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FGF1 Mediates Overnutrition-Induced Compensatory β-Cell Differentiation.
Li M, Page-McCaw P, Chen W
(2016) Diabetes 65: 96-109
MeSH Terms: Animals, Animals, Genetically Modified, Cell Differentiation, Cell Line, Tumor, Endoplasmic Reticulum Stress, Fibroblast Growth Factor 1, Flow Cytometry, Humans, Insulin-Secreting Cells, Overnutrition, RNA, Messenger, Rats, Reverse Transcriptase Polymerase Chain Reaction, Signal Transduction, Zebrafish, Zebrafish Proteins
Show Abstract · Added February 15, 2016
Increased insulin demand resulting from insulin resistance and/or overnutrition induces a compensatory increase in β-cell mass. The physiological factors responsible for the compensation have not been fully characterized. In zebrafish, overnutrition rapidly induces compensatory β-cell differentiation through triggering the release of a paracrine signal from persistently activated β-cells. We identified Fgf1 signaling as a key component of the overnutrition-induced β-cell differentiation signal in a small molecule screen. Fgf1 was confirmed as the overnutrition-induced β-cell differentiation signal, as inactivation of fgf1 abolished the compensatory β-cell differentiation. Furthermore, expression of human FGF1 solely in β-cells in fgf1(-/-) animals rescued the compensatory response, indicating that β-cells can be the source of FGF1. Additionally, constitutive secretion of FGF1 with an exogenous signal peptide increased β-cell number in the absence of overnutrition. These results demonstrate that fgf1 is necessary and FGF1 expression in β-cells is sufficient for the compensatory β-cell differentiation. We further show that FGF1 is secreted during prolonged activation of cultured mammalian β-cells and that endoplasmic reticulum stress acts upstream of FGF1 release. Thus, the recently discovered antidiabetes function of FGF1 may act partially through increasing β-cell differentiation.
© 2016 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.
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16 MeSH Terms
Generation of Targeted Mutations in Zebrafish Using the CRISPR/Cas System.
Yin L, Jao LE, Chen W
(2015) Methods Mol Biol 1332: 205-17
MeSH Terms: Animals, Animals, Genetically Modified, CRISPR-Cas Systems, Gene Targeting, Mutagenesis, Site-Directed, Mutation, RNA Editing, RNA, Guide, RNA, Messenger, Zebrafish
Show Abstract · Added February 15, 2016
Several strategies have been developed to generate targeted gene disruptions in zebrafish.Here we developed a simple targeted gene inactivation strategy in zebrafish using a clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system. By injecting two simple in vitro-synthesized components [Cas9 mRNA and single guide (sgRNA)] into one-cell-stage embryos, mutations of the target gene could be efficiently generated. We used a codon-optimized version of Cas9 to improve its translation efficiency in zebrafish. In addition, we designed a cloning-free strategy to facilitate the synthesis of sgRNA. The system allows biallelic inactivation of multiple genes simultaneously by co-injecting a mix of sgRNAs with a single Cas9 construct. This flexible strategy of gene inactivation provides an efficient way to interrogate gene functions and genetic interactions in zebrafish.
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10 MeSH Terms
A conserved role of αA-crystallin in the development of the zebrafish embryonic lens.
