Defining the Regulation of Telomerase Through Identification of Mammary-Specific Telomerase Interacting Proteins

DTIC Science & Technology

2007-06-01

Pharmacology and Toxicology , Virginia Commonwealth University, Richmond, VA 2002-present Member, Molecular Biology and Genetics Program, Virginia...Studying Telomeres and Telomerase. Zebrafish , 1:349-355. Jones,K.R., L.W.Elmore, L.Povirk, S.E.Holt, and D.A.Gewirtz. 2005. Reciprocal regulation... Zebrafish Blastula Cell Line on Rainbow Trout Stromal Cells and Subsequent Development under Feeder-Free Conditions into a Cell Line, ZEB2J. Zebrafish 5: 49

There and Back Again: Development and Regeneration of the Zebrafish Lateral Line System

PubMed Central

Thomas, Eric D.; Cruz, Ivan A.; Hailey, Dale W.; Raible, David W.

2014-01-01

The zebrafish lateral line is a sensory system used to detect changes in water flow. It is comprised of clusters of mechanosensory hair cells called neuromasts. The lateral line is initially established by a migratory group of cells, called a primordium, that deposits neuromasts at stereotyped locations along the surface of the fish. Wnt, FGF, and Notch signaling are all important regulators of various aspects of lateral line development, from primordium migration to hair cell specification. As zebrafish age, the organization of the lateral line becomes more complex in order to accommodate the fish’s increased size. This expansion is regulated by many of the same factors involved in the initial development. Furthermore, unlike mammalian hair cells, lateral line hair cells have the capacity to regenerate after damage. New hair cells arise from the proliferation and differentiation of surrounding support cells, and the molecular and cellular pathways regulating this are beginning to be elucidated. All in all, the zebrafish lateral line has proven to be an excellent model in which to study a diverse array of processes, including collective cell migration, cell polarity, cell fate, and regeneration. PMID:25330982

Development of a new canine osteosarcoma cell line.

PubMed

Séguin, B; Zwerdling, T; McCallan, J L; DeCock, H E V; Dewe, L L; Naydan, D K; Young, A E; Bannasch, D L; Foreman, O; Kent, M S

2006-12-01

Establishing a canine osteosarcoma (OSA) cell line can be useful to develop in vivo and in vitro models of OSA. The goal of this study was to develop, characterize and authenticate a new canine OSA cell line and a clone. A cell line and a clone were developed with standard cell culture techniques from a naturally occurring OSA in a dog. The clonal cell line induced a tumour after injection in RAG 1-deficient mouse. Histology was consistent with OSA. The original tumour from the dog and the tumour induced in the mouse were both reactive with vimentin and osteonectin (ON). The parent cell line and clonal cell line were reactive with ON, osteocalcin and alkaline phosphatase. Loss of heterozygosity was found in the same three microsatellite markers in the parent and clonal cell lines, and the tumour tissue grown in the mouse.

Functional mechanotransduction is required for cisplatin-induced hair cell death in the zebrafish lateral line.

PubMed

Thomas, Andrew J; Hailey, Dale W; Stawicki, Tamara M; Wu, Patricia; Coffin, Allison B; Rubel, Edwin W; Raible, David W; Simon, Julian A; Ou, Henry C

2013-03-06

Cisplatin, one of the most commonly used anticancer drugs, is known to cause inner ear hair cell damage and hearing loss. Despite much investigation into mechanisms of cisplatin-induced hair cell death, little is known about the mechanism whereby cisplatin is selectively toxic to hair cells. Using hair cells of the zebrafish lateral line, we found that chemical inhibition of mechanotransduction with quinine and EGTA protected against cisplatin-induced hair cell death. Furthermore, we found that the zebrafish mutants mariner (myo7aa) and sputnik (cad23) that lack functional mechanotransduction were resistant to cisplatin-induced hair cell death. Using a fluorescent analog of cisplatin, we found that chemical or genetic inhibition of mechanotransduction prevented its uptake. These findings demonstrate that cisplatin-induced hair cell death is dependent on functional mechanotransduction in the zebrafish lateral line.

Muscle Stem Cells Undergo Extensive Clonal Drift during Tissue Growth via Meox1-Mediated Induction of G2 Cell-Cycle Arrest.

PubMed

Nguyen, Phong Dang; Gurevich, David Baruch; Sonntag, Carmen; Hersey, Lucy; Alaei, Sara; Nim, Hieu Tri; Siegel, Ashley; Hall, Thomas Edward; Rossello, Fernando Jaime; Boyd, Sarah Elizabeth; Polo, Jose Maria; Currie, Peter David

2017-07-06

Organ growth requires a careful balance between stem cell self-renewal and lineage commitment to ensure proper tissue expansion. The cellular and molecular mechanisms that mediate this balance are unresolved in most organs, including skeletal muscle. Here we identify a long-lived stem cell pool that mediates growth of the zebrafish myotome. This population exhibits extensive clonal drift, shifting from random deployment of stem cells during development to reliance on a small number of dominant clones to fuel the vast majority of muscle growth. This clonal drift requires Meox1, a homeobox protein that directly inhibits the cell-cycle checkpoint gene ccnb1. Meox1 initiates G 2 cell-cycle arrest within muscle stem cells, and disrupting this G 2 arrest causes premature lineage commitment and the resulting defects in muscle growth. These findings reveal that distinct regulatory mechanisms orchestrate stem cell dynamics during organ growth, beyond the G 0 /G 1 cell-cycle inhibition traditionally associated with maintaining tissue-resident stem cells. Copyright © 2017. Published by Elsevier Inc.

SENSORY HAIR CELL REGENERATION IN THE ZEBRAFISH LATERAL LINE

PubMed Central

Lush, Mark E.; Piotrowski, Tatjana

2014-01-01

Damage or destruction of sensory hair cells in the inner ear leads to hearing or balance deficits that can be debilitating, especially in older adults. Unfortunately, the damage is permanent, as regeneration of the inner ear sensory epithelia does not occur in mammals. Zebrafish and other non-mammalian vertebrates have the remarkable ability to regenerate sensory hair cells and understanding the molecular and cellular basis for this regenerative ability will hopefully aid us in designing therapies to induce regeneration in mammals. Zebrafish not only possess hair cells in the ear but also in the sensory lateral line system. Hair cells in both organs are functionally analogous to hair cells in the inner ear of mammals. The lateral line is a mechanosensory system found in most aquatic vertebrates that detects water motion and aids in predator avoidance, prey capture, schooling and mating. Although hair cell regeneration occurs in both the ear and lateral line, most research to date has focused on the lateral line due to its relatively simple structure and accessibility. Here we review the recent discoveries made during the characterization of hair cell regeneration in zebrafish. PMID:25045019

Production of Zebrafish Offspring from Cultured Female Germline Stem Cells

PubMed Central

Wong, Ten-Tsao; Tesfamichael, Abraham; Collodi, Paul

2013-01-01

Zebrafish female germline stem cell (FGSC) cultures were generated from a transgenic line of fish that expresses Neo and DsRed under the control of the germ cell specific promoter, ziwi [Tg(ziwi:neo);Tg(ziwi:DsRed)]. Homogeneous FGSC cultures were established by G418 selection and continued to express ziwi for more than 6 weeks along with the germ cell markers nanos3, dnd, dazl and vasa. A key component of the cell culture system was the use of a feeder cell line that was initiated from ovaries of a transgenic line of fish [Tg(gsdf:neo)] that expresses Neo controlled by the zebrafish gonadal soma derived factor (gsdf) promoter. The feeder cell line was selected in G418 and engineered to express zebrafish leukemia inhibitory factor (Lif), basic fibroblast growth factor (Fgf2) and glial-cell-line derived neurotrophic factor (Gdnf). These factors were shown to significantly enhance FGSC growth, survival and germline competency in culture. Results from cell transplantation experiments revealed that the cultured FGSCs were able to successfully colonize the gonad of sterile recipient fish and generate functional gametes. Up to 20% of surviving recipient fish that were injected with the cultured FGSCs were fertile and generated multiple batches of normal offspring for at least 6 months. The FGSC cultures will provide an in vitro system for studies of zebrafish germ cell growth and differentiation and their high frequency of germline transmission following transplantation could form the basis of a stem cell-mediated strategy for gene transfer and manipulation of the zebrafish genome. PMID:23671620

Humanizing the zebrafish liver shifts drug metabolic profiles and improves pharmacokinetics of CYP3A4 substrates.

PubMed

Poon, Kar Lai; Wang, Xingang; Ng, Ashley S; Goh, Wei Huang; McGinnis, Claudia; Fowler, Stephen; Carney, Tom J; Wang, Haishan; Ingham, Phillip W

2017-03-01

Understanding and predicting whether new drug candidates will be safe in the clinic is a critical hurdle in pharmaceutical development, that relies in part on absorption, distribution, metabolism, excretion and toxicology studies in vivo. Zebrafish is a relatively new model system for drug metabolism and toxicity studies, offering whole organism screening coupled with small size and potential for high-throughput screening. Through toxicity and absorption analyses of a number of drugs, we find that zebrafish is generally predictive of drug toxicity, although assay outcomes are influenced by drug lipophilicity which alters drug uptake. In addition, liver microsome assays reveal specific differences in metabolism of compounds between human and zebrafish livers, likely resulting from the divergence of the cytochrome P450 superfamily between species. To reflect human metabolism more accurately, we generated a transgenic "humanized" zebrafish line that expresses the major human phase I detoxifying enzyme, CYP3A4, in the liver. Here, we show that this humanized line shows an elevated metabolism of CYP3A4-specific substrates compared to wild-type zebrafish. The generation of this first described humanized zebrafish liver suggests such approaches can enhance the accuracy of the zebrafish model for toxicity prediction.

Functional mechanotransduction is required for cisplatin-induced hair cell death in the zebrafish lateral line

PubMed Central

Thomas, Andrew J.; Hailey, Dale W.; Stawicki, Tamara M.; Wu, Patricia; Coffin, Allison B.; Rubel, Edwin W.; Raible, David W.; Simon, Julian A.; Ou, Henry C.

2013-01-01

Cisplatin, one of the most commonly used anti-cancer drugs, is known to cause inner ear hair cell damage and hearing loss. Despite much investigation into mechanisms of cisplatin-induced hair cell death, little is known about the mechanism whereby cisplatin is selectively toxic to hair cells. Using hair cells of the zebrafish lateral line, we found that chemical inhibition of mechanotransduction with quinine and EGTA protected against cisplatin-induced hair cell death. Furthermore, we found that the zebrafish mutants mariner (myo7aa) and sputnik (cad23) that lack functional mechanotransduction were resistant to cisplatin-induced hair cell death. Using a fluorescent analogue of cisplatin, we found that chemical or genetic inhibition of mechanotransduction prevented its uptake. These findings demonstrate that cisplatin-induced hair cell death is dependent on functional mechanotransduction in the zebrafish lateral line. PMID:23467357

Establishment of a Transgenic Zebrafish Line for Superficial Skin Ablation and Functional Validation of Apoptosis Modulators In Vivo

PubMed Central

Chen, Chi-Fang; Chu, Che-Yu; Chen, Te-Hao; Lee, Shyh-Jye; Shen, Chia-Ning; Hsiao, Chung-Der

2011-01-01

Background Zebrafish skin is composed of enveloping and basal layers which form a first-line defense system against pathogens. Zebrafish epidermis contains ionocytes and mucous cells that aid secretion of acid/ions or mucous through skin. Previous studies demonstrated that fish skin is extremely sensitive to external stimuli. However, little is known about the molecular mechanisms that modulate skin cell apoptosis in zebrafish. Methodology/Principal Findings This study aimed to create a platform to conduct conditional skin ablation and determine if it is possible to attenuate apoptotic stimuli by overexpressing potential apoptosis modulating genes in the skin of live animals. A transgenic zebrafish line of Tg(krt4:NTR-hKikGR)cy17 (killer line), which can conditionally trigger apoptosis in superficial skin cells, was first established. When the killer line was incubated with the prodrug metrodinazole, the superficial skin displayed extensive apoptosis as judged by detection of massive TUNEL- and active caspase 3-positive signals. Great reductions in NTR-hKikGR+ fluorescent signals accompanied epidermal cell apoptosis. This indicated that NTR-hKikGR+ signal fluorescence can be utilized to evaluate apoptotic events in vivo. After removal of metrodinazole, the skin integrity progressively recovered and NTR-hKikGR+ fluorescent signals gradually restored. In contrast, either crossing the killer line with testing lines or transiently injecting the killer line with testing vectors that expressed human constitutive active Akt1, mouse constitutive active Stat3, or HPV16 E6 element displayed apoptosis-resistant phenotypes to cytotoxic metrodinazole as judged by the loss of reduction in NTR-hKikGR+ fluorescent signaling. Conclusion/Significance The killer/testing line binary system established in the current study demonstrates a nitroreductase/metrodinazole system that can be utilized to conditionally perform skin ablation in a real-time manner, and provides a valuable tool to visualize and quantify the anti-apoptotic potential of interesting target genes in vivo. The current work identifies a potential use for transgenic zebrafish as a high-throughput platform to validate potential apoptosis modulators in vivo. PMID:21655190

In vivo and in vitro biophysical properties of hair cells from the lateral line and inner ear of developing and adult zebrafish.

PubMed

Olt, Jennifer; Johnson, Stuart L; Marcotti, Walter

2014-05-15

Hair cells detect and process sound and movement information, and transmit this with remarkable precision and efficiency to afferent neurons via specialized ribbon synapses. The zebrafish is emerging as a powerful model for genetic analysis of hair cell development and function both in vitro and in vivo. However, the full exploitation of the zebrafish is currently limited by the difficulty in obtaining systematic electrophysiological recordings from hair cells under physiological recording conditions. Thus, the biophysical properties of developing and adult zebrafish hair cells are largely unknown. We investigated potassium and calcium currents, voltage responses and synaptic activity in hair cells from the lateral line and inner ear in vivo and using near-physiological in vitro recordings. We found that the basolateral current profile of hair cells from the lateral line becomes more segregated with age, and that cells positioned in the centre of the neuromast show more mature characteristics and those towards the edge retain a more immature phenotype. The proportion of mature-like hair cells within a given neuromast increased with zebrafish development. Hair cells from the inner ear showed a developmental change in current profile between the juvenile and adult stages. In lateral line hair cells from juvenile zebrafish, exocytosis also became more efficient and required less calcium for vesicle fusion. In hair cells from mature zebrafish, the biophysical characteristics of ion channels and exocytosis resembled those of hair cells from other lower vertebrates and, to some extent, those in the immature mammalian vestibular and auditory systems. We show that although the zebrafish provides a suitable animal model for studies on hair cell physiology, it is advisable to consider that the age at which the majority of hair cells acquire a mature-type configuration is reached only in the juvenile lateral line and in the inner ear from >2 months after hatching. © 2014 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.

Generation of genome-modified Drosophila cell lines using SwAP.

PubMed

Franz, Alexandra; Brunner, Erich; Basler, Konrad

2017-10-02

The ease of generating genetically modified animals and cell lines has been markedly increased by the recent development of the versatile CRISPR/Cas9 tool. However, while the isolation of isogenic cell populations is usually straightforward for mammalian cell lines, the generation of clonal Drosophila cell lines has remained a longstanding challenge, hampered by the difficulty of getting Drosophila cells to grow at low densities. Here, we describe a highly efficient workflow to generate clonal Cas9-engineered Drosophila cell lines using a combination of cell pools, limiting dilution in conditioned medium and PCR with allele-specific primers, enabling the efficient selection of a clonal cell line with a suitable mutation profile. We validate the protocol by documenting the isolation, selection and verification of eight independently Cas9-edited armadillo mutant Drosophila cell lines. Our method provides a powerful and simple workflow that improves the utility of Drosophila cells for genetic studies with CRISPR/Cas9.

Isolation of Novel CreERT2-Driver Lines in Zebrafish Using an Unbiased Gene Trap Approach

PubMed Central

Jungke, Peggy; Hammer, Juliane; Hans, Stefan; Brand, Michael

2015-01-01

Gene manipulation using the Cre/loxP-recombinase system has been successfully employed in zebrafish to study gene functions and lineage relationships. Recently, gene trapping approaches have been applied to produce large collections of transgenic fish expressing conditional alleles in various tissues. However, the limited number of available cell- and tissue-specific Cre/CreERT2-driver lines still constrains widespread application in this model organism. To enlarge the pool of existing CreERT2-driver lines, we performed a genome-wide gene trap screen using a Tol2-based mCherry-T2a-CreERT2 (mCT2aC) gene trap vector. This cassette consists of a splice acceptor and a mCherry-tagged variant of CreERT2 which enables simultaneous labeling of the trapping event, as well as CreERT2 expression from the endogenous promoter. Using this strategy, we generated 27 novel functional CreERT2-driver lines expressing in a cell- and tissue-specific manner during development and adulthood. This study summarizes the analysis of the generated CreERT2-driver lines with respect to functionality, expression, integration, as well as associated phenotypes. Our results significantly enlarge the existing pool of CreERT2-driver lines in zebrafish and combined with Cre–dependent effector lines, the new CreERT2-driver lines will be important tools to manipulate the zebrafish genome. PMID:26083735

Sensory hair cell regeneration in the zebrafish lateral line.

PubMed

Lush, Mark E; Piotrowski, Tatjana

2014-10-01

Damage or destruction of sensory hair cells in the inner ear leads to hearing or balance deficits that can be debilitating, especially in older adults. Unfortunately, the damage is permanent, as regeneration of the inner ear sensory epithelia does not occur in mammals. Zebrafish and other non-mammalian vertebrates have the remarkable ability to regenerate sensory hair cells and understanding the molecular and cellular basis for this regenerative ability will hopefully aid us in designing therapies to induce regeneration in mammals. Zebrafish not only possess hair cells in the ear but also in the sensory lateral line system. Hair cells in both organs are functionally analogous to hair cells in the inner ear of mammals. The lateral line is a mechanosensory system found in most aquatic vertebrates that detects water motion and aids in predator avoidance, prey capture, schooling, and mating. Although hair cell regeneration occurs in both the ear and lateral line, most research to date has focused on the lateral line due to its relatively simple structure and accessibility. Here we review the recent discoveries made during the characterization of hair cell regeneration in zebrafish. Copyright © 2014 Wiley Periodicals, Inc.

Combined toxicity of silica nanoparticles and methylmercury on cardiovascular system in zebrafish (Danio rerio) embryos.

PubMed

Duan, Junchao; Hu, Hejing; Li, Qiuling; Jiang, Lizhen; Zou, Yang; Wang, Yapei; Sun, Zhiwei

2016-06-01

This study was to investigate the combined toxicity of silica nanoparticles (SiNPs) and methylmercury (MeHg) on cardiovascular system in zebrafish (Danio rerio) embryos. Ultraviolet absorption analysis showed that the co-exposure system had high absorption and stability. The dosages used in this study were based on the NOAEL level. Zebrafish embryos exposed to the co-exposure of SiNPs and MeHg did not show any cardiovascular malformation or atrioventricular block, but had an inhibition effect on bradycardia. Using o-Dianisidine for erythrocyte staining, the cardiac output of zebrafish embryos was decreased gradually in SiNPs, MeHg, co-exposure groups, respectively. Co-exposure of SiNPs and MeHg enhanced the vascular endothelial damage in Tg(fli-1:EGFP) transgenic zebrafish line. Moreover, the co-exposure significantly activated the oxidative stress and inflammatory response in neutrophils-specific Tg(mpo:GFP) transgenic zebrafish line. This study suggested that the combined toxic effects of SiNPs and MeHg on cardiovascular system had more severe toxicity than the single exposure alone. Copyright © 2016 Elsevier B.V. All rights reserved.

Photoluminescent graphene quantum dots for in vivo imaging of apoptotic cells

NASA Astrophysics Data System (ADS)

Roy, Prathik; Periasamy, Arun Prakash; Lin, Chiu-Ya; Her, Guor-Mour; Chiu, Wei-Jane; Li, Chi-Lin; Shu, Chia-Lun; Huang, Chih-Ching; Liang, Chi-Te; Chang, Huan-Tsung

2015-01-01

Apoptosis (programmed cell death) is linked to many incurable neurodegenerative, cardiovascular and cancer causing diseases. Numerous methods have been developed for imaging apoptotic cells in vitro; however, there are few methods available for imaging apoptotic cells in live animals (in vivo). Here we report a novel method utilizing the unique photoluminescence properties of plant leaf-derived graphene quantum dots (GQDs) modified with annexin V antibody (AbA5) to form (AbA5)-modified GQDs (AbA5-GQDs) enabling us to label apoptotic cells in live zebrafish (Danio rerio). The key is that zebrafish shows bright red photoluminescence in the presence of apoptotic cells. The toxicity of the GQDs has also been investigated with the GQDs exhibiting high biocompatibility as they were excreted from the zebrafish's body without affecting its growth significantly at a concentration lower than 2 mg mL-1 over a period of 4 to 72 hour post fertilization. The GQDs have further been used to image human breast adenocarcinoma cell line (MCF-7 cells), human cervical cancer cell line (HeLa cells), and normal human mammary epithelial cell line (MCF-10A). These results are indispensable to further the advance of graphene-based nanomaterials for biomedical applications.Apoptosis (programmed cell death) is linked to many incurable neurodegenerative, cardiovascular and cancer causing diseases. Numerous methods have been developed for imaging apoptotic cells in vitro; however, there are few methods available for imaging apoptotic cells in live animals (in vivo). Here we report a novel method utilizing the unique photoluminescence properties of plant leaf-derived graphene quantum dots (GQDs) modified with annexin V antibody (AbA5) to form (AbA5)-modified GQDs (AbA5-GQDs) enabling us to label apoptotic cells in live zebrafish (Danio rerio). The key is that zebrafish shows bright red photoluminescence in the presence of apoptotic cells. The toxicity of the GQDs has also been investigated with the GQDs exhibiting high biocompatibility as they were excreted from the zebrafish's body without affecting its growth significantly at a concentration lower than 2 mg mL-1 over a period of 4 to 72 hour post fertilization. The GQDs have further been used to image human breast adenocarcinoma cell line (MCF-7 cells), human cervical cancer cell line (HeLa cells), and normal human mammary epithelial cell line (MCF-10A). These results are indispensable to further the advance of graphene-based nanomaterials for biomedical applications. Electronic supplementary information (ESI) available: Experimental discussion on synthesis, characterization, cellular imaging, cytotoxicity of GQDs in addition to its effect on zebrafish embryos, preparation of annexin V (A5)-modified GQDs (AbA5-GQDs), staining procedures and imaging are given. Figures for XRD, UV-vis absorption, photoluminescence of GQDs, mortality of zebrafish, time course recording of morphology of zebrafish embryos and morphology of adult zebrafish exposed to GQDs are illustrated. See DOI: 10.1039/c4nr07005d

The ciliary marginal zone of the zebrafish retina: clonal and time-lapse analysis of a continuously growing tissue.

PubMed

Wan, Yinan; Almeida, Alexandra D; Rulands, Steffen; Chalour, Naima; Muresan, Leila; Wu, Yunmin; Simons, Benjamin D; He, Jie; Harris, William A

2016-04-01

Clonal analysis is helping us understand the dynamics of cell replacement in homeostatic adult tissues (Simons and Clevers, 2011). Such an analysis, however, has not yet been achieved for continuously growing adult tissues, but is essential if we wish to understand the architecture of adult organs. The retinas of lower vertebrates grow throughout life from retinal stem cells (RSCs) and retinal progenitor cells (RPCs) at the rim of the retina, called the ciliary marginal zone (CMZ). Here, we show that RSCs reside in a niche at the extreme periphery of the CMZ and divide asymmetrically along a radial (peripheral to central) axis, leaving one daughter in the peripheral RSC niche and the other more central where it becomes an RPC. We also show that RPCs of the CMZ have clonal sizes and compositions that are statistically similar to progenitor cells of the embryonic retina and fit the same stochastic model of proliferation. These results link embryonic and postembryonic cell behaviour, and help to explain the constancy of tissue architecture that has been generated over a lifetime. © 2016. Published by The Company of Biologists Ltd.

The Prx1 limb enhancers: targeted gene expression in developing zebrafish pectoral fins.

PubMed

Hernández-Vega, Amayra; Minguillón, Carolina

2011-08-01

Limbs represent an excellent model to study the induction, growth, and patterning of several organs. A breakthrough to study gene function in various tissues has been the characterization of regulatory elements that allow tissue-specific interference of gene function. The mouse Prx1 promoter has been used to generate limb-specific mutants and overexpress genes in tetrapod limbs. Although zebrafish possess advantages that favor their use to study limb morphogenesis, there is no driver described suitable for specifically interfering with gene function in developing fins. We report the generation of zebrafish lines that express enhanced green fluorescent protein (EGFP) driven by the mouse Prx1 enhancer in developing pectoral fins. We also describe the expression pattern of the zebrafish prrx1 genes and identify three conserved non-coding elements (CNEs) that we use to generate fin-specific EGFP reporter lines. Finally, we show that the mouse and zebrafish regulatory elements may be used to modify gene function in pectoral fins. Copyright © 2011 Wiley-Liss, Inc.

Comparison of stem morphology and anatomy of two alfalfa clonal lines exhibiting divergent cell wall composition

USDA-ARS?s Scientific Manuscript database

In previous research, two alfalfa clonal lines (252, 1283) were identified that exhibited environmentally stable differences in stem cell walls. Compared to stems of 1283, stems of 252 have a higher cell wall concentration and greater amounts of lignin and cellulose but reduced levels of pectic suga...

Macrophage–Microbe Interactions: Lessons from the Zebrafish Model

PubMed Central

Yoshida, Nagisa; Frickel, Eva-Maria; Mostowy, Serge

2017-01-01

Macrophages provide front line defense against infections. The study of macrophage–microbe interplay is thus crucial for understanding pathogenesis and infection control. Zebrafish (Danio rerio) larvae provide a unique platform to study macrophage–microbe interactions in vivo, from the level of the single cell to the whole organism. Studies using zebrafish allow non-invasive, real-time visualization of macrophage recruitment and phagocytosis. Furthermore, the chemical and genetic tractability of zebrafish has been central to decipher the complex role of macrophages during infection. Here, we discuss the latest developments using zebrafish models of bacterial and fungal infection. We also review novel aspects of macrophage biology revealed by zebrafish, which can potentiate development of new therapeutic strategies for humans. PMID:29250076

Editor's Highlight: Transgenic Zebrafish Reporter Lines as Alternative In Vivo Organ Toxicity Models.

PubMed

Poon, Kar Lai; Wang, Xingang; Lee, Serene G P; Ng, Ashley S; Goh, Wei Huang; Zhao, Zhonghua; Al-Haddawi, Muthafar; Wang, Haishan; Mathavan, Sinnakaruppan; Ingham, Philip W; McGinnis, Claudia; Carney, Tom J

2017-03-01

Organ toxicity, particularly liver toxicity, remains one of the major reasons for the termination of drug candidates in the development pipeline as well as withdrawal or restrictions of marketed drugs. A screening-amenable alternative in vivo model such as zebrafish would, therefore, find immediate application in the early prediction of unacceptable organ toxicity. To identify highly upregulated genes as biomarkers of toxic responses in the zebrafish model, a set of well-characterized reference drugs that cause drug-induced liver injury (DILI) in the clinic were applied to zebrafish larvae and adults. Transcriptome microarray analysis was performed on whole larvae or dissected adult livers. Integration of data sets from different drug treatments at different stages identified common upregulated detoxification pathways. Within these were candidate biomarkers which recurred in multiple treatments. We prioritized 4 highly upregulated genes encoding enzymes acting in distinct phases of the drug metabolism pathway. Through promoter isolation and fosmid recombineering, eGFP reporter transgenic zebrafish lines were generated and evaluated for their response to DILI drugs. Three of the 4 generated reporter lines showed a dose and time-dependent induction in endodermal organs to reference drugs and an expanded drug set. In conclusion, through integrated transcriptomics and transgenic approaches, we have developed parallel independent zebrafish in vivo screening platforms able to predict organ toxicities of preclinical drugs. © The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Development of Cre-loxP technology in zebrafish to study the regulation of fish reproduction.

PubMed

Lin, Heng-Ju; Lee, Shu-Hua; Wu, Jen-Leih; Duann, Yeh-Fang; Chen, Jyh-Yih

2013-12-01

One cannot seek permission to market transgenic fish mainly because there is no field test or any basic research on technological developments for evaluating their biosafety. Infertility is a necessary adjunct to exploiting transgenic fish unless completely secure land-locked facilities are available. In this study, we report the generation of a Cre transgenic zebrafish line using a cytomegalovirus promoter. We also produced fish carrying the Bax1 and Bax2 plasmids; these genes were separated by two loxP sites under a zona pellucida C promoter or were driven by an anti-Müllerian hormone promoter. We inserted a red fluorescent protein gene between the two loxP sites. After obtaining transgenic lines with the two transgenic fish crossed with each other (Cre transgenic zebrafish x loxP transgenic zebrafish), the floxed DNA was found to be specifically eliminated from the female or male zebrafish, and apoptosis gene expressions caused ovarian and testicular growth cessation and degeneration. Overexpression of the Bax1 and Bax2 genes caused various expression levels of apoptosis-related genes. Accordingly, this transgenic zebrafish model system provides a method to produce infertile fish and may be useful for application to genetically modified fish.

Neoplasia and Neoplasm Associated Lesions in Laboratory Colonies of Zebrafish Emphasizing Key Influences of Diet and Aquaculture System Design

PubMed Central

Spitsbergen, Jan M.; Buhler, Donald R.; Peterson, Tracy S.

2014-01-01

During the past decade the zebrafish has emerged as a leading model for mechanistic cancer research due to its sophisticated genetic and genomic resources, its tractability for tissue targeting of transgene expression, its efficiency for forward genetic approaches to cancer model development, and its cost-effectiveness for enhancer and suppressor screens once a cancer model is established. However, in contrast to other laboratory animal species widely used as cancer models, much basic cancer biology information is lacking in zebrafish. As yet data are not published regarding dietary influences on neoplasm incidences in zebrafish. Little information is available regarding spontaneous tumor incidences or histologic types in wild-type (wt) lines of zebrafish. So far a comprehensive database documenting the full spectrum of neoplasia in various organ systems and tissues in not available for zebrafish as it is for other intensely studied laboratory animal species. This manuscript confirms that as in other species diet and husbandry can profoundly influence tumor incidences and histologic spectra in zebrafish. We show that in many laboratory colonies wt lines of zebrafish exhibit elevated neoplasm incidences and neoplasm associated lesions such as heptocyte megalocytosis. We present experimental evidence showing that certain diet and water management regimens can result in high incidences of neoplasia and neoplasm associated lesions. We document the wide array of benign and malignant neoplasms affecting nearly every organ, tissue and cell type in zebrafish, in some cases as a spontaneous aging change, and in other cases due to carcinogen treatment or genetic manipulation. PMID:23382343

RELATIVE POTENCY VALUES FOR POLYCHLORINATED DIBENZO-P-DIOXIN, DIBENZOFURAN AND BIPHENYL CONGENERS TO INDUCE CYTOCHROME P4501A MRNA IN A ZEBRAFISH LIVER CELL LINE (ZF-L)

EPA Science Inventory

Induction of cytochrome P450 (CYPIA) mRNA by polychlorinated dibenzo-p-dioxin (PCDD), polychlorinated dibenzofuran (PCDF) and polychlorinated biphenyl (PCB) congeners was measured in a zebrafish liver cell line (ZF-L). ZF-L cells were far less sensitive to PCDD, PCDF and PCB cong...

Casein gene expression in mouse mammary epithelial cell lines: Dependence upon extracellular matrix and cell type

DOE Office of Scientific and Technical Information (OSTI.GOV)

Medina, D.; Oborn, C.J.; Li, M.L.

1987-09-01

The COMMA-D mammary cell line exhibits mammary-specific functional differentiation under appropriate conditions in cell culture. The cytologically heterogeneous COMMA-D parental line and the clonal lines DB-1, TA-5, and FA-1 derived from the COMMA-D parent were examined for similar properties of functional differentiation. In monolayer cell culture, the cell lines DB-1, TA-5, FA-1, and MA-4 were examined for expression of mammary-specific and epithelial-specific proteins by an indirect immunofluorescence assay. The clonal cell lines were relatively homogeneous in their respective staining properties and seemed to represent three subpopulations found in the heterogeneous parental COMMA-D lines. None of the four clonal lines appearedmore » to represent myoepithelial cells. The cell lines were examined for expression of {beta}-casein mRNA in the presence or absence of prolactin. The inducibility of {beta}-casein in the COMMA-D cell line was further enhanced by a reconstituted basement membrane preparation enriched in laminin, collagen IV, and proteoglycans. These results support the hypothesis that the functional response of inducible mammary cell populations is a result of interaction among hormones, multiple extracellular matrix components, and specific cell types.« less

Different osteochondral potential of clonal cell lines derived from adult human trabecular bone.

PubMed

Osyczka, Anna M; Nöth, Ulrich; Danielson, Keith G; Tuan, Rocky S

2002-06-01

Cells derived from human trabecular bones have been shown to have multipotential differentiation ability along osteogenic, chondrogenic, and adipogenic lineages. In this study, we have derived two clonal sublines of human trabecular bone cells by means of stable transduction with human papilloma virus E6/E7 genes. Our results showed that these clonal sublines differ in their osteochondral potential, but are equally adipogenic, indicative of the heterogeneous nature of the parental cell population. The availability of these cell lines should be useful for the analysis of the mechanisms regulating the differentiation of adult mesenchymal progenitor cells.

Whole-organism clone tracing using single-cell sequencing.

PubMed

Alemany, Anna; Florescu, Maria; Baron, Chloé S; Peterson-Maduro, Josi; van Oudenaarden, Alexander

2018-04-05

Embryonic development is a crucial period in the life of a multicellular organism, during which limited sets of embryonic progenitors produce all cells in the adult body. Determining which fate these progenitors acquire in adult tissues requires the simultaneous measurement of clonal history and cell identity at single-cell resolution, which has been a major challenge. Clonal history has traditionally been investigated by microscopically tracking cells during development, monitoring the heritable expression of genetically encoded fluorescent proteins and, more recently, using next-generation sequencing technologies that exploit somatic mutations, microsatellite instability, transposon tagging, viral barcoding, CRISPR-Cas9 genome editing and Cre-loxP recombination. Single-cell transcriptomics provides a powerful platform for unbiased cell-type classification. Here we present ScarTrace, a single-cell sequencing strategy that enables the simultaneous quantification of clonal history and cell type for thousands of cells obtained from different organs of the adult zebrafish. Using ScarTrace, we show that a small set of multipotent embryonic progenitors generate all haematopoietic cells in the kidney marrow, and that many progenitors produce specific cell types in the eyes and brain. In addition, we study when embryonic progenitors commit to the left or right eye. ScarTrace reveals that epidermal and mesenchymal cells in the caudal fin arise from the same progenitors, and that osteoblast-restricted precursors can produce mesenchymal cells during regeneration. Furthermore, we identify resident immune cells in the fin with a distinct clonal origin from other blood cell types. We envision that similar approaches will have major applications in other experimental systems, in which the matching of embryonic clonal origin to adult cell type will ultimately allow reconstruction of how the adult body is built from a single cell.

Inhibition of H3K9me2 Reduces Hair Cell Regeneration after Hair Cell Loss in the Zebrafish Lateral Line by Down-Regulating the Wnt and Fgf Signaling Pathways

PubMed Central

Tang, Dongmei; Lin, Qin; He, Yingzi; Chai, Renjie; Li, Huawei

2016-01-01

The activation of neuromast (NM) supporting cell (SC) proliferation leads to hair cell (HC) regeneration in the zebrafish lateral line. Epigenetic mechanisms have been reported that regulate HC regeneration in the zebrafish lateral line, but the role of H3K9me2 in HC regeneration after HC loss remains poorly understood. In this study, we focused on the role of H3K9me2 in HC regeneration following neomycin-induced HC loss. To investigate the effects of H3K9me2 in HC regeneration, we took advantage of the G9a/GLP-specific inhibitor BIX01294 that significantly reduces the dimethylation of H3K9. We found that BIX01294 significantly reduced HC regeneration after neomycin-induced HC loss in the zebrafish lateral line. BIX01294 also significantly reduced the proliferation of NM cells and led to fewer SCs in the lateral line. In situ hybridization showed that BIX01294 significantly down-regulated the Wnt and Fgf signaling pathways, which resulted in reduced SC proliferation and HC regeneration in the NMs of the lateral line. Altogether, our results suggest that down-regulation of H3K9me2 significantly decreases HC regeneration after neomycin-induced HC loss through inactivation of the Wnt/β-catenin and Fgf signaling pathways. Thus H3K9me2 plays a critical role in HC regeneration. PMID:27303264

Dynamic and differential expression of the gonadal aromatase during the process of sexual differentiation in a novel transgenic cyp19a1a-eGFP zebrafish line.

PubMed

Hinfray, Nathalie; Sohm, Frédéric; Caulier, Morgane; Chadili, Edith; Piccini, Benjamin; Torchy, Camille; Porcher, Jean-Marc; Guiguen, Yann; Brion, François

2018-05-15

In zebrafish, there exists a clear need for new tools to study sex differentiation dynamic and its perturbation by endocrine disrupting chemicals. In this context, we developed and characterized a novel transgenic zebrafish line expressing green fluorescent protein (GFP) under the control of the zebrafish cyp19a1a (gonadal aromatase) promoter. In most gonochoristic fish species including zebrafish, cyp19a1a, the enzyme responsible for the synthesis of estrogens, has been shown to play a critical role in the processes of reproduction and sexual differentiation. This novel cyp19a1a-eGFP transgenic line allowed a deeper characterization of expression and localization of cyp19a1a gene in zebrafish gonads both at the adult stage and during development. At the adult stage, GFP expression was higher in ovaries than in testis. We showed a perfect co-expression of GFP and endogenous Cyp19a1a protein in gonads that was mainly localized in the cytoplasm of peri-follicular cells in the ovary and of Leydig and germ cells in the testis. During development, GFP was expressed in all immature gonads of 20 dpf-old zebrafish. Then, GFP expression increased in early differentiated female at 30 and 35dpf to reach a high GFP intensity in well-differentiated ovaries at 40dpf. On the contrary, males consistently displayed low GFP expression as compared to female whatever their stage of development, resulting in a clear dimorphic expression between both sexes. Interestingly, fish that undergoes ovary-to-testis transition (35 and 40dpf) presented GFP levels similar to males or intermediate between females and males. In this transgenic line our results confirm that cyp19a1a is expressed early during development, before the histological differentiation of the gonads, and that the down-regulation of cyp19a1a expression is likely responsible for the testicular differentiation. Moreover, we show that although cyp19a1a expression exhibits a clear dimorphic expression pattern in gonads during sexual differentiation, its expression persists whatever the sex suggesting that estradiol synthesis is important for gonadal development of both sexes. Monitoring the expression of GFP in control and exposed-fish will help determine the sensitivity of this transgenic line to EDCs and to refine mechanistic based-assays for the study of EDCs. In fine, this transgenic zebrafish line will be a useful tool to study physiological processes such as reproduction and sexual differentiation, and their perturbations by EDCs. Copyright © 2017 Elsevier Inc. All rights reserved.

A novel model for development, organization, and function of gonadotropes in fish pituitary.

PubMed

Golan, Matan; Biran, Jakob; Levavi-Sivan, Berta

2014-01-01

The gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are key regulators of the reproductive axis in vertebrates. Despite the high popularity of zebrafish as a model organism for studying reproductive functions, to date no transgenic zebrafish with labeled gonadotropes have been introduced. Using gonadotropin regulatory elements from tilapia, we generated two transgenic zebrafish lines with labeled gonadotropes. The tilapia and zebrafish regulatory sequences were highly divergent but several conserved elements allowed the tilapia promoters to correctly drive the transgenes in zebrafish pituitaries. FSH cells reacted to stimulation with gonadotropin releasing hormone by proliferating and showing increased transgene fluorescence, whereas estrogen exposure caused a decrease in cell number and transgene fluorescence. Transgene fluorescence reflected the expression pattern of the endogenous fshb gene. Ontogenetic expression of the transgenes followed typical patterns, with FSH cells appearing early in development, and LH cells appearing later and increasing dramatically in number with the onset of puberty. Our transgenic lines provide a powerful tool for investigating the development, anatomy, and function of the reproductive axis in lower vertebrates.

Defective calmodulin-dependent rapid apical endocytosis in zebrafish sensory hair cell mutants.

PubMed

Seiler, C; Nicolson, T

1999-11-15

Vertebrate mechanosensory hair cells contain a narrow "pericuticular" zone which is densely populated with small vesicles between the cuticular plate and cellular junctions near the apical surface. The presence of many cytoplasmic vesicles suggests that the apical surface of hair cells has a high turnover rate. The significance of intense membrane trafficking at the apical surface is not known. Using a marker of endocytosis, the styryl dye FM1-43, this report shows that rapid apical endocytosis in zebrafish lateral line sensory hair cells is calcium and calmodulin dependent and is partially blocked by the presence of amiloride and dihydrostreptomycin, known inhibitors of mechanotransduction channels. As seen in lateral line hair cells, sensory hair cells within the larval otic capsule also exhibit rapid apical endocytosis. Defects in internalization of the dye in both lateral line and inner ear hair cells were found in five zebrafish auditory/vestibular mutants: sputnik, mariner, orbiter, mercury, and skylab. In addition, lateral line hair cells in these mutants were not sensitive to prolonged exposure to streptomycin, which is toxic to hair cells. The presence of endocytic defects in the majority of zebrafish mechanosensory mutants points to a important role of apical endocytosis in hair cell function. Copyright 1999 John Wiley & Sons, Inc.

Effects of nicotine on zebrafish: A comparative response between a newly established gill cell line and whole gills.

PubMed

Nathiga Nambi, K S; Abdul Majeed, S; Taju, G; Sivasubbu, Sridhar; Sarath Babu, V; Sahul Hameed, A S

2017-05-01

A novel cell line, Danio rerio gill (DrG), derived from the gill tissue of zebrafish, was established and characterized. The cells were able to grow at a wide range of temperatures from 25°C to 32°C in Leibovitz's L-15 medium. The DrG cell line consists of epithelial-like cells with a diameter of 18-22μm. The cell line was characterized by mitochondrial 12S rRNA gene. Acute toxicity tests were conducted on D. rerio by exposing them to nicotine for 96h under static conditions. In vitro cytotoxicity of nicotine was assessed in DrG cell line using multiple endpoints such as 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), Neutral Red assay, Alamar Blue assay and Coomassie Blue protein assay. Linear correlations between each in vitro cytotoxicity assay and the in vivo mortality data were highly significant. Nicotine induced intracellular reactive oxygen species generation in DrG cell line in a concentration dependent manner. DrG cell line and zebrafish exposed to nicotine significantly increased the elevation of lipid peroxidation (LPO) while depletion of reduced glutathione (GSH), manganese superoxide dismutase (MnSOD), catalase (CAT), glutathione S-transferase (GST) and glutathione peroxidise(GPx1a) was observed. In nicotine treated fish and cells a negative correlation between reduced glutathione and LPO was observed. In addition, the production of ROS and the resulting oxidative stress resulted in increased expression of apoptosis related genes p53 and cas3.Collectively, our result suggests that nicotine has the potential to induce reactive oxygen species (ROS) production, oxidative stress and apoptosis in DrG cell line and zebrafish. Copyright © 2017 Elsevier Inc. All rights reserved.

Genetic lineage labeling in zebrafish uncovers novel neural crest contributions to the head, including gill pillar cells.

PubMed

Mongera, Alessandro; Singh, Ajeet P; Levesque, Mitchell P; Chen, Yi-Yen; Konstantinidis, Peter; Nüsslein-Volhard, Christiane

2013-02-01

At the protochordate-vertebrate transition, a new predatory lifestyle and increased body size coincided with the appearance of a true head. Characteristic innovations of this head are a skull protecting and accommodating a centralized nervous system, a jaw for prey capture and gills as respiratory organs. The neural crest (NC) is a major ontogenetic source for the 'new head' of vertebrates and its contribution to the cranial skeleton has been intensively studied in different model organisms. However, the role of NC in the expansion of the respiratory surface of the gills has been neglected. Here, we use genetic lineage labeling to address the contribution of NC to specific head structures, in particular to the gills of adult zebrafish. We generated a sox10:ER(T2)-Cre line and labeled NC cells by inducing Cre/loxP recombination with tamoxifen at embryonic stages. In juvenile and adult fish, we identified numerous established NC derivatives and, in the cranium, we precisely defined the crest/mesoderm interface of the skull roof. We show the NC origin of the opercular bones and of multiple cell types contributing to the barbels, chemosensory organs located in the mouth region. In the gills, we observed labeled primary and secondary lamellae. Clonal analysis reveals that pillar cells, a craniate innovation that mechanically supports the filaments and forms gill-specific capillaries, have a NC origin. Our data point to a crucial role for the NC in enabling more efficient gas exchange, thus uncovering a novel, direct involvement of this embryonic tissue in the evolution of respiratory systems at the protochordate-vertebrate transition.

Long-term drug administration in the adult zebrafish using oral gavage for cancer preclinical studies

PubMed Central

Dang, Michelle; Henderson, Rachel E.; Garraway, Levi A.

2016-01-01

ABSTRACT Zebrafish are a major model for chemical genetics, and most studies use embryos when investigating small molecules that cause interesting phenotypes or that can rescue disease models. Limited studies have dosed adults with small molecules by means of water-borne exposure or injection techniques. Challenges in the form of drug delivery-related trauma and anesthesia-related toxicity have excluded the adult zebrafish from long-term drug efficacy studies. Here, we introduce a novel anesthetic combination of MS-222 and isoflurane to an oral gavage technique for a non-toxic, non-invasive and long-term drug administration platform. As a proof of principle, we established drug efficacy of the FDA-approved BRAFV600E inhibitor, Vemurafenib, in adult zebrafish harboring BRAFV600E melanoma tumors. In the model, adult casper zebrafish intraperitoneally transplanted with a zebrafish melanoma cell line (ZMEL1) and exposed to daily sub-lethal dosing at 100 mg/kg of Vemurafenib for 2 weeks via oral gavage resulted in an average 65% decrease in tumor burden and a 15% mortality rate. In contrast, Vemurafenib-resistant ZMEL1 cell lines, generated in culture from low-dose drug exposure for 4 months, did not respond to the oral gavage treatment regimen. Similarly, this drug treatment regimen can be applied for treatment of primary melanoma tumors in the zebrafish. Taken together, we developed an effective long-term drug treatment system that will allow the adult zebrafish to be used to identify more effective anti-melanoma combination therapies and opens up possibilities for treating adult models of other diseases. PMID:27482819

The State of the Art of the Zebrafish Model for Toxicology and Toxicologic Pathology Research—Advantages and Current Limitations

PubMed Central

Spitsbergen, Jan M.; Kent, Michael L.

2007-01-01

The zebrafish (Danio rerio) is now the pre-eminent vertebrate model system for clarification of the roles of specific genes and signaling pathways in development. The zebrafish genome will be completely sequenced within the next 1–2 years. Together with the substantial historical database regarding basic developmental biology, toxicology, and gene transfer, the rich foundation of molecular genetic and genomic data makes zebrafish a powerful model system for clarifying mechanisms in toxicity. In contrast to the highly advanced knowledge base on molecular developmental genetics in zebrafish, our database regarding infectious and noninfectious diseases and pathologic lesions in zebrafish lags far behind the information available on most other domestic mammalian and avian species, particularly rodents. Currently, minimal data are available regarding spontaneous neoplasm rates or spontaneous aging lesions in any of the commonly used wild-type or mutant lines of zebrafish. Therefore, to fully utilize the potential of zebrafish as an animal model for understanding human development, disease, and toxicology we must greatly advance our knowledge on zebrafish diseases and pathology. PMID:12597434

Involvement of histone methyltransferase GLP in HIV-1 latency through catalysis of H3K9 dimethylation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ding, Donglin; Qu, Xiying; Li, Lin

Understanding the mechanism of HIV-1 latency is crucial to eradication of the viral reservoir in HIV-1-infected individuals. However, the role of histone methyltransferase (HMT) G9a-like protein (GLP) in HIV-1 latency is still unclear. In the present work, we established four clonal cell lines containing HIV-1 vector. We found that the integration sites of most clonal cell lines favored active gene regions. However, we also observed hypomethylation of CpG of HIV 5′LTR in all four clonal cell lines. Additionally, 5′-deoxy-5′-methylthioadenosine (MTA), a broad-spectrum histone methyltransferase inhibitor, was used to examine the role of histone methylation in HIV-1 latency. MTA was foundmore » to decrease the level of H3K9 dimethylation, causing reactivation of latent HIV-1 in C11 cells. GLP knockdown by small interfering RNA clearly induced HIV-1 LTR expression. Results suggest that GLP may play a significant role in the maintenance of HIV-1 latency by catalyzing dimethylation of H3K9. - Highlights: ► We have established an in vitro model of HIV-1 latency. ► The integration sites of most clonal cell lines favor in active gene regions. ► Hypomethylation occurs in CpG islands of HIV 5′LTR in all four clonal cell lines. ► MTA can reactivate latent HIV-1 by decreasing the level of H3K9 me2 in C11 cells. ► HMT GLP may play a significant role in the maintenance of HIV-1 latency.« less

Acetyl-CoA carboxylase in Reuber hepatoma cells: variation in enzyme activity, insulin regulation, and cellular lipid content.

PubMed

Bianchi, A; Evans, J L; Nordlund, A C; Watts, T D; Witters, L A

1992-01-01

Reuber hepatoma cells are useful cultured lines for the study of insulin action, lipid and lipoprotein metabolism, and the regulation of acetyl-CoA carboxylase (ACC), the rate-limiting enzyme of fatty acid biosynthesis. During investigations in different clonal lines of these cells, we have uncovered marked intercellular variability in the activity, enzyme content, and insulin regulation of ACC paralleled by differences in cellular neutral lipid (triglyceride) content. Two contrasting clonal lines, Fao and H356A-1, have been studied in detail. Several features distinguish these two lines, including differences in ACC activity and enzyme kinetics, the content of the two major hepatic ACC isozymes (Mr 280,000 and 265,000 Da) and their heteroisozymic complex, the extent of ACC phosphorylation, and the ability of ACC to be activated on stimulation by insulin and insulinomimetic agonists. As studied by Nile Red staining and fluorescence-activated cell sorting, these two lines also display marked differences in neutral lipid content, which correlates with both basal levels of ACC activity and inhibition of ACC by the fatty acid analog, 5-(tetradecyloxy)-2-furoic acid (TOFA). These results emphasize the importance of characterization of any particular clonal line of Reuber cells for studies of enzyme regulation, substrate metabolism, and hormone action. With respect to ACC, studies in contrasting clonal lines of Reuber cells could provide valuable clues to understanding both the complex mechanisms of intracellular ACC regulation in the absence and presence of hormones and its regulatory role(s) in overall hepatic lipid metabolism.

A zebrafish model of chordoma initiated by notochord-driven expression of HRASV12

PubMed Central

Burger, Alexa; Vasilyev, Aleksandr; Tomar, Ritu; Selig, Martin K.; Nielsen, G. Petur; Peterson, Randall T.; Drummond, Iain A.; Haber, Daniel A.

2014-01-01

Chordoma is a malignant tumor thought to arise from remnants of the embryonic notochord, with its origin in the bones of the axial skeleton. Surgical resection is the standard treatment, usually in combination with radiation therapy, but neither chemotherapeutic nor targeted therapeutic approaches have demonstrated success. No animal model and only few chordoma cell lines are available for preclinical drug testing, and, although no druggable genetic drivers have been identified, activation of EGFR and downstream AKT-PI3K pathways have been described. Here, we report a zebrafish model of chordoma, based on stable transgene-driven expression of HRASV12 in notochord cells during development. Extensive intra-notochordal tumor formation is evident within days of transgene expression, ultimately leading to larval death. The zebrafish tumors share characteristics of human chordoma as demonstrated by immunohistochemistry and electron microscopy. The mTORC1 inhibitor rapamycin, which has some demonstrated activity in a chordoma cell line, delays the onset of tumor formation in our zebrafish model, and improves survival of tumor-bearing fish. Consequently, the HRASV12-driven zebrafish model of chordoma could enable high-throughput screening of potential therapeutic agents for the treatment of this refractory cancer. PMID:24311731

A Fully Automated High-Throughput Zebrafish Behavioral Ototoxicity Assay.

PubMed

Todd, Douglas W; Philip, Rohit C; Niihori, Maki; Ringle, Ryan A; Coyle, Kelsey R; Zehri, Sobia F; Zabala, Leanne; Mudery, Jordan A; Francis, Ross H; Rodriguez, Jeffrey J; Jacob, Abraham

2017-08-01

Zebrafish animal models lend themselves to behavioral assays that can facilitate rapid screening of ototoxic, otoprotective, and otoregenerative drugs. Structurally similar to human inner ear hair cells, the mechanosensory hair cells on their lateral line allow the zebrafish to sense water flow and orient head-to-current in a behavior called rheotaxis. This rheotaxis behavior deteriorates in a dose-dependent manner with increased exposure to the ototoxin cisplatin, thereby establishing itself as an excellent biomarker for anatomic damage to lateral line hair cells. Building on work by our group and others, we have built a new, fully automated high-throughput behavioral assay system that uses automated image analysis techniques to quantify rheotaxis behavior. This novel system consists of a custom-designed swimming apparatus and imaging system consisting of network-controlled Raspberry Pi microcomputers capturing infrared video. Automated analysis techniques detect individual zebrafish, compute their orientation, and quantify the rheotaxis behavior of a zebrafish test population, producing a powerful, high-throughput behavioral assay. Using our fully automated biological assay to test a standardized ototoxic dose of cisplatin against varying doses of compounds that protect or regenerate hair cells may facilitate rapid translation of candidate drugs into preclinical mammalian models of hearing loss.

Protein PSMD8 may mediate microgravity-induced cell cycle arrest

NASA Astrophysics Data System (ADS)

Hang, Xiaoming; Sun, Yeqing; Xu, Dan; Wu, Di; Chen, Xiaoning

Microgravity environment of space can induce a serial of changes in cells, such as morphology alterations, cytoskeleton disorder and cell cycle disturbance. Our previous study of simulated-microgravity on zebrafish (Danio rerio) embryos demonstrated 26s proteasome non-ATPase regulatory subunit 8 (PSMD8) might be a microgravity sensitive gene. However, functional study on PSMD8 is very limited and it has not been cloned in zebrafish till now. In this study, we tried to clone PSMD8 gene in zebrafish, quantify its protein expression level in zebrafish embryos after simulated microgravity and identify its possible function in cell cycle regulation. A rotary cell culture system (RCCS) designed by national aeronautics and apace administration (NASA) of America was used to simulate microgravity. The full-length of psmd8 gene in zebrafish was cloned. Preliminary analysis on its sequence and phylogenetic tree construction were carried out subsequently. Quantitative analysis by western blot showed that PSMD8 protein expression levels were significantly increased 1.18 and 1.22 times after 24-48hpf and 24-72hpf simulated microgravity, respectively. Moreover, a significant delay on zebrafish embryo development was found in simulated-microgravity exposed group. Inhibition of PSMD8 protein in zebrafish embryonic cell lines ZF4 could block cell cycle in G1 phase, which indicated that PSMD8 may play a role in cell cycle regulation. Interestingly, simulated-microgravity could also block ZF4 cell in G1 phase. Whether it is PSMD8 mediated cell cycle regulation result in the zebrafish embryo development delay after simulated microgravity exposure still needs further study. Key Words: PSMD8; Simulated-microgravity; Cell cycle; ZF4 cell line

Toxicity Assessment of 17 alpha-ethinylestradiol by Cell-Culture Based NMR Metabolomics

EPA Science Inventory

A zebrafish liver cell line (ZFL) established from adult zebrafish has been used in a variety of biological research, including toxicology, pharmacology, developmental biology and molecular genetics. The goal of this study is to develop an in vitro approach to identify the respo...

Differences in toxicity of anionic and cationic PAMAM and PPI dendrimers in zebrafish embryos and cancer cell lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bodewein, Lambert

Dendrimers are an emerging class of polymeric nanoparticles with beneficial biomedical applications like early diagnostics, in vitro gene transfection or controlled drug delivery. However, the potential toxic impact of exposure on human health or the environment is often inadequately defined. Thus, polyamidoamine (PAMAM) dendrimers of generations G3.0, 3.5, 4.0, 4.5 and 5.0 and polypropylenimine (PPI) dendrimers G3.0, 4.0 and 5.0 were tested in zebrafish embryos for 96 h and human cancer cell lines for 24 h, to assess and compare developmental in vivo toxicity with cytotoxicity. The zebrafish embryo toxicity of cationic PAMAM and PPI dendrimers increased over time, withmore » EC50 values ranging from 0.16 to just below 1.7 μM at 24 and 48 hpf. The predominant effects were mortality, plus reduced heartbeat and blood circulation for PPI dendrimers. Apoptosis in the embryos increased in line with the general toxicity concentration-dependently. Hatch and dechorionation of the embryos increased the toxicity, suggesting a protective role of the chorion. Lower generation dendrimers were more toxic in the embryos whereas the toxicity in the HepG2 and DU145 cell lines increased with increasing generation of cationic PAMAMs and PPI dendrimers. HepG2 were less sensitive than DU145 cells, with IC50 values ≥ 402 μM (PAMAMs) and ≤ 240 μM (PPIs) for HepG2 and ≤ 13.24 μM (PAMAMs) and ≤ 12.84 μM (PPIs) for DU145. Neither in fish embryos nor cells toxicity thresholds were determinable for anionic PAMAM G3.5 and G4.5. The study demonstrated that the cytotoxicity underestimated the in-vivo toxicity of the dendrimers in the fish embryos. - Highlights: • Zebrafish embryo toxicity of cationic PAMAM and PPI dendrimers increased over time. • Zebrafish embryo toxicity of cationic dendrimers did not increase with generation. • Cationic dendrimers induced apoptosis in zebrafish embryos. • Toxicity of cationic dendrimers was lower in HepG2 and DU145 than zebrafish embryos. • Anionic PAMAM dendrimers showed little to no toxicity in fish embryos and cells.« less

Mismatch repair deficiency does not enhance ENU mutagenesis in the zebrafish germ line.

