in the concentration of 5.0 mM that were higher than the frequency for the same concentration in the ST cross. In the highest concentrations the result was negative, which means that the difference observed can be attributed, in part, to the high levels of P450, suggesting that increasing the metabolic capacity maximized the toxic effect of these doses. In the evaluation of carcinogenesis using the wts test, the results obtained for the same concentrations of UA show a positive result for the presence of tumors when compared to the negative control. We conclude that UA has recombinogenic, mutagenic and carcinogenic effects on somatic cells in D. melanogaster. PMID:27497765

  19. Recombinogenic activity of Pantoprazole(®) in somatic cells of Drosophila melanogaster.

    PubMed

    Lopes, Jeyson Césary; Machado, Nayane Moreira; Saturnino, Rosiane Soares; Nepomuceno, Júlio César

    2015-03-01

    Pantoprazole(®) is one of the leading proton pump inhibitors (PPIs) used in the treatment of a variety of diseases related to the upper gastrointestinal tract. However, studies have shown an increased risk of developing gastric cancer, intestinal metaplasia and hyperplasia of endocrine cells with prolonged use. In the present study, the somatic mutation and recombination test (SMART) was employed to determine the mutagenic effects of Pantoprazole on Drosophila melanogaster. Repeated treatments with Pantoprazole were performed on 72-hour larvae of the standard (ST) and high bioactivation (HB) crosses at concentrations of 2.5, 5.0, and 10.0 μM. In addition, doxorubicin (DXR) was administered at 0.4 mM, as a positive control. When administered to ST descendants, total number of spots were statistically significant at 2.5 and 5.0 μM concentrations. For HB descendants, a significant increase in the total number of spots was observed among the marked transheterozygous (MH) flies. Through analysis of balancer heterozygous (BH) descendants, recombinogenic effects were observed at all concentrations in descendants of the HB cross. In view of these experimental conditions and results, it was concluded that Pantoprazole is associated with recombinogenic effects in Drosophila melanogaster. PMID:25983631

  20. Waves of Cdk1 Activity in S Phase Synchronize the Cell Cycle in Drosophila Embryos.

    PubMed

    Deneke, Victoria E; Melbinger, Anna; Vergassola, Massimo; Di Talia, Stefano

    2016-08-22

    Embryos of most metazoans undergo rapid and synchronous cell cycles following fertilization. While diffusion is too slow for synchronization of mitosis across large spatial scales, waves of Cdk1 activity represent a possible process of synchronization. However, the mechanisms regulating Cdk1 waves during embryonic development remain poorly understood. Using biosensors of Cdk1 and Chk1 activities, we dissect the regulation of Cdk1 waves in the Drosophila syncytial blastoderm. We show that Cdk1 waves are not controlled by the mitotic switch but by a double-negative feedback between Cdk1 and Chk1. Using mathematical modeling and surgical ligations, we demonstrate a fundamental distinction between S phase Cdk1 waves, which propagate as active trigger waves in an excitable medium, and mitotic Cdk1 waves, which propagate as passive phase waves. Our findings show that in Drosophila embryos, Cdk1 positive feedback serves primarily to ensure the rapid onset of mitosis, while wave propagation is regulated by S phase events.

  1. Ensconsin/Map7 promotes microtubule growth and centrosome separation in Drosophila neural stem cells

    PubMed Central

    Gallaud, Emmanuel; Caous, Renaud; Pascal, Aude; Bazile, Franck; Gagné, Jean-Philippe; Huet, Sébastien; Poirier, Guy G.; Chrétien, Denis; Richard-Parpaillon, Laurent

    2014-01-01

    The mitotic spindle is crucial to achieve segregation of sister chromatids. To identify new mitotic spindle assembly regulators, we isolated 855 microtubule-associated proteins (MAPs) from Drosophila melanogaster mitotic or interphasic embryos. Using RNAi, we screened 96 poorly characterized genes in the Drosophila central nervous system to establish their possible role during spindle assembly. We found that Ensconsin/MAP7 mutant neuroblasts display shorter metaphase spindles, a defect caused by a reduced microtubule polymerization rate and enhanced by centrosome ablation. In agreement with a direct effect in regulating spindle length, Ensconsin overexpression triggered an increase in spindle length in S2 cells, whereas purified Ensconsin stimulated microtubule polymerization in vitro. Interestingly, ensc-null mutant flies also display defective centrosome separation and positioning during interphase, a phenotype also detected in kinesin-1 mutants. Collectively, our results suggest that Ensconsin cooperates with its binding partner Kinesin-1 during interphase to trigger centrosome separation. In addition, Ensconsin promotes microtubule polymerization during mitosis to control spindle length independent of Kinesin-1. PMID:24687279

  2. Establishing and maintaining cell polarity with mRNA localization in Drosophila

    PubMed Central

    Barr, Justinn; Yakovlev, Konstantin V.; Shidlovskii, Yulii; Schedl, Paul

    2016-01-01

    How cell polarity is established and maintained is an important question in diverse biological contexts. Molecular mechanisms used to localize polarity proteins to distinct domains are likely context-dependent and provide a feedback loop in order to maintain polarity. One such mechanism is the localized translation of mRNAs encoding polarity proteins, which will be the focus of this review and may play a more important role in the establishment and maintenance of polarity than is currently known. Localized translation of mRNAs encoding polarity proteins can be used to establish polarity in response to an external signal, and to maintain polarity by local production of polarity determinants. The importance of this mechanism is illustrated by recent findings, including orb2-dependent localized translation of aPKC mRNA at the apical end of elongating spermatid tails in the Drosophila testis, and the apical localization of stardust A mRNA in Drosophila follicle and embryonic epithelia. PMID:26773560

  3. Cellular Defense and Sensory Cell Survival Require Distinct Functions of ebi in Drosophila

    PubMed Central

    Lim, Young-Mi; Yagi, Yoshimasa; Tsuda, Leo

    2015-01-01

    The innate immune response and stress-induced apoptosis are well-established signaling pathways related to cellular defense. NF-κB and AP-1 are redox-sensitive transcription factors that play important roles in those pathways. Here we show that Ebi, a Drosophila homolog of the mammalian co-repressor molecule transducin β-like 1 (TBL1), variously regulates the expression of specific genes that are targets of redox-sensitive transcription factors. In response to different stimuli, Ebi activated gene expression to support the acute immune response in fat bodies, whereas Ebi repressed genes that are involved in apoptosis in photoreceptor cells. Thus, Ebi seems to act as a regulatory switch for genes that are activated or repressed in response to different external stimuli. Our results offer clear in vivo evidence that the Ebi-containing co-repressor complex acts in a distinct manner to regulate transcription that is required for modulating the output of various processes during Drosophila development. PMID:26524764

  4. Waves of Cdk1 Activity in S Phase Synchronize the Cell Cycle in Drosophila Embryos.

    PubMed

    Deneke, Victoria E; Melbinger, Anna; Vergassola, Massimo; Di Talia, Stefano

    2016-08-22

    Embryos of most metazoans undergo rapid and synchronous cell cycles following fertilization. While diffusion is too slow for synchronization of mitosis across large spatial scales, waves of Cdk1 activity represent a possible process of synchronization. However, the mechanisms regulating Cdk1 waves during embryonic development remain poorly understood. Using biosensors of Cdk1 and Chk1 activities, we dissect the regulation of Cdk1 waves in the Drosophila syncytial blastoderm. We show that Cdk1 waves are not controlled by the mitotic switch but by a double-negative feedback between Cdk1 and Chk1. Using mathematical modeling and surgical ligations, we demonstrate a fundamental distinction between S phase Cdk1 waves, which propagate as active trigger waves in an excitable medium, and mitotic Cdk1 waves, which propagate as passive phase waves. Our findings show that in Drosophila embryos, Cdk1 positive feedback serves primarily to ensure the rapid onset of mitosis, while wave propagation is regulated by S phase events. PMID:27554859

  5. Recombinogenic activity of Pantoprazole® in somatic cells of Drosophila melanogaster

    PubMed Central

    Lopes, Jeyson Césary; Machado, Nayane Moreira; Saturnino, Rosiane Soares; Nepomuceno, Júlio César

    2015-01-01

    Pantoprazole® is one of the leading proton pump inhibitors (PPIs) used in the treatment of a variety of diseases related to the upper gastrointestinal tract. However, studies have shown an increased risk of developing gastric cancer, intestinal metaplasia and hyperplasia of endocrine cells with prolonged use. In the present study, the somatic mutation and recombination test (SMART) was employed to determine the mutagenic effects of Pantoprazole on Drosophila melanogaster. Repeated treatments with Pantoprazole were performed on 72-hour larvae of the standard (ST) and high bioactivation (HB) crosses at concentrations of 2.5, 5.0, and 10.0 μM. In addition, doxorubicin (DXR) was administered at 0.4 mM, as a positive control. When administered to ST descendants, total number of spots were statistically significant at 2.5 and 5.0 μM concentrations. For HB descendants, a significant increase in the total number of spots was observed among the marked transheterozygous (MH) flies. Through analysis of balancer heterozygous (BH) descendants, recombinogenic effects were observed at all concentrations in descendants of the HB cross. In view of these experimental conditions and results, it was concluded that Pantoprazole is associated with recombinogenic effects in Drosophila melanogaster. PMID:25983631

  6. Nonautonomous Apoptosis Is Triggered by Local Cell Cycle Progression during Epithelial Replacement in Drosophila ▿ †

    PubMed Central

    Nakajima, Yu-ichiro; Kuranaga, Erina; Sugimura, Kaoru; Miyawaki, Atsushi; Miura, Masayuki

    2011-01-01

    Tissue remodeling involves collective cell movement, and cell proliferation and apoptosis are observed in both development and disease. Apoptosis and proliferation are considered to be closely correlated, but little is known about their coordinated regulation in physiological tissue remodeling in vivo. The replacement of larval abdominal epidermis with adult epithelium in Drosophila pupae is a simple model of tissue remodeling. During this process, larval epidermal cells (LECs) undergo apoptosis and are replaced by histoblasts, which are adult precursor cells. By analyzing caspase activation at the single-cell level in living pupae, we found that caspase activation in LECs is induced at the LEC/histoblast boundary, which expands as the LECs die. Manipulating histoblast proliferation at the LEC/histoblast boundary, either genetically or by UV illumination, indicated that local interactions with proliferating histoblasts triggered caspase activation in the boundary LECs. Finally, by monitoring the spatiotemporal dynamics of the S/G2/M phase in histoblasts in vivo, we found that the transition from S/G2 phases is necessary to induce nonautonomous LEC apoptosis at the LEC/histoblast boundary. The replacement boundary, formed as caspase activation is regulated locally by cell-cell communication, may drive the dynamic orchestration of cell replacement during tissue remodeling. PMID:21482673

  7. The Hippo pathway polarizes the actin cytoskeleton during collective migration of Drosophila border cells.

    PubMed

    Lucas, Eliana P; Khanal, Ichha; Gaspar, Pedro; Fletcher, Georgina C; Polesello, Cedric; Tapon, Nicolas; Thompson, Barry J

    2013-06-10

    Collective migration of Drosophila border cells depends on a dynamic actin cytoskeleton that is highly polarized such that it concentrates around the outer rim of the migrating cluster of cells. How the actin cytoskeleton becomes polarized in these cells to enable collective movement remains unknown. Here we show that the Hippo signaling pathway links determinants of cell polarity to polarization of the actin cytoskeleton in border cells. Upstream Hippo pathway components localize to contacts between border cells inside the cluster and signal through the Hippo and Warts kinases to polarize actin and promote border cell migration. Phosphorylation of the transcriptional coactivator Yorkie (Yki)/YAP by Warts does not mediate the function of this pathway in promoting border cell migration, but rather provides negative feedback to limit the speed of migration. Instead, Warts phosphorylates and inhibits the actin regulator Ena to activate F-actin Capping protein activity on inner membranes and thereby restricts F-actin polymerization mainly to the outer rim of the migrating cluster.

  8. Surface apposition and multiple cell contacts promote myoblast fusion in Drosophila flight muscles.

    PubMed

    Dhanyasi, Nagaraju; Segal, Dagan; Shimoni, Eyal; Shinder, Vera; Shilo, Ben-Zion; VijayRaghavan, K; Schejter, Eyal D

    2015-10-12

    Fusion of individual myoblasts to form multinucleated myofibers constitutes a widely conserved program for growth of the somatic musculature. We have used electron microscopy methods to study this key form of cell-cell fusion during development of the indirect flight muscles (IFMs) of Drosophila melanogaster. We find that IFM myoblast-myotube fusion proceeds in a stepwise fashion and is governed by apparent cross talk between transmembrane and cytoskeletal elements. Our analysis suggests that cell adhesion is necessary for bringing myoblasts to within a minimal distance from the myotubes. The branched actin polymerization machinery acts subsequently to promote tight apposition between the surfaces of the two cell types and formation of multiple sites of cell-cell contact, giving rise to nascent fusion pores whose expansion establishes full cytoplasmic continuity. Given the conserved features of IFM myogenesis, this sequence of cell interactions and membrane events and the mechanistic significance of cell adhesion elements and the actin-based cytoskeleton are likely to represent general principles of the myoblast fusion process.

  9. Mushroom body miscellanea: transgenic Drosophila strains expressing anatomical and physiological sensor proteins in Kenyon cells

    PubMed Central

    Pech, Ulrike; Dipt, Shubham; Barth, Jonas; Singh, Priyanka; Jauch, Mandy; Thum, Andreas S.; Fiala, André; Riemensperger, Thomas

    2013-01-01

    The fruit fly Drosophila melanogaster represents a key model organism for analyzing how neuronal circuits regulate behavior. The mushroom body in the central brain is a particularly prominent brain region that has been intensely studied in several insect species and been implicated in a variety of behaviors, e.g., associative learning, locomotor activity, and sleep. Drosophila melanogaster offers the advantage that transgenes can be easily expressed in neuronal subpopulations, e.g., in intrinsic mushroom body neurons (Kenyon cells). A number of transgenes has been described and engineered to visualize the anatomy of neurons, to monitor physiological parameters of neuronal activity, and to manipulate neuronal function artificially. To target the expression of these transgenes selectively to specific neurons several sophisticated bi- or even multipartite transcription systems have been invented. However, the number of transgenes that can be combined in the genome of an individual fly is limited in practice. To facilitate the analysis of the mushroom body we provide a compilation of transgenic fruit flies that express transgenes under direct control of the Kenyon-cell specific promoter, mb247. The transgenes expressed are fluorescence reporters to analyze neuroanatomical aspects of the mushroom body, proteins to restrict ectopic gene expression to mushroom bodies, or fluorescent sensors to monitor physiological parameters of neuronal activity of Kenyon cells. Some of the transgenic animals compiled here have been published already, whereas others are novel and characterized here for the first time. Overall, the collection of transgenic flies expressing sensor and reporter genes in Kenyon cells facilitates combinations with binary transcription systems and might, ultimately, advance the physiological analysis of mushroom body function. PMID:24065891

  10. Condensin II Subunit dCAP-D3 Restricts Retrotransposon Mobilization in Drosophila Somatic Cells

    PubMed Central

    Schuster, Andrew T.; Sarvepalli, Kavitha; Murphy, Eain A.; Longworth, Michelle S.

