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1.
Zebrafish ; 13 Suppl 1: S150-2, 2016 07.
Article in English | MEDLINE | ID: mdl-27158771

ABSTRACT

The National Institutes of Health: Final Report to Office of Laboratory Animal Welfare (OLAW) on Euthanasia of Zebrafish (2009) established for the first time a policy for the developmental stage at which zebrafish would qualify for animal oversight by OLAW interpretation of Public Health Service policy. This policy established the time point based on a comparison with chicken/avian hatching. For zebrafish, this is 3 days postfertilization (dpf). This is in contrast to the traditional time established within the community as 7 dpf. There are significant implications for this policy not the least of which is the demand to account for all embryo and larvae at all stages. This narrative provides a situational context based on a synthesis of real experience with this policy. The hope is that it provides a starting point for a community conversation on the hatching time point as the appropriate established policy for the future.


Subject(s)
Animal Care Committees/legislation & jurisprudence , Animal Welfare/legislation & jurisprudence , Zebrafish , Animals , Animals, Laboratory , United States
2.
Zebrafish ; 10(2): 211-7, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23668934

ABSTRACT

In the polycystic liver diseases (PLD), genetic defects initiate the formation of cysts in the liver and kidney. In rodent models of PLD (i.e., the PCK rat and Pkd2(WS25/-) mouse), we have studied hepatorenal cystic disease and therapeutic approaches. In this study, we employed zebrafish injected with morpholinos against genes involved in the PLD, including sec63, prkcsh, and pkd1a. We calculated the liver cystic area, and based on our rodent studies, we exposed the embryos to pasireotide [1 µM] or vitamin K3 [100 µM] and assessed the endoplasmic reticulum (ER) in cholangiocytes in embryos treated with 4-phenylbutyrate (4-PBA). Our results show that (a) morpholinos against sec63, prkcsh, and pkd1a eliminate expression of the respective proteins; (b) phenotypic body changes included curved tail and the formation of hepatic cysts in zebrafish larvae; (c) exposure of embryos to pasireotide inhibited hepatic cystogenesis in the zebrafish models; and (d) exposure of embryos to 4-PBA resulted in the ER in cholangiocytes resolving from a curved to a smooth appearance. Our results suggest that the zebrafish model of PLD may provide a means to screen drugs that could inhibit hepatic cystogenesis.


Subject(s)
Cysts/drug therapy , Cysts/genetics , Disease Models, Animal , Gene Expression Regulation, Neoplastic , Liver Diseases/drug therapy , Liver Diseases/genetics , Polycystic Kidney, Autosomal Dominant/drug therapy , Polycystic Kidney, Autosomal Dominant/genetics , Zebrafish , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/therapeutic use , Calcium-Binding Proteins , Cysts/etiology , Cysts/physiopathology , DNA Helicases/genetics , DNA Helicases/metabolism , Glucosidases/genetics , Glucosidases/metabolism , Humans , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Larva/metabolism , Liver Diseases/etiology , Liver Diseases/physiopathology , Morpholinos/administration & dosage , Morpholinos/metabolism , Phenylbutyrates/administration & dosage , Phenylbutyrates/therapeutic use , Polycystic Kidney, Autosomal Dominant/etiology , Polycystic Kidney, Autosomal Dominant/physiopathology , Somatostatin/administration & dosage , Somatostatin/analogs & derivatives , Somatostatin/therapeutic use , TRPP Cation Channels/genetics , TRPP Cation Channels/metabolism , Vitamin K 3/administration & dosage , Vitamin K 3/therapeutic use
3.
J Mol Biol ; 403(4): 516-28, 2010 Nov 05.
Article in English | MEDLINE | ID: mdl-20850453

ABSTRACT

The actin binding protein α-actinin is a major component of focal adhesions found in vertebrate cells and of focal-adhesion-like structures found in the body wall muscle of the nematode Caenorhabditis elegans. To study its in vivo function in this genetic model system, we isolated a strain carrying a deletion of the single C. elegans α-actinin gene. We assessed the cytological organization of other C. elegans focal adhesion proteins and the ultrastructure of the mutant. The mutant does not have normal dense bodies, as observed by electron microscopy; however, these dense-body-like structures still contain the focal adhesion proteins integrin, talin, and vinculin, as observed by immunofluorescence microscopy. Actin is found in normal-appearing I-bands, but with abnormal accumulations near muscle cell membranes. Although swimming in water appeared grossly normal, use of automated methods for tracking the locomotion of individual worms revealed a defect in bending. We propose that the reduced motility of α-actinin null is due to abnormal dense bodies that are less able to transmit the forces generated by actin/myosin interactions.