Zou P, Wu SY, Koteiche HA, Mishra S, Levic DS, Knapik E, Chen W, Mchaourab HS
(2015) Exp Eye Res 138: 104-13
MeSH Terms: Animals, Animals, Genetically Modified, Blotting, Western, Electrophoresis, Polyacrylamide Gel, Embryo, Nonmammalian, Gene Expression Regulation, Developmental, Gene Knockout Techniques, Lens, Crystalline, Real-Time Polymerase Chain Reaction, Zebrafish, alpha-Crystallin A Chain
Show Abstract · Added July 23, 2015
αA- and αB-crystallins are small heat shock proteins that bind thermodynamically destabilized proteins thereby inhibiting their aggregation. Highly expressed in the mammalian lens, the α-crystallins have been postulated to play a critical role in the maintenance of lens optical properties by sequestering age-damaged proteins prone to aggregation as well as through a multitude of roles in lens epithelial cells. Here, we have examined the role of α-crystallins in the development of the vertebrate zebrafish lens. For this purpose, we have carried out morpholino-mediated knockdown of αA-, αBa- and αBb-crystallin and characterized the gross morphology of the lens. We observed lens abnormalities, including increased reflectance intensity, as a consequence of the interference with expression of these proteins. These abnormalities were less frequent in transgenic zebrafish embryos expressing rat αA-crystallin suggesting a specific role of α-crystallins in embryonic lens development. To extend and confirm these findings, we generated an αA-crystallin knockout zebrafish line. A more consistent and severe lens phenotype was evident in maternal/zygotic αA-crystallin mutants compared to those observed by morpholino knockdown. The penetrance of the lens phenotype was reduced by transgenic expression of rat αA-crystallin and its severity was attenuated by maternal αA-crystallin expression. These findings demonstrate that the role of α-crystallins in lens development is conserved from mammals to zebrafish and set the stage for using the embryonic lens as a model system to test mechanistic aspects of α-crystallin chaperone activity and to develop strategies to fine-tune protein-protein interactions in aging and cataracts.
Copyright © 2015 Elsevier Ltd. All rights reserved.
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11 MeSH Terms
Glial Expression of the Caenorhabditis elegans Gene swip-10 Supports Glutamate Dependent Control of Extrasynaptic Dopamine Signaling.
Hardaway JA, Sturgeon SM, Snarrenberg CL, Li Z, Xu XZ, Bermingham DP, Odiase P, Spencer WC, Miller DM, Carvelli L, Hardie SL, Blakely RD
(2015) J Neurosci 35: 9409-23
MeSH Terms: Animals, Animals, Genetically Modified, Caenorhabditis elegans, Caenorhabditis elegans Proteins, Dopamine, Dopamine Plasma Membrane Transport Proteins, Glutamic Acid, Microscopy, Confocal, Motor Activity, Nerve Tissue Proteins, Neuroglia, Neurons, Reverse Transcriptase Polymerase Chain Reaction, Signal Transduction
Show Abstract · Added September 28, 2015
Glial cells play a critical role in shaping neuronal development, structure, and function. In a screen for Caenorhabditis elegans mutants that display dopamine (DA)-dependent, Swimming-Induced Paralysis (Swip), we identified a novel gene, swip-10, the expression of which in glia is required to support normal swimming behavior. swip-10 mutants display reduced locomotion rates on plates, consistent with our findings of elevated rates of presynaptic DA vesicle fusion using fluorescence recovery after photobleaching. In addition, swip-10 mutants exhibit elevated DA neuron excitability upon contact with food, as detected by in vivo Ca(2+) monitoring, that can be rescued by glial expression of swip-10. Mammalian glia exert powerful control of neuronal excitability via transporter-dependent buffering of extracellular glutamate (Glu). Consistent with this idea, swip-10 paralysis was blunted in mutants deficient in either vesicular Glu release or Glu receptor expression and could be phenocopied by mutations that disrupt the function of plasma membrane Glu transporters, most noticeably glt-1, the ortholog of mammalian astrocytic GLT1 (EAAT2). swip-10 encodes a protein containing a highly conserved metallo-β-lactamase domain, within which our swip-10 mutations are located and where engineered mutations disrupt Swip rescue. Sequence alignments identify the CNS-expressed gene MBLAC1 as a putative mammalian ortholog. Together, our studies provide evidence of a novel pathway in glial cells regulated by swip-10 that limits DA neuron excitability, DA secretion, and DA-dependent behaviors through modulation of Glu signaling.
Copyright © 2015 the authors 0270-6474/15/359409-15$15.00/0.
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14 MeSH Terms
Multiplex Conditional Mutagenesis Using Transgenic Expression of Cas9 and sgRNAs.