    2013-01-01

    Retrotransposon sequences are positioned throughout the genome of almost every eukaryote that has been sequenced. As mobilization of these elements can have detrimental effects on the transcriptional regulation and stability of an organism's genome, most organisms have evolved mechanisms to repress their movement. Here, we identify a novel role for the Drosophila melanogaster Condensin II subunit, dCAP-D3 in preventing the mobilization of retrotransposons located in somatic cell euchromatin. dCAP-D3 regulates transcription of euchromatic gene clusters which contain or are proximal to retrotransposon sequence. ChIP experiments demonstrate that dCAP-D3 binds to these loci and is important for maintaining a repressed chromatin structure within the boundaries of the retrotransposon and for repressing retrotransposon transcription. We show that dCAP-D3 prevents accumulation of double stranded DNA breaks within retrotransposon sequence, and decreased dCAP-D3 levels leads to a precise loss of retrotransposon sequence at some dCAP-D3 regulated gene clusters and a gain of sequence elsewhere in the genome. Homologous chromosomes exhibit high levels of pairing in Drosophila somatic cells, and our FISH analyses demonstrate that retrotransposon-containing euchromatic loci are regions which are actually less paired than euchromatic regions devoid of retrotransposon sequences. Decreased dCAP-D3 expression increases pairing of homologous retrotransposon-containing loci in tissue culture cells. We propose that the combined effects of dCAP-D3 deficiency on double strand break levels, chromatin structure, transcription and pairing at retrotransposon-containing loci may lead to 1) higher levels of homologous recombination between repeats flanking retrotransposons in dCAP-D3 deficient cells and 2) increased retrotransposition. These findings identify a novel role for the anti-pairing activities of dCAP-D3/Condensin II and uncover a new way in which dCAP-D3/Condensin II influences local

  11. Condensin II subunit dCAP-D3 restricts retrotransposon mobilization in Drosophila somatic cells.

    PubMed

    Schuster, Andrew T; Sarvepalli, Kavitha; Murphy, Eain A; Longworth, Michelle S

    2013-10-01

    Retrotransposon sequences are positioned throughout the genome of almost every eukaryote that has been sequenced. As mobilization of these elements can have detrimental effects on the transcriptional regulation and stability of an organism's genome, most organisms have evolved mechanisms to repress their movement. Here, we identify a novel role for the Drosophila melanogaster Condensin II subunit, dCAP-D3 in preventing the mobilization of retrotransposons located in somatic cell euchromatin. dCAP-D3 regulates transcription of euchromatic gene clusters which contain or are proximal to retrotransposon sequence. ChIP experiments demonstrate that dCAP-D3 binds to these loci and is important for maintaining a repressed chromatin structure within the boundaries of the retrotransposon and for repressing retrotransposon transcription. We show that dCAP-D3 prevents accumulation of double stranded DNA breaks within retrotransposon sequence, and decreased dCAP-D3 levels leads to a precise loss of retrotransposon sequence at some dCAP-D3 regulated gene clusters and a gain of sequence elsewhere in the genome. Homologous chromosomes exhibit high levels of pairing in Drosophila somatic cells, and our FISH analyses demonstrate that retrotransposon-containing euchromatic loci are regions which are actually less paired than euchromatic regions devoid of retrotransposon sequences. Decreased dCAP-D3 expression increases pairing of homologous retrotransposon-containing loci in tissue culture cells. We propose that the combined effects of dCAP-D3 deficiency on double strand break levels, chromatin structure, transcription and pairing at retrotransposon-containing loci may lead to 1) higher levels of homologous recombination between repeats flanking retrotransposons in dCAP-D3 deficient cells and 2) increased retrotransposition. These findings identify a novel role for the anti-pairing activities of dCAP-D3/Condensin II and uncover a new way in which dCAP-D3/Condensin II influences local

  12. Decellularized and Engineered Tendons as Biological Substitutes: A Critical Review

    PubMed Central

    Lovati, Arianna B.; Bottagisio, Marta; Moretti, Matteo

    2016-01-01

    Tendon ruptures are a great burden in clinics. Finding a proper graft material as a substitute for tendon repair is one of the main challenges in orthopaedics, for which the requirement of a biological scaffold would be different for each clinical application. Among biological scaffolds, the use of decellularized tendon-derived matrix increasingly represents an interesting approach to treat tendon ruptures. We analyzed in vitro and in vivo studies focused on the development of efficient protocols for the decellularization and for the cell reseeding of the tendon matrix to obtain medical devices for tendon substitution. Our review considered also the proper tendon source and preclinical animal models with the aim of entering into clinical trials. The results highlight a wide panorama in terms of allogenic or xenogeneic tendon sources, specimen dimensions, physical or chemical decellularization techniques, and the cell type variety for reseeding from terminally differentiated to undifferentiated mesenchymal stem cells and their static or dynamic culture employed to generate implantable constructs tested in different animal models. We try to identify the most efficient approach to achieve an optimal biological scaffold for biomechanics and intrinsic properties, resembling the native tendon and being applicable in clinics in the near future, with particular attention to the Achilles tendon substitution. PMID:26880985

  13. Exocyst-Dependent Membrane Addition Is Required for Anaphase Cell Elongation and Cytokinesis in Drosophila

    PubMed Central

    Giansanti, Maria Grazia; Vanderleest, Timothy E.; Jewett, Cayla E.; Sechi, Stefano; Frappaolo, Anna; Fabian, Lacramioara; Robinett, Carmen C.; Brill, Julie A.; Loerke, Dinah; Fuller, Margaret T.; Blankenship, J. Todd

    2015-01-01

    Mitotic and cytokinetic processes harness cell machinery to drive chromosomal segregation and the physical separation of dividing cells. Here, we investigate the functional requirements for exocyst complex function during cell division in vivo, and demonstrate a common mechanism that directs anaphase cell elongation and cleavage furrow progression during cell division. We show that onion rings (onr) and funnel cakes (fun) encode the Drosophila homologs of the Exo84 and Sec8 exocyst subunits, respectively. In onr and fun mutant cells, contractile ring proteins are recruited to the equatorial region of dividing spermatocytes. However, cytokinesis is disrupted early in furrow ingression, leading to cytokinesis failure. We use high temporal and spatial resolution confocal imaging with automated computational analysis to quantitatively compare wild-type versus onr and fun mutant cells. These results demonstrate that anaphase cell elongation is grossly disrupted in cells that are compromised in exocyst complex function. Additionally, we observe that the increase in cell surface area in wild type peaks a few minutes into cytokinesis, and that onr and fun mutant cells have a greatly reduced rate of surface area growth specifically during cell division. Analysis by transmission electron microscopy reveals a massive build-up of cytoplasmic astral membrane and loss of normal Golgi architecture in onr and fun spermatocytes, suggesting that exocyst complex is required for proper vesicular trafficking through these compartments. Moreover, recruitment of the small GTPase Rab11 and the PITP Giotto to the cleavage site depends on wild-type function of the exocyst subunits Exo84 and Sec8. Finally, we show that the exocyst subunit Sec5 coimmunoprecipitates with Rab11. Our results are consistent with the exocyst complex mediating an essential, coordinated increase in cell surface area that potentiates anaphase cell elongation and cleavage furrow ingression. PMID:26528720

  14. Exocyst-Dependent Membrane Addition Is Required for Anaphase Cell Elongation and Cytokinesis in Drosophila.

    PubMed

    Giansanti, Maria Grazia; Vanderleest, Timothy E; Jewett, Cayla E; Sechi, Stefano; Frappaolo, Anna; Fabian, Lacramioara; Robinett, Carmen C; Brill, Julie A; Loerke, Dinah; Fuller, Margaret T; Blankenship, J Todd

    2015-11-01

    Mitotic and cytokinetic processes harness cell machinery to drive chromosomal segregation and the physical separation of dividing cells. Here, we investigate the functional requirements for exocyst complex function during cell division in vivo, and demonstrate a common mechanism that directs anaphase cell elongation and cleavage furrow progression during cell division. We show that onion rings (onr) and funnel cakes (fun) encode the Drosophila homologs of the Exo84 and Sec8 exocyst subunits, respectively. In onr and fun mutant cells, contractile ring proteins are recruited to the equatorial region of dividing spermatocytes. However, cytokinesis is disrupted early in furrow ingression, leading to cytokinesis failure. We use high temporal and spatial resolution confocal imaging with automated computational analysis to quantitatively compare wild-type versus onr and fun mutant cells. These results demonstrate that anaphase cell elongation is grossly disrupted in cells that are compromised in exocyst complex function. Additionally, we observe that the increase in cell surface area in wild type peaks a few minutes into cytokinesis, and that onr and fun mutant cells have a greatly reduced rate of surface area growth specifically during cell division. Analysis by transmission electron microscopy reveals a massive build-up of cytoplasmic astral membrane and loss of normal Golgi architecture in onr and fun spermatocytes, suggesting that exocyst complex is required for proper vesicular trafficking through these compartments. Moreover, recruitment of the small GTPase Rab11 and the PITP Giotto to the cleavage site depends on wild-type function of the exocyst subunits Exo84 and Sec8. Finally, we show that the exocyst subunit Sec5 coimmunoprecipitates with Rab11. Our results are consistent with the exocyst complex mediating an essential, coordinated increase in cell surface area that potentiates anaphase cell elongation and cleavage furrow ingression. PMID:26528720

  15. Active tendon implants in flexor tendon reconstruction.

    PubMed

    Hunter, J M; Singer, D I; Jaeger, S H; Mackin, E J

    1988-11-01

    Forty-five active flexor tendon implants were evaluated after placement in scarred tendon beds of digits II through V. The implant is constructed of silicone rubber with a Dacron core, terminating in a loop proximally and a metal plate distally. Modification of the implant during the period of study has improved its reliability and longevity. The improvement in total active motion (TAM) averaged 72 degrees during implant functioning (stage I) in a group of digits that before operation were classified as 78% Boyes grade 5 (salvage). Complication rate during stage I was 11% (5 out of 45). Of the 27 digits evaluated after implant replacement by tendon autograft (stage II), there was an overall improvement in 62 degrees total active motion with 70% of digits being Boyes grade 5. Many of the complications were believed to be avoidable with experience. This study demonstrates the feasibility of an active tendon implant and the possibility of a permanent prosthesis. PMID:2976074

  16. A biomechanical model for cell polarization and intercalation during Drosophila germband extension

    NASA Astrophysics Data System (ADS)

    Lan, Haihan; Wang, Qiming; Fernandez-Gonzalez, Rodrigo; Feng, James J.

    2015-10-01

    Germband extension during Drosophila development features the merging of cells along the dorsal-ventral (DV) axis and their separation along the anterior-posterior (AP) axis. This intercalation process involves planar cell polarity, anisotropic contractile forces along cell edges, and concerted cell deformation and movement. Although prior experiments have probed each of these factors separately, the connection among them remains unclear. This paper presents a chemo-mechanical model that integrates the three factors into a coherent framework. The model predicts the polarization of Rho-kinase, myosin and Bazooka downstream of an anisotropic Shroom distribution. In particular, myosin accumulates on cell edges along the DV axis, causing them to contract into a vertex. Subsequently, medial myosin in the cells anterior and posterior to the vertex helps to elongate it into a new edge parallel to the body axis. Thus, the tissue extends along the AP axis and narrows in the transverse direction through neighbor exchange. Model predictions of the polarity of the proteins and cell and tissue deformation are in good agreement with experimental observations.

  17. Tousled-like kinase mediated a new type of cell death pathway in Drosophila.

    PubMed

    Zhang, Y; Cai, R; Zhou, R; Li, Y; Liu, L

    2016-01-01

    Programmed cell death (PCD) has an important role in sculpting organisms during development. However, much remains to be learned about the molecular mechanism of PCD. We found that ectopic expression of tousled-like kinase (tlk) in Drosophila initiated a new type of cell death. Furthermore, the TLK-induced cell death is likely to be independent of the canonical caspase pathway and other known caspase-independent pathways. Genetically, atg2 RNAi could rescue the TLK-induced cell death, and this function of atg2 was likely distinct from its role in autophagy. In the developing retina, loss of tlk resulted in reduced PCD in the interommatidial cells (IOCs). Similarly, an increased number of IOCs was present in the atg2 deletion mutant clones. However, double knockdown of tlk and atg2 by RNAi did not have a synergistic effect. These results suggested that ATG2 may function downstream of TLK. In addition to a role in development, tlk and atg2 RNAi could rescue calcium overload-induced cell death. Together, our results suggest that TLK mediates a new type of cell death pathway that occurs in both development and calcium cytotoxicity.

  18. Tousled-like kinase mediated a new type of cell death pathway in Drosophila.

    PubMed

    Zhang, Y; Cai, R; Zhou, R; Li, Y; Liu, L

    2016-01-01

    Programmed cell death (PCD) has an important role in sculpting organisms during development. However, much remains to be learned about the molecular mechanism of PCD. We found that ectopic expression of tousled-like kinase (tlk) in Drosophila initiated a new type of cell death. Furthermore, the TLK-induced cell death is likely to be independent of the canonical caspase pathway and other known caspase-independent pathways. Genetically, atg2 RNAi could rescue the TLK-induced cell death, and this function of atg2 was likely distinct from its role in autophagy. In the developing retina, loss of tlk resulted in reduced PCD in the interommatidial cells (IOCs). Similarly, an increased number of IOCs was present in the atg2 deletion mutant clones. However, double knockdown of tlk and atg2 by RNAi did not have a synergistic effect. These results suggested that ATG2 may function downstream of TLK. In addition to a role in development, tlk and atg2 RNAi could rescue calcium overload-induced cell death. Together, our results suggest that TLK mediates a new type of cell death pathway that occurs in both development and calcium cytotoxicity. PMID:26088162

  19. miR-965 controls cell proliferation and migration during tissue morphogenesis in the Drosophila abdomen

    PubMed Central

    Verma, Pushpa; Cohen, Stephen M

    2015-01-01

    Formation of the Drosophila adult abdomen involves a process of tissue replacement in which larval epidermal cells are replaced by adult cells. The progenitors of the adult epidermis are specified during embryogenesis and, unlike the imaginal discs that make up the thoracic and head segments, they remain quiescent during larval development. During pupal development, the abdominal histoblast cells proliferate and migrate to replace the larval epidermis. Here, we provide evidence that the microRNA, miR-965, acts via string and wingless to control histoblast proliferation and migration. Ecdysone signaling downregulates miR-965 at the onset of pupariation, linking activation of the histoblast nests to the hormonal control of metamorphosis. Replacement of the larval epidermis by adult epidermal progenitors involves regulation of both cell-intrinsic events and cell communication. By regulating both cell proliferation and cell migration, miR-965 contributes to the robustness of this morphogenetic system. DOI: http://dx.doi.org/10.7554/eLife.07389.001 PMID:26226636