Subject(s)
Actinin/physiology , Caenorhabditis elegans Proteins/physiology , Caenorhabditis elegans/physiology , Focal Adhesions/physiology , Actinin/antagonists & inhibitors , Actinin/genetics , Animals , Caenorhabditis elegans/genetics , Caenorhabditis elegans Proteins/antagonists & inhibitors , Caenorhabditis elegans Proteins/genetics , Focal Adhesions/genetics , Gene Deletion , Genes, Helminth , Locomotion/physiology , Microscopy, Fluorescence , Muscles/physiology , Mutation , Phenotype , RNA Interference
4.
Proc Natl Acad Sci U S A ; 106(44): 18662-7, 2009 Nov 03.
Article in English | MEDLINE | ID: mdl-19858493

ABSTRACT

Tobacco use is predicted to result in over 1 billion deaths worldwide by the end of the 21(st) century. How genetic variation contributes to the observed differential predisposition in the human population to drug dependence is unknown. The zebrafish (Danio rerio) is an emerging vertebrate model system for understanding the genetics of behavior. We developed a nicotine behavioral assay in zebrafish and applied it in a forward genetic screen using gene-breaking transposon mutagenesis. We used this method to molecularly characterize bdav/cct8 and hbog/gabbr1.2 as mutations with altered nicotine response. Each have a single human ortholog, identifying two points for potential scientific, diagnostic, and drug development for nicotine biology and cessation therapeutics. We show this insertional method generates mutant alleles that are reversible through Cre-mediated recombination, representing a conditional mutation system for the zebrafish. The combination of this reporter-tagged insertional mutagen approach and zebrafish provides a powerful platform for a rich array of questions amenable to genetic-based scientific inquiry, including the basis of behavior, epigenetics, plasticity, stress, memory, and learning.


Subject(s)
Nicotine/pharmacology , Zebrafish/genetics , Animals , Behavior, Animal/drug effects , Gene Expression Profiling , Gene Expression Regulation , Larva/drug effects , Larva/genetics , Mutagenesis, Insertional/drug effects , Mutation/genetics
5.
Cell ; 130(6): 1108-19, 2007 Sep 21.
Article in English | MEDLINE | ID: mdl-17889653

ABSTRACT

Extracellular serpins such as antithrombin and alpha1-antitrypsin are the quintessential regulators of proteolytic pathways. In contrast, the biological functions of the intracellular serpins remain obscure. We now report that the C. elegans intracellular serpin, SRP-6, exhibits a prosurvival function by blocking necrosis. Minutes after hypotonic shock, srp-6 null animals underwent a catastrophic series of events culminating in lysosomal disruption, cytoplasmic proteolysis, and death. This newly defined hypo-osmotic stress lethal (Osl) phenotype was dependent upon calpains and lysosomal cysteine peptidases, two in vitro targets of SRP-6. By protecting against both the induction of and the lethal effects from lysosomal injury, SRP-6 also blocked death induced by heat shock, oxidative stress, hypoxia, and cation channel hyperactivity. These findings suggest that multiple noxious stimuli converge upon a peptidase-driven, core stress response pathway that, in the absence of serpin regulation, triggers a lysosomal-dependent necrotic cell death routine.


