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Articles by Halyna R. Shcherbata in JoVE
JoVE General
Paraffininbäddade och frysta snitt av Drosophila Vuxen Muskler
Gene Expression and Signaling Research Group, Max Planck Institute for Biophysical Chemistry
Identifiering av mekanismerna bakom muskelskador är avgörande. Här presenterar vi de histologiska teknik för att förbereda paraffininbäddade och frysta delar av Drosophila bröstkorg muskler. Detta möjliggör analys av muskel morfologi och lokalisering av protein och andra muskel-komponenter cell.
Other articles by Halyna R. Shcherbata on PubMed
Notch-dependent Fizzy-related/Hec1/Cdh1 Expression is Required for the Mitotic-to-endocycle Transition in Drosophila Follicle Cells
During Drosophila oogenesis, Notch function regulates the transition from mitotic cell cycle to endocycle in follicle cells at stage 6. Loss of either Notch function or its ligand Delta (Dl) disrupts the normal transition; this disruption causes mitotic cycling to continue and leads to an overproliferation phenotype. In this context, the only known cell cycle component that responds to the Notch pathway is String/Cdc25 (Stg), a G2/M cell cycle regulator. We found that prolonged expression of string is not sufficient to keep cells efficiently in mitotic cell cycle past stage 6, suggesting that Notch also regulates other cell cycle components in the transition. By using an expression screen, we found such a component: Fizzy-related/Hec1/Cdh1 (Fzr), a WD40 repeat protein. Fzr regulates the anaphase-promoting complex/cyclosome (APC/C) and is expressed at the mitotic-to-endocycle transition in a Notch-dependent manner. Mutant clones of Fzr revealed that Fzr is dispensable for mitosis but essential for endocycles. Unlike in Notch clones, in Fzr mutant cells mitotic markers are absent past stage 6. Only a combined reduction of Fzr and ectopic Stg expression prolongs mitotic cycles in follicle cells, suggesting that these two cell cycle regulators, Fzr and Stg, are important mediators of the Notch pathway in the mitotic-to-endocycle transition.
The Mitotic-to-endocycle Switch in Drosophila Follicle Cells is Executed by Notch-dependent Regulation of G1/S, G2/M and M/G1 Cell-cycle Transitions
The Notch signaling pathway controls the follicle cell mitotic-to-endocycle transition in Drosophila oogenesis by stopping the mitotic cycle and promoting the endocycle. To understand how the Notch pathway coordinates this process, we have identified and performed a functional analysis of genes whose transcription is responsive to the Notch pathway at this transition. These genes include the G2/M regulator Cdc25 phosphatase, String; a regulator of the APC ubiquitination complex Hec/CdhFzr and an inhibitor of the CyclinE/CDK complex, Dacapo. Notch activity leads to downregulation of String and Dacapo, and activation of Fzr. All three genes are independently responsive to Notch. In addition, CdhFzr, an essential gene for endocycles, is sufficient to stop mitotic cycle and promote precocious endocycles when expressed prematurely during mitotic stages. In contrast, overexpression of the growth controller Myc does not induce premature endocycles but accelerates the kinetics of normal endocycles. We also show that Archipelago (Ago), a SCF-regulator is dispensable for mitosis, but crucial for endocycle progression in follicle epithelium. The results support a model in which Notch activity executes the mitotic-to-endocycle switch by regulating all three major cell cycle transitions. Repression of String blocks the M-phase, activation of Fzr allows G1 progression and repression of Dacapo assures entry into the S-phase. This study provides a comprehensive picture of the logic that external signaling pathways may use to control cell cycle transitions by the coordinated regulation of the cell cycle.