  20. Dissecting the Function and Assembly of Acentriolar Microtubule Organizing Centers in Drosophila Cells In Vivo

    PubMed Central

    Baumbach, Janina; Novak, Zsofia Anna; Raff, Jordan W.; Wainman, Alan

    2015-01-01

    Acentriolar microtubule organizing centers (aMTOCs) are formed during meiosis and mitosis in several cell types, but their function and assembly mechanism is unclear. Importantly, aMTOCs can be overactive in cancer cells, enhancing multipolar spindle formation, merotelic kinetochore attachment and aneuploidy. Here we show that aMTOCs can form in acentriolar Drosophila somatic cells in vivo via an assembly pathway that depends on Asl, Cnn and, to a lesser extent, Spd-2—the same proteins that appear to drive mitotic centrosome assembly in flies. This finding enabled us to ablate aMTOC formation in acentriolar cells, and so perform a detailed genetic analysis of the contribution of aMTOCs to acentriolar mitotic spindle formation. Here we show that although aMTOCs can nucleate microtubules, they do not detectably increase the efficiency of acentriolar spindle assembly in somatic fly cells. We find that they are required, however, for robust microtubule array assembly in cells without centrioles that also lack microtubule nucleation from around the chromatin. Importantly, aMTOCs are also essential for dynein-dependent acentriolar spindle pole focusing and for robust cell proliferation in the absence of centrioles and HSET/Ncd (a kinesin essential for acentriolar spindle pole focusing in many systems). We propose an updated model for acentriolar spindle pole coalescence by the molecular motors Ncd/HSET and dynein in conjunction with aMTOCs. PMID:26020779

  1. Pelle Modulates dFoxO-Mediated Cell Death in Drosophila.

    PubMed

    Wu, Chenxi; Chen, Yujun; Wang, Feng; Chen, Changyan; Zhang, Shiping; Li, Chaojie; Li, Wenzhe; Wu, Shian; Xue, Lei

    2015-10-01

    Interleukin-1 receptor-associated kinases (IRAKs) are crucial mediators of the IL-1R/TLR signaling pathways that regulate the immune and inflammation response in mammals. Recent studies also suggest a critical role of IRAKs in tumor development, though the underlying mechanism remains elusive. Pelle is the sole Drosophila IRAK homolog implicated in the conserved Toll pathway that regulates Dorsal/Ventral patterning, innate immune response, muscle development and axon guidance. Here we report a novel function of pll in modulating apoptotic cell death, which is independent of the Toll pathway. We found that loss of pll results in reduced size in wing tissue, which is caused by a reduction in cell number but not cell size. Depletion of pll up-regulates the transcription of pro-apoptotic genes, and triggers caspase activation and cell death. The transcription factor dFoxO is required for loss-of-pll induced cell death. Furthermore, loss of pll activates dFoxO, promotes its translocation from cytoplasm to nucleus, and up-regulates the transcription of its target gene Thor/4E-BP. Finally, Pll physically interacts with dFoxO and phosphorylates dFoxO directly. This study not only identifies a previously unknown physiological function of pll in cell death, but also shed light on the mechanism of IRAKs in cell survival/death during tumorigenesis.

  2. Pelle Modulates dFoxO-Mediated Cell Death in Drosophila

    PubMed Central

    Chen, Changyan; Zhang, Shiping; Li, Chaojie; Li, Wenzhe; Wu, Shian; Xue, Lei

    2015-01-01

    Interleukin-1 receptor-associated kinases (IRAKs) are crucial mediators of the IL-1R/TLR signaling pathways that regulate the immune and inflammation response in mammals. Recent studies also suggest a critical role of IRAKs in tumor development, though the underlying mechanism remains elusive. Pelle is the sole Drosophila IRAK homolog implicated in the conserved Toll pathway that regulates Dorsal/Ventral patterning, innate immune response, muscle development and axon guidance. Here we report a novel function of pll in modulating apoptotic cell death, which is independent of the Toll pathway. We found that loss of pll results in reduced size in wing tissue, which is caused by a reduction in cell number but not cell size. Depletion of pll up-regulates the transcription of pro-apoptotic genes, and triggers caspase activation and cell death. The transcription factor dFoxO is required for loss-of-pll induced cell death. Furthermore, loss of pll activates dFoxO, promotes its translocation from cytoplasm to nucleus, and up-regulates the transcription of its target gene Thor/4E-BP. Finally, Pll physically interacts with dFoxO and phosphorylates dFoxO directly. This study not only identifies a previously unknown physiological function of pll in cell death, but also shed light on the mechanism of IRAKs in cell survival/death during tumorigenesis. PMID:26474173

  3. Characterization of proteins and RNAs present on polysomes from normal and heat shocked Drosophila cells

    SciTech Connect

    Sanders, M.M.; Bruederle, L.P.

    1987-05-01

    Translational regulation in heat shocked Drosophila cells is characterized by a shutdown in translation of normal cell mRNA and selective translation of mRNAs coding for heat shock polypeptides. They have characterized the sedimentation profiles and macromolecular compositions of ribosomes and polysomes in normal and heat shocked cells in order to implicate factors responsible for the changes in regulation of translation in heat shocked cells. Sucrose gradient analysis of polysome sedimentation carried out under physiological salt conditions confirms the previously reported decrease in polysomes and increase in 80s monosomes in heat shock. The size of the 40s and 60s subunit peaks and the levels of /sup 35/S-methionyl-tRNA associated with the 43s preinitiation complex do not change significantly in heat shock. Titration of specific mRNA levels across the gradients shows that normal cell mRNAs accumulate in the 40 to 80s region of heat shock profiles while heat shock mRNAs are distributed on heat shock polysomes. Analysis of proteins present on the gradients shows a 46 kD protein is present on polysomes from normal cells and polymerized into the insoluble cytoskeleton in heat shocked cells. The rearrangement of the insoluble cytoskeleton follows the same time course as changes in translation regulation in induction and recovery from heat shock and is accompanied by phosphorylation of the 46 kD polypeptide.

  4. Proliferation of Double-Strand Break-Resistant Polyploid Cells Requires Drosophila FANCD2.

    PubMed

    Bretscher, Heidi S; Fox, Donald T

    2016-06-01

    Conserved DNA-damage responses (DDRs) sense genome damage and prevent mitosis of broken chromosomes. How cells lacking DDRs cope with broken chromosomes during mitosis is poorly understood. DDRs are frequently inactivated in cells with extra genomes (polyploidy), suggesting that study of polyploidy can reveal how cells with impaired DDRs/genome damage continue dividing. Here, we show that continued division and normal organ development occurs in polyploid, DDR-impaired Drosophila papillar cells. As papillar cells become polyploid, they naturally accumulate broken acentric chromosomes but do not apoptose/arrest the cell cycle. To survive mitosis with acentric chromosomes, papillar cells require Fanconi anemia proteins FANCD2 and FANCI, as well as Blm helicase, but not canonical DDR signaling. FANCD2 acts independently of previous S phases to promote alignment and segregation of acentric DNA produced by double-strand breaks, thus avoiding micronuclei and organ malformation. Because polyploidy and impaired DDRs can promote cancer, our findings provide insight into disease-relevant DNA-damage tolerance mechanisms. PMID:27270041

  5. A biomechanical model for cell polarization and intercalation during Drosophila germband extension.

    PubMed

    Lan, Haihan; Wang, Qiming; Fernandez-Gonzalez, Rodrigo; Feng, James J

    2015-10-01

    Germband extension during Drosophila development features the merging of cells along the dorsal-ventral (DV) axis and their separation along the anterior-posterior (AP) axis. This intercalation process involves planar cell polarity, anisotropic contractile forces along cell edges, and concerted cell deformation and movement. Although prior experiments have probed each of these factors separately, the connection among them remains unclear. This paper presents a chemo-mechanical model that integrates the three factors into a coherent framework. The model predicts the polarization of Rho-kinase, myosin and Bazooka downstream of an anisotropic Shroom distribution. In particular, myosin accumulates on cell edges along the DV axis, causing them to contract into a vertex. Subsequently, medial myosin in the cells anterior and posterior to the vertex helps to elongate it into a new edge parallel to the body axis. Thus, the tissue extends along the AP axis and narrows in the transverse direction through neighbor exchange. Model predictions of the polarity of the proteins and cell and tissue deformation are in good agreement with experimental observations. PMID:26356256

  6. Inhibition of RNA interference and modulation of transposable element expression by cell death in Drosophila.

    PubMed

    Xie, Weiwu; Liang, Chengzhi; Birchler, James A

    2011-08-01

    RNA interference (RNAi) regulates gene expression by sequence-specific destruction of RNA. It acts as a defense mechanism against viruses and represses the expression of transposable elements (TEs) and some endogenous genes. We report that mutations and transgene constructs that condition cell death suppress RNA interference in adjacent cells in Drosophila melanogaster. The reversal of RNAi is effective for both the white (w) eye color gene and green fluorescent protein (GFP), indicating the generality of the inhibition. Antiapoptotic transgenes that reverse cell death will also reverse the inhibition of RNAi. Using GFP and a low level of cell death produced by a heat shock-head involution defective (hs-hid) transgene, the inhibition appears to occur by blocking the conversion of double-stranded RNA (dsRNA) to short interfering RNA (siRNA). We also demonstrate that the mus308 gene and endogenous transposable elements, which are both regularly silenced by RNAi, are increased in expression and accompanied by a reduced level of siRNA, when cell death occurs. The finding that chronic ectopic cell death affects RNAi is critical for an understanding of the application of the technique in basic and applied studies. These results also suggest that developmental perturbations, disease states, or environmental insults that cause ectopic cell death would alter transposon and gene expression patterns in the organism by the inhibition of small RNA silencing processes. PMID:21596898

  7. THE ROLE OF MECHANOBIOLOGY IN TENDON HEALING

    PubMed Central

    Killian, Megan L.; Cavinatto, Leonardo; Galatz, Leesa M.; Thomopoulos, Stavros

    2011-01-01

    Mechanical cues affect tendon healing, homeostasis, and development in a variety of settings. Alterations in the mechanical environment are known to result in changes in the expression of extracellular matrix proteins, growth factors, transcription factors, and cytokines that can alter tendon structure and cell viability. Loss of muscle force in utero or in the immediate postnatal period delays tendon and enthesis development. The response of healing tendons to mechanical load varies depending on anatomic location. Flexor tendons require motion to prevent adhesion formation, yet excessive force results in gap formation and subsequent weakening of the repair. Excessive motion in the setting of anterior cruciate ligament reconstruction causes accumulation of macrophages, which are detrimental to tendon graft healing. Complete removal of load is detrimental to rotator cuff healing, yet large forces are also harmful. Controlled loading can enhance healing in most settings; however, a fine balance must be reached between loads that are too low (leading to a catabolic state) and too high (leading to micro-damage). This review will summarize existing knowledge of the mechanobiology of tendon development, homeostasis, and healing. PMID:22244066

  8. Atraumatic quadriceps tendon tear associated with calcific tendonitis.

    PubMed

    Abram, Simon G F; Sharma, Akash D; Arvind, Chinnakonda

    2012-11-27

    Calcific tendonitis of the quadriceps tendon is an uncommon condition. We present the first case of a quadriceps tendon tear associated with calcific tendonitis. In this case, the patient presented with symptoms mimicking a rupture of the quadriceps tendon. This case illustrates that although calcific tendonitis of the quadriceps is a rare condition it is not benign and should be considered when investigating acute symptoms associated with the extensor mechanism of the knee.

  9. Bazooka/PAR3 is dispensable for polarity in Drosophila follicular epithelial cells.

    PubMed

    Shahab, Jaffer; Tiwari, Manu D; Honemann-Capito, Mona; Krahn, Michael P; Wodarz, Andreas

    2015-03-13

    Apico-basal polarity is the defining characteristic of epithelial cells. In Drosophila, apical membrane identity is established and regulated through interactions between the highly conserved Par complex (Bazooka/Par3, atypical protein kinase C and Par6), and the Crumbs complex (Crumbs, Stardust and PATJ). It has been proposed that Bazooka operates at the top of a genetic hierarchy in the establishment and maintenance of apico-basal polarity. However, there is still ambiguity over the correct sequence of events and cross-talk with other pathways during this process. In this study, we reassess this issue by comparing the phenotypes of the commonly used baz(4) and baz(815-8) alleles with those of the so far uncharacterized baz(XR11) and baz(EH747) null alleles in different Drosophila epithelia. While all these baz alleles display identical phenotypes during embryonic epithelial development, we observe strong discrepancies in the severity and penetrance of polarity defects in the follicular epithelium: polarity is mostly normal in baz(EH747) and baz(XR11) while baz(4) and baz(815) (-8) show loss of polarity, severe multilayering and loss of epithelial integrity throughout the clones. Further analysis reveals that the chromosomes carrying the baz(4) and baz(815-8) alleles may contain additional mutations that enhance the true baz loss-of-function phenotype in the follicular epithelium. This study clearly shows that Baz is dispensable for the regulation of polarity in the follicular epithelium, and that the requirement for key regulators of cell polarity is highly dependent on developmental context and cell type.

  10. Bazooka/PAR3 is dispensable for polarity in Drosophila follicular epithelial cells

    PubMed Central

    Shahab, Jaffer; Tiwari, Manu D.; Honemann-Capito, Mona; Krahn, Michael P.; Wodarz, Andreas

    2015-01-01

    Apico-basal polarity is the defining characteristic of epithelial cells. In Drosophila, apical membrane identity is established and regulated through interactions between the highly conserved Par complex (Bazooka/Par3, atypical protein kinase C and Par6), and the Crumbs complex (Crumbs, Stardust and PATJ). It has been proposed that Bazooka operates at the top of a genetic hierarchy in the establishment and maintenance of apico-basal polarity. However, there is still ambiguity over the correct sequence of events and cross-talk with other pathways during this process. In this study, we reassess this issue by comparing the phenotypes of the commonly used baz4 and baz815-8 alleles with those of the so far uncharacterized bazXR11 and bazEH747 null alleles in different Drosophila epithelia. While all these baz alleles display identical phenotypes during embryonic epithelial development, we observe strong discrepancies in the severity and penetrance of polarity defects in the follicular epithelium: polarity is mostly normal in bazEH747 and bazXR11 while baz4 and baz815-8 show loss of polarity, severe multilayering and loss of epithelial integrity throughout the clones. Further analysis reveals that the chromosomes carrying the baz4 and baz815-8 alleles may contain additional mutations that enhance the true baz loss-of-function phenotype in the follicular epithelium. This study clearly shows that Baz is dispensable for the regulation of polarity in the follicular epithelium, and that the requirement for key regulators of cell polarity is highly dependent on developmental context and cell type. PMID:25770183

  11. FACS Purification and Transcriptome Analysis of Drosophila Neural Stem Cells Reveals a Role for Klumpfuss in Self-Renewal

    PubMed Central

    Berger, Christian; Harzer, Heike; Burkard, Thomas R.; Steinmann, Jonas; van der Horst, Suzanne; Laurenson, Anne-Sophie; Novatchkova, Maria; Reichert, Heinrich; Knoblich, Juergen A.