Subject(s)
Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/metabolism , Lysosomes/metabolism , Serpins/metabolism , Animals , Caenorhabditis elegans/enzymology , Caenorhabditis elegans/genetics , Caenorhabditis elegans/ultrastructure , Caenorhabditis elegans Proteins/genetics , Calcium/metabolism , Calcium Channels/metabolism , Calpain/genetics , Calpain/metabolism , Cell Hypoxia , Cell Size , Cysteine Endopeptidases/genetics , Cysteine Endopeptidases/metabolism , Genotype , Hot Temperature , Lysosomes/enzymology , Lysosomes/ultrastructure , Mutation , Necrosis , Osmotic Pressure , Oxidative Stress , Phenotype , RNA Interference , RNA, Small Interfering/metabolism , Serpins/genetics , Time Factors
6.
Mol Biol Cell ; 17(7): 3021-30, 2006 Jul.
Article in English | MEDLINE | ID: mdl-16641366

ABSTRACT

Sodium-dependent neurotransmitter transporters participate in the clearance and/or recycling of neurotransmitters from synaptic clefts. The snf-11 gene in Caenorhabditis elegans encodes a protein of high similarity to mammalian GABA transporters (GATs). We show here that snf-11 encodes a functional GABA transporter; SNF-11-mediated GABA transport is Na+ and Cl- dependent, has an EC50 value of 168 microM, and is blocked by the GAT1 inhibitor SKF89976A. The SNF-11 protein is expressed in seven GABAergic neurons, several additional neurons in the head and retrovesicular ganglion, and three groups of muscle cells. Therefore, all GABAergic synapses are associated with either presynaptic or postsynaptic (or both) expression of SNF-11. Although a snf-11 null mutation has no obvious effects on GABAergic behaviors, it leads to resistance to inhibitors of acetylcholinesterase. In vivo, a snf-11 null mutation blocks GABA uptake in at least a subset of GABAergic cells; in a cell culture system, all GABA uptake is abolished by the snf-11 mutation. We conclude that GABA transport activity is not essential for normal GABAergic function in C. elegans and that the localization of SNF-11 is consistent with a GABA clearance function rather than recycling.


Subject(s)
Caenorhabditis elegans Proteins/physiology , Caenorhabditis elegans/metabolism , GABA Plasma Membrane Transport Proteins/physiology , Genes, Helminth/physiology , Synapses/metabolism , gamma-Aminobutyric Acid/metabolism , Animals , Caenorhabditis elegans/chemistry , Caenorhabditis elegans Proteins/analysis , Caenorhabditis elegans Proteins/genetics , GABA Agents/pharmacology , GABA Plasma Membrane Transport Proteins/analysis , GABA Plasma Membrane Transport Proteins/genetics , Mutation , Nipecotic Acids/pharmacology , Phenotype , Phylogeny , Sodium/metabolism , Synaptic Transmission
7.
J Mol Biol ; 332(5): 1037-46, 2003 Oct 03.
Article in English | MEDLINE | ID: mdl-14499607

ABSTRACT

Syntrophins are a family of PDZ domain-containing adaptor proteins required for receptor localization. Syntrophins are also associated with the dystrophin complex in muscles. We report here the molecular and functional characterization of the Caenorhabditis elegans gene stn-1 (F30A10.8), which encodes a syntrophin with homology to vertebrate alpha and beta-syntrophins. stn-1 is expressed in neurons and in muscles of C.elegans. stn-1 mutants resemble dystrophin (dys-1) and dystrobrevin (dyb-1) mutants: they are hyperactive, bend their heads when they move forward, tend to hypercontract, and are hypersensitive to the acetylcholinesterase inhibitor aldicarb. These phenotypes are suppressed when stn-1 is expressed under the control of a muscular promoter, indicating that they are caused by the absence of stn-1 in muscles. These results suggest that the role of syntrophin is linked to dystrophin function in C.elegans.


Subject(s)
Caenorhabditis elegans Proteins , Dystrophin-Associated Proteins , Dystrophin/physiology , Membrane Proteins/genetics , Membrane Proteins/physiology , Muscle Proteins/genetics , Muscle Proteins/physiology , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/physiology , Neuropeptides/physiology , Aldicarb/pharmacology , Animals , Caenorhabditis elegans , Calcium Channels/metabolism , Calcium-Binding Proteins , Databases as Topic , Genome , Genotype , Green Fluorescent Proteins , Humans , Luminescent Proteins/metabolism , Models, Biological , Muscles/pathology , Muscular Dystrophy, Duchenne/metabolism , Mutation , Phenotype , Phylogeny , Promoter Regions, Genetic , Protein Binding , Protein Structure, Tertiary , Sensitivity and Specificity , Two-Hybrid System Techniques
8.
Neuron ; 35(2): 307-18, 2002 Jul 18.
Article in English | MEDLINE | ID: mdl-12160748