Stem Cells Signal to the Niche Through the Notch Pathway in the Drosophila Ovary
Stem cells are maintained and retain their capacity to continue dividing because of the influence of a niche. Although niches are important to maintain "stemness" in a wide variety of tissues, control of these niches is poorly understood. The Drosophila germline stem cells (GSCs) reside in a somatic cell niche. We show that Notch activation can induce the expression of niche-cell markers even in an adult fly; overexpression of Delta in the germline, or activated Notch in the somatic cells, results in extra niche cells, up to 10-fold over the normal number. In turn, these ectopic niche cells induce ectopic GSCs. Conversely, when GCSs do not produce functional Notch ligands, Delta and Serrate, the TGF-beta pathway is not activated in the GSCs, and they differentiate and subsequently leave the niche. Importantly, clonal analysis reveals that the receiving end of the Notch pathway is required in the somatic cells. These data show that a feedback loop exists between the stem cells and niche cells. Demonstration that stem cells can contribute to niche function has far-reaching consequences for stem cell therapies and may provide insight into how cancer can spread throughout an organism via populations of cancer stem cells.
Dissecting Muscle and Neuronal Disorders in a Drosophila Model of Muscular Dystrophy
Perturbation in the Dystroglycan (Dg)-Dystrophin (Dys) complex results in muscular dystrophies and brain abnormalities in human. Here we report that Drosophila is an excellent genetically tractable model to study muscular dystrophies and neuronal abnormalities caused by defects in this complex. Using a fluorescence polarization assay, we show a high conservation in Dg-Dys interaction between human and Drosophila. Genetic and RNAi-induced perturbations of Dg and Dys in Drosophila cause cell polarity and muscular dystrophy phenotypes: decreased mobility, age-dependent muscle degeneration and defective photoreceptor path-finding. Dg and Dys are required in targeting glial cells and neurons for correct neuronal migration. Importantly, we now report that Dg interacts with insulin receptor and Nck/Dock SH2/SH3-adaptor molecule in photoreceptor path-finding. This is the first demonstration of a genetic interaction between Dg and InR.
A Putative Src Homology 3 Domain Binding Motif but Not the C-terminal Dystrophin WW Domain Binding Motif is Required for Dystroglycan Function in Cellular Polarity in Drosophila
The conserved dystroglycan-dystrophin (Dg.Dys) complex connects the extracellular matrix to the cytoskeleton. In humans as well as Drosophila, perturbation of this complex results in muscular dystrophies and brain malformations and in some cases cellular polarity defects. However, the regulation of the Dg.Dys complex is poorly understood in any cell type. We now find that in loss-of-function and overexpression studies more than half (34 residues) of the Dg proline-rich conserved C-terminal regions can be truncated without significantly compromising its function in regulating cellular polarity in Drosophila. Notably, the truncation eliminates the WW domain binding motif at the very C terminus of the protein thought to mediate interactions with dystrophin, suggesting that a second, internal WW binding motif can also mediate this interaction. We confirm this hypothesis by using a sensitive fluorescence polarization assay to show that both WW domain binding sites of Dg bind to Dys in humans (K(d) = 7.6 and 81 microM, respectively) and Drosophila (K(d) = 16 and 46 microM, respectively). In contrast to the large deletion mentioned above, a single proline to an alanine point mutation within a predicted Src homology 3 domain (SH3) binding site abolishes Dg function in cellular polarity. This suggests that an SH3-containing protein, which has yet to be identified, functionally interacts with Dg.
Stage-specific Differences in the Requirements for Germline Stem Cell Maintenance in the Drosophila Ovary
In this study, we uncover a role for microRNAs in Drosophila germline stem cell (GSC) maintenance. Disruption of Dicer-1 function in GSCs during adult life results in GSC loss. Surprisingly, however, loss of Dicer-1 during development does not result in a GSC maintenance defect, although a defect is seen if both Dicer-1 and Dicer-2 function are disrupted. Loss of the bantam microRNA mimics the Dicer-1 maintenance defect when induced in adult GSCs, suggesting that bantam plays a key role in GSC self-renewal. Mad, a component of the TGF-beta pathway, behaves similarly to Dicer-1: adult GSC maintenance requires Mad if it is lost during adult life, but not if it is lost during pupal development. Overall, these results show stage-specific differential sensitivity of GSC maintenance to certain perturbations, and suggest that there may be Dcr-2 dependent redundancy of GSC maintenance mechanisms during development that is lost in later life.