    2012-01-01

    Summary Drosophila neuroblasts (NBs) have emerged as a model for stem cell biology that is ideal for genetic analysis but is limited by the lack of cell-type-specific gene expression data. Here, we describe a method for isolating large numbers of pure NBs and differentiating neurons that retain both cell-cycle and lineage characteristics. We determine transcriptional profiles by mRNA sequencing and identify 28 predicted NB-specific transcription factors that can be arranged in a network containing hubs for Notch signaling, growth control, and chromatin regulation. Overexpression and RNA interference for these factors identify Klumpfuss as a regulator of self-renewal. We show that loss of Klumpfuss function causes premature differentiation and that overexpression results in the formation of transplantable brain tumors. Our data represent a valuable resource for investigating Drosophila developmental neurobiology, and the described method can be applied to other invertebrate stem cell lineages as well. PMID:22884370

  12. Pak3 regulates apical-basal polarity in migrating border cells during Drosophila oogenesis.

    PubMed

    Felix, Martina; Chayengia, Mrinal; Ghosh, Ritabrata; Sharma, Aditi; Prasad, Mohit

    2015-11-01

    Group cell migration is a highly coordinated process that is involved in a number of physiological events such as morphogenesis, wound healing and tumor metastasis. Unlike single cells, collectively moving cells are physically attached to each other and retain some degree of apical-basal polarity during the migratory phase. Although much is known about direction sensing, how polarity is regulated in multicellular movement remains unclear. Here we report the role of the protein kinase Pak3 in maintaining apical-basal polarity in migrating border cell clusters during Drosophila oogenesis. Pak3 is enriched in border cells and downregulation of its function impedes border cell movement. Time-lapse imaging suggests that Pak3 affects protrusive behavior of the border cell cluster, specifically regulating the stability and directionality of protrusions. Pak3 functions downstream of guidance receptor signaling to regulate the level and distribution of F-actin in migrating border cells. We also provide evidence that Pak3 genetically interacts with the lateral polarity marker Scribble and that it regulates JNK signaling in the moving border cells. Since Pak3 depletion results in mislocalization of several apical-basal polarity markers and overexpression of Jra rescues the polarity of the Pak3-depleted cluster, we propose that Pak3 functions through JNK signaling to modulate apical-basal polarity of the migrating border cell cluster. We also observe loss of apical-basal polarity in Rac1-depleted border cell clusters, suggesting that guidance receptor signaling functions through Rac GTPase and Pak3 to regulate the overall polarity of the cluster and mediate efficient collective movement of the border cells to the oocyte boundary.

  13. Chloride channels in stellate cells are essential for uniquely high secretion rates in neuropeptide-stimulated Drosophila diuresis.

    PubMed

    Cabrero, Pablo; Terhzaz, Selim; Romero, Michael F; Davies, Shireen A; Blumenthal, Edward M; Dow, Julian A T

    2014-09-30

    Epithelia frequently segregate transport processes to specific cell types, presumably for improved efficiency and control. The molecular players underlying this functional specialization are of particular interest. In Drosophila, the renal (Malpighian) tubule displays the highest per-cell transport rates known and has two main secretory cell types, principal and stellate. Electrogenic cation transport is known to reside in the principal cells, whereas stellate cells control the anion conductance, but by an as-yet-undefined route. Here, we resolve this issue by showing that a plasma membrane chloride channel, encoded by ClC-a, is exclusively expressed in the stellate cell and is required for Drosophila kinin-mediated induction of diuresis and chloride shunt conductance, evidenced by chloride ion movement through the stellate cells, leading to depolarization of the transepithelial potential. By contrast, ClC-a knockdown had no impact on resting secretion levels. Knockdown of a second CLC gene showing highly abundant expression in adult Malpighian tubules, ClC-c, did not impact depolarization of transepithelial potential after kinin stimulation. Therefore, the diuretic action of kinin in Drosophila can be explained by an increase in ClC-a-mediated chloride conductance, over and above a resting fluid transport level that relies on other (ClC-a-independent) mechanisms or routes. This key segregation of cation and anion transport could explain the extraordinary fluid transport rates displayed by some epithelia.

  14. A Sensitized PiggyBac-Based Screen for Regulators of Border Cell Migration in Drosophila

    PubMed Central

    Mathieu, Juliette; Sung, Hsin-Ho; Pugieux, Céline; Soetaert, Jan; Rorth, Pernille

    2007-01-01

    Migration of border cells during Drosophila melanogaster oogenesis is a good model system for investigating the genetic requirements for cell migration in vivo. We present a sensitized loss-of-function screen used to identify new genes required in border cells for their migration. Chromosomes bearing FRTs on all four major autosomal arms were mutagenized by insertions of the transposable element PiggyBac, allowing multiple parallel clonal screens and easy identification of the mutated gene. For border cells, we analyzed homozygous mutant clones positively marked with lacZ and sensitized by expression of dominant-negative PVR, the guidance receptor. We identified new alleles of genes already known to be required for border cell migration, including aop/yan, DIAP1, and taiman as well as a conserved Slbo-regulated enhancer downstream of shg/DE–cadherin. Mutations in genes not previously described to be required in border cells were also uncovered: hrp48, vir, rme-8, kismet, and puckered. puckered was unique in that the migration defects were observed only when PVR signaling was reduced. We present evidence that an excess of JNK signaling is deleterious for migration in the absence of PVR activity at least in part through Fos transcriptional activity and possibly through antagonistic effects on DIAP1. PMID:17483425

  15. Interplay of cell dynamics and epithelial tension during morphogenesis of the Drosophila pupal wing

    PubMed Central

    Etournay, Raphaël; Popović, Marko; Merkel, Matthias; Nandi, Amitabha; Blasse, Corinna; Aigouy, Benoît; Brandl, Holger; Myers, Gene; Salbreux, Guillaume; Jülicher, Frank; Eaton, Suzanne

    2015-01-01

    How tissue shape emerges from the collective mechanical properties and behavior of individual cells is not understood. We combine experiment and theory to study this problem in the developing wing epithelium of Drosophila. At pupal stages, the wing-hinge contraction contributes to anisotropic tissue flows that reshape the wing blade. Here, we quantitatively account for this wing-blade shape change on the basis of cell divisions, cell rearrangements and cell shape changes. We show that cells both generate and respond to epithelial stresses during this process, and that the nature of this interplay specifies the pattern of junctional network remodeling that changes wing shape. We show that patterned constraints exerted on the tissue by the extracellular matrix are key to force the tissue into the right shape. We present a continuum mechanical model that quantitatively describes the relationship between epithelial stresses and cell dynamics, and how their interplay reshapes the wing. DOI: http://dx.doi.org/10.7554/eLife.07090.001 PMID:26102528

  16. Nanotubes mediate niche-stem cell signaling in the Drosophila testis

    PubMed Central

    Inaba, Mayu; Buszczak, Michael; Yamashita, Yukiko M.

    2015-01-01

    Stem cell niches provide resident stem cells with signals that specify their identity. Niche signals act over a short-range such that only stem cells but not their differentiating progeny receive the self-renewing signals1. However, the cellular mechanisms that limit niche signaling to stem cells remain poorly understood. Here we show that the Drosophila male germline stem cells (GSCs) form previously unrecognized structures, microtubule-based (MT)-nanotubes, which extend into the hub, a major niche component. MT-nanotubes are observed specifically within GSC populations, and require IFT (intraflagellar transport) proteins for their formation. The BMP receptor Tkv localizes to MT-nanotubes. Perturbation of MT-nanotubes compromises activation of Dpp signaling within GSCs, leading to GSC loss. Moreover, Dpp ligand and Tkv receptor interaction is necessary and sufficient for MT-nanotube formation. We propose that MT-nanotubes provide a novel mechanism for selective receptor-ligand interaction, contributing to the short-range nature of niche-stem cell signaling. PMID:26131929

  17. Surface apposition and multiple cell contacts promote myoblast fusion in Drosophila flight muscles

    PubMed Central

    Dhanyasi, Nagaraju; Segal, Dagan; Shimoni, Eyal; Shinder, Vera

    2015-01-01

    Fusion of individual myoblasts to form multinucleated myofibers constitutes a widely conserved program for growth of the somatic musculature. We have used electron microscopy methods to study this key form of cell–cell fusion during development of the indirect flight muscles (IFMs) of Drosophila melanogaster. We find that IFM myoblast–myotube fusion proceeds in a stepwise fashion and is governed by apparent cross talk between transmembrane and cytoskeletal elements. Our analysis suggests that cell adhesion is necessary for bringing myoblasts to within a minimal distance from the myotubes. The branched actin polymerization machinery acts subsequently to promote tight apposition between the surfaces of the two cell types and formation of multiple sites of cell–cell contact, giving rise to nascent fusion pores whose expansion establishes full cytoplasmic continuity. Given the conserved features of IFM myogenesis, this sequence of cell interactions and membrane events and the mechanistic significance of cell adhesion elements and the actin-based cytoskeleton are likely to represent general principles of the myoblast fusion process. PMID:26459604

  18. The novel tumour suppressor Madm regulates stem cell competition in the Drosophila testis.

    PubMed

    Singh, Shree Ram; Liu, Ying; Zhao, Jiangsha; Zeng, Xiankun; Hou, Steven X

    2016-01-01

    Stem cell competition has emerged as a mechanism for selecting fit stem cells/progenitors and controlling tumourigenesis. However, little is known about the underlying molecular mechanism. Here we identify Mlf1-adaptor molecule (Madm), a novel tumour suppressor that regulates the competition between germline stem cells (GSCs) and somatic cyst stem cells (CySCs) for niche occupancy. Madm knockdown results in overexpression of the EGF receptor ligand vein (vn), which further activates EGF receptor signalling and integrin expression non-cell autonomously in CySCs to promote their overproliferation and ability to outcompete GSCs for niche occupancy. Conversely, expressing a constitutively activated form of the Drosophila JAK kinase (hop(Tum-l)) promotes Madm nuclear translocation, and suppresses vn and integrin expression in CySCs that allows GSCs to outcompete CySCs for niche occupancy and promotes GSC tumour formation. Tumour suppressor-mediated stem cell competition presented here could be a mechanism of tumour initiation in mammals. PMID:26792023

  19. The progeny of wingless-expressing cells deliver the signal at a distance in Drosophila embryos.

    PubMed

    Pfeiffer, S; Alexandre, C; Calleja, M; Vincent, J P

    2000-03-23

    Pattern formation in developing animals requires that cells exchange signals mediated by secreted proteins. How these signals spread is still unclear. It is generally assumed that they reach their target site either by diffusion or active transport (reviewed in [1] [2]). Here, we report an alternative mode of transport for Wingless (Wg), a member of the Wnt family of signaling molecules. In embryos of the fruit fly Drosophila, the wingless (wg) gene is transcribed in narrow stripes of cells abutting the source of Hedgehog protein. We found that these cells or their progeny are free to roam towards the anterior. As they do so, they no longer receive the Hedgehog signal and stop transcribing wg. The cells leaving the expression domain retain inherited Wg protein in secretory vesicles, however, and carry it forwards over a distance of up to four cell diameters. Experiments using a membrane-tethered form of Wg showed that this mechanism is sufficient to account for the normal range of Wg. Nevertheless, evidence exists that Wg can also reach distant target cells independently of protein inheritance, possibly by restricted diffusion. We suggest that both transport mechanisms operate in wild-type embryos.

  20. Transient junction anisotropies orient annular cell polarization in the Drosophila airway tubes.

    PubMed

    Hosono, Chie; Matsuda, Ryo; Adryan, Boris; Samakovlis, Christos

    2015-12-01

    In contrast to planes, three-dimensional (3D) structures such as tubes are physically anisotropic. Tubular organs exhibit a striking orientation of landmarks according to the physical anisotropy of the 3D shape, in addition to planar cell polarization. However, the influence of 3D tissue topography on the constituting cells remains underexplored. Here, we identify a regulatory network polarizing cellular biochemistry according to the physical anisotropy of the 3D tube geometry (tube cell polarization) by a genome-wide, tissue-specific RNAi screen. During Drosophila airway remodelling, each apical cellular junction is equipotent to establish perpendicular actomyosin cables, irrespective of the longitudinal or transverse tube axis. A dynamic transverse enrichment of atypical protein kinase C (aPKC) shifts the balance and transiently targets activated small GTPase RhoA, myosin phosphorylation and Rab11 vesicle trafficking to longitudinal junctions. We propose that the PAR complex translates tube physical anisotropy into longitudinal junctional anisotropy, where cell-cell communication aligns the contractile cytoskeleton of neighbouring cells.

  1. The let-7-Imp axis regulates ageing of the Drosophila testis stem-cell niche.

    PubMed

    Toledano, Hila; D'Alterio, Cecilia; Czech, Benjamin; Levine, Erel; Jones, D Leanne

    2012-05-31

    Adult stem cells support tissue homeostasis and repair throughout the life of an individual. During ageing, numerous intrinsic and extrinsic changes occur that result in altered stem-cell behaviour and reduced tissue maintenance and regeneration. In the Drosophila testis, ageing results in a marked decrease in the self-renewal factor Unpaired (Upd), leading to a concomitant loss of germline stem cells. Here we demonstrate that IGF-II messenger RNA binding protein (Imp) counteracts endogenous small interfering RNAs to stabilize upd (also known as os) RNA. However, similar to upd, Imp expression decreases in the hub cells of older males, which is due to the targeting of Imp by the heterochronic microRNA let-7. In the absence of Imp, upd mRNA therefore becomes unprotected and susceptible to degradation. Understanding the mechanistic basis for ageing-related changes in stem-cell behaviour will lead to the development of strategies to treat age-onset diseases and facilitate stem-cell-based therapies in older individuals.

  2. Principles of Tendon Transfer.

    PubMed

    Wilbur, Danielle; Hammert, Warren C

    2016-08-01

    Tendon transfers provide a substitute, either temporary or permanent, when function is lost due to neurologic injury in stroke, cerebral palsy or central nervous system lesions, peripheral nerve injuries, or injuries to the musculotendinous unit itself. This article reviews the basic principles of tendon transfer, which are important when planning surgery and essential for an optimal outcome. In addition, concepts for coapting the tendons during surgery and general principles to be followed during the rehabilitation process are discussed. PMID:27387072

  3. Interaction between genes Mos and mwh expressed in somatic cells of Drosophila melanogaster

    SciTech Connect

    Vaisman, N.Ya.; Zakharov, I.K.

    1995-07-01

    Gene Mosaic (Mos) of chromosome 3 of Drosophila melanogaster was located by means of dominant markers Ly, Sb, and Dr. This gene was shown to be located between Ly and Sb in the centromeric region (45-50 map units). An analysis of interaction between Mos and mwh genes in cis- and trans-heterozygotes showed a significant effect of the Mos gene on mutability (recombinogenesis) of chromosome mwh in somatic cells. In the cis heterozygote mwh Mos/++, the frequency of small mutant clones on wings of flies increased. In mwh/Mos heterozygotes, the Mos gene caused a significant reduction of dorsocentral and scutellar bristles (78% in mwh/Mos, 85% in mwh +/+ Mos, and 98% in mwh Mos/mwh +). 20 refs., 3 tabs.

  4. Pigment-Dispersing Factor Modulates Pheromone Production in Clock Cells that Influence Mating in Drosophila

    PubMed Central

    Krupp, Joshua J.; Billeter, Jean-Christophe; Wong, Amy; Choi, Charles; Nitabach, Michael N.; Levine, Joel D.