Yin L, Maddison LA, Li M, Kara N, LaFave MC, Varshney GK, Burgess SM, Patton JG, Chen W
(2015) Genetics 200: 431-41
MeSH Terms: Animals, Animals, Genetically Modified, CRISPR-Cas Systems, Gene Expression, Gene Order, Gene Silencing, Gene Targeting, Genetic Vectors, Glucose, Hypopigmentation, Mutagenesis, Phenotype, RNA, Guide, Transgenes, Zebrafish
Show Abstract · Added July 23, 2015
Determining the mechanism of gene function is greatly enhanced using conditional mutagenesis. However, generating engineered conditional alleles is inefficient and has only been widely used in mice. Importantly, multiplex conditional mutagenesis requires extensive breeding. Here we demonstrate a system for one-generation multiplex conditional mutagenesis in zebrafish (Danio rerio) using transgenic expression of both cas9 and multiple single guide RNAs (sgRNAs). We describe five distinct zebrafish U6 promoters for sgRNA expression and demonstrate efficient multiplex biallelic inactivation of tyrosinase and insulin receptor a and b, resulting in defects in pigmentation and glucose homeostasis. Furthermore, we demonstrate temporal and tissue-specific mutagenesis using transgenic expression of Cas9. Heat-shock-inducible expression of cas9 allows temporal control of tyr mutagenesis. Liver-specific expression of cas9 disrupts insulin receptor a and b, causing fasting hypoglycemia and postprandial hyperglycemia. We also show that delivery of sgRNAs targeting ascl1a into the eye leads to impaired damage-induced photoreceptor regeneration. Our findings suggest that CRISPR/Cas9-based conditional mutagenesis in zebrafish is not only feasible but rapid and straightforward.
Copyright © 2015 by the Genetics Society of America.
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15 MeSH Terms
Circadian modulation of dopamine levels and dopaminergic neuron development contributes to attention deficiency and hyperactive behavior.
Huang J, Zhong Z, Wang M, Chen X, Tan Y, Zhang S, He W, He X, Huang G, Lu H, Wu P, Che Y, Yan YL, Postlethwait JH, Chen W, Wang H
(2015) J Neurosci 35: 2572-87
MeSH Terms: Animals, Animals, Genetically Modified, Attention Deficit Disorder with Hyperactivity, Avoidance Learning, Behavior, Animal, Circadian Rhythm, Dopamine, Dopaminergic Neurons, Impulsive Behavior, Larva, Mice, Motor Activity, NIH 3T3 Cells, Period Circadian Proteins, Tyrosine 3-Monooxygenase, Zebrafish, Zebrafish Proteins
Show Abstract · Added February 20, 2015
Attention-deficit/hyperactivity disorder (ADHD) is one of the most prevalent psychiatric disorders in children and adults. While ADHD patients often display circadian abnormalities, the underlying mechanisms are unclear. Here we found that the zebrafish mutant for the circadian gene period1b (per1b) displays hyperactive, impulsive-like, and attention deficit-like behaviors and low levels of dopamine, reminiscent of human ADHD patients. We found that the circadian clock directly regulates dopamine-related genes monoamine oxidase and dopamine β hydroxylase, and acts via genes important for the development or maintenance of dopaminergic neurons to regulate their number and organization in the ventral diencephalic posterior tuberculum. We then found that Per1 knock-out mice also display ADHD-like symptoms and reduced levels of dopamine, thereby showing highly conserved roles of the circadian clock in ADHD. Our studies demonstrate that disruption of a circadian clock gene elicits ADHD-like syndrome. The circadian model for attention deficiency and hyperactive behavior sheds light on ADHD pathogenesis and opens avenues for exploring novel targets for diagnosis and therapy for this common psychiatric disorder.
Copyright © 2015 the authors 0270-6474/15/352572-16$15.00/0.
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17 MeSH Terms
Skeletal muscle insulin resistance in zebrafish induces alterations in β-cell number and glucose tolerance in an age- and diet-dependent manner.