PubMed

Feitsma, Harma; de Bruijn, Ewart; van de Belt, Jose; Nijman, Isaac J; Cuppen, Edwin

2008-07-01

S(N)1-type alkylating agents such as N-ethyl-N-nitrosourea (ENU) are very potent mutagens. They act by transferring their alkyl group to DNA bases, which, upon mispairing during replication, can cause single base pair mutations in the next replication cycle. As DNA mismatch repair (MMR) proteins are involved in the recognition of alkylation damage, we hypothesized that ENU-induced mutation rates could be increased in a MMR-deficient background, which would be beneficial for mutagenesis approaches. We applied a standard ENU mutagenesis protocol to adult zebrafish deficient in the MMR gene msh6 and heterozygous controls to study the effect of MMR on ENU-induced DNA damage. Dose-dependent lethality was found to be similar for homozygous and heterozygous mutants, indicating that there is no difference in ENU resistance. Mutation discovery by high-throughput dideoxy resequencing of genomic targets in outcrossed progeny of the mutagenized fish did also not reveal any differences in germ line mutation frequency. These results may indicate that the maximum mutation load for zebrafish has been reached with the currently used, highly optimized ENU mutagenesis protocol. Alternatively, the MMR system in the zebrafish germ line may be saturated very rapidly, thereby having a limited effect on high-dose ENU mutagenesis.

Seven diverse human embryonic stem cell-derived chondrogenic clonal embryonic progenitor cell lines display site-specific cell fates.

PubMed

Sternberg, Hal; Kidd, Jennifer; Murai, James T; Jiang, Jianjie; Rinon, Ariel; Erickson, Isaac E; Funk, Walter D; Wang, Qian; Chapman, Karen B; Vangsness, C Thomas; West, Michael D

2013-03-01

The transcriptomes of seven diverse clonal human embryonic progenitor cell lines with chondrogenic potential were compared with that of bone marrow-derived mesenchymal stem cells (MSCs). The cell lines 4D20.8, 7PEND24, 7SMOO32, E15, MEL2, SK11 and SM30 were compared with MSCs using immunohistochemical methods, gene expression microarrays and quantitative real-time PCR. In the undifferentiated progenitor state, each line displayed unique combinations of site-specific markers, including AJAP1, ALDH1A2, BMP5, BARX1, HAND2, HOXB2, LHX1, LHX8, PITX1, TBX15 and ZIC2, but none of the lines expressed the MSC marker CD74. The lines showed diverse responses when differentiated in the presence of combinations of TGF-β3, BMP2, 4, 6 and 7 and GDF5, with the lines 4D20.8, SK11, SM30 and MEL2 showing osteogenic markers in some differentiation conditions. The line 7PEND24 showed evidence of regenerating articular cartilage and, in some conditions, markers of tendon differentiation. The scalability of site-specific clonal human embryonic stem cell-derived embryonic progenitor cell lines may provide novel models for the study of differentiation and methods for preparing purified and identified cells types for use in therapy.

A privileged intraphagocyte niche is responsible for disseminated infection of Staphylococcus aureus in a zebrafish model

PubMed Central

Prajsnar, Tomasz K; Hamilton, Ruth; Garcia-Lara, Jorge; McVicker, Gareth; Williams, Alexander; Boots, Michael; Foster, Simon J; Renshaw, Stephen A

2012-01-01

The innate immune system is the primary defence against the versatile pathogen, Staphylococcus aureus. How this organism is able to avoid immune killing and cause infections is poorly understood. Using an established larval zebrafish infection model, we have shown that overwhelming infection is due to subversion of phagocytes by staphylococci, allowing bacteria to evade killing and found foci of disease. Larval zebrafish coinfected with two S. aureus strains carrying different fluorescent reporter gene fusions (but otherwise isogenic) had bacterial lesions, at the time of host death, containing predominantly one strain. Quantitative data using two marked strains revealed that the strain ratios, during overwhelming infection, were often skewed towards the extremes, with one strain predominating. Infection with passaged bacterial clones revealed the phenomenon not to bedue to adventitious mutations acquired by the pathogen. After infection of the host, all bacteria are internalized by phagocytes and the skewing of population ratios is absolutely dependent on the presence of phagocytes. Mathematical modelling of pathogen population dynamics revealed the data patterns are consistent with the hypothesis that a small number of infected phagocytes serve as an intracellular reservoir for S. aureus, which upon release leads to disseminated infection. Strategies to specifically alter neutrophil/macrophage numbers were used to map the potential subpopulation of phagocytes acting as a pathogen reservoir, revealing neutrophils as the likely ‘niche’. Subsequently in a murine sepsis model, S. aureus abscesses in kidneys were also found to be predominantly clonal, therefore likely founded by an individual cell, suggesting a potential mechanism analogous to the zebrafish model with few protected niches. These findings add credence to the argument that S. aureus control regimes should recognize both the intracellular as well as extracellular facets of the S. aureus life cycle. PMID:22694745

Arrested development of the myxozoan parasite, Myxobolus cerebralis, in certain populations of mitochondrial 16S lineage III Tubifex tubifex.

PubMed

Baxa, D V; Kelley, G O; Mukkatira, K S; Beauchamp, K A; Rasmussen, C; Hedrick, R P

2008-01-01

Laboratory populations of Tubifex tubifex from mitochondrial (mt)16S ribosomal DNA (rDNA) lineage III were generated from single cocoons of adult worms releasing the triactinomyxon stages (TAMs) of the myxozoan parasite, Myxobolus cerebralis. Subsequent worm populations from these cocoons, referred to as clonal lines, were tested for susceptibility to infection with the myxospore stages of M. cerebralis. Development and release of TAMs occurred in five clonal lines, while four clonal lines showed immature parasitic forms that were not expelled from the worm (non-TAM producers). Oligochaetes from TAM- and non-TAM-producing clonal lines were confirmed as lineage III based on mt16S rDNA and internal transcribed spacer region 1 (ITS1) sequences, but these genes did not differentiate these phenotypes. In contrast, random amplified polymorphic DNA analyses of genomic DNA demonstrated unique banding patterns that distinguished the phenotypes. Cohabitation of parasite-exposed TAM- and non-TAM-producing phenotypes showed an overall decrease in expected TAM production compared to the same exposure dose of the TAM-producing phenotype without cohabitation. These studies suggest that differences in susceptibility to parasite infection can occur in genetically similar T. tubifex populations, and their coexistence may affect overall M. cerebralis production, a factor that may influence the severity of whirling disease in wild trout populations.

Arrested development of the myxozoan parasite, Myxobolus cerebralis, in certain populations of mitochondrial 16S lineage III Tubifex tubifex

USGS Publications Warehouse

Baxa, D.V.; Kelley, G.O.; Mukkatira, K.S.; Beauchamp, K.A.; Rasmussen, C.; Hedrick, R.P.

2008-01-01

Laboratory populations of Tubifex tubifex from mitochondrial (mt)16S ribosomal DNA (rDNA) lineage III were generated from single cocoons of adult worms releasing the triactinomyxon stages (TAMs) of the myxozoan parasite, Myxobolus cerebralis. Subsequent worm populations from these cocoons, referred to as clonal lines, were tested for susceptibility to infection with the myxospore stages of M. cerebralis. Development and release of TAMs occurred in five clonal lines, while four clonal lines showed immature parasitic forms that were not expelled from the worm (non-TAM producers). Oligochaetes from TAM- and non-TAM-producing clonal lines were confirmed as lineage III based on mt16S rDNA and internal transcribed spacer region 1 (ITS1) sequences, but these genes did not differentiate these phenotypes. In contrast, random amplified polymorphic DNA analyses of genomic DNA demonstrated unique banding patterns that distinguished the phenotypes. Cohabitation of parasite-exposed TAM- and non-TAM-producing phenotypes showed an overall decrease in expected TAM production compared to the same exposure dose of the TAM-producing phenotype without cohabitation. These studies suggest that differences in susceptibility to parasite infection can occur in genetically similar T. tubifex populations, and their coexistence may affect overall M. cerebralis production, a factor that may influence the severity of whirling disease in wild trout populations. ?? 2007 Springer-Verlag.

Pineal-specific expression of green fluorescent protein under the control of the serotonin-N-acetyltransferase gene regulatory regions in transgenic zebrafish.

PubMed

Gothilf, Yoav; Toyama, Reiko; Coon, Steven L; Du, Shao-Jun; Dawid, Igor B; Klein, David C

2002-11-01

Zebrafish serotonin-N-acetyltransferase-2 (zfAANAT-2) mRNA is exclusively expressed in the pineal gland (epiphysis) at the embryonic stage. Here, we have initiated an effort to study the mechanisms underlying tissue-specific expression of this gene. DNA constructs were prepared in which green fluorescent protein (GFP) is driven by regulatory regions of the zfAANAT-2 gene. In vivo transient expression analysis in zebrafish embryos indicated that in addition to the 5'-flanking region, a regulatory sequence in the 3'-flanking region is required for pineal-specific expression. This finding led to an effort to produce transgenic lines expressing GFP under the control of the 5' and 3' regulatory regions of the zfAANAT-2 gene. Embryos transiently expressing GFP were raised to maturity and tested for germ cell transmission of the transgene. Three transgenic lines were produced in which GFP fluorescence in the pineal was detected starting 1 to 2 days after fertilization. One line was crossed with mindbomb and floating head mutants that cause abnormal development of the pineal and an elevation or reduction of zfAANAT-2 mRNA levels, respectively. Homozygous mutant transgenic embryos exhibited similar effects on GFP expression in the pineal gland. These observations indicate that the transgenic lines described here will be useful in studying the development of the pineal gland and the mechanisms that determine pineal-specific gene expression in the zebrafish. Published 2002 Wiley-Liss, Inc.

Surface Defects on Plate-Shaped Silver Nanoparticles Contribute to Its Hazard Potential in a Fish Gill Cell Line and Zebrafish Embyos

PubMed Central

George, Saji; Lin, Sijie; Ji, Zhaoxia; Thomas, Courtney; Li, LinJiang; Mecklenburg, Mathew; Meng, Huan; Wang, Xiang; Zhang, Haiyuan; Xia, Tian; Lin, Shuo; Hohman, J. Nathan; Zink, Jeffrey I.; Weiss, Paul; Nel, André E.

2014-01-01

We investigated and compared nano-size Ag spheres, plates, and wires in a fish gill epithelial cell line (RT-W1) and in zebrafish embryos to understand the mechanism of toxicity of an engineered nanomaterial raising considerable environmental concern. While most of the Ag nanoparticles induced N-acetyl cysteine sensitive toxic oxidative stress effects in RT-W1, Ag nanoplates were considerably more toxic than other particle shapes. Interestingly, while Ag ion shedding and bioavailability failed to explain the high toxicity of the nanoplates, cellular injury required direct particle contact, resulting in cell membrane lysis in RT-W1 as well as red blood cells (RBC). Ag nanoplates were also considerably more toxic in zebrafish embryos in spite of their lesser ability to shed Ag into the exposure medium. In order to elucidate the “surface reactivity” of Ag nanoplates, high-resolution transmission electron microscopy was performed and demonstrated a high level of crystal defects (stacking faults and point defects) on the nanoplate surfaces. Surface coating with cysteine was used to passivate the surface defects and demonstrated a reduction of toxicity in RT-W1 cells, RBC, and zebrafish embryos. This study demonstrates the important role of crystal defects in contributing to Ag nanoparticle toxicity in addition to the established roles of Ag ion shed from spherical nanoparticles. The excellent correlation between the in vitro and in vivo toxicological assessment illustrates the utility of using a fish cell line in parallel with zebrafish embryos to perform a predictive environmental toxicological paradigm. PMID:22482460

Enhanced Cell-Specific Ablation in Zebrafish Using a Triple Mutant of Escherichia Coli Nitroreductase

PubMed Central

Mathias, Jonathan R.; Zhang, Zhanying; Saxena, Meera T.

2014-01-01

Abstract Transgenic expression of bacterial nitroreductase (NTR) facilitates chemically-inducible targeted cell ablation. In zebrafish, the NTR system enables studies of cell function and cellular regeneration. Metronidazole (MTZ) has become the most commonly used prodrug substrate for eliciting cell loss in NTR-expressing transgenic zebrafish due to the cell-specific nature of its cytotoxic derivatives. Unfortunately, MTZ treatments required for effective cell ablation border toxic effects, and, thus, likely incur undesirable nonspecific effects. Here, we tested whether a triple mutant variant of NTR, previously shown to display improved activity in bacterial assays, can solve this issue by promoting cell ablation in zebrafish using reduced prodrug treatment regimens. We generated several complementary transgenic zebrafish lines expressing either wild-type or mutant NTR (mutNTR) in specific neural cell types, and assayed prodrug-induced cell ablation kinetics using confocal time series imaging and plate reader-based quantification of fluorescent reporters expressed in targeted cell types. The results show that cell ablation can be achieved in mutNTR expressing transgenic lines with markedly shortened prodrug exposure times and/or at lower prodrug concentrations. The mutNTR variant characterized here can circumvent problematic nonspecific/toxic effects arising from low prodrug conversion efficiency, thus increasing the effectiveness and versatility of this selective cell ablation methodology. PMID:24428354

Enhanced cell-specific ablation in zebrafish using a triple mutant of Escherichia coli nitroreductase.

PubMed

Mathias, Jonathan R; Zhang, Zhanying; Saxena, Meera T; Mumm, Jeff S

2014-04-01

Transgenic expression of bacterial nitroreductase (NTR) facilitates chemically-inducible targeted cell ablation. In zebrafish, the NTR system enables studies of cell function and cellular regeneration. Metronidazole (MTZ) has become the most commonly used prodrug substrate for eliciting cell loss in NTR-expressing transgenic zebrafish due to the cell-specific nature of its cytotoxic derivatives. Unfortunately, MTZ treatments required for effective cell ablation border toxic effects, and, thus, likely incur undesirable nonspecific effects. Here, we tested whether a triple mutant variant of NTR, previously shown to display improved activity in bacterial assays, can solve this issue by promoting cell ablation in zebrafish using reduced prodrug treatment regimens. We generated several complementary transgenic zebrafish lines expressing either wild-type or mutant NTR (mutNTR) in specific neural cell types, and assayed prodrug-induced cell ablation kinetics using confocal time series imaging and plate reader-based quantification of fluorescent reporters expressed in targeted cell types. The results show that cell ablation can be achieved in mutNTR expressing transgenic lines with markedly shortened prodrug exposure times and/or at lower prodrug concentrations. The mutNTR variant characterized here can circumvent problematic nonspecific/toxic effects arising from low prodrug conversion efficiency, thus increasing the effectiveness and versatility of this selective cell ablation methodology.

A zebrafish model of chordoma initiated by notochord-driven expression of HRASV12.

PubMed

Burger, Alexa; Vasilyev, Aleksandr; Tomar, Ritu; Selig, Martin K; Nielsen, G Petur; Peterson, Randall T; Drummond, Iain A; Haber, Daniel A

2014-07-01

Chordoma is a malignant tumor thought to arise from remnants of the embryonic notochord, with its origin in the bones of the axial skeleton. Surgical resection is the standard treatment, usually in combination with radiation therapy, but neither chemotherapeutic nor targeted therapeutic approaches have demonstrated success. No animal model and only few chordoma cell lines are available for preclinical drug testing, and, although no druggable genetic drivers have been identified, activation of EGFR and downstream AKT-PI3K pathways have been described. Here, we report a zebrafish model of chordoma, based on stable transgene-driven expression of HRASV12 in notochord cells during development. Extensive intra-notochordal tumor formation is evident within days of transgene expression, ultimately leading to larval death. The zebrafish tumors share characteristics of human chordoma as demonstrated by immunohistochemistry and electron microscopy. The mTORC1 inhibitor rapamycin, which has some demonstrated activity in a chordoma cell line, delays the onset of tumor formation in our zebrafish model, and improves survival of tumor-bearing fish. Consequently, the HRASV12-driven zebrafish model of chordoma could enable high-throughput screening of potential therapeutic agents for the treatment of this refractory cancer. © 2014. Published by The Company of Biologists Ltd.

Single-cell transcriptional analysis of normal, aberrant, and malignant hematopoiesis in zebrafish.

PubMed

Moore, Finola E; Garcia, Elaine G; Lobbardi, Riadh; Jain, Esha; Tang, Qin; Moore, John C; Cortes, Mauricio; Molodtsov, Aleksey; Kasheta, Melissa; Luo, Christina C; Garcia, Amaris J; Mylvaganam, Ravi; Yoder, Jeffrey A; Blackburn, Jessica S; Sadreyev, Ruslan I; Ceol, Craig J; North, Trista E; Langenau, David M

2016-05-30

Hematopoiesis culminates in the production of functionally heterogeneous blood cell types. In zebrafish, the lack of cell surface antibodies has compelled researchers to use fluorescent transgenic reporter lines to label specific blood cell fractions. However, these approaches are limited by the availability of transgenic lines and fluorescent protein combinations that can be distinguished. Here, we have transcriptionally profiled single hematopoietic cells from zebrafish to define erythroid, myeloid, B, and T cell lineages. We also used our approach to identify hematopoietic stem and progenitor cells and a novel NK-lysin 4(+) cell type, representing a putative cytotoxic T/NK cell. Our platform also quantified hematopoietic defects in rag2(E450fs) mutant fish and showed that these fish have reduced T cells with a subsequent expansion of NK-lysin 4(+) cells and myeloid cells. These data suggest compensatory regulation of the innate immune system in rag2(E450fs) mutant zebrafish. Finally, analysis of Myc-induced T cell acute lymphoblastic leukemia showed that cells are arrested at the CD4(+)/CD8(+) cortical thymocyte stage and that a subset of leukemia cells inappropriately reexpress stem cell genes, including bmi1 and cmyb In total, our experiments provide new tools and biological insights into single-cell heterogeneity found in zebrafish blood and leukemia. © 2016 Moore et al.

Single-cell transcriptional analysis of normal, aberrant, and malignant hematopoiesis in zebrafish

PubMed Central

Garcia, Elaine G.; Lobbardi, Riadh; Jain, Esha; Tang, Qin; Moore, John C.; Cortes, Mauricio; Molodtsov, Aleksey; Kasheta, Melissa; Luo, Christina C.; Garcia, Amaris J.; Mylvaganam, Ravi; Yoder, Jeffrey A.; Blackburn, Jessica S.; Sadreyev, Ruslan I.; Ceol, Craig J.; North, Trista E.

2016-01-01

Hematopoiesis culminates in the production of functionally heterogeneous blood cell types. In zebrafish, the lack of cell surface antibodies has compelled researchers to use fluorescent transgenic reporter lines to label specific blood cell fractions. However, these approaches are limited by the availability of transgenic lines and fluorescent protein combinations that can be distinguished. Here, we have transcriptionally profiled single hematopoietic cells from zebrafish to define erythroid, myeloid, B, and T cell lineages. We also used our approach to identify hematopoietic stem and progenitor cells and a novel NK-lysin 4+ cell type, representing a putative cytotoxic T/NK cell. Our platform also quantified hematopoietic defects in rag2E450fs mutant fish and showed that these fish have reduced T cells with a subsequent expansion of NK-lysin 4+ cells and myeloid cells. These data suggest compensatory regulation of the innate immune system in rag2E450fs mutant zebrafish. Finally, analysis of Myc-induced T cell acute lymphoblastic leukemia showed that cells are arrested at the CD4+/CD8+ cortical thymocyte stage and that a subset of leukemia cells inappropriately reexpress stem cell genes, including bmi1 and cmyb. In total, our experiments provide new tools and biological insights into single-cell heterogeneity found in zebrafish blood and leukemia. PMID:27139488

Zebrafish xenograft models of cancer and metastasis for drug discovery.

PubMed

Brown, Hannah K; Schiavone, Kristina; Tazzyman, Simon; Heymann, Dominique; Chico, Timothy Ja

2017-04-01

Patients with metastatic cancer suffer the highest rate of cancer-related death, but existing animal models of metastasis have disadvantages that limit our ability to understand this process. The zebrafish is increasingly used for cancer modelling, particularly xenografting of human cancer cell lines, and drug discovery, and may provide novel scientific and therapeutic insights. However, this model system remains underexploited. Areas covered: The authors discuss the advantages and disadvantages of the zebrafish xenograft model for the study of cancer, metastasis and drug discovery. They summarise previous work investigating the metastatic cascade, such as tumour-induced angiogenesis, intravasation, extravasation, dissemination and homing, invasion at secondary sites, assessing metastatic potential and evaluation of cancer stem cells in zebrafish. Expert opinion: The practical advantages of zebrafish for basic biological study and drug discovery are indisputable. However, their ability to sufficiently reproduce and predict the behaviour of human cancer and metastasis remains unproven. For this to be resolved, novel mechanisms must to be discovered in zebrafish that are subsequently validated in humans, and for therapeutic interventions that modulate cancer favourably in zebrafish to successfully translate to human clinical studies. In the meantime, more work is required to establish the most informative methods in zebrafish.

A Retrospective Study of the Prevalence and Classification of Intestinal Neoplasia in Zebrafish (Danio Rerio)

PubMed Central

Paquette, Colleen E.; Buchner, Cari; Tanguay, Robert L.; Guillemin, Karen; Mason, Timothy J.; Peterson, Tracy S.

2013-01-01

Abstract For over a decade, spontaneous intestinal neoplasia has been observed in zebrafish (Danio rerio) submitted to the ZIRC (Zebrafish International Resource Center) diagnostic service. In addition, zebrafish displayed preneoplastic intestinal changes including hyperplasia, dysplasia, and enteritis. A total of 195 zebrafish, representing 2% of the total fish submitted to the service, were diagnosed with these lesions. Neoplastic changes were classified either as adenocarcinoma or small cell carcinoma, with a few exceptions (carcinoma not otherwise specified, tubular adenoma, and tubulovillous adenoma). Tumor prevalence appeared similarly distributed between sexes and generally occurred in zebrafish greater than 1 year of age, although neoplastic changes were observed in fish 6 months of age. Eleven lines displayed these preneoplastic and neoplastic changes, including wild-types and mutants. Affected zebrafish originated from 18 facilities, but the majority of fish were from a single zebrafish research facility (hereafter referred to as the primary facility) that has submitted numerous samples to the ZIRC diagnostic service. Zebrafish from the primary facility submitted as normal sentinel fish demonstrate that these lesions are most often subclinical. Fish fed the diet from the primary facility and held at another location did not develop intestinal lesions, indicating that diet is not the etiologic agent. PMID:23544991

Functional Development of the Circadian Clock in the Zebrafish Pineal Gland

PubMed Central

Ben-Moshe, Zohar; Foulkes, Nicholas S.

2014-01-01

The zebrafish constitutes a powerful model organism with unique advantages for investigating the vertebrate circadian timing system and its regulation by light. In particular, the remarkably early and rapid development of the zebrafish circadian system has facilitated exploring the factors that control the onset of circadian clock function during embryogenesis. Here, we review our understanding of the molecular basis underlying functional development of the central clock in the zebrafish pineal gland. Furthermore, we examine how the directly light-entrainable clocks in zebrafish cell lines have facilitated unravelling the general mechanisms underlying light-induced clock gene expression. Finally, we summarize how analysis of the light-induced transcriptome and miRNome of the zebrafish pineal gland has provided insight into the regulation of the circadian system by light, including the involvement of microRNAs in shaping the kinetics of light- and clock-regulated mRNA expression. The relative contributions of the pineal gland central clock and the distributed peripheral oscillators to the synchronization of circadian rhythms at the whole animal level are a crucial question that still remains to be elucidated in the zebrafish model. PMID:24839600

Oceans of opportunity: exploring vertebrate hematopoiesis in zebrafish.

PubMed

Carroll, Kelli J; North, Trista E

2014-08-01

Exploitation of the zebrafish model in hematology research has surged in recent years, becoming one of the most useful and tractable systems for understanding regulation of hematopoietic development, homeostasis, and malignancy. Despite the evolutionary distance between zebrafish and humans, remarkable genetic and phenotypic conservation in the hematopoietic system has enabled significant advancements in our understanding of blood stem and progenitor cell biology. The strengths of zebrafish in hematology research lie in the ability to perform real-time in vivo observations of hematopoietic stem, progenitor, and effector cell emergence, expansion, and function, as well as the ease with which novel genetic and chemical modifiers of specific hematopoietic processes or cell types can be identified and characterized. Further, myriad transgenic lines have been developed including fluorescent reporter systems to aid in the visualization and quantification of specified cell types of interest and cell-lineage relationships, as well as effector lines that can be used to implement a wide range of experimental manipulations. As our understanding of the complex nature of blood stem and progenitor cell biology during development, in response to infection or injury, or in the setting of hematologic malignancy continues to deepen, zebrafish will remain essential for exploring the spatiotemporal organization and integration of these fundamental processes, as well as the identification of efficacious small molecule modifiers of hematopoietic activity. In this review, we discuss the biology of the zebrafish hematopoietic system, including similarities and differences from mammals, and highlight important tools currently utilized in zebrafish embryos and adults to enhance our understanding of vertebrate hematology, with emphasis on findings that have impacted our understanding of the onset or treatment of human hematologic disorders and disease. Copyright © 2014 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

Oceans of Opportunity: Exploring Vertebrate Hematopoiesis in Zebrafish

PubMed Central

Carroll, Kelli J.; North, Trista E.

2015-01-01

Exploitation of the zebrafish model in hematology research has surged in recent years, becoming one of the most useful and tractable systems for understanding regulation of hematopoietic development, homeostasis, and malignancy. Despite the evolutionary distance between zebrafish and humans, remarkable genetic and phenotypic conservation in the hematopoietic system has enabled significant advancements in our understanding of blood stem and progenitor cell (HSPC) biology. The strengths of zebrafish in hematology research lie in the ability to perform real-time in vivo observations of hematopoietic stem, progenitor and effector cell emergence, expansion and function, as well as the ease with which novel genetic and chemical modifiers of specific hematopoietic processes or cell-types can be identified and characterized. Further, a myriad of transgenic lines have been developed including fluorescent reporter systems to aid in the visualization and quantification of specified cell types of interest and cell-lineage relationships, as well as effector lines that can be used to implement a wide range of experimental manipulations. As our understanding of the complex nature of HSPC biology during development, in response to infection or injury, or in the setting of hematological malignancy, continues to deepen, zebrafish will remain essential for exploring the spatio-temporal organization and integration of these fundamental processes, as well as the identification of efficacious small molecule modifiers of hematopoietic activity. In this review, we discuss the biology of the zebrafish hematopoietic system, including similarities and differences from mammals, and highlight important tools currently utilized in zebrafish embryos and adults to enhance our understanding of vertebrate hematology, with emphasis on findings that have impacted our understanding of the onset or treatment of human hematologic disorders and disease. PMID:24816275

Using Zebrafish Models of Human Influenza A Virus Infections to Screen Antiviral Drugs and Characterize Host Immune Cell Responses.

PubMed

Sullivan, Con; Jurcyzszak, Denise; Goody, Michelle F; Gabor, Kristin A; Longfellow, Jacob R; Millard, Paul J; Kim, Carol H

2017-01-20

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare burden. The principle strategy to curtail infections is yearly vaccination. In individuals who have contracted influenza, antiviral drugs can mitigate symptoms. There is a clear and unmet need to develop alternative strategies to combat influenza. Several animal models have been created to model host-influenza interactions. Here, protocols for generating zebrafish models for systemic and localized human influenza A virus (IAV) infection are described. Using a systemic IAV infection model, small molecules with potential antiviral activity can be screened. As a proof-of-principle, a protocol that demonstrates the efficacy of the antiviral drug Zanamivir in IAV-infected zebrafish is described. It shows how disease phenotypes can be quantified to score the relative efficacy of potential antivirals in IAV-infected zebrafish. In recent years, there has been increased appreciation for the critical role neutrophils play in the human host response to influenza infection. The zebrafish has proven to be an indispensable model for the study of neutrophil biology, with direct impacts on human medicine. A protocol to generate a localized IAV infection in the Tg(mpx:mCherry) zebrafish line to study neutrophil biology in the context of a localized viral infection is described. Neutrophil recruitment to localized infection sites provides an additional quantifiable phenotype for assessing experimental manipulations that may have therapeutic applications. Both zebrafish protocols described faithfully recapitulate aspects of human IAV infection. The zebrafish model possesses numerous inherent advantages, including high fecundity, optical clarity, amenability to drug screening, and availability of transgenic lines, including those in which immune cells such as neutrophils are labeled with fluorescent proteins. The protocols detailed here exploit these advantages and have the potential to reveal critical insights into host-IAV interactions that may ultimately translate into the clinic.

Grouper tshβ Promoter-Driven Transgenic Zebrafish Marks Proximal Kidney Tubule Development

PubMed Central

Wang, Yang; Sun, Zhi-Hui; Zhou, Li; Li, Zhi; Gui, Jian-Fang

2014-01-01

Kidney tubule plays a critical role in recovering or secreting solutes, but the detailed morphogenesis remains unclear. Our previous studies have found that grouper tshβ (gtshβ) is also expressed in kidney, however, the distribution significance is still unknown. To understand the gtshβ role and kidney tubule morphogenesis, here, we have generated a transgenic zebrafish line Tg(gtshβ:GFP) with green fluorescent protein driven by the gtshβ promoter. Similar to the endogenous tshβ in zebrafish or in grouper, the gtshβ promoter-driven GFP is expressed in pituitary and kidney, and the developing details of proximal kidney tubule are marked in the transgenic zebrafish line. The gfp initially transcribes at 16 hours post fertilization (hpf) above the dorsal mesentery, and partially co-localizes with pronephric tubular markers slc20a1a and cdh17. Significantly, the GFP specifically localizes in proximal pronephric segments during embryogenesis and resides at kidney duct epithelium in adult fish. To test whether the gtshβ promoter-driven GFP may serve as a readout signal of the tubular development, we have treated the embryos with retinoic acid signaing (RA) reagents, in which exogenous RA addition results in a distal extension of the proximal segments, while RA inhibition induces a weakness and shortness of the proximal segments. Therefore, this transgenic line provides a useful tool for genetic or chemical analysis of kidney tubule. PMID:24905828

Morphologic analysis of the zebrafish digestive system.

PubMed

Trotter, Andrew J; Parslow, Adam C; Heath, Joan K

2009-01-01

The zebrafish provides an ideal model for the study of vertebrate organogenesis, including the formation of the digestive tract and its associated organs. Despite optical transparency of embryos, the internal position of the developing digestive system and its close juxtaposition with the yolk initially made morphological analysis relatively challenging, particularly during the first 3 d of development. However, methodologies have been successfully developed to address these problems and comprehensive morphologic analysis of the developing digestive system has now been achieved using a combination of light and fluorescence microscope approaches-including confocal analysis-to visualize wholemount and histological preparations of zebrafish embryos. Furthermore, the expanding number of antibodies that cross-react with zebrafish proteins and the generation of tissue-specific transgenic green fluorescent protein reporter lines that mark specific cell and tissue compartments have greatly enhanced our ability to successfully image the developing zebrafish digestive system.

A TALEN-Exon Skipping Design for a Bethlem Myopathy Model in Zebrafish.

PubMed

Radev, Zlatko; Hermel, Jean-Michel; Elipot, Yannick; Bretaud, Sandrine; Arnould, Sylvain; Duchateau, Philippe; Ruggiero, Florence; Joly, Jean-Stéphane; Sohm, Frédéric

2015-01-01

Presently, human collagen VI-related diseases such as Ullrich congenital muscular dystrophy (UCMD) and Bethlem myopathy (BM) remain incurable, emphasizing the need to unravel their etiology and improve their treatments. In UCMD, symptom onset occurs early, and both diseases aggravate with ageing. In zebrafish fry, morpholinos reproduced early UCMD and BM symptoms but did not allow to study the late phenotype. Here, we produced the first zebrafish line with the human mutation frequently found in collagen VI-related disorders such as UCMD and BM. We used a transcription activator-like effector nuclease (TALEN) to design the col6a1ama605003-line with a mutation within an essential splice donor site, in intron 14 of the col6a1 gene, which provoke an in-frame skipping of exon 14 in the processed mRNA. This mutation at a splice donor site is the first example of a template-independent modification of splicing induced in zebrafish using a targetable nuclease. This technique is readily expandable to other organisms and can be instrumental in other disease studies. Histological and ultrastructural analyzes of homozygous and heterozygous mutant fry and 3 months post-fertilization (mpf) fish revealed co-dominantly inherited abnormal myofibers with disorganized myofibrils, enlarged sarcoplasmic reticulum, altered mitochondria and misaligned sarcomeres. Locomotion analyzes showed hypoxia-response behavior in 9 mpf col6a1 mutant unseen in 3 mpf fish. These symptoms worsened with ageing as described in patients with collagen VI deficiency. Thus, the col6a1ama605003-line is the first adult zebrafish model of collagen VI-related diseases; it will be instrumental both for basic research and drug discovery assays focusing on this type of disorders.

A TALEN-Exon Skipping Design for a Bethlem Myopathy Model in Zebrafish

PubMed Central

Elipot, Yannick; Bretaud, Sandrine; Arnould, Sylvain; Duchateau, Philippe; Ruggiero, Florence; Joly, Jean-Stéphane; Sohm, Frédéric

2015-01-01

Presently, human collagen VI-related diseases such as Ullrich congenital muscular dystrophy (UCMD) and Bethlem myopathy (BM) remain incurable, emphasizing the need to unravel their etiology and improve their treatments. In UCMD, symptom onset occurs early, and both diseases aggravate with ageing. In zebrafish fry, morpholinos reproduced early UCMD and BM symptoms but did not allow to study the late phenotype. Here, we produced the first zebrafish line with the human mutation frequently found in collagen VI-related disorders such as UCMD and BM. We used a transcription activator-like effector nuclease (TALEN) to design the col6a1ama605003-line with a mutation within an essential splice donor site, in intron 14 of the col6a1 gene, which provoke an in-frame skipping of exon 14 in the processed mRNA. This mutation at a splice donor site is the first example of a template-independent modification of splicing induced in zebrafish using a targetable nuclease. This technique is readily expandable to other organisms and can be instrumental in other disease studies. Histological and ultrastructural analyzes of homozygous and heterozygous mutant fry and 3 months post-fertilization (mpf) fish revealed co-dominantly inherited abnormal myofibers with disorganized myofibrils, enlarged sarcoplasmic reticulum, altered mitochondria and misaligned sarcomeres. Locomotion analyzes showed hypoxia-response behavior in 9 mpf col6a1 mutant unseen in 3 mpf fish. These symptoms worsened with ageing as described in patients with collagen VI deficiency. Thus, the col6a1ama605003-line is the first adult zebrafish model of collagen VI-related diseases; it will be instrumental both for basic research and drug discovery assays focusing on this type of disorders. PMID:26221953

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

PubMed Central

Bernut, Audrey; Dupont, Christian; Sahuquet, Alain; Herrmann, Jean-Louis; Lutfalla, Georges; Kremer, Laurent

2015-01-01

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen interactions. The ease of obtaining large numbers of embryos, their accessibility due to external development, their optical transparency as well as the availability of a wide panoply of genetic/immunological tools and transgenic reporter line collections, contribute to the versatility of this model. In this respect, the present manuscript describes the use of zebrafish as an in vivo model system to investigate the chronology of Mycobacterium abscessus infection. This human pathogen can exist either as smooth (S) or rough (R) variants, depending on cell wall composition, and their respective virulence can be imaged and compared in zebrafish embryos and larvae. Micro-injection of either S or R fluorescent variants directly in the blood circulation via the caudal vein, leads to chronic or acute/lethal infections, respectively. This biological system allows high resolution visualization and analysis of the role of mycobacterial cording in promoting abscess formation. In addition, the use of fluorescent bacteria along with transgenic zebrafish lines harbouring fluorescent macrophages produces a unique opportunity for multi-color imaging of the host-pathogen interactions. This article describes detailed protocols for the preparation of homogenous M. abscessus inoculum and for intravenous injection of zebrafish embryos for subsequent fluorescence imaging of the interaction with macrophages. These techniques open the avenue to future investigations involving mutants defective in cord formation and are dedicated to understand how this impacts on M. abscessus pathogenicity in a whole vertebrate. PMID:26382225

The kiss/kissr Systems Are Dispensable for Zebrafish Reproduction: Evidence From Gene Knockout Studies

PubMed Central

Tang, Haipei; Liu, Yun; Luo, Daji; Ogawa, Satoshi; Yin, Yike; Li, Shuisheng; Zhang, Yong; Hu, Wei; Parhar, Ishwar S.; Lin, Haoran

2015-01-01

The kiss1/gpr54 signaling system is considered to be a critical regulator of reproduction in most vertebrates. However, this presumption has not been tested vigorously in nonmammalian vertebrates. Distinct from mammals, multiple kiss1/gpr54 paralogous genes (kiss/kissr) have been identified in nonmammalian vertebrates, raising the possibility of functional redundancy among these genes. In this study, we have systematically generated the zebrafish kiss1−/−, kiss2−/−, and kiss1−/−;kiss2−/− mutant lines as well as the kissr1−/−, kissr2−/−, and kissr1−/−;kissr2−/− mutant lines using transcription activator-like effector nucleases. We have demonstrated that spermatogenesis and folliculogenesis as well as reproductive capability are not impaired in all of these 6 mutant lines. Collectively, our results indicate that kiss/kissr signaling is not absolutely required for zebrafish reproduction, suggesting that the kiss/kissr systems play nonessential roles for reproduction in certain nonmammalian vertebrates. These findings also demonstrated that fish and mammals have evolved different strategies for neuroendocrine control of reproduction. PMID:25406015

Dihydroartemisinin promotes angiogenesis during the early embryonic development of zebrafish

PubMed Central

Ba, Qian; Duan, Juan; Tian, Jia-qiang; Wang, Zi-liang; Chen, Tao; Li, Xiao-guang; Chen, Pei-zhan; Wu, Song-jie; Xiang, Li; Li, Jing-quan; Chu, Rui-ai; Wang, Hui

2013-01-01

Aim: To investigate the embryotoxicity of dihydroartemisinin (DHA), the main active metabolite of artemisinin, in zebrafish, and explore the corresponding mechanisms. Methods: The embryos of wild type and TG (flk1:GFP) transgenic zebrafish were exposed to DHA. Developmental phenotypes of the embryos were observed. Development of blood vessels was directly observed in living embryos of TG (flk1:GFP) transgenic zebrafish under fluorescence microscope. The expression of angiogenesis marker genes vegfa, flk1, and flt1 in the embryos was detected using real-time PCR and RNA in situ hybridization assays. Results: Exposure to DHA (1–10 mg/L) dose-dependently caused abnormal zebrafish embryonic phenotypes in the early developmental stage. Furthermore, exposure to DHA (10 mg/L) resulted in more pronounced embryonic angiogenesis in TG (flk1:GFP) zebrafish line. Exposure to DHA (10 mg/L) significantly increased the mRNA expression of vegfa, flk1, and flt1 in the embryos. Knockdown of the flk1 protein partially blocked the effects of DHA on embryogenesis. Conclusion: DHA causes abnormal embryonic phenotypes and promotes angiogenesis in zebrafish early embryonic development, demonstrating the potential embryotoxicity of DHA. PMID:23708556

CHROMOSOME 11 ABERRATIONS IN SMALL COLONY L5178Y TK-/-MUTANTS EARLY IN THEIR CLONAL HISTORY

EPA Science Inventory

The authors have developed a cytogenetic technique that allows observation of chromosome rearrangements associated with TK-/- mutagenesis of the L5178Y/TK+/-3.7.2C cell line early in mutant clonal history. For a series of mutagenic treatments they show that the major proportion (...

Selection of Phage Display Peptides Targeting Human Pluripotent Stem Cell-Derived Progenitor Cell Lines.

PubMed

Bignone, Paola A; Krupa, Rachel A; West, Michael D; Larocca, David

2016-01-01

The ability of human pluripotent stem cells (hPS) to both self-renew and differentiate into virtually any cell type makes them a promising source of cells for cell-based regenerative therapies. However, stem cell identity, purity, and scalability remain formidable challenges that need to be overcome for translation of pluripotent stem cell research into clinical applications. Directed differentiation from hPS cells is inefficient and residual contamination with pluripotent cells that have the potential to form tumors remains problematic. The derivation of scalable (self-renewing) embryonic progenitor stem cell lines offers a solution because they are well defined and clonally pure. Clonally pure progenitor stem cell lines also provide a means for identifying cell surface targeting reagents that are useful for identification, tracking, and repeated derivation of the corresponding progenitor stem cell types from additional hPS cell sources. Such stem cell targeting reagents can then be applied to the manufacture of genetically diverse banks of human embryonic progenitor cell lines for drug screening, disease modeling, and cell therapy. Here we present methods to identify human embryonic progenitor stem cell targeting peptides by selection of phage display libraries on clonal embryonic progenitor cell lines and demonstrate their use for targeting quantum dots (Qdots) for stem cell labeling.

Sox-2 in taste bud and lateral line system of zebrafish during development.

PubMed

Germanà, A; Montalbano, G; Guerrera, M C; Laura, R; Levanti, M; Abbate, F; de Carlos, F; Vega, J A; Ciriaco, E

2009-12-18

The Sox-2 is a transcription factor involved in adult neurogenesis in different vertebrate species, including fishes. Sox-2 also participates in growth and renewal on sensory cells in neuromasts of the fish lateral line system, and it is essential for development of taste buds in mammals. Using immunohistochemistry and Western blot we have investigated the occurrence and localization of Sox-2 taste buds and neuromast of zebrafish from 10 days post-fertilization to adult stage (1 year). The antibody used identifies two protein bands with estimated molecular weights of 34 and 37kDa which are consistent with those predicted for Sox-2. Sensory cells in taste buds displayed Sox-2 immunoreactivity at all the ages sampled, whereas in the neuromasts Sox-2 expression was restricted to the basal non-sensory cells. Interestingly Sox-2 immunoreactivity was observed in epithelial cells associated with both taste buds and neuromasts. Present results demonstrate that Sox-2 expressed in taste buds and neuromasts of zebrafish during the whole lifespan. Nevertheless, whereas the role of Sox-2 in taste buds of zebrafish remains to be established, the results in neuromast suggest that Sox-2 could participate in cell renewal of the mechanosensory cells.

Pharmacological analyses of learning and memory in zebrafish (Danio rerio).

PubMed

Bailey, Jordan M; Oliveri, Anthony N; Levin, Edward D

2015-12-01

Over the last decade, zebrafish (Danio rerio) have become valuable as a complementary model in behavioral pharmacology, opening a new avenue for understanding the relationships between drug action and behavior. This species offers a useful intermediate approach bridging the gap between in vitro studies and traditional mammalian models. Zebrafish offer great advantages of economy compared to their rodent counterparts, their complex brains and behavioral repertoire offer great translational potential relative to in vitro models. The development and validation of a variety of tests to measure behavior, including cognition, in zebrafish have set the stage for the use of this animal for behavioral pharmacology studies. This has led to research into the basic mechanisms of cognitive function as well as screening for potential cognition-improving drug therapies, among other lines of research. As with all models, zebrafish have limitations, which span pharmacokinetic challenges to difficulties quantifying behavior. The use, efficacy and limitations associated with a zebrafish model of cognitive function are discussed in this review, within the context of behavioral pharmacology. Copyright © 2015 Elsevier Inc. All rights reserved.

Enumerating Hematopoietic Stem and Progenitor Cells in Zebrafish Embryos.

PubMed

Esain, Virginie; Cortes, Mauricio; North, Trista E

2016-01-01

Over the past 20 years, zebrafish have proven to be a valuable model to dissect the signaling pathways involved in hematopoiesis, including Hematopoietic Stem and Progenitor Cell (HSPC) formation and homeostasis. Despite tremendous efforts to generate the tools necessary to characterize HSPCs in vitro and in vivo the zebrafish community still lacks standardized methods to quantify HSPCs across laboratories. Here, we describe three methods used routinely in our lab, and in others, to reliably enumerate HSPCs in zebrafish embryos: large-scale live imaging of transgenic reporter lines, Fluorescence-Activated Cell Sorting (FACS), and in vitro cell culture. While live imaging and FACS analysis allows enumeration of total or site-specific HSPCs, the cell culture assay provides the unique opportunity to test the functional potential of isolated HSPCs, similar to those employed in mammals.

Neuronal expression of fibroblast growth factor receptors in zebrafish.

PubMed

Rohs, Patricia; Ebert, Alicia M; Zuba, Ania; McFarlane, Sarah

2013-12-01

Fibroblast growth factor (FGF) signaling is important for a host of developmental processes such as proliferation, differentiation, tissue patterning, and morphogenesis. In vertebrates, FGFs signal through a family of four fibroblast growth factor receptors (FGFR 1-4), one of which is duplicated in zebrafish (FGFR1). Here we report the mRNA expression of the five known zebrafish fibroblast growth factor receptors at five developmental time points (24, 36, 48, 60, and 72h postfertilization), focusing on expression within the central nervous system. We show that the receptors have distinct and dynamic expression in the developing zebrafish brain, eye, inner ear, lateral line, and pharynx. In many cases, the expression patterns are similar to those of homologous FGFRs in mouse, chicken, amphibians, and other teleosts. Copyright © 2013 Elsevier B.V. All rights reserved.

Toxicity of chlorine to zebrafish embryos

PubMed Central

Kent, Michael L.; Buchner, Cari; Barton, Carrie; Tanguay, Robert L.

2014-01-01

Surface disinfection of fertilized fish eggs is widely used in aquaculture to reduce extraovum pathogens that may be released from brood fish during spawning, and this is routinely used in zebrafish (Danio rerio) research laboratories. Most laboratories use approximately 25 – 50 ppm unbuffered chlorine solution for 5 – 10 min. Treatment of embryos with chlorine has significant germicidal effects for many Gram-negative bacteria, viruses, and trophozoite stages of protozoa, it has reduced efficacy against cyst or spore stages of protozoa and certain Mycobacterium spp. Therefore, we evaluated the toxicity of unbufferred and buffered chlorine solution to embryos exposed at 6 or 24 hours post-fertilization (hpf) to determine if higher concentrations can be used for treating zebrafish embryos. Most of our experiments entailed using an outbred line (5D), with both mortality and malformations as endpoints. We found that 6 hpf embryos consistently were more resistant than 24 hpf embryos to the toxic effects of chlorine. Chlorine is more toxic and germicidal at lower pHs, and chlorine causes elevated pH. Consistent with this, we found that unbufferred chlorine solutions (pH ca 8–9) were less toxic at corresponding concentrations than solutions buffered to pH 7. Based on our findings here, we recommend treating 6 hpf embryos for 10 min and 24 hpf for 5 min with unbuffered chlorine solution at 100 ppm. One trial indicated that AB fish, a popular outbred line, are more susceptible to toxicity than 5Ds. This suggests that variability between zebrafish lines occurs, and researchers should evaluate each line or strain under their particular laboratory conditions for selection of the optimum chlorine treatment procedure. PMID:24429474

Dopamine receptor antagonism disrupts social preference in zebrafish, a strain comparison study

PubMed Central

Scerbina, Tanya; Chatterjee, Diptendu; Gerlai, Robert

2012-01-01

Zebrafish form shoals in nature and in the laboratory. The sight of conspecifics has been found reinforcing in zebrafish learning tasks. However, the mechanisms of shoaling, and those of its reinforcing properties, are not known. The dopaminergic system has been implicated in reward among other functions and it is also engaged by drugs of abuse as shown in a variety of vertebrates including zebrafish. The ontogenetic changes in dopamine levels and, to a lesser degree, in serotonin levels, have been found to accompany the maturation of shoaling in zebrafish. Thus, we hypothesized that the dopaminergic system may contribute to shoaling in zebrafish. To test this we employed a D1-receptor antagonist and quantified behavioral responses of our subjects using a social preference (shoaling) paradigm. We found significant reduction of social preference induced by the D1-R antagonist, SCH23390, in the AB strain of zebrafish, an alteration that was not accompanied by changes in motor function or vision. We also detected D1-R antagonist induced changes in the level of dopamine, DOPAC, serotonin and 5HIAA, respectively, in the brain of AB zebrafish as quantified by HPLC with electrochemical detection. We found the antagonist induced behavioral changes to be absent and the levels of these neurochemicals to be lower in another zebrafish population, SF, demonstrating naturally occurring genetic variability in these traits. We conclude that this variability may be utilized to unravel the mechanisms of social behavior in zebrafish, a line of research that may be extended to other vertebrates including our own species. PMID:22491827

Development of a transgenic zebrafish model expressing GFP in the notochord, somite and liver directed by the hfe2 gene promoter.

PubMed

Bian, Yue-Hong; Xu, Cheng; Li, Junling; Xu, Jin; Zhang, Hongwei; Du, Shao Jun

2011-08-01

Hemojuvelin, also known as RGMc, is encoded by hfe2 gene that plays an important role in iron homeostasis. hfe2 is specifically expressed in the notochord, developing somite and skeletal muscles during development. The molecular regulation of hfe2 expression is, however, not clear. We reported here the characterization of hfe2 gene expression and the regulation of its tissue-specific expression in zebrafish embryos. We demonstrated that the 6 kb 5'-flanking sequence upstream of the ATG start codon in the zebrafish hfe2 gene could direct GFP specific expression in the notochord, somites, and skeletal muscle of zebrafish embryos, recapitulating the expression pattern of the endogenous gene. However, the Tg(hfe2:gfp) transgene is also expressed in the liver of fish embryos, which did not mimic the expression of the endogenous hfe2 at the early stage. Nevertheless, the Tg(hfe2:gfp) transgenic zebrafish provides a useful model to study liver development. Treating Tg(hfe2:gfp) transgenic zebrafish embryos with valproic acid, a liver development inhibitor, significantly inhibited GFP expression in zebrafish. Together, these data indicate that the tissue specific expression of hfe2 in the notochord, somites and muscles is regulated by regulatory elements within the 6 kb 5'-flanking sequence of the hfe2 gene. Moreover, the Tg(hfe2:gfp) transgenic zebrafish line provides a useful model system for analyzing liver development in zebrafish.

Fusion of locomotor maneuvers, and improving sensory capabilities, give rise to the flexible homing strikes of juvenile zebrafish

PubMed Central

Westphal, Rebecca E.; O'Malley, Donald M.

2013-01-01

At 5 days post-fertilization and 4 mm in length, zebrafish larvae are successful predators of mobile prey items. The tracking and capture of 200 μm long Paramecia requires efficient sensorimotor transformations and precise neural controls that activate axial musculature for orientation and propulsion, while coordinating jaw muscle activity to engulf them. Using high-speed imaging, we report striking changes across ontogeny in the kinematics, structure and efficacy of zebrafish feeding episodes. Most notably, the discrete tracking maneuvers used by larval fish (turns, forward swims) become fused with prey capture swims to form the continuous, fluid homing strikes of juvenile and adult zebrafish. Across this same developmental time frame, the duration of feeding episodes become much shorter, with strikes occurring at broader angles and from much greater distances than seen with larval zebrafish. Moreover, juveniles use a surprisingly diverse array of motor patterns that constitute a flexible predatory strategy. This enhances the ability of zebrafish to capture more mobile prey items such as Artemia. Visually-guided tracking is complemented by the mechanosensory lateral line system. Neomycin ablation of lateral line hair cells reduced the accuracy of strikes and overall feeding rates, especially when neomycin-treated larvae and juveniles were placed in the dark. Darkness by itself reduced the distance from which strikes were launched, as visualized by infrared imaging. Rapid growth and changing morphology, including ossification of skeletal elements and differentiation of control musculature, present challenges for sustaining and enhancing predatory capabilities. The concurrent expansion of the cerebellum and subpallium (an ancestral basal ganglia) may contribute to the emergence of juvenile homing strikes, whose ontogeny possibly mirrors a phylogenetic expansion of motor capabilities. PMID:23761739

Generation and characterization of gsuα:EGFP transgenic zebrafish for evaluating endocrine-disrupting effects

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cheng, Xiaoxia; University of Chinese Academy of Sciences, Beijing; Chen, Xiaowen

The glycoprotein subunit α (gsuα) gene encodes the shared α subunit of the three pituitary heterodimeric glycoprotein hormones: follicle-stimulating hormone β (Fshβ), luteinizing hormone β (Lhβ) and thyroid stimulating hormone β (Tshβ). In our current study, we identified and characterized the promoter region of zebrafish gsuα and generated a stable gsuα:EGFP transgenic line, which recapitulated the endogenous gsuα expression in the early developing pituitary gland. A relatively conserved regulatory element set is presented in the promoter regions of zebrafish and three other known mammalian gsuα promoters. Our results also demonstrated that the expression patterns of the gsuα:EGFP transgene were allmore » identical to those expression patterns of the endogenous gsuα expression in the pituitary tissue when our transgenic fish were treated with various endocrine chemicals, including forskolin (FSK), SP600125, trichostatin A (TSA), KClO{sub 4}, dexamethasone (Dex), β-estradiol and progesterone. Thus, this gsuα:EGFP transgenic fish reporter line provides another valuable tool for investigating the lineage development of gsuα-expressing gonadotrophins and the coordinated regulation of various glycoprotein hormone subunit genes. These reporter fish can serve as a novel platform to perform screenings of endocrine-disrupting chemicals (EDCs) in vivo as well. - Highlights: • Identification of the promoter of zebrafish glycoprotein subunit α (gsuα) gene • Generation of stable transmission gsuα:EGFP transgenic zebrafish reporter • Demonstration of the recapitulation of the gsuα:EGFP and endogenous gsuα expression • Suggestion of the gsuα:EGFP transgenic zebrafish as a novel platform for EDC study.« less

Multifocal clonal evolution characterized using circulating tumour DNA in a case of metastatic breast cancer

PubMed Central

Murtaza, Muhammed; Dawson, Sarah-Jane; Pogrebniak, Katherine; Rueda, Oscar M.; Provenzano, Elena; Grant, John; Chin, Suet-Feung; Tsui, Dana W. Y.; Marass, Francesco; Gale, Davina; Ali, H. Raza; Shah, Pankti; Contente-Cuomo, Tania; Farahani, Hossein; Shumansky, Karey; Kingsbury, Zoya; Humphray, Sean; Bentley, David; Shah, Sohrab P.; Wallis, Matthew; Rosenfeld, Nitzan; Caldas, Carlos

2015-01-01

Circulating tumour DNA analysis can be used to track tumour burden and analyse cancer genomes non-invasively but the extent to which it represents metastatic heterogeneity is unknown. Here we follow a patient with metastatic ER-positive and HER2-positive breast cancer receiving two lines of targeted therapy over 3 years. We characterize genomic architecture and infer clonal evolution in eight tumour biopsies and nine plasma samples collected over 1,193 days of clinical follow-up using exome and targeted amplicon sequencing. Mutation levels in the plasma samples reflect the clonal hierarchy inferred from sequencing of tumour biopsies. Serial changes in circulating levels of sub-clonal private mutations correlate with different treatment responses between metastatic sites. This comparison of biopsy and plasma samples in a single patient with metastatic breast cancer shows that circulating tumour DNA can allow real-time sampling of multifocal clonal evolution. PMID:26530965

Multifocal clonal evolution characterized using circulating tumour DNA in a case of metastatic breast cancer.

PubMed

Murtaza, Muhammed; Dawson, Sarah-Jane; Pogrebniak, Katherine; Rueda, Oscar M; Provenzano, Elena; Grant, John; Chin, Suet-Feung; Tsui, Dana W Y; Marass, Francesco; Gale, Davina; Ali, H Raza; Shah, Pankti; Contente-Cuomo, Tania; Farahani, Hossein; Shumansky, Karey; Kingsbury, Zoya; Humphray, Sean; Bentley, David; Shah, Sohrab P; Wallis, Matthew; Rosenfeld, Nitzan; Caldas, Carlos

2015-11-04

Circulating tumour DNA analysis can be used to track tumour burden and analyse cancer genomes non-invasively but the extent to which it represents metastatic heterogeneity is unknown. Here we follow a patient with metastatic ER-positive and HER2-positive breast cancer receiving two lines of targeted therapy over 3 years. We characterize genomic architecture and infer clonal evolution in eight tumour biopsies and nine plasma samples collected over 1,193 days of clinical follow-up using exome and targeted amplicon sequencing. Mutation levels in the plasma samples reflect the clonal hierarchy inferred from sequencing of tumour biopsies. Serial changes in circulating levels of sub-clonal private mutations correlate with different treatment responses between metastatic sites. This comparison of biopsy and plasma samples in a single patient with metastatic breast cancer shows that circulating tumour DNA can allow real-time sampling of multifocal clonal evolution.