    2014-01-01

    Summary Social cues contribute to the circadian entrainment of physiological and behavioral rhythms. These cues supplement the influence of daily and seasonal cycles in light and temperature. In Drosophila, the social environment modulates circadian mechanisms that regulate sex pheromone production and mating behavior. Here we demonstrate that a neuroendocrine pathway, defined by the neuropeptide Pigment-Dispersing Factor (PDF), couples the central nervous system (CNS) to the physiological output of peripheral clock cells that produce pheromones, the oenocytes. PDF signaling from the CNS modulates the phase of the oenocyte clock. Despite its requirement for sustaining free-running locomoter activity rhythms, PDF is not necessary to sustain molecular rhythms in the oenocytes. Interestingly, disruption of the PDF signaling pathway reduces male sex pheromones and results in sex-specific differences in mating behavior. Our findings highlight the role of neuropeptide signaling and the circadian system in synchronizing the physiological and behavioral processes which govern social interactions. PMID:23849197

  5. Pigment-dispersing factor modulates pheromone production in clock cells that influence mating in drosophila.

    PubMed

    Krupp, Joshua J; Billeter, Jean-Christophe; Wong, Amy; Choi, Charles; Nitabach, Michael N; Levine, Joel D

    2013-07-10

    Social cues contribute to the circadian entrainment of physiological and behavioral rhythms. These cues supplement the influence of daily and seasonal cycles in light and temperature. In Drosophila, the social environment modulates circadian mechanisms that regulate sex pheromone production and mating behavior. Here we demonstrate that a neuroendocrine pathway, defined by the neuropeptide Pigment-Dispersing Factor (PDF), couples the CNS to the physiological output of peripheral clock cells that produce pheromones, the oenocytes. PDF signaling from the CNS modulates the phase of the oenocyte clock. Despite its requirement for sustaining free-running locomoter activity rhythms, PDF is not necessary to sustain molecular rhythms in the oenocytes. Interestingly, disruption of the PDF signaling pathway reduces male sex pheromones and results in sex-specific differences in mating behavior. Our findings highlight the role of neuropeptide signaling and the circadian system in synchronizing the physiological and behavioral processes that govern social interactions.

  6. Bacterial autolysins trim cell surface peptidoglycan to prevent detection by the Drosophila innate immune system

    PubMed Central

    Atilano, Magda Luciana; Pereira, Pedro Matos; Vaz, Filipa; Catalão, Maria João; Reed, Patricia; Grilo, Inês Ramos; Sobral, Rita Gonçalves; Ligoxygakis, Petros; Pinho, Mariana Gomes; Filipe, Sérgio Raposo

    2014-01-01

    Bacteria have to avoid recognition by the host immune system in order to establish a successful infection. Peptidoglycan, the principal constituent of virtually all bacterial surfaces, is a specific molecular signature recognized by dedicated host receptors, present in animals and plants, which trigger an immune response. Here we report that autolysins from Gram-positive pathogenic bacteria, enzymes capable of hydrolyzing peptidoglycan, have a major role in concealing this inflammatory molecule from Drosophila peptidoglycan recognition proteins (PGRPs). We show that autolysins trim the outermost peptidoglycan fragments and that in their absence bacterial virulence is impaired, as PGRPs can directly recognize leftover peptidoglycan extending beyond the external layers of bacterial proteins and polysaccharides. The activity of autolysins is not restricted to the producer cells but can also alter the surface of neighboring bacteria, facilitating the survival of the entire population in the infected host. DOI: http://dx.doi.org/10.7554/eLife.02277.001 PMID:24692449

  7. The decapping activator HPat a novel factor co-purifying with GW182 from Drosophila cells

    PubMed Central

    Jäger, Elisabeth; Dorner, Silke

    2011-01-01

    miRNAs post-transcriptionally regulate gene expression in many eukaryotes and thereby affect a wide range of biological processes. GW182 is a key factor in translation repression and mRNA degradation by miRNAs. In this study we investigate the potential interaction of GW182 and translation or mRNA degradation factors in Drosophila S2 cells. We have identified the decapping activator HPat as a novel factor co-purifying with GW182. Furthermore, we show that the C-terminal domain of GW182, important for gene silencing, is sufficient to form a complex with HPat. Our findings implicate a potential interaction of the miRNA effector component GW182 with the decapping machinery. PMID:20458171

  8. Riser and tendon management system

    SciTech Connect

    Devlin, P.V.

    1992-02-18

    This patent describes a riser and tendon management system. It comprises means to set nominal conditions for the risers and tendons; means to measure actual riser and tendon conditions; means to compare the actual and nominal conditions of the risers and tendons; and means responsive to a differential between the actual and nominal riser and tendon conditions, which difference exceeds specified limits, and recommending corrective action to bring the risers and tendons back to within nominal conditions.

  9. The HLH protein Extramacrochaetae is required for R7 cell and cone cell fates in the Drosophila eye

    PubMed Central

    Bhattacharya, Abhishek; Baker, Nicholas E.

    2010-01-01

    Summary Notch signaling is one of the most important pathways in development and homeostasis, and is altered in multiple pathologies. Study of Drosophila eye development shows that Notch signaling depends on the HLH protein Extramacrochaetae. Null mutant clones show that extramacrochaetae is required for multiple aspects of eye development that depend on Notch signaling, including morphogenetic furrow progression, differentiation of R4, R7 and cone cell types, and rotation of ommatidial clusters. Detailed analysis of R7 and cone cell specification reveals that extramacrochaetae acts cell autonomously and epistatically to Notch, and is required for normal expression of bHLH genes encoded by the E(spl)-C which are effectors of most Notch signaling. A model is proposed in which Extramacrochaetae acts in parallel to or as a feed-forward regulator of the E(spl)-Complex to promote Notch signaling in particular cellular contexts. PMID:19118542

  10. Tendon and ligament imaging

    PubMed Central

    Hodgson, R J; O'Connor, P J; Grainger, A J

    2012-01-01

    MRI and ultrasound are now widely used for the assessment of tendon and ligament abnormalities. Healthy tendons and ligaments contain high levels of collagen with a structured orientation, which gives rise to their characteristic normal imaging appearances as well as causing particular imaging artefacts. Changes to ligaments and tendons as a result of disease and injury can be demonstrated using both ultrasound and MRI. These have been validated against surgical and histological findings. Novel imaging techniques are being developed that may improve the ability of MRI and ultrasound to assess tendon and ligament disease. PMID:22553301

  11. Tendon Homeostasis in Hypercholesterolemia.

    PubMed

    Soslowsky, Louis J; Fryhofer, George W

    2016-01-01

    Hypercholesterolemia is a serious health problem that is associated not only with heart disease, but also tendon pathology. In high cholesterol environments (e.g. familial hyperlipidemia), lipids accumulate within the tendon extracellular matrix and form deposits called xanthomas. Lipid-related changes are known to affect several tendon mechanical properties, including stiffness and modulus, in uninjured and injured tendons, alike. Mechanisms to explain these cholesterol-related changes are multiple, including alterations in tenocyte gene and protein expression, matrix turnover, tissue vascularity, and cytokine production. Clinically, rotator cuff tear and Achilles tendon rupture are clearly associated with metabolic derangements, and elevated total cholesterol is often among the specific metabolic parameters implicated. Treatment of hypercholesterolemia using statin medications has also been shown to affect tendon properties, resulting in normalization of tendon thickness and improved tendon healing. Despite current work, the pathophysiology of lipid-related tendon pathology remains incompletely understood, and additional hypothesis-generating studies, including those incorporating whole-genome and whole-transcriptome technologies, will help to point the field in new directions. PMID:27535257

  12. Inflammation in Tendon Disorders.

    PubMed

    Speed, Cathy

    2016-01-01

    The role of inflammation in tendon disorders has long been a subject of considerable debate. Developments in our understanding of the basic science of inflammation have provided further insight into its potential role in specific forms of tendon disease, and the circumstances that may potentiate this. Such circumstances include excessive mechanical stresses on tendon and the presence of systemic inflammation associated with chronic diseases. In this chapter a brief review of the basic science of inflammation is provided and the influence that it may play on tendons is discussed. PMID:27535263

  13. Remote Control of Intestinal Stem Cell Activity by Haemocytes in Drosophila

    PubMed Central

    Chakrabarti, Sveta; Li, Xiaoxue; Collas, Esther Jeanne; Boquete, Jean-Phillipe; Lemaitre, Bruno

    2016-01-01

    The JAK/STAT pathway is a key signaling pathway in the regulation of development and immunity in metazoans. In contrast to the multiple combinatorial JAK/STAT pathways in mammals, only one canonical JAK/STAT pathway exists in Drosophila. It is activated by three secreted proteins of the Unpaired family (Upd): Upd1, Upd2 and Upd3. Although many studies have established a link between JAK/STAT activation and tissue damage, the mode of activation and the precise function of this pathway in the Drosophila systemic immune response remain unclear. In this study, we used mutations in upd2 and upd3 to investigate the role of the JAK/STAT pathway in the systemic immune response. Our study shows that haemocytes express the three upd genes and that injury markedly induces the expression of upd3 by the JNK pathway in haemocytes, which in turn activates the JAK/STAT pathway in the fat body and the gut. Surprisingly, release of Upd3 from haemocytes upon injury can remotely stimulate stem cell proliferation and the expression of Drosomycin-like genes in the intestine. Our results also suggest that a certain level of intestinal epithelium renewal is required for optimal survival to septic injury. While haemocyte-derived Upd promotes intestinal stem cell activation and survival upon septic injury, haemocytes are dispensable for epithelium renewal upon oral bacterial infection. Our study also indicates that intestinal epithelium renewal is sensitive to insults from both the lumen and the haemocoel. It also reveals that release of Upds by haemocytes coordinates the wound-healing program in multiple tissues, including the gut, an organ whose integrity is critical to fly survival. PMID:27231872

  14. Why Adult Stem Cell Functionality Declines with Age? Studies from the Fruit Fly Drosophila Melanogaster Model Organism

    PubMed Central

    Gonen, Oren; Toledano, Hila

    2014-01-01

    Highly regenerative adult tissues are supported by rare populations of stem cells that continuously divide to self-renew and generate differentiated progeny. This process is tightly regulated by signals emanating from surrounding cells to fulfill the dynamic demands of the tissue. One of the hallmarks of aging is slow and aberrant tissue regeneration due to deteriorated function of stem and supporting cells. Several Drosophila regenerative tissues are unique in that they provide exact identification of stem and neighboring cells in whole-tissue anatomy. This allows for precise tracking of age-related changes as well as their targeted manipulation within the tissue. In this review we present the stem cell niche of Drosophila testis, ovary and intestine and describe the major changes and phenotypes that occur in the course of aging. Specifically we discuss changes in both intrinsic properties of stem cells and their microenvironment that contribute to the decline in tissue functionality. Understanding these mechanisms in adult Drosophila tissues will likely provide new paradigms in the field of aging. PMID:24955030

  15. Conditional knockout of retinal determination genes in differentiating cells in Drosophila.

    PubMed

    Jin, Meng; Eblimit, Aiden; Pulikkathara, Merlyn; Corr, Stuart; Chen, Rui; Mardon, Graeme

    2016-08-01

    Conditional gene knockout in postmitotic cells is a valuable technique which allows the study of gene function with spatiotemporal control. Surprisingly, in contrast to its long-term and extensive use in mouse studies, this technology is lacking in Drosophila. Here, we use a novel method for generating complete loss of eyes absent (eya) or sine oculis (so) function in postmitotic cells posterior to the morphogenetic furrow (MF). Specifically, genomic rescue constructs with flippase recognition target (FRT) sequences flanking essential exons are used to generate conditional null alleles. By removing gene function in differentiating cells, we show that eya and so are dispensable for larval photoreceptor differentiation, but are required for differentiation during pupal development. Both eya and so are necessary for photoreceptor survival and the apoptosis caused by loss of eya or so function is likely a secondary consequence of inappropriate differentiation. We also confirm their requirement for cone cell development and reveal a novel role in interommatidial bristle (IOB) formation. In addition, so is required for normal eye disc morphology. This is the first report of a knockout method to study eya and so function in postmitotic cells. This technology will open the door to a large array of new functional studies in virtually any tissue and at any stage of development or in adults. PMID:27257739

  16. Translational repression determines a neuronal potential in Drosophila asymmetric cell division.

    PubMed

    Okabe, M; Imai, T; Kurusu, M; Hiromi, Y; Okano, H

    2001-05-01

    Asymmetric cell division is a fundamental strategy for generating cellular diversity during animal development. Daughter cells manifest asymmetry in their differential gene expression. Transcriptional regulation of this process has been the focus of many studies, whereas cell-type-specific 'translational' regulation has been considered to have a more minor role. During sensory organ development in Drosophila, Notch signalling directs the asymmetry between neuronal and non-neuronal lineages, and a zinc-finger transcriptional repressor Tramtrack69 (TTK69) acts downstream of Notch as a determinant of non-neuronal identity. Here we show that repression of TTK69 protein expression in the neuronal lineage occurs translationally rather than transcriptionally. This translational repression is achieved by a direct interaction between cis-acting sequences in the 3' untranslated region of ttk69 messenger RNA and its trans-acting repressor, the RNA-binding protein Musashi (MSI). Although msi can act downstream of Notch, Notch signalling does not affect MSI expression. Thus, Notch signalling is likely to regulate MSI activity rather than its expression. Our results define cell-type-specific translational control of ttk69 by MSI as a downstream event of Notch signalling in asymmetric cell division.

  17. Drosophila Stardust is a partner of Crumbs in the control of epithelial cell polarity.

    PubMed

    Bachmann, A; Schneider, M; Theilenberg, E; Grawe, F; Knust, E

    2001-12-01

    The polarized architecture of epithelial cells depends on the highly stereotypic distribution of cellular junctions and other membrane-associated protein complexes. In epithelial cells of the Drosophila embryo, three distinct domains subdivide the lateral plasma membrane. The most apical one comprises the subapical complex (SAC). It is followed by the zonula adherens (ZA) and, further basally, by the septate junction. A core component of the SAC is the transmembrane protein Crumbs, the cytoplasmic domain of which recruits the PDZ-protein Discs Lost into the complex. Cells lacking crumbs or the functionally related gene stardust fail to organize a continuous ZA and to maintain cell polarity. Here we show that stardust provides an essential component of the SAC. Stardust proteins colocalize with Crumbs and bind to the carboxy-terminal amino acids of its cytoplasmic tail. We introduce two different Stardust proteins here: one MAGUK protein, characterized by a PDZ domain, an SH3 domain and a guanylate kinase domain; and a second isoform comprising only the guanylate kinase domain. The Stardust proteins represent versatile candidates as structural and possibly regulatory constituents of the SAC, a crucial element in the control of epithelial cell polarity.

  18. Jak-STAT regulation of male germline stem cell establishment during Drosophila embryogenesis

    PubMed Central

    Sheng, X. Rebecca; Posenau, Trevor; Gumulak-Smith, Juliann J.; Matunis, Erika; Van Doren, Mark; Wawersik, Matthew

    2009-01-01

    Summary Germline stem cells (GSCs) in Drosophila are descendants of primordial germ cells (PGCs) specified during embryogenesis. The precise timing of GSC establishment in the testis has not been determined, nor is it known whether mechanisms that control GSC maintenance in the adult are involved in GSC establishment. Here, we determine that PGCs in the developing male gonad first become GSCs at the embryo to larval transition. This coincides with formation of the embryonic hub; the critical signaling center that regulates adult GSC behavior within the stem cell microenvironment (niche). We find that the Jak-STAT signaling pathway is activated in a subset of PGCs that associate with the newly-formed embryonic hub. These PGCs express GSC markers and function like GSCs, while PGCs that do not associate with the hub begin to differentiate. In the absence of Jak-STAT activation, PGCs adjacent to the hub fail to exhibit the characteristics of GSCs, while ectopic activation of the Jak-STAT pathway prevents differentiation. These findings show that stem cell formation is closely linked to development of the stem cell niche, and suggest that Jak-STAT signaling is required for initial establishment of the GSC population in developing testes. PMID:19643104

  19. Kinetic response of a Drosophila melanogaster cell line to different medium formulations and culture conditions

    PubMed Central

    Bovo, R.; Galesi, A. L. L; Jorge, S. A. C.; Piccoli, R. A. M.; Moraes, A. M.; Pereira, C. A.