Genetic Modifier Screens Reveal New Components That Interact with the Drosophila Dystroglycan-dystrophin Complex
The Dystroglycan-Dystrophin (Dg-Dys) complex has a capacity to transmit information from the extracellular matrix to the cytoskeleton inside the cell. It is proposed that this interaction is under tight regulation; however the signaling/regulatory components of Dg-Dys complex remain elusive. Understanding the regulation of the complex is critical since defects in this complex cause muscular dystrophy in humans. To reveal new regulators of the Dg-Dys complex, we used a model organism Drosophila melanogaster and performed genetic interaction screens to identify modifiers of Dg and Dys mutants in Drosophila wing veins. These mutant screens revealed that the Dg-Dys complex interacts with genes involved in muscle function and components of Notch, TGF-beta and EGFR signaling pathways. In addition, components of pathways that are required for cellular and/or axonal migration through cytoskeletal regulation, such as Semaphorin-Plexin, Frazzled-Netrin and Slit-Robo pathways show interactions with Dys and/or Dg. These data suggest that the Dg-Dys complex and the other pathways regulating extracellular information transfer to the cytoskeletal dynamics are more intercalated than previously thought.
Dicer-1-dependent Dacapo Suppression Acts Downstream of Insulin Receptor in Regulating Cell Division of Drosophila Germline Stem Cells
It is important to understand the regulation of stem cell division because defects in this process can cause altered tissue homeostasis or cancer. The cyclin-dependent kinase inhibitor Dacapo (Dap), a p21/p27 homolog, acts downstream of the microRNA (miRNA) pathway to regulate the cell cycle in Drosophila melanogaster germline stem cells (GSCs). Tissue-extrinsic signals, including insulin, also regulate cell division of GSCs. We report that intrinsic and extrinsic regulators intersect in GSC division control; the Insulin receptor (InR) pathway regulates Dap levels through miRNAs, thereby controlling GSC division. Using GFP-dap 3'UTR sensors in vivo, we show that in GSCs the dap 3'UTR is responsive to Dicer-1, an RNA endonuclease III required for miRNA processing. Furthermore, the dap 3'UTR can be directly targeted by miR-7, miR-278 and miR-309 in luciferase assays. Consistent with this, miR-278 and miR-7 mutant GSCs are partially defective in GSC division and show abnormal cell cycle marker expression, respectively. These data suggest that the GSC cell cycle is regulated via the dap 3'UTR by multiple miRNAs. Furthermore, the GFP-dap 3'UTR sensors respond to InR but not to TGF-beta signaling, suggesting that InR signaling utilizes Dap for GSC cell cycle regulation. We further demonstrate that the miRNA-based Dap regulation may act downstream of InR signaling; Dcr-1 and Dap are required for nutrition-dependent cell cycle regulation in GSCs and reduction of dap partially rescues the cell cycle defect of InR-deficient GSCs. These data suggest that miRNA- and Dap-based cell cycle regulation in GSCs can be controlled by InR signaling.
MicroRNAs Regulate Human Embryonic Stem Cell Division
microRNAs (miRNAs) regulate numerous physiological processes such as cell division and differentiation in many tissue types including stem cells. To probe the role that miRNAs play in regulating processes relevant to embryonic stem cell biology, we used RNA interference to silence DICER and DROSHA, the two main miRNA processing enzymes. Consistent with a role for miRNAs in maintaining normal stem cell division and renewal, we found that perturbation of miRNA pathway function in human embryonic stem cells (hESCs) attenuates cell proliferation. Normal cell growth can be partially restored by introduction of the mature miRNAs miR-195 and miR-372. These miRNAs regulate two tumor suppressor genes, respectively: WEE1, which encodes a negative G2/M kinase modulator of the CycB/CDK complex and CDKN1A, which encodes p21, a CycE/CDK cyclin dependent kinase inhibitor that regulates the G1/S transition. We show that in wild-type hESCs, WEE 1 levels control the rate of hESC division, whereas p21 levels must be maintained at a low level for hESC division to proceed. These data support a model for hESC cell cycle control in which miRNAs regulate negative cell cycle modulators at two phases of the cell cycle to ensure proper replenishment of the stem cell population.