Using local chromatin structure to improve CRISPR/Cas9 efficiency in zebrafish.

PubMed

Chen, Yunru; Zeng, Shiyang; Hu, Ruikun; Wang, Xiangxiu; Huang, Weilai; Liu, Jiangfang; Wang, Luying; Liu, Guifen; Cao, Ying; Zhang, Yong

2017-01-01

Although the CRISPR/Cas9 has been successfully applied in zebrafish, considerable variations in efficiency have been observed for different gRNAs. The workload and cost of zebrafish mutant screening is largely dependent on the mutation rate of injected embryos; therefore, selecting more effective gRNAs is especially important for zebrafish mutant construction. Besides the sequence features, local chromatin structures may have effects on CRISPR/Cas9 efficiency, which remain largely unexplored. In the only related study in zebrafish, nucleosome organization was not found to have an effect on CRISPR/Cas9 efficiency, which is inconsistent with recent studies in vitro and in mammalian cell lines. To understand the effects of local chromatin structure on CRISPR/Cas9 efficiency in zebrafish, we first determined that CRISPR/Cas9 introduced genome editing mainly before the dome stage. Based on this observation, we reanalyzed our published nucleosome organization profiles and generated chromatin accessibility profiles in the 256-cell and dome stages using ATAC-seq technology. Our study demonstrated that chromatin accessibility showed positive correlation with CRISPR/Cas9 efficiency, but we did not observe a clear correlation between nucleosome organization and CRISPR/Cas9 efficiency. We constructed an online database for zebrafish gRNA selection based on local chromatin structure features that could prove beneficial to zebrafish homozygous mutant construction via CRISPR/Cas9.

Two clonal cell lines of immortalized human corneal endothelial cells show either differentiated or precursor cell characteristics.

PubMed

Valtink, Monika; Gruschwitz, Rita; Funk, Richard H W; Engelmann, Katrin

2008-01-01

Access to primary human corneal endothelial cells (HCEC) is limited and donor-derived differences between cultures exacerbate the issue of data reproducibility, whereas cell lines can provide sufficient numbers of homogenous cells for multiple experiments. An immortalized HCEC population was adapted to serum-free culture medium and repeated cloning was performed. Clonally grown cells were propagated under serum-free conditions and growth curves were recorded. Cells were characterized immunocytochemically for junctional proteins, collagens, Na,K-ATPase and HCEC-specific 9.3.E-antigen. Ultrastructure was monitored by scanning and transmission electron microscopy. Two clonal cell lines, HCEC-B4G12 and HCEC-H9C1, could be isolated and expanded, which differed morphologically: B4G12 cells were polygonal, strongly adherent and formed a strict monolayer, H9C1 cells were less adherent and formed floating spheres. The generation time of B4G12 cells was 62.26 +/- 14.5 h and that of H9C1 cells 44.05 +/- 5.05 h. Scanning electron microscopy revealed that B4G12 cells had a smooth cell surface, while H9C1 cells had numerous thin filopodia. Both cell lines expressed ZO-1 and occludin adequately, and little but well detectable amounts of connexin-43. Expression of HCEC-specific 9.3.E-antigen was found commensurately in both cell lines, while expression of Na,K-ATPase alpha1 was higher in H9C1 cells than in B4G12 cells. B4G12 cells expressed collagen IV abundantly and almost no collagen III, while H9C1 cells expressed both collagens at reasonable amounts. It is concluded that the clonal cell line B4G12 represents an ideal model of differentiated HCEC, while H9C1 may reflect features of developing or transitional HCEC. Copyright 2008 S. Karger AG, Basel.

Senescence marker protein 30 (SMP30)/regucalcin (RGN) expression decreases with aging, acute liver injuries and tumors in zebrafish

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fujisawa, Koichi; Terai, Shuji, E-mail: terais@yamaguchi-u.ac.jp; Hirose, Yoshikazu

2011-10-22

Highlights: {yields} Zebrafish SMP30/RGN mRNA expression decreases with aging. {yields} Decreased expression was observed in liver tumors as compared to the surrounding area. {yields} SMP30/RGN is important for liver proliferation and tumorigenesis. -- Abstract: Senescence marker protein 30 (SMP30)/regucalcin (RGN) is known to be related to aging, hepatocyte proliferation and tumorigenesis. However, expression and function of non-mammalian SMP30/RGN is poorly understood. We found that zebrafish SMP30/RGN mRNA expression decreases with aging, partial hepatectomy and thioacetamide-induced acute liver injury. SMP30/RGN expression was also greatly decreased in a zebrafish liver cell line. In addition, we induced liver tumors in adult zebrafish bymore » administering diethylnitrosamine. Decreased expression was observed in foci, hepatocellular carcinomas, cholangiocellular carcinomas and mixed tumors as compared to the surrounding area. We thus showed the importance of SMP30/RGN in liver proliferation and tumorigenesis.« less

Zebrabow: multispectral cell labeling for cell tracing and lineage analysis in zebrafish

PubMed Central

Pan, Y. Albert; Freundlich, Tom; Weissman, Tamily A.; Schoppik, David; Wang, X. Cindy; Zimmerman, Steve; Ciruna, Brian; Sanes, Joshua R.; Lichtman, Jeff W.; Schier, Alexander F.

2013-01-01

Advances in imaging and cell-labeling techniques have greatly enhanced our understanding of developmental and neurobiological processes. Among vertebrates, zebrafish is uniquely suited for in vivo imaging owing to its small size and optical translucency. However, distinguishing and following cells over extended time periods remains difficult. Previous studies have demonstrated that Cre recombinase-mediated recombination can lead to combinatorial expression of spectrally distinct fluorescent proteins (RFP, YFP and CFP) in neighboring cells, creating a ‘Brainbow’ of colors. The random combination of fluorescent proteins provides a way to distinguish adjacent cells, visualize cellular interactions and perform lineage analyses. Here, we describe Zebrabow (Zebrafish Brainbow) tools for in vivo multicolor imaging in zebrafish. First, we show that the broadly expressed ubi:Zebrabow line provides diverse color profiles that can be optimized by modulating Cre activity. Second, we find that colors are inherited equally among daughter cells and remain stable throughout embryonic and larval stages. Third, we show that UAS:Zebrabow lines can be used in combination with Gal4 to generate broad or tissue-specific expression patterns and facilitate tracing of axonal processes. Fourth, we demonstrate that Zebrabow can be used for long-term lineage analysis. Using the cornea as a model system, we provide evidence that embryonic corneal epithelial clones are replaced by large, wedge-shaped clones formed by centripetal expansion of cells from the peripheral cornea. The Zebrabow tool set presented here provides a resource for next-generation color-based anatomical and lineage analyses in zebrafish. PMID:23757414

Extensive conversion of hepatic biliary epithelial cells to hepatocytes after near total loss of hepatocytes in zebrafish.

PubMed

Choi, Tae-Young; Ninov, Nikolay; Stainier, Didier Y R; Shin, Donghun

2014-03-01

Biliary epithelial cells (BECs) are considered to be a source of regenerating hepatocytes when hepatocyte proliferation is compromised. However, there is still controversy about the extent to which BECs can contribute to the regenerating hepatocyte population, and thereby to liver recovery. To investigate this issue, we established a zebrafish model of liver regeneration in which the extent of hepatocyte ablation can be controlled. Hepatocytes were depleted by administration of metronidazole to Tg(fabp10a:CFP-NTR) animals. We traced the origin of regenerating hepatocytes using short-term lineage-tracing experiments, as well as the inducible Cre/loxP system; specifically, we utilized both a BEC tracer line Tg(Tp1:CreER(T2)) and a hepatocyte tracer line Tg(fabp10a:CreER(T2)). We also examined BEC and hepatocyte proliferation and liver marker gene expression during liver regeneration. BECs gave rise to most of the regenerating hepatocytes in larval and adult zebrafish after severe hepatocyte depletion. After hepatocyte loss, BECs proliferated as they dedifferentiated into hepatoblast-like cells; they subsequently differentiated into highly proliferative hepatocytes that restored the liver mass. This process was impaired in zebrafish wnt2bb mutants; in these animals, hepatocytes regenerated but their proliferation was greatly reduced. BECs contribute to regenerating hepatocytes after substantial hepatocyte depletion in zebrafish, thereby leading to recovery from severe liver damage. Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.

Effect of histone deacetylase inhibitors trichostatin A and valproic acid on hair cell regeneration in zebrafish lateral line neuromasts

PubMed Central

He, Yingzi; Cai, Chengfu; Tang, Dongmei; Sun, Shan; Li, Huawei

2014-01-01

In humans, auditory hair cells are not replaced when injured. Thus, cochlear hair cell loss causes progressive and permanent hearing loss. Conversely, non-mammalian vertebrates are capable of regenerating lost sensory hair cells. The zebrafish lateral line has numerous qualities that make it well-suited for studying hair cell development and regeneration. Histone deacetylase (HDAC) activity has been shown to have an important role in regenerative processes in vertebrates, but its function in hair cell regeneration in vivo is not fully understood. Here, we have examined the role of HDAC activity in hair cell regeneration in the zebrafish lateral line. We eliminated lateral line hair cells of 5-day post-fertilization larvae using neomycin and then treated the larvae with HDAC inhibitors. To assess hair cell regeneration, we used 5-bromo-2-deoxyuridine (BrdU) incorporation in zebrafish larvae to label mitotic cells after hair cell loss. We found that pharmacological inhibition of HDACs using trichostatin A (TSA) or valproic acid (VPA) increased histone acetylation in the regenerated neuromasts following neomycin-induced damage. We also showed that treatment with TSA or VPA decreased the number of supporting cells and regenerated hair cells in response to hair cell damage. Additionally, BrdU immunostaining and western blot analysis showed that TSA or VPA treatment caused a significant decrease in the percentage of S-phase cells and induced p21Cip1 and p27Kip1 expression, both of which are likely to explain the decrease in the amount of newly regenerated hair cells in treated embryos. Finally, we showed that HDAC inhibitors induced no observable cell death in neuromasts as measured by cleaved caspase-3 immunohistochemistry and western blot analysis. Taken together, our results demonstrate that HDAC activity has an important role in the regeneration of hair cells in the lateral line. PMID:25431550

Heart-specific expression of laminopathic mutations in transgenic zebrafish.

PubMed

Verma, Ajay D; Parnaik, Veena K

2017-07-01

Lamins are key determinants of nuclear organization and function in the metazoan nucleus. Mutations in human lamin A cause a spectrum of genetic diseases that affect cardiac muscle and skeletal muscle as well as other tissues. A few laminopathies have been modeled using the mouse. As zebrafish is a well established model for the study of cardiac development and disease, we have investigated the effects of heart-specific lamin A mutations in transgenic zebrafish. We have developed transgenic lines of zebrafish expressing conserved lamin A mutations that cause cardiac dysfunction in humans. Expression of zlamin A mutations Q291P and M368K in the heart was driven by the zebrafish cardiac troponin T2 promoter. Homozygous mutant embryos displayed nuclear abnormalities in cardiomyocyte nuclei. Expression analysis showed the upregulation of genes involved in heart regeneration in transgenic mutant embryos and a cell proliferation marker was increased in adult heart tissue. At the physiological level, there was deviation of up to 20% from normal heart rate in transgenic embryos expressing mutant lamins. Adult homozygous zebrafish were fertile and did not show signs of early mortality. Our results suggest that transgenic zebrafish models of heart-specific laminopathies show cardiac regeneration and moderate deviations in heart rate during embryonic development. © 2017 International Federation for Cell Biology.

Expression of CALR mutants causes mpl-dependent thrombocytosis in zebrafish.

PubMed

Lim, K-H; Chang, Y-C; Chiang, Y-H; Lin, H-C; Chang, C-Y; Lin, C-S; Huang, L; Wang, W-T; Gon-Shen Chen, C; Chou, W-C; Kuo, Y-Y

2016-10-07

CALR mutations are identified in about 30% of JAK2/MPL-unmutated myeloproliferative neoplasms (MPNs) including essential thrombocythemia (ET) and primary myelofibrosis. Although the molecular pathogenesis of CALR mutations leading to MPNs has been studied using in vitro cell lines models, how mutant CALR may affect developmental hematopoiesis remains unknown. Here we took advantage of the zebrafish model to examine the effects of mutant CALR on early hematopoiesis and model human CALR-mutated MPNs. We identified three zebrafish genes orthologous to human CALR, referred to as calr, calr3a and calr3b. The expression of CALR-del52 and CALR-ins5 mutants caused an increase in the hematopoietic stem/progenitor cells followed by thrombocytosis without affecting normal angiogenesis. The expression of CALR mutants also perturbed early developmental hematopoiesis in zebrafish. Importantly, morpholino knockdown of mpl but not epor or csf3r could significantly attenuate the effects of mutant CALR. Furthermore, the expression of mutant CALR caused jak-stat signaling activation in zebrafish that could be blocked by JAK inhibitors (ruxolitinib and fedratinib). These findings showed that mutant CALR activates jak-stat signaling through an mpl-dependent mechanism to mediate pathogenic thrombopoiesis in zebrafish, and illustrated that the signaling machinery related to mutant CALR tumorigenesis are conserved between human and zebrafish.

Production of maternal-zygotic mutant zebrafish by germ-line replacement.

PubMed

Ciruna, Brian; Weidinger, Gilbert; Knaut, Holger; Thisse, Bernard; Thisse, Christine; Raz, Erez; Schier, Alexander F

2002-11-12

We report a generally applicable strategy for transferring zygotic lethal mutations through the zebrafish germ line. By using a morpholino oligonucleotide that blocks primordial germ cell (PGC) development, we generate embryos devoid of endogenous PGCs to serve as hosts for the transplantation of germ cells derived from homozygous mutant donors. Successful transfers are identified by the localization of specifically labeled donor PGCs to the region of the developing gonad in chimeric embryos. This strategy, which results in the complete replacement of the host germ line with donor PGCs, was validated by the generation of maternal and maternal-zygotic mutants for the miles apart locus. This germ-line replacement technique provides a powerful tool for studying the maternal effects of zygotic lethal mutations. Furthermore, the ability to generate large clutches of purely mutant embryos will greatly facilitate embryological, genetic, genomic, and biochemical studies.

Production of maternal-zygotic mutant zebrafish by germ-line replacement

PubMed Central

Ciruna, Brian; Weidinger, Gilbert; Knaut, Holger; Thisse, Bernard; Thisse, Christine; Raz, Erez; Schier, Alexander F.

2002-01-01

We report a generally applicable strategy for transferring zygotic lethal mutations through the zebrafish germ line. By using a morpholino oligonucleotide that blocks primordial germ cell (PGC) development, we generate embryos devoid of endogenous PGCs to serve as hosts for the transplantation of germ cells derived from homozygous mutant donors. Successful transfers are identified by the localization of specifically labeled donor PGCs to the region of the developing gonad in chimeric embryos. This strategy, which results in the complete replacement of the host germ line with donor PGCs, was validated by the generation of maternal and maternal-zygotic mutants for the miles apart locus. This germ-line replacement technique provides a powerful tool for studying the maternal effects of zygotic lethal mutations. Furthermore, the ability to generate large clutches of purely mutant embryos will greatly facilitate embryological, genetic, genomic, and biochemical studies. PMID:12397179

Genetic Analysis of earl field growth of loblolly pine clones and seedlings from the same full-sib families

Treesearch

Brian Baltunis; Dudley Huber; Tim Wite

2006-01-01

The Forest Biology Research Cooperative recently established a series of loblolly pine clonal trials known as CCLONES (Comparing Clonal Lines on Experimental Sites). There are three primary levels of genetic structure in this study (parental, full-sib family, clone) that strengthen the power of CCLONES for examining genetic mechanisms and interactions with cultural...

Lineage tracing of dlx1a/2a and dlx5a/6a expressing cells in the developing zebrafish brain.

PubMed

Solek, Cynthia M; Feng, Shengrui; Perin, Sofia; Weinschutz Mendes, Hellen; Ekker, Marc

2017-07-01

Lineage tracing of specific populations of progenitor cells provides crucial information about developmental programs. Four members of the Dlx homeobox gene family, Dlx1,2, 5 and 6, are involved in the specification of γ-aminobutyric acid (GABA)ergic neurons in the vertebrate forebrain. Orthologous genes in mammals and teleost show similarities in expression patterns and transcriptional regulation mechanisms. We have used lineage tracing to permanently label dlx-expressing cells in the zebrafish and have characterized the progeny of these cells in the larva and in the juvenile and adult brain. We have found that dlx1a/2a and dlx5a/6a expressing progenitors give rise, for the most part, to small populations of cells which constitute only a small proportion of GABAergic cells in the adult brain tissue. Moreover, some of the cells do not acquire a neuronal phenotype suggesting that, regardless of the time a cell expresses dlx genes in the brain, it can potentially give rise to cells other than neurons. In some instances, labeling larval dlx5a/6a-expressing cells, but not dlx1a/2a-expressing cells, results in massively expanding, widespread clonal expansion throughout the adult brain. Our data provide a detailed lineage analysis of the dlx1a/2a and dlx5a/6a expressing progenitors in the zebrafish brain and lays the foundation for further characterization of the role of these transcription factors beyond the specification of GABAergic neurons. Copyright © 2017 Elsevier Inc. All rights reserved.

Xenogenesis in teleost fish through generation of germ-line chimeras by single primordial germ cell transplantation.

PubMed

Saito, Taiju; Goto-Kazeto, Rie; Arai, Katsutoshi; Yamaha, Etsuro

2008-01-01

Primordial germ cells (PGCs) are the only cells in developing embryos with the potential to transmit genetic information to the next generation. PGCs therefore have the potential to be of value for gene banking and cryopreservation, particularly via the production of donor gametes with germ-line chimeras. Currently, it is not clear how many PGCs are required for germ-line differentiation and formation of gonadal structures. In the present study, we achieved complete germ-line replacement between two related teleost species, the pearl danio (Danio albolineatus) and the zebrafish (Danio rerio), with transplantation of a single PGC into each host embryo. We isolated and transplanted a single PGC into each blastula-stage, zebrafish embryo. Development of host germ-line cells was prevented by an antisense dead end morpholino oligonucleotide. In many host embryos, the transplanted donor PGC successfully migrated toward the gonadal anlage without undergoing cell division. At the gonadal anlage, the PGC differentiated to form one normally sized gonad rather than the pair of gonads usually present. Offspring were obtained from natural spawning of these chimeras. Analyses of morphology and DNA showed that the offspring were of donor origin. We extended our study to confirm that transplanted single PGCs of goldfish (Carassius auratus) and loach (Misgurnus anguillicaudatus) can similarly differentiate into sperm in zebrafish host embryos. Our results show that xenogenesis is realistic and practical across species, genus, and family barriers and can be achieved by the transplantation of a single PGC from a donor species.

Zebrafish hair cell mechanics and physiology through the lens of noise-induced hair cell death

NASA Astrophysics Data System (ADS)

Coffin, Allison B.; Xu, Jie; Uribe, Phillip M.

2018-05-01

Hair cells are exquisitely sensitive to auditory stimuli, but also to damage from a variety of sources including noise trauma and ototoxic drugs. Mammals cannot regenerate cochlear hair cells, while non-mammalian vertebrates exhibit robust regenerative capacity. Our research group uses the lateral line system of larval zebrafish to explore the mechanisms underlying hair cell damage, identify protective therapies, and determine molecular drivers of innate regeneration. The lateral line system contains externally located sensory organs called neuromasts, each composed of ˜8-20 hair cells. Lateral line hair cells are homologous to vertebrate inner ear hair cells and share similar susceptibility to ototoxic damage. In the last decade, the lateral line has emerged as a powerful model system for understanding hair cell death mechanisms and for identifying novel protective compounds. Here we demonstrate that the lateral line is a tractable model for noise-induced hair cell death. We have developed a novel noise damage system capable of inducing over 50% loss of lateral line hair cells, with hair cell death occurring in a dose- and time-dependent manner. Cell death is greatest 72 hours post-exposure. However, early signs of hair cell damage, including changes in membrane integrity and reduced mechanotransduction, are apparent within hours of noise exposure. These features, early signs of damage followed by delayed hair cell death, are consistent with mammalian data, suggesting that noise acts similarly on zebrafish and mammalian hair cells. In our future work we will use our new model system to investigate noise damage events in real time, and to develop protective therapies for future translational research.

Quaternary and tertiary aldoxime antidotes for organophosphate exposure in a zebrafish model system

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schmidt, Hayden R.; Radić, Zoran; Taylor, Palmer

The zebrafish is rapidly becoming an important model system for screening of new therapeutics. Here we evaluated the zebrafish as a potential pharmacological model for screening novel oxime antidotes to organophosphate (OP)-inhibited acetylcholinesterase (AChE). The k{sub i} values determined for chlorpyrifos oxon (CPO) and dichlorvos (DDVP) showed that CPO was a more potent inhibitor of both human and zebrafish AChE, but overall zebrafish AChE was less sensitive to OP inhibition. In contrast, aldoxime antidotes, the quaternary ammonium 2-PAM and tertiary amine RS-194B, showed generally similar overall reactivation kinetics, k{sub r}, in both zebrafish and human AChE. However, differences between themore » K{sub ox} and k{sub 2} constants suggest that zebrafish AChE associates more tightly with oximes, but has a slower maximal reactivation rate than human AChE. Homology modeling suggests that these kinetic differences result from divergences in the amino acids lining the entrance to the active site gorge. Although 2-PAM had the more favorable in vitro reactivation kinetics, RS-194B was more effective antidote in vivo. In intact zebrafish embryos, antidotal treatment with RS-194B rescued embryos from OP toxicity, whereas 2-PAM had no effect. Dechorionation of the embryos prior to antidotal treatment allowed both 2-PAM and RS-194B to rescue zebrafish embryos from OP toxicity. Interestingly, RS-194B and 2-PAM alone increased cholinergic motor activity in dechorionated embryos possibly due to the reversible inhibition kinetics, K{sub i} and αK{sub i}, of the oximes. Together these results demonstrate that the zebrafish at various developmental stages provides an excellent model for investigating membrane penetrant antidotes to OP exposure. - Highlights: • Zebrafish AChE shares significant structural similarities with human AChE. • OP-inhibited zebrafish and human AChE exhibit similar reactivation kinetics. • The zebrafish chorion is permeable to BBB penetrant and not charged aldoximes. • Zebrafish are a good aquatic model for studying centrally acting antidotes.« less

Generation of Rab-based transgenic lines for in vivo studies of endosome biology in zebrafish

PubMed Central

Clark, Brian S.; Winter, Mark; Cohen, Andrew R.; Link, Brian A.

2011-01-01

The Rab family of small GTPases function as molecular switches regulating membrane and protein trafficking. Individual Rab isoforms define and are required for specific endosomal compartments. To facilitate in vivo investigation of specific Rab proteins, and endosome biology in general, we have generated transgenic zebrafish lines to mark and manipulate Rab proteins. We also developed software to track and quantify endosome dynamics within time-lapse movies. The established transgenic lines ubiquitously express EGFP fusions of Rab5c (early endosomes), Rab11a (recycling endosomes), and Rab7 (late endosomes) to study localization and dynamics during development. Additionally, we generated UAS-based transgenic lines expressing constitutive active (CA) and dominant negative (DN) versions for each of these Rab proteins. Predicted localization and functional consequences for each line were verified through a variety of assays, including lipophilic dye uptake and Crumbs2a localization. In summary, we have established a toolset for in vivo analyses of endosome dynamics and functions. PMID:21976318

Retinoid regulation of the zebrafish cyp26a1 promoter.

PubMed

Hu, Ping; Tian, Miao; Bao, Jie; Xing, Guangdong; Gu, Xingxing; Gao, Xiang; Linney, Elwood; Zhao, Qingshun

2008-12-01

Cyp26A1 is a major enzyme that controls retinoic acid (RA) homeostasis by metabolizing RA into bio-inactive metabolites. Previous research revealed that the mouse Cyp26A1 promoter has two canonical RA response elements (RAREs) that underlie the regulation of the gene by RA. Analyzing the 2,533-base pairs (2.5 k) genomic sequence upstream of zebrafish cyp26a1 start codon, we report that the two RAREs are conserved in zebrafish cyp26a1 promoter. Mutagenesis demonstrated that the two RAREs work synergistically in RA inducibility of cyp26a1. Fusing the 2.5 k (kilobase pairs) fragment to the enhanced yellow fluorescent protein (eYFP) reporter gene, we have generated two transgenic lines of zebrafish [Tg(cyp26a1:eYFP)]. The transgenic zebrafish display expression patterns similar to that of cyp26a1 gene in vivo. Consistent with the in vitro results, the reporter activity is RA inducible in embryos. Taken together, our results demonstrate that the 2.5 k fragment underlies the regulation of the zebrafish cyp26a1 gene by RA. (c) 2008 Wiley-Liss, Inc.

Polygenic Sex Determination System in Zebrafish

PubMed Central

Liew, Woei Chang; Bartfai, Richard; Lim, Zijie; Sreenivasan, Rajini; Siegfried, Kellee R.; Orban, Laszlo

2012-01-01

Background Despite the popularity of zebrafish as a research model, its sex determination (SD) mechanism is still unknown. Most cytogenetic studies failed to find dimorphic sex chromosomes and no primary sex determining switch has been identified even though the assembly of zebrafish genome sequence is near to completion and a high resolution genetic map is available. Recent publications suggest that environmental factors within the natural range have minimal impact on sex ratios of zebrafish populations. The primary aim of this study is to find out more about how sex is determined in zebrafish. Methodology/Principal Findings Using classical breeding experiments, we found that sex ratios across families were wide ranging (4.8% to 97.3% males). On the other hand, repeated single pair crossings produced broods of very similar sex ratios, indicating that parental genotypes have a role in the sex ratio of the offspring. Variation among family sex ratios was reduced after selection for breeding pairs with predominantly male or female offspring, another indication that zebrafish sex is regulated genetically. Further examinations by a PCR-based “blind assay" and array comparative genomic hybridization both failed to find universal sex-linked differences between the male and female genomes. Together with the ability to increase the sex bias of lines by selective breeding, these data suggest that zebrafish is unlikely to utilize a chromosomal sex determination (CSD) system. Conclusions/Significance Taken together, our study suggests that zebrafish sex is genetically determined with limited, secondary influences from the environment. As we have not found any sign for CSD in the species, we propose that the zebrafish has a polygenic sex determination system. PMID:22506019

Halogenated Bisphenol-A Analogs Act as Obesogens in Zebrafish Larvae (Danio rerio)

PubMed Central

Pinto, Caroline L.; Grimaldi, Marina; Hillenweck, Anne; Perdu, Elisabeth; Zalko, Daniel; Bernard, Laure; Laudet, Vincent; Balaguer, Patrick; Bondesson, Maria; Gustafsson, Jan-Ake

2014-01-01

Obesity has increased dramatically over the past decades, reaching epidemic proportions. The reasons are likely multifactorial. One of the suggested causes is the accelerated exposure to obesity-inducing chemicals (obesogens). However, out of the tens of thousands of industrial chemicals humans are exposed to, very few have been tested for their obesogenic potential, mostly due to the limited availability of appropriate in vivo screening models. In this study, we investigated whether two commonly used flame retardants, the halogenated bisphenol-A (BPA) analogs tetrabromobisphenol-A (TBBPA) and tetrachlorobisphenol-A (TCBPA), could act as obesogens using zebrafish larvae as an in vivo animal model. The effect of embryonic exposure to these chemicals on lipid accumulation was analyzed by Oil Red-O staining, and correlated to their capacity to activate human and zebrafish peroxisome proliferator-activated receptor gamma (PPARγ) in zebrafish and in reporter cell lines. Then, the metabolic fate of TBBPA and TCBPA in zebrafish larvae was analyzed by high-performance liquid chromatography (HPLC) . TBBPA and TCBPA were readily taken up by the fish embryo and both compounds were biotransformed to sulfate-conjugated metabolites. Both halogenated-BPAs, as well as TBBPA-sulfate induced lipid accumulation in zebrafish larvae. TBBPA and TCBPA also induced late-onset weight gain in juvenile zebrafish. These effects correlated to their capacity to act as zebrafish PPARγ agonists. Screening of chemicals for inherent obesogenic capacities through the zebrafish lipid accumulation model could facilitate prioritizing chemicals for further investigations in rodents, and ultimately, help protect humans from exposure to environmental obesogens. PMID:24591153

GACD: Integrated Software for Genetic Analysis in Clonal F1 and Double Cross Populations.

PubMed

Zhang, Luyan; Meng, Lei; Wu, Wencheng; Wang, Jiankang

2015-01-01

Clonal species are common among plants. Clonal F1 progenies are derived from the hybridization between 2 heterozygous clones. In self- and cross-pollinated species, double crosses can be made from 4 inbred lines. A clonal F1 population can be viewed as a double cross population when the linkage phase is determined. The software package GACD (Genetic Analysis of Clonal F1 and Double cross) is freely available public software, capable of building high-density linkage maps and mapping quantitative trait loci (QTL) in clonal F1 and double cross populations. Three functionalities are integrated in GACD version 1.0: binning of redundant markers (BIN); linkage map construction (CDM); and QTL mapping (CDQ). Output of BIN can be directly used as input of CDM. After adding the phenotypic data, the output of CDM can be used as input of CDQ. Thus, GACD acts as a pipeline for genetic analysis. GACD and example datasets are freely available from www.isbreeding.net. © The American Genetic Association. 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

In Vivo Time-Lapse Imaging in the Zebrafish Lateral Line: A Flexible, Open-Ended Research Project for an Undergraduate Neurobiology Laboratory Course.

PubMed

Marra, Molly H; Tobias, Zachary J C; Cohen, Hannah R; Glover, Greta; Weissman, Tamily A

2015-01-01

The lateral line sensory system in fish detects movements in the water and allows fish to respond to predators, prey, and other stimuli. As the lateral line forms in the first two days of zebrafish development, axons extend caudally along the lateral surface of the fish, eventually forming synapses with hair cells of neuromasts. Growing lateral line axons are located superficially under the skin and can be labeled in living zebrafish using fluorescent protein expression. This system provides a relatively straightforward approach for in vivo time-lapse imaging of neuronal development in an undergraduate setting. Here we describe an upper-level neurobiology laboratory module in which students investigate aspects of axonal development in the zebrafish lateral line system. Students learn to handle and image living fish, collect time-lapse videos of moving mitochondria, and quantitatively measure mitochondrial dynamics by generating and analyzing kymographs of their movements. Energy demands may differ between axons with extending growth cones versus axons that have already reached their targets and are forming synapses. Since relatively little is known about this process in developing lateral line axons, students generate and test their own hypotheses regarding how mitochondrial dynamics may differ at two different time points in axonal development. Students also learn to incorporate into their analysis a powerful yet accessible quantitative tool, the kymograph, which is used to graph movement over time. After students measure and quantify dynamics in living fish at 1 and 2 days post fertilization, this module extends into independent projects, in which students can expand their studies in a number of different, inquiry-driven directions. The project can also be pared down for courses that wish to focus solely on the quantitative analysis (without fish handling), or vice versa. This research module provides a useful approach for the design of open-ended laboratory research projects that integrate the scientific process into undergraduate Biology courses, as encouraged by the AAAS and NSF Vision and Change Initiative.

Bromodomain and Extra-terminal (BET) Protein Inhibitors Suppress Chondrocyte Differentiation and Restrain Bone Growth.

PubMed

Niu, Ningning; Shao, Rui; Yan, Guang; Zou, Weiguo

2016-12-23

Small molecule inhibitors for bromodomain and extra-terminal (BET) proteins have recently emerged as potential therapeutic agents in clinical trials for various cancers. However, to date, it is unknown whether these inhibitors have side effects on bone structures. Here, we report that inhibition of BET bromodomain proteins may suppress chondrocyte differentiation and restrain bone growth. We generated a luciferase reporter system using the chondrogenic cell line ATDC5 in which the luciferase gene was driven by the promoter of Col2a1, an elementary collagen of the chondrocyte. The Col2a1-luciferase ATDC5 system was used for rapidly screening both activators and repressors of human collagen Col2a1 gene expression, and we found that BET bromodomain inhibitors reduce the Col2a1-luciferase. Consistent with the luciferase assay, BET inhibitors decrease the expression of Col2a1 Furthermore, we constructed a zebrafish line in which the enhanced green fluorescent protein (EGFP) expression was driven by col2a1 promoter. The transgenic (col2a1-EGFP) zebrafish line demonstrated that BET inhibitors I-BET151 and (+)-JQ1 may affect EGFP expression in zebrafish. Furthermore, we found that I-BET151 and (+)-JQ1 may affect chondrocyte differentiation in vitro and inhibit zebrafish growth in vivo Mechanistic analysis revealed that BET inhibitors influenced the depletion of RNA polymerase II from the Col2a1 promoter. Collectively, these results suggest that BET bromodomain inhibition may have side effects on skeletal bone structures. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Optimization and Pharmacological Validation of a Leukocyte Migration Assay in Zebrafish Larvae for the Rapid In Vivo Bioactivity Analysis of Anti-Inflammatory Secondary Metabolites

PubMed Central

Vicet-Muro, Liliana; Wilches-Arizábala, Isabel María; Esguerra, Camila V.; de Witte, Peter A. M.; Crawford, Alexander D.

2013-01-01

Over the past decade, zebrafish (Danio rerio) have emerged as an attractive model for in vivo drug discovery. In this study, we explore the suitability of zebrafish larvae to rapidly evaluate the anti-inflammatory activity of natural products (NPs) and medicinal plants used in traditional medicine for the treatment of inflammatory disorders. First, we optimized a zebrafish assay for leukocyte migration. Inflammation was induced in four days post-fertilization (dpf) zebrafish larvae by tail transection and co-incubation with bacterial lipopolysaccharides (LPS), resulting in a robust recruitment of leukocytes to the zone of injury. Migrating zebrafish leukocytes were detected in situ by myeloperoxidase (MPO) staining, and anti-inflammatory activity was semi-quantitatively scored using a standardized scale of relative leukocyte migration (RLM). Pharmacological validation of this optimized assay was performed with a panel of anti-inflammatory drugs, demonstrating a concentration-responsive inhibition of leukocyte migration for both steroidal and non-steroidal anti-inflammatory drugs (SAIDs and NSAIDs). Subsequently, we evaluated the bioactivity of structurally diverse NPs with well-documented anti-inflammatory properties. Finally, we further used this zebrafish-based assay to quantify the anti-inflammatory activity in the aqueous and methanolic extracts of several medicinal plants. Our results indicate the suitability of this LPS-enhanced leukocyte migration assay in zebrafish larvae as a front-line screening platform in NP discovery, including for the bioassay-guided isolation of anti-inflammatory secondary metabolites from complex NP extracts. PMID:24124487

Advancements in zebrafish applications for 21st century toxicology.

PubMed

Garcia, Gloria R; Noyes, Pamela D; Tanguay, Robert L

2016-05-01

The zebrafish model is the only available high-throughput vertebrate assessment system, and it is uniquely suited for studies of in vivo cell biology. A sequenced and annotated genome has revealed a large degree of evolutionary conservation in comparison to the human genome. Due to our shared evolutionary history, the anatomical and physiological features of fish are highly homologous to humans, which facilitates studies relevant to human health. In addition, zebrafish provide a very unique vertebrate data stream that allows researchers to anchor hypotheses at the biochemical, genetic, and cellular levels to observations at the structural, functional, and behavioral level in a high-throughput format. In this review, we will draw heavily from toxicological studies to highlight advances in zebrafish high-throughput systems. Breakthroughs in transgenic/reporter lines and methods for genetic manipulation, such as the CRISPR-Cas9 system, will be comprised of reports across diverse disciplines. Copyright © 2016 Elsevier Inc. All rights reserved.

Advancements in zebrafish applications for 21st century toxicology

PubMed Central

Garcia, Gloria R.; Noyes, Pamela D.; Tanguay, Robert L.

2016-01-01

The zebrafish model is the only available high-throughput vertebrate assessment system, and it is uniquely suited for studies of in vivo cell biology. A sequenced and annotated genome has revealed a large degree of evolutionary conservation in comparison to the human genome. Due to our shared evolutionary history, the anatomical and physiological features of fish are highly homologous to humans, which facilitates studies relevant to human health. In addition, zebrafish provide a very unique vertebrate data stream that allows researchers to anchor hypotheses at the biochemical, genetic, and cellular levels to observations at the structural, functional, and behavioral level in a high-throughput format. In this review, we will draw heavily from toxicological studies to highlight advances in zebrafish high-throughput systems. Breakthroughs in transgenic/reporter lines and methods for genetic manipulation, such as the CRISPR-Cas9 system, will be comprised of reports across diverse disciplines. PMID:27016469

Transient receptor potential channel M5 and phospholipaseC-beta2 colocalizing in zebrafish taste receptor cells.

PubMed

Yoshida, Yuki; Saitoh, Kana; Aihara, Yoshiko; Okada, Shinji; Misaka, Takumi; Abe, Keiko

2007-10-08

In mammals, transient receptor potential (TRP) channel M5 (TRPM5) is coexpressed with phospholipaseC-beta2 (PLC-beta2) in the taste receptor cells, and both PLC-beta2 and TRPM5 are essential elements in the signal transduction of sweet, bitter and umami stimuli. In this study, we identified the zebrafish homologue of TRPM5 (zfTRPM5) and examined its expression in the gustatory system by in-situ hybridization. Using a transgenic zebrafish line that expressed green fluorescent protein under the control of the PLC-beta2 promoter, we showed that zfTRPM5 is expressed in green fluorescent protein-labeled cells of the taste buds. These results demonstrate that zfTRPM5 and PLC-beta2 colocalize in zebrafish taste receptor cells, suggesting their crucial roles in taste signaling via the fish taste receptors.

Whole-body and multispectral photoacoustic imaging of adult zebrafish

NASA Astrophysics Data System (ADS)

Huang, Na; Xi, Lei

2016-10-01

Zebrafish is a top vertebrate model to study developmental biology and genetics, and it is becoming increasingly popular for studying human diseases due to its high genome similarity to that of humans and the optical transparency in embryonic stages. However, it becomes difficult for pure optical imaging techniques to volumetric visualize the internal organs and structures of wild-type zebrafish in juvenile and adult stages with excellent resolution and penetration depth. Even with the establishment of mutant lines which remain transparent over the life cycle, it is still a challenge for pure optical imaging modalities to image the whole body of adult zebrafish with micro-scale resolution. However, the method called photoacoustic imaging that combines all the advantages of the optical imaging and ultrasonic imaging provides a new way to image the whole body of the zebrafish. In this work, we developed a non-invasive photoacoustic imaging system with optimized near-infrared illumination and cylindrical scanning to image the zebrafish. The lateral and axial resolution yield to 80 μm and 600 μm, respectively. Multispectral strategy with wavelengths from 690 nm to 930 nm was employed to image various organs inside the zebrafish. From the reconstructed images, most major organs and structures inside the body can be precisely imaged. Quantitative and statistical analysis of absorption for organs under illumination with different wavelengths were carried out.

Alkbh4 and Atrn Act Maternally to Regulate Zebrafish Epiboly

PubMed Central

Sun, Qingrui; Liu, Xingfeng; Gong, Bo; Wu, Di; Meng, Anming; Jia, Shunji

2017-01-01

During embryonic gastrulation, coordinated cell movements occur to bring cells to their correct position. Among them, epiboly produces the first distinct morphological changes, which is essential for the early development of zebrafish. Despite its fundamental importance, little is known to understand the underlying molecular mechanisms. By generating maternal mutant lines with CRISPR/Cas9 technology and using morpholino knockdown strategy, we showed that maternal Alkbh4 depletion leads to severe epiboly defects in zebrafish. Immunofluorescence assays revealed that Alkbh4 promotes zebrafish embryonic epiboly through regulating actomyosin contractile ring formation, which is composed of Actin and non-muscular myosin II (NMII). To further investigate this process, yeast two hybridization assay was performed and Atrn was identified as a binding partner of Alkbh4. Combining with the functional results of Alkbh4, we found that maternal Atrn plays a similar role in zebrafish embryonic morphogenesis by regulating actomyosin formation. On the molecular level, our data revealed that Atrn prefers to interact with the active form of Alkbh4 and functions together with it to regulate the demethylation of Actin, the actomyosin formation, and subsequently the embryonic epiboly. PMID:28924386

It's time to swim! Zebrafish and the circadian clock.

PubMed

Vatine, Gad; Vallone, Daniela; Gothilf, Yoav; Foulkes, Nicholas S

2011-05-20

The zebrafish represents a fascinating model for studying key aspects of the vertebrate circadian timing system. Easy access to early embryonic development has made this species ideal for investigating how the clock is first established during embryogenesis. In particular, the molecular basis for the functional development of the zebrafish pineal gland has received much attention. In addition to this dedicated clock and photoreceptor organ, and unlike the situation in mammals, the clocks in zebrafish peripheral tissues and even cell lines are entrainable by direct exposure to light thus providing unique insight into the function and evolution of the light input pathway. Finally, the small size, low maintenance costs and high fecundity of this fish together with the availability of genetic tools make this an attractive model for forward genetic analysis of the circadian clock. Here, we review the work that has established the zebrafish as a valuable clock model organism and highlight the key questions that will shape the future direction of research. Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Progenitor potential of nkx6.1-expressing cells throughout zebrafish life and during beta cell regeneration.

PubMed

Ghaye, Aurélie P; Bergemann, David; Tarifeño-Saldivia, Estefania; Flasse, Lydie C; Von Berg, Virginie; Peers, Bernard; Voz, Marianne L; Manfroid, Isabelle

2015-09-02

In contrast to mammals, the zebrafish has the remarkable capacity to regenerate its pancreatic beta cells very efficiently. Understanding the mechanisms of regeneration in the zebrafish and the differences with mammals will be fundamental to discovering molecules able to stimulate the regeneration process in mammals. To identify the pancreatic cells able to give rise to new beta cells in the zebrafish, we generated new transgenic lines allowing the tracing of multipotent pancreatic progenitors and endocrine precursors. Using novel bacterial artificial chromosome transgenic nkx6.1 and ascl1b reporter lines, we established that nkx6.1-positive cells give rise to all the pancreatic cell types and ascl1b-positive cells give rise to all the endocrine cell types in the zebrafish embryo. These two genes are initially co-expressed in the pancreatic primordium and their domains segregate, not as a result of mutual repression, but through the opposite effects of Notch signaling, maintaining nkx6.1 expression while repressing ascl1b in progenitors. In the adult zebrafish, nkx6.1 expression persists exclusively in the ductal tree at the tip of which its expression coincides with Notch active signaling in centroacinar/terminal end duct cells. Tracing these cells reveals that they are able to differentiate into other ductal cells and into insulin-expressing cells in normal (non-diabetic) animals. This capacity of ductal cells to generate endocrine cells is supported by the detection of ascl1b in the nkx6.1:GFP ductal cell transcriptome. This transcriptome also reveals, besides actors of the Notch and Wnt pathways, several novel markers such as id2a. Finally, we show that beta cell ablation in the adult zebrafish triggers proliferation of ductal cells and their differentiation into insulin-expressing cells. We have shown that, in the zebrafish embryo, nkx6.1+ cells are bona fide multipotent pancreatic progenitors, while ascl1b+ cells represent committed endocrine precursors. In contrast to the mouse, pancreatic progenitor markers nkx6.1 and pdx1 continue to be expressed in adult ductal cells, a subset of which we show are still able to proliferate and undergo ductal and endocrine differentiation, providing robust evidence of the existence of pancreatic progenitor/stem cells in the adult zebrafish. Our findings support the hypothesis that nkx6.1+ pancreatic progenitors contribute to beta cell regeneration. Further characterization of these cells will open up new perspectives for anti-diabetic therapies.

Acute multi-sgRNA knockdown of KEOPS complex genes reproduces the microcephaly phenotype of the stable knockout zebrafish model.

PubMed

Jobst-Schwan, Tilman; Schmidt, Johanna Magdalena; Schneider, Ronen; Hoogstraten, Charlotte A; Ullmann, Jeremy F P; Schapiro, David; Majmundar, Amar J; Kolb, Amy; Eddy, Kaitlyn; Shril, Shirlee; Braun, Daniela A; Poduri, Annapurna; Hildebrandt, Friedhelm

2018-01-01

Until recently, morpholino oligonucleotides have been widely employed in zebrafish as an acute and efficient loss-of-function assay. However, off-target effects and reproducibility issues when compared to stable knockout lines have compromised their further use. Here we employed an acute CRISPR/Cas approach using multiple single guide RNAs targeting simultaneously different positions in two exemplar genes (osgep or tprkb) to increase the likelihood of generating mutations on both alleles in the injected F0 generation and to achieve a similar effect as morpholinos but with the reproducibility of stable lines. This multi single guide RNA approach resulted in median likelihoods for at least one mutation on each allele of >99% and sgRNA specific insertion/deletion profiles as revealed by deep-sequencing. Immunoblot showed a significant reduction for Osgep and Tprkb proteins. For both genes, the acute multi-sgRNA knockout recapitulated the microcephaly phenotype and reduction in survival that we observed previously in stable knockout lines, though milder in the acute multi-sgRNA knockout. Finally, we quantify the degree of mutagenesis by deep sequencing, and provide a mathematical model to quantitate the chance for a biallelic loss-of-function mutation. Our findings can be generalized to acute and stable CRISPR/Cas targeting for any zebrafish gene of interest.

Zebrafish and Medaka: new model organisms for modern biomedical research.

PubMed

Lin, Cheng-Yung; Chiang, Cheng-Yi; Tsai, Huai-Jen

2016-01-28

Although they are primitive vertebrates, zebrafish (Danio rerio) and medaka (Oryzias latipes) have surpassed other animals as the most used model organisms based on their many advantages. Studies on gene expression patterns, regulatory cis-elements identification, and gene functions can be facilitated by using zebrafish embryos via a number of techniques, including transgenesis, in vivo transient assay, overexpression by injection of mRNAs, knockdown by injection of morpholino oligonucleotides, knockout and gene editing by CRISPR/Cas9 system and mutagenesis. In addition, transgenic lines of model fish harboring a tissue-specific reporter have become a powerful tool for the study of biological sciences, since it is possible to visualize the dynamic expression of a specific gene in the transparent embryos. In particular, some transgenic fish lines and mutants display defective phenotypes similar to those of human diseases. Therefore, a wide variety of fish model not only sheds light on the molecular mechanisms underlying disease pathogenesis in vivo but also provides a living platform for high-throughput screening of drug candidates. Interestingly, transgenic model fish lines can also be applied as biosensors to detect environmental pollutants, and even as pet fish to display beautiful fluorescent colors. Therefore, transgenic model fish possess a broad spectrum of applications in modern biomedical research, as exampled in the following review.

Live imaging of collagen deposition during skin development and repair in a collagen I - GFP fusion transgenic zebrafish line.

PubMed

Morris, Josephine L; Cross, Stephen J; Lu, Yinhui; Kadler, Karl E; Lu, Yongbo; Dallas, Sarah L; Martin, Paul

2018-06-06

Fibrillar collagen is a major component of many tissues but has been difficult to image in vivo using transgenic approaches because of problems associated with establishing cells and organisms that generate GFP-fusion collagens that can polymerise into functional fibrils. Here we have developed and characterised GFP and mCherry collagen-I fusion zebrafish lines with basal epidermal-specific expression. We use these lines to reveal the dynamic nature of collagen-I fibril deposition beneath the developing embryonic epidermis, as well as the repair of this collagen meshwork following wounding. Transmission electron microscope studies show that these transgenic lines faithfully reproduce the collagen ultrastructure present in wild type larval skin. During skin development we show that collagen I is deposited by basal epidermal cells initially in fine filaments that are largely randomly orientated but are subsequently aligned into a cross-hatch, orthogonal sub-epithelial network by embryonic day 4. Following skin wounding, we see that sub-epidermal collagen is re-established in the denuded domain, initially as randomly orientated wisps that subsequently become bonded to the undamaged collagen and aligned in a way that recapitulates developmental deposition of sub-epidermal collagen. Crossing our GFP-collagen line against one with tdTomato marking basal epidermal cell membranes reveals how much more rapidly wound re-epithelialisation occurs compared to the re-deposition of collagen beneath the healed epidermis. By use of other tissue specific drivers it will be possible to establish zebrafish lines to enable live imaging of collagen deposition and its remodelling in various other organs in health and disease. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Conditionally immortalized stem cell lines from human spinal cord retain regional identity and generate functional V2a interneurons and motorneurons.

PubMed

Cocks, Graham; Romanyuk, Nataliya; Amemori, Takashi; Jendelova, Pavla; Forostyak, Oksana; Jeffries, Aaron R; Perfect, Leo; Thuret, Sandrine; Dayanithi, Govindan; Sykova, Eva; Price, Jack

2013-06-07

The use of immortalized neural stem cells either as models of neural development in vitro or as cellular therapies in central nervous system (CNS) disorders has been controversial. This controversy has centered on the capacity of immortalized cells to retain characteristic features of the progenitor cells resident in the tissue of origin from which they were derived, and the potential for tumorogenicity as a result of immortalization. Here, we report the generation of conditionally immortalized neural stem cell lines from human fetal spinal cord tissue, which addresses these issues. Clonal neural stem cell lines were derived from 10-week-old human fetal spinal cord and conditionally immortalized with an inducible form of cMyc. The derived lines were karyotyped, transcriptionally profiled by microarray, and assessed against a panel of spinal cord progenitor markers with immunocytochemistry. In addition, the lines were differentiated and assessed for the presence of neuronal fate markers and functional calcium channels. Finally, a clonal line expressing eGFP was grafted into lesioned rat spinal cord and assessed for survival, differentiation characteristics, and tumorogenicity. We demonstrate that these clonal lines (a) retain a clear transcriptional signature of ventral spinal cord progenitors and a normal karyotype after extensive propagation in vitro, (b) differentiate into relevant ventral neuronal subtypes with functional T-, L-, N-, and P/Q-type Ca(2+) channels and spontaneous calcium oscillations, and (c) stably engraft into lesioned rat spinal cord without tumorogenicity. We propose that these cells represent a useful tool both for the in vitro study of differentiation into ventral spinal cord neuronal subtypes, and for examining the potential of conditionally immortalized neural stem cells to facilitate functional recovery after spinal cord injury or disease.

Somatically Acquired LINE-1 Insertions in Normal Esophagus Undergo Clonal Expansion in Esophageal Squamous Cell Carcinoma.

PubMed

Doucet-O'Hare, Tara T; Sharma, Reema; Rodić, Nemanja; Anders, Robert A; Burns, Kathleen H; Kazazian, Haig H

2016-09-01

Squamous cell carcinoma of the esophagus (SCC) is the most common form of esophageal cancer in the world and is typically diagnosed at an advanced stage when successful treatment is challenging. Understanding the mutational profile of this cancer may identify new treatment strategies. Because somatic retrotransposition has been shown in tumors of the gastrointestinal system, we focused on LINE-1 (L1) mobilization as a source of genetic instability in this cancer. We hypothesized that retrotransposition is ongoing in SCC patients. The expression of L1 encoded proteins is necessary for retrotransposition to occur; therefore, we evaluated the expression of L1 open reading frame 1 protein (ORF1p). Using immunohistochemistry, we detected ORF1p expression in all four SCC cases evaluated. Using L1-seq, we identified and validated 74 somatic insertions in eight tumors of the nine evaluated. Of these, 12 insertions appeared to be somatic, not genetically inherited, and sub-clonal (i.e., present in less than one copy per genome equivalent) in the adjacent normal esophagus (NE), while clonal in the tumor. Our results indicate that L1 retrotransposition is active in SCC of the esophagus and that insertion events are present in histologically NE that expands clonally in the subsequent tumor. © 2016 WILEY PERIODICALS, INC.

Production of zebrafish cardiospheres and cardiac progenitor cells in vitro and three-dimensional culture of adult zebrafish cardiac tissue in scaffolds.

PubMed

Zeng, Wendy R; Beh, Siew-Joo; Bryson-Richardson, Robert J; Doran, Pauline M

2017-09-01

The hearts of adult zebrafish (Danio rerio) are capable of complete regeneration in vivo even after major injury, making this species of particular interest for understanding the growth and differentiation processes required for cardiac tissue engineering. To date, little research has been carried out on in vitro culture of adult zebrafish cardiac cells. In this work, progenitor-rich cardiospheres suitable for cardiomyocyte differentiation and myocardial regeneration were produced from adult zebrafish hearts. The cardiospheres contained a mixed population of c-kit + and Mef2c + cells; proliferative peripheral cells of possible mesenchymal lineage were also observed. Cellular outgrowth from cardiac explants and cardiospheres was enhanced significantly using conditioned medium harvested from cultures of a rainbow trout cell line, suggesting that fish-specific trophic factors are required for zebrafish cardiac cell expansion. Three-dimensional culture of zebrafish heart cells in fibrous polyglycolic acid (PGA) scaffolds was carried out under dynamic fluid flow conditions. High levels of cell viability and cardiomyocyte differentiation were maintained within the scaffolds. Expression of cardiac troponin T, a marker of differentiated cardiomyocytes, increased during the first 7 days of scaffold culture; after 15 days, premature disintegration of the biodegradable scaffolds led to cell detachment and a decline in differentiation status. This work expands our technical capabilities for three-dimensional zebrafish cardiac cell culture with potential applications in tissue engineering, drug and toxicology screening, and ontogeny research. Biotechnol. Bioeng. 2017;114: 2142-2148. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Systematic gene tagging using CRISPR/Cas9 in human stem cells to illuminate cell organization

PubMed Central

Roberts, Brock; Haupt, Amanda; Tucker, Andrew; Grancharova, Tanya; Arakaki, Joy; Fuqua, Margaret A.; Nelson, Angelique; Hookway, Caroline; Ludmann, Susan A.; Mueller, Irina A.; Yang, Ruian; Horwitz, Rick; Rafelski, Susanne M.; Gunawardane, Ruwanthi N.

2017-01-01

We present a CRISPR/Cas9 genome-editing strategy to systematically tag endogenous proteins with fluorescent tags in human induced pluripotent stem cells (hiPSC). To date, we have generated multiple hiPSC lines with monoallelic green fluorescent protein tags labeling 10 proteins representing major cellular structures. The tagged proteins include alpha tubulin, beta actin, desmoplakin, fibrillarin, nuclear lamin B1, nonmuscle myosin heavy chain IIB, paxillin, Sec61 beta, tight junction protein ZO1, and Tom20. Our genome-editing methodology using Cas9/crRNA ribonuclear protein and donor plasmid coelectroporation, followed by fluorescence-based enrichment of edited cells, typically resulted in <0.1–4% homology-directed repair (HDR). Twenty-five percent of clones generated from each edited population were precisely edited. Furthermore, 92% (36/39) of expanded clonal lines displayed robust morphology, genomic stability, expression and localization of the tagged protein to the appropriate subcellular structure, pluripotency-marker expression, and multilineage differentiation. It is our conclusion that, if cell lines are confirmed to harbor an appropriate gene edit, pluripotency, differentiation potential, and genomic stability are typically maintained during the clonal line–generation process. The data described here reveal general trends that emerged from this systematic gene-tagging approach. Final clonal lines corresponding to each of the 10 cellular structures are now available to the research community. PMID:28814507

Conservation/Mutation in the Splice Sites of Mitochondrial Solute Carrier Genes of Vertebrates.

PubMed

Calvello, Rosa; Panaro, Maria A; Salvatore, Rosaria; Mitolo, Vincenzo; Cianciulli, Antonia

2016-10-01

The "canonical" introns begin by the dinucleotide GT and end by the dinucleotide AG. GT, together with a few downstream nucleotides, and AG, with a few of the immediately preceding nucleotides, are thought to be the strongest splicing signals (5'ss and 3'ss, respectively). We examined the composition of the intronic initial and terminal hexanucleotides of the mitochondrial solute carrier genes (SLC25A's) of zebrafish, chicken, mouse, and human. These genes are orthologous and we selected the transcripts in which the arrangement of exons and introns was superimposable in the species considered. Both 5'ss and 3'ss were highly polymorphic, with 104 and 126 different configurations, respectively, in our sample. In the line of evolution from zebrafish to chicken, as well as in that from zebrafish to mammals, the average nucleotide conservation in the four variable nucleotides was about 50 % at 5' and 40 % at 3'. In the divergent evolution of mouse and human, the conservation was about 80 % at 5' and 70 % at 3'. Despite these changes, the splicing signals remain strong enough to operate at the same site. At both 5' and 3', the frequency of a nucleotide at a given position in the zebrafish sequence is positively correlated with its conservation in chicken and mammals, suggesting that selection continued to operate in birds and mammals along similar lines.

Knockout of the Gnrh genes in zebrafish: effects on reproduction and potential compensation by reproductive and feeding-related neuropeptides.