ABSTRACT

C. elegans OSM-9 is a TRPV channel protein involved in sensory transduction and adaptation. Here, we show that distinct sensory functions arise from different combinations of OSM-9 and related OCR TRPV proteins. Both OSM-9 and OCR-2 are essential for several forms of sensory transduction, including olfaction, osmosensation, mechanosensation, and chemosensation. In neurons that express both OSM-9 and OCR-2, tagged OCR-2 and OSM-9 proteins reside in sensory cilia and promote each other's localization to cilia. In neurons that express only OSM-9, tagged OSM-9 protein resides in the cell body and acts in sensory adaptation rather than sensory transduction. Thus, alternative combinations of TRPV proteins may direct different functions in distinct subcellular locations. Animals expressing the mammalian TRPV1 (VR1) channel in ASH nociceptor neurons avoid the TRPV1 ligand capsaicin, allowing selective, drug-inducible activation of a specific behavior.


Subject(s)
Caenorhabditis elegans Proteins/isolation & purification , Caenorhabditis elegans/metabolism , Cell Membrane/metabolism , Ion Channels/isolation & purification , Ion Channels/metabolism , Nerve Tissue Proteins/isolation & purification , Nerve Tissue Proteins/metabolism , Nervous System/metabolism , Neurons, Afferent/metabolism , Sensation/genetics , Amino Acid Sequence/genetics , Animals , Base Sequence/genetics , Behavior, Animal/drug effects , Behavior, Animal/physiology , Caenorhabditis elegans/cytology , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , Capsaicin/pharmacology , Cell Compartmentation/genetics , Cell Membrane/drug effects , Cell Membrane/ultrastructure , Gene Expression Regulation/physiology , Ion Channels/genetics , Ion Channels/ultrastructure , Molecular Sequence Data , Mutation/genetics , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/ultrastructure , Nervous System/cytology , Nervous System/drug effects , Neurons, Afferent/cytology , Neurons, Afferent/drug effects , Pain/genetics , Pain/metabolism , Pain/physiopathology , Phylogeny , Receptors, Drug/drug effects , Receptors, Drug/metabolism , Receptors, Drug/ultrastructure , Sensation/drug effects , Signal Transduction/genetics , TRPV Cation Channels , Transient Receptor Potential Channels
9.
Nature ; 417(6889): 660-3, 2002 Jun 06.
Article in English | MEDLINE | ID: mdl-12050669

ABSTRACT

Germline stem cells are defined by their unique ability to generate more of themselves as well as differentiated gametes. The molecular mechanisms controlling the decision between self-renewal and differentiation are central unsolved problems in developmental biology with potentially broad medical implications. In Caenorhabditis elegans, germline stem cells are controlled by the somatic distal tip cell. FBF-1 and FBF-2, two nearly identical proteins, which together are called FBF ('fem-3 mRNA binding factor'), were originally discovered as regulators of germline sex determination. Here we report that FBF also controls germline stem cells: in an fbf-1 fbf-2 double mutant, germline proliferation is initially normal, but stem cells are not maintained. We suggest that FBF controls germline stem cells, at least in part, by repressing gld-1, which itself promotes commitment to the meiotic cell cycle. FBF belongs to the PUF family ('Pumilio and FBF') of RNA-binding proteins. Pumilio controls germline stem cells in Drosophila females, and, in lower eukaryotes, PUF proteins promote continued mitoses. We suggest that regulation by PUF proteins may be an ancient and widespread mechanism for control of stem cells.