Stress and Muscular Dystrophy: a Genetic Screen for Dystroglycan and Dystrophin Interactors in Drosophila Identifies Cellular Stress Response Components
In Drosophila, like in humans, Dystrophin Glycoprotein Complex (DGC) deficiencies cause a life span shortening disease, associated with muscle dysfunction. We performed the first in vivo genetic interaction screen in ageing dystrophic muscles and identified genes that have not been shown before to have a role in the development of muscular dystrophy and interact with dystrophin and/or dystroglycan. Mutations in many of the found interacting genes cause age-dependent morphological and heat-induced physiological defects in muscles, suggesting their importance in the tissue. Majority of them is phylogenetically conserved and implicated in human disorders, mainly tumors and myopathies. Functionally they can be divided into three main categories: proteins involved in communication between muscle and neuron, and interestingly, in mechanical and cellular stress response pathways. Our data show that stress induces muscle degeneration and accelerates age-dependent muscular dystrophy. Dystrophic muscles are already compromised; and as a consequence they are less adaptive and more sensitive to energetic stress and to changes in the ambient temperature. However, only dystroglycan, but not dystrophin deficiency causes extreme myodegeneration induced by energetic stress suggesting that dystroglycan might be a component of the low-energy pathway and act as a transducer of energetic stress in normal and dystrophic muscles.
Ecdysteroids Affect Drosophila Ovarian Stem Cell Niche Formation and Early Germline Differentiation
Previously, it has been shown that in Drosophila steroid hormones are required for progression of oogenesis during late stages of egg maturation. Here, we show that ecdysteroids regulate progression through the early steps of germ cell lineage. Upon ecdysone signalling deficit germline stem cell progeny delay to switch on a differentiation programme. This differentiation impediment is associated with reduced TGF-β signalling in the germline and increased levels of cell adhesion complexes and cytoskeletal proteins in somatic escort cells. A co-activator of the ecdysone receptor, Taiman is the spatially restricted regulator of the ecdysone signalling pathway in soma. Additionally, when ecdysone signalling is perturbed during the process of somatic stem cell niche establishment enlarged functional niches able to host additional stem cells are formed.
New Dystrophin/Dystroglycan Interactors Control Neuron Behavior in Drosophila Eye
The Dystrophin Glycoprotein Complex (DGC) is a large multi-component complex that is well known for its function in muscle tissue. When the main components of the DGC, Dystrophin (Dys) and Dystroglycan (Dg) are affected cognitive impairment and mental retardation in addition to muscle degeneration can occur. Previously we performed an array of genetic screens using a Drosophila model for muscular dystrophy in order to find novel DGC interactors aiming to elucidate the signaling role(s) in which the complex is involved. Since the function of the DGC in the brain and nervous system has not been fully defined, we have here continued to analyze the DGC modifiers' function in the developing Drosophila brain and eye.
Dystrophin Orchestrates the Epigenetic Profile of Muscle Cells Via MiRNAs
Mammalian musculature is a very robust and dynamic tissue that goes through many rounds of degeneration and regeneration in an individual's lifetime. There is a biological program that maintains muscle progenitor cells that, when activated, give rise to intermediate myoblast progeny that consequently differentiate into mature muscle cells. Recent works have provided a picture of the role that microRNAs (miRNAs) play in maintaining aspects of this program. Intriguingly, a subset of these miRNAs is de-regulated in muscular dystrophies (MDs), a group of fatal inherited neuromuscular disorders that are often associated with deficiencies in the Dystrophin (Dys) complex. Apparently, transcriptional expression of many of the muscle specific genes and miRNAs is dependent on chromatin state regulated by the Dys-Syn-nNOS pathway. This puts Dystrophin at the epicenter of a highly regulated program of muscle gene expression in which miRNAs help to coordinate networking between multiple phases of muscle maintenance, degeneration, and regeneration. Therefore, understanding the role of miRNAs in physiology of normal and diseased muscle tissue could be useful for future applications in improving the MD therapies and could open new clinical perspectives.