PubMed

Marvel, Miranda; Spicer, Olivia Smith; Wong, Ten-Tsao; Zmora, Nilli; Zohar, Yonathan

2018-04-04

Gonadotropin-releasing hormone (GnRH) is known as a pivotal upstream regulator of reproduction in vertebrates. However, reproduction is not compromised in the hypophysiotropic Gnrh3 knockout line in zebrafish (gnrh3-/-). In order to determine if Gnrh2, the only other Gnrh isoform in zebrafish brains, is compensating for the loss of Gnrh3, we generated a double Gnrh knockout zebrafish line. Surprisingly, the loss of both Gnrh isoforms resulted in no major impact on reproduction, indicating that a compensatory response, outside of the Gnrh system, was evoked. A plethora of factors acting along the reproductive hypothalamus-pituitary axis were evaluated as possible compensators based on neuroanatomical and differential gene expression studies. In addition, we also examined the involvement of feeding factors in the brain as potential compensators for Gnrh2, which has known anorexigenic effects. We found that the double knockout fish exhibited upregulation of several genes in the brain, specifically gonadotropin-inhibitory hormone (gnih), secretogranin 2 (scg2), tachykinin 3a (tac3a), and pituitary adenylate cyclase-activating peptide 1 (pacap1), and downregulation of agouti-related peptide 1 (agrp1), indicating the compensation occurs outside of Gnrh cells and therefore is a non-cell autonomous response to the loss of Gnrh. While the differential expression of gnih and agrp1 in the double knockout line was confined to the periventricular nucleus and hypothalamus, respectively, the upregulation of scg2 corresponded with a broader neuronal redistribution in the lateral hypothalamus and hindbrain. In conclusion, our results demonstrate the existence of a redundant reproductive regulatory system that comes into play when Gnrh2 and Gnrh3 are lost.

The basic helix-loop-helix transcription factor, heart and neural crest derivatives expressed transcript 2, marks hepatic stellate cells in zebrafish: analysis of stellate cell entry into the developing liver.

PubMed

Yin, Chunyue; Evason, Kimberley J; Maher, Jacquelyn J; Stainier, Didier Y R

2012-11-01

Hepatic stellate cells (HSCs) are liver-specific mesenchymal cells that play vital roles in liver development and injury. Our knowledge of HSC biology is limited by the paucity of in vivo data. HSCs and sinusoidal endothelial cells (SECs) reside in close proximity, and interactions between these two cell types are potentially critical for their development and function. Here, we introduce a transgenic zebrafish line, Tg(hand2:EGFP), that labels HSCs. We find that zebrafish HSCs share many similarities with their mammalian counterparts, including morphology, location, lipid storage, gene-expression profile, and increased proliferation and matrix production, in response to an acute hepatic insult. Using the Tg(hand2:EGFP) line, we conducted time-course analyses during development to reveal that HSCs invade the liver after SECs do. However, HSCs still enter the liver in mutants that lack most endothelial cells, including SECs, indicating that SECs are not required for HSC differentiation or their entry into the liver. In the absence of SECs, HSCs become abnormally associated with hepatic biliary cells, suggesting that SECs influence HSC localization during liver development. We analyzed factors that regulate HSC development and show that inhibition of vascular endothelial growth factor signaling significantly reduces the number of HSCs that enter the liver. We also performed a pilot chemical screen and identified two compounds that affect HSC numbers during development. Our work provides the first comprehensive description of HSC development in zebrafish and reveals the requirement of SECs in HSC localization. The Tg(hand2:EGFP) line represents a unique tool for in vivo analysis and molecular dissection of HSC behavior. Copyright © 2012 American Association for the Study of Liver Diseases.

Next-generation sequencing traces human induced pluripotent stem cell lines clonally generated from heterogeneous cancer tissue.

PubMed

Ishikawa, Tetsuya

2017-05-26

To investigate genotype variation among induced pluripotent stem cell (iPSC) lines that were clonally generated from heterogeneous colon cancer tissues using next-generation sequencing. Human iPSC lines were clonally established by selecting independent single colonies expanded from heterogeneous primary cells of S-shaped colon cancer tissues by retroviral gene transfer ( OCT3/4 , SOX2 , and KLF4 ). The ten iPSC lines, their starting cancer tissues, and the matched adjacent non-cancerous tissues were analyzed using next-generation sequencing and bioinformatics analysis using the human reference genome hg19. Non-synonymous single-nucleotide variants (SNVs) (missense, nonsense, and read-through) were identified within the target region of 612 genes related to cancer and the human kinome. All SNVs were annotated using dbSNP135, CCDS, RefSeq, GENCODE, and 1000 Genomes. The SNVs of the iPSC lines were compared with the genotypes of the cancerous and non-cancerous tissues. The putative genotypes were validated using allelic depth and genotype quality. For final confirmation, mutated genotypes were manually curated using the Integrative Genomics Viewer. In eight of the ten iPSC lines, one or two non-synonymous SNVs in EIF2AK2 , TTN , ULK4 , TSSK1B , FLT4 , STK19 , STK31 , TRRAP , WNK1 , PLK1 or PIK3R5 were identified as novel SNVs and were not identical to the genotypes found in the cancer and non-cancerous tissues. This result suggests that the SNVs were de novo or pre-existing mutations that originated from minor populations, such as multifocal pre-cancer (stem) cells or pre-metastatic cancer cells from multiple, different clonal evolutions, present within the heterogeneous cancer tissue. The genotypes of all ten iPSC lines were different from the mutated ERBB2 and MKNK2 genotypes of the cancer tissues and were identical to those of the non-cancerous tissues and that found in the human reference genome hg19. Furthermore, two of the ten iPSC lines did not have any confirmed mutated genotypes, despite being derived from cancerous tissue. These results suggest that the traceability and preference of the starting single cells being derived from pre-cancer (stem) cells, stroma cells such as cancer-associated fibroblasts, and immune cells that co-existed in the tissues along with the mature cancer cells. The genotypes of iPSC lines derived from heterogeneous cancer tissues can provide information on the type of starting cell that the iPSC line was generated from.

Mutagenesis Screen Identifies agtpbp1 and eps15L1 as Essential for T lymphocyte Development in Zebrafish.

PubMed

Seiler, Christoph; Gebhart, Nichole; Zhang, Yong; Shinton, Susan A; Li, Yue-sheng; Ross, Nicola L; Liu, Xingjun; Li, Qin; Bilbee, Alison N; Varshney, Gaurav K; LaFave, Matthew C; Burgess, Shawn M; Balciuniene, Jorune; Balciunas, Darius; Hardy, Richard R; Kappes, Dietmar J; Wiest, David L; Rhodes, Jennifer

2015-01-01

Genetic screens are a powerful tool to discover genes that are important in immune cell development and function. The evolutionarily conserved development of lymphoid cells paired with the genetic tractability of zebrafish make this a powerful model system for this purpose. We used a Tol2-based gene-breaking transposon to induce mutations in the zebrafish (Danio rerio, AB strain) genome, which served the dual purpose of fluorescently tagging cells and tissues that express the disrupted gene and provided a means of identifying the disrupted gene. We identified 12 lines in which hematopoietic tissues expressed green fluorescent protein (GFP) during embryonic development, as detected by microscopy. Subsequent analysis of young adult fish, using a novel approach in which single cell suspensions of whole fish were analyzed by flow cytometry, revealed that 8 of these lines also exhibited GFP expression in young adult cells. An additional 15 lines that did not have embryonic GFP+ hematopoietic tissue by microscopy, nevertheless exhibited GFP+ cells in young adults. RT-PCR analysis of purified GFP+ populations for expression of T and B cell-specific markers identified 18 lines in which T and/or B cells were fluorescently tagged at 6 weeks of age. As transposon insertion is expected to cause gene disruption, these lines can be used to assess the requirement for the disrupted genes in immune cell development. Focusing on the lines with embryonic GFP+ hematopoietic tissue, we identified three lines in which homozygous mutants exhibited impaired T cell development at 6 days of age. In two of the lines we identified the disrupted genes, agtpbp1 and eps15L1. Morpholino-mediated knockdown of these genes mimicked the T cell defects in the corresponding mutant embryos, demonstrating the previously unrecognized, essential roles of agtpbp1 and eps15L1 in T cell development.

BAC Recombineering of the Agouti Loci from Spotted Gar and Zebrafish Reveals the Evolutionary Ancestry of Dorsal-Ventral Pigment Asymmetry in Fish.

PubMed

Cal, Laura; MegÍas, Manuel; Cerdá-Reverter, José Miguel; Postlethwait, John H; Braasch, Ingo; Rotllant, Josep

2017-11-01

Dorsoventral pigment patterning, characterized by a light ventrum and a dark dorsum, is one of the most widespread chromatic adaptations in vertebrate body coloration. In mammals, this countershading depends on differential expression of agouti-signaling protein (ASIP), which drives a switch of synthesis of one type of melanin to another within melanocytes. Teleost fish share countershading, but the pattern results from a differential distribution of multiple types of chromatophores, with black-brown melanophores most abundant in the dorsal body and reflective iridophores most abundant in the ventral body. We previously showed that Asip1 (a fish ortholog of mammalian ASIP) plays a role in patterning melanophores. This observation leads to the surprising hypothesis that agouti may control an evolutionarily conserved pigment pattern by regulating different mechanisms in mammals and fish. To test this hypothesis, we compared two ray-finned fishes: the teleost zebrafish and the nonteleost spotted gar (Lepisosteus oculatus). By examining the endogenous pattern of asip1 expression in gar, we demonstrate a dorsoventral-graded distribution of asip1 expression that is highest ventrally, similar to teleosts. Additionally, in the first reported experiments to generate zebrafish transgenic lines carrying a bacterial artificial chromosome (BAC) from spotted gar, we show that both transgenic zebrafish lines embryos replicate the endogenous asip1 expression pattern in adult zebrafish, showing that BAC transgenes from both species contain all of the regulatory elements required for regular asip1 expression within adult ray-finned fishes. These experiments provide evidence that the mechanism leading to an environmentally important pigment pattern was likely in place before the origin of teleosts. © 2017 Wiley Periodicals, Inc.

The Zebrafish Xenograft Platform: Evolution of a Novel Cancer Model and Preclinical Screening Tool.

PubMed

Wertman, Jaime; Veinotte, Chansey J; Dellaire, Graham; Berman, Jason N

2016-01-01

Animal xenografts of human cancers represent a key preclinical tool in the field of cancer research. While mouse xenografts have long been the gold standard, investigators have begun to use zebrafish (Danio rerio) xenotransplantation as a relatively rapid, robust and cost-effective in vivo model of human cancers. There are several important methodological considerations in the design of an informative and efficient zebrafish xenotransplantation experiment. Various transgenic fish strains have been created that facilitate microscopic observation, ranging from the completely transparent casper fish to the Tg(fli1:eGFP) fish that expresses fluorescent GFP protein in its vascular tissue. While human cancer cell lines have been used extensively in zebrafish xenotransplantation studies, several reports have also used primary patient samples as the donor material. The zebrafish is ideally suited for transplanting primary patient material by virtue of the relatively low number of cells required for each embryo (between 50 and 300 cells), the absence of an adaptive immune system in the early zebrafish embryo, and the short experimental timeframe (5-7 days). Following xenotransplantation into the fish, cells can be tracked using in vivo or ex vivo measures of cell proliferation and migration, facilitated by fluorescence or human-specific protein expression. Importantly, assays have been developed that allow for the reliable detection of in vivo human cancer cell growth or inhibition following administration of drugs of interest. The zebrafish xenotransplantation model is a unique and effective tool for the study of cancer cell biology.

Turning rice meiosis into mitosis

PubMed Central

Mieulet, Delphine; Jolivet, Sylvie; Rivard, Maud; Cromer, Laurence; Vernet, Aurore; Mayonove, Pauline; Pereira, Lucie; Droc, Gaëtan; Courtois, Brigitte; Guiderdoni, Emmanuel; Mercier, Raphael

2016-01-01

Introduction of clonal reproduction through seeds (apomixis) in crops has the potential to revolutionize agriculture by allowing self-propagation of any elite variety, in particular F1 hybrids. In the sexual model plant Arabidopsis thaliana synthetic clonal reproduction through seeds can be artificially implemented by (i) combining three mutations to turn meiosis into mitosis (MiMe) and (ii) crossing the obtained clonal gametes with a line expressing modified CENH3 and whose genome is eliminated in the zygote. Here we show that additional combinations of mutations can turn Arabidopsis meiosis into mitosis and that a combination of three mutations in rice (Oryza sativa) efficiently turns meiosis into mitosis, leading to the production of male and female clonal diploid gametes in this major crop. Successful implementation of the MiMe technology in the phylogenetically distant eudicot Arabidopsis and monocot rice opens doors for its application to any flowering plant and paves the way for introducing apomixis in crop species. PMID:27767093

Hypothalamic Projections to the Optic Tectum in Larval Zebrafish

PubMed Central

Heap, Lucy A.; Vanwalleghem, Gilles C.; Thompson, Andrew W.; Favre-Bulle, Itia; Rubinsztein-Dunlop, Halina; Scott, Ethan K.

2018-01-01

The optic tectum of larval zebrafish is an important model for understanding visual processing in vertebrates. The tectum has been traditionally viewed as dominantly visual, with a majority of studies focusing on the processes by which tectal circuits receive and process retinally-derived visual information. Recently, a handful of studies have shown a much more complex role for the optic tectum in larval zebrafish, and anatomical and functional data from these studies suggest that this role extends beyond the visual system, and beyond the processing of exclusively retinal inputs. Consistent with this evolving view of the tectum, we have used a Gal4 enhancer trap line to identify direct projections from rostral hypothalamus (RH) to the tectal neuropil of larval zebrafish. These projections ramify within the deepest laminae of the tectal neuropil, the stratum album centrale (SAC)/stratum griseum periventriculare (SPV), and also innervate strata distinct from those innervated by retinal projections. Using optogenetic stimulation of the hypothalamic projection neurons paired with calcium imaging in the tectum, we find rebound firing in tectal neurons consistent with hypothalamic inhibitory input. Our results suggest that tectal processing in larval zebrafish is modulated by hypothalamic inhibitory inputs to the deep tectal neuropil. PMID:29403362

Zebrafish tissue injury causes upregulation of interleukin-1 and caspase-dependent amplification of the inflammatory response.

PubMed

Ogryzko, Nikolay V; Hoggett, Emily E; Solaymani-Kohal, Sara; Tazzyman, Simon; Chico, Timothy J A; Renshaw, Stephen A; Wilson, Heather L

2014-02-01

Interleukin-1 (IL-1), the 'gatekeeper' of inflammation, is the apical cytokine in a signalling cascade that drives the early response to injury or infection. Expression, processing and secretion of IL-1 are tightly controlled, and dysregulated IL-1 signalling has been implicated in a number of pathologies ranging from atherosclerosis to complications of infection. Our understanding of these processes comes from in vitro monocytic cell culture models as lines or primary isolates, in which a range and spectra of IL-1 secretion mechanisms have been described. We therefore investigated whether zebrafish embryos provide a suitable in vivo model for studying IL-1-mediated inflammation. Structurally, zebrafish IL-1β shares a β-sheet-rich trefoil structure with its human counterpart. Functionally, leukocyte expression of IL-1β was detectable only following injury, which activated leukocytes throughout zebrafish embryos. Migration of macrophages and neutrophils was attenuated by inhibitors of either caspase-1 or P2X7, which similarly inhibited the activation of NF-κB at the site of injury. Zebrafish offer a new and versatile model to study the IL-1β pathway in inflammatory disease and should offer unique insights into IL-1 biology in vivo.

Hypothalamic Projections to the Optic Tectum in Larval Zebrafish.

PubMed

Heap, Lucy A; Vanwalleghem, Gilles C; Thompson, Andrew W; Favre-Bulle, Itia; Rubinsztein-Dunlop, Halina; Scott, Ethan K

2017-01-01

The optic tectum of larval zebrafish is an important model for understanding visual processing in vertebrates. The tectum has been traditionally viewed as dominantly visual, with a majority of studies focusing on the processes by which tectal circuits receive and process retinally-derived visual information. Recently, a handful of studies have shown a much more complex role for the optic tectum in larval zebrafish, and anatomical and functional data from these studies suggest that this role extends beyond the visual system, and beyond the processing of exclusively retinal inputs. Consistent with this evolving view of the tectum, we have used a Gal4 enhancer trap line to identify direct projections from rostral hypothalamus (RH) to the tectal neuropil of larval zebrafish. These projections ramify within the deepest laminae of the tectal neuropil, the stratum album centrale (SAC)/stratum griseum periventriculare (SPV), and also innervate strata distinct from those innervated by retinal projections. Using optogenetic stimulation of the hypothalamic projection neurons paired with calcium imaging in the tectum, we find rebound firing in tectal neurons consistent with hypothalamic inhibitory input. Our results suggest that tectal processing in larval zebrafish is modulated by hypothalamic inhibitory inputs to the deep tectal neuropil.

Genetic Disruption of 21-Hydroxylase in Zebrafish Causes Interrenal Hyperplasia.

PubMed

Eachus, Helen; Zaucker, Andreas; Oakes, James A; Griffin, Aliesha; Weger, Meltem; Güran, Tülay; Taylor, Angela; Harris, Abigail; Greenfield, Andy; Quanson, Jonathan L; Storbeck, Karl-Heinz; Cunliffe, Vincent T; Müller, Ferenc; Krone, Nils

2017-12-01

Congenital adrenal hyperplasia is a group of common inherited disorders leading to glucocorticoid deficiency. Most cases are caused by 21-hydroxylase deficiency (21OHD). The systemic consequences of imbalanced steroid hormone biosynthesis due to severe 21OHD remains poorly understood. Therefore, we developed a zebrafish model for 21OHD, which focuses on the impairment of glucocorticoid biosynthesis. A single 21-hydroxylase gene (cyp21a2) is annotated in the zebrafish genome based on sequence homology. Our in silico analysis of the 21-hydroxylase (Cyp21a2) protein sequence suggests a sufficient degree of similarity for the usage of zebrafish cyp21a2 to model aspects of human 21OHD in vivo. We determined the spatiotemporal expression patterns of cyp21a2 by whole-mount in situ hybridization and reverse transcription polymerase chain reaction throughout early development. Early cyp21a2 expression is restricted to the interrenal gland (zebrafish adrenal counterpart) and the brain. To further explore the in vivo consequences of 21OHD we created several cyp21a2 null-allele zebrafish lines by using a transcription activator-like effector nuclease genomic engineering strategy. Homozygous mutant zebrafish larvae showed an upregulation of the hypothalamic-pituitary-interrenal (HPI) axis and interrenal hyperplasia. Furthermore, Cyp21a2-deficient larvae had a typical steroid profile, with reduced concentrations of cortisol and increased concentrations of 17-hydroxyprogesterone and 21-deoxycortisol. Affected larvae showed an upregulation of the HPI axis and interrenal hyperplasia. Downregulation of the glucocorticoid-responsive genes pck1 and fkbp5 indicated systemic glucocorticoid deficiency. Our work demonstrates the crucial role of Cyp21a2 in glucocorticoid biosynthesis in zebrafish larvae and establishes an in vivo model allowing studies of systemic consequences of altered steroid hormone synthesis.

Auditory sensitivity of larval zebrafish (Danio rerio) measured using a behavioral prepulse inhibition assay

PubMed Central

Bhandiwad, Ashwin A.; Zeddies, David G.; Raible, David W.; Rubel, Edwin W.; Sisneros, Joseph A.

2013-01-01

SUMMARY Zebrafish (Danio rerio) have become a valuable model for investigating the molecular genetics and development of the inner ear in vertebrates. In this study, we employed a prepulse inhibition (PPI) paradigm to assess hearing in larval wild-type (AB) zebrafish during early development at 5–6 days post-fertilization (d.p.f.). We measured the PPI of the acoustic startle response in zebrafish using a 1-dimensional shaker that simulated the particle motion component of sound along the fish's dorsoventral axis. The thresholds to startle-inducing stimuli were determined in 5–6 d.p.f. zebrafish, and their hearing sensitivity was then characterized using the thresholds of prepulse tone stimuli (90–1200 Hz) that inhibited the acoustic startle response to a reliable startle stimulus (820 Hz at 20 dB re. 1 m s−2). Hearing thresholds were defined as the minimum prepulse tone level required to significantly reduce the startle response probability compared with the baseline (no-prepulse) condition. Larval zebrafish showed greatest auditory sensitivity from 90 to 310 Hz with corresponding mean thresholds of −19 to −10 dB re. 1 m s−2, respectively. Hearing thresholds of prepulse tones were considerably lower than previously predicted by startle response assays. The PPI assay was also used to investigate the relative contribution of the lateral line to the detection of acoustic stimuli. After aminoglycoside-induced neuromast hair-cell ablation, we found no difference in PPI thresholds between treated and control fish. We propose that this PPI assay can be used to screen for novel zebrafish hearing mutants and to investigate the ontogeny of hearing in zebrafish and other fishes. PMID:23966590

Sub-clonal analysis of the murine C1498 acute myeloid leukaemia cell line reveals genomic and immunogenic diversity.

PubMed

Driss, Virginie; Leprêtre, Frédéric; Briche, Isabelle; Mopin, Alexia; Villenet, Céline; Figeac, Martin; Quesnel, Bruno; Brinster, Carine

2017-12-01

In acute myeloid leukaemia (AML)-affected patients, the presence of heterogeneous sub-clones at diagnosis has been shown to be responsible for minimal residual disease and relapses. The role played by the immune system in this leukaemic sub-clonal hierarchy and maintenance remains unknown. As leukaemic sub-clone immunogenicity could not be evaluated in human AML xenograft models, we assessed the sub-clonal diversity of the murine C1498 AML cell line and the immunogenicity of its sub-clones in immune-competent syngeneic mice. The murine C1498 cell line was cultured in vitro and sub-clonal cells were generated after limiting dilution. The genomic profiles of 6 different sub-clones were analysed by comparative genomic hybridization arrays (CGH). The sub-clones were then injected into immune-deficient and - competent syngeneic mice. The immunogenicities of the sub-clones was evaluated through 1) assessment of mouse survival, 2) determination of leukaemic cell infiltration into organs by flow cytometry and the expression of a fluorescent reporter gene, 3) assessment of the CTL response ex vivo and 4) detection of residual leukaemic cells in the organs via amplification of the genomic reporter gene by real-time PCR (qPCR). Genomic analyses revealed heterogeneity among the parental cell line and its derived sub-clones. When injected individually into immune-deficient mice, all sub-clones induced cases of AML with different kinetics. However, when administered into immune-competent animals, some sub-clones triggered AML in which no mice survived, whereas others elicited reduced lethality rates. The AML-surviving mice presented efficient anti-leukaemia CTL activity ex vivo and eliminated the leukaemic cells in vivo. We showed that C1498 cell sub-clones presented genomic heterogeneity and differential immunogenicity resulting either in immune escape or elimination. Such findings could have potent implications for new immunotherapeutic strategies in patients with AML. Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

Real-time quantification of subcellular H2O2 and glutathione redox potential in living cardiovascular tissues.

PubMed

Panieri, Emiliano; Millia, Carlo; Santoro, Massimo M

2017-08-01

Detecting and measuring the dynamic redox events that occur in vivo is a prerequisite for understanding the impact of oxidants and redox events in normal and pathological conditions. These aspects are particularly relevant in cardiovascular tissues wherein alterations of the redox balance are associated with stroke, aging, and pharmacological intervention. An ambiguous aspect of redox biology is how redox events occur in subcellular organelles including mitochondria, and nuclei. Genetically-encoded Rogfp2 fluorescent probes have become powerful tools for real-time detection of redox events. These probes detect hydrogen peroxide (H 2 O 2 ) levels and glutathione redox potential (E GSH ), both with high spatiotemporal resolution. By generating novel transgenic (Tg) zebrafish lines that express compartment-specific Rogfp2-Orp1 and Grx1-Rogfp2 sensors we analyzed cytosolic, mitochondrial, and the nuclear redox state of endothelial cells and cardiomyocytes of living zebrafish embryos. We provide evidence for the usefulness of these Tg lines for pharmacological compounds screening by addressing the blocking of pentose phosphate pathways (PPP) and glutathione synthesis, thus altering subcellular redox state in vivo. Rogfp2-based transgenic zebrafish lines represent valuable tools to characterize the impact of redox changes in living tissues and offer new opportunities for studying metabolic driven antioxidant response in biomedical research. Copyright © 2017 Elsevier Inc. All rights reserved.

Acute multi-sgRNA knockdown of KEOPS complex genes reproduces the microcephaly phenotype of the stable knockout zebrafish model

PubMed Central

Schneider, Ronen; Hoogstraten, Charlotte A.; Schapiro, David; Majmundar, Amar J.; Kolb, Amy; Eddy, Kaitlyn; Shril, Shirlee; Braun, Daniela A.; Poduri, Annapurna

2018-01-01

Until recently, morpholino oligonucleotides have been widely employed in zebrafish as an acute and efficient loss-of-function assay. However, off-target effects and reproducibility issues when compared to stable knockout lines have compromised their further use. Here we employed an acute CRISPR/Cas approach using multiple single guide RNAs targeting simultaneously different positions in two exemplar genes (osgep or tprkb) to increase the likelihood of generating mutations on both alleles in the injected F0 generation and to achieve a similar effect as morpholinos but with the reproducibility of stable lines. This multi single guide RNA approach resulted in median likelihoods for at least one mutation on each allele of >99% and sgRNA specific insertion/deletion profiles as revealed by deep-sequencing. Immunoblot showed a significant reduction for Osgep and Tprkb proteins. For both genes, the acute multi-sgRNA knockout recapitulated the microcephaly phenotype and reduction in survival that we observed previously in stable knockout lines, though milder in the acute multi-sgRNA knockout. Finally, we quantify the degree of mutagenesis by deep sequencing, and provide a mathematical model to quantitate the chance for a biallelic loss-of-function mutation. Our findings can be generalized to acute and stable CRISPR/Cas targeting for any zebrafish gene of interest. PMID:29346415

The zebrafish lysozyme C promoter drives myeloid-specific expression in transgenic fish

PubMed Central

Hall, Chris; Flores, Maria Vega; Storm, Thilo; Crosier, Kathy; Crosier, Phil

2007-01-01

Background How different immune cell compartments contribute to a successful immune response is central to fully understanding the mechanisms behind normal processes such as tissue repair and the pathology of inflammatory diseases. However, the ability to observe and characterize such interactions, in real-time, within a living vertebrate has proved elusive. Recently, the zebrafish has been exploited to model aspects of human disease and to study specific immune cell compartments using fluorescent reporter transgenic lines. A number of blood-specific lines have provided a means to exploit the exquisite optical clarity that this vertebrate system offers and provide a level of insight into dynamic inflammatory processes previously unavailable. Results We used regulatory regions of the zebrafish lysozyme C (lysC) gene to drive enhanced green fluorescent protein (EGFP) and DsRED2 expression in a manner that completely recapitulated the endogenous expression profile of lysC. Labeled cells were shown by co-expression studies and FACS analysis to represent a subset of macrophages and likely also granulocytes. Functional assays within transgenic larvae proved that these marked cells possess hallmark traits of myelomonocytic cells, including the ability to migrate to inflammatory sources and phagocytose bacteria. Conclusion These reporter lines will have utility in dissecting the genetic determinants of commitment to the myeloid lineage and in further defining how lysozyme-expressing cells participate during inflammation. PMID:17477879

Significant genetic and phenotypic changes arising from clonal growth of a single spore of an arbuscular mycorrhizal fungus over multiple generations.

PubMed

Ehinger, Martine O; Croll, Daniel; Koch, Alexander M; Sanders, Ian R

2012-11-01

Arbuscular mycorrhizal fungi (AMF) are highly successful plant symbionts. They reproduce clonally producing multinucleate spores. It has been suggested that some AMF harbor genetically different nuclei. However, recent advances in sequencing the Glomus irregulare genome have indicated very low within-fungus polymorphism. We tested the null hypothesis that, with no genetic differences among nuclei, no significant genetic or phenotypic variation would occur among clonal single spore lines generated from one initial AMF spore. Furthermore, no additional variation would be expected in the following generations of single spore lines. Genetic diversity contained in one initial spore repeatedly gave rise to genetically different variants of the fungus with novel phenotypes. The genetic changes represented quantitative changes in allele frequencies, most probably as a result of changes in the frequency of genetic variation partitioned on different nuclei. The genetic and phenotypic variation is remarkable, given that it arose repeatedly from one clonal individual. Our results highlight the dynamic nature of AMF genetics. Even though within-fungus genetic variation is low, some is probably partitioned among nuclei and potentially causes changes in the phenotype. Our results are important for understanding AMF genetics, as well as for researchers and biotechnologists hoping to use AMF genetic diversity for the improvement of AMF inoculum. © 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.

Changes of heterogeneous cell populations in the Ishikawa cell line during long-term culture: Proposal for an in vitro clonal evolution model of tumor cells.

PubMed

Kasai, Fumio; Hirayama, Noriko; Ozawa, Midori; Iemura, Masashi; Kohara, Arihiro

2016-06-01

Genomic changes in tumor cell lines can occur during culture, leading to differences between cell lines carrying the same name. In this study, genome profiles between low and high passages were investigated in the Ishikawa 3-H-12 cell line (JCRB1505). Cells contained between 43 and 46 chromosomes and the modal number changed from 46 to 45 during culture. Cytogenetic analysis revealed that a translocation t(9;14), observed in all metaphases, is a robust marker for this cell line. Single-nucleotide polymorphism microarrays showed a heterogeneous copy number in the early passages and distinct profiles at late passages. These results demonstrate that cell culture can lead to elimination of ancestral clones by sequential selection, resulting in extensive replacement with a novel clone. Our observations on Ishikawa cells in vitro are different from the in vivo heterogeneity in which ancestral clones are often retained during tumor evolution and suggest a model for in vitro clonal evolution. Copyright © 2016 Elsevier Inc. All rights reserved.

Focal gene misexpression in zebrafish embryos induced by local heat shock using a modified soldering iron.

PubMed

Hardy, Melissa E; Ross, Louis V; Chien, Chi-Bin

2007-11-01

Misexpression of genes in a temporally and spatially controlled fashion is an important tool for assessing gene function during development. Because few tissue-specific promoters have been identified in zebrafish, inducible systems such as the Cre/LoxP and Tet repressor systems are of limited utility. Here we describe a new method of misexpression: local heat shock using a modified soldering iron. Zebrafish carrying transgenes under the control of a heat shock promoter (hsp70) are focally heated with the soldering iron to induce gene expression in a small area of the embryo. We have validated this method in three stable transgenic lines and at three developmental timepoints. Local heat shock is a fast, easy, and inexpensive method for gene misexpression. Copyright 2007 Wiley-Liss, Inc.

Loss of Smyhc1 or Hsp90α1 Function Results in Different Effects on Myofibril Organization in Skeletal Muscles of Zebrafish Embryos

PubMed Central

Codina, Marta; Li, Junling; Gutiérrez, Joaquim; Kao, Joseph P. Y.; Du, Shao Jun

2010-01-01

Background Myofibrillogenesis requires the correct folding and assembly of sarcomeric proteins into highly organized sarcomeres. Heat shock protein 90α1 (Hsp90α1) has been implicated as a myosin chaperone that plays a key role in myofibrillogenesis. Knockdown or mutation of hsp90α1 resulted in complete disorganization of thick and thin filaments and M- and Z-line structures. It is not clear whether the disorganization of these sarcomeric structures is due to a direct effect from loss of Hsp90α1 function or indirectly through the disorganization of myosin thick filaments. Methodology/Principal Findings In this study, we carried out a loss-of-function analysis of myosin thick filaments via gene-specific knockdown or using a myosin ATPase inhibitor BTS (N-benzyl-p-toluene sulphonamide) in zebrafish embryos. We demonstrated that knockdown of myosin heavy chain 1 (myhc1) resulted in sarcomeric defects in the thick and thin filaments and defective alignment of Z-lines. Similarly, treating zebrafish embryos with BTS disrupted thick and thin filament organization, with little effect on the M- and Z-lines. In contrast, loss of Hsp90α1 function completely disrupted all sarcomeric structures including both thick and thin filaments as well as the M- and Z-lines. Conclusion/Significance Together, these studies indicate that the hsp90α1 mutant phenotype is not simply due to disruption of myosin folding and assembly, suggesting that Hsp90α1 may play a role in the assembly and organization of other sarcomeric structures. PMID:20049323

Expression of sall4 in taste buds of zebrafish.

PubMed

Jackson, Robyn; Braubach, Oliver R; Bilkey, Jessica; Zhang, Jing; Akimenko, Marie-Andrée; Fine, Alan; Croll, Roger P; Jonz, Michael G

2013-07-01

We characterized the expression of sall4, a gene encoding a zinc finger transcription factor involved in the maintenance of embryonic stem cells, in taste buds of zebrafish (Danio rerio). Using an enhancer trap line (ET5), we detected enhanced green fluorescent protein (EGFP) in developing and adult transgenic zebrafish in regions containing taste buds: the lips, branchial arches, and the nasal and maxillary barbels. Localization of EGFP to taste cells of the branchial arches and lips was confirmed by co-immunolabeling with antibodies against calretinin and serotonin, and a zebrafish-derived neuronal marker (zn-12). Transgenic insertion of the ET construct into the zebrafish genome was evaluated and mapped to chromosome 23 in proximity (i.e. 23 kb) to the sall4 gene. In situ hybridization and expression analysis between 24 and 96 h post-fertilization (hpf) demonstrated that transgenic egfp expression in ET5 zebrafish was correlated with the spatial and temporal pattern of expression of sall4 in the wild-type. Expression was first observed in the central nervous system and branchial arches at 24 hpf. At 48 hpf, sall4 and egfp expression was observed in taste bud primordia surrounding the mouth and branchial arches. At 72 and 96 hpf, expression was detected in the upper and lower lips and branchial arches. Double fluorescence in situ hybridization at 3 and 10 dpf confirmed colocalization of sall4 and egfp in the lips and branchial arches. These studies reveal sall4 expression in chemosensory cells and implicate this transcription factor in the development and renewal of taste epithelia in zebrafish. Copyright © 2013 Wiley Periodicals, Inc.

Systematic gene tagging using CRISPR/Cas9 in human stem cells to illuminate cell organization.

PubMed

Roberts, Brock; Haupt, Amanda; Tucker, Andrew; Grancharova, Tanya; Arakaki, Joy; Fuqua, Margaret A; Nelson, Angelique; Hookway, Caroline; Ludmann, Susan A; Mueller, Irina A; Yang, Ruian; Horwitz, Rick; Rafelski, Susanne M; Gunawardane, Ruwanthi N

2017-10-15

We present a CRISPR/Cas9 genome-editing strategy to systematically tag endogenous proteins with fluorescent tags in human induced pluripotent stem cells (hiPSC). To date, we have generated multiple hiPSC lines with monoallelic green fluorescent protein tags labeling 10 proteins representing major cellular structures. The tagged proteins include alpha tubulin, beta actin, desmoplakin, fibrillarin, nuclear lamin B1, nonmuscle myosin heavy chain IIB, paxillin, Sec61 beta, tight junction protein ZO1, and Tom20. Our genome-editing methodology using Cas9/crRNA ribonuclear protein and donor plasmid coelectroporation, followed by fluorescence-based enrichment of edited cells, typically resulted in <0.1-4% homology-directed repair (HDR). Twenty-five percent of clones generated from each edited population were precisely edited. Furthermore, 92% (36/39) of expanded clonal lines displayed robust morphology, genomic stability, expression and localization of the tagged protein to the appropriate subcellular structure, pluripotency-marker expression, and multilineage differentiation. It is our conclusion that, if cell lines are confirmed to harbor an appropriate gene edit, pluripotency, differentiation potential, and genomic stability are typically maintained during the clonal line-generation process. The data described here reveal general trends that emerged from this systematic gene-tagging approach. Final clonal lines corresponding to each of the 10 cellular structures are now available to the research community. © 2017 Roberts, Haupt, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Establishment and Characterization of a New Muscle Cell Line of Zebrafish (Danio rerio) as an In Vitro Model for Gene Expression Studies.

PubMed

Kumar, Amit; Singh, Neha; Goswami, Mukunda; Srivastava, J K; Mishra, Akhilesh K; Lakra, W S

2016-01-01

A new continuous fibroblast cell line was established from the muscle tissue of healthy juvenile Danio rerio (Zebrafish) through explant method. Fish cell lines serve as useful tool for investigating basic fish biology, as a model for bioassay of environmental toxicant, toxicity ranking, and for developing molecular biomarkers. The cell line was continuously subcultured for a period of 12 months (61 passages) and maintained at 28 °C in L-15 medium supplemented with 10% FBS and 10 ng/mL of basic fibroblastic growth factor (bFGF) without use of antibiotics. Its growth rate was proportional to the FBS concentration, with optimum growth at 15% FBS. DNA barcoding (16SrRNA and COX1) was used to authenticate the cell line. Cells were incubated with propidium iodide and sorted via flow cytometry to calculate the DNA content to confirm the genetic stability. Significant green fluorescent protein (GFP) signals confirmed the utility of cell line in transgenic and genetic manipulation studies. In vitro assay was performed with MTT to examine the growth potential of the cell line. The muscle cell line would provide a novel invaluable in vitro model to identify important genes to understand regulatory mechanisms that govern the molecular regulation of myogenesis and should be useful in biomedical research.

SERCA directs cell migration and branching across species and germ layers

PubMed Central

Lansdale, Nick; Navarro, Sonia; Truong, Thai V.; Bower, Dan J.; Featherstone, Neil C.; Connell, Marilyn G.; Al Alam, Denise; Frey, Mark R.; Trinh, Le A.; Fernandez, G. Esteban; Warburton, David; Fraser, Scott E.; Bennett, Daimark; Jesudason, Edwin C.

2017-01-01

ABSTRACT Branching morphogenesis underlies organogenesis in vertebrates and invertebrates, yet is incompletely understood. Here, we show that the sarco-endoplasmic reticulum Ca2+ reuptake pump (SERCA) directs budding across germ layers and species. Clonal knockdown demonstrated a cell-autonomous role for SERCA in Drosophila air sac budding. Live imaging of Drosophila tracheogenesis revealed elevated Ca2+ levels in migratory tip cells as they form branches. SERCA blockade abolished this Ca2+ differential, aborting both cell migration and new branching. Activating protein kinase C (PKC) rescued Ca2+ in tip cells and restored cell migration and branching. Likewise, inhibiting SERCA abolished mammalian epithelial budding, PKC activation rescued budding, while morphogens did not. Mesoderm (zebrafish angiogenesis) and ectoderm (Drosophila nervous system) behaved similarly, suggesting a conserved requirement for cell-autonomous Ca2+ signaling, established by SERCA, in iterative budding. PMID:28821490

Zebrafish tissue injury causes upregulation of interleukin-1 and caspase-dependent amplification of the inflammatory response

PubMed Central

Ogryzko, Nikolay V.; Hoggett, Emily E.; Solaymani-Kohal, Sara; Tazzyman, Simon; Chico, Timothy J. A.; Renshaw, Stephen A.; Wilson, Heather L.

2014-01-01

ABSTRACT Interleukin-1 (IL-1), the ‘gatekeeper’ of inflammation, is the apical cytokine in a signalling cascade that drives the early response to injury or infection. Expression, processing and secretion of IL-1 are tightly controlled, and dysregulated IL-1 signalling has been implicated in a number of pathologies ranging from atherosclerosis to complications of infection. Our understanding of these processes comes from in vitro monocytic cell culture models as lines or primary isolates, in which a range and spectra of IL-1 secretion mechanisms have been described. We therefore investigated whether zebrafish embryos provide a suitable in vivo model for studying IL-1-mediated inflammation. Structurally, zebrafish IL-1β shares a β-sheet-rich trefoil structure with its human counterpart. Functionally, leukocyte expression of IL-1β was detectable only following injury, which activated leukocytes throughout zebrafish embryos. Migration of macrophages and neutrophils was attenuated by inhibitors of either caspase-1 or P2X7, which similarly inhibited the activation of NF-κB at the site of injury. Zebrafish offer a new and versatile model to study the IL-1β pathway in inflammatory disease and should offer unique insights into IL-1 biology in vivo. PMID:24203886

Role of brain-derived neurotrophic factor during the regenerative response after traumatic brain injury in adult zebrafish.

PubMed

Cacialli, Pietro; Palladino, Antonio; Lucini, Carla

2018-06-01

Several mammalian animal models of traumatic brain injury have been used, mostly rodents. However, reparative mechanisms in mammalian brain are very limited, and newly formed neurons do not survive for long time. The brain of adult zebrafish, a teleost fish widely used as vertebrate model, possesses high regenerative properties after injury due to the presence of numerous stem cells niches. The ventricular lining of the zebrafish dorsal telencephalon is the most studied neuronal stem cell niche because its dorso-lateral zone is considered the equivalent to the hippocampus of mammals which contains one of the two constitutive neurogenic niches of mammals. To mimic TBI, stab wound in the dorso-lateral telencephalon of zebrafish was used in studies devoted to fish regenerative properties. Brain-derived neurotrophic factor, which is known to play key roles in the repair process after traumatic brain lesions, persists around the lesioned area of injured telencephalon of adult zebrafish. These results are extensively compared to reparative processes in rodent brain. Considering the complete repair of the damaged area in fish, it could be tempting to consider brain-derived neurotrophic factor as a factor contributing to create a permissive environment that enables the establishment of new neuronal population in damaged brain.

Fluorescent transgenic mice suitable for multi-color aggregation chimera studies.

PubMed

Ohtsuka, Masato; Miura, Hiromi; Gurumurthy, Channabasavaiah B; Kimura, Minoru; Inoko, Hidetoshi; Yoshimura, Shinichi; Sato, Masahiro

2012-11-01

We recently reported a novel method of mouse transgenesis called Pronuclear Injection-based Targeted Transgenisis (PITT) using which a series of fluorescent transgenic (Tg) mice lines were generated. These lines, unlike those generated using conventional random integration methods, express the transgenes faithfully and reproducibly generation after generation. Because of this superior nature, these lines are ideal for the generation of multi-colored aggregation chimeras that can be used to study cell-cell interactions and lineage analyses in living embryos/organs, where the transgenes can be detected and the clonal origin of a given cell population easily traced by its distinct fluorescence. In this study, to verify if Tg fluorescent mice generated through PITT were suitable for such applications, we sought to generate chimeric blastocysts and chimeric-Tg mice by aggregating two- or three-colored 8-cell embryos. Our analyses using these models led to the following observations. First, we noticed that cell mixing was infrequent during the stages of morula to early blastocyst. Second, chimeric fetuses obtained after aggregation of the two-colored 8-cell embryos exhibited uniform cell mixing. And third, in the organs of adult chimeric mice, the mode of cell distribution could be either clonal or polyclonal, as previously pointed out by others. Implications of our novel and improved Tg-chimeric mice approach for clonal cell lineage and developmental studies are discussed.

Patient-derived xenograft in zebrafish embryos: a new platform for translational research in neuroendocrine tumors.

PubMed

Gaudenzi, Germano; Albertelli, Manuela; Dicitore, Alessandra; Würth, Roberto; Gatto, Federico; Barbieri, Federica; Cotelli, Franco; Florio, Tullio; Ferone, Diego; Persani, Luca; Vitale, Giovanni

2017-08-01

Preclinical research on neuroendocrine tumors usually involves immortalized cell lines and few animal models. In the present study we described an in vivo model based on patient-derived xenografts of neuroendocrine tumor cells in zebrafish (Danio rerio) embryos, allowing a rapid analysis of the angiogenic and invasive potential. Patient-derived neuroendocrine tumor cells were transplanted in 48 hours post-fertilization Tg(fli1a:EGFP) y1 zebrafish embryos that express enhanced green fluorescent protein in the entire vasculature. Neuroendocrine tumor cells, stained with CM-Dil, were injected into the subperidermal (perivitelline) space, close to the developing subintestinal venous plexus. A proper control group, represented by zebrafish injected with only D-PBS, was included in this study. Angiogenic and invasive potentials of each patient-derived xenograft were evaluated by both epifluorescence and confocal microscopes. Six out of eight neuroendocrine tumor samples were successfully transplanted in zebrafish embryos. Although the implanted tumor mass had a limited size (about 100 cells for embryos), patient-derived xenografts showed pro-angiogenic (5 cases) and invasive (6 cases) behaviors within 48 hours post injection. Patient-derived xenograft in zebrafish embryos appears to be a reliable in vivo preclinical model for neuroendocrine tumors, tumors with often limited cell availability. The rapidity of this procedure makes our model a promising platform to perform preclinical drug screening and opens a new scenario for personalized treatment in patients with neuroendocrine tumors.

Zebrafish in the sea of mineral (iron, zinc, and copper) metabolism

PubMed Central

Zhao, Lu; Xia, Zhidan; Wang, Fudi

2014-01-01

Iron, copper, zinc, and eight other minerals are classified as essential trace elements because they present in minute in vivo quantities and are essential for life. Because either excess or insufficient levels of trace elements can be detrimental to life (causing human diseases such as iron-deficiency anemia, hemochromatosis, Menkes syndrome and Wilson's disease), the endogenous levels of trace minerals must be tightly regulated. Many studies have demonstrated the existence of systems that maintain trace element homeostasis, and these systems are highly conserved in multiple species ranging from yeast to mice. As a model for studying trace mineral metabolism, the zebrafish is indispensable to researchers. Several large-scale mutagenesis screens have been performed in zebrafish, and these screens led to the identification of a series of metal transporters and the generation of several mutagenesis lines, providing an in-depth functional analysis at the system level. Moreover, because of their developmental advantages, zebrafish have also been used in mineral metabolism-related chemical screens and toxicology studies. Here, we systematically review the major findings of trace element homeostasis studies using the zebrafish model, with a focus on iron, zinc, copper, selenium, manganese, and iodine. We also provide a homology analysis of trace mineral transporters in fish, mice and humans. Finally, we discuss the evidence that zebrafish is an ideal experimental tool for uncovering novel mechanisms of trace mineral metabolism and for improving approaches to treat mineral imbalance-related diseases. PMID:24639652

Etiology and functional validation of gastrointestinal motility dysfunction in a zebrafish model of CHARGE syndrome.

PubMed

Cloney, Kellie; Steele, Shelby L; Stoyek, Matthew R; Croll, Roger P; Smith, Frank M; Prykhozhij, Sergey V; Brown, Mary M; Midgen, Craig; Blake, Kim; Berman, Jason N

2018-06-01

CHARGE syndrome is linked to autosomal-dominant mutations in the CHD7 gene and results in a number of physiological and structural abnormalities, including heart defects, hearing and vision loss, and gastrointestinal (GI) problems. Of these challenges, GI problems have a profound impact throughout an individual's life, resulting in increased morbidity and mortality. A homolog of CHD7 has been identified in the zebrafish, the loss of which recapitulates many of the features of the human disease. Using a morpholino chd7 knockdown model complemented by a chd7 null mutant zebrafish line, we examined GI structure, innervation, and motility in larval zebrafish. Loss of chd7 resulted in physically smaller GI tracts with normal epithelial and muscular histology, but decreased and disorganized vagal projections, particularly in the foregut. chd7 morphant larvae had significantly less ability to empty their GI tract of gavaged fluorescent beads, and this condition was only minimally improved by the prokinetic agents, domperidone and erythromycin, in keeping with mixed responses to these agents in patients with CHARGE syndrome. The conserved genetics and transparency of the zebrafish have provided new insights into the consequences of chd7 gene dysfunction on the GI system and cranial nerve patterning. These findings highlight the opportunity of the zebrafish to serve as a preclinical model for studying compounds that may improve GI motility in individuals with CHARGE syndrome. © 2018 Federation of European Biochemical Societies.

Analysis of proviral integration in human mammary epithelial cell lines immortalized by retroviral infection with a temperature-sensitive SV40 T-antigen construct.

PubMed

Stamps, A C; Davies, S C; Burman, J; O'Hare, M J

1994-06-15

A panel of eight conditionally immortal lines derived by infection of human breast epithelial cells with an amphotropic retrovirus transducing a ts mutant of SV40 large T-antigen was analyzed with respect to individual retroviral integration patterns. Each line contained multiple integration sites which were clonal and stable over extended passage. Similar integration patterns were observed between individual lines arising separately from the same stock of pre-immortal cells, suggesting a common progenitor. Retroviral integration analysis of pre-immortal cells at different stages of pre-crisis growth showed changes indicative of a progressive transition from polyclonality to clonality as the cells approached crisis. Each of the immortal lines contained a sub-set of the integration sites of their pre-immortal progenitors, with individual combinations and copy numbers of sites. Since all the cell lines appeared to originate from single foci in separate flasks, it is likely that each set arose from a common clone of pre-immortal cells as the result of separate genetic events. There was no evidence from this analysis to suggest that specific integration sites played any part either in the selection of pre-crisis clones or in the subsequent establishment of immortal lines.

PI3K and Inhibitor of Apoptosis Proteins Modulate Gentamicin- Induced Hair Cell Death in the Zebrafish Lateral Line

PubMed Central

Wiedenhoft, Heather; Hayashi, Lauren; Coffin, Allison B.

2017-01-01

Inner ear hair cell death leads to sensorineural hearing loss and can be a direct consequence of aminoglycoside antibiotic treatment. Aminoglycosides such as gentamicin are effective therapy for serious Gram-negative bacterial infections such as some forms of meningitis, pneumonia, and sepsis. Aminoglycosides enter hair cells through mechanotransduction channels at the apical end of hair bundles and initiate intrinsic cell death cascades, but the precise cell signaling that leads to hair cell death is incompletely understood. Here, we examine the cell death pathways involved in aminoglycoside damage using the zebrafish (Danio rerio). The zebrafish lateral line contains hair cell-bearing organs called neuromasts that are homologous to hair cells of the mammalian inner ear and represents an excellent model to study ototoxicity. Based on previous research demonstrating a role for p53, Bcl2 signaling, autophagy, and proteasomal degradation in aminoglycoside-damaged hair cells, we used the Cytoscape GeneMANIA Database to identify additional proteins that might play a role in neomycin or gentamicin ototoxicity. Our bioinformatics analysis identified the pro-survival proteins phosphoinositide-dependent kinase-1 (PDK1) and X-linked inhibitor of apoptosis protein (Xiap) as potential mediators of gentamicin-induced hair cell damage. Pharmacological inhibition of PDK1 or its downstream mediator protein kinase C facilitated gentamicin toxicity, as did Xiap mutation, suggesting that both PI3K and endogenous Xiap confer protection. Surprisingly, aminoglycoside-induced hair cell death was highly attenuated in wild type Tupfel long-fin (TL fish; the background strain for the Xiap mutant line) compared to wild type ∗AB zebrafish. Pharmacologic manipulation of p53 suggested that the strain difference might result from decreased p53 in TL hair cells, allowing for increased hair cell survival. Overall, our studies identified additional steps in the cell death cascade triggered by aminoglycoside damage, suggesting possible drug targets to combat hearing loss resulting from aminoglycoside exposure. PMID:29093665

Characterizing the mechanical behavior of the zebrafish germ layers

NASA Astrophysics Data System (ADS)

Kealhofer, David; Serwane, Friedhelm; Mongera, Alessandro; Rowghanian, Payam; Lucio, Adam; Campàs, Otger

Organ morphogenesis and the development of the animal body plan involve complex spatial and temporal control of tissue- and cell-level mechanics. A prime example is the generation of stresses by individual cells to reorganize the tissue. These processes have remained poorly understood due to a lack of techniques to characterize the local constitutive law of the material, which relates local cellular forces to the resulting tissue flows. We have developed a method for quantitative, local in vivo study of material properties in living tissue using magnetic droplet probes. We use this technique to study the material properties of the different zebrafish germ layers using aggregates of zebrafish mesendodermal and ectodermal cells as a model system. These aggregates are ideal for controlled studies of the mechanics of individual germ layers because of the homogeneity of the cell type and the simple spherical geometry. Furthermore, the numerous molecular tools and transgenic lines already developed for this model organism can be applied to these aggregates, allowing us to characterize the contributions of cell cortex tension and cell adhesion to the mechanical properties of the zebrafish germ layers.

Graph theoretical model of a sensorimotor connectome in zebrafish.

PubMed

Stobb, Michael; Peterson, Joshua M; Mazzag, Borbala; Gahtan, Ethan

2012-01-01

Mapping the detailed connectivity patterns (connectomes) of neural circuits is a central goal of neuroscience. The best quantitative approach to analyzing connectome data is still unclear but graph theory has been used with success. We present a graph theoretical model of the posterior lateral line sensorimotor pathway in zebrafish. The model includes 2,616 neurons and 167,114 synaptic connections. Model neurons represent known cell types in zebrafish larvae, and connections were set stochastically following rules based on biological literature. Thus, our model is a uniquely detailed computational representation of a vertebrate connectome. The connectome has low overall connection density, with 2.45% of all possible connections, a value within the physiological range. We used graph theoretical tools to compare the zebrafish connectome graph to small-world, random and structured random graphs of the same size. For each type of graph, 100 randomly generated instantiations were considered. Degree distribution (the number of connections per neuron) varied more in the zebrafish graph than in same size graphs with less biological detail. There was high local clustering and a short average path length between nodes, implying a small-world structure similar to other neural connectomes and complex networks. The graph was found not to be scale-free, in agreement with some other neural connectomes. An experimental lesion was performed that targeted three model brain neurons, including the Mauthner neuron, known to control fast escape turns. The lesion decreased the number of short paths between sensory and motor neurons analogous to the behavioral effects of the same lesion in zebrafish. This model is expandable and can be used to organize and interpret a growing database of information on the zebrafish connectome.

A zebrafish transgenic model of Ewing's sarcoma reveals conserved mediators of EWS-FLI1 tumorigenesis.

PubMed

Leacock, Stefanie W; Basse, Audrey N; Chandler, Garvin L; Kirk, Anne M; Rakheja, Dinesh; Amatruda, James F

2012-01-01

Ewing's sarcoma, a malignant bone tumor of children and young adults, is a member of the small-round-blue-cell tumor family. Ewing's sarcoma family tumors (ESFTs), which include peripheral primitive neuroectodermal tumors (PNETs), are characterized by chromosomal translocations that generate fusions between the EWS gene and ETS-family transcription factors, most commonly FLI1. The EWS-FLI1 fusion oncoprotein represents an attractive therapeutic target for treatment of Ewing's sarcoma. The cell of origin of ESFT and the molecular mechanisms by which EWS-FLI1 mediates tumorigenesis remain unknown, and few animal models of Ewing's sarcoma exist. Here, we report the use of zebrafish as a vertebrate model of EWS-FLI1 function and tumorigenesis. Mosaic expression of the human EWS-FLI1 fusion protein in zebrafish caused the development of tumors with histology strongly resembling that of human Ewing's sarcoma. The incidence of tumors increased in a p53 mutant background, suggesting that the p53 pathway suppresses EWS-FLI1-driven tumorigenesis. Gene expression profiling of the zebrafish tumors defined a set of genes that might be regulated by EWS-FLI1, including the zebrafish ortholog of a crucial EWS-FLI1 target gene in humans. Stable zebrafish transgenic lines expressing EWS-FLI1 under the control of the heat-shock promoter exhibit altered embryonic development and defective convergence and extension, suggesting that EWS-FLI1 interacts with conserved developmental pathways. These results indicate that functional targets of EWS-FLI1 that mediate tumorigenesis are conserved from zebrafish to human and provide a novel context in which to study the function of this fusion oncogene.

Miniaturized Embryo Array for Automated Trapping, Immobilization and Microperfusion of Zebrafish Embryos

PubMed Central

Akagi, Jin; Khoshmanesh, Khashayar; Evans, Barbara; Hall, Chris J.; Crosier, Kathryn E.; Cooper, Jonathan M.; Crosier, Philip S.; Wlodkowic, Donald

2012-01-01

Zebrafish (Danio rerio) has recently emerged as a powerful experimental model in drug discovery and environmental toxicology. Drug discovery screens performed on zebrafish embryos mirror with a high level of accuracy the tests usually performed on mammalian animal models, and fish embryo toxicity assay (FET) is one of the most promising alternative approaches to acute ecotoxicity testing with adult fish. Notwithstanding this, automated in-situ analysis of zebrafish embryos is still deeply in its infancy. This is mostly due to the inherent limitations of conventional techniques and the fact that metazoan organisms are not easily susceptible to laboratory automation. In this work, we describe the development of an innovative miniaturized chip-based device for the in-situ analysis of zebrafish embryos. We present evidence that automatic, hydrodynamic positioning, trapping and long-term immobilization of single embryos inside the microfluidic chips can be combined with time-lapse imaging to provide real-time developmental analysis. Our platform, fabricated using biocompatible polymer molding technology, enables rapid trapping of embryos in low shear stress zones, uniform drug microperfusion and high-resolution imaging without the need of manual embryo handling at various developmental stages. The device provides a highly controllable fluidic microenvironment and post-analysis eleuthero-embryo stage recovery. Throughout the incubation, the position of individual embryos is registered. Importantly, we also for first time show that microfluidic embryo array technology can be effectively used for the analysis of anti-angiogenic compounds using transgenic zebrafish line (fli1a:EGFP). The work provides a new rationale for rapid and automated manipulation and analysis of developing zebrafish embryos at a large scale. PMID:22606275

Additive effects of levonorgestrel and ethinylestradiol on brain aromatase (cyp19a1b) in zebrafish specific in vitro and in vivo bioassays.