Maddison LA, Joest KE, Kammeyer RM, Chen W
(2015) Am J Physiol Endocrinol Metab 308: E662-9
MeSH Terms: Aging, Animals, Animals, Genetically Modified, Biological Transport, Cell Count, Disease Progression, Glucose, Glucose Intolerance, Green Fluorescent Proteins, Hyperglycemia, Insulin, Insulin Resistance, Insulin-Like Growth Factor I, Insulin-Secreting Cells, Luminescent Proteins, Muscle, Skeletal, Overnutrition, Receptor, IGF Type 1, Recombinant Fusion Proteins, Zebrafish, Zebrafish Proteins
Show Abstract · Added February 12, 2015
Insulin resistance creates an environment that promotes β-cell failure and development of diabetes. Understanding the events that lead from insulin resistance to diabetes is necessary for development of effective preventional and interventional strategies, and model systems that reflect the pathophysiology of disease progression are an important component toward this end. We have confirmed that insulin enhances glucose uptake in zebrafish skeletal muscle and have developed a zebrafish model of skeletal muscle insulin resistance using a dominant-negative IGF-IR. These zebrafish exhibit blunted insulin signaling and glucose uptake in the skeletal muscle, confirming insulin resistance. In young animals, we observed an increase in the number of β-cells and normal glucose tolerance that was indicative of compensation for insulin resistance. In older animals, the β-cell mass was reduced to that of control with the appearance of impaired glucose clearance but no elevation in fasting blood glucose. Combined with overnutrition, the insulin-resistant animals have an increased fasting blood glucose compared with the control animals, demonstrating that the β-cells in the insulin-resistant fish are in a vulnerable state. The relatively slow progression from insulin resistance to glucose intolerance in this model system has the potential in the future to test cooperating genes or metabolic conditions that may accelerate the development of diabetes and provide new therapeutic targets.
Copyright © 2015 the American Physiological Society.
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21 MeSH Terms
Oncogenic KRAS promotes malignant brain tumors in zebrafish.
Ju B, Chen W, Orr BA, Spitsbergen JM, Jia S, Eden CJ, Henson HE, Taylor MR
(2015) Mol Cancer 14: 18
MeSH Terms: Animals, Animals, Genetically Modified, Brain, Brain Neoplasms, Cell Transformation, Neoplastic, Disease Models, Animal, Drug Screening Assays, Antitumor, Gene Expression, Humans, Immunohistochemistry, Keratin-5, Nerve Tissue Proteins, Promoter Regions, Genetic, Proto-Oncogene Proteins, Proto-Oncogene Proteins p21(ras), Signal Transduction, TOR Serine-Threonine Kinases, Transgenes, Zebrafish, ras Proteins
Show Abstract · Added February 12, 2015
BACKGROUND - Zebrafish have been used as a vertebrate model to study human cancers such as melanoma, rhabdomyosarcoma, liver cancer, and leukemia as well as for high-throughput screening of small molecules of therapeutic value. However, they are just emerging as a model for human brain tumors, which are among the most devastating and difficult to treat. In this study, we evaluated zebrafish as a brain tumor model by overexpressing a human version of oncogenic KRAS (KRAS(G12V)).
METHODS - Using zebrafish cytokeratin 5 (krt5) and glial fibrillary acidic protein (gfap) gene promoters, we activated Ras signaling in the zebrafish central nervous system (CNS) through transient and stable transgenic overexpression. Immunohistochemical analyses were performed to identify activated pathways in the resulting brain tumors. The effects of the MEK inhibitor U0126 on oncogenic KRAS were evaluated.