    2008-01-01

    In the past few years, Drosophila melanogaster cells have been employed for recombinant protein production purposes, and a comprehensive knowledge of their metabolism is essential for process optimization. In this work, the kinetic response of a Schneider S2 cell line, grown in shake flasks, in two different culture media, the serum-free SF900-II® and the serum-supplemented TC-100, was evaluated. Cell growth, amino acids and glucose uptake, and lactate synthesis were measured allowing the calculation of kinetic parameters. The results show that S2 cells metabolism was able to adjust to different environmental situations, as determined by medium formulation, as well as by the particular situation resulting from the culture conditions. Cells attained a 163% higher final cell concentration (1.4 × 107 cells mL−1) in SF900 II® medium, when compared to serum-supplemented TC-100 medium. Also, a maximum specific cell growth rate 52% higher in SF900 II® medium, when compared to serum-supplemented TC-100 one, was observed. Glutamine was the growth limiting factor in SF900 II® medium, while glucose, sometimes associated with glutamine, controlled growth in serum-supplemented TC-100 medium based formulation. The different pattern of lactate production is an example of the versatility of the metabolism of these cells. This by-product was produced only in glutamine limitation, but the amount synthesized depended not only on the excess glucose, but on other medium components. Therefore, in serum-supplemented TC-100 medium a much smaller lactate amount was generated. Besides, glucose was identified not only as a growth limiting factor, but also as a viability limiting factor, since its depletion accelerated cell death. PMID:19003169

  20. ECM-Regulator timp Is Required for Stem Cell Niche Organization and Cyst Production in the Drosophila Ovary

    PubMed Central

    Pearson, John R.; Zurita, Federico; Tomás-Gallardo, Laura; Díaz-Torres, Alfonsa; Díaz de la Loza, María del Carmen; Franze, Kristian; Martín-Bermudo, María D.; González-Reyes, Acaimo

    2016-01-01

    The extracellular matrix (ECM) is a pivotal component adult tissues and of many tissue-specific stem cell niches. It provides structural support and regulates niche signaling during tissue maintenance and regeneration. In many tissues, ECM remodeling depends on the regulation of MMP (matrix metalloproteinase) activity by inhibitory TIMP (tissue inhibitors of metalloproteinases) proteins. Here, we report that the only Drosophila timp gene is required for maintaining the normal organization and function of the germline stem cell niche in adult females. timp mutant ovaries show reduced levels of both Drosophila Collagen IV α chains. In addition, tissue stiffness and the cellular organization of the ovarian niche are affected in timp mutants. Finally, loss of timp impairs the ability of the germline stem cell niche to generate new cysts. Our results demonstrating a crucial role for timp in tissue organization and gamete production thus provide a link between the regulation of ECM metabolism and tissue homeostasis. PMID:26808525

  1. Loss of l(3)mbt leads to acquisition of the ping-pong cycle in Drosophila ovarian somatic cells

    PubMed Central

    Sumiyoshi, Tetsutaro; Sato, Kaoru; Yamamoto, Hitomi; Iwasaki, Yuka W.; Siomi, Haruhiko; Siomi, Mikiko C.

    2016-01-01

    In Drosophila germ cells, PIWI-interacting RNAs (piRNAs) are amplified through a PIWI slicer-dependent feed-forward loop termed the ping-pong cycle, yielding secondary piRNAs. However, the detailed mechanism remains poorly understood, largely because an ex vivo model system amenable to biochemical analyses has not been available. Here, we show that CRISPR-mediated loss of function of lethal (3) malignant brain tumor [l(3)mbt] leads to ectopic activation of the germ-specific ping-pong cycle in ovarian somatic cells. Perinuclear foci resembling nuage, the ping-pong center, appeared following l(3)mbt mutation. This activation of the ping-pong machinery in cultured cells will greatly facilitate elucidation of the mechanism underlying secondary piRNA biogenesis in Drosophila. PMID:27474440

  2. Glycosaminoglycans in Tendon Physiology, Pathophysiology, and Therapy.

    PubMed

    Ryan, Christina N M; Sorushanova, Anna; Lomas, Alex J; Mullen, Anne Maria; Pandit, Abhay; Zeugolis, Dimitrios I

    2015-07-15

    Although glycosaminoglycans constitute a minor portion of native tissues, they play a crucial role in various physiological processes, while their abnormal expression is associated with numerous pathophysiologies. Glycosaminoglycans have become increasingly prevalent in biomaterial design for tendon repair, given their low immunogenicity and their inherent capacity to stimulate the regenerative processes, while maintaining resident cell phenotype and function. Further, their incorporation into three-dimensional scaffold conformations significantly improves their mechanical properties, while reducing the formation of peritendinous adhesions. Herein, we discuss the role of glycosaminoglycans in tendon physiology and pathophysiology and the advancements achieved to date using glycosaminoglycan-functionalized scaffolds for tendon repair and regeneration. It is evidenced that glycosaminoglycan functionalization has led to many improvements in tendon tissue engineering and it is anticipated to play a pivotal role in future reparative therapies.

  3. MicroRNA-Dependent Transcriptional Silencing of Transposable Elements in Drosophila Follicle Cells

    PubMed Central

    Mugat, Bruno; Akkouche, Abdou; Serrano, Vincent; Armenise, Claudia; Li, Blaise; Brun, Christine; Fulga, Tudor A.; Van Vactor, David; Pélisson, Alain; Chambeyron, Séverine

    2015-01-01

    RNA interference-related silencing mechanisms concern very diverse and distinct biological processes, from gene regulation (via the microRNA pathway) to defense against molecular parasites (through the small interfering RNA and the Piwi-interacting RNA pathways). Small non-coding RNAs serve as specificity factors that guide effector proteins to ribonucleic acid targets via base-pairing interactions, to achieve transcriptional or post-transcriptional regulation. Because of the small sequence complementarity required for microRNA-dependent post-transcriptional regulation, thousands of microRNA (miRNA) putative targets have been annotated in Drosophila. In Drosophila somatic ovarian cells, genomic parasites, such as transposable elements (TEs), are transcriptionally repressed by chromatin changes induced by Piwi-interacting RNAs (piRNAs) that prevent them from invading the germinal genome. Here we show, for the first time, that a functional miRNA pathway is required for the piRNA-mediated transcriptional silencing of TEs in this tissue. Global miRNA depletion, caused by tissue- and stage-specific knock down of drosha (involved in miRNA biogenesis), AGO1 or gawky (both responsible for miRNA activity), resulted in loss of TE-derived piRNAs and chromatin-mediated transcriptional de-silencing of TEs. This specific TE de-repression was also observed upon individual titration (by expression of the complementary miRNA sponge) of two miRNAs (miR-14 and miR-34) as well as in a miR-14 loss-of-function mutant background. Interestingly, the miRNA defects differentially affected TE- and 3' UTR-derived piRNAs. To our knowledge, this is the first indication of possible differences in the biogenesis or stability of TE- and 3' UTR-derived piRNAs. This work is one of the examples of detectable phenotypes caused by loss of individual miRNAs in Drosophila and the first genetic evidence that miRNAs have a role in the maintenance of genome stability via piRNA-mediated TE repression. PMID

  4. MicroRNA-Dependent Transcriptional Silencing of Transposable Elements in Drosophila Follicle Cells.

    PubMed

    Mugat, Bruno; Akkouche, Abdou; Serrano, Vincent; Armenise, Claudia; Li, Blaise; Brun, Christine; Fulga, Tudor A; Van Vactor, David; Pélisson, Alain; Chambeyron, Séverine

    2015-05-01

    RNA interference-related silencing mechanisms concern very diverse and distinct biological processes, from gene regulation (via the microRNA pathway) to defense against molecular parasites (through the small interfering RNA and the Piwi-interacting RNA pathways). Small non-coding RNAs serve as specificity factors that guide effector proteins to ribonucleic acid targets via base-pairing interactions, to achieve transcriptional or post-transcriptional regulation. Because of the small sequence complementarity required for microRNA-dependent post-transcriptional regulation, thousands of microRNA (miRNA) putative targets have been annotated in Drosophila. In Drosophila somatic ovarian cells, genomic parasites, such as transposable elements (TEs), are transcriptionally repressed by chromatin changes induced by Piwi-interacting RNAs (piRNAs) that prevent them from invading the germinal genome. Here we show, for the first time, that a functional miRNA pathway is required for the piRNA-mediated transcriptional silencing of TEs in this tissue. Global miRNA depletion, caused by tissue- and stage-specific knock down of drosha (involved in miRNA biogenesis), AGO1 or gawky (both responsible for miRNA activity), resulted in loss of TE-derived piRNAs and chromatin-mediated transcriptional de-silencing of TEs. This specific TE de-repression was also observed upon individual titration (by expression of the complementary miRNA sponge) of two miRNAs (miR-14 and miR-34) as well as in a miR-14 loss-of-function mutant background. Interestingly, the miRNA defects differentially affected TE- and 3' UTR-derived piRNAs. To our knowledge, this is the first indication of possible differences in the biogenesis or stability of TE- and 3' UTR-derived piRNAs. This work is one of the examples of detectable phenotypes caused by loss of individual miRNAs in Drosophila and the first genetic evidence that miRNAs have a role in the maintenance of genome stability via piRNA-mediated TE repression. PMID

  5. MicroRNA-Dependent Transcriptional Silencing of Transposable Elements in Drosophila Follicle Cells.

    PubMed

    Mugat, Bruno; Akkouche, Abdou; Serrano, Vincent; Armenise, Claudia; Li, Blaise; Brun, Christine; Fulga, Tudor A; Van Vactor, David; Pélisson, Alain; Chambeyron, Séverine

    2015-05-01

    RNA interference-related silencing mechanisms concern very diverse and distinct biological processes, from gene regulation (via the microRNA pathway) to defense against molecular parasites (through the small interfering RNA and the Piwi-interacting RNA pathways). Small non-coding RNAs serve as specificity factors that guide effector proteins to ribonucleic acid targets via base-pairing interactions, to achieve transcriptional or post-transcriptional regulation. Because of the small sequence complementarity required for microRNA-dependent post-transcriptional regulation, thousands of microRNA (miRNA) putative targets have been annotated in Drosophila. In Drosophila somatic ovarian cells, genomic parasites, such as transposable elements (TEs), are transcriptionally repressed by chromatin changes induced by Piwi-interacting RNAs (piRNAs) that prevent them from invading the germinal genome. Here we show, for the first time, that a functional miRNA pathway is required for the piRNA-mediated transcriptional silencing of TEs in this tissue. Global miRNA depletion, caused by tissue- and stage-specific knock down of drosha (involved in miRNA biogenesis), AGO1 or gawky (both responsible for miRNA activity), resulted in loss of TE-derived piRNAs and chromatin-mediated transcriptional de-silencing of TEs. This specific TE de-repression was also observed upon individual titration (by expression of the complementary miRNA sponge) of two miRNAs (miR-14 and miR-34) as well as in a miR-14 loss-of-function mutant background. Interestingly, the miRNA defects differentially affected TE- and 3' UTR-derived piRNAs. To our knowledge, this is the first indication of possible differences in the biogenesis or stability of TE- and 3' UTR-derived piRNAs. This work is one of the examples of detectable phenotypes caused by loss of individual miRNAs in Drosophila and the first genetic evidence that miRNAs have a role in the maintenance of genome stability via piRNA-mediated TE repression.

  6. Using Drosophila Larval Imaginal Discs to Study Low-Dose Radiation-Induced Cell Cycle Arrest

    PubMed Central

    Yan, Shian-Jang; Li, Willis X.

    2012-01-01

    Under genotoxic stress, activation of cell cycle checkpoint responses leads to cell cycle arrest, which allows cells to repair DNA damage before continuing to cycle. Drosophila larval epithelial sacs, called imaginal discs, are an excellent in vivo model system for studying radiation-induced cell cycle arrest. Larval imaginal discs go into cell cycle arrest after being subjected to low-dose irradiation, are subject to easy genetic manipulation, are not crucial for survival of the organism, and can be dissected easily for further molecular or cellular analysis. In this chapter, we describe methods for assessing low-dose irradiation-induced cell cycle arrest. Mitotic cells are identified by immunofluorescence staining for the mitotic marker phosphorylated histone H3 (phospho-histone H3 or pH3). When wandering third-instar control larvae, without transgene expression, are exposed to 500 rads of X-ray or γ-ray irradiation, the number of pH3-positive cells in wing imaginal discs is reduced from hundreds before irradiation to approximately 30 after irradiation, with an equal distribution between the anterior and posterior compartments (Yan et al., 2011, FASEB J). Using the GAL4/UAS system, RNAi, cDNA, or microRNA sponge transgenes can be expressed in the posterior compartment of the wing disc using drivers such as engrailed (en)-Gal4, while the anterior compartment serves as an internal control. This approach makes it possible to do genome-wide genetic screening for molecules involved in radiation-induced cell cycle arrest. PMID:21870287

  7. Unipolar distributions of junctional Myosin II identify cell stripe boundaries that drive cell intercalation throughout Drosophila axis extension

    PubMed Central

    Tetley, Robert J; Blanchard, Guy B; Fletcher, Alexander G; Adams, Richard J; Sanson, Bénédicte

    2016-01-01

    Convergence and extension movements elongate tissues during development. Drosophila germ-band extension (GBE) is one example, which requires active cell rearrangements driven by Myosin II planar polarisation. Here, we develop novel computational methods to analyse the spatiotemporal dynamics of Myosin II during GBE, at the scale of the tissue. We show that initial Myosin II bipolar cell polarization gives way to unipolar enrichment at parasegmental boundaries and two further boundaries within each parasegment, concomitant with a doubling of cell number as the tissue elongates. These boundaries are the primary sites of cell intercalation, behaving as mechanical barriers and providing a mechanism for how cells remain ordered during GBE. Enrichment at parasegment boundaries during GBE is independent of Wingless signaling, suggesting pair-rule gene control. Our results are consistent with recent work showing that a combinatorial code of Toll-like receptors downstream of pair-rule genes contributes to Myosin II polarization via local cell-cell interactions. We propose an updated cell-cell interaction model for Myosin II polarization that we tested in a vertex-based simulation. DOI: http://dx.doi.org/10.7554/eLife.12094.001 PMID:27183005

  8. The Drosophila F-box protein dSkp2 regulates cell proliferation by targeting Dacapo for degradation.

    PubMed

    Dui, Wen; Wei, Bin; He, Feng; Lu, Wei; Li, Changqing; Liang, Xuehong; Ma, Jun; Jiao, Renjie

    2013-06-01

    Cell cycle progression is controlled by a complex regulatory network consisting of interacting positive and negative factors. In humans, the positive regulator Skp2, an F-box protein, has been a subject of intense investigation in part because of its oncogenic activity. By contrast, the molecular and developmental functions of its Drosophila homologue, dSkp2, are poorly understood. Here we investigate the role of dSkp2 by focusing on its functional relationship with Dacapo (Dap), the Drosophila homologue of the cyclin-dependent kinase inhibitors p21(cip1)/p27(kip1)/p57(kip2). We show that dSkp2 interacts physically with Dap and has a role in targeting Dap for ubiquitination and proteasome-mediated degradation. We present evidence that dSkp2 regulates cell cycle progression by antagonizing Dap in vivo. dSkp2 knockdown reduces cell density in the wing by prolonging the cell doubling time. In addition, the wing phenotype caused by dSkp2 knockdown resembles that caused by dap overexpression and can be partially suppressed by reducing the gene dose of dap. Our study thus documents a conserved functional relationship between dSkp2 and Dap in their control of cell cycle progression, suggesting the possibility of using Drosophila as a model system to study Skp2-mediated tumorigenesis.