PubMed

Hinfray, N; Tebby, C; Garoche, C; Piccini, B; Bourgine, G; Aït-Aïssa, S; Kah, O; Pakdel, F; Brion, F

2016-09-15

Estrogens and progestins are widely used in combination in human medicine and both are present in aquatic environment. Despite the joint exposure of aquatic wildlife to estrogens and progestins, very little information is available on their combined effects. In the present study we investigated the effect of ethinylestradiol (EE2) and Levonorgestrel (LNG), alone and in mixtures, on the expression of the brain specific ER-regulated cyp19a1b gene. For that purpose, recently established zebrafish-derived tools were used: (i) an in vitro transient reporter gene assay in a human glial cell line (U251-MG) co-transfected with zebrafish estrogen receptors (zfERs) and the luciferase gene under the control of the zebrafish cyp19a1b gene promoter and (ii) an in vivo bioassay using a transgenic zebrafish expressing GFP under the control of the zebrafish cyp19a1b gene promoter (cyp19a1b-GFP). Concentration-response relationships for single chemicals were modeled and used to design the mixture experiments following a ray design. The results from mixture experiments were analyzed to predict joint effects according to concentration addition and statistical approaches were used to characterize the potential interactions between the components of the mixtures (synergism/antagonism). We confirmed that some progestins could elicit estrogenic effects in fish brain. In mixtures, EE2 and LNG exerted additive estrogenic effects both in vitro and in vivo, suggesting that some environmental progestin could exert effects that will add to those of environmental (xeno-)estrogens. Moreover, our zebrafish specific assays are valuable tools that could be used in risk assessment for both single chemicals and their mixtures. Copyright © 2016 Elsevier Inc. All rights reserved.

The notochord breaks bilateral symmetry by controlling cell shapes in the zebrafish laterality organ.

PubMed

Compagnon, Julien; Barone, Vanessa; Rajshekar, Srivarsha; Kottmeier, Rita; Pranjic-Ferscha, Kornelija; Behrndt, Martin; Heisenberg, Carl-Philipp

2014-12-22

Kupffer's vesicle (KV) is the zebrafish organ of laterality, patterning the embryo along its left-right (LR) axis. Regional differences in cell shape within the lumen-lining KV epithelium are essential for its LR patterning function. However, the processes by which KV cells acquire their characteristic shapes are largely unknown. Here, we show that the notochord induces regional differences in cell shape within KV by triggering extracellular matrix (ECM) accumulation adjacent to anterior-dorsal (AD) regions of KV. This localized ECM deposition restricts apical expansion of lumen-lining epithelial cells in AD regions of KV during lumen growth. Our study provides mechanistic insight into the processes by which KV translates global embryonic patterning into regional cell shape differences required for its LR symmetry-breaking function. Copyright © 2014 Elsevier Inc. All rights reserved.

Persistent Oxytetracycline Exposure Induces an Inflammatory Process That Improves Regenerative Capacity in Zebrafish Larvae

PubMed Central

Barros-Becker, Francisco; Romero, Jaime; Pulgar, Alvaro; Feijóo, Carmen G.

2012-01-01

Background The excessive use of antibiotics in aquaculture can adversely affect not only the environment, but also fish themselves. In this regard, there is evidence that some antibiotics can activate the immune system and reduce their effectiveness. None of those studies consider in detail the adverse inflammatory effect that the antibiotic remaining in the water may cause to the fish. In this work, we use the zebrafish to analyze quantitatively the effects of persistent exposure to oxytetracycline, the most common antibiotic used in fish farming. Methodology We developed a quantitative assay in which we exposed zebrafish larvae to oxytetracycline for a period of 24 to 96 hrs. In order to determinate if the exposure causes any inflammation reaction, we evaluated neutrophils infiltration and quantified their total number analyzing the Tg(mpx:GFP)i114 transgenic line by fluorescence stereoscope, microscope and flow cytometry respectively. On the other hand, we characterized the process at a molecular level by analyzing several immune markers (il-1β, il-10, lysC, mpx, cyp1a) at different time points by qPCR. Finally, we evaluated the influence of the inflammation triggered by oxytetracycline on the regeneration capacity in the lateral line. Conclusions Our results suggest that after 48 hours of exposure, the oxytetracycline triggered a widespread inflammation process that persisted until 96 hours of exposure. Interestingly, larvae that developed an inflammation process showed an improved regeneration capacity in the mechanosensory system lateral line. PMID:22590621

Ethanol affects the development of sensory hair cells in larval zebrafish (Danio rerio).

PubMed

Uribe, Phillip M; Asuncion, James D; Matsui, Jonathan I

2013-01-01

Children born to mothers with substantial alcohol consumption during pregnancy can present a number of morphological, cognitive, and sensory abnormalities, including hearing deficits, collectively known as fetal alcohol syndrome (FAS). The goal of this study was to determine if the zebrafish lateral line could be used to study sensory hair cell abnormalities caused by exposure to ethanol during embryogenesis. Some lateral line sensory hair cells are present at 2 days post-fertilization (dpf) and are functional by 5 dpf. Zebrafish embryos were raised in fish water supplemented with varying concentrations of ethanol (0.75%-1.75% by volume) from 2 dpf through 5 dpf. Ethanol treatment during development resulted in many physical abnormalities characteristic of FAS in humans. Also, the number of sensory hair cells decreased as the concentration of ethanol increased in a dose-dependent manner. The dye FM 1-43FX was used to detect the presence of functional mechanotransduction channels. The percentage of FM 1-43-labeled hair cells decreased as the concentration of ethanol increased. Methanol treatment did not affect the development of hair cells. The cell cycle markers proliferating cell nuclear antigen (PCNA) and bromodeoxyuridine (BrdU) demonstrated that ethanol reduced the number of sensory hair cells, as a consequence of decreased cellular proliferation. There was also a significant increase in the rate of apoptosis, as determined by TUNEL-labeling, in neuromasts following ethanol treatment during larval development. Therefore, zebrafish are a useful animal model to study the effects of hair cell developmental disorders associated with FAS.

The plant decapeptide OSIP108 prevents copper-induced toxicity in various models for Wilson disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Spincemaille, Pieter; Pham, Duc-Hung; Chandhok, Gursimran

2014-10-15

Background: Wilson disease (WD) is caused by accumulation of excess copper (Cu) due to a mutation in the gene encoding the liver Cu transporter ATP7B, and is characterized by acute liver failure or cirrhosis and neuronal cell death. We investigated the effect of OSIP108, a plant derived decapeptide that prevents Cu-induced apoptosis in yeast and human cells, on Cu-induced toxicity in various mammalian in vitro models relevant for WD and in a Cu-toxicity zebrafish larvae model applicable to WD. Methods: The effect of OSIP108 was evaluated on viability of various cell lines in the presence of excess Cu, on livermore » morphology of a Cu-treated zebrafish larvae strain that expresses a fluorescent reporter in hepatocytes, and on oxidative stress levels in wild type AB zebrafish larvae. Results: OSIP108 increased not only viability of Cu-treated CHO cells transgenically expressing ATP7B and the common WD-causing mutant ATP7B{sup H1069Q}, but also viability of Cu-treated human glioblastoma U87 cells. Aberrancies in liver morphology of Cu-treated zebrafish larvae were observed, which were further confirmed as Cu-induced hepatotoxicity by liver histology. Injections of OSIP108 into Cu-treated zebrafish larvae significantly increased the amount of larvae with normal liver morphology and decreased Cu-induced production of reactive oxygen species. Conclusions: OSIP108 prevents Cu-induced toxicity in in vitro models and in a Cu-toxicity zebrafish larvae model applicable to WD. General significance: All the above data indicate the potential of OSIP108 as a drug lead for further development as a novel WD treatment. - Highlights: • Wilson disease (WD) is characterized by accumulation of toxic copper (Cu). • OSIP108 increases viability of Cu-treated cellular models applicable to WD. • OSIP108 injections preserve liver morphology of Cu-treated zebrafish larvae. • OSIP108 injections into zebrafish larvae abrogates Cu-induced oxidative stress.« less

A transgenic zebrafish model for monitoring xbp1 splicing and endoplasmic reticulum stress in vivo.

PubMed

Li, Junling; Chen, Zhiliang; Gao, Lian-Yong; Colorni, Angelo; Ucko, Michal; Fang, Shengyun; Du, Shao Jun

2015-08-01

Accumulation of misfolded or unfolded proteins in the endoplasmic reticulum (ER) triggers ER stress that initiates unfolded protein response (UPR). XBP1 is a transcription factor that mediates one of the key signaling pathways of UPR to cope with ER stress through regulating gene expression. Activation of XBP1 involves an unconventional mRNA splicing catalyzed by IRE1 endonuclease that removes an internal 26 nucleotides from xbp1 mRNA transcripts in the cytoplasm. Researchers have taken advantage of this unique activation mechanism to monitor XBP1 activation, thereby UPR, in cell culture and transgenic models. Here we report a Tg(ef1α:xbp1δ-gfp) transgenic zebrafish line to monitor XBP1 activation using GFP as a reporter especially in zebrafish oocytes and developing embryos. The Tg(ef1α:xbp1δ-gfp) transgene was constructed using part of the zebrafish xbp1 cDNA containing the splicing element. ER stress induced splicing results in the cDNA encoding a GFP-tagged partial XBP1 without the transactivation activation domain (XBP1Δ-GFP). The results showed that xbp1 transcripts mainly exist as the spliced active isoform in unfertilized oocytes and zebrafish embryos prior to zygotic gene activation at 3 hours post fertilization. A strong GFP expression was observed in unfertilized oocytes, eyes, brain and skeletal muscle in addition to a weak expression in the hatching gland. Incubation of transgenic zebrafish embryos with (dithiothreitol) DTT significantly induced XBP1Δ-GFP expression. Collectively, these studies unveil the presence of maternal xbp1 splicing in zebrafish oocytes, fertilized eggs and early stage embryos. The Tg(ef1α:xbp1δ-gfp) transgenic zebrafish provides a useful model for in vivo monitoring xbp1 splicing during development and under ER stress conditions. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

The maintenance of sex: Ronald Fisher meets the Red Queen.

PubMed

Green, David; Mason, Chris

2013-08-21

Sex in higher diploids carries a two-fold cost of males that should reduce its fitness relative to cloning, and result in its extinction. Instead, sex is widespread and clonal species face early obsolescence. One possible reason is that sex is an adaptation that allows organisms to respond more effectively to endless changes in their environment. The purpose of this study was to model mutation and selection in a diploid organism in an evolving environment and ascertain their support for sex. We used a computational approach to model finite populations where a haploid environment subjects a diploid host to endlessly evolving change. Evolution in both populations is primarily through adoption of novel advantageous mutations within a large allele space. Sex outcompetes cloning by two complementary mechanisms. First, sexual diploids adopt advantageous homozygous mutations more rapidly than clonal ones under conditions of lag load (the gap between the actual adaptation of the diploid population and its theoretical optimum). This rate advantage can offset the higher fecundity of cloning. Second, a relative advantage to sex emerges where populations are significantly polymorphic, because clonal polymorphism runs the risk of clonal interference caused by selection on numerous lines of similar adaptation. This interference extends allele lifetime and reduces the rate of adaptation. Sex abolishes the interference, making selection faster and elevating population fitness. Differences in adaptation between sexual and clonal populations increase markedly with the number of loci under selection, the rate of mutation in the host, and a rapidly evolving environment. Clonal interference in these circumstances leads to conditions where the greater fecundity of clones is unable to offset their poor adaptation. Sexual and clonal populations then either co-exist, or sex emerges as the more stable evolutionary strategy. Sex can out-compete clones in a rapidly evolving environment, such as that characterized by pathogens, where clonal interference reduces the adaptation of clonal populations and clones adopt advantageous mutations more slowly. Since all organisms carry parasitic loads, the model is of potentially general applicability.

Dynamic neuroanatomy at subcellular resolution in the zebrafish.

PubMed

Faucherre, Adèle; López-Schier, Hernán

2014-01-01

Genetic means to visualize and manipulate neuronal circuits in the intact animal have revolutionized neurobiology. "Dynamic neuroanatomy" defines a range of approaches aimed at quantifying the architecture or subcellular organization of neurons over time during their development, regeneration, or degeneration. A general feature of these approaches is their reliance on the optical isolation of defined neurons in toto by genetically expressing markers in one or few cells. Here we use the afferent neurons of the lateral line as an example to describe a simple method for the dynamic neuroanatomical study of axon terminals in the zebrafish by laser-scanning confocal microscopy.

The cytokine macrophage migration inhibitory factor (MIF) acts as a neurotrophin in the developing inner ear of the zebrafish, Danio rerio

PubMed Central

Shen, Yu-chi; Thompson, Deborah L.; Kuah, Meng-Kiat; Wong, Kah-Loon; Wu, Karen L.; Linn, Stephanie A.; Jewett, Ethan M.; Shu-Chien, Alexander Chong; Barald, Kate F.

2012-01-01

Macrophage migration inhibitory factor (MIF) plays versatile roles in the immune system. MIF is also widely expressed during embryonic development, particularly in the nervous system, although its roles in neural development are only beginning to be understood. Evidence from frogs, mice and zebrafish suggests that MIF has a major role as a neurotrophin in the early development of sensory systems, including the auditory system. Here we show that the zebrafish mif pathway is required for both sensory hair cell (HC) and sensory neuronal cell survival in the ear, for HC differentiation, semicircular canal formation, statoacoustic ganglion (SAG) development, and lateral line HC differentiation. This is consistent with our findings that MIF is expressed in the developing mammalian and avian auditory systems and promotes mouse and chick SAG neurite outgrowth and neuronal survival, demonstrating key instructional roles for MIF in vertebrate otic development. PMID:22210003

High resolution fate map of the zebrafish diencephalon.

PubMed

Russek-Blum, Niva; Nabel-Rosen, Helit; Levkowitz, Gil

2009-07-01

The diencephalon acts as an interactive site between the sensory, central, and endocrine systems and is one of the most elaborate structures in the vertebrate brain. To better understand the embryonic development and morphogenesis of the diencephalon, we developed an improved photoactivation (uncaging)-based lineage tracing strategy. To determine the exact position of a given diencephalic progenitor domain, we used a transgenic line driving green fluorescent protein (GFP) in cells expressing the proneural protein, Neurogenin1 (Neurog1), which was used as a visible neural plate landmark. This approach facilitated precise labeling of defined groups of cells in the prospective diencephalon of the zebrafish neural plate. In this manner, we labeled multiple overlapping areas of the diencephalon, thereby ensuring both accuracy and reproducibility of our lineage tracing regardless of the dynamic changes of the developing neural plate. We present a fate map of the zebrafish diencephalon at a higher spatial resolution than previously described. (c) 2009 Wiley-Liss, Inc.

Gold Nanobeacons for Tracking Gene Silencing in Zebrafish

PubMed Central

Cordeiro, Milton; Carvalho, Lara; Silva, Joana; Saúde, Leonor; Fernandes, Alexandra R.; Baptista, Pedro V.

2017-01-01

The use of gold nanoparticles for effective gene silencing has demonstrated its potential as a tool for gene expression experiments and for the treatment of several diseases. Here, we used a gold nanobeacon designed to specifically silence the enhanced green fluorescence protein (EGFP) mRNA in embryos of a fli-EGFP transgenic zebrafish line, while simultaneously allowing the tracking and localization of the silencing events via the beacon’s emission. Fluorescence imaging measurements demonstrated a decrease of the EGFP emission with a concomitant increase in the fluorescence of the Au-nanobeacon. Furthermore, microinjection of the Au-nanobeacon led to a negligible difference in mortality and malformations in comparison to the free oligonucleotide, indicating that this system is a biocompatible platform for the administration of gene silencing moieties. Together, these data illustrate the potential of Au-nanobeacons as tools for in vivo zebrafish gene modulation with low toxicity which may be used towards any gene of interest. PMID:28336844

Automated deep-phenotyping of the vertebrate brain

PubMed Central

Allalou, Amin; Wu, Yuelong; Ghannad-Rezaie, Mostafa; Eimon, Peter M; Yanik, Mehmet Fatih

2017-01-01

Here, we describe an automated platform suitable for large-scale deep-phenotyping of zebrafish mutant lines, which uses optical projection tomography to rapidly image brain-specific gene expression patterns in 3D at cellular resolution. Registration algorithms and correlation analysis are then used to compare 3D expression patterns, to automatically detect all statistically significant alterations in mutants, and to map them onto a brain atlas. Automated deep-phenotyping of a mutation in the master transcriptional regulator fezf2 not only detects all known phenotypes but also uncovers important novel neural deficits that were overlooked in previous studies. In the telencephalon, we show for the first time that fezf2 mutant zebrafish have significant patterning deficits, particularly in glutamatergic populations. Our findings reveal unexpected parallels between fezf2 function in zebrafish and mice, where mutations cause deficits in glutamatergic neurons of the telencephalon-derived neocortex. DOI: http://dx.doi.org/10.7554/eLife.23379.001 PMID:28406399

Analyses of pancreas development by generation of gfp transgenic zebrafish using an exocrine pancreas-specific elastaseA gene promoter

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wan Haiyan; Korzh, Svitlana; Li Zhen

2006-05-15

In contrast to what we know on development of endocrine pancreas, the formation of exocrine pancreas remains poorly understood. To create an animal model that allows observation of exocrine cell differentiation, proliferation, and morphogenesis in living animals, we used the zebrafish elastaseA (elaA) regulatory sequence to develop transgenic zebrafish that display highly specific exocrine pancreas expression of GFP in both larvae and adult. By following GFP expression, we found that the pancreas in early development was a relatively compact organ and later extended posterior along the intestine. By transferring the elaA:gfp transgene into slow muscle omitted mutant that is deficientmore » in receiving Hedgehog signals, we further showed that Hedgehog signaling is required for exocrine morphogenesis but not for cell differentiation. We also applied the morpholino knockdown and toxin-mediated cell ablation approaches to this transgenic line. We showed that the development of exocrine pancreas is Islet-1 dependent. Injection of the diphtheria toxin A (DTA) construct under the elastaseA promoter resulted in selective ablation of exocrine cells while the endocrine cells and other endodermal derivatives (liver and intestine) were not affected. Thus, our works demonstrated the new transgenic line provided a useful experimental tool in analyzing exocrine pancreas development.« less

Inhibition of H3K27me3 Histone Demethylase Activity Prevents the Proliferative Regeneration of Zebrafish Lateral Line Neuromasts

PubMed Central

Bao, Beier; He, Yingzi; Tang, Dongmei; Li, Wenyan; Li, Huawei

2017-01-01

The H3K27 demethylases are involved in a variety of biological processes, including cell differentiation, proliferation, and cell death by regulating transcriptional activity. However, the function of H3K27 demethylation in the field of hearing research is poorly understood. Here, we investigated the role of H3K27me3 histone demethylase activity in hair cell regeneration using an in vivo animal model. Our data showed that pharmacologic inhibition of H3K27 demethylase activity with the specific small-molecule inhibitor GSK-J4 decreased the number of regenerated hair cells in response to neomycin damage. Furthermore, inhibition of H3K27me3 histone demethylase activity dramatically suppressed cell proliferation and activated caspase-3 levels in the regenerating neuromasts of the zebrafish lateral line. GSK-J4 administration also increased the expression of p21 and p27 in neuromast cells and inhibited the ERK signaling pathway. Collectively, our findings indicate that H3K27me3 demethylation is a key epigenetic regulator in the process of hair cell regeneration in zebrafish and suggest that H3K27me3 histone demethylase activity might be a novel therapeutic target for the treatment of hearing loss. PMID:28348517

Aberrant AKT activation drives well-differentiated liposarcoma

PubMed Central

Gutierrez, Alejandro; Snyder, Eric L.; Marino-Enriquez, Adrian; Zhang, Yi-Xiang; Sioletic, Stefano; Kozakewich, Elena; Grebliunaite, Ruta; Ou, Wen-bin; Sicinska, Ewa; Raut, Chandrajit P.; Demetri, George D.; Perez-Atayde, Antonio R.; Wagner, Andrew J.; Fletcher, Jonathan A.; Fletcher, Christopher D. M.; Look, A. Thomas

2011-01-01

Well-differentiated liposarcoma (WDLPS), one of the most common human sarcomas, is poorly responsive to radiation and chemotherapy, and the lack of animal models suitable for experimental analysis has seriously impeded functional investigation of its pathobiology and development of effective targeted therapies. Here, we show that zebrafish expressing constitutively active Akt2 in mesenchymal progenitors develop WDLPS that closely resembles the human disease. Tumor incidence rates were 8% in p53 wild-type zebrafish, 6% in p53 heterozygotes, and 29% in p53-homozygous mutant zebrafish (P = 0.013), indicating that aberrant Akt activation collaborates with p53 mutation in WDLPS pathogenesis. Analysis of primary clinical specimens of WDLPS, and of the closely related dedifferentiated liposarcoma (DDLPS) subtype, revealed immunohistochemical evidence of AKT activation in 27% of cases. Western blot analysis of a panel of cell lines derived from patients with WDLPS or DDLPS revealed robust AKT phosphorylation in all cell lines examined, even when these cells were cultured in serum-free media. Moreover, BEZ235, a small molecule inhibitor of PI3K and mammalian target of rapamycin that effectively inhibits AKT activation in these cells, impaired viability at nanomolar concentrations. Our findings are unique in providing an animal model to decipher the molecular pathogenesis of WDLPS, and implicate AKT as a previously unexplored therapeutic target in this chemoresistant sarcoma. PMID:21930930

Protective effects of edaravone against cisplatin-induced hair cell damage in zebrafish.

PubMed

Hong, Seok Jin; Im, Gi Jung; Chang, Jiwon; Chae, Sung Won; Lee, Seung Hoon; Kwon, Soon Young; Jung, Hak Hyun; Chung, Ah Young; Park, Hae Chul; Choi, June

2013-06-01

Edaravone is known to have a potent free radical scavenging effect. The objective of the present study was to evaluate the effects of edaravone on cisplatin-induced ototoxicity in transgenic zebrafish (Brn3C: EGFP). Five day post-fertilization zebrafish larvae were exposed to 1000 μM cisplatin and 50 μM, 100 μM, 250 μM, 500 μM, 750 μM, and 1000 μM concentrations of edaravone for 4h. Hair cells within neuromasts of the supraorbital (SO1 and SO2), otic (O1), and occipital (OC1) lateral lines were analyzed by fluorescence microscopy and confocal microscopy (n=10). Hair cell survival was calculated as a percentage of the hair cells in the control group that were not exposed to cisplatin. Ultrastructural changes were evaluated using scanning electron microscopy and transmission electron microscopy. Edaravone protected cisplatin-induced hair cell loss of neuromasts (edaravone 750 μM: 8.7 ± 1.5 cells, cisplatin 1000 μM only: 3.7 ± 0.9 cells; n=10, p<0.0001) and decreased the Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) reaction. Structures of mitochondria and hair cell within neuromasts in ultrastructural analysis were preserved in zebrafish exposed to 1000 μM cisplatin and 750 μM edaravone for 4h. Edaravone attenuated cisplatin-induced hair cell damage in zebrafish. The results of the current study suggest that cisplatin induces apoptosis, and the apoptotic cell death can be prevented by treatment with edaravone in zebrafish. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Comparison of cannabinoids with known analgesics using a novel high throughput zebrafish larval model of nociception.

PubMed

Ellis, L D; Berrue, F; Morash, M; Achenbach, J C; Hill, J; McDougall, J J

2018-01-30

It has been established that both adult and larval zebrafish are capable of showing nociceptive responses to noxious stimuli; however, the use of larvae to test novel analgesics has not been fully explored. Zebrafish larvae represent a low-cost, high-throughput alternative to traditional mammalian models for the assessment of product efficacy during the initial stages of drug development. In the current study, a novel model of nociception using zebrafish larvae is described. During the recovery from an acute exposure to low levels of acetic acid, larvae display innate changes in behaviour that may be indicative of nociception. To assess the usefulness of this model for testing potential analgesics, three known synthetic pain medications were assessed (ibuprofen, acetaminophen and tramadol) along with three naturally occurring products (honokiol, tetrahydrocannabinol and cannabidiol). When the effect of each compound on both the acetic acid recovery and control activity was compared there appeared to be both similarities and differences between the compounds. One of the most interesting effects was found for cannabidiol which appeared to oppose the activity change during the recovery period of AA exposed larvae while having a nominal effect on control activity. This would appear to be in line with current research that has demonstrated the nociceptive properties of cannabidiol. Here we have provided a novel model that will complement existing zebrafish models and will expand on the potential use of zebrafish larvae for studying both nociception and new analgesics. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

Genome editing using CRISPR/Cas9-based knock-in approaches in zebrafish.

PubMed

Albadri, Shahad; Del Bene, Filippo; Revenu, Céline

2017-05-15

With its variety of applications, the CRISPR/Cas9 genome editing technology has been rapidly evolving in the last few years. In the zebrafish community, knock-out reports are constantly increasing but insertion studies have been so far more challenging. With this review, we aim at giving an overview of the homologous directed repair (HDR)-based knock-in generation in zebrafish. We address the critical points and limitations of the procedure such as cutting efficiency of the chosen single guide RNA, use of cas9 mRNA or Cas9 protein, homology arm size etc. but also ways to circumvent encountered issues with HDR insertions by the development of non-homologous dependent strategies. While imprecise, these homology-independent mechanisms based on non-homologous-end-joining (NHEJ) repair have been employed in zebrafish to generate reporter lines or to accurately edit an open reading frame by the use of intron-targeting modifications. Therefore, with higher efficiency and insertion rate, NHEJ-based knock-in seems to be a promising approach to target endogenous loci and to circumvent the limitations of HDR whenever it is possible and appropriate. In this perspective, we propose new strategies to generate cDNA edited or tagged insertions, which once established will constitute a new and versatile toolbox for CRISPR/Cas9-based knock-ins in zebrafish. Copyright © 2017 Elsevier Inc. All rights reserved.

High-Resolution Tissue Doppler Imaging of the Zebrafish Heart During Its Regeneration

PubMed Central

Su, Ta-Han; Shih, Cho-Chiang

2015-01-01

Abstract The human heart cannot regenerate after injury, whereas the adult zebrafish can fully regenerate its heart even after 20% of the ventricle is amputated. Many studies have begun to reveal the cellular and molecular mechanisms underlying this regenerative process, which have exciting implications for human cardiac diseases. However, the dynamic functions of the zebrafish heart during regeneration are not yet understood. This study established a high-resolution echocardiography for tissue Doppler imaging (TDI) of the zebrafish heart to explore the cardiac functions during different regeneration phases. Experiments were performed on AB-line adult zebrafish (n=40) in which 15% of the ventricle was surgically removed. An 80-MHz ultrasound TDI based on color M-mode imaging technology was employed. The cardiac flow velocities and patterns from both the ventricular chamber and myocardium were measured at different regeneration phases relative to the day of amputation. The peak velocities of early diastolic inflow, early diastolic myocardial motion, late diastolic myocardial motion, early diastolic deceleration slope, and heart rate were increased at 3 days after the myocardium amputation, but these parameters gradually returned to close to their baseline values for the normal heart at 7 days after amputation. The peak velocities of late diastolic inflow, ventricular systolic outflow, and systolic myocardial motion did not significantly differ during the heart regeneration. PMID:25517185

Variable effects of dexamethasone on protein synthesis in clonal rat osteosarcoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hodge, B.O.; Kream, B.E.

1988-05-01

We examined the effects of dexamethasone on protein synthesis in clonal rat osteoblastic osteosarcoma (ROS) cell lines by measuring the incorporation of (/sup 3/H)proline into collagenase-digestible and noncollagen protein in the cell layer and medium of the cultures. In ROS 17/2 and subclone C12 of ROS 17/2.8, dexamethasone decreased collagen synthesis with no change in DNA content of the cultures. In ROS 17/2.8 and its subclone G2, dexamethasone stimulated collagen and noncollagen protein synthesis, with a concomitant decrease in the DNA content of the cells. These data indicate that ROS cell lines are phenotypically heterogeneous and suggest that in normalmore » bone there may be distinct subpopulations of osteoblasts with varying phenotypic traits with respect to the regulation of protein synthesis.« less

Abcb4 acts as multixenobiotic transporter and active barrier against chemical uptake in zebrafish (Danio rerio) embryos

PubMed Central

2013-01-01

Background In mammals, ABCB1 constitutes a cellular “first line of defense” against a wide array of chemicals and drugs conferring cellular multidrug or multixenobiotic resistance (MDR/MXR). We tested the hypothesis that an ABCB1 ortholog serves as protection for the sensitive developmental processes in zebrafish embryos against adverse compounds dissolved in the water. Results Indication for ABCB1-type efflux counteracting the accumulation of chemicals in zebrafish embryos comes from experiments with fluorescent and toxic transporter substrates and inhibitors. With inhibitors present, levels of fluorescent dyes in embryo tissue and sensitivity of embryos to toxic substrates were generally elevated. We verified two predicted sequences from zebrafish, previously annotated as abcb1, by cloning; our synteny analyses, however, identified them as abcb4 and abcb5, respectively. The abcb1 gene is absent in the zebrafish genome and we explored whether instead Abcb4 and/or Abcb5 show toxicant defense properties. Quantitative real-time polymerase chain reaction (qPCR) analyses showed the presence of transcripts of both genes throughout the first 48 hours of zebrafish development. Similar to transporter inhibitors, morpholino knock-down of Abcb4 increased accumulation of fluorescent substrates in embryo tissue and sensitivity of embryos toward toxic compounds. In contrast, morpholino knock-down of Abcb5 did not exert this effect. ATPase assays with recombinant protein obtained with the baculovirus expression system confirmed that dye and toxic compounds act as substrates of zebrafish Abcb4 and inhibitors block its function. The compounds tested comprised model substrates of human ABCB1, namely the fluorescent dyes rhodamine B and calcein-am and the toxic compounds vinblastine, vincristine and doxorubicin; cyclosporin A, PSC833, MK571 and verapamil were applied as inhibitors. Additionally, tests were performed with ecotoxicologically relevant compounds: phenanthrene (a polycyclic aromatic hydrocarbon) and galaxolide and tonalide (two polycyclic musks). Conclusions We show that zebrafish Abcb4 is a cellular toxicant transporter and provides protection of embryos against toxic chemicals dissolved in the water. Zebrafish Abcb4 thus is functionally similar to mammalian ABCB1, but differs from mammalian ABCB4, which is not involved in cellular resistance to chemicals but specifically transports phospholipids in the liver. Our data have important implications: Abcb4 could affect bioavailability - and thus toxicologic and pharmacologic potency - of chemicals to zebrafish embryos and inhibition of Abcb4 therefore causes chemosensitization, that is, enhanced sensitivity of embryos to toxicants. These aspects should be considered in (eco)toxicologic and pharmacologic chemical screens with the zebrafish embryo, a major vertebrate model. PMID:23773777

Food-borne pathogens, health and role of dietary phytochemicals.

PubMed

Shetty, K; Labbe, R G

1998-12-01

Infectious diseases transmitted by food have become a major public health concern in recent years. In the USA alone, there are an estimated 6-33 million cases each year. The list of responsible agents continues to grow. In the past 20 years some dozen new pathogens that are primarily food-borne have been identified. Fruits and vegetables, often from the global food market, have been added to the traditional vehicles of food-borne illness; that is, undercooked meat, poultry, seafood, or unpasteurized milk. Such products are minimally processed and have fewer barriers to microbial growth such as salt, sugar or preservatives. The evolution of the epidemiology of food-borne illness requires a rethinking of traditional, though still valid, solutions for their prevention. Among various strategies to prevent food-borne pathogens, use of dietary phytochemicals is promising. The major obstacle in the use of dietary phytochemical is the consistency of phytochemicals in different foods due to their natural genetic variation. We have developed a novel tissue-culture-based selection strategy to isolate elite phenolic phytochemical-producing clonal lines of species belonging to the family Lamiaceae. Among several species we have targeted elite clonal lines of thyme (Thymus vulgaris) and oregano (Origanum vulgare) against Escherichia coli and Clostridium perfrigens in fresh and processed meats. We are also evaluating high phenolic profile-containing clonal lines of basil (Ocimum basilicum) to inhibit gastric ulcer-causing Helicobacter pylori. Other elite lines of the members of the family Lamiaceae, rosemary (Rosmarinus officinalis) and salvia (Salvia officinalis) also hold promise against a wide range of food pathogens such as Salmonella species in poultry products and Vibrio species in seafood.

Osteoblasts are target cells for transformation in c-fos transgenic mice

PubMed Central

1993-01-01

We have generated transgenic mice expressing the proto-oncogene c-fos from an H-2Kb class I MHC promoter as a tool to identify and isolate cell populations which are sensitive to altered levels of Fos protein. All homozygous H2-c-fosLTR mice develop osteosarcomas with a short latency period. This phenotype is specific for c-fos as transgenic mice expressing the fos- and jun-related genes, fosB and c-jun, from the same regulatory elements do not develop any pathology despite high expression in bone tissues. The c-fos transgene is not expressed during embryogenesis but is expressed after birth in bone tissues before the onset of tumor formation, specifically in putative preosteoblasts, bone- forming osteoblasts, osteocytes, as well as in osteoblastic cells present within the tumors. Primary and clonal cell lines established from c-fos-induced tumors expressed high levels of exogenous c-fos as well as the bone cell marker genes, type I collagen, alkaline phosphatase, and osteopontin/2ar. In contrast, osteocalcin/BGP expression was either low or absent. All cell lines were tumorigenic in vivo, some of which gave rise to osteosarcomas, expressing exogenous c- fos mRNA, and Fos protein in osteoblastic cells. Detailed analysis of one osteogenic cell line, P1, and several P1-derived clonal cell lines indicated that bone-forming osteoblastic cells were transformed by Fos. The regulation of osteocalcin/BGP and alkaline phosphatase gene expression by 1,25-dihydroxyvitamin D3 was abrogated in P1-derived clonal cells, whereas glucocorticoid responsiveness was unaltered. These results suggest that high levels of Fos perturb the normal growth control of osteoblastic cells and exert specific effects on the expression of the osteoblast phenotype. PMID:8335693

Physiological concentrations of interleukin-6 directly promote insulin secretion, signal transduction, nitric oxide release, and redox status in a clonal pancreatic β-cell line and mouse islets.

PubMed

da Silva Krause, Mauricio; Bittencourt, Aline; Homem de Bittencourt, Paulo Ivo; McClenaghan, Neville H; Flatt, Peter R; Murphy, Colin; Newsholme, Philip

2012-09-01

Interleukin-6 (IL6) has recently been reported to promote insulin secretion in a glucagon-like peptide-1-dependent manner. Herein, the direct effects of IL6 (at various concentrations from 0 to 1000 pg/ml) on pancreatic β-cell metabolism, AMP-activated protein kinase (AMPK) signaling, insulin secretion, nitrite release, and redox status in a rat clonal β-cell line and mouse islets are reported. Chronic insulin secretion (in μg/mg protein per 24  h) was increased from 128·7±7·3 (no IL6) to 178·4±7·7 (at 100  pg/ml IL6) in clonal β-cells and increased significantly in islets incubated in the presence of 5·5  mM glucose for 2  h, from 0·148 to 0·167±0·003  ng/islet. Pretreatment with IL6 also induced a twofold increase in basal and nutrient-stimulated insulin secretion in subsequent 20 min static incubations. IL6 enhanced both glutathione (GSH) and glutathione disulphide (GSSG) by nearly 20% without changing intracellular redox status (GSSG/GSH). IL6 dramatically increased iNOS expression (by ca. 100-fold) with an accompanying tenfold rise in nitrite release in clonal β-cells. Phosphorylated AMPK levels were elevated approximately twofold in clonal β-cells and mouse islet cells. Calmodulin-dependent protein kinase kinase levels (CaMKK), an upstream kinase activator of AMPK, were also increased by 50% after IL6 exposure (in β-cells and islets). Our data have demonstrated that IL6 can stimulate β-cell-dependent insulin secretion via direct cell-based mechanisms. AMPK, CaMKK (an upstream kinase activator of AMPK), and the synthesis of nitric oxide appear to alter cell metabolism to benefit insulin secretion. In summary, IL6 exerts positive effects on β-cell signaling, metabolism, antioxidant status, and insulin secretion.

Development without germ cells: the role of the germ line in zebrafish sex differentiation.

PubMed

Slanchev, Krasimir; Stebler, Jürg; de la Cueva-Méndez, Guillermo; Raz, Erez

2005-03-15

The progenitors of the gametes, the primordial germ cells (PGCs) are typically specified early in the development in positions, which are distinct from the gonad. These cells then migrate toward the gonad where they differentiate into sperms and eggs. Here, we study the role of the germ cells in somatic development and particularly the role of the germ line in the sex differentiation in zebrafish. To this end, we ablated the germ cells using two independent methods and followed the development of the experimental fish. First, PGCs were ablated by knocking down the function of dead end, a gene important for the survival of this lineage. Second, a method to eliminate the PGCs using the toxin-antitoxin components of the parD bacterial genetic system was used. Specifically, we expressed a bacterial toxin Kid preferentially in the PGCs and at the same time protected somatic cells by uniformly expressing the specific antidote Kis. Our results demonstrate an unexpected role for the germ line in promoting female development because PGC-ablated fish invariably developed as males.

Development without germ cells: The role of the germ line in zebrafish sex differentiation

PubMed Central

Slanchev, Krasimir; Stebler, Jürg; de la Cueva-Méndez, Guillermo; Raz, Erez

2005-01-01

The progenitors of the gametes, the primordial germ cells (PGCs) are typically specified early in the development in positions, which are distinct from the gonad. These cells then migrate toward the gonad where they differentiate into sperms and eggs. Here, we study the role of the germ cells in somatic development and particularly the role of the germ line in the sex differentiation in zebrafish. To this end, we ablated the germ cells using two independent methods and followed the development of the experimental fish. First, PGCs were ablated by knocking down the function of dead end, a gene important for the survival of this lineage. Second, a method to eliminate the PGCs using the toxin–antitoxin components of the parD bacterial genetic system was used. Specifically, we expressed a bacterial toxin Kid preferentially in the PGCs and at the same time protected somatic cells by uniformly expressing the specific antidote Kis. Our results demonstrate an unexpected role for the germ line in promoting female development because PGC-ablated fish invariably developed as males. PMID:15728735

Insights into electrosensory organ development, physiology and evolution from a lateral line-enriched transcriptome

PubMed Central

Modrell, Melinda S; Lyne, Mike; Carr, Adrian R; Zakon, Harold H; Buckley, David; Campbell, Alexander S; Davis, Marcus C; Micklem, Gos; Baker, Clare VH

2017-01-01

The anamniote lateral line system, comprising mechanosensory neuromasts and electrosensory ampullary organs, is a useful model for investigating the developmental and evolutionary diversification of different organs and cell types. Zebrafish neuromast development is increasingly well understood, but neither zebrafish nor Xenopus is electroreceptive and our molecular understanding of ampullary organ development is rudimentary. We have used RNA-seq to generate a lateral line-enriched gene-set from late-larval paddlefish (Polyodon spathula). Validation of a subset reveals expression in developing ampullary organs of transcription factor genes critical for hair cell development, and genes essential for glutamate release at hair cell ribbon synapses, suggesting close developmental, physiological and evolutionary links between non-teleost electroreceptors and hair cells. We identify an ampullary organ-specific proneural transcription factor, and candidates for the voltage-sensing L-type Cav channel and rectifying Kv channel predicted from skate (cartilaginous fish) ampullary organ electrophysiology. Overall, our results illuminate ampullary organ development, physiology and evolution. DOI: http://dx.doi.org/10.7554/eLife.24197.001 PMID:28346141

CRISPR/Cas9-Mediated Zebrafish Knock-in as a Novel Strategy to Study Midbrain-Hindbrain Boundary Development

PubMed Central

Kesavan, Gokul; Chekuru, Avinash; Machate, Anja; Brand, Michael

2017-01-01

The midbrain-hindbrain boundary (MHB) acts as an organizer and controls the fate of neighboring cells to develop into either mesencephalic (midbrain) or metencephalic (hindbrain) cells by secreting signaling molecules like Wnt1 and Fgf8. The zebrafish is an excellent vertebrate model for studying MHB development due to the ease of gene manipulation and the possibility of following cellular dynamics and morphogenetic processes using live imaging. Currently, only very few reporter and/or Cre-driver lines are available to study gene expression at the MHB, hampering the understanding of MHB development, and traditional transgenic technologies using promoter/enhancer fragments or bacterial artificial chromosome (BAC)-mediated transgenesis often do not faithfully recapitulate endogenous expression patterns. In contrast, CRISPR/Cas9-mediated genome editing technology now provides a great opportunity to efficiently knock-in or knock-out genes. We have generated four CRISPR/Cas9-based knock-in fluorescent reporter lines for two crucial genes involved in MHB development, namely otx2 and pax2a. The coding sequences of the reporters were knocked-in upstream of the corresponding ATG and are, thus, under the control of the endogenous promoter/enhancer elements. Interestingly, this strategy does not disturb endogenous gene expression. Using the fast maturing fluorescent protein reporter, Venus, enabled us to follow MHB development using cell tracking and live imaging. In addition, we show that these reporter lines label various neuronal and glial cell types in the adult zebrafish brain, making them highly suitable for investigating embryonic and adult midbrain, hindbrain, and MHB development. PMID:28713249

CRISPR/Cas9-Mediated Zebrafish Knock-in as a Novel Strategy to Study Midbrain-Hindbrain Boundary Development.

PubMed

Kesavan, Gokul; Chekuru, Avinash; Machate, Anja; Brand, Michael

2017-01-01

The midbrain-hindbrain boundary (MHB) acts as an organizer and controls the fate of neighboring cells to develop into either mesencephalic (midbrain) or metencephalic (hindbrain) cells by secreting signaling molecules like Wnt1 and Fgf8. The zebrafish is an excellent vertebrate model for studying MHB development due to the ease of gene manipulation and the possibility of following cellular dynamics and morphogenetic processes using live imaging. Currently, only very few reporter and/or Cre-driver lines are available to study gene expression at the MHB, hampering the understanding of MHB development, and traditional transgenic technologies using promoter/enhancer fragments or bacterial artificial chromosome (BAC)-mediated transgenesis often do not faithfully recapitulate endogenous expression patterns. In contrast, CRISPR/Cas9-mediated genome editing technology now provides a great opportunity to efficiently knock-in or knock-out genes. We have generated four CRISPR/Cas9-based knock-in fluorescent reporter lines for two crucial genes involved in MHB development, namely otx2 and pax2a . The coding sequences of the reporters were knocked-in upstream of the corresponding ATG and are, thus, under the control of the endogenous promoter/enhancer elements. Interestingly, this strategy does not disturb endogenous gene expression. Using the fast maturing fluorescent protein reporter, Venus, enabled us to follow MHB development using cell tracking and live imaging. In addition, we show that these reporter lines label various neuronal and glial cell types in the adult zebrafish brain, making them highly suitable for investigating embryonic and adult midbrain, hindbrain, and MHB development.

In vivo alternative assessment of the chemicals that interfere with anterior pituitary POMC expression and interrenal steroidogenesis in POMC: EGFP transgenic zebrafish

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun Lingli; Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei, 430072; Xu Wei

2010-11-01

Adrenocorticotropin (ACTH) has been considered a classic adrenocorticotropic hormone and the key pituitary-derived peptide controlling steroidogenesis in the adult adrenal. ACTH is encoded by the propiomelanocortin (POMC) gene, and its active form is mainly synthesized and processed from the POMC-encoded multihormone precursor in the anterior pituitary. The ACTH level has always been precisely controlled in the signaling cascade of the hypothalamo-pituitary-adrenal (HPA) axis due to its central role. The purpose of this study was to investigate whether the transgenic zebrafish line with EGFP driven by the POMC promoter can be used as a surrogate marker to detect the interference effectsmore » on anterior pituitary POMC expression caused by chemicals in teleost. The Tg (POMC:EGFP) fish treated for 4 days with the known adrenergic agents, dexamethasone (Dex) or aminoglutethimide (AG), exhibited altered levels of EGFP and POMC expression in the anterior domain of pituitary corticotrophs. Whole-mount in situ hybridization revealed impaired patterns of expression of the zebrafish ftz-fl gene (ff1b), a key molecular marker for early interrenal development. Next, several chemicals and six commonly used organophosphorus compounds (OPs) were tested for their effects on anterior pituitary POMC expression and early interrenal development. Our preliminary screening analyses indicated that simazine and 3,3',4,4'5-pentachlorobiphenyl (PCB126) could interfere with anterior pituitary POMC expression and interrenal development in fish. In summary, our results demonstrated that the Tg (POMC:EGFP) zebrafish line might be employed as a specific and reproductive in vivo assessment model for the effects of endocrine disruption on HPA signaling.« less

Zebrafish Staufen1 and Staufen2 are required for the survival and migration of primordial germ cells.

PubMed

Ramasamy, Srinivas; Wang, Hui; Quach, Helen Ngoc Bao; Sampath, Karuna

2006-04-15

In sexually reproducing organisms, primordial germ cells (PGCs) give rise to the cells of the germ line, the gametes. In many animals, PGCs are set apart from somatic cells early during embryogenesis. Work in Drosophila, C. elegans, Xenopus, and zebrafish has shown that maternally provided localized cytoplasmic determinants specify the germ line in these organisms (Raz, E., 2003. Primordial germ-cell development: the zebrafish perspective. Nat. Rev., Genet. 4, 690--700; Santos, A.C., Lehmann, R., 2004. Germ cell specification and migration in Drosophila and beyond. Curr. Biol. 14, R578-R589). The Drosophila RNA-binding protein, Staufen is required for germ cell formation, and mutations in stau result in a maternal effect grandchild-less phenotype (Schupbach,T., Weischaus, E., 1989. Female sterile mutations on the second chromosome of Drosophila melanogaster:1. Maternal effect mutations. Genetics 121, 101-17). Here we describe the functions of two zebrafish Staufen-related proteins, Stau1 and Stau2. When Stau1 or Stau2 functions are compromised in embryos by injecting antisense morpholino modified oligonucleotides or dominant-negative Stau peptides, germ layer patterning is not affected. However, expression of the PGC marker vasa is not maintained. Furthermore, expression of a green fluorescent protein (GFP):nanos 3'UTR fusion protein in germ cells shows that PGC migration is aberrant, and the mis-migrating PGCs do not survive in Stau-compromised embryos. Stau2 is also required for survival of neurons in the central nervous system (CNS). These phenotypes are rescued by co-injection of Drosophila stau mRNA. Thus, staufen has an evolutionarily conserved function in germ cells. In addition, we have identified a function for Stau proteins in PGC migration.

Functional Analysis of Nuclear Estrogen Receptors in Zebrafish Reproduction by Genome Editing Approach.

PubMed

Lu, Huijie; Cui, Yong; Jiang, Liwen; Ge, Wei

2017-07-01

Estrogens signal through both nuclear and membrane receptors with most reported effects being mediated via the nuclear estrogen receptors (nERs). Although much work has been reported on nERs in the zebrafish, there is a lack of direct genetic evidence for their functional roles and importance in reproduction. To address this issue, we undertook this study to disrupt all three nERs in the zebrafish, namely esr1 (ERα), esr2a (ERβII), and esr2b (ERβI), by the genome-editing technology clustered regularly interspaced short palindromic repeats and its associated nuclease (CRISPR/Cas9). Using this loss-of-function genetic approach, we successfully created three mutant zebrafish lines with each nER knocked out. In addition, we also generated all possible double and triple knockouts of the three nERs. The phenotypes of these mutants in reproduction were analyzed in all single, double, and triple nER knockouts in both females and males. Surprisingly, all three single nER mutant fish lines display normal reproductive development and function in both females and males, suggesting functional redundancy among these nERs. Further analysis of double and triple knockouts showed that nERs, especially Esr2a and Esr2b, were essential for female reproduction, and loss of these two nERs led to an arrest of folliculogenesis at previtellogenic stage II followed by sex reversal from female to male. In addition, the current study also revealed a unique role for Esr2a in follicle cell proliferation and transdifferentiation, follicle growth, and chorion formation. Taken together, this study provides the most comprehensive genetic analysis for differential functions of esr1, esr2a, and esr2b in fish reproduction. Copyright © 2017 Endocrine Society.

Cell Migration During Heart Regeneration in Zebrafish

PubMed Central

Tahara, Naoyuki; Brush, Michael; Kawakami, Yasuhiko

2018-01-01

Zebrafish possess the remarkable ability to regenerate injured hearts as adults, which contrasts the very limited ability in mammals. Although very limited, mammalian hearts do in fact have measurable levels of cardiomyocyte regeneration. Therefore, elucidating mechanisms of zebrafish heart regeneration would provide information of naturally occurring regeneration to potentially apply to mammalian studies, in addition to addressing this biologically interesting phenomenon in itself. Studies over the past 13 years have identified processes and mechanisms of heart regeneration in zebrafish. After heart injury, preexisting cardiomyocytes dedifferentiate, enter the cell cycle, and repair the injured myocardium. This process requires interaction with epicardial cells, endocardial cells, and vascular endothelial cells. Epicardial cells envelope the heart, while endocardial cells make up the inner lining of the heart. They provide paracrine signals to cardiomyocytes to regenerate the injured myocardium, which is vascularized during heart regeneration. In addition, accumulating results suggest that local migration of these major cardiac cell types have roles in heart regeneration. In this review, we summarize the characteristics of various heart injury methods used in the research community and regeneration of the major cardiac cell types. Then, we discuss local migration of these cardiac cell types and immune cells during heart regeneration. PMID:27085002

A novel keratin18 promoter that drives reporter gene expression in the intrahepatic and extrahepatic biliary system allows isolation of cell-type specific transcripts from zebrafish liver

PubMed Central

Wilkins, Benjamin J.; Gong, Weilong; Pack, Michael

2015-01-01

Heritable and acquired biliary disorders are an important cause of acute and chronic human liver disease. Biliary development and physiology have been studied extensively in rodent models and more recently, zebrafish have been used to uncover pathogenic mechanisms and potential therapies for these conditions. Here we report development of novel transgenic lines labeling the intrahepatic and extrahepatic biliary system of zebrafish larvae that can be used for lineage tracing and isolation of biliary-specific RNAs from mixed populations of liver cells. We show that GFP expression driven by a 4.4 kilobase promoter fragment from the zebrafish keratin18 (krt18) gene allows visualization of all components of the developing biliary system as early as 3 days post-fertilization. In addition, expression of a ribosomal fusion protein (EGFP-Rpl10a) in krt18:TRAP transgenic fish allows for enrichment of translated biliary cell mRNAs via translating ribosome affinity purification (TRAP). Future studies utilizing these reagents will enhance our understanding of the morphologic and molecular processes involved in biliary development and disease. PMID:24394404

Clonal Expansion during Staphylococcus aureus Infection Dynamics Reveals the Effect of Antibiotic Intervention

PubMed Central

McVicker, Gareth; Prajsnar, Tomasz K.; Williams, Alexander; Wagner, Nelly L.; Boots, Michael; Renshaw, Stephen A.; Foster, Simon J.

2014-01-01

To slow the inexorable rise of antibiotic resistance we must understand how drugs impact on pathogenesis and influence the selection of resistant clones. Staphylococcus aureus is an important human pathogen with populations of antibiotic-resistant bacteria in hospitals and the community. Host phagocytes play a crucial role in controlling S. aureus infection, which can lead to a population “bottleneck” whereby clonal expansion of a small fraction of the initial inoculum founds a systemic infection. Such population dynamics may have important consequences on the effect of antibiotic intervention. Low doses of antibiotics have been shown to affect in vitro growth and the generation of resistant mutants over the long term, however whether this has any in vivo relevance is unknown. In this work, the population dynamics of S. aureus pathogenesis were studied in vivo using antibiotic-resistant strains constructed in an isogenic background, coupled with systemic models of infection in both the mouse and zebrafish embryo. Murine experiments revealed unexpected and complex bacterial population kinetics arising from clonal expansion during infection in particular organs. We subsequently elucidated the effect of antibiotic intervention within the host using mixed inocula of resistant and sensitive bacteria. Sub-curative tetracycline doses support the preferential expansion of resistant microorganisms, importantly unrelated to effects on growth rate or de novo resistance acquisition. This novel phenomenon is generic, occurring with methicillin-resistant S. aureus (MRSA) in the presence of β-lactams and with the unrelated human pathogen Pseudomonas aeruginosa. The selection of resistant clones at low antibiotic levels can result in a rapid increase in their prevalence under conditions that would previously not be thought to favor them. Our results have key implications for the design of effective treatment regimes to limit the spread of antimicrobial resistance, where inappropriate usage leading to resistance may reduce the efficacy of life-saving drugs. PMID:24586163

Cell lines derived from feline fibrosarcoma display unstable chromosomal aneuploidy and additionally centrosome number aberrations.

PubMed

von Erichsen, J; Hecht, W; Löhberg-Gruene, C; Reinacher, M

2012-07-01

The purpose of the study was to evaluate clonality and presence of numerical chromosomal and centrosomal aberrations in 5 established feline fibrosarcoma cell lines and in a fetal dermal fibroblast cell line as a control. The clonality of all cell lines was examined using limited-dilution cloning. The number of chromosomes was counted in metaphase spreads. The immunocytochemical analysis of centrosome numbers was performed by indirect immunofluorescence using a monoclonal antibody that targets γ-tubulin, a well-characterized component of centrosomes. Monoclonal cell populations could be established from all cell lines. In all feline fibrosarcoma cell lines, the number of chromosomes deviated abnormally from the normal feline chromosome number of 2n = 38, ranging from 19 to 155 chromosomes per cell. Centrosome hyperamplification was observed in all 5 feline fibrosarcoma cell lines with a proportion of cells (5.7 to 15.2%) having more than 2 centrosomes. In the control cell line, only 0.6% of the cells had more than 2 centrosomes. In conclusion, the examinations revealed that centrosome hyperamplification occurs in feline fibrosarcoma cell lines. The feline fibrosarcoma cell lines possessed 10 to 25 times as many cells with centrosome hyperamplification as the control cell line. These observations suggest an association of numerical centrosome aberrations with karyotype instability by increasing the frequency of chromosome missegregation. The results of this study may be helpful for further characterization of feline fibrosarcomas and may contribute to the knowledge of cytogenetic factors that may be important for the pathogenesis of feline fibrosarcomas.

Culturable Gut Microbiota Diversity in Zebrafish

PubMed Central

Sørby, Jan Roger Torp; Aleström, Peter; Sørum, Henning

2012-01-01

Abstract The zebrafish (Danio rerio) is an increasingly used laboratory animal model in basic biology and biomedicine, novel drug development, and toxicology. The wide use has increased the demand for optimized husbandry protocols to ensure animal health care and welfare. The knowledge about the correlation between culturable zebrafish intestinal microbiota and health in relation to environmental factors and management procedures is very limited. A semi-quantitative level of growth of individual types of bacteria was determined and associated with sampling points. A total of 72 TAB line zebrafish from four laboratories (Labs A–D) in the Zebrafish Network Norway were used. Diagnostic was based on traditional bacterial culture methods and biochemical characterization using commercial kits, followed by 16S rDNA gene sequencing from pure subcultures. Also selected Gram-negative isolates were analyzed for antibiotic susceptibility to 8 different antibiotics. A total of 13 morphologically different bacterial species were the most prevalent: Aeromonas hydrophila, Aeromonas sobria, Vibrio parahaemolyticus, Photobacterium damselae, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas luteola, Comamonas testosteroni, Ochrobactrum anthropi, Staphylococcus cohnii, Staphylococcus epidermidis, Staphylococcus capitis, and Staphylococcus warneri. Only Lab B had significantly higher levels of total bacterial growth (OR=2.03), whereas numbers from Lab C (OR=1.01) and Lab D (OR=1.12) were found to be similar to the baseline Lab A. Sexually immature individuals had a significantly higher level of harvested total bacterial growth than mature fish (OR=0.82), no statistically significant differences were found between male and female fish (OR=1.01), and the posterior intestinal segment demonstrated a higher degree of culturable bacteria than the anterior segment (OR=4.1). Multiple antibiotic (>3) resistance was observed in 17% of the strains. We propose that a rapid conventional diagnostic bacteriological assay on the culturable microbiota profiles can be designed and used as quality measure of the husbandry routines of a zebrafish facility to ensure a bacterial standard safeguarding the zebrafish health and welfare. PMID:22428747

Culturable gut microbiota diversity in zebrafish.