RESULTS - We demonstrated that transient transgenic expression of KRAS(G12V) in putative neural stem and/or progenitor cells induced brain tumorigenesis. When expressed under the control of the krt5 gene promoter, KRAS(G12V) induced brain tumors in ventricular zones (VZ) at low frequency. The majority of other tumors were composed mostly of spindle and epithelioid cells, reminiscent of malignant peripheral nerve sheath tumors (MPNSTs). In contrast, when expressed under the control of the gfap gene promoter, KRAS(G12V) induced brain tumors in both VZs and brain parenchyma at higher frequency. Immunohistochemical analyses indicated prominent activation of the canonical RAS-RAF-ERK pathway, variable activation of the mTOR pathway, but no activation of the PI3K-AKT pathway. In a krt5-derived stable and inducible transgenic line, expression of oncogenic KRAS resulted in skin hyperplasia, and the MEK inhibitor U0126 effectively suppressed this pro-proliferative effects. In a gfap-derived stable and inducible line, expression of oncogenic KRAS led to significantly increased mitotic index in the spinal cord.
CONCLUSIONS - Our studies demonstrate that zebrafish could be explored to study cellular origins and molecular mechanisms of brain tumorigenesis and could also be used as a platform for studying human oncogene function and for discovering oncogenic RAS inhibitors.
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20 MeSH Terms
High content screening for modulators of cardiovascular or global developmental pathways in zebrafish.
Williams CH, Hong CC
(2015) Methods Mol Biol 1263: 167-74
MeSH Terms: Animals, Animals, Genetically Modified, Cardiovascular System, Disease Models, Animal, Drug Evaluation, Preclinical, High-Throughput Screening Assays, Humans, Organogenesis, Phenotype, Signal Transduction, Small Molecule Libraries, Zebrafish
Show Abstract · Added February 3, 2015
Major developmental pathways play critical roles in wide array of human pathologies. Chemical genomic screening allows for the discovery of novel tools not only to target known pathway interactors but also to discover new, chemically tractable targets for known pathways. The zebrafish has emerged as a useful model for developmental biology and has been well characterized. The zebrafish represents a hardy conglomerate of totipotent cells that are massively and simultaneously assessing all significant pathways in parallel in an endogenous context. This represents a gold standard for biological assays, chemically targeting select pathways without causing pleiotropic effects. Here, we describe methods used to develop high content screening assays implementing transgenic zebrafish to assess phenotypic changes that have identified several classes of novel compounds that effect developmental pathways.
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12 MeSH Terms
Glycolytic enzymes localize to ribonucleoprotein granules in Drosophila germ cells, bind Tudor and protect from transposable elements.
Gao M, Thomson TC, Creed TM, Tu S, Loganathan SN, Jackson CA, McCluskey P, Lin Y, Collier SE, Weng Z, Lasko P, Ohi MD, Arkov AL
(2015) EMBO Rep 16: 379-86
MeSH Terms: Animals, Animals, Genetically Modified, Base Sequence, Cytoplasmic Granules, DNA Transposable Elements, Drosophila, Drosophila Proteins, Germ Cells, Glycolysis, Membrane Transport Proteins, MicroRNAs, Molecular Sequence Data, Ribonucleoproteins, Sequence Analysis, DNA
Show Abstract · Added January 5, 2016
Germ cells give rise to all cell lineages in the next-generation and are responsible for the continuity of life. In a variety of organisms, germ cells and stem cells contain large ribonucleoprotein granules. Although these particles were discovered more than 100 years ago, their assembly and functions are not well understood. Here we report that glycolytic enzymes are components of these granules in Drosophila germ cells and both their mRNAs and the enzymes themselves are enriched in germ cells. We show that these enzymes are specifically required for germ cell development and that they protect their genomes from transposable elements, providing the first link between metabolism and transposon silencing. We further demonstrate that in the granules, glycolytic enzymes associate with the evolutionarily conserved Tudor protein. Our biochemical and single-particle EM structural analyses of purified Tudor show a flexible molecule and suggest a mechanism for the recruitment of glycolytic enzymes to the granules. Our data indicate that germ cells, similarly to stem cells and tumor cells, might prefer to produce energy through the glycolytic pathway, thus linking a particular metabolism to pluripotency.
© 2015 The Authors.
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14 MeSH Terms