  9. The frizzled/stan Pathway and Planar Cell Polarity in the Drosophila Wing

    PubMed Central

    Adler, Paul N.

    2013-01-01

    Drosophila has been the key model system for studies on Planar Cell Polarity (PCP). The rich morphology of the insect exoskeleton contains many structures that display PCP. Among these are the trichomes (cuticular hairs) that cover much of the exoskeleton, sensory bristles and ommatidia. Many genes have been identified that must function for the development of normal PCP. Among these are the genes that comprise the frizzled/starry night (fz/stan) and dachsous/fat pathways. The mechanisms that underlie the function of the fz/stan pathway are best understood. All of the protein products of these genes accumulate asymmetrically in wing cells and there is good evidence that this involves local intercellular signaling between protein complexes on the distal edge of one cell and the juxtaposed proximal edge of its neighbor. It is thought that a feedback system, directed transport and stabilizing protein-protein interactions mediate the formation of distal and proximal protein complexes. These complexes appear to recruit downstream proteins that function to spatially restrict the activation of the cytoskeleton in wing cells. This leads to the formation of the array of distally pointing hairs found on wings. PMID:23140623

  10. Mechanochemical regulation of oscillatory follicle cell dynamics in the developing Drosophila egg chamber.

    PubMed

    Koride, Sarita; He, Li; Xiong, Li-Ping; Lan, Ganhui; Montell, Denise J; Sun, Sean X

    2014-11-01

    During tissue elongation from stage 9 to stage 10 in Drosophila oogenesis, the egg chamber increases in length by ∼1.7-fold while increasing in volume by eightfold. During these stages, spontaneous oscillations in the contraction of cell basal surfaces develop in a subset of follicle cells. This patterned activity is required for elongation of the egg chamber; however, the mechanisms generating the spatiotemporal pattern have been unclear. Here we use a combination of quantitative modeling and experimental perturbation to show that mechanochemical interactions are sufficient to generate oscillations of myosin contractile activity in the observed spatiotemporal pattern. We propose that follicle cells in the epithelial layer contract against pressure in the expanding egg chamber. As tension in the epithelial layer increases, Rho kinase signaling activates myosin assembly and contraction. The activation process is cooperative, leading to a limit cycle in the myosin dynamics. Our model produces asynchronous oscillations in follicle cell area and myosin content, consistent with experimental observations. In addition, we test the prediction that removal of the basal lamina will increase the average oscillation period. The model demonstrates that in principle, mechanochemical interactions are sufficient to drive patterning and morphogenesis, independent of patterned gene expression. PMID:24943847

  11. PERK Limits Drosophila Lifespan by Promoting Intestinal Stem Cell Proliferation in Response to ER Stress

    PubMed Central

    Wang, Lifen; Ryoo, Hyung Don; Qi, Yanyan; Jasper, Heinrich

    2015-01-01

    Intestinal homeostasis requires precise control of intestinal stem cell (ISC) proliferation. In Drosophila, this control declines with age largely due to chronic activation of stress signaling and associated chronic inflammatory conditions. An important contributor to this condition is the age-associated increase in endoplasmic reticulum (ER) stress. Here we show that the PKR-like ER kinase (PERK) integrates both cell-autonomous and non-autonomous ER stress stimuli to induce ISC proliferation. In addition to responding to cell-intrinsic ER stress, PERK is also specifically activated in ISCs by JAK/Stat signaling in response to ER stress in neighboring cells. The activation of PERK is required for homeostatic regeneration, as well as for acute regenerative responses, yet the chronic engagement of this response becomes deleterious in aging flies. Accordingly, knocking down PERK in ISCs is sufficient to promote intestinal homeostasis and extend lifespan. Our studies highlight the significance of the PERK branch of the unfolded protein response of the ER (UPRER) in intestinal homeostasis and provide a viable strategy to improve organismal health- and lifespan. PMID:25945494

  12. Mechanochemical regulation of oscillatory follicle cell dynamics in the developing Drosophila egg chamber.

    PubMed

    Koride, Sarita; He, Li; Xiong, Li-Ping; Lan, Ganhui; Montell, Denise J; Sun, Sean X

    2014-11-01

    During tissue elongation from stage 9 to stage 10 in Drosophila oogenesis, the egg chamber increases in length by ∼1.7-fold while increasing in volume by eightfold. During these stages, spontaneous oscillations in the contraction of cell basal surfaces develop in a subset of follicle cells. This patterned activity is required for elongation of the egg chamber; however, the mechanisms generating the spatiotemporal pattern have been unclear. Here we use a combination of quantitative modeling and experimental perturbation to show that mechanochemical interactions are sufficient to generate oscillations of myosin contractile activity in the observed spatiotemporal pattern. We propose that follicle cells in the epithelial layer contract against pressure in the expanding egg chamber. As tension in the epithelial layer increases, Rho kinase signaling activates myosin assembly and contraction. The activation process is cooperative, leading to a limit cycle in the myosin dynamics. Our model produces asynchronous oscillations in follicle cell area and myosin content, consistent with experimental observations. In addition, we test the prediction that removal of the basal lamina will increase the average oscillation period. The model demonstrates that in principle, mechanochemical interactions are sufficient to drive patterning and morphogenesis, independent of patterned gene expression.

  13. Oncogenic transformation of Drosophila somatic cells induces a functional piRNA pathway.

    PubMed

    Fagegaltier, Delphine; Falciatori, Ilaria; Czech, Benjamin; Castel, Stephane; Perrimon, Norbert; Simcox, Amanda; Hannon, Gregory J

    2016-07-15

    Germline genes often become re-expressed in soma-derived human cancers as "cancer/testis antigens" (CTAs), and piRNA (PIWI-interacting RNA) pathway proteins are found among CTAs. However, whether and how the piRNA pathway contributes to oncogenesis in human neoplasms remain poorly understood. We found that oncogenic Ras combined with loss of the Hippo tumor suppressor pathway reactivates a primary piRNA pathway in Drosophila somatic cells coincident with oncogenic transformation. In these cells, Piwi becomes loaded with piRNAs derived from annotated generative loci, which are normally restricted to either the germline or the somatic follicle cells. Negating the pathway leads to increases in the expression of a wide variety of transposons and also altered expression of some protein-coding genes. This correlates with a reduction in the proliferation of the transformed cells in culture, suggesting that, at least in this context, the piRNA pathway may play a functional role in cancer. PMID:27474441

  14. The Drosophila Actin Regulator ENABLED Regulates Cell Shape and Orientation during Gonad Morphogenesis

    PubMed Central

    Sano, Hiroko; Barbosa, Vitor; Clark, Ivan B. N.; Ishihara, Shuji; Sugimura, Kaoru; Lehmann, Ruth

    2012-01-01

    Organs develop distinctive morphologies to fulfill their unique functions. We used Drosophila embryonic gonads as a model to study how two different cell lineages, primordial germ cells (PGCs) and somatic gonadal precursors (SGPs), combine to form one organ. We developed a membrane GFP marker to image SGP behaviors live. These studies show that a combination of SGP cell shape changes and inward movement of anterior and posterior SGPs leads to the compaction of the spherical gonad. This process is disrupted in mutants of the actin regulator, enabled (ena). We show that Ena coordinates these cell shape changes and the inward movement of the SGPs, and Ena affects the intracellular localization of DE-cadherin (DE-cad). Mathematical simulation based on these observations suggests that changes in DE-cad localization can generate the forces needed to compact an elongated structure into a sphere. We propose that Ena regulates force balance in the SGPs by sequestering DE-cad, leading to the morphogenetic movement required for gonad compaction. PMID:23300733

  15. PERK Limits Drosophila Lifespan by Promoting Intestinal Stem Cell Proliferation in Response to ER Stress.

    PubMed

    Wang, Lifen; Ryoo, Hyung Don; Qi, Yanyan; Jasper, Heinrich

    2015-05-01

    Intestinal homeostasis requires precise control of intestinal stem cell (ISC) proliferation. In Drosophila, this control declines with age largely due to chronic activation of stress signaling and associated chronic inflammatory conditions. An important contributor to this condition is the age-associated increase in endoplasmic reticulum (ER) stress. Here we show that the PKR-like ER kinase (PERK) integrates both cell-autonomous and non-autonomous ER stress stimuli to induce ISC proliferation. In addition to responding to cell-intrinsic ER stress, PERK is also specifically activated in ISCs by JAK/Stat signaling in response to ER stress in neighboring cells. The activation of PERK is required for homeostatic regeneration, as well as for acute regenerative responses, yet the chronic engagement of this response becomes deleterious in aging flies. Accordingly, knocking down PERK in ISCs is sufficient to promote intestinal homeostasis and extend lifespan. Our studies highlight the significance of the PERK branch of the unfolded protein response of the ER (UPRER) in intestinal homeostasis and provide a viable strategy to improve organismal health- and lifespan. PMID:25945494

  16. PERK Limits Drosophila Lifespan by Promoting Intestinal Stem Cell Proliferation in Response to ER Stress.

    PubMed

    Wang, Lifen; Ryoo, Hyung Don; Qi, Yanyan; Jasper, Heinrich

    2015-05-01

    Intestinal homeostasis requires precise control of intestinal stem cell (ISC) proliferation. In Drosophila, this control declines with age largely due to chronic activation of stress signaling and associated chronic inflammatory conditions. An important contributor to this condition is the age-associated increase in endoplasmic reticulum (ER) stress. Here we show that the PKR-like ER kinase (PERK) integrates both cell-autonomous and non-autonomous ER stress stimuli to induce ISC proliferation. In addition to responding to cell-intrinsic ER stress, PERK is also specifically activated in ISCs by JAK/Stat signaling in response to ER stress in neighboring cells. The activation of PERK is required for homeostatic regeneration, as well as for acute regenerative responses, yet the chronic engagement of this response becomes deleterious in aging flies. Accordingly, knocking down PERK in ISCs is sufficient to promote intestinal homeostasis and extend lifespan. Our studies highlight the significance of the PERK branch of the unfolded protein response of the ER (UPRER) in intestinal homeostasis and provide a viable strategy to improve organismal health- and lifespan.

  17. Diversity of miRNAs, siRNAs, and piRNAs across 25 Drosophila cell lines

    PubMed Central

    Wen, Jiayu; Mohammed, Jaaved; Bortolamiol-Becet, Diane; Tsai, Harrison; Robine, Nicolas; Westholm, Jakub O.; Ladewig, Erik; Dai, Qi; Okamura, Katsutomo; Flynt, Alex S.; Zhang, Dayu; Andrews, Justen; Cherbas, Lucy; Kaufman, Thomas C.; Cherbas, Peter; Siepel, Adam; Lai, Eric C.

    2014-01-01

    We expanded the knowledge base for Drosophila cell line transcriptomes by deeply sequencing their small RNAs. In total, we analyzed more than 1 billion raw reads from 53 libraries across 25 cell lines. We verify reproducibility of biological replicate data sets, determine common and distinct aspects of miRNA expression across cell lines, and infer the global impact of miRNAs on cell line transcriptomes. We next characterize their commonalities and differences in endo-siRNA populations. Interestingly, most cell lines exhibit enhanced TE-siRNA production relative to tissues, suggesting this as a common aspect of cell immortalization. We also broadly extend annotations of cis-NAT-siRNA loci, identifying ones with common expression across diverse cells and tissues, as well as cell-restricted loci. Finally, we characterize small RNAs in a set of ovary-derived cell lines, including somatic cells (OSS and OSC) and a mixed germline/somatic cell population (fGS/OSS) that exhibits ping-pong piRNA signatures. Collectively, the ovary data reveal new genic piRNA loci, including unusual configurations of piRNA-generating regions. Together with the companion analysis of mRNAs described in a previous study, these small RNA data provide comprehensive information on the transcriptional landscape of diverse Drosophila cell lines. These data should encourage broader usage of fly cell lines, beyond the few that are presently in common usage. PMID:24985917

  18. Diversity of miRNAs, siRNAs, and piRNAs across 25 Drosophila cell lines.

    PubMed

    Wen, Jiayu; Mohammed, Jaaved; Bortolamiol-Becet, Diane; Tsai, Harrison; Robine, Nicolas; Westholm, Jakub O; Ladewig, Erik; Dai, Qi; Okamura, Katsutomo; Flynt, Alex S; Zhang, Dayu; Andrews, Justen; Cherbas, Lucy; Kaufman, Thomas C; Cherbas, Peter; Siepel, Adam; Lai, Eric C

    2014-07-01

    We expanded the knowledge base for Drosophila cell line transcriptomes by deeply sequencing their small RNAs. In total, we analyzed more than 1 billion raw reads from 53 libraries across 25 cell lines. We verify reproducibility of biological replicate data sets, determine common and distinct aspects of miRNA expression across cell lines, and infer the global impact of miRNAs on cell line transcriptomes. We next characterize their commonalities and differences in endo-siRNA populations. Interestingly, most cell lines exhibit enhanced TE-siRNA production relative to tissues, suggesting this as a common aspect of cell immortalization. We also broadly extend annotations of cis-NAT-siRNA loci, identifying ones with common expression across diverse cells and tissues, as well as cell-restricted loci. Finally, we characterize small RNAs in a set of ovary-derived cell lines, including somatic cells (OSS and OSC) and a mixed germline/somatic cell population (fGS/OSS) that exhibits ping-pong piRNA signatures. Collectively, the ovary data reveal new genic piRNA loci, including unusual configurations of piRNA-generating regions. Together with the companion analysis of mRNAs described in a previous study, these small RNA data provide comprehensive information on the transcriptional landscape of diverse Drosophila cell lines. These data should encourage broader usage of fly cell lines, beyond the few that are presently in common usage.

  19. Regional adaptations in three rat tendons.

    PubMed

    Covizi, D Z; Felisbino, S L; Gomes, L; Pimentel, E R; Carvalho, H F

    2001-10-01

    Although detailed histological and immunocytochemical studies have been published for the rat calcanear tendon (CT), little is known of the structure, composition and biomechanics of the deep (DFT) and superficial (SFT) flexor tendons. In this study, we examined the structural specialization of these three tendons in 90-day-old rats by applying histochemical and biochemical assays to different tendon regions (proximal, intermediate and distal regions of the DFT and SFT, and proximal and distal regions of the CT). There were regional differences in tissue structure, glycosaminoglycan type and content, swelling properties and in the amount and distribution of elastic fibers. Dermatan sulfate occurred in all regions, but chondroitin sulfate predominated in the intermediate region of the DFT and in the distal region of the CT. These two chondroitin sulfate-bearing regions showed swelling in water, while all other regions lost fluid in water. Fibrocartilaginous sites were observed on the CT, one at the insertion to the bone and another distally at the innermost area of the tendon. The intermediate region of the DFT showed round cells disposed in lacunae, while the proximal and distal regions were typically fibrous. The intermediate region of the SFT showed a wavy array of collagen bundles but neither toluidine blue staining in the matrix nor round cells. Elastic fibers were present in each region of the three tendons, but were more prominent in the intermediate zone of the SFT. These results demonstrate regional variation in the three tendons. Tendon differentiation may occur by an increase in the number of elastic fibers and by variations in the arrangement of collagen fibers, without fibrocartilage formation.