PubMed

Cantas, Leon; Sørby, Jan Roger Torp; Aleström, Peter; Sørum, Henning

2012-03-01

The zebrafish (Danio rerio) is an increasingly used laboratory animal model in basic biology and biomedicine, novel drug development, and toxicology. The wide use has increased the demand for optimized husbandry protocols to ensure animal health care and welfare. The knowledge about the correlation between culturable zebrafish intestinal microbiota and health in relation to environmental factors and management procedures is very limited. A semi-quantitative level of growth of individual types of bacteria was determined and associated with sampling points. A total of 72 TAB line zebrafish from four laboratories (Labs A-D) in the Zebrafish Network Norway were used. Diagnostic was based on traditional bacterial culture methods and biochemical characterization using commercial kits, followed by 16S rDNA gene sequencing from pure subcultures. Also selected Gram-negative isolates were analyzed for antibiotic susceptibility to 8 different antibiotics. A total of 13 morphologically different bacterial species were the most prevalent: Aeromonas hydrophila, Aeromonas sobria, Vibrio parahaemolyticus, Photobacterium damselae, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas luteola, Comamonas testosteroni, Ochrobactrum anthropi, Staphylococcus cohnii, Staphylococcus epidermidis, Staphylococcus capitis, and Staphylococcus warneri. Only Lab B had significantly higher levels of total bacterial growth (OR=2.03), whereas numbers from Lab C (OR=1.01) and Lab D (OR=1.12) were found to be similar to the baseline Lab A. Sexually immature individuals had a significantly higher level of harvested total bacterial growth than mature fish (OR=0.82), no statistically significant differences were found between male and female fish (OR=1.01), and the posterior intestinal segment demonstrated a higher degree of culturable bacteria than the anterior segment (OR=4.1). Multiple antibiotic (>3) resistance was observed in 17% of the strains. We propose that a rapid conventional diagnostic bacteriological assay on the culturable microbiota profiles can be designed and used as quality measure of the husbandry routines of a zebrafish facility to ensure a bacterial standard safeguarding the zebrafish health and welfare.

Light directs zebrafish period2 expression via conserved D and E boxes.

PubMed

Vatine, Gad; Vallone, Daniela; Appelbaum, Lior; Mracek, Philipp; Ben-Moshe, Zohar; Lahiri, Kajori; Gothilf, Yoav; Foulkes, Nicholas S

2009-10-01

For most species, light represents the principal environmental signal for entraining the endogenous circadian clock. The zebrafish is a fascinating vertebrate model for studying this process since unlike mammals, direct exposure of most of its tissues to light leads to local clock entrainment. Importantly, light induces the expression of a set of genes including certain clock genes in most zebrafish cell types in vivo and in vitro. However, the mechanism linking light to gene expression remains poorly understood. To elucidate this key mechanism, here we focus on how light regulates transcription of the zebrafish period2 (per2) gene. Using transgenic fish and stably transfected cell line-based assays, we define a Light Responsive Module (LRM) within the per2 promoter. The LRM lies proximal to the transcription start site and is both necessary and sufficient for light-driven gene expression and also for a light-dependent circadian clock regulation. Curiously, the LRM sequence is strongly conserved in other vertebrate per2 genes, even in species lacking directly light-sensitive peripheral clocks. Furthermore, we reveal that the human LRM can substitute for the zebrafish LRM to confer light-regulated transcription in zebrafish cells. The LRM contains E- and D-box elements that are critical for its function. While the E-box directs circadian clock regulation by mediating BMAL/CLOCK activity, the D-box confers light-driven expression. The zebrafish homolog of the thyrotroph embryonic factor binds efficiently to the LRM D-box and transactivates expression. We demonstrate that tef mRNA levels are light inducible and that knock-down of tef expression attenuates light-driven transcription from the per2 promoter in vivo. Together, our results support a model where a light-dependent crosstalk between E- and D-box binding factors is a central determinant of per2 expression. These findings extend the general understanding of the mechanism whereby the clock is entrained by light and how the regulation of clock gene expression by light has evolved in vertebrates.

Rapid high-throughput cloning and stable expression of antibodies in HEK293 cells.

PubMed

Spidel, Jared L; Vaessen, Benjamin; Chan, Yin Yin; Grasso, Luigi; Kline, J Bradford

2016-12-01

Single-cell based amplification of immunoglobulin variable regions is a rapid and powerful technique for cloning antigen-specific monoclonal antibodies (mAbs) for purposes ranging from general laboratory reagents to therapeutic drugs. From the initial screening process involving small quantities of hundreds or thousands of mAbs through in vitro characterization and subsequent in vivo experiments requiring large quantities of only a few, having a robust system for generating mAbs from cloning through stable cell line generation is essential. A protocol was developed to decrease the time, cost, and effort required by traditional cloning and expression methods by eliminating bottlenecks in these processes. Removing the clonal selection steps from the cloning process using a highly efficient ligation-independent protocol and from the stable cell line process by utilizing bicistronic plasmids to generate stable semi-clonal cell pools facilitated an increased throughput of the entire process from plasmid assembly through transient transfections and selection of stable semi-clonal cell pools. Furthermore, the time required by a single individual to clone, express, and select stable cell pools in a high-throughput format was reduced from 4 to 6months to only 4 to 6weeks. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Development of an Automated Imaging Pipeline for the Analysis of the Zebrafish Larval Kidney

PubMed Central

Westhoff, Jens H.; Giselbrecht, Stefan; Schmidts, Miriam; Schindler, Sebastian; Beales, Philip L.; Tönshoff, Burkhard; Liebel, Urban; Gehrig, Jochen

2013-01-01

The analysis of kidney malformation caused by environmental influences during nephrogenesis or by hereditary nephropathies requires animal models allowing the in vivo observation of developmental processes. The zebrafish has emerged as a useful model system for the analysis of vertebrate organ development and function, and it is suitable for the identification of organotoxic or disease-modulating compounds on a larger scale. However, to fully exploit its potential in high content screening applications, dedicated protocols are required allowing the consistent visualization of inner organs such as the embryonic kidney. To this end, we developed a high content screening compatible pipeline for the automated imaging of standardized views of the developing pronephros in zebrafish larvae. Using a custom designed tool, cavities were generated in agarose coated microtiter plates allowing for accurate positioning and orientation of zebrafish larvae. This enabled the subsequent automated acquisition of stable and consistent dorsal views of pronephric kidneys. The established pipeline was applied in a pilot screen for the analysis of the impact of potentially nephrotoxic drugs on zebrafish pronephros development in the Tg(wt1b:EGFP) transgenic line in which the developing pronephros is highlighted by GFP expression. The consistent image data that was acquired allowed for quantification of gross morphological pronephric phenotypes, revealing concentration dependent effects of several compounds on nephrogenesis. In addition, applicability of the imaging pipeline was further confirmed in a morpholino based model for cilia-associated human genetic disorders associated with different intraflagellar transport genes. The developed tools and pipeline can be used to study various aspects in zebrafish kidney research, and can be readily adapted for the analysis of other organ systems. PMID:24324758

Transient Exposure to Ethanol during Zebrafish Embryogenesis Results in Defects in Neuronal Differentiation: An Alternative Model System to Study FASD

PubMed Central

Joya, Xavier; Garcia-Algar, Oscar; Vall, Oriol; Pujades, Cristina

2014-01-01

Background The exposure of the human embryo to ethanol results in a spectrum of disorders involving multiple organ systems, including the impairment of the development of the central nervous system (CNS). In spite of the importance for human health, the molecular basis of prenatal ethanol exposure remains poorly understood, mainly to the difficulty of sample collection. Zebrafish is now emerging as a powerful organism for the modeling and the study of human diseases. In this work, we have assessed the sensitivity of specific subsets of neurons to ethanol exposure during embryogenesis and we have visualized the sensitive embryonic developmental periods for specific neuronal groups by the use of different transgenic zebrafish lines. Methodology/Principal Findings In order to evaluate the teratogenic effects of acute ethanol exposure, we exposed zebrafish embryos to ethanol in a given time window and analyzed the effects in neurogenesis, neuronal differentiation and brain patterning. Zebrafish larvae exposed to ethanol displayed small eyes and/or a reduction of the body length, phenotypical features similar to the observed in children with prenatal exposure to ethanol. When neuronal populations were analyzed, we observed a clear reduction in the number of differentiated neurons in the spinal cord upon ethanol exposure. There was a decrease in the population of sensory neurons mainly due to a decrease in cell proliferation and subsequent apoptosis during neuronal differentiation, with no effect in motoneuron specification. Conclusion Our investigation highlights that transient exposure to ethanol during early embryonic development affects neuronal differentiation although does not result in defects in early neurogenesis. These results establish the use of zebrafish embryos as an alternative research model to elucidate the molecular mechanism(s) of ethanol-induced developmental toxicity at very early stages of embryonic development. PMID:25383948

In vitro and in vivo estrogenic activity of BPA, BPF and BPS in zebrafish-specific assays.

PubMed

Le Fol, Vincent; Aït-Aïssa, Selim; Sonavane, Manoj; Porcher, Jean-Marc; Balaguer, Patrick; Cravedi, Jean-Pierre; Zalko, Daniel; Brion, François

2017-08-01

Bisphenol A (BPA) is a widely used chemical that has been extensively studied as an endocrine-disrupting chemical (EDC). Other bisphenols sharing close structural features with BPA, are increasingly being used as alternatives, increasing the need to assess associated hazards to the endocrine system. In the present study, the estrogenic activity of BPA, bisphenol S (BPS) and bisphenol F (BPF) was assessed by using a combination of zebrafish-specific mechanism-based in vitro and in vivo assays. The three bisphenols were found to efficiently transactivate all zebrafish estrogen receptor (zfER) subtypes in zebrafish hepatic reporter cell lines (ZELH-zfERs). BPA was selective for zfERα while BPS and BPF were slightly more potent on zfERβ subtypes. We further documented the estrogenic effect in vivo by quantifying the expression of brain aromatase using a transgenic cyp19a1b-GFP zebrafish embryo assay. All three bisphenols induced GFP in a concentration-dependent manner. BPS only partially induced brain aromatase at the highest tested concentrations (>30µM) while BPA and BPF strongly induced GFP, in an ER-dependent manner, at 1-10µM. Furthermore, we show that BPF strongly induced vitellogenin synthesis in adult male zebrafish. Overall, this study demonstrates the estrogenic activity of BPA, BPF and BPS in different cell- and tissue-contexts and at different stages of development. Differences between in vitro and in vivo responses are discussed in light of selective ER activation and the fate of the compounds in the models. This study confirms the relevance of combining cellular and whole-organism bioassays in a unique model species for the hazard assessment of candidate EDCs. Copyright © 2017 Elsevier Inc. All rights reserved.

Small GTPase R-Ras participates in neural tube formation in zebrafish embryonic spinal cord.

PubMed

Ohata, Shinya; Uga, Hideko; Okamoto, Hitoshi; Katada, Toshiaki

2018-06-27

Ras related (R-Ras), a small GTPase, is involved in the maintenance of apico-basal polarity in neuroepithelial cells of the zebrafish hindbrain, axonal collapse in cultured murine hippocampal neurons, and maturation of blood vessels in adult mice. However, the role of R-Ras in neural tube formation remains unknown. Using antisense morpholino oligonucleotides (AMOs), we found that in the spinal cord of zebrafish embryos, the lumen was formed bilaterally in rras morphants, whereas it was formed at the midline in control embryos. As AMO can cause off-target effects, we generated rras mutant zebrafish lines using CRISPR/Cas9 technology. Although these rras mutant embryos did not have a bilateral lumen in the spinal cord, the following findings suggest that the phenotype is unlikely due to an off-target effect of rras AMO: 1) The rras morphant phenotype was rescued by an injection of AMO-resistant rras mRNA, and 2) a bilaterally segregated spinal cord was not observed in rras mutant embryos injected with rras AMO. The results suggest that the function of other ras family genes may be redundant in rras mutants. Previous research reported a bilaterally formed lumen in the spinal cord of zebrafish embryos with a mutation in a planar cell polarity (PCP) gene, van gogh-like 2 (vangl2). In the present study, in cultured cells, R-Ras was co-immunoprecipitated with Vangl2 but not with another PCP regulator, Pricke1. Interestingly, the interaction between R-Ras and Vangl2 was stronger in guanine-nucleotide free point mutants of R-Ras than in wild-type or constitutively active (GTP-bound) forms of R-Ras. R-Ras may regulate neural tube formation in cooperation with Vangl2 in the developing zebrafish spinal cord. Copyright © 2018 Elsevier Inc. All rights reserved.

Brief embryonic cadmium exposure induces a stress response and cell death in the developing olfactory system followed by long-term olfactory deficits in juvenile zebrafish.

PubMed

Blechinger, Scott R; Kusch, Robin C; Haugo, Kristine; Matz, Carlyn; Chivers, Douglas P; Krone, Patrick H

2007-10-01

The toxic effects of cadmium and other metals have been well established. A primary target of these metals is known to be the olfactory system, and fish exposed to a number of different waterborne metals display deficiencies in olfaction. Importantly, exposure over embryonic/larval development periods can cause deficits in chemosensory function in juvenile fish, but the specific cell types affected are unknown. We have previously characterized a transgenic zebrafish strain expressing the green fluorescent protein (eGFP) gene linked to the hsp70 gene promoter, and shown it to be a useful tool for examining cell-specific toxicity in living embryos and larvae. Here we show that the hsp70/eGFP transgene is strongly and specifically upregulated within the olfactory sensory neurons (OSNs) of transgenic zebrafish larvae following a brief 3-h exposure to water-borne cadmium. This molecular response was closely correlated to an endpoint for tissue damage within the olfactory placode, namely cell death. Furthermore, cadmium-induced olfactory cytotoxicity in zebrafish larvae gives rise to more permanent effects. Juvenile zebrafish briefly exposed to cadmium during early larval development display deficits in olfactory-dependent predator avoidance behaviors 4-6 weeks after a return to clean water. Lateral line neuromasts of exposed zebrafish larvae also activate both the endogenous hsp70 gene and the hsp70/eGFP transgene. The data reveal that even a very brief exposure period that gives rise to cell death within the developing olfactory placode results in long-term deficits in olfaction, and that hsp70/eGFP may serve as an effective indicator of sublethal cadmium exposure in sensory cells.

Brief embryonic cadmium exposure induces a stress response and cell death in the developing olfactory system followed by long-term olfactory deficits in juvenile zebrafish

DOE Office of Scientific and Technical Information (OSTI.GOV)

Blechinger, Scott R.; Toxicology Group, University of Saskatchewan, Saskatoon, Saskatchewan; Kusch, Robin C.

2007-10-01

The toxic effects of cadmium and other metals have been well established. A primary target of these metals is known to be the olfactory system, and fish exposed to a number of different waterborne metals display deficiencies in olfaction. Importantly, exposure over embryonic/larval development periods can cause deficits in chemosensory function in juvenile fish, but the specific cell types affected are unknown. We have previously characterized a transgenic zebrafish strain expressing the green fluorescent protein (eGFP) gene linked to the hsp70 gene promoter, and shown it to be a useful tool for examining cell-specific toxicity in living embryos and larvae.more » Here we show that the hsp70/eGFP transgene is strongly and specifically upregulated within the olfactory sensory neurons (OSNs) of transgenic zebrafish larvae following a brief 3-h exposure to water-borne cadmium. This molecular response was closely correlated to an endpoint for tissue damage within the olfactory placode, namely cell death. Furthermore, cadmium-induced olfactory cytotoxicity in zebrafish larvae gives rise to more permanent effects. Juvenile zebrafish briefly exposed to cadmium during early larval development display deficits in olfactory-dependent predator avoidance behaviors 4-6 weeks after a return to clean water. Lateral line neuromasts of exposed zebrafish larvae also activate both the endogenous hsp70 gene and the hsp70/eGFP transgene. The data reveal that even a very brief exposure period that gives rise to cell death within the developing olfactory placode results in long-term deficits in olfaction, and that hsp70/eGFP may serve as an effective indicator of sublethal cadmium exposure in sensory cells.« less

Development of an automated imaging pipeline for the analysis of the zebrafish larval kidney.

PubMed

Westhoff, Jens H; Giselbrecht, Stefan; Schmidts, Miriam; Schindler, Sebastian; Beales, Philip L; Tönshoff, Burkhard; Liebel, Urban; Gehrig, Jochen

2013-01-01

The analysis of kidney malformation caused by environmental influences during nephrogenesis or by hereditary nephropathies requires animal models allowing the in vivo observation of developmental processes. The zebrafish has emerged as a useful model system for the analysis of vertebrate organ development and function, and it is suitable for the identification of organotoxic or disease-modulating compounds on a larger scale. However, to fully exploit its potential in high content screening applications, dedicated protocols are required allowing the consistent visualization of inner organs such as the embryonic kidney. To this end, we developed a high content screening compatible pipeline for the automated imaging of standardized views of the developing pronephros in zebrafish larvae. Using a custom designed tool, cavities were generated in agarose coated microtiter plates allowing for accurate positioning and orientation of zebrafish larvae. This enabled the subsequent automated acquisition of stable and consistent dorsal views of pronephric kidneys. The established pipeline was applied in a pilot screen for the analysis of the impact of potentially nephrotoxic drugs on zebrafish pronephros development in the Tg(wt1b:EGFP) transgenic line in which the developing pronephros is highlighted by GFP expression. The consistent image data that was acquired allowed for quantification of gross morphological pronephric phenotypes, revealing concentration dependent effects of several compounds on nephrogenesis. In addition, applicability of the imaging pipeline was further confirmed in a morpholino based model for cilia-associated human genetic disorders associated with different intraflagellar transport genes. The developed tools and pipeline can be used to study various aspects in zebrafish kidney research, and can be readily adapted for the analysis of other organ systems.

Developmental exposure to acetaminophen does not induce hyperactivity in zebrafish larvae.

PubMed

Reuter, Isabel; Knaup, Sabine; Romanos, Marcel; Lesch, Klaus-Peter; Drepper, Carsten; Lillesaar, Christina

2016-08-01

First line pain relief medication during pregnancy relies nearly entirely on the over-the-counter analgesic acetaminophen, which is generally considered safe to use during gestation. However, recent epidemiological studies suggest a risk of developing attention-deficit/hyperactivity disorder (ADHD)-like symptoms in children if mothers use acetaminophen during pregnancy. Currently, there are no experimental proofs that prenatal acetaminophen exposure causes developmental brain alterations of progeny. Exposure to high acetaminophen concentrations causes liver toxicity, which is well investigated in different model organisms. However, sub-liver-toxic concentrations have not been experimentally investigated with respect to ADHD endophenotypes such as hyperactivity. We used zebrafish to investigate the potential impact of acetaminophen exposure on locomotor activity levels, and compared it to the established zebrafish Latrophilin 3 (Lphn3) ADHD-model. We determined the sub-liver-toxic concentration of acetaminophen in zebrafish larvae and treated wild-type and lphn3.1 knockdown larvae with increasing concentrations of acetaminophen. We were able to confirm that lphn3.1 knockdown alone causes hyperactivity, strengthening the implication of Lphn3 dysfunction as an ADHD risk factor. Neither acute nor chronic exposure to acetaminophen at sub-liver-toxic concentrations in wild-type or lphn3.1 knock-downs increases locomotor activity levels. Together our findings show that embryonic to larval exposure to acetaminophen does not cause hyperactivity in zebrafish larvae. Furthermore, there are no additive and/or synergistic effects of acetaminophen exposure in a susceptible background induced by knock-down of lphn3.1. Our experimental study suggests that there is, at least in zebrafish larvae, no direct link between embryonic acetaminophen exposure and hyperactivity. Further work is necessary to clarify this issue in humans.

Triphasic low-dose response in zebrafish embryos irradiated by microbeam protons.

PubMed

Choi, Viann Wing Yan; Yum, Emily Hoi Wa; Konishi, Teruaki; Oikawa, Masakazu; Cheng, Shuk Han; Yu, Kwan Ngok

2012-01-01

The microbeam irradiation system (Single-Particle Irradiation System to Cell, acronym as SPICE) at the National Institute of Radiological Sciences (NIRS), Japan, was employed to irradiate dechorionated zebrafish embryos at the 2-cell stage at 0.75 h post fertilization (hpf) by microbeam protons. Either one or both of the cells of the embryos were irradiated with 10, 20, 40, 50, 80, 100, 160, 200, 300 and 2000 protons each with an energy of 3.37 MeV. The embryos were then returned back to the incubator until 24 hpf for analyses. The levels of apoptosis in zebrafish embryos at 25 hpf were quantified through terminal dUTP transferase-mediated nick end-labeling (TUNEL) assay, with the apoptotic signals captured by a confocal microscope. The results revealed a triphasic dose-response for zebrafish embryos with both cells irradiated at the 2-cell stage, namely, (1) increase in apoptotic signals for < 200 protons (< 30 mGy), (2) hormesis to reduce the apoptotic signals below the spontaneous number for 200-400 protons (at doses of 30-60 mGy), and (3) increase in apoptotic signals again for > 600 protons (at doses > 90 mGy). The dose response for zebrafish embryos with only one cell irradiated at the 2-cell stage was also likely a triphasic one, but the apoptotic signals in the first zone (< 200 protons or < 30 mGy) did not have significant differences from those of the background. At the same time, the experimental data were in line with induction of radiation-induced bystander effect as well as rescue effect in the zebrafish embryos, particular in those embryos with unirradiated cells.

ZeBase: an open-source relational database for zebrafish laboratories.

PubMed

Hensley, Monica R; Hassenplug, Eric; McPhail, Rodney; Leung, Yuk Fai

2012-03-01

Abstract ZeBase is an open-source relational database for zebrafish inventory. It is designed for the recording of genetic, breeding, and survival information of fish lines maintained in a single- or multi-laboratory environment. Users can easily access ZeBase through standard web-browsers anywhere on a network. Convenient search and reporting functions are available to facilitate routine inventory work; such functions can also be automated by simple scripting. Optional barcode generation and scanning are also built-in for easy access to the information related to any fish. Further information of the database and an example implementation can be found at http://zebase.bio.purdue.edu.

From fish embryos to human patients: lymphangiogenesis in development and disease.

PubMed

Mauri, Cristina; Wang, Guangxia; Schulte-Merker, Stefan

2018-05-22

The lymphatic vasculature plays vital roles in immune surveillance, fluid homeostasis and fat absorption in the body. Lined by endothelial cells, the lymphatic system is functionally distinct from the blood vasculature, and fulfills different physiological functions. In recent years, insight from zebrafish, mice and human patients have improved our understanding of lymphatics, and the interplay between zebrafish genetics, studies in mice and GWAS analysis in human patients have identified genes that, when mutated, will lead to lymphedema formation. Here, we focus on components of the Vegfr3 pathway, and how they are connected to Milroy disease and Hennekam syndrome. Copyright © 2018 Elsevier Ltd. All rights reserved.

Additive effects of levonorgestrel and ethinylestradiol on brain aromatase (cyp19a1b) in zebrafish specific in vitro and in vivo bioassays

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hinfray, N., E-mail: nathalie.hinfray@ineris.fr

Estrogens and progestins are widely used in combination in human medicine and both are present in aquatic environment. Despite the joint exposure of aquatic wildlife to estrogens and progestins, very little information is available on their combined effects. In the present study we investigated the effect of ethinylestradiol (EE2) and Levonorgestrel (LNG), alone and in mixtures, on the expression of the brain specific ER-regulated cyp19a1b gene. For that purpose, recently established zebrafish-derived tools were used: (i) an in vitro transient reporter gene assay in a human glial cell line (U251-MG) co-transfected with zebrafish estrogen receptors (zfERs) and the luciferase genemore » under the control of the zebrafish cyp19a1b gene promoter and (ii) an in vivo bioassay using a transgenic zebrafish expressing GFP under the control of the zebrafish cyp19a1b gene promoter (cyp19a1b-GFP). Concentration-response relationships for single chemicals were modeled and used to design the mixture experiments following a ray design. The results from mixture experiments were analyzed to predict joint effects according to concentration addition and statistical approaches were used to characterize the potential interactions between the components of the mixtures (synergism/antagonism). We confirmed that some progestins could elicit estrogenic effects in fish brain. In mixtures, EE2 and LNG exerted additive estrogenic effects both in vitro and in vivo, suggesting that some environmental progestin could exert effects that will add to those of environmental (xeno-)estrogens. Moreover, our zebrafish specific assays are valuable tools that could be used in risk assessment for both single chemicals and their mixtures. - Highlights: • Combined effects of EE2 and LNG were assessed on ER-dependent cyp19a1b expression. • EE2 and LNG alone induced brain aromatase in zebrafish specific bioassays. • Experimental ray design allowed complete concentration-response surfaces modeling. • EE2 and LNG exerted additive effects on brain aromatase in radial glial cells.« less

Retinoic Acid Signaling Mediates Hair Cell Regeneration by Repressing p27kip and sox2 in Supporting Cells.

PubMed

Rubbini, Davide; Robert-Moreno, Àlex; Hoijman, Esteban; Alsina, Berta

2015-11-25

During development, otic sensory progenitors give rise to hair cells and supporting cells. In mammalian adults, differentiated and quiescent sensory cells are unable to generate new hair cells when these are lost due to various insults, leading to irreversible hearing loss. Retinoic acid (RA) has strong regenerative capacity in several organs, but its role in hair cell regeneration is unknown. Here, we use genetic and pharmacological inhibition to show that the RA pathway is required for hair cell regeneration in zebrafish. When regeneration is induced by laser ablation in the inner ear or by neomycin treatment in the lateral line, we observe rapid activation of several components of the RA pathway, with dynamics that position RA signaling upstream of other signaling pathways. We demonstrate that blockade of the RA pathway impairs cell proliferation of supporting cells in the inner ear and lateral line. Moreover, in neuromast, RA pathway regulates the transcription of p27(kip) and sox2 in supporting cells but not fgf3. Finally, genetic cell-lineage tracing using Kaede photoconversion demonstrates that de novo hair cells derive from FGF-active supporting cells. Our findings reveal that RA has a pivotal role in zebrafish hair cell regeneration by inducing supporting cell proliferation, and shed light on the underlying transcriptional mechanisms involved. This signaling pathway might be a promising approach for hearing recovery. Hair cells are the specialized mechanosensory cells of the inner ear that capture auditory and balance sensory input. Hair cells die after acoustic trauma, ototoxic drugs or aging diseases, leading to progressive hearing loss. Mammals, in contrast to zebrafish, lack the ability to regenerate hair cells. Here, we find that retinoic acid (RA) pathway is required for hair cell regeneration in vivo in the zebrafish inner ear and lateral line. RA pathway is activated very early upon hair cell loss, promotes cell proliferation of progenitor cells, and regulates two key genes, p27(kip) and sox2. Our results position RA as an essential signal for hair cell regeneration with relevance in future regenerative strategies in mammals. Copyright © 2015 the authors 0270-6474/15/3515752-15$15.00/0.

Establishment of oct4:gfp transgenic zebrafish line for monitoring cellular multipotency by GFP fluorescence.

PubMed

Kato, Hiroyuki; Abe, Kota; Yokota, Shinpei; Matsuno, Rinta; Mikekado, Tsuyoshi; Yokoi, Hayato; Suzuki, Tohru

2015-01-01

The establishment of induced pluripotent stem (iPS) cell technology in fish could facilitate the establishment of novel cryopreservation techniques for storing selected aquaculture strains as frozen cells. In order to apply iPS cell technology to fish, we established a transgenic zebrafish line, Tg(Tru.oct4:EGFP), using green fluorescent protein (GFP) expression under the control of the oct4 gene promoter as a marker to evaluate multipotency in iPS cell preparations. We used the oct4 promoter from fugu (Takifugu rubripes) due to the compact nature of the fugu genome and to facilitate future applications of this technology in marine fishes. During embryogenesis, maternal GFP fluorescence was observed at the cleavage stage and zygotic GFP expression was observed from the start of the shield stage until approximately 24 h after fertilization. gfp messenger RNA (mRNA) was expressed by whole embryonic cells at the shield stage, and then restricted to the caudal neural tube in the latter stages of embryogenesis. These observations showed that GFP fluorescence and the regulation of gfp mRNA expression by the exogenous fugu oct4 promoter are well suited for monitoring endogenous oct4 mRNA expression in embryos. Bisulfite sequencing revealed that the rate of CpG methylation in the transgenic oct4 promoter was high in adult cells (98%) and low in embryonic cells (37%). These findings suggest that, as with the endogenous oct4 promoter, demethylation and methylation both take place normally in the transgenic oct4 promoter during embryogenesis. The embryonic cells harvested at the shield stage formed embryonic body-like cellular aggregates and maintained GFP fluorescence for 6 d when cultured on Transwell-COL Permeable Supports or a feeder layer of adult fin cells. Loss of GFP fluorescence by cultured cells was correlated with cellular differentiation. We consider that the Tg(Tru.oct4:EGFP) zebrafish line established here is well suited for monitoring multipotency in multipotent zebrafish cell cultures and for iPS cell preparation.

Excitation-contraction coupling in zebrafish ventricular myocardium is regulated by trans-sarcolemmal Ca2+ influx and sarcoplasmic reticulum Ca2+ release.

PubMed

Haustein, Moritz; Hannes, Tobias; Trieschmann, Jan; Verhaegh, Rabea; Köster, Annette; Hescheler, Jürgen; Brockmeier, Konrad; Adelmann, Roland; Khalil, Markus

2015-01-01

Zebrafish (Danio rerio) have become a popular model in cardiovascular research mainly due to identification of a large number of mutants with structural defects. In recent years, cardiomyopathies and other diseases influencing contractility of the heart have been studied in zebrafish mutants. However, little is known about the regulation of contractility of the zebrafish heart on a tissue level. The aim of the present study was to elucidate the role of trans-sarcolemmal Ca(2+)-flux and sarcoplasmic reticulum Ca(2+)-release in zebrafish myocardium. Using isometric force measurements of fresh heart slices, we characterised the effects of changes of the extracellular Ca(2+)-concentration, trans-sarcolemmal Ca(2+)-flux via L-type Ca(2+)-channels and Na(+)-Ca(2+)-exchanger, and Ca(2+)-release from the sarcoplasmic reticulum as well as beating frequency and β-adrenergic stimulation on contractility of adult zebrafish myocardium. We found an overall negative force-frequency relationship (FFR). Inhibition of L-type Ca(2+)-channels by verapamil (1 μM) decreased force of contraction to 22 ± 7% compared to baseline (n=4, p<0.05). Ni(2+) was the only substance to prolong relaxation (5 mM, time after peak to 50% relaxation: 73 ± 3 ms vs. 101 ± 8 ms, n=5, p<0.05). Surprisingly though, inhibition of the sarcoplasmic Ca(2+)-release decreased force development to 54 ± 3% in ventricular (n=13, p<0.05) and to 52 ± 8% in atrial myocardium (n=5, p<0.05) suggesting a substantial role of SR Ca(2+)-release in force generation. In line with this finding, we observed significant post pause potentiation after pauses of 5 s (169 ± 7% force compared to baseline, n=8, p<0.05) and 10 s (198 ± 9% force compared to baseline, n=5, p<0.05) and mildly positive lusitropy after β-adrenergic stimulation. In conclusion, force development in adult zebrafish ventricular myocardium requires not only trans-sarcolemmal Ca2+-flux, but also intact sarcoplasmic reticulum Ca(2+)-cycling. In contrast to mammals, FFR is strongly negative in the zebrafish heart. These aspects need to be considered when using zebrafish to model human diseases of myocardial contractility.

Excitation-Contraction Coupling in Zebrafish Ventricular Myocardium Is Regulated by Trans-Sarcolemmal Ca2+ Influx and Sarcoplasmic Reticulum Ca2+ Release

PubMed Central

Trieschmann, Jan; Verhaegh, Rabea; Köster, Annette; Hescheler, Jürgen; Brockmeier, Konrad; Adelmann, Roland; Khalil, Markus

2015-01-01

Zebrafish (Danio rerio) have become a popular model in cardiovascular research mainly due to identification of a large number of mutants with structural defects. In recent years, cardiomyopathies and other diseases influencing contractility of the heart have been studied in zebrafish mutants. However, little is known about the regulation of contractility of the zebrafish heart on a tissue level. The aim of the present study was to elucidate the role of trans-sarcolemmal Ca2+-flux and sarcoplasmic reticulum Ca2+-release in zebrafish myocardium. Using isometric force measurements of fresh heart slices, we characterised the effects of changes of the extracellular Ca2+-concentration, trans-sarcolemmal Ca2+-flux via L-type Ca2+-channels and Na+-Ca2+-exchanger, and Ca2+-release from the sarcoplasmic reticulum as well as beating frequency and β-adrenergic stimulation on contractility of adult zebrafish myocardium. We found an overall negative force-frequency relationship (FFR). Inhibition of L-type Ca2+-channels by verapamil (1 μM) decreased force of contraction to 22±7% compared to baseline (n=4, p<0.05). Ni2+ was the only substance to prolong relaxation (5 mM, time after peak to 50% relaxation: 73±3 ms vs. 101±8 ms, n=5, p<0.05). Surprisingly though, inhibition of the sarcoplasmic Ca2+-release decreased force development to 54±3% in ventricular (n=13, p<0.05) and to 52±8% in atrial myocardium (n=5, p<0.05) suggesting a substantial role of SR Ca2+-release in force generation. In line with this finding, we observed significant post pause potentiation after pauses of 5 s (169±7% force compared to baseline, n=8, p<0.05) and 10 s (198±9% force compared to baseline, n=5, p<0.05) and mildly positive lusitropy after β-adrenergic stimulation. In conclusion, force development in adult zebrafish ventricular myocardium requires not only trans-sarcolemmal Ca2+-flux, but also intact sarcoplasmic reticulum Ca2+-cycling. In contrast to mammals, FFR is strongly negative in the zebrafish heart. These aspects need to be considered when using zebrafish to model human diseases of myocardial contractility. PMID:25938412

Glyphosate and Roundup® alter morphology and behavior in zebrafish.

PubMed

Bridi, Daiane; Altenhofen, Stefani; Gonzalez, Jonas Brum; Reolon, Gustavo Kellermann; Bonan, Carla Denise

2017-12-01

Glyphosate has become the most widely used herbicide in the world, due to the wide scale adoption of transgenic glyphosate resistant crops after its introduction in 1996. Glyphosate may be used alone, but it is commonly applied as an active ingredient of the herbicide Roundup ® . This pesticide contains several adjuvants, which may promote an unknown toxicity. The indiscriminate application poses numerous problems, both for the health of the applicators and consumers, and for the environment, contaminating the soil, water and leading to the death of plants and animals. Zebrafish (Danio rerio) is quickly gaining popularity in behavioral research, because of physiological similarity to mammals, sensitivity to pharmacological factors, robust performance, low cost, short spawning intervals, external fertilization, transparency of embryos through larval stages, and rapid development. The aim of this study was evaluate the effects of glyphosate and Roundup ® on behavioral and morphological parameters in zebrafish larvae and adults. Zebrafish larvae at 3days post-fertilization and adults were exposed to glyphosate (0.01, 0.065, and 0.5mg/L) or Roundup ® (0.01, 0.065, and 0.5mg/L) for 96h. Immediately after the exposure, we performed the analysis of locomotor activity, aversive behavior, and morphology for the larvae and exploratory behavior, aggression and inhibitory avoidance memory for adult zebrafish. In zebrafish larvae, there were significant differences in the locomotor activity and aversive behavior after glyphosate or Roundup ® exposure when compared to the control group. Our findings demonstrated that exposure to glyphosate at the concentration of 0.5mg/L, Roundup ® at 0.065 or 0.5mg/L reduced the distance traveled, the mean speed and the line crossings in adult zebrafish. A decreased ocular distance was observed for larvae exposed at 0.5mg/L of glyphosate. We verified that at 0.5mg/L of Roundup ® -treated adult zebrafish demonstrated a significant impairment in memory. Both glyphosate and Roundup ® reduced aggressive behavior. Our data suggest that there are small differences between the effects induced by glyphosate and Roundup ® , altering morphological and behavioral parameters in zebrafish, suggesting common mechanisms of toxicity and cellular response. Copyright © 2017 Elsevier B.V. All rights reserved.

Immortality of cancers

PubMed Central

Duesberg, Peter; McCormack, Amanda

2013-01-01

Immortality is a common characteristic of cancers, but its origin and purpose are still unclear. Here we advance a karyotypic theory of immortality based on the theory that carcinogenesis is a form of speciation. Accordingly, cancers are generated from normal cells by random karyotypic rearrangements and selection for cancer-specific reproductive autonomy. Since such rearrangements unbalance long-established mitosis genes, cancer karyotypes vary spontaneously but are stabilized perpetually by clonal selections for autonomy. To test this theory we have analyzed neoplastic clones, presumably immortalized by transfection with overexpressed telomerase or with SV40 tumor virus, for the predicted clonal yet flexible karyotypes. The following results were obtained: (1) All immortal tumorigenic lines from cells transfected with overexpressed telomerase had clonal and flexible karyotypes; (2) Searching for the origin of such karyotypes, we found spontaneously increasing, random aneuploidy in human fibroblasts early after transfection with overexpressed telomerase; (3) Late after transfection, new immortal tumorigenic clones with new clonal and flexible karyotypes were found; (4) Testing immortality of one clone during 848 unselected generations showed the chromosome number was stable, but the copy numbers of 36% of chromosomes drifted ± 1; (5) Independent immortal tumorigenic clones with individual, flexible karyotypes arose after individual latencies; (6) Immortal tumorigenic clones with new flexible karyotypes also arose late from cells of a telomerase-deficient mouse rendered aneuploid by SV40 virus. Because immortality and tumorigenicity: (1) correlated exactly with individual clonal but flexible karyotypes; (2) originated simultaneously with such karyotypes; and (3) arose in the absence of telomerase, we conclude that clonal and flexible karyotypes generate the immortality of cancers. PMID:23388461

Stimulus-triggered enhancement of chilling tolerance in zebrafish embryos

PubMed Central

Szabó, Katalin; Budai, Csilla; Losonczi, Eszter; Bernáth, Gergely; Csenki-Bakos, Zsolt; Urbányi, Béla; Pribenszky, Csaba; Horváth, Ákos; Cserepes, Judit

2017-01-01

Background Cryopreservation of zebrafish embryos is still an unsolved problem despite market demand and massive efforts to preserve genetic variation among numerous existing lines. Chilled storage of embryos might be a step towards developing successful cryopreservation, but no methods to date have worked. Methods In the present study, we applied a novel strategy to improve the chilling tolerance of zebrafish embryos by introducing a preconditioning hydrostatic pressure treatment to the embryos. In our experiments, 26-somites and Prim-5 stage zebrafish embryos were chilled at 0°C for 24 hours after preconditioning. Embryo survival rate, ability to reach maturation and fertilizing capacity were tested. Results Our results indicate that applied preconditioning technology made it possible for the chilled embryos to develop normally until maturity, and to produce healthy offspring as normal, thus passing on their genetic material successfully. Treated embryos had a significantly higher survival and better developmental rate, moreover the treated group had a higher ratio of normal morphology during continued development. While all controls from chilled embryos died by 30 day-post-fertilization, the treated group reached maturity (~90–120 days) and were able to reproduce, resulting in offspring in expected quantity and quality. Conclusions Based on our results, we conclude that the preconditioning technology represents a significant improvement in zebrafish embryo chilling tolerance, thus enabling a long-time survival. Furthermore, as embryonic development is arrested during chilled storage this technology also provides a solution to synchronize or delay the development. PMID:28166301

Chlorpyrifos is estrogenic and alters embryonic hatching, cell proliferation and apoptosis in zebrafish.

PubMed

Yu, Kaimin; Li, Guochao; Feng, Weimin; Liu, Lili; Zhang, Jiayu; Wu, Wei; Xu, Lei; Yan, Yanchun

2015-09-05

The potential interference of endocrine disrupting chemicals (EDCs) on aquatic animals and humans has drawn wide attention in recent years. Reports have shown that some organophosphorus pesticides were a kind of EDCs, but their effects on fish species are still under research. In present study, flow cytometry data of HEC-1B cell line showed that chlorpyrifos (CPF) could increase cell proliferation index like 17β-estradiol (E2), but the effect of CPF was weaker than of E2 in the same concentration. Moreover, CPF altered the expression pattern of estrogen-responsive gene VTG and ERα in zebrafish embryos. When exposed to CPF at various concentrations (0, 0.10, 0.25, 0.50, 0.75 and 1.00mg/L) for 48h during the embryo stage, compared with controls, the hatching rate of treated groups significantly increased at the same time and the hatching rate of embryos was proportional to CPF concentration. The mRNA expression levels of c-myc, cyclin D1, Bax and Bcl-2, which are closely related to cell proliferation and cell apoptosis, were disturbed by CPF in zebrafish embryos after exposure treated for 48h. In addition, acridine orange (AO) staining of zebrafish embryos showed that cell apoptosis was appeared in the 0.75, 1.00mg/L CPF treated groups. Taken together, the results obtained in the present study indicated that chlorpyrifos is estrogenic and alters embryonic hatching, cell proliferation and apoptosis in zebrafish. Copyright © 2015. Published by Elsevier Ireland Ltd.

PhOTO Zebrafish: A Transgenic Resource for In Vivo Lineage Tracing during Development and Regeneration

PubMed Central

Dempsey, William P.; Fraser, Scott E.; Pantazis, Periklis

2012-01-01

Background Elucidating the complex cell dynamics (divisions, movement, morphological changes, etc.) underlying embryonic development and adult tissue regeneration requires an efficient means to track cells with high fidelity in space and time. To satisfy this criterion, we developed a transgenic zebrafish line, called PhOTO, that allows photoconvertible optical tracking of nuclear and membrane dynamics in vivo. Methodology PhOTO zebrafish ubiquitously express targeted blue fluorescent protein (FP) Cerulean and photoconvertible FP Dendra2 fusions, allowing for instantaneous, precise targeting and tracking of any number of cells using Dendra2 photoconversion while simultaneously monitoring global cell behavior and morphology. Expression persists through adulthood, making the PhOTO zebrafish an excellent tool for studying tissue regeneration: after tail fin amputation and photoconversion of a ∼100µm stripe along the cut area, marked differences seen in how cells contribute to the new tissue give detailed insight into the dynamic process of regeneration. Photoconverted cells that contributed to the regenerate were separated into three distinct populations corresponding to the extent of cell division 7 days after amputation, and a subset of cells that divided the least were organized into an evenly spaced, linear orientation along the length of the newly regenerating fin. Conclusions/Significance PhOTO zebrafish have wide applicability for lineage tracing at the systems-level in the early embryo as well as in the adult, making them ideal candidate tools for future research in development, traumatic injury and regeneration, cancer progression, and stem cell behavior. PMID:22431986

Gene-expression analysis of hair cell regeneration in the zebrafish lateral line

PubMed Central

Jiang, Linjia; Romero-Carvajal, Andres; Haug, Jeff S.; Seidel, Christopher W.; Piotrowski, Tatjana

2014-01-01

Deafness caused by the terminal loss of inner ear hair cells is one of the most common sensory diseases. However, nonmammalian animals (e.g., birds, amphibians, and fish) regenerate damaged hair cells. To understand better the reasons underpinning such disparities in regeneration among vertebrates, we set out to define at high resolution the changes in gene expression associated with the regeneration of hair cells in the zebrafish lateral line. We performed RNA-Seq analyses on regenerating support cells purified by FACS. The resulting expression data were subjected to pathway enrichment analyses, and the differentially expressed genes were validated in vivo via whole-mount in situ hybridizations. We discovered that cell cycle regulators are expressed hours before the activation of Wnt/β-catenin signaling following hair cell death. We propose that Wnt/β-catenin signaling is not involved in regulating the onset of proliferation but governs proliferation at later stages of regeneration. In addition, and in marked contrast to mammals, our data clearly indicate that the Notch pathway is significantly down-regulated shortly after injury, thus uncovering a key difference between the zebrafish and mammalian responses to hair cell injury. Taken together, our findings lay the foundation for identifying differences in signaling pathway regulation that could be exploited as potential therapeutic targets to promote either sensory epithelium or hair cell regeneration in mammals. PMID:24706903

Natural Bizbenzoquinoline Derivatives Protect Zebrafish Lateral Line Sensory Hair Cells from Aminoglycoside Toxicity.

PubMed

Kruger, Matthew; Boney, Robert; Ordoobadi, Alexander J; Sommers, Thomas F; Trapani, Josef G; Coffin, Allison B

2016-01-01

Moderate to severe hearing loss affects 360 million people worldwide and most often results from damage to sensory hair cells. Hair cell damage can result from aging, genetic mutations, excess noise exposure, and certain medications including aminoglycoside antibiotics. Aminoglycosides are effective at treating infections associated with cystic fibrosis and other life-threatening conditions such as sepsis, but cause hearing loss in 20-30% of patients. It is therefore imperative to develop new therapies to combat hearing loss and allow safe use of these potent antibiotics. We approach this drug discovery question using the larval zebrafish lateral line because zebrafish hair cells are structurally and functionally similar to mammalian inner ear hair cells and respond similarly to toxins. We screened a library of 502 natural compounds in order to identify novel hair cell protectants. Our screen identified four bisbenzylisoquinoline derivatives: berbamine, E6 berbamine, hernandezine, and isotetrandrine, each of which robustly protected hair cells from aminoglycoside-induced damage. Using fluorescence microscopy and electrophysiology, we demonstrated that the natural compounds confer protection by reducing antibiotic uptake into hair cells and showed that hair cells remain functional during and after incubation in E6 berbamine. We also determined that these natural compounds do not reduce antibiotic efficacy. Together, these natural compounds represent a novel source of possible otoprotective drugs that may offer therapeutic options for patients receiving aminoglycoside treatment.

Natural Bizbenzoquinoline Derivatives Protect Zebrafish Lateral Line Sensory Hair Cells from Aminoglycoside Toxicity

PubMed Central

Kruger, Matthew; Boney, Robert; Ordoobadi, Alexander J.; Sommers, Thomas F.; Trapani, Josef G.; Coffin, Allison B.

2016-01-01

Moderate to severe hearing loss affects 360 million people worldwide and most often results from damage to sensory hair cells. Hair cell damage can result from aging, genetic mutations, excess noise exposure, and certain medications including aminoglycoside antibiotics. Aminoglycosides are effective at treating infections associated with cystic fibrosis and other life-threatening conditions such as sepsis, but cause hearing loss in 20–30% of patients. It is therefore imperative to develop new therapies to combat hearing loss and allow safe use of these potent antibiotics. We approach this drug discovery question using the larval zebrafish lateral line because zebrafish hair cells are structurally and functionally similar to mammalian inner ear hair cells and respond similarly to toxins. We screened a library of 502 natural compounds in order to identify novel hair cell protectants. Our screen identified four bisbenzylisoquinoline derivatives: berbamine, E6 berbamine, hernandezine, and isotetrandrine, each of which robustly protected hair cells from aminoglycoside-induced damage. Using fluorescence microscopy and electrophysiology, we demonstrated that the natural compounds confer protection by reducing antibiotic uptake into hair cells and showed that hair cells remain functional during and after incubation in E6 berbamine. We also determined that these natural compounds do not reduce antibiotic efficacy. Together, these natural compounds represent a novel source of possible otoprotective drugs that may offer therapeutic options for patients receiving aminoglycoside treatment. PMID:27065807

HIV integration sites in latently infected cell lines: evidence of ongoing replication.

PubMed

Symons, Jori; Chopra, Abha; Malatinkova, Eva; De Spiegelaere, Ward; Leary, Shay; Cooper, Don; Abana, Chike O; Rhodes, Ajantha; Rezaei, Simin D; Vandekerckhove, Linos; Mallal, Simon; Lewin, Sharon R; Cameron, Paul U

2017-01-13

Assessing the location and frequency of HIV integration sites in latently infected cells can potentially inform our understanding of how HIV persists during combination antiretroviral therapy. We developed a novel high throughput sequencing method to evaluate HIV integration sites in latently infected cell lines to determine whether there was virus replication or clonal expansion in these cell lines observed as multiple integration events at the same position. We modified a previously reported method using random DNA shearing and PCR to allow for high throughput robotic processing to identify the site and frequency of HIV integration in latently infected cell lines. Latently infected cell lines infected with intact virus demonstrated multiple distinct HIV integration sites (28 different sites in U1, 110 in ACH-2 and 117 in J1.1 per 150,000 cells). In contrast, cell lines infected with replication-incompetent viruses (J-Lat cells) demonstrated single integration sites. Following in vitro passaging of the ACH-2 cell line, we observed a significant increase in the frequency of unique HIV integration sites and there were multiple mutations and large deletions in the proviral DNA. When the ACH-2 cell line was cultured with the integrase inhibitor raltegravir, there was a significant decrease in the number of unique HIV integration sites and a transient increase in the frequency of 2-LTR circles consistent with virus replication in these cells. Cell lines latently infected with intact HIV demonstrated multiple unique HIV integration sites indicating that these cell lines are not clonal and in the ACH-2 cell line there was evidence of low level virus replication. These findings have implications for the use of latently infected cell lines as models of HIV latency and for the use of these cells as standards.

Functional and genetic analysis of haplotypic sequence variation at the nicastrin genomic locus

PubMed Central

Hamilton, Gillian; Killick, Richard; Lambert, Jean-Charles; Amouyel, Philippe; Carrasquillo, Minerva M.; Pankratz, V. Shane; Graff-Radford, Neill R.; Dickson, Dennis W.; Petersen, Ronald C.; Younkin, Steven G.; Powell, John F.; Wade-Martins, Richard

2013-01-01

Nicastrin (NCSTN) is a component of the γ-secretase complex and therefore potentially a candidate risk gene for Alzheimer's disease. Here, we have developed a novel functional genomics methodology to express common locus haplotypes to assess functional differences. DNA recombination was used to engineer 5 bacterial artificial chromosomes (BACs) to each express a different haplotype of the NCSTN locus. Each NCSTN-BAC was delivered to knockout nicastrin (Ncstn−/−) cells and clonal NCSTN-BAC+/Ncstn−/− cell lines were created for functional analyses. We showed that all NCSTN-BAC haplotypes expressed nicastrin protein and rescued γ-secretase activity and amyloid beta (Aβ) production in NCSTN-BAC+/Ncstn−/− lines. We then showed that genetic variation at the NCSTN locus affected alternative splicing in human postmortem brain tissue. However, there was no robust functional difference between clonal cell lines rescued by each of the 5 different haplotypes. Finally, there was no statistically significant association of NCSTN with disease risk in the 4 cohorts. We therefore conclude that it is unlikely that common variation at the NCSTN locus is a risk factor for Alzheimer's disease. PMID:22405046

Differences in toxicity of anionic and cationic PAMAM and PPI dendrimers in zebrafish embryos and cancer cell lines.

PubMed

Bodewein, Lambert; Schmelter, Frank; Di Fiore, Stefano; Hollert, Henner; Fischer, Rainer; Fenske, Martina

2016-08-15

Dendrimers are an emerging class of polymeric nanoparticles with beneficial biomedical applications like early diagnostics, in vitro gene transfection or controlled drug delivery. However, the potential toxic impact of exposure on human health or the environment is often inadequately defined. Thus, polyamidoamine (PAMAM) dendrimers of generations G3.0, 3.5, 4.0, 4.5 and 5.0 and polypropylenimine (PPI) dendrimers G3.0, 4.0 and 5.0 were tested in zebrafish embryos for 96h and human cancer cell lines for 24h, to assess and compare developmental in vivo toxicity with cytotoxicity. The zebrafish embryo toxicity of cationic PAMAM and PPI dendrimers increased over time, with EC50 values ranging from 0.16 to just below 1.7μM at 24 and 48hpf. The predominant effects were mortality, plus reduced heartbeat and blood circulation for PPI dendrimers. Apoptosis in the embryos increased in line with the general toxicity concentration-dependently. Hatch and dechorionation of the embryos increased the toxicity, suggesting a protective role of the chorion. Lower generation dendrimers were more toxic in the embryos whereas the toxicity in the HepG2 and DU145 cell lines increased with increasing generation of cationic PAMAMs and PPI dendrimers. HepG2 were less sensitive than DU145 cells, with IC50 values≥402μM (PAMAMs) and ≤240μM (PPIs) for HepG2 and ≤13.24μM (PAMAMs) and ≤12.84μM (PPIs) for DU145. Neither in fish embryos nor cells toxicity thresholds were determinable for anionic PAMAM G3.5 and G4.5. The study demonstrated that the cytotoxicity underestimated the in-vivo toxicity of the dendrimers in the fish embryos. Copyright © 2016 Elsevier Inc. All rights reserved.

Persistence, period and precision of autonomous cellular oscillators from the zebrafish segmentation clock

PubMed Central

Webb, Alexis B; Lengyel, Iván M; Jörg, David J; Valentin, Guillaume; Jülicher, Frank; Morelli, Luis G; Oates, Andrew C

2016-01-01

In vertebrate development, the sequential and rhythmic segmentation of the body axis is regulated by a “segmentation clock”. This clock is comprised of a population of coordinated oscillating cells that together produce rhythmic gene expression patterns in the embryo. Whether individual cells autonomously maintain oscillations, or whether oscillations depend on signals from neighboring cells is unknown. Using a transgenic zebrafish reporter line for the cyclic transcription factor Her1, we recorded single tailbud cells in vitro. We demonstrate that individual cells can behave as autonomous cellular oscillators. We described the observed variability in cell behavior using a theory of generic oscillators with correlated noise. Single cells have longer periods and lower precision than the tissue, highlighting the role of collective processes in the segmentation clock. Our work reveals a population of cells from the zebrafish segmentation clock that behave as self-sustained, autonomous oscillators with distinctive noisy dynamics. DOI: http://dx.doi.org/10.7554/eLife.08438.001 PMID:26880542

Reprogramming the Maternal Zebrafish Genome after Fertilization to Match the Paternal Methylation Pattern

PubMed Central

Potok, Magdalena E.; Nix, David A.; Parnell, Timothy J.; Cairns, Bradley R.

2014-01-01

SUMMARY Early vertebrate embryos must achieve totipotency and prepare for zygotic genome activation (ZGA). To understand this process, we determined the DNA methylation (DNAme) profiles of zebrafish gametes, embryos at different stages, and somatic muscle and compared them to gene activity and histone modifications. Sperm chromatin patterns are virtually identical to those at ZGA. Unexpectedly, the DNA of many oocyte genes important for germ-line functions (i.e., piwil1) or early development (i.e., hox genes) is methylated, but the loci are demethylated during zygotic cleavage stages to precisely the state observed in sperm, even in parthenogenetic embryos lacking a replicating paternal genome. Furthermore, this cohort constitutes the genes and loci that acquire DNAme during development (i.e., ZGA to muscle). Finally, DNA methyltransferase inhibition experiments suggest that DNAme silences particular gene and chromatin cohorts at ZGA, preventing their precocious expression. Thus, zebrafish achieve a totipotent chromatin state at ZGA through paternal genome competency and maternal genome DNAme reprogramming. PMID:23663776

A live zebrafish-based screening system for human nuclear receptor ligand and cofactor discovery.

PubMed

Tiefenbach, Jens; Moll, Pamela R; Nelson, Meryl R; Hu, Chun; Baev, Lilia; Kislinger, Thomas; Krause, Henry M

2010-03-22

Nuclear receptors (NRs) belong to a superfamily of transcription factors that regulate numerous homeostatic, metabolic and reproductive processes. Taken together with their modulation by small lipophilic molecules, they also represent an important and successful class of drug targets. Although many NRs have been targeted successfully, the majority have not, and one third are still orphans. Here we report the development of an in vivo GFP-based reporter system suitable for monitoring NR activities in all cells and tissues using live zebrafish (Danio rerio). The human NR fusion proteins used also contain a new affinity tag cassette allowing the purification of receptors with bound molecules from responsive tissues. We show that these constructs 1) respond as expected to endogenous zebrafish hormones and cofactors, 2) facilitate efficient receptor and cofactor purification, 3) respond robustly to NR hormones and drugs and 4) yield readily quantifiable signals. Transgenic lines representing the majority of human NRs have been established and are available for the investigation of tissue- and isoform-specific ligands and cofactors.

Novel immortal human cell lines reveal subpopulations in the nucleus pulposus

PubMed Central

2014-01-01

Introduction Relatively little is known about cellular subpopulations in the mature nucleus pulposus (NP). Detailed understanding of the ontogenetic, cellular and molecular characteristics of functional intervertebral disc (IVD) cell populations is pivotal to the successful development of cell replacement therapies and IVD regeneration. In this study, we aimed to investigate whether phenotypically distinct clonal cell lines representing different subpopulations in the human NP could be generated using immortalization strategies. Methods Nondegenerate healthy disc material (age range, 8 to 15 years) was obtained as surplus surgical material. Early passage NP monolayer cell cultures were initially characterized using a recently established NP marker set. NP cells were immortalized by simian virus 40 large T antigen (SV40LTag) and human telomerase reverse transcriptase expression. Immortalized cells were clonally expanded and characterized based on collagen type I, collagen type II, α1 (COL2A1), and SRY-box 9 (SOX9) protein expression profiles, as well as on expression of a subset of established in vivo NP cell lineage markers. Results A total of 54 immortal clones were generated. Profiling of a set of novel NP markers (CD24, CA12, PAX1, PTN, FOXF1 and KRT19 mRNA) in a representative set of subclones substantiated successful immortalization of multiple cellular subpopulations from primary isolates and confirmed their NP origin and/or phenotype. We were able to identify two predominant clonal NP subtypes based on their morphological characteristics and their ability to induce SOX9 and COL2A1 under conventional differentiation conditions. In addition, cluster of differentiation 24 (CD24)–negative NP responder clones formed spheroid structures in various culture systems, suggesting the preservation of a more immature phenotype compared to CD24-positive nonresponder clones. Conclusions Here we report the generation of clonal NP cell lines from nondegenerate human IVD tissue and present a detailed characterization of NP cellular subpopulations. Differential cell surface marker expression and divergent responses to differentiation conditions suggest that the NP subtypes may correspond to distinct maturation stages and represent distinct NP cell subpopulations. Hence, we provide evidence that the immortalization strategy that we applied is capable of detecting cell heterogeneity in the NP. Our cell lines yield novel insights into NP biology and provide promising new tools for studies of IVD development, cell function and disease. PMID:24972717

Novel immortal human cell lines reveal subpopulations in the nucleus pulposus.