Subject(s)
Caenorhabditis elegans/cytology , Caenorhabditis elegans/genetics , Conserved Sequence , Gene Expression Regulation , Germ Cells/cytology , Helminth Proteins/metabolism , RNA-Binding Proteins/metabolism , Stem Cells/cytology , 3' Untranslated Regions/genetics , 3' Untranslated Regions/metabolism , Animals , Base Sequence , Caenorhabditis elegans/growth & development , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , Cell Differentiation , Cell Division , Cell Lineage , Disorders of Sex Development/genetics , Electrophoretic Mobility Shift Assay , Female , Genes, Helminth/genetics , Germ Cells/metabolism , Helminth Proteins/genetics , Male , Meiosis , Mutation/genetics , Protein Binding , RNA-Binding Proteins/genetics , Repressor Proteins/genetics , Repressor Proteins/metabolism , Response Elements/genetics , Stem Cells/metabolism , Two-Hybrid System Techniques
10.
Nucleic Acids Res ; 30(12): e52, 2002 Jun 15.
Article in English | MEDLINE | ID: mdl-12060690

ABSTRACT

About 40% of the genes in the nematode Caenorhabditis elegans have homologs in humans. Based on the history of this model system, it is clear that the application of genetic methods to the study of this set of genes would provide important clues to their function in humans. To facilitate such genetic studies, we are engaged in a project to derive deletion alleles in every gene in this set. Our standard methods make use of nested PCR to hunt for animals in mutagenized populations that carry deletions at a given locus. The deletion bearing animals exist initially in mixed populations where the majority of the animals are wild type at the target. Therefore, the production of the PCR fragment representing the deletion allele competes with the production of the wild type fragment. The size of the deletion fragment relative to wild type determines whether it can compete to a level where it can be detected above the background. Using our standard conditions, we have found that when the deletion is <600 bp, the deletion fragment does not compete effectively with the production of the wild type fragment in PCR. Therefore, although our standard methods work well to detect mutants with deletions >600 bp, they do not work well to detect mutants with smaller deletions. Here we report a new strategy to detect small deletion alleles in complex DNA pools. Our new strategy is a modification of our standard PCR based screens. In the first round of the nested PCR, we include a third PCR primer between the two external primers. The presence of this third primer leads to the production of three fragments from wild type DNA. We configure the system so that two of these three fragments cannot serve as a template in the second round of the nested PCR. The addition of this third primer, therefore, handicaps the amplification from wild type template. On the other hand, the amplification of mutant fragments where the binding site for the third primer is deleted is unabated. Overall, we see at least a 500-fold increase in the sensitivity for small deletion fragments using our new method. Using this new method, we report the recovery of new deletion alleles within 12 C.elegans genes.


Subject(s)
Caenorhabditis elegans/genetics , DNA Mutational Analysis/methods , DNA, Helminth , Polymerase Chain Reaction/methods , Sequence Deletion , Alleles , Animals , DNA , Genes, Helminth , Sensitivity and Specificity
11.
RNA ; 8(6): 725-39, 2002 Jun.
Article in English | MEDLINE | ID: mdl-12088146

ABSTRACT

Cell fates in the Caenorhabditis elegans germline are regulated, at least in part, at the posttranscriptional level. For example, the switch from spermatogenesis to oogenesis in the hermaphrodite relies on posttranscriptional repression of the fem-3 mRNA via its 3' untranslated region (UTR). Previous studies identified three DEAH box proteins, MOG-1, MOG-4, and MOG-5, that are critical for the fem-3 3' UTR control. Here we describe MEP-1, a zinc-finger protein that binds specifically to each of these three MOG proteins and that is required for repression by the fem-3 3' UTR in vivo. To investigate its in vivo function, we generated a mep-1 deletion mutant. The mep-1 null phenotype suggests a broad role for MEP-1 in C. elegans development, as it is associated with early larval arrest. In addition, mep-1 mutants can be defective in gonadogenesis and oocyte production when derived from a heterozygous mother. We suggest that MEP-1 acts together with the MOG proteins to repress fem-3 mRNA and that it also functions in other pathways to control development more broadly.


Subject(s)
3' Untranslated Regions , Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/genetics , Gene Expression Regulation , Helminth Proteins/genetics , Helminth Proteins/metabolism , RNA Helicases , Transcription Factors/metabolism , Zinc Fingers , Amino Acid Sequence , Animals , Base Sequence , Caenorhabditis elegans Proteins/chemistry , Caenorhabditis elegans Proteins/genetics , DEAD-box RNA Helicases , DNA Primers , Genes, Essential , Molecular Sequence Data , RNA Processing, Post-Transcriptional , RNA Splicing Factors , RNA, Messenger/genetics , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Transcription Factors/chemistry , Transcription Factors/genetics
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