  20. Loss of Drosophila pseudouridine synthase triggers apoptosis-induced proliferation and promotes cell-nonautonomous EMT.

    PubMed

    Vicidomini, R; Di Giovanni, A; Petrizzo, A; Iannucci, L F; Benvenuto, G; Nagel, A C; Preiss, A; Furia, M

    2015-03-26

    Many developing tissues display regenerative capability that allows them to compensate cell loss and preserve tissue homeostasis. Because of their remarkable regenerative capability, Drosophila wing discs are extensively used for the study of regenerative phenomena. We thus used the developing wing to investigate the role played in tissue homeostasis by the evolutionarily conserved eukaryotic H/ACA small nucleolar ribonucleoprotein pseudouridine synthase. Here we show that localized depletion of this enzyme can act as an endogenous stimulus capable of triggering apoptosis-induced proliferation, and that context-dependent effects are elicited in different sub-populations of the silenced cells. In fact, some cells undergo apoptosis, whereas those surrounding the apoptotic foci, although identically depleted, overproliferate. This overproliferation correlates with ectopic induction of the Wg and JAK-STAT (Janus kinase-signal transducer and activator of transcription) mitogenic pathways. Expression of a p35 transgene, which blocks the complete execution of the death program and generates the so-called 'undead cells', amplifies the proliferative response. Pseudouridine synthase depletion also causes loss of apicobasal polarity, disruption of adherens cell junctions and ectopic induction of JNK (c-Jun N-terminal kinase) and Mmp1 (matrix metalloproteinase-1) activity, leading to a significant epithelial reorganization. Unexpectedly, cell-nonautonomous effects, such as epithelial mesenchymal transition in the contiguous unsilenced squamous epithelium, are also promoted. Collectively, these data point out that cell-cell communication and long-range signaling can take a relevant role in the response to pseudouridine synthase decline. Considering that all the affected pathways are highly conserved throughout evolution, it is plausible that the response to pseudouridine synthase depletion has been widely preserved. On this account, our results can add new light on the still

  1. Fatigue loading of tendon

    PubMed Central

    Shepherd, Jennifer H; Screen, Hazel R C

    2013-01-01

    Tendon injuries, often called tendinopathies, are debilitating and painful conditions, generally considered to develop as a result of tendon overuse. The aetiology of tendinopathy remains poorly understood, and whilst tendon biopsies have provided some information concerning tendon appearance in late-stage disease, there is still little information concerning the mechanical and cellular events associated with disease initiation and progression. Investigating this in situ is challenging, and numerous models have been developed to investigate how overuse may generate tendon fatigue damage and how this may relate to tendinopathy conditions. This article aims to review these models and our current understanding of tendon fatigue damage. We review the strengths and limitations of different methodologies for characterizing tendon fatigue, considering in vitro methods that adopt both viable and non-viable samples, as well as the range of different in vivo approaches. By comparing data across model systems, we review the current understanding of fatigue damage development. Additionally, we compare these findings with data from tendinopathic tissue biopsies to provide some insights into how these models may relate to the aetiology of tendinopathy. Fatigue-induced damage consistently highlights the same microstructural, biological and mechanical changes to the tendon across all model systems and also correlates well with the findings from tendinopathic biopsy tissue. The multiple testing routes support matrix damage as an important contributor to tendinopathic conditions, but cellular responses to fatigue appear complex and often contradictory. PMID:23837793

  2. Tendon Transfers for Tetraplegia.

    PubMed

    Bednar, Michael S

    2016-08-01

    It is estimated that 65% to 75% of patients with cervical spinal cord injuries could benefit from upper extremity tendon transfer surgery. The goals of surgery are to restore elbow extension, as well as hand pinch, grasp, and release. Patients who have defined goals, actively participate in therapy, and understand expected outcomes, appear to have the highest satisfaction following tendon transfer procedures. PMID:27387082

  3. Mechanical characteristics of native tendon slices for tissue engineering scaffold.

    PubMed

    Qin, Ting-Wu; Chen, Qingshan; Sun, Yu-Long; Steinmann, Scott P; Amadio, Peter C; An, Kai-Nan; Zhao, Chunfeng

    2012-04-01

    The purpose of this study was to characterize the mechanical behavior of tendon slices with different thicknesses. Tendon slices of 100, 200, 300, 400, and 500 μm thickness were mechanically tested. The 300 μm slices were further tested for strength and modulus after 21,000-cycle fatigue testing under different applied strain levels (0, 1, 3, 5, 8, 10, and 12%). The tendon slice structure, morphology, and viability of bone marrow stromal cells (BMSCs) seeded onto the slices were also examined with histology, scanning electron microscopy, and vital cell labeling, respectively. Tendon slices 300 μm or more in thickness had similar ultimate tensile strength and Young's modulus to the intact tendon bundle. A strain of 5% or less did not cause any structural damage, nor did it change the mechanical properties of a 300 μm-thick tendon slice after 21,000-cycle fatigue testing. BMSCs were viable between and on the tendon slices after 2 weeks in tissue culture. This study demonstrated that, if tendon slices are used as a scaffold for tendon tissue engineering, slices 300 μm or more in thickness would be preferable from a mechanical strength point of view. If mechanical stimulation is performed for seeded-cell preparations, 5% strain or less would be appropriate.

  4. Enhancement of tendon-to-bone healing after anterior cruciate ligament reconstruction using bone marrow-derived mesenchymal stem cells genetically modified with bFGF/BMP2

    PubMed Central

    Chen, Biao; Li, Bin; Qi, Yong-Jian; Ni, Qu-Bo; Pan, Zheng-Qi; Wang, Hui; Chen, Liao-Bin

    2016-01-01

    Many strategies, including various growth factors and gene transfer, have been used to augment healing after anterior cruciate ligament (ACL) reconstruction. The biological environment regulated by the growth factors during the stage of tendon-bone healing was considered important in controlling the integrating process. The purpose of this study was to evaluate the effects of bone marrow-derived mesenchymal stem cells (BMSCs) genetically modified with bone morphogenetic protein 2 (BMP2) and basic fibroblast growth factor (bFGF) on healing after ACL reconstruction. BMSCs were infected with an adenoviral vector encoding BMP2 (AdBMP2) or bFGF (AdbFGF). Then, the infected BMSCs were surgically implanted into the tendon-bone interface. At 12 weeks postoperatively, the formation of abundant cartilage-like cells, smaller tibial bone tunnel and significantly higher ultimate load and stiffness levels, through histological analysis, micro-computed tomography and biomechanical testing, were observed. In addition, the AdBMP2-plus-AdbFGF group had the smallest bone tunnel and the best mechanical properties among all the groups. The addition of BMP2 or bFGF by gene transfer resulted in better cellularity, new bone formation and higher mechanical property, which contributed to the healing process after ACL reconstruction. Furthermore, the co-application of these two genes was more powerful and efficient than either single gene therapy. PMID:27173013

  5. Enhancement of tendon-to-bone healing after anterior cruciate ligament reconstruction using bone marrow-derived mesenchymal stem cells genetically modified with bFGF/BMP2.

    PubMed

    Chen, Biao; Li, Bin; Qi, Yong-Jian; Ni, Qu-Bo; Pan, Zheng-Qi; Wang, Hui; Chen, Liao-Bin

    2016-01-01

    Many strategies, including various growth factors and gene transfer, have been used to augment healing after anterior cruciate ligament (ACL) reconstruction. The biological environment regulated by the growth factors during the stage of tendon-bone healing was considered important in controlling the integrating process. The purpose of this study was to evaluate the effects of bone marrow-derived mesenchymal stem cells (BMSCs) genetically modified with bone morphogenetic protein 2 (BMP2) and basic fibroblast growth factor (bFGF) on healing after ACL reconstruction. BMSCs were infected with an adenoviral vector encoding BMP2 (AdBMP2) or bFGF (AdbFGF). Then, the infected BMSCs were surgically implanted into the tendon-bone interface. At 12 weeks postoperatively, the formation of abundant cartilage-like cells, smaller tibial bone tunnel and significantly higher ultimate load and stiffness levels, through histological analysis, micro-computed tomography and biomechanical testing, were observed. In addition, the AdBMP2-plus-AdbFGF group had the smallest bone tunnel and the best mechanical properties among all the groups. The addition of BMP2 or bFGF by gene transfer resulted in better cellularity, new bone formation and higher mechanical property, which contributed to the healing process after ACL reconstruction. Furthermore, the co-application of these two genes was more powerful and efficient than either single gene therapy. PMID:27173013

  6. Role of JAK/STAT signaling in neuroepithelial stem cell maintenance and proliferation in the Drosophila optic lobe

    SciTech Connect

    Wang, Wei; Li, Yonggang; Zhou, Liya; Yue, Haitao; Luo, Hong

    2011-07-15

    Highlights: {yields} JAK/STAT activity is graded in the Drosophila optic lobe neuroepithelium. {yields} Inactivation of JAK signaling causes disintegration of the optic lobe neuroepithelium and depletion of the neuroepithelial stem cells. {yields} JAK pathway overactivation promotes neuroepithelial overgrowth. {yields} Notch signaling acts downstream of JAK/STAT to promote neuroepithelial growth and expansion. -- Abstract: During Drosophila optic lobe development, proliferation and differentiation must be tightly modulated to reach its normal size for proper functioning. The JAK/STAT pathway plays pleiotropic roles in Drosophila development and in the larval brain, has been shown to inhibit medulla neuroblast formation. In this study, we find that JAK/STAT activity is required for the maintenance and proliferation of the neuroepithelial stem cells in the optic lobe. In loss-of-function JAK/STAT mutant brains, the neuroepithelial cells lose epithelial cell characters and differentiate prematurely while ectopic activation of this pathway is sufficient to induce neuroepithelial overgrowth in the optic lobe. We further show that Notch signaling acts downstream of JAK/STAT to control the maintenance and growth of the optic lobe neuroepithelium. Thus, in addition to its role in suppression of neuroblast formation, the JAK/STAT pathway is necessary and sufficient for optic lobe neuroepithelial growth.

  7. Metformin inhibits age-related centrosome amplification in Drosophila midgut stem cells through AKT/TOR pathway.

    PubMed

    Na, Hyun-Jin; Park, Joung-Sun; Pyo, Jung-Hoon; Jeon, Ho-Jun; Kim, Young-Shin; Arking, Robert; Yoo, Mi-Ae

    2015-07-01

    We delineated the mechanism regulating the inhibition of centrosome amplification by metformin in Drosophila intestinal stem cells (ISCs). Age-related changes in tissue-resident stem cells may be closely associated with tissue aging and age-related diseases, such as cancer. Centrosome amplification is a hallmark of cancers. Our recent work showed that Drosophila ISCs are an excellent model for stem cell studies evaluating age-related increase in centrosome amplification. Here, we showed that metformin, a recognized anti-cancer drug, inhibits age- and oxidative stress-induced centrosome amplification in ISCs. Furthermore, we revealed that this effect is mediated via down-regulation of AKT/target of rapamycin (TOR) activity, suggesting that metformin prevents centrosome amplification by inhibiting the TOR signaling pathway. Additionally, AKT/TOR signaling hyperactivation and metformin treatment indicated a strong correlation between DNA damage accumulation and centrosome amplification in ISCs, suggesting that DNA damage might mediate centrosome amplification. Our study reveals the beneficial and protective effects of metformin on centrosome amplification via AKT/TOR signaling modulation. We identified a new target for the inhibition of age- and oxidative stress-induced centrosome amplification. We propose that the Drosophila ISCs may be an excellent model system for in vivo studies evaluating the effects of anti-cancer drugs on tissue-resident stem cell aging.

  8. The tibialis posterior tendon.

    PubMed

    Lhoste-Trouilloud, A

    2012-02-01

    The tibialis posterior tendon is the largest and anteriormost tendon in the medial ankle. It produces plantar flexion and supination of the ankle and stabilizes the plantar vault. Sonographic assessment of this tendon is done with high-frequency, linear-array transducers; an optimal examination requires transverse retromalleolar, longitudinal retromalleolar, and distal longitudinal scans, as well as dynamic studies. Disorders of the posterior tibial tendon include chronic tendinopathy with progressive rupture, tenosynovitis, acute rupture, dislocation and instability, enthesopathies. The most common lesion is a progressive "chewing gum" lesion that develops in a setting of chronic tendinopathy; it is usually seen in overweight women over 50 years of age with valgus flat feet. Medial ankle pain must also be carefully investigated, and the presence of instability assessed with dynamic maneuvers (forced inversion, or dorsiflexion) of the foot. Sonography plays an important role in the investigation of disorders involving the posterior tibial tendon.

  9. Functions of maternal mRNA as a cytoplasmic factor responsible for pole cell formation in Drosophila embryos

    SciTech Connect

    Togashi, S.; Kobayashi, S.; Okada, M.

    1986-12-01

    Injection of mRNA extracted from Drosophila cleavage embryos or mature oocytes restored pole cell-forming ability to embryos that had been deprived of this ability by uv irradiation. However, mRNA extracted from blastoderms did not show the restoration activity. Pole cells thus formed in uv-irradiated embryos bear similarities to normal pole cells both in their morphology and their ability to migrate to the gonadal rudiments. But this mRNA does not appear to be capable of rescuing uv-induced sterility, or inducing pole cells in the anterior polar region.

  10. Loss of Drosophila pseudouridine synthase triggers apoptosis-induced proliferation and promotes cell-nonautonomous EMT

    PubMed Central

    Vicidomini, R; Di Giovanni, A; Petrizzo, A; Iannucci, L F; Benvenuto, G; Nagel, A C; Preiss, A; Furia, M

    2015-01-01

    Many developing tissues display regenerative capability that allows them to compensate cell loss and preserve tissue homeostasis. Because of their remarkable regenerative capability, Drosophila wing discs are extensively used for the study of regenerative phenomena. We thus used the developing wing to investigate the role played in tissue homeostasis by the evolutionarily conserved eukaryotic H/ACA small nucleolar ribonucleoprotein pseudouridine synthase. Here we show that localized depletion of this enzyme can act as an endogenous stimulus capable of triggering apoptosis-induced proliferation, and that context-dependent effects are elicited in different sub-populations of the silenced cells. In fact, some cells undergo apoptosis, whereas those surrounding the apoptotic foci, although identically depleted, overproliferate. This overproliferation correlates with ectopic induction of the Wg and JAK-STAT (Janus kinase-signal transducer and activator of transcription) mitogenic pathways. Expression of a p35 transgene, which blocks the complete execution of the death program and generates the so-called ‘undead cells', amplifies the proliferative response. Pseudouridine synthase depletion also causes loss of apicobasal polarity, disruption of adherens cell junctions and ectopic induction of JNK (c-Jun N-terminal kinase) and Mmp1 (matrix metalloproteinase-1) activity, leading to a significant epithelial reorganization. Unexpectedly, cell