PubMed

van den Akker, Guus G H; Surtel, Don A M; Cremers, Andy; Rodrigues-Pinto, Ricardo; Richardson, Stephen M; Hoyland, Judith A; van Rhijn, Lodewijk W; Welting, Tim J M; Voncken, Jan Willem

2014-06-27

Relatively little is known about cellular subpopulations in the mature nucleus pulposus (NP). Detailed understanding of the ontogenetic, cellular and molecular characteristics of functional intervertebral disc (IVD) cell populations is pivotal to the successful development of cell replacement therapies and IVD regeneration. In this study, we aimed to investigate whether phenotypically distinct clonal cell lines representing different subpopulations in the human NP could be generated using immortalization strategies. Nondegenerate healthy disc material (age range, 8 to 15 years) was obtained as surplus surgical material. Early passage NP monolayer cell cultures were initially characterized using a recently established NP marker set. NP cells were immortalized by simian virus 40 large T antigen (SV40LTag) and human telomerase reverse transcriptase expression. Immortalized cells were clonally expanded and characterized based on collagen type I, collagen type II, α1 (COL2A1), and SRY-box 9 (SOX9) protein expression profiles, as well as on expression of a subset of established in vivo NP cell lineage markers. A total of 54 immortal clones were generated. Profiling of a set of novel NP markers (CD24, CA12, PAX1, PTN, FOXF1 and KRT19 mRNA) in a representative set of subclones substantiated successful immortalization of multiple cellular subpopulations from primary isolates and confirmed their NP origin and/or phenotype. We were able to identify two predominant clonal NP subtypes based on their morphological characteristics and their ability to induce SOX9 and COL2A1 under conventional differentiation conditions. In addition, cluster of differentiation 24 (CD24)-negative NP responder clones formed spheroid structures in various culture systems, suggesting the preservation of a more immature phenotype compared to CD24-positive nonresponder clones. Here we report the generation of clonal NP cell lines from nondegenerate human IVD tissue and present a detailed characterization of NP cellular subpopulations. Differential cell surface marker expression and divergent responses to differentiation conditions suggest that the NP subtypes may correspond to distinct maturation stages and represent distinct NP cell subpopulations. Hence, we provide evidence that the immortalization strategy that we applied is capable of detecting cell heterogeneity in the NP. Our cell lines yield novel insights into NP biology and provide promising new tools for studies of IVD development, cell function and disease.

Establishing Clonal Cell Lines with Endothelial-Like Potential from CD9hi, SSEA-1− Cells in Embryonic Stem Cell-Derived Embryoid Bodies

PubMed Central

Lian, Qizhou; Yeo, KengSuan; Que, Jianwen; Tan, EileenKhiaWay; Yu, Fenggang; Yin, Yijun; Salto-Tellez, Manuel; Oakley, Reida Menshawe El; Lim, Sai-Kiang

2006-01-01

Background Differentiation of embryonic stem cells (ESCs) into specific cell types with minimal risk of teratoma formation could be efficiently directed by first reducing the differentiation potential of ESCs through the generation of clonal, self-renewing lineage-restricted stem cell lines. Efforts to isolate these stem cells are, however, mired in an impasse where the lack of purified lineage-restricted stem cells has hindered the identification of defining markers for these rare stem cells and, in turn, their isolation. Methodology/Principal Findings We describe here a method for the isolation of clonal lineage-restricted cell lines with endothelial potential from ESCs through a combination of empirical and rational evidence-based methods. Using an empirical protocol that we have previously developed to generate embryo-derived RoSH lines with endothelial potential, we first generated E-RoSH lines from mouse ESC-derived embryoid bodies (EBs). Despite originating from different mouse strains, RoSH and E- RoSH lines have similar gene expression profiles (r2 = 0.93) while that between E-RoSH and ESCs was 0.83. In silico gene expression analysis predicted that like RoSH cells, E-RoSH cells have an increased propensity to differentiate into vasculature. Unlike their parental ESCs, E-RoSH cells did not form teratomas and differentiate efficiently into endothelial-like cells in vivo and in vitro. Gene expression and FACS analysis revealed that RoSH and E-RoSH cells are CD9hi, SSEA-1− while ESCs are CD9lo, SSEA-1+. Isolation of CD9hi, SSEA-1− cells that constituted 1%–10% of EB-derived cultures generated an E-RoSH-like culture with an identical E-RoSH-like gene expression profile (r2 = 0.95) and a propensity to differentiate into endothelial-like cells. Conclusions By combining empirical and rational evidence-based methods, we identified definitive selectable surface antigens for the isolation and propagation of lineage-restricted stem cells with endothelial-like potential from mouse ESCs. PMID:17183690

Developmental stage-specific regulation of the circadian clock by temperature in zebrafish.

PubMed

Lahiri, Kajori; Froehlich, Nadine; Heyd, Andreas; Foulkes, Nicholas S; Vallone, Daniela

2014-01-01

The circadian clock enables animals to adapt their physiology and behaviour in anticipation of the day-night cycle. Light and temperature represent two key environmental timing cues (zeitgebers) able to reset this mechanism and so maintain its synchronization with the environmental cycle. One key challenge is to unravel how the regulation of the clock by zeitgebers matures during early development. The zebrafish is an ideal model for studying circadian clock ontogeny since the process of development occurs ex utero in an optically transparent chorion and many tools are available for genetic analysis. However, the role played by temperature in regulating the clock during zebrafish development is poorly understood. Here, we have established a clock-regulated luciferase reporter transgenic zebrafish line (Tg (-3.1) per1b::luc) to study the effects of temperature on clock entrainment. We reveal that under complete darkness, from an early developmental stage onwards (48 to 72 hpf), exposure to temperature cycles is a prerequisite for the establishment of self-sustaining rhythms of zfper1b, zfaanat2, and zfirbp expression and also for circadian cell cycle rhythms. Furthermore, we show that following the 5-9 somite stage, the expression of zfper1b is regulated by acute temperature shifts.

A gene trap transposon eliminates haematopoietic expression of zebrafish Gfi1aa, but does not interfere with haematopoiesis.

PubMed

Thambyrajah, Roshana; Ucanok, Deniz; Jalali, Maryam; Hough, Yasmin; Wilkinson, Robert Neil; McMahon, Kathryn; Moore, Chris; Gering, Martin

2016-09-01

A transposon-mediated gene trap screen identified the zebrafish line qmc551 that expresses a GFP reporter in primitive erythrocytes and also in haemogenic endothelial cells, which give rise to haematopoietic stem and progenitor cells (HSPCs) that seed sites of larval and adult haematopoiesis. The transposon that mediates this GFP expression is located in intron 1 of the gfi1aa gene, one of three zebrafish paralogs that encode transcriptional repressors homologous to mammalian Gfi1 and Gfi1b proteins. In qmc551 transgenics, GFP expression is under the control of the endogenous gfi1aa promoter, recapitulates early gfi1aa expression and allows live observation of gfi1aa promoter activity. While the transposon integration interferes with the expression of gfi1aa mRNA in haematopoietic cells, homozygous qmc551 fish are viable and fertile, and display normal primitive and definitive haematopoiesis. Retained expression of Gfi1b in primitive erythrocytes and up-regulation of Gfi1ab at the onset of definitive haematopoiesis in homozygous qmc551 carriers, are sufficient to allow normal haematopoiesis. This finding contradicts previously published morpholino data that suggested an essential role for zebrafish Gfi1aa in primitive erythropoiesis. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Genetically Blocking the Zebrafish Pineal Clock Affects Circadian Behavior.

PubMed

Ben-Moshe Livne, Zohar; Alon, Shahar; Vallone, Daniela; Bayleyen, Yared; Tovin, Adi; Shainer, Inbal; Nisembaum, Laura G; Aviram, Idit; Smadja-Storz, Sima; Fuentes, Michael; Falcón, Jack; Eisenberg, Eli; Klein, David C; Burgess, Harold A; Foulkes, Nicholas S; Gothilf, Yoav

2016-11-01

The master circadian clock in fish has been considered to reside in the pineal gland. This dogma is challenged, however, by the finding that most zebrafish tissues contain molecular clocks that are directly reset by light. To further examine the role of the pineal gland oscillator in the zebrafish circadian system, we generated a transgenic line in which the molecular clock is selectively blocked in the melatonin-producing cells of the pineal gland by a dominant-negative strategy. As a result, clock-controlled rhythms of melatonin production in the adult pineal gland were disrupted. Moreover, transcriptome analysis revealed that the circadian expression pattern of the majority of clock-controlled genes in the adult pineal gland is abolished. Importantly, circadian rhythms of behavior in zebrafish larvae were affected: rhythms of place preference under constant darkness were eliminated, and rhythms of locomotor activity under constant dark and constant dim light conditions were markedly attenuated. On the other hand, global peripheral molecular oscillators, as measured in whole larvae, were unaffected in this model. In conclusion, characterization of this novel transgenic model provides evidence that the molecular clock in the melatonin-producing cells of the pineal gland plays a key role, possibly as part of a multiple pacemaker system, in modulating circadian rhythms of behavior.

Cell migration during heart regeneration in zebrafish.

PubMed

Tahara, Naoyuki; Brush, Michael; Kawakami, Yasuhiko

2016-07-01

Zebrafish possess the remarkable ability to regenerate injured hearts as adults, which contrasts the very limited ability in mammals. Although very limited, mammalian hearts do in fact have measurable levels of cardiomyocyte regeneration. Therefore, elucidating mechanisms of zebrafish heart regeneration would provide information of naturally occurring regeneration to potentially apply to mammalian studies, in addition to addressing this biologically interesting phenomenon in itself. Studies over the past 13 years have identified processes and mechanisms of heart regeneration in zebrafish. After heart injury, pre-existing cardiomyocytes dedifferentiate, enter the cell cycle, and repair the injured myocardium. This process requires interaction with epicardial cells, endocardial cells, and vascular endothelial cells. Epicardial cells envelope the heart, while endocardial cells make up the inner lining of the heart. They provide paracrine signals to cardiomyocytes to regenerate the injured myocardium, which is vascularized during heart regeneration. In addition, accumulating results suggest that local migration of these major cardiac cell types have roles in heart regeneration. In this review, we summarize the characteristics of various heart injury methods used in the research community and regeneration of the major cardiac cell types. Then, we discuss local migration of these cardiac cell types and immune cells during heart regeneration. Developmental Dynamics 245:774-787, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Genetically Blocking the Zebrafish Pineal Clock Affects Circadian Behavior

PubMed Central

Alon, Shahar; Vallone, Daniela; Tovin, Adi; Shainer, Inbal; Nisembaum, Laura G.; Aviram, Idit; Smadja-Storz, Sima; Fuentes, Michael; Falcón, Jack; Eisenberg, Eli; Klein, David C.; Burgess, Harold A.; Foulkes, Nicholas S.; Gothilf, Yoav

2016-01-01

The master circadian clock in fish has been considered to reside in the pineal gland. This dogma is challenged, however, by the finding that most zebrafish tissues contain molecular clocks that are directly reset by light. To further examine the role of the pineal gland oscillator in the zebrafish circadian system, we generated a transgenic line in which the molecular clock is selectively blocked in the melatonin-producing cells of the pineal gland by a dominant-negative strategy. As a result, clock-controlled rhythms of melatonin production in the adult pineal gland were disrupted. Moreover, transcriptome analysis revealed that the circadian expression pattern of the majority of clock-controlled genes in the adult pineal gland is abolished. Importantly, circadian rhythms of behavior in zebrafish larvae were affected: rhythms of place preference under constant darkness were eliminated, and rhythms of locomotor activity under constant dark and constant dim light conditions were markedly attenuated. On the other hand, global peripheral molecular oscillators, as measured in whole larvae, were unaffected in this model. In conclusion, characterization of this novel transgenic model provides evidence that the molecular clock in the melatonin-producing cells of the pineal gland plays a key role, possibly as part of a multiple pacemaker system, in modulating circadian rhythms of behavior. PMID:27870848

Protection of silver nanoparticles using Eysenhardtia polystachya in peroxide-induced pancreatic β-Cell damage and their antidiabetic properties in zebrafish.

PubMed

Garcia Campoy, Abraham Heriberto; Perez Gutierrez, Rosa Martha; Manriquez-Alvirde, Gabriela; Muñiz Ramirez, Alethia

2018-01-01

The aim was to explore the efficacy of extract of Eysenhardtia polystachya -loaded silver nanoparticles (EP/AgNPs) on pancreatic β cells, INS-1 cells, and zebrafish as a valuable model for the study of diabetes mellitus. EP/AgNPs were synthesized using methanol/water bark extract of E. polystachya and characterized using various physicochemical techniques. Immersion of adult zebrafish in 111 mM glucose solution resulted in a sustained hyperglycemic, hyperlipidemic state, and serum insulin levels decreased. The synthesized EP/AgNPs showed an absorption peak at 413 nm on ultraviolet-visible spectroscopy, revealing the surface plasmon resonance of the nanoparticles. Transmission electron microscopy indicated that most of the particles were spherical, with a diameter of 10-12 nm, a polydispersity index of 0.197, and a zeta potential of -32.25 mV, suggesting high stability of the nanoparticles. EP/AgNPs promote pancreatic β-cell survival, insulin secretion, enhanced hyperglycemia, and hyperlipidemia in glucose-induced diabetic zebrafish. EP/AgNPs also showed protection of the pancreatic β-cell line INS-1 against hydrogen peroxide-induced oxidative injury. The results indicate that EP/AgNPs have good antidiabetic activity and therefore could be used to prevent the development of diabetes.

Fishing on chips: up-and-coming technological advances in analysis of zebrafish and Xenopus embryos.

PubMed

Zhu, Feng; Skommer, Joanna; Huang, Yushi; Akagi, Jin; Adams, Dany; Levin, Michael; Hall, Chris J; Crosier, Philip S; Wlodkowic, Donald

2014-11-01

Biotests performed on small vertebrate model organisms provide significant investigative advantages as compared with bioassays that employ cell lines, isolated primary cells, or tissue samples. The main advantage offered by whole-organism approaches is that the effects under study occur in the context of intact physiological milieu, with all its intercellular and multisystem interactions. The gap between the high-throughput cell-based in vitro assays and low-throughput, disproportionally expensive and ethically controversial mammal in vivo tests can be closed by small model organisms such as zebrafish or Xenopus. The optical transparency of their tissues, the ease of genetic manipulation and straightforward husbandry, explain the growing popularity of these model organisms. Nevertheless, despite the potential for miniaturization, automation and subsequent increase in throughput of experimental setups, the manipulation, dispensing and analysis of living fish and frog embryos remain labor-intensive. Recently, a new generation of miniaturized chip-based devices have been developed for zebrafish and Xenopus embryo on-chip culture and experimentation. In this work, we review the critical developments in the field of Lab-on-a-Chip devices designed to alleviate the limits of traditional platforms for studies on zebrafish and clawed frog embryo and larvae. © 2014 International Society for Advancement of Cytometry. © 2014 International Society for Advancement of Cytometry.

CNS angiogenesis and barriergenesis occur simultaneously.

PubMed

Umans, Robyn A; Henson, Hannah E; Mu, Fangzhou; Parupalli, Chaithanyarani; Ju, Bensheng; Peters, Jennifer L; Lanham, Kevin A; Plavicki, Jessica S; Taylor, Michael R

2017-05-15

The blood-brain barrier (BBB) plays a vital role in the central nervous system (CNS). A comprehensive understanding of BBB development has been hampered by difficulties in observing the differentiation of brain endothelial cells (BECs) in real-time. Here, we generated two transgenic zebrafish line, Tg(glut1b:mCherry) and Tg(plvap:EGFP), to serve as in vivo reporters of BBB development. We showed that barriergenesis (i.e. the induction of BEC differentiation) occurs immediately as endothelial tips cells migrate into the brain parenchyma. Using the Tg(glut1b:mCherry) transgenic line, we performed a genetic screen and identified a zebrafish mutant with a nonsense mutation in gpr124, a gene known to play a role in CNS angiogenesis and BBB development. We also showed that our transgenic plvap:EGFP line, a reporter of immature brain endothelium, is initially expressed in newly formed brain endothelial cells, but subsides during BBB maturation. Our results demonstrate the ability to visualize the in vivo differentiation of brain endothelial cells into the BBB phenotype and establish that CNS angiogenesis and barriergenesis occur simultaneously. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Infection of zebrafish embryos with live fluorescent Streptococcus pneumoniae as a real-time pneumococcal meningitis model.

PubMed

Jim, Kin Ki; Engelen-Lee, JooYeon; van der Sar, Astrid M; Bitter, Wilbert; Brouwer, Matthijs C; van der Ende, Arie; Veening, Jan-Willem; van de Beek, Diederik; Vandenbroucke-Grauls, Christina M J E

2016-08-19

Streptococcus pneumoniae is one of the most important causes of bacterial meningitis, an infection where unfavourable outcome is driven by bacterial and host-derived toxins. In this study, we developed and characterized a pneumococcal meningitis model in zebrafish embryos that allows for real-time investigation of early host-microbe interaction. Zebrafish embryos were infected in the caudal vein or hindbrain ventricle with green fluorescent wild-type S. pneumoniae D39 or a pneumolysin-deficient mutant. The kdrl:mCherry transgenic zebrafish line was used to visualize the blood vessels, whereas phagocytic cells were visualized by staining with far red anti-L-plastin or in mpx:GFP/mpeg1:mCherry zebrafish, that have green fluorescent neutrophils and red fluorescent macrophages. Imaging was performed by fluorescence confocal and time-lapse microscopy. After infection by caudal vein, we saw focal clogging of the pneumococci in the blood vessels and migration of bacteria through the blood-brain barrier into the subarachnoid space and brain tissue. Infection with pneumolysin-deficient S. pneumoniae in the hindbrain ventricle showed attenuated growth and migration through the brain as compared to the wild-type strain. Time-lapse and confocal imaging revealed that the initial innate immune response to S. pneumoniae in the subarachnoid space mainly consisted of neutrophils and that pneumolysin-mediated cytolytic activity caused a marked reduction of phagocytes. This new meningitis model permits detailed analysis and visualization of host-microbe interaction in pneumococcal meningitis in real time and is a very promising tool to further our insights in the pathogenesis of pneumococcal meningitis.

Simple, economical heat-shock devices for zebrafish housing racks.

PubMed

Duszynski, Robert J; Topczewski, Jacek; LeClair, Elizabeth E

2011-12-01

One reason for the popularity of the zebrafish (Danio rerio) as a model vertebrate is the ability to manipulate gene expression in this organism. A common method is to induce gene expression transiently under control of a heat-shock promoter (e.g., hsp70l). By making simple mechanical adjustments to small aquarium heaters (25-50W), we were able to produce consistent and reliable heat-shock conditions within a conventional zebrafish housing system. Up to two heat-shock intervals per day (>37°C) could be maintained under conditions of continuous flow (5-25 mL/min). Temperature logging every 30 s indicated rapid warm up times, consistent heat-shock lengths, and accurate and precise peak water temperatures (mean±SD=38°C±0.2°C). The biological effects of these heat-shock treatments were confirmed by observing inducible expression of enhanced green fluorescent protein (EGFP) and inhibition of caudal fin regeneration in a transgenic fish line expressing a dominant negative fibroblast growth factor receptor (Tg(hsp70l:dnfgfr1-EGFP)(pd1)). These devices are inexpensive, easily modified, and can be calibrated to accommodate a variety of experimental designs. After setup on a programmable timer, the heaters require no intervention to produce consistent daily heat shocks, and all other standard care protocols can be followed in the fish facility. The simplicity and stability of these devices make them suitable for long-term heat shocks at any stage of the zebrafish lifecycle (>7 days postfertilization), and useful for both laboratory and classroom experiments on transgenic zebrafish.

Streptococcus dysgalactiae subsp. dysgalactiae isolated from milk of the bovine udder as emerging pathogens: In vitro and in vivo infection of human cells and zebrafish as biological models.

PubMed

Alves-Barroco, Cinthia; Roma-Rodrigues, Catarina; Raposo, Luís R; Brás, Catarina; Diniz, Mário; Caço, João; Costa, Pedro M; Santos-Sanches, Ilda; Fernandes, Alexandra R

2018-03-25

Streptococcus dysgalactiae subsp. dysgalactiae (SDSD) is a major cause of bovine mastitis and has been regarded as an animal-restricted pathogen, although rare infections have been described in humans. Previous studies revealed the presence of virulence genes encoded by phages of the human pathogen Group A Streptococcus pyogenes (GAS) in SDSD isolated from the milk of bovine udder with mastitis. The isolates SDSD VSD5 and VSD13 could adhere and internalize human primary keratinocyte cells, suggesting a possible human infection potential of bovine isolates. In this work, the in vitro and in vivo potential of SDSD to internalize/adhere human cells of the respiratory track and zebrafish as biological models was evaluated. Our results showed that, in vitro, bovine SDSD strains could interact and internalize human respiratory cell lines and that this internalization was dependent on an active transport mechanism and that, in vivo, SDSD are able to cause invasive infections producing zebrafish morbidity and mortality. The infectious potential of these isolates showed to be isolate-specific and appeared to be independent of the presence or absence of GAS phage-encoded virulence genes. Although the infection ability of the bovine SDSD strains was not as strong as the human pathogenic S. pyogenes in the zebrafish model, results suggested that these SDSD isolates are able to interact with human cells and infect zebrafish, a vertebrate infectious model, emerging as pathogens with zoonotic capability. © 2018 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

Hypoxic Signaling and the Cellular Redox Tumor Environment Determine Sensitivity to MTH1 Inhibition.

PubMed

Bräutigam, Lars; Pudelko, Linda; Jemth, Ann-Sofie; Gad, Helge; Narwal, Mohit; Gustafsson, Robert; Karsten, Stella; Carreras Puigvert, Jordi; Homan, Evert; Berndt, Carsten; Berglund, Ulrika Warpman; Stenmark, Pål; Helleday, Thomas

2016-04-15

Cancer cells are commonly in a state of redox imbalance that drives their growth and survival. To compensate for oxidative stress induced by the tumor redox environment, cancer cells upregulate specific nononcogenic addiction enzymes, such as MTH1 (NUDT1), which detoxifies oxidized nucleotides. Here, we show that increasing oxidative stress in nonmalignant cells induced their sensitization to the effects of MTH1 inhibition, whereas decreasing oxidative pressure in cancer cells protected against inhibition. Furthermore, we purified zebrafish MTH1 and solved the crystal structure of MTH1 bound to its inhibitor, highlighting the zebrafish as a relevant tool to study MTH1 biology. Delivery of 8-oxo-dGTP and 2-OH-dATP to zebrafish embryos was highly toxic in the absence of MTH1 activity. Moreover, chemically or genetically mimicking activated hypoxia signaling in zebrafish revealed that pathologic upregulation of the HIF1α response, often observed in cancer and linked to poor prognosis, sensitized embryos to MTH1 inhibition. Using a transgenic zebrafish line, in which the cellular redox status can be monitored in vivo, we detected an increase in oxidative pressure upon activation of hypoxic signaling. Pretreatment with the antioxidant N-acetyl-L-cysteine protected embryos with activated hypoxia signaling against MTH1 inhibition, suggesting that the aberrant redox environment likely causes sensitization. In summary, MTH1 inhibition may offer a general approach to treat cancers characterized by deregulated hypoxia signaling or redox imbalance. Cancer Res; 76(8); 2366-75. ©2016 AACR. ©2016 American Association for Cancer Research.

Preleukaemic clonal haemopoiesis and risk of therapy-related myeloid neoplasms: a case-control study.

PubMed

Takahashi, Koichi; Wang, Feng; Kantarjian, Hagop; Doss, Denaha; Khanna, Kanhav; Thompson, Erika; Zhao, Li; Patel, Keyur; Neelapu, Sattva; Gumbs, Curtis; Bueso-Ramos, Carlos; DiNardo, Courtney D; Colla, Simona; Ravandi, Farhad; Zhang, Jianhua; Huang, Xuelin; Wu, Xifeng; Samaniego, Felipe; Garcia-Manero, Guillermo; Futreal, P Andrew

2017-01-01

Therapy-related myeloid neoplasms are secondary malignancies that are often fatal, but their risk factors are not well understood. Evidence suggests that individuals with clonal haemopoiesis have increased risk of developing haematological malignancies. We aimed to identify whether patients with cancer who have clonal haemopoiesis are at an increased risk of developing therapy-related myeloid neoplasms. We did this retrospective case-control study to compare the prevalence of clonal haemopoiesis between patients treated for cancer who later developed therapy-related myeloid neoplasms (cases) and patients who did not develop these neoplasms (controls). All patients in both case and control groups were treated at MD Anderson Cancer Center (Houston, TX, USA) from 1997 to 2015. We used the institutional medical database to locate these patients. Patients were included as cases if they were treated for a primary cancer, subsequently developed therapy-related myeloid neoplasms, and had available paired samples of bone marrow from the time of therapy-related myeloid neoplasm diagnosis and peripheral blood from the time of primary cancer diagnosis. Patients were eligible for inclusion as age-matched controls if they were treated for lymphoma, received combination chemotherapy, and did not develop therapy-related myeloid neoplasms after at least 5 years of follow-up. We used molecular barcode sequencing of 32 genes on the pretreatment peripheral blood samples to detect clonal haemopoiesis. For cases, we also used targeted gene sequencing on bone marrow samples and investigated clonal evolution from clonal haemopoiesis to the development of therapy-related myeloid neoplasms. To further clarify the association between clonal haemopoiesis and therapy-related myeloid neoplasm development, we also analysed the prevalence of clonal haemopoiesis in an external cohort of patients with lymphoma who were treated in a randomised trial of front-line chemotherapy with cyclophosphamide, doxorubicin, vincristine, and prednisone, with or without melatonin. This trial was done at MD Anderson Cancer Center between 1999 and 2001 (protocol number 98-009). We identified 14 cases and 54 controls. Of the 14 cases, we detected clonal haemopoiesis in the peripheral blood samples of ten (71%) patients. We detected clonal haemopoiesis in 17 (31%) of the 54 controls. The cumulative incidence of therapy-related myeloid neoplasms in both cases and controls at 5 years was significantly higher in patients with clonal haemopoiesis (30%, 95% CI 16-51) than in those without (7%, 2-21; p=0·016). In the external cohort, five (7%) of 74 patients developed therapy-related myeloid neoplasms, of whom four (80%) had clonal haemopoiesis; 11 (16%) of 69 patients who did not develop therapy-related myeloid neoplasms had clonal haemopoiesis. In the external cohort, the cumulative incidence of therapy-related myeloid neoplasms at 10 years was significantly higher in patients with clonal haemopoiesis (29%, 95% CI 8-53) than in those without (0%, 0-0; p=0·0009). In a multivariate Fine and Gray model based on the external cohort, the presence of clonal haemopoiesis significantly increased the risk of therapy-related myeloid neoplasm development (hazard ratio 13·7, 95% CI 1·7-108·7; p=0·013). Preleukaemic clonal haemopoiesis is common in patients with therapy-related myeloid neoplasms at the time of their primary cancer diagnosis and before they have been exposed to treatment. Our results suggest that clonal haemopoiesis could be used as a predictive marker to identify patients with cancer who are at risk of developing therapy-related myeloid neoplasms. A prospective trial to validate this concept is warranted. Cancer Prevention Research Institute of Texas, Red and Charline McCombs Institute for the Early Detection and Treatment of Cancer, NIH through MD Anderson Cancer Center Support Grant, and the MD Anderson MDS & AML Moon Shots Program. Copyright © 2017 Elsevier Ltd. All rights reserved.

Telomeric fusion and chromosome instability in multiple tissues of a patient with mosaic Ullrich-Turner syndrome

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sawyer, J.R.; North, P.E.; Hassed, S.J.

1997-04-14

We describe the cytogenetic evolution of multiple cell lines in the gonadal tissue of a 10-year-old girl with mosaic Ullrich-Turner syndrome (UTS) involving clonal telomeric associations (tas) of the Y chromosome. G-band analysis of all tissues showed at least 2 cell lines; 45,X and 46,X,tas(Y;21)(q12;p13). However, analysis of left gonadal tissue of this patient showed the evolution of 2 additional cell lines, one designated 45,X,tas(Y;21)(q12;p13),-22 and the other 46,X,tas(Y;21)(q12;p13),+tas(Y;14)(q12;p13),-22. Fluorescence in situ hybridization (FISH) analysis of interphase nuclei from uncultured gonadal tissue confirmed the findings of aneuploidy in the left gonadal tissue and extended the findings of aneuploidy to themore » tissue of the right gonad. The chromosome findings in the gonadal tissue of this patient suggest a preneoplastic karyotype relating to several distinct tumor associations. The clonal evolution of telomeric fusions indicates chromosome instability and suggests the extra copy of the Y chromosome may have resulted from a fusion-related malsegregation. In addition, the extra Y suggests low-level amplification of a putative gonadoblastoma gene, while the loss of chromosome 22 suggests the loss of heterozygosity for genes on chromosome 22. This case demonstrates the utility of the study of gonadal tissue in 45X46,XY UTS patients, and provides evidence that clonal telomeric fusions may, in rare cases, be associated with chromosomal malsegregation and with the subsequent evolution of unstable karyotypes. 27 refs., 3 figs.« less

Establishment and characterization of scleroderma fibroblast clonal cell lines by introduction of the hTERT gene

PubMed Central

Kapanadze, Bagrat; Morris, Erin; Smith, Edwin; Trojanowska, Maria

2010-01-01

Abstract Lack of an adequate experimental model has hindered the ability to fully understand scleroderma (SSc) pathogenesis. Current SSc research is based on the study of cultured fibroblasts from skin biopsies. In depth characterization of the SSc fibroblast phenotype is hindered by the limited lifespan and heterogeneity of these cells. The goal of this study was to isolate high collagen-producing fibroblasts from SSc biopsies and extend their lifespan with hTERT immortalization to enable characterization of their phenotype. Fibroblasts from two pairs of closely matched normal and SSc biopsies were infected with an hTERT lentivirus. Infected colonies were isolated, cultured into clonal cell lines and analysed with respect to profibrotic gene expression. The mRNA levels of nine profibrotic genes were measured by quantitative real-time PCR. Protein levels were assessed by Western blot. The hTERT SSc clones were heterogeneous with regards to expression of the profibrotic genes measured. A subset of the SSc clones showed elevated expression levels of collagen I, connective tissue growth factor and thrombospondin 1 mRNA, while expression of other genes was not significantly changed. Elevated expression of collagen I protein and mRNA was correlative with elevated expression of connective tissue growth factor. Several hTERT clones expressed high levels of pSmad1, Smad1 and TGF-βRI indicative of altered TGF-β signalling. A portion of SSc clones expressed several profibrotic genes. This study demonstrates that select characteristics of the SSc phenotype are expressed in a subset of activated fibroblasts in culture. The clonal SSc cell lines may present a new and useful model to investigate the mechanisms involved in SSc fibrosis. PMID:19432820

Establishment and characterization of scleroderma fibroblast clonal cell lines by introduction of the hTERT gene.

PubMed

Kapanadze, Bagrat; Morris, Erin; Smith, Edwin; Trojanowska, Maria

2010-05-01

Lack of an adequate experimental model has hindered the ability to fully understand scleroderma (SSc) pathogenesis. Current SSc research is based on the study of cultured fibroblasts from skin biopsies. In depth characterization of the SSc fibroblast phenotype is hindered by the limited lifespan and heterogeneity of these cells. The goal of this study was to isolate high collagen-producing fibroblasts from SSc biopsies and extend their lifespan with hTERT immortalization to enable characterization of their phenotype. Fibroblasts from two pairs of closely matched normal and SSc biopsies were infected with an hTERT lentivirus. Infected colonies were isolated, cultured into clonal cell lines and analysed with respect to profibrotic gene expression. The mRNA levels of nine profibrotic genes were measured by quantitative real-time PCR. Protein levels were assessed by Western blot. The hTERT SSc clones were heterogeneous with regards to expression of the profibrotic genes measured. A subset of the SSc clones showed elevated expression levels of collagen I, connective tissue growth factor and thrombospondin 1 mRNA, while expression of other genes was not significantly changed. Elevated expression of collagen I protein and mRNA was correlative with elevated expression of connective tissue growth factor. Several hTERT clones expressed high levels of pSmad1, Smad1 and TGF-betaRI indicative of altered TGF-beta signalling. A portion of SSc clones expressed several profibrotic genes. This study demonstrates that select characteristics of the SSc phenotype are expressed in a subset of activated fibroblasts in culture. The clonal SSc cell lines may present a new and useful model to investigate the mechanisms involved in SSc fibrosis.

Selectivity of natural, synthetic and environmental estrogens for zebrafish estrogen receptors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pinto, Caroline; Grimaldi, Marina; Boulahtouf, Abdelhay

2014-10-01

Zebrafish, Danio rerio, is increasingly used as an animal model to study the effects of pharmaceuticals and environmental estrogens. As most of these estrogens have only been tested on human estrogen receptors (ERs), it is necessary to measure their effects on zebrafish ERs. In humans there are two distinct nuclear ERs (hERα and hERβ), whereas the zebrafish genome encodes three ERs, zfERα and two zfERβs (zfERβ1 and zfERβ2). In this study, we established HeLa-based reporter cell lines stably expressing each of the three zfERs. We first reported that estrogens more efficiently activate the zfERs at 28 °C as compared tomore » 37 °C, thus reflecting the physiological temperature of zebrafish in wildlife. We then showed significant differences in the ability of agonist and antagonist estrogens to modulate activation of the three zfER isotypes in comparison to hERs. Environmental compounds (bisphenol A, alkylphenols, mycoestrogens) which are hER panagonists and hERβ selective agonists displayed greater potency for zfERα as compared to zfERβs. Among hERα selective synthetic agonists, PPT did not activate zfERα while 16α-LE2 was the most zfERα selective compound. Altogether, these results confirm that all hER ligands control in a similar manner the transcriptional activity of zfERs although significant differences in selectivity were observed among subtypes. The zfER subtype selective ligands that we identified thus represent new valuable tools to dissect the physiological roles of the different zfERs. Finally, our work also points out that care has to be taken in transposing the results obtained using the zebrafish as a model for human physiopathology. - Highlights: • Zebrafish is increasingly used to study the effects of estrogens. • We assessed the activity of pharmaceutical and environmental estrogens on zfERs. • Environmental estrogens displayed greater potency for zfERα compared to zfERβs. • hERβ selective agonists displayed greater potency for zfERα compared to zfERβs. • The hERα selective agonist 16αL-E2 is the most zfERα selective compound.« less

Inducible Sterilization of Zebrafish by Disruption of Primordial Germ Cell Migration

PubMed Central

Wong, Ten-Tsao; Collodi, Paul

2013-01-01

During zebrafish development, a gradient of stromal-derived factor 1a (Sdf1a) provides the directional cue that guides the migration of the primordial germ cells (PGCs) to the gonadal tissue. Here we describe a method to produce large numbers of infertile fish by inducing ubiquitous expression of Sdf1a in zebrafish embryos resulting in disruption of the normal PGC migration pattern. A transgenic line of zebrafish, Tg(hsp70:sdf1a-nanos3, EGFP), was generated that expresses Sdf1a under the control of the heat-shock protein 70 (hsp70) promoter and nanos3 3?UTR. To better visualize the PGCs, the Tg(hsp70:sdf1a-nanos3, EGFP) fish were crossed with another transgenic line, Tg(kop:DsRed-nanos3), that expresses DsRed driven by the PGC-specific kop promoter. Heat treatment of the transgenic embryos caused an induction of Sdf1a expression throughout the embryo resulting in the disruption of their normal migration. Optimal embryo survival and disruption of PGC migration was achieved when transgenic embryos at the 4- to 8-cell stage were incubated at 34.5°C for 18 hours. Under these conditions, disruption of PGC migration was observed in 100% of the embryos. Sixty-four adult fish were developed from three separate batches of heat-treated embryos and all were found to be infertile males. When each male was paired with a wild-type female, only unfertilized eggs were produced and histological examination revealed that each of the adult male fish possessed severely under-developed gonads that lacked gametes. The results demonstrate that inducible Sdf1a expression is an efficient and reliable strategy to produce infertile fish. This approach makes it convenient to generate large numbers of infertile adult fish while also providing the capability to maintain a fertile brood stock. PMID:23826390

Cytoskeleton disorder and cell cycle arrest may be associated with the alteration of protein CEP135 by microgravity

NASA Astrophysics Data System (ADS)

Hang, Xiaoming; Sun, Yeqing; Wu, Di; Li, Yixiao; Liu, Zhiyuan

In the past decades, alterations in the morphology, cytoskeleton and cell cycle have been observed in cells in vitro under microgravity conditions. But the underlying mechanisms are not absolutely identified yet. Our previous study on proteomic and microRNA expression profiles of zebrafish embryos exposed to simulated-microgravity has demonstrated a serial of microgravity-sensitive molecules. Centrosomal protein of 135 kDa (CEP135) was found down-regulated, but the mRNA expression level of it was up-regulated in zebrafish embryos after simulated-microgravity. However, the functional study on CEP135 is very limited and it has not been cloned in zebrafish till now. In this study, we try to determine whether the cytoskeleton disorder and cell cycle arrest is associated with the alteration of CEP135 by microgravity. Full-length cDNA of cep135 gene was firstly cloned from mitosis phase of ZF4. The sequence was analyzed and the phylogenetic tree was constructed based on the similarity to other species. Zebrafish embryonic cell line ZF4 were exposed to simulated microgravity for 24 and 48 hours, using a rotary cell culture system (RCCS) designed by NASA. Quantitative analysis by western blot showed that CEP135 expression level was significantly decreased two times after 24 hour simulated microgravity. Cell cycle detection by flow cytometer indicated ZF4 cells were blocked in G1 phase after 24 and 48 hour simulated microgravity. Moreover, double immunostained ZF4 cells with anti-tubulin and anti-CEP135antibodies demonstrated simulated microgravity could lead to cytoskeleton disorder and CEP135 abnormality. Further investigations are currently being carried out to determine whether knockdown and over-expression of CEP135 will modulate cytoskeleton and cell cycle. In vitro data in combination within vivo results might, at least in part, explain the dramatic effects of microgravity. Key Words: microgravity; CEP135; Cytoskeleton disorder; G1 arrest; ZF4 cell line

DOE Office of Scientific and Technical Information (OSTI.GOV)

Leite, Carlos Eduardo, E-mail: carlos.leite@pucrs.br; Programa de Pós-Graduação em Medicina: Ciências Médicas, Universidade Federal do Rio Grande do Sul, Porto Alegre, CEP 90035-003; Maboni, Lucas de Oliveira

The use of zebrafish (Danio rerio) is increasing as an intermediate preclinical model, to prioritize drug candidates for mammalian testing. As the immune system of the zebrafish is quite similar to that of mammals, models of inflammation are being developed for the screening of new drugs. The characterization of these models is crucial for studies that seek for mechanisms of action and specific pharmacological targets. It is well known that copper is a metal that induces damage and cell migration to hair cells of lateral line of zebrafish. Extracellular nucleotides/nucleosides, as ATP and adenosine (ADO), act as endogenous signaling moleculesmore » during tissue damage by exerting effects on inflammatory and immune responses. The present study aimed to characterize the inflammatory status, and to investigate the involvement of the purinergic system in copper-induced inflammation in zebrafish larvae. Fishes of 7 days post-fertilization were exposed to 10 μM of copper for a period of 24 h. The grade of oxidative stress, inflammatory status, copper uptake, the activity and the gene expression of the enzymes responsible for controlling the levels of nucleotides and adenosine were evaluated. Due to the copper accumulation in zebrafish larvae tissues, the damage and oxidative stress were exacerbated over time, resulting in an inflammatory process involving IL-1β, TNF-α, COX-2 and PGE{sub 2}. Within the purinergic system, the mechanisms that control the ADO levels were the most involved, mainly the reactions performed by the isoenzyme ADA 2. In conclusion, our data shed new lights on the mechanisms related to copper-induced inflammation in zebrafish larvae. - Graphical abstract: This scheme provides a chronological proposition for the biochemical events induced by copper in zebrafish larvae. The dashed line shows the absorption of copper over the exposure time. After 1 h of exposure to copper, the release of PGE{sub 2} occurs, followed by an increase of MPO (as a consequence of neutrophil migration), increased expression of genes involved in inflammatory events (IL-1β and TNF-α) and, reduction of the anti-inflammatory cytokine IL-10 at 4 h. At 24 h, the copper concentration is found highly increased, what is coincident with oxidative stress. Regarding the purinergic system, it is possible to observe an inhibition of ecto-5′-NT and ADA, with the consequent increase of AMP and ADA, respectively, at 24 h. The expression of enzyme-related genes shows a decrease in the expression of ecto-5′-NT and variable expressions of ADA subfamily enzymes. - Highlights: • Copper led to increased oxidative stress, and decreased the antioxidants' defenses. • Copper induced time-related changes of IL-1β, TNF-α, IL-10 and PGE{sub 2} levels. • ADA activity controls the levels of adenosine in copper-induced inflammation. • ADA 2 is the main ADA subfamily involved. • The purinergic system seems to be involved in the resolution of inflammation.« less

Mechanism of Action of Presynaptic Neurotoxins

DTIC Science & Technology

1986-07-01

Green, 1981;. (2) There are no consistent effeects of the toxin on neurotransmftter synthesis , storage, degradation, or uptake (Collingrldge et al...Transport by Nitrendipine fit a Clonal Cell Line. ýL Biol. Chem,. 257, 13189-13192. Van Heyningen W.E. (1963) The Fixation of Tetanus Toxin, Strychnine

Endocrine-Therapy-Resistant ESR1 Variants Revealed by Genomic Characterization of Breast-Cancer-Derived Xenografts

PubMed Central

Li, Shunqiang; Shen, Dong; Shao, Jieya; Crowder, Robert; Liu, Wenbin; Prat, Aleix; He, Xiaping; Liu, Shuying; Hoog, Jeremy; Lu, Charles; Ding, Li; Griffith, Obi L.; Miller, Christopher; Larson, Dave; Fulton, Robert S.; Harrison, Michelle; Mooney, Tom; McMichael, Joshua F.; Luo, Jingqin; Tao, Yu; Goncalves, Rodrigo; Schlosberg, Christopher; Hiken, Jeffrey F.; Saied, Laila; Sanchez, Cesar; Giuntoli, Therese; Bumb, Caroline; Cooper, Crystal; Kitchens, Robert T.; Lin, Austin; Phommaly, Chanpheng; Davies, Sherri R.; Zhang, Jin; Kavuri, Megha Shyam; McEachern, Donna; Dong, Yi Yu; Ma, Cynthia; Pluard, Timothy; Naughton, Michael; Bose, Ron; Suresh, Rama; McDowell, Reida; Michel, Loren; Aft, Rebecca; Gillanders, William; DeSchryver, Katherine; Wilson, Richard K.; Wang, Shaomeng; Mills, Gordon B.; Gonzalez-Angulo, Ana; Edwards, John R.; Maher, Christopher; Perou, Charles M.; Mardis, Elaine R.; Ellis, Matthew J.

2013-01-01

SUMMARY To characterize patient-derived xenografts (PDXs) for functional studies, we made whole-genome comparisons with originating breast cancers representative of the major intrinsic subtypes. Structural and copy number aberrations were found to be retained with high fidelity. However, at the single-nucleotide level, variable numbers of PDX-specific somatic events were documented, although they were only rarely functionally significant. Variant allele frequencies were often preserved in the PDXs, demonstrating that clonal representation can be transplantable. Estrogen-receptor-positive PDXs were associated with ESR1 ligand-binding-domain mutations, gene amplification, or an ESR1/YAP1 translocation. These events produced different endocrine-therapy-response phenotypes in human, cell line, and PDX endocrine-response studies. Hence, deeply sequenced PDX models are an important resource for the search for genome-forward treatment options and capture endocrine-drug-resistance etiologies that are not observed in standard cell lines. The originating tumor genome provides a benchmark for assessing genetic drift and clonal representation after transplantation. PMID:24055055

Mixed epithelial and stromal tumor of the middle ear: The first case report.

PubMed

Michal, Michael; Skálová, Alena; Kazakov, Dmitry V; Pecková, Květoslava; Heidenreich, Filip; Grossmann, Petr; Michal, Michal

2017-03-01

We report a tumor arising in the middle ear of a 65-year-old female patient that was composed of an ovarian-type stroma (OS) and an epithelial component. The tumor consisted of irregular, polypoid masses containing multiple variably sized cystic spaces, which were invariably surrounded by the OS. The cystic spaces were lined by flat, cuboidal, or columnar epithelial cells, in most parts showing mucinous differentiation. The epithelial lining of the cysts strongly expressed cytokeratins AE1-3, CK7, CK8, CK18, CK19, EMA, and S100 protein. The stroma expressed CD34 and smooth muscle actin. No cytological atypia or mitoses were present, and the proliferative activity was less than 1% in both components. The clonality analysis proved the clonal nature of the neoplasm. We believe that this tumor is a new member in the family of neoplasms containing the OS, and therefore we propose the term mixed epithelial and stromal tumor of the middle ear. Copyright © 2017 Elsevier Inc. All rights reserved.

Towards an alternative for the acute fish LC(50) test in chemical assessment: the fish embryo toxicity test goes multi-species -- an update.

PubMed

Braunbeck, Thomas; Boettcher, Melanie; Hollert, Henner; Kosmehl, Thomas; Lammer, Eva; Leist, Erik; Rudolf, Mark; Seitz, Nadja

2005-01-01

After its standardisation at the national level in Germany (DIN 38415-6, 2001, 2001), the 48 h sewage testing assay with zebrafish (Danio rerio) embryos has been submitted for standardisation to ISO. As an alternative to the conventional acute (96 h) fish test, a modified fish embryo test will be submitted to the OECD for chemical testing in late 2005. For this, a protocol originally designed for zebrafish was adapted to fit also the requirements of other OECD species, namely medaka (Oryzias latipes) and fathead minnow (Pimephales promelas). Results document that the transfer of the protocol is possible with only minor modifications. Data obtained from embryo tests with the three species are comparable. Statistical analysis of existing zebrafish embryo toxicity data resulted in the conclusions (1) that there is a reliable correlation between the fish embryo test and the acute fish test, (2) that the confidence belt of the regression line was relatively small, but that the prediction range was relatively wide. The regression thus seems appropriate to describe the relationship between acute fish and embryo LC(50) with good confidence, but is less appropriate as a prediction model. Investigations into oxygen requirements of zebrafish embryos reveal that they adapt to a broad range of oxygen levels and survive at concentrations of 2 mg/l without malformations. Zebrafish embryos can thus be exposed in very small toxicant volumes (100 microl), which is of particular interest for the testing of metabolites. Dechorionation studies with 48 h old zebrafish embryos indicate that the barrier function of the chorion increases with the lipophilicity of the test compound. Finally, examples are given as to how additional endpoints can be incorporated into the fish embryo test protocol to extend its scope, e.g. to sediment toxicity assessment or genotoxicity and mutagenicity testing.

An integrated approach with the zebrafish model for biomonitoring of municipal wastewater effluent and receiving waters.

PubMed

Li, Caixia; Chen, Qiyu; Zhang, Xiaoyan; Snyder, Shane A; Gong, Zhiyuan; Lam, Siew Hong

2017-12-11

Comprehensive monitoring of water pollution is challenging. With the increasing amount and types of anthropogenic compounds being released into water, there are rising concerns of undetected toxicity. This is especially true for municipal wastewater effluents that are discharged to surface waters. This study was designed to integrate zebrafish toxicogenomics, targeted gene expression, and morphological analyses, for toxicity evaluation of effluent discharged from two previously characterized wastewater treatment plants (WWTPs) in Pima County, Arizona, and their receiving surface water. Zebrafish embryos were exposed to organic extracts from the WWTP1 effluent that were reconstituted to represent 1× and 0.5× of the original concentration. Microarray analyses identified deregulated gene probes that mapped to 1666, 779, and 631 unique human homologs in the 1×, 0.5×, and the intersection of both groups, respectively. These were associated with 18 cellular and molecular functions ranging from cell cycle to metabolism and are involved in the development and function of 10 organ systems including nervous, cardiovascular, haematological, reproductive, and hepatic systems. Superpathway of cholesterol biosynthesis, retinoic acid receptor activation, glucocorticoid receptor and prolactin signaling were among the top 11 perturbed canonical pathways. Real-time quantitative PCR validated the expression changes of 12 selected genes. These genes were then tested on zebrafish embryos exposed to the reconstituted extract of water sampled downstream of WWTP1 and another nearby WWTP2. The expression of several targeted genes were significantly affected by the WWTP effluents and some of the downstream receiving waters. Morphological analyses using four transgenic zebrafish lines revealed potential toxicity associated with nervous, hepatic, endothelial-vascular and myeloid systems. This study demonstrated how information can be obtained using adverse outcome pathway framework to derive biological effect-based monitoring tools. This integrated approach using zebrafish can supplement analytical chemistry to provide more comprehensive monitoring of discharged effluents and their receiving waters. Copyright © 2017 Elsevier Ltd. All rights reserved.

Label retention identifies a multipotent mesenchymal stem cell-like population in the postnatal thymus.

PubMed

Osada, Masako; Singh, Varan J; Wu, Kenmin; Sant'Angelo, Derek B; Pezzano, Mark

2013-01-01

Thymic microenvironments are essential for the proper development and selection of T cells critical for a functional and self-tolerant adaptive immune response. While significant turnover occurs, it is unclear whether populations of adult stem cells contribute to the maintenance of postnatal thymic epithelial microenvironments. Here, the slow cycling characteristic of stem cells and their property of label-retention were used to identify a K5-expressing thymic stromal cell population capable of generating clonal cell lines that retain the capacity to differentiate into a number of mesenchymal lineages including adipocytes, chondrocytes and osteoblasts suggesting a mesenchymal stem cell-like phenotype. Using cell surface analysis both culture expanded LRCs and clonal thymic mesenchymal cell lines were found to express Sca1, PDGFRα, PDGFRβ,CD29, CD44, CD49F, and CD90 similar to MSCs. Sorted GFP-expressing stroma, that give rise to TMSC lines, contribute to thymic architecture when reaggregated with fetal stroma and transplanted under the kidney capsule of nude mice. Together these results show that the postnatal thymus contains a population of mesenchymal stem cells that can be maintained in culture and suggests they may contribute to the maintenance of functional thymic microenvironments.

Multiplexing clonality: combining RGB marking and genetic barcoding

PubMed Central

Cornils, Kerstin; Thielecke, Lars; Hüser, Svenja; Forgber, Michael; Thomaschewski, Michael; Kleist, Nadja; Hussein, Kais; Riecken, Kristoffer; Volz, Tassilo; Gerdes, Sebastian; Glauche, Ingmar; Dahl, Andreas; Dandri, Maura; Roeder, Ingo; Fehse, Boris

2014-01-01

RGB marking and DNA barcoding are two cutting-edge technologies in the field of clonal cell marking. To combine the virtues of both approaches, we equipped LeGO vectors encoding red, green or blue fluorescent proteins with complex DNA barcodes carrying color-specific signatures. For these vectors, we generated highly complex plasmid libraries that were used for the production of barcoded lentiviral vector particles. In proof-of-principle experiments, we used barcoded vectors for RGB marking of cell lines and primary murine hepatocytes. We applied single-cell polymerase chain reaction to decipher barcode signatures of individual RGB-marked cells expressing defined color hues. This enabled us to prove clonal identity of cells with one and the same RGB color. Also, we made use of barcoded vectors to investigate clonal development of leukemia induced by ectopic oncogene expression in murine hematopoietic cells. In conclusion, by combining RGB marking and DNA barcoding, we have established a novel technique for the unambiguous genetic marking of individual cells in the context of normal regeneration as well as malignant outgrowth. Moreover, the introduction of color-specific signatures in barcodes will facilitate studies on the impact of different variables (e.g. vector type, transgenes, culture conditions) in the context of competitive repopulation studies. PMID:24476916

Effect of HSP27 on Human Breast Tumor Cell Growth and Motility

DTIC Science & Technology

1999-08-01

the small stress protein, HSP27 , on growth and motility characteristics of normal and tumor-derived human mammary cell lines. We hypothesized that...cells overexpressing HSP27 would show increased motility, altered chemotactic properties, increased resistance to heat killing and to certain drugs...Donna has prepared and studied 19 clonal MDA23 1 breast tumor cell lines that overexpress human HSP27 , and determined that, while heat resistance is

Regeneration of multiple shoots from transgenic potato events facilitates the recovery of phenotypically normal lines: assessing a cry9Aa2 gene conferring insect resistance

PubMed Central

2011-01-01

Background The recovery of high performing transgenic lines in clonal crops is limited by the occurrence of somaclonal variation during the tissue culture phase of transformation. This is usually circumvented by developing large populations of transgenic lines, each derived from the first shoot to regenerate from each transformation event. This study investigates a new strategy of assessing multiple shoots independently regenerated from different transformed cell colonies of potato (Solanum tuberosum L.). Results A modified cry9Aa2 gene, under the transcriptional control of the CaMV 35S promoter, was transformed into four potato cultivars using Agrobacterium-mediated gene transfer using a nptII gene conferring kanamycin resistance as a selectable marker gene. Following gene transfer, 291 transgenic lines were grown in greenhouse experiments to assess somaclonal variation and resistance to potato tuber moth (PTM), Phthorimaea operculella (Zeller). Independently regenerated lines were recovered from many transformed cell colonies and Southern analysis confirmed whether they were derived from the same transformed cell. Multiple lines regenerated from the same transformed cell exhibited a similar response to PTM, but frequently exhibited a markedly different spectrum of somaclonal variation. Conclusions A new strategy for the genetic improvement of clonal crops involves the regeneration and evaluation of multiple shoots from each transformation event to facilitate the recovery of phenotypically normal transgenic lines. Most importantly, regenerated lines exhibiting the phenotypic appearance most similar to the parental cultivar are not necessarily derived from the first shoot regenerated from a transformed cell colony, but can frequently be a later regeneration event. PMID:21995716

Dimethyl sulfoxide (DMSO) exacerbates cisplatin-induced sensory hair cell death in zebrafish (Danio rerio).

PubMed

Uribe, Phillip M; Mueller, Melissa A; Gleichman, Julia S; Kramer, Matthew D; Wang, Qi; Sibrian-Vazquez, Martha; Strongin, Robert M; Steyger, Peter S; Cotanche, Douglas A; Matsui, Jonathan I

2013-01-01

Inner ear sensory hair cells die following exposure to aminoglycoside antibiotics or chemotherapeutics like cisplatin, leading to permanent auditory and/or balance deficits in humans. Zebrafish (Danio rerio) are used to study drug-induced sensory hair cell death since their hair cells are similar in structure and function to those found in humans. We developed a cisplatin dose-response curve using a transgenic line of zebrafish that expresses membrane-targeted green fluorescent protein under the control of the Brn3c promoter/enhancer. Recently, several small molecule screens have been conducted using zebrafish to identify potential pharmacological agents that could be used to protect sensory hair cells in the presence of ototoxic drugs. Dimethyl sulfoxide (DMSO) is typically used as a solvent for many pharmacological agents in sensory hair cell cytotoxicity assays. Serendipitously, we found that DMSO potentiated the effects of cisplatin and killed more sensory hair cells than treatment with cisplatin alone. Yet, DMSO alone did not kill hair cells. We did not observe the synergistic effects of DMSO with the ototoxic aminoglycoside antibiotic neomycin. Cisplatin treatment with other commonly used organic solvents (i.e. ethanol, methanol, and polyethylene glycol 400) also did not result in increased cell death compared to cisplatin treatment alone. Thus, caution should be exercised when interpreting data generated from small molecule screens since many compounds are dissolved in DMSO.

Dimethyl Sulfoxide (DMSO) Exacerbates Cisplatin-induced Sensory Hair Cell Death in Zebrafish (Danio rerio)

PubMed Central

Gleichman, Julia S.; Kramer, Matthew D.; Wang, Qi; Sibrian-Vazquez, Martha; Strongin, Robert M.; Steyger, Peter S.; Cotanche, Douglas A.; Matsui, Jonathan I.

2013-01-01

Inner ear sensory hair cells die following exposure to aminoglycoside antibiotics or chemotherapeutics like cisplatin, leading to permanent auditory and/or balance deficits in humans. Zebrafish (Danio rerio) are used to study drug-induced sensory hair cell death since their hair cells are similar in structure and function to those found in humans. We developed a cisplatin dose-response curve using a transgenic line of zebrafish that expresses membrane-targeted green fluorescent protein under the control of the Brn3c promoter/enhancer. Recently, several small molecule screens have been conducted using zebrafish to identify potential pharmacological agents that could be used to protect sensory hair cells in the presence of ototoxic drugs. Dimethyl sulfoxide (DMSO) is typically used as a solvent for many pharmacological agents in sensory hair cell cytotoxicity assays. Serendipitously, we found that DMSO potentiated the effects of cisplatin and killed more sensory hair cells than treatment with cisplatin alone. Yet, DMSO alone did not kill hair cells. We did not observe the synergistic effects of DMSO with the ototoxic aminoglycoside antibiotic neomycin. Cisplatin treatment with other commonly used organic solvents (i.e. ethanol, methanol, and polyethylene glycol 400) also did not result in increased cell death compared to cisplatin treatment alone. Thus, caution should be exercised when interpreting data generated from small molecule screens since many compounds are dissolved in DMSO. PMID:23383324

Ectopic expression of cone-specific G-protein-coupled receptor kinase GRK7 in zebrafish rods leads to lower photosensitivity and altered responses

PubMed Central

Vogalis, F; Shiraki, T; Kojima, D; Wada, Y; Nishiwaki, Y; Jarvinen, J L P; Sugiyama, J; Kawakami, K; Masai, I; Kawamura, S; Fukada, Y; Lamb, T D

2011-01-01

Abstract To investigate the roles of G-protein receptor kinases (GRKs) in the light responses of vertebrate photoreceptors, we generated transgenic zebrafish lines, the rods of which express either cone GRK (GRK7) or rod GRK (GRK1) in addition to the endogenous GRK1, and we then measured the electrophysiological characteristics of single-cell responses and the behavioural responses of intact animals. Our study establishes the zebrafish expression system as a convenient platform for the investigation of specific components of the phototransduction cascade. The addition of GRK1 led to minor changes in rod responses. However, exogenous GRK7 in GRK7-tg animals led to lowered rod sensitivity, as occurs in cones, but surprisingly to slower response kinetics. Examination of responses to long series of very dim flashes suggested the possibility that the GRK7-tg rods generated two classes of single-photon response, perhaps corresponding to the interaction of activated rhodopsin with GRK1 (giving a standard response) or with GRK7 (giving a very small response). Behavioural measurement of optokinetic responses (OKR) in intact GRK7-tg zebrafish larvae showed that the overall rod visual pathway was less sensitive, in accord with the lowered sensitivity of the rods. These results help provide an understanding for the molecular basis of the electrophysiological differences between cones and rods. PMID:21486791

Silica nanoparticles inhibit macrophage activity and angiogenesis via VEGFR2-mediated MAPK signaling pathway in zebrafish embryos.

PubMed

Duan, Junchao; Hu, Hejing; Feng, Lin; Yang, Xiaozhe; Sun, Zhiwei

2017-09-01

The safety evaluation of silica nanoparticles (SiNPs) are getting great attention due to its widely-used in food sciences, chemical industry and biomedicine. However, the adverse effect and underlying mechanisms of SiNPs on cardiovascular system, especially on angiogenesis is still unclear. This study was aimed to illuminate the possible mechanisms of SiNPs on angiogenesis in zebrafish transgenic lines, Tg(fli-1:EGFP) and Albino. SiNPs caused the cardiovascular malformations in a dose-dependent manner via intravenous microinjection. The incidences of cardiovascular malformations were observed as: Pericardial edema > Bradycardia > Blood deficiency. The area of subintestinal vessels (SIVs) was significant reduced in SiNPs-treated groups, accompanied with the weaken expression of vascular endothelial cells in zebrafish embryos. Using neutral red staining, the quantitative number of macrophage was declined; whereas macrophage inhibition rate was elevated in a dose-dependent way. Furthermore, SiNPs significantly decreased the mRNA expression of macrophage activity related gene, macrophage migration inhibitory factor (MIF) and the angiogenesis related gene, vascular endothelial growth factor receptor 2 (VEGFR2). The protein levels of p-Erk1/2 and p-p38 MAPK were markedly decreased in zebrafish exposed to SiNPs. Our results implicate that SiNPs inhibited the macrophage activity and angiogenesis via the downregulation of MAPK singaling pathway. Copyright © 2017 Elsevier Ltd. All rights reserved.

Reduced synaptic density and deficient locomotor response in neuronal activity-regulated pentraxin 2a mutant zebrafish.

PubMed

Elbaz, Idan; Lerer-Goldshtein, Tali; Okamoto, Hitoshi; Appelbaum, Lior

2015-04-01

Neuronal-activity-regulated pentraxin (NARP/NPTX2/NP2) is a secreted synaptic protein that regulates the trafficking of glutamate receptors and mediates learning, memory, and drug addiction. The role of NPTX2 in regulating structural synaptic plasticity and behavior in a developing vertebrate is indefinite. We characterized the expression of nptx2a in larvae and adult zebrafish and established a transcription activator-like effector nuclease (TALEN)-mediated nptx2a mutant (nptx2a(-/-)) to study the role of Nptx2a in regulating structural synaptic plasticity and behavior. Similar to mammals, the zebrafish nptx2a was expressed in excitatory neurons in the brain and spinal cord. Its expression was induced in response to a mechanosensory stimulus but did not change during day and night. Behavioral assays showed that loss of Nptx2a results in reduced locomotor response to light-to-dark transition states and to a sound stimulus. Live imaging of synapses using the transgenic nptx2a:GAL4VP16 zebrafish and a fluorescent presynaptic synaptophysin (SYP) marker revealed reduced synaptic density in the axons of the spinal motor neurons and the anterodorsal lateral-line ganglion (gAD), which regulate locomotor activity and locomotor response to mechanosensory stimuli, respectively. These results suggest that Nptx2a affects locomotor response to external stimuli by mediating structural synaptic plasticity in excitatory neuronal circuits. © FASEB.

Sustainable, Rapid Synthesis of Bright-Luminescent CuInS2-ZnS Alloyed Nanocrystals: Multistage Nano-xenotoxicity Assessment and Intravital Fluorescence Bioimaging in Zebrafish-Embryos

NASA Astrophysics Data System (ADS)

Chetty, S. Shashank; Praneetha, S.; Basu, Sandeep; Sachidanandan, Chetana; Murugan, A. Vadivel

2016-05-01

Near-infrared (NIR) luminescent CuInS2-ZnS alloyed nanocrystals (CIZS-NCs) for highly fluorescence bioimaging have received considerable interest in recent years. Owing, they became a desirable alternative to heavy-metal based-NCs and organic dyes with unique optical properties and low-toxicity for bioimaging and optoelectronic applications. In the present study, bright and robust CIZS-NCs have been synthesized within 5 min, as-high-as 230 °C without requiring any inert-gas atmosphere via microwave-solvothermal (MW-ST) method. Subsequently, the in vitro and in vivo nano-xenotoxicity and cellular uptake of the MUA-functionalized CIZS-NCs were investigated in L929, Vero, MCF7 cell lines and zebrafish-embryos. We observed minimal toxicity and acute teratogenic consequences upto 62.5 μg/ml of the CIZS-NCs in zebrafish-embryos. We also observed spontaneous uptake of the MUA-functionalized CIZS-NCs by 3 dpf older zebrafish-embryos that are evident through bright red fluorescence-emission at a low concentration of 7.8 μg/mL. Hence, we propose that the rapid, low-cost, large-scale “sustainable” MW-ST synthesis of CIZS-NCs, is an ideal bio-nanoprobe with good temporal and spatial resolution for rapid labeling, long-term in vivo tracking and intravital-fluorescence-bioimaging (IVBI).

Protection of silver nanoparticles using Eysenhardtia polystachya in peroxide-induced pancreatic β-Cell damage and their antidiabetic properties in zebrafish

PubMed Central

Garcia Campoy, Abraham Heriberto; Perez Gutierrez, Rosa Martha; Manriquez-Alvirde, Gabriela; Muñiz Ramirez, Alethia

2018-01-01

Background The aim was to explore the efficacy of extract of Eysenhardtia polystachya-loaded silver nanoparticles (EP/AgNPs) on pancreatic β cells, INS-1 cells, and zebrafish as a valuable model for the study of diabetes mellitus. Materials and methods EP/AgNPs were synthesized using methanol/water bark extract of E. polystachya and characterized using various physicochemical techniques. Results Immersion of adult zebrafish in 111 mM glucose solution resulted in a sustained hyperglycemic, hyperlipidemic state, and serum insulin levels decreased. The synthesized EP/AgNPs showed an absorption peak at 413 nm on ultraviolet–visible spectroscopy, revealing the surface plasmon resonance of the nanoparticles. Transmission electron microscopy indicated that most of the particles were spherical, with a diameter of 10–12 nm, a polydispersity index of 0.197, and a zeta potential of −32.25 mV, suggesting high stability of the nanoparticles. EP/AgNPs promote pancreatic β-cell survival, insulin secretion, enhanced hyperglycemia, and hyperlipidemia in glucose-induced diabetic zebrafish. EP/AgNPs also showed protection of the pancreatic β-cell line INS-1 against hydrogen peroxide-induced oxidative injury. Conclusion The results indicate that EP/AgNPs have good antidiabetic activity and therefore could be used to prevent the development of diabetes. PMID:29750032

Simultaneous mapping of membrane voltage and calcium in zebrafish heart in vivo reveals chamber-specific developmental transitions in ionic currents

PubMed Central

Hou, Jennifer H.; Kralj, Joel M.; Douglass, Adam D.; Engert, Florian; Cohen, Adam E.

2014-01-01

The cardiac action potential (AP) and the consequent cytosolic Ca2+ transient are key indicators of cardiac function. Natural developmental processes, as well as many drugs and pathologies change the waveform, propagation, or variability (between cells or over time) of these parameters. Here we apply a genetically encoded dual-function calcium and voltage reporter (CaViar) to study the development of the zebrafish heart in vivo between 1.5 and 4 days post fertilization (dpf). We developed a high-sensitivity spinning disk confocal microscope and associated software for simultaneous three-dimensional optical mapping of voltage and calcium. We produced a transgenic zebrafish line expressing CaViar under control of the heart-specific cmlc2 promoter, and applied ion channel blockers at a series of developmental stages to map the maturation of the action potential in vivo. Early in development, the AP initiated via a calcium current through L-type calcium channels. Between 90 and 102 h post fertilization (hpf), the ventricular AP switched to a sodium-driven upswing, while the atrial AP remained calcium driven. In the adult zebrafish heart, a sodium current drives the AP in both the atrium and ventricle. Simultaneous voltage and calcium imaging with genetically encoded reporters provides a new approach for monitoring cardiac development, and the effects of drugs on cardiac function. PMID:25309445

Simultaneous mapping of membrane voltage and calcium in zebrafish heart in vivo reveals chamber-specific developmental transitions in ionic currents.

PubMed

Hou, Jennifer H; Kralj, Joel M; Douglass, Adam D; Engert, Florian; Cohen, Adam E

2014-01-01

The cardiac action potential (AP) and the consequent cytosolic Ca(2+) transient are key indicators of cardiac function. Natural developmental processes, as well as many drugs and pathologies change the waveform, propagation, or variability (between cells or over time) of these parameters. Here we apply a genetically encoded dual-function calcium and voltage reporter (CaViar) to study the development of the zebrafish heart in vivo between 1.5 and 4 days post fertilization (dpf). We developed a high-sensitivity spinning disk confocal microscope and associated software for simultaneous three-dimensional optical mapping of voltage and calcium. We produced a transgenic zebrafish line expressing CaViar under control of the heart-specific cmlc2 promoter, and applied ion channel blockers at a series of developmental stages to map the maturation of the action potential in vivo. Early in development, the AP initiated via a calcium current through L-type calcium channels. Between 90 and 102 h post fertilization (hpf), the ventricular AP switched to a sodium-driven upswing, while the atrial AP remained calcium driven. In the adult zebrafish heart, a sodium current drives the AP in both the atrium and ventricle. Simultaneous voltage and calcium imaging with genetically encoded reporters provides a new approach for monitoring cardiac development, and the effects of drugs on cardiac function.

The pre-rRNA processing factor DEF is rate limiting for the pathogenesis of MYCN-driven neuroblastoma.

PubMed

Tao, T; Sondalle, S B; Shi, H; Zhu, S; Perez-Atayde, A R; Peng, J; Baserga, S J; Look, A T

2017-07-06

The nucleolar factor, digestive organ expansion factor (DEF), has a key role in ribosome biogenesis, functioning in pre-ribosomal RNA (pre-rRNA) processing as a component of the small ribosomal subunit (SSU) processome. Here we show that the peripheral sympathetic nervous system (PSNS) is very underdeveloped in def-deficient zebrafish, and that def haploinsufficiency significantly decreases disease penetrance and tumor growth rate in a MYCN-driven transgenic zebrafish model of neuroblastoma that arises in the PSNS. Consistent with these findings, DEF is highly expressed in human neuroblastoma, and its depletion in human neuroblastoma cell lines induces apoptosis. Interestingly, overexpression of MYCN in zebrafish and in human neuroblastoma cells results in the appearance of intermediate pre-rRNAs species that reflect the processing of pre-rRNAs through Pathway 2, a pathway that processes pre-rRNAs in a different temporal order than the more often used Pathway 1. Our results indicate that DEF and possibly other components of the SSU processome provide a novel site of vulnerability in neuroblastoma cells that could be exploited for targeted therapy.

Earth-strength magnetic field affects the rheotactic threshold of zebrafish swimming in shoals.

PubMed

Cresci, Alessandro; De Rosa, Rosario; Putman, Nathan F; Agnisola, Claudio

2017-02-01

Rheotaxis, the unconditioned orienting response to water currents, is a main component of fish behavior. Rheotaxis is achieved using multiple sensory systems, including visual and tactile cues. Rheotactic orientation in open or low-visibility waters might also benefit from the stable frame of reference provided by the geomagnetic field, but this possibility has not been explored before. Zebrafish (Danio rerio) form shoals living in freshwater systems with low visibility, show a robust positive rheotaxis, and respond to geomagnetic fields. Here, we investigated whether a static magnetic field in the Earth-strength range influenced the rheotactic threshold of zebrafish in a swimming tunnel. The direction of the horizontal component of the magnetic field relative to water flow influenced the rheotactic threshold of fish as part of a shoal, but not of fish tested alone. Results obtained after disabling the lateral line of shoaling individuals with Co 2+ suggest that this organ system is involved in the observed magneto-rheotactic response. These findings constitute preliminary evidence that magnetic fields influence rheotaxis and suggest new avenues for further research. Copyright © 2016 Elsevier Inc. All rights reserved.

Transcriptome analysis of genes and gene networks involved in aggressive behavior in mouse and zebrafish.

PubMed

Malki, Karim; Du Rietz, Ebba; Crusio, Wim E; Pain, Oliver; Paya-Cano, Jose; Karadaghi, Rezhaw L; Sluyter, Frans; de Boer, Sietse F; Sandnabba, Kenneth; Schalkwyk, Leonard C; Asherson, Philip; Tosto, Maria Grazia

2016-09-01

Despite moderate heritability estimates, the molecular architecture of aggressive behavior remains poorly characterized. This study compared gene expression profiles from a genetic mouse model of aggression with zebrafish, an animal model traditionally used to study aggression. A meta-analytic, cross-species approach was used to identify genomic variants associated with aggressive behavior. The Rankprod algorithm was used to evaluated mRNA differences from prefrontal cortex tissues of three sets of mouse lines (N = 18) selectively bred for low and high aggressive behavior (SAL/LAL, TA/TNA, and NC900/NC100). The same approach was used to evaluate mRNA differences in zebrafish (N = 12) exposed to aggressive or non-aggressive social encounters. Results were compared to uncover genes consistently implicated in aggression across both studies. Seventy-six genes were differentially expressed (PFP < 0.05) in aggressive compared to non-aggressive mice. Seventy genes were differentially expressed in zebrafish exposed to a fight encounter compared to isolated zebrafish. Seven genes (Fos, Dusp1, Hdac4, Ier2, Bdnf, Btg2, and Nr4a1) were differentially expressed across both species 5 of which belonging to a gene-network centred on the c-Fos gene hub. Network analysis revealed an association with the MAPK signaling cascade. In human studies HDAC4 haploinsufficiency is a key genetic mechanism associated with brachydactyly mental retardation syndrome (BDMR), which is associated with aggressive behaviors. Moreover, the HDAC4 receptor is a drug target for valproic acid, which is being employed as an effective pharmacological treatment for aggressive behavior in geriatric, psychiatric, and brain-injury patients. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Meisoindigo, but not its core chemical structure indirubin, inhibits zebrafish interstitial leukocyte chemotactic migration.

PubMed

Ye, Baixin; Xiong, Xiaoxing; Deng, Xu; Gu, Lijuan; Wang, Qiongyu; Zeng, Zhi; Gao, Xiang; Gao, Qingping; Wang, Yueying

2017-12-01

Inflammatory disease is a big threat to human health. Leukocyte chemotactic migration is required for efficient inflammatory response. Inhibition of leukocyte chemotactic migration to the inflammatory site has been shown to provide therapeutic targets for treating inflammatory diseases. Our study was designed to discover effective and safe compounds that can inhibit leukocyte chemotactic migration, thus providing possible novel therapeutic strategy for treating inflammatory diseases. In this study, we used transgenic zebrafish model (Tg:zlyz-EGFP line) to visualize the process of leukocyte chemotactic migration. Then, we used this model to screen the hit compound and evaluate its biological activity on leukocyte chemotactic migration. Furthermore, western blot analysis was performed to evaluate the effect of the hit compound on the AKT or ERK-mediated pathway, which plays an important role in leukocyte chemotactic migration. In this study, using zebrafish-based chemical screening, we identified that the hit compound meisoindigo (25 μM, 50 μM, 75 μM) can significantly inhibit zebrafish leukocyte chemotactic migration in a dose-dependent manner (p = 0.01, p = 0.0006, p < 0.0001). Also, we found that meisoindigo did not affect the process of leukocyte reverse migration (p = 0.43). Furthermore, our results unexpectedly showed that indirubin, the core structure of meisoindigo, had no significant effect on zebrafish leukocyte chemotactic migration (p = 0.6001). Additionally, our results revealed that meisoindigo exerts no effect on the Akt or Erk-mediated signalling pathway. Our results suggest that meisoindigo, but not indirubin, is effective for inhibiting leukocyte chemotactic migration, thus providing a potential therapeutic agent for treating inflammatory diseases.

Interleukin 8 mediates bcl-xL-induced enhancement of human melanoma cell dissemination and angiogenesis in a zebrafish xenograft model.

PubMed

Gabellini, Chiara; Gómez-Abenza, Elena; Ibáñez-Molero, Sofia; Tupone, Maria Grazia; Pérez-Oliva, Ana B; de Oliveira, Sofia; Del Bufalo, Donatella; Mulero, Victoriano

2018-02-01

The protein bcl-xL is able to enhance the secretion of the proinflammatory chemokine interleukin 8 (CXCL8) in human melanoma lines. In this study, we investigate whether the bcl-xL/CXCL8 axis is important for promoting melanoma angiogenesis and aggressiveness in vivo, using angiogenesis and xenotransplantation assays in zebrafish embryos. When injected into wild-type embryos, bcl-xL-overexpressing melanoma cells showed enhanced dissemination and angiogenic activity compared with control cells. Human CXCL8 protein elicited a strong proangiogenic activity in zebrafish embryos and zebrafish Cxcr2 receptor was identified as the mediator of CXCL8 proangiogenic activity using a morpholino-mediated gene knockdown. However, human CXCL8 failed to induce neutrophil recruitment in contrast to its zebrafish homolog. Interestingly, the greater aggressiveness of bcl-xL-overexpressing melanoma cells was mediated by an autocrine effect of CXCL8 on its CXCR2 receptor, as confirmed by an shRNA approach. Finally, correlation studies of gene expression and survival analyses using microarray and RNA-seq public databases of human melanoma biopsies revealed that bcl-xL expression significantly correlated with the expression of CXCL8 and other markers of melanoma progression. More importantly, a high level of co-expression of bcl-xL and CXCL8 was associated with poor prognosis in melanoma patients. In conclusion, these data demonstrate the existence of an autocrine CXCL8/CXCR2 signaling pathway in the bcl-xL-induced melanoma aggressiveness, encouraging the development of novel therapeutic approaches for high bcl-xL-expressing melanoma. © 2017 UICC.

Zebrafish as an early stage screening tool to study the systemic circulation of nanoparticulate drug delivery systems in vivo.

PubMed

Sieber, Sandro; Grossen, Philip; Detampel, Pascal; Siegfried, Salome; Witzigmann, Dominik; Huwyler, Jörg

2017-10-28

Nanomedicines have gained much attention for the delivery of small molecules or nucleic acids as treatment options for many diseases. However, the transfer from experimental systems to in vivo applications remains a challenge since it is difficult to assess their circulation behavior in the body at an early stage of drug discovery. Thus, innovative and improved concepts are urgently needed to overcome this issue and to close the gap between empiric nanoparticle design, in vitro assessment, and first in vivo experiments using rodent animal models. This study was focused on the zebrafish as a vertebrate screening model to assess the circulation in blood and extravasation behavior of nanoparticulate drug delivery systems in vivo. To validate this novel approach, monodisperse preparations of fluorescently labeled liposomes with similar size and zeta potential were injected into transgenic zebrafish lines expressing green fluorescent protein in their vasculature. Phosphatidylcholine-based lipids differed by fatty acid chain length and saturation. Circulation behavior and vascular distribution pattern were evaluated qualitatively and semi-quantitatively using image analysis. Liposomes composed of lipids with lower transition temperature (<28°C) as well as PEGylated liposomes showed longer circulation times and extravasation. In contrast, liposomes composed of lipids with transition temperatures>28°C bound to venous parts of the vasculature. This circulation patterns in the zebrafish model did correlate with published and experimental pharmacokinetic data from mice and rats. Our findings indicate that the zebrafish model is a useful vertebrate screening tool for nanoparticulate drug delivery systems to predict their in vivo circulation behavior with respect to systemic circulation time and exposure. Copyright © 2017 Elsevier B.V. All rights reserved.

The physiological role of CTGF/CCN2 in zebrafish notochond development and biological analysis of the proximal promoter region.

PubMed

Chiou, Ming-Jyun; Chao, Tsung-Tai; Wu, Jen-Leih; Kuo, Ching-Ming; Chen, Jyh-Yih

2006-10-20

During mouse embryogenesis, CTGF/CCN2 is expressed in zones containing hypertrophic chondroctyes and calcifying cartilage such as long bones, ribs, vertebral column, and phalanges. But in fish, its expression is yet unclear. Development of the vertebrae is morphologically similar among vertebrates, indicating that the underlying mechanism regulating the process is highly conserved during evolution. Analysis of 3.2kb of the CTGF/CCN2 proximal promoter sequence revealed a consensus TATAA box, putative AP1, Brn-2, CdxA, C/EBP alpha, C/EBP beta, C-Ets-, delta E, HFH-2, and HSF2 binding sites. Transient expression experiments with a 5'-deletion revealed at least 4 regulatory regions in the zebrafish CTGF/CCN2 gene, 2 with a stimulatory effect on transcription and 2 with an apparent inhibitory effect after IGF-I treatment in the ZFL cell line. To study the promoter-specific expression, we constructed a series of CTGF/CCN2 (3.0-, 2.5-, 2.0-, 1.5-, 1.0-, and 0.4-kb) promoter-driven green fluorescent protein (GFP) fragments encoding the GFP cDNA transgene which was microinjected into zebrafish embryos. Morphological studies of transgenic zebrafish indicated that the CTGF/CCN2 promoter-driven GFP transcripts appeared in the notochord. Targeted knockdown of the CTGF/CCN2 gene by two antisense morpholino oligonucleotides resulted in disruptions to notochord development. From a comparative point of view, this study of the CTGF/CCN2 gene in zebrafish may correlate well with those previously published on the mouse. These molecular results suggest that CTGF/CCN2 plays an important role in notochord development and is required for general embryonic development.

A conserved role of αA-crystallin in the development of the zebrafish embryonic lens.

PubMed

Zou, Ping; Wu, Shu-Yu; Koteiche, Hanane A; Mishra, Sanjay; Levic, Daniel S; Knapik, Ela; Chen, Wenbiao; Mchaourab, Hassane S

2015-09-01

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.

Cytogenetic and molecular genetic characterization of immortalized human ovarian surface epithelial cell lines: consistent loss of chromosome 13 and amplification of chromosome 20.

PubMed

Jin, Yuesheng; Zhang, Hao; Tsao, Sai Wah; Jin, Charlotte; Lv, Mei; Strömbeck, Bodil; Wiegant, Joop; Wan, Thomas Shek Kong; Yuen, Po Wing; Kwong, Yok-Lam

2004-01-01

This study aimed at identifying the genetic events involved in immortalization of ovarian epithelial cells, which might be important steps in ovarian carcinogenesis. The genetic profiles of five human ovarian surface epithelial (HOSE) cell lines immortalized by retroviral transfection of the human papillomavirus (HPV) E6/E7 genes were thoroughly characterized by chromosome banding and fluorescence in situ hybridization (FISH), at various passages pre- and post-crisis. In pre-crisis, most cells had simple, non-clonal karyotypic changes. Telomere association was the commonest aberration, suggesting that tolermase dysfunction might be an important genetic event leading to cellular crisis. After immortalization post-crisis, however, the karyotypic patterns were non-random. Loss of genetic materials was a characteristic feature. The commonest numerical aberrations were -13, -14, -16, -17, -18, and +5. Among them, loss of chromosome 13 was common change observed in all lines. The only recurrent structural aberration was homogeneously staining regions (hsr) observed in three lines. FISH and combined binary ratio labeling (COBRA)-FISH showed in two cases that the hsrs were derived from chromosome 20. Clonal evolution was observed in four of the lines. In one line, hsr was the only change shared by all subclones, suggesting that it might be a primary event in cell immortalization. The results of the present study suggested that loss of chromosome 13 and the amplification of chromosome 20 might be early genetic events involved in ovarian cell immortalization, and might be useful targets for the study of genomic aberrations in ovarian carcinogenesis.

A functional Bucky ball-GFP transgene visualizes germ plasm in living zebrafish.

PubMed

Riemer, Stephan; Bontems, Franck; Krishnakumar, Pritesh; Gömann, Jasmin; Dosch, Roland

2015-01-01

In many animals, the germline is specified by maternal RNA-granules termed germ plasm. The correct localization of germ plasm during embryogenesis is therefore crucial for the specification of germ cells. In zebrafish, we previously identified Bucky ball (Buc) as a key regulator of germ plasm formation. Here, we used a Buc antibody to describe its continuous germ plasm localization. Moreover, we generated a transgenic Buc-GFP line for live imaging, which visualizes germ plasm from its assembly during oogenesis up to the larval stages. Live imaging of Buc-GFP generated stunning movies, as they highlighted the dynamic details of germ plasm movements. Moreover, we discovered that Buc was still detected in primordial germ cells 2 days after fertilization. Interestingly, the transgene rescued buc mutants demonstrating genetically that the Buc-GFP fusion protein is functional. These results show that Buc-GFP exerts all biochemical interactions essential for germline development and highlight the potential of this line to analyze the molecular regulation of germ plasm formation. Copyright © 2015 Elsevier B.V. All rights reserved.

Reporter gene expression in fish following cutaneous infection with pantropic retroviral vectors.

PubMed

Paul, T A; Burns, J C; Shike, H; Getchell, R; Bowser, P R; Whitlock, K E; Casey, J W

2001-06-01

A central issue in gene delivery systems is choosing promoters that will direct defined and sustainable levels of gene expression. Pantropic retroviral vectors provide a means to insert genes into either somatic or germline cells. In this study, we focused on somatic cell infection by evaluating the activity of 3 promoters inserted by vectors into fish cell lines and fish skin using pantropic retroviruses. In bluegill and zebrafish cell lines, the highest levels of luciferase expression were observed from the 5' murine leukemia virus long terminal repeat of the retroviral vector. The Rous sarcoma virus long terminal repeat and cytomegalovirus early promoter, as internal promoters, generated lower levels of luciferase. Luciferase reporter vectors infected zebrafish skin, as measured by the presence of viral DNA, and expressed luciferase. We infected developing walleye dermal sarcomas with retroviral vectors to provide an environment with enhanced cell proliferation, a condition necessary for integration of the provirus into the host genome. We demonstrated a 4-fold to 7-fold increase in luciferase gene expression in tumor tissue over infections in normal walleye skin.

The resurrection genome of Boea hygrometrica: A blueprint for survival of dehydration

USDA-ARS?s Scientific Manuscript database

The dicot, Boea hygrometrica, is an important plant model for the discovery of genes central to desiccation tolerance in plants; an essential trait in land plant evolution and the foundation for global agriculture. We used a vegetative clonal line to uncover the sequence and characterize the respons...

Lamarck Will Not Lie Down.

ERIC Educational Resources Information Center

Lewin, Roger

1981-01-01

Describes recent research by Edward Steele appearing to support the Lamarckian theory of inheritance. Steele suggests that a mutant somatic cell favored by the environment will undergo clonal expansion. Altered genetic materials from these cells is then picked up by C-type viruses and inserted into the germ line genome. (CS)

Geno- and phenotypic characteristics of a transfected babesia bovis 6-Cys-E knockout clonal line

USDA-ARS?s Scientific Manuscript database

Babesia bovis is an intra-erythrocytic tick transmitted apicomplexan protozoan parasite. It has a complex life style including asexual replication in the mammalian host and sexual replication occurring in the midgut of host tick vector, typically, Rhipicephalus microplus. Previous evidence showed th...

Afferent and motoneuron activity in response to single neuromast stimulation in the posterior lateral line of larval zebrafish

PubMed Central

Haehnel-Taguchi, Melanie; Akanyeti, Otar

2014-01-01

The lateral line system of fishes contains mechanosensory receptors along the body surface called neuromasts, which can detect water motion relative to the body. The ability to sense flow informs many behaviors, such as schooling, predator avoidance, and rheotaxis. Here, we developed a new approach to stimulate individual neuromasts while either recording primary sensory afferent neuron activity or swimming motoneuron activity in larval zebrafish (Danio rerio). Our results allowed us to characterize the transfer functions between a controlled lateral line stimulus, its representation by primary sensory neurons, and its subsequent behavioral output. When we deflected the cupula of a neuromast with a ramp command, we found that the connected afferent neuron exhibited an adapting response which was proportional in strength to deflection velocity. The maximum spike rate of afferent neurons increased sigmoidally with deflection velocity, with a linear range between 0.1 and 1.0 μm/ms. However, spike rate did not change when the cupula was deflected below 8 μm, regardless of deflection velocity. Our findings also reveal an unexpected sensitivity in the larval lateral line system: stimulation of a single neuromast could elicit a swimming response which increased in reliability with increasing deflection velocities. At high deflection velocities, we observed that lateral line evoked swimming has intermediate values of burst frequency and duty cycle that fall between electrically evoked and spontaneous swimming. An understanding of the sensory capabilities of a single neuromast will help to build a better picture of how stimuli are encoded at the systems level and ultimately translated into behavior. PMID:24966296

Zebrafish as a model for monocarboxyl transporter 8-deficiency.

PubMed

Vatine, Gad David; Zada, David; Lerer-Goldshtein, Tali; Tovin, Adi; Malkinson, Guy; Yaniv, Karina; Appelbaum, Lior

2013-01-04

Allan-Herndon-Dudley syndrome (AHDS) is a severe psychomotor retardation characterized by neurological impairment and abnormal thyroid hormone (TH) levels. Mutations in the TH transporter, monocarboxylate transporter 8 (MCT8), are associated with AHDS. MCT8 knock-out mice exhibit impaired TH levels; however, they lack neurological defects. Here, the zebrafish mct8 gene and promoter were isolated, and mct8 promoter-driven transgenic lines were used to show that, similar to humans, mct8 is primarily expressed in the nervous and vascular systems. Morpholino-based knockdown and rescue experiments revealed that MCT8 is strictly required for neural development in the brain and spinal cord. This study shows that MCT8 is a crucial regulator during embryonic development and establishes the first vertebrate model for MCT8 deficiency that exhibits a neurological phenotype.

FUNCTIONAL SUBCLONE PROFILING FOR PREDICTION OF TREATMENT-INDUCED INTRA-TUMOR POPULATION SHIFTS AND DISCOVERY OF RATIONAL DRUG COMBINATIONS IN HUMAN GLIOBLASTOMA

PubMed Central

Reinartz, Roman; Wang, Shanshan; Kebir, Sied; Silver, Daniel J.; Wieland, Anja; Zheng, Tong; Küpper, Marius; Rauschenbach, Laurèl; Fimmers, Rolf; Shepherd, Timothy M.; Trageser, Daniel; Till, Andreas; Schäfer, Niklas; Glas, Martin; Hillmer, Axel M.; Cichon, Sven; Smith, Amy A.; Pietsch, Torsten; Liu, Ying; Reynolds, Brent A.; Yachnis, Anthony; Pincus, David W.; Simon, Matthias; Brüstle, Oliver; Steindler, Dennis A.; Scheffler, Björn

2016-01-01

Purpose Investigation of clonal heterogeneity may be key to understanding mechanisms of therapeutic failure in human cancer. However, little is known on the consequences of therapeutic intervention on the clonal composition of solid tumors. Experimental Design Here, we used 33 single cell-derived subclones generated from five clinical glioblastoma specimens for exploring intra- and inter-individual spectra of drug resistance profiles in vitro. In a personalized setting, we explored whether differences in pharmacological sensitivity among subclones could be employed to predict drug-dependent changes to the clonal composition of tumors. Results Subclones from individual tumors exhibited a remarkable heterogeneity of drug resistance to a library of potential anti-glioblastoma compounds. A more comprehensive intra-tumoral analysis revealed that stable genetic and phenotypic characteristics of co-existing subclones could be correlated with distinct drug sensitivity profiles. The data obtained from differential drug response analysis could be employed to predict clonal population shifts within the naïve parental tumor in vitro and in orthotopic xenografts. Furthermore, the value of pharmacological profiles could be shown for establishing rational strategies for individualized secondary lines of treatment. Conclusions Our data provide a previously unrecognized strategy for revealing functional consequences of intra-tumor heterogeneity by enabling predictive modeling of treatment-related subclone dynamics in human glioblastoma. PMID:27521447

Method for rapid isolation of sensitive mutants

DOEpatents

Freyer, James P.

1997-01-01

Sensitive mammalian cell mutants are rapidly isolated using flow cytometry. A first population of clonal spheroids is established to contain both normal and mutant cells. The population may be naturally occurring or may arise from mutagenized cells. The first population is then flow sorted by size to obtain a second population of clonal spheroids of a first uniform size. The second population is then exposed to a DNA-damaging agent that is being investigated. The exposed second population is placed in a growth medium to form a third population of clonal spheroids comprising spheroids of increased size from the mammalian cells that are resistant to the DNA-damaging agent and spheroids of substantially the first uniform size formed from the mammalian cells that are sensitive to the DNA-damaging agent. The third population is not flow sorted to differentiate the spheroids formed from resistant mammalian cells from spheroids formed from sensitive mammalian cells. The spheroids formed from sensitive mammalian cells are now treated to recover viable sensitive cells from which a sensitive cell line can be cloned.

Method for rapid isolation of sensitive mutants

DOEpatents

Freyer, J.P.

1997-07-29

Sensitive mammalian cell mutants are rapidly isolated using flow cytometry. A first population of clonal spheroids is established to contain both normal and mutant cells. The population may be naturally occurring or may arise from mutagenized cells. The first population is then flow sorted by size to obtain a second population of clonal spheroids of a first uniform size. The second population is then exposed to a DNA-damaging agent that is being investigated. The exposed second population is placed in a growth medium to form a third population of clonal spheroids comprising spheroids of increased size from the mammalian cells that are resistant to the DNA-damaging agent and spheroids of substantially the first uniform size formed from the mammalian cells that are sensitive to the DNA-damaging agent. The third population is not flow sorted to differentiate the spheroids formed from resistant mammalian cells from spheroids formed from sensitive mammalian cells. The spheroids formed from sensitive mammalian cells are now treated to recover viable sensitive cells from which a sensitive cell line can be cloned. 15 figs.

Systemic Mastocytosis with Smoldering Multiple Myeloma: Report of a Case

PubMed Central

Garcia, Gwenalyn; Ying, Liu; Hurford, Matthew; Odaimi, Marcel

2016-01-01

Systemic mastocytosis (SM) is a disease characterized by a clonal infiltration of mast cells affecting various tissues of the body. It is grouped into six different subtypes according to the World Health Organization classification. It is called indolent systemic mastocytosis (ISM) when there is no evidence of end organ dysfunction, while the presence of end organ dysfunction defines aggressive systemic mastocytosis (ASM). When SM coexists with a clonal hematological disorder, it is classified as systemic mastocytosis with associated clonal hematological nonmast cell lineage disease (SM-AHNMD). Over 80% of SM-AHNMD cases involve disorders of the myeloid cell lines. To our knowledge, there are only 8 reported cases to date of SM associated with a plasma cell disorder. We report a patient with ISM who was found to have concomitant smoldering multiple myeloma. His disease later progressed to ASM. We discuss this rare association between SM and a plasma cell disorder, and potential common pathophysiologic mechanisms linking the two disorders will be reviewed. We also discuss prognostic factors in SM as well as the management options considered during the evolution of the patient's disease. PMID:27293930

The Therapeutic Effect of the Antitumor Drug 11 Beta and Related Molecules on Polycystic Kidney Disease

DTIC Science & Technology

2017-10-01

structure central to the pathogenesis of ADPKD. The team at Yale employed CRISPR - Cas9 genome editing technology to generate two isogenic cell lines...possibilities may have contributed to this result, including a clonal effect perhaps from off target CRISPR /Cas9 activity leading to a rescue...Pkd1 knockout background; this cell line is another control for any off-target genetic in the CRISPR /Cas9 editing procedure. This will establish

Embryonic fate map of first pharyngeal arch structures in the sox10: kaede zebrafish transgenic model.

PubMed

Dougherty, Max; Kamel, George; Shubinets, Valeriy; Hickey, Graham; Grimaldi, Michael; Liao, Eric C

2012-09-01

Cranial neural crest cells follow stereotypic patterns of migration to form craniofacial structures. The zebrafish is a powerful vertebrate genetic model where transgenics with reporter proteins under the transcriptional regulation of lineage-specific promoters can be generated. Numerous studies demonstrate that the zebrafish ethmoid plate is embryologically analogous to the mammalian palate. A fate map correlating embryonic cranial neural crest to defined jaw structures would provide a useful context for the morphogenetic analysis of craniofacial development. To that end, the sox10:kaede transgenic was generated, where sox10 provides lineage restriction to the neural crest. Specific regions of neural crest were labeled at the 10-somite stage by photoconversion of the kaede reporter protein. Lineage analysis was carried out during pharyngeal development in wild-type animals, after miR140 injection, and after estradiol treatment. At the 10-somite stage, cranial neural crest cells anterior of the eye contributed to the median ethmoid plate, whereas cells medial to the eye formed the lateral ethmoid plate and trabeculae and a posterior population formed the mandible. miR-140 overexpression and estradiol inhibition of Hedgehog signaling resulted in cleft development, with failed migration of the anterior cell population to form the median ethmoid plate. The sox10:kaede transgenic line provides a useful tool for neural crest lineage analysis. These studies illustrate the advantages of the zebrafish model for application in morphogenetic studies of vertebrate craniofacial development.

Cftr controls lumen expansion and function of Kupffer’s vesicle in zebrafish

PubMed Central

Navis, Adam; Marjoram, Lindsay; Bagnat, Michel

2013-01-01

Regulated fluid secretion is crucial for the function of most organs. In vertebrates, the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) is a master regulator of fluid secretion. Although the biophysical properties of CFTR have been well characterized in vitro, little is known about its in vivo role during development. Here, we investigated the function of Cftr during zebrafish development by generating several cftr mutant alleles using TAL effector nucleases. We found that loss of cftr function leads to organ laterality defects. In zebrafish, left-right (LR) asymmetry requires cilia-driven fluid flow within the lumen of Kupffer’s vesicle (KV). Using live imaging we found that KV morphogenesis is disrupted in cftr mutants. Loss of Cftr-mediated fluid secretion impairs KV lumen expansion leading to defects in organ laterality. Using bacterial artificial chromosome recombineering, we generated transgenic fish expressing functional Cftr fusion proteins with fluorescent tags under the control of the cftr promoter. The transgenes completely rescued the cftr mutant phenotype. Live imaging of these transgenic lines showed that Cftr is localized to the apical membrane of the epithelial cells in KV during lumen formation. Pharmacological stimulation of Cftr-dependent fluid secretion led to an expansion of the KV lumen. Conversely, inhibition of ion gradient formation impaired KV lumen inflation. Interestingly, cilia formation and motility in KV were not affected, suggesting that fluid secretion and flow are independently controlled in KV. These findings uncover a new role for cftr in KV morphogenesis and function during zebrafish development. PMID:23487313

Studying disorders of vertebrate iron and heme metabolism using zebrafish

PubMed Central

van der Vorm, Lisa N.; Paw, Barry H.

2017-01-01

Iron is a crucial component of heme- and iron-sulfur clusters, involved in vital cellular functions such as oxygen transport, DNA synthesis, and respiration. Both excess and insufficient levels of iron and heme-precursors cause human disease, such as iron-deficiency anemia, hemochromatosis, and porphyrias. Hence, their levels must be tightly regulated, requiring a complex network of transporters and feedback mechanisms. The use of zebrafish to study these pathways and the underlying genetics offers many advantages, among others their optical transparency, ex-vivo development and high genetic and physiological conservations. This chapter first reviews well-established methods, such as large-scale mutagenesis screens that have led to the initial identification of a series of iron and heme transporters and the generation of a variety of mutant lines. Other widely used techniques are based on injection of RNA, including complementary morpholino knockdown and gene overexpression. In addition, we highlight several recently developed approaches, most notably endonuclease-based gene knockouts such as TALENs or the CRISPR/Cas9 system that have been used to study how loss of function can induce human disease phenocopies in zebrafish. Rescue by chemical complementation with iron-based compounds or small molecules can subsequently be used to confirm causality of the genetic defect for the observed phenotype. All together, zebrafish have proven to be – and will continue to serve as an ideal model to advance our understanding of the pathogenesis of human iron and heme-related diseases and to develop novel therapies to treat these conditions. PMID:28129844

Clonal Propagation of Walnut Rootstock Genotypes for Genetic Improvement 2009

USDA-ARS?s Scientific Manuscript database

We produced over 3000 liner-sized plantlets of 26 genotypes for use in greenhouse screens and for growing in nurseries to a size large enough for grafting and use in orchard trials. In addition, over 1400 liner plantlets of 15 lines transformed for resistance to crown gall were produced for greenhou...

Molecular and functional characterization of the scavenger receptor CD36 in zebrafish and common carp.

PubMed

Fink, Inge R; Benard, Erica L; Hermsen, Trudi; Meijer, Annemarie H; Forlenza, Maria; Wiegertjes, Geert F

2015-02-01

CD36 is a scavenger receptor which has been studied closely in mammals where it is expressed by many different cell types and plays a role in highly diverse processes, both homeostatic and pathologic. It is among other things important in the innate immune system, in angiogenesis, and in clearance of apoptotic cells, and it is also involved in lipid metabolism and atherosclerosis. Recently, in the cephalochordate amphioxus a primitive CD36 family member was described, which was present before the divergence of CD36 from other scavenger receptor B family members, SCARB1 and SCARB2. Not much is known on the Cd36 molecule in teleost fish. We therefore studied Cd36 in both zebrafish and common carp, two closely related cyprinid fish species. Whereas a single cd36 gene is present in zebrafish, carp has two cd36 genes, and all show conserved synteny compared to mammalian CD36. The gene expression of carp cd36 is high in brain, ovary and testis but absent in immune organs. Although in mammals CD36 expression in erythrocytes, monocytes and macrophages is high, gene expression studies in leukocyte subtypes of adult carp and zebrafish larvae, including thrombocytes and macrophages provided no indication for any substantial expression of cd36 in immune cell types. Surprisingly, analysis of the cd36 promoter region does show the presence of several binding sites for transcription factors known to regulate immune responses. Overexpression of carp cd36 locates the receptor on the cell surface of mammalian cell lines consistent with the predicted topology of cyprinid Cd36 with a large extracellular domain, two transmembrane domains, and short cytoplasmic tails at both ends. Gene expression of cd36 is down-regulated during infection of zebrafish with Mycobacterium marinum, whereas knockdown of cd36 in zebrafish larvae led to higher bacterial burden upon such infection. We discuss the putative role for Cd36 in immune responses of fish in the context of other members of the scavenger receptor class B family. Copyright © 2014 Elsevier Ltd. All rights reserved.

Histological and Transcriptomic Changes in Male Zebrafish Testes Due to Early Life Exposure to Low Level 2,3,7,8-Tetrachlorodibenzo-p-Dioxin.

PubMed

Baker, Bridget B; Yee, Jeremiah S; Meyer, Danielle N; Yang, Doris; Baker, Tracie R

2016-10-01

We have shown that zebrafish (Danio rerio) are an excellent model for evaluating the link between early life stage exposure to environmental chemicals and disease in adulthood and subsequent unexposed generations. Previously, we used this model to identify transgenerational effects of dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin [TCDD]) on skeletal development, sex ratio, and reproductive capacity. Transgenerational inheritance of TCDD toxicity, notably decreased reproductive capacity, appears to be mediated through the male germ line. Thus, we examine testicular tissue for structural and gene expression changes using histology, microarray, and quantitative reverse transcription polymerase chain reaction (qRT-PCR). Histological analysis revealed decreased spermatozoa with concurrent increase in spermatogonia, and decreased germinal epithelium thickness in TCDD-exposed males compared with controls. We also identified altered expression of genes associated with testis development, steroidogenesis, spermatogenesis, hormone metabolism, and xenobiotic response. Altered genes are in pathways involving lipid metabolism, molecular transport, small molecule biochemistry, cell morphology, and metabolism of vitamins and minerals. These data will inform future investigations to elucidate the mechanism of adult-onset and transgenerational infertility due to TCDD exposure in zebrafish.

Zebrafish Caudal Haematopoietic Embryonic Stromal Tissue (CHEST) Cells Support Haematopoiesis.

PubMed

Wolf, Anja; Aggio, Julian; Campbell, Clyde; Wright, Francis; Marquez, Gabriel; Traver, David; Stachura, David L

2017-03-16

Haematopoiesis is an essential process in early vertebrate development that occurs in different distinct spatial locations in the embryo that shift over time. These different sites have distinct functions: in some anatomical locations specific hematopoietic stem and progenitor cells (HSPCs) are generated de novo. In others, HSPCs expand. HSPCs differentiate and renew in other locations, ensuring homeostatic maintenance. These niches primarily control haematopoiesis through a combination of cell-to-cell signalling and cytokine secretion that elicit unique biological effects in progenitors. To understand the molecular signals generated by these niches, we report the generation of caudal hematopoietic embryonic stromal tissue (CHEST) cells from 72-hours post fertilization (hpf) caudal hematopoietic tissue (CHT), the site of embryonic HSPC expansion in fish. CHEST cells are a primary cell line with perivascular endothelial properties that expand hematopoietic cells in vitro. Morphological and transcript analysis of these cultures indicates lymphoid, myeloid, and erythroid differentiation, indicating that CHEST cells are a useful tool for identifying molecular signals critical for HSPC proliferation and differentiation in the zebrafish. These findings permit comparison with other temporally and spatially distinct haematopoietic-supportive zebrafish niches, as well as with mammalian haematopoietic-supportive cells to further the understanding of the evolution of the vertebrate hematopoietic system.

Identification of ion-channel modulators that protect against aminoglycoside-induced hair cell death

PubMed Central

Kenyon, Emma J.; Kirkwood, Nerissa K.; Kitcher, Siân R.; O’Reilly, Molly; Cantillon, Daire M.; Goodyear, Richard J.; Secker, Abigail; Baxendale, Sarah; Bull, James C.; Waddell, Simon J.; Whitfield, Tanya T.; Ward, Simon E.; Kros, Corné J.; Richardson, Guy P.

2017-01-01

Aminoglycoside antibiotics are used to treat life-threatening bacterial infections but can cause deafness due to hair cell death in the inner ear. Compounds have been described that protect zebrafish lateral line hair cells from aminoglycosides, but few are effective in the cochlea. As the aminoglycosides interact with several ion channels, including the mechanoelectrical transducer (MET) channels by which they can enter hair cells, we screened 160 ion-channel modulators, seeking compounds that protect cochlear outer hair cells (OHCs) from aminoglycoside-induced death in vitro. Using zebrafish, 72 compounds were identified that either reduced loading of the MET-channel blocker FM 1-43FX, decreased Texas red–conjugated neomycin labeling, or reduced neomycin-induced hair cell death. After testing these 72 compounds, and 6 structurally similar compounds that failed in zebrafish, 13 were found that protected against gentamicin-induced death of OHCs in mouse cochlear cultures, 6 of which are permeant blockers of the hair cell MET channel. None of these compounds abrogated aminoglycoside antibacterial efficacy. By selecting those without adverse effects at high concentrations, 5 emerged as leads for developing pharmaceutical otoprotectants to alleviate an increasing clinical problem. PMID:29263311

Predicting adult fish acute lethality with the zebrafish embryo: relevance of test duration, endpoints, compound properties, and exposure concentration analysis.

PubMed

Knöbel, Melanie; Busser, Frans J M; Rico-Rico, Angeles; Kramer, Nynke I; Hermens, Joop L M; Hafner, Christoph; Tanneberger, Katrin; Schirmer, Kristin; Scholz, Stefan

2012-09-04

The zebrafish embryo toxicity test has been proposed as an alternative for the acute fish toxicity test, which is required by various regulations for environmental risk assessment of chemicals. We investigated the reliability of the embryo test by probing organic industrial chemicals with a wide range of physicochemical properties, toxicities, and modes of toxic action. Moreover, the relevance of using measured versus nominal (intended) exposure concentrations, inclusion of sublethal endpoints, and different exposure durations for the comparability with reported fish acute toxicity was explored. Our results confirm a very strong correlation of zebrafish embryo to fish acute toxicity. When toxicity values were calculated based on measured exposure concentrations, the slope of the type II regression line was 1 and nearly passed through the origin (1 to 1 correlation). Measured concentrations also explained several apparent outliers. Neither prolonged exposure (up to 120 h) nor consideration of sublethal effects led to a reduced number of outliers. Yet, two types of compounds were less lethal to embryos than to adult fish: a neurotoxic compound acting via sodium channels (permethrin) and a compound requiring metabolic activation (allyl alcohol).

Identification of ion-channel modulators that protect against aminoglycoside-induced hair cell death.

PubMed

Kenyon, Emma J; Kirkwood, Nerissa K; Kitcher, Siân R; O'Reilly, Molly; Derudas, Marco; Cantillon, Daire M; Goodyear, Richard J; Secker, Abigail; Baxendale, Sarah; Bull, James C; Waddell, Simon J; Whitfield, Tanya T; Ward, Simon E; Kros, Corné J; Richardson, Guy P

2017-12-21

Aminoglycoside antibiotics are used to treat life-threatening bacterial infections but can cause deafness due to hair cell death in the inner ear. Compounds have been described that protect zebrafish lateral line hair cells from aminoglycosides, but few are effective in the cochlea. As the aminoglycosides interact with several ion channels, including the mechanoelectrical transducer (MET) channels by which they can enter hair cells, we screened 160 ion-channel modulators, seeking compounds that protect cochlear outer hair cells (OHCs) from aminoglycoside-induced death in vitro. Using zebrafish, 72 compounds were identified that either reduced loading of the MET-channel blocker FM 1-43FX, decreased Texas red-conjugated neomycin labeling, or reduced neomycin-induced hair cell death. After testing these 72 compounds, and 6 structurally similar compounds that failed in zebrafish, 13 were found that protected against gentamicin-induced death of OHCs in mouse cochlear cultures, 6 of which are permeant blockers of the hair cell MET channel. None of these compounds abrogated aminoglycoside antibacterial efficacy. By selecting those without adverse effects at high concentrations, 5 emerged as leads for developing pharmaceutical otoprotectants to alleviate an increasing clinical problem.

Gene miles-apart is required for formation of otic vesicle and hair cells in zebrafish.

PubMed

Hu, Z-y; Zhang, Q-y; Qin, W; Tong, J-w; Zhao, Q; Han, Y; Meng, J; Zhang, J-p

2013-10-31

Hearing loss is a serious burden to physical and mental health worldwide. Aberrant development and damage of hearing organs are recognized as the causes of hearing loss, the molecular mechanisms underlining these pathological processes remain elusive. Investigation of new molecular mechanisms involved in proliferation, differentiation, migration and maintenance of neuromast primordium and hair cells will contribute to better understanding of hearing loss pathology. This knowledge will enable the development of protective agents and mechanism study of drug ototoxicity. In this study, we demonstrate that the zebrafish gene miles-apart, a homolog of sphingosine-1-phosphate receptor 2 (s1pr2) in mammals, has an important role in the development of otic vesicle, neuromasts and survival of hair cells. Whole-mount in situ hybridization of embryos showed that miles-apart expression occurred mainly in the encephalic region and the somites at 24 h.p.f. (hour post fertilization), in the midbrain/hindbrain boundary, the brainstem and the pre-neuromast of lateral line at 48 h.p.f. in a strict spatiotemporal regulation. Both up- and downregulation of miles-apart led to abnormal otoliths and semicircular canals, excess or few hair cells and neuromasts, and their disarranged depositions in the lateral lines. Miles-apart (Mil) dysregulation also caused abnormal expression of hearing-associated genes, including hmx2, fgf3, fgf8a, foxi1, otop1, pax2.1 and tmieb during zebrafish organogenesis. Moreover, in larvae miles-apart gene knockdown significantly upregulated proapoptotic gene zBax2 and downregulated prosurvival gene zMcl1b; in contrast, the level of zBax2 was decreased and of zMcl1b enhanced by miles-apart overexpression. Collectively, Mil activity is linked to organization and number decision of hair cells within a neuromast, also to deposition of neuromasts and formation of otic vesicle during zebrafish organogenesis. At the larva stage, Mil as an upstream regulator of bcl-2 gene family has a role in protection of hair cells against apoptosis by promoting expression of prosurvival gene zMcl1b and suppressing proapoptotic gene zBax2.

Knock-down of pantothenate kinase 2 severely affects the development of the nervous and vascular system in zebrafish, providing new insights into PKAN disease

PubMed Central

Zizioli, Daniela; Tiso, Natascia; Guglielmi, Adele; Saraceno, Claudia; Busolin, Giorgia; Giuliani, Roberta; Khatri, Deepak; Monti, Eugenio; Borsani, Giuseppe; Argenton, Francesco; Finazzi, Dario

2016-01-01

Pantothenate Kinase Associated Neurodegeneration (PKAN) is an autosomal recessive disorder with mutations in the pantothenate kinase 2 gene (PANK2), encoding an essential enzyme for Coenzyme A (CoA) biosynthesis. The molecular connection between defects in this enzyme and the neurodegenerative phenotype observed in PKAN patients is still poorly understood. We exploited the zebrafish model to study the role played by the pank2 gene during embryonic development and get new insight into PKAN pathogenesis. The zebrafish orthologue of hPANK2 lies on chromosome 13, is a maternal gene expressed in all development stages and, in adult animals, is highly abundant in CNS, dorsal aorta and caudal vein. The injection of a splice-inhibiting morpholino induced a clear phenotype with perturbed brain morphology and hydrocephalus; edema was present in the heart region and caudal plexus, where hemorrhages with reduction of blood circulation velocity were detected. We characterized the CNS phenotype by studying the expression pattern of wnt1 and neurog1 neural markers and by use of the Tg(neurod:EGFP/sox10:dsRed) transgenic line. The results evidenced that downregulation of pank2 severely impairs neuronal development, particularly in the anterior part of CNS (telencephalon). Whole-mount in situ hybridization analysis of the endothelial markers cadherin-5 and fli1a, and use of Tg(fli1a:EGFP/gata1a:dsRed) transgenic line, confirmed the essential role of pank2 in the formation of the vascular system. The specificity of the morpholino-induced phenotype was proved by the restoration of a normal development in a high percentage of embryos co-injected with pank2 mRNA. Also, addition of pantethine or CoA, but not of vitamin B5, to pank2 morpholino-injected embryos rescued the phenotype with high efficiency. The zebrafish model indicates the relevance of pank2 activity and CoA homeostasis for normal neuronal development and functioning and provides evidence of an unsuspected role for this enzyme and its product in vascular development. PMID:26476142

BDE-99, but not BDE-47, is a transient aryl hydrocarbon receptor agonist in zebrafish liver cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Jie; Zhu, Jinyong; Chan, King Ming, E-mail:

Polybrominated diphenyl ethers (PBDEs) are endocrine-disrupting chemicals that affect the environment and the health of humans and wildlife. In this study, the zebrafish liver (ZFL) cell line was used in vitro to investigate two major PBDE contaminants: 2, 2′, 4, 4′, 5-pentabromodiphenyl ether (BDE-99) and 2, 2′, 4, 4′-tetrabromodiphenyl ether (BDE-47). BDE-99 was found to significantly induce cytochrome P450 (CYP1A), uridine diphosphate glucuronosyl transferase 1 family a, b (ugt1ab), 7-ethoxyresorufin-O-deethylase activity and an aryl hydrocarbon receptor (Ahr) dependent xenobiotic response element luciferase reporter system, confirming the Ahr-mediated activation of CYP1A by BDE-99. The time-course effect indicated that the role ofmore » BDE-99 in Ahr-mediated signaling is likely to be transient and highly dependent on the ability of BDE-99 to induce CYP1A and ugt1ab, and presumably its metabolism. BDE-99 also exhibited a significant dose-response effect on a developed zebrafish pregnane X receptor luciferase reporter gene system. However, the other abundant contaminant under study, BDE-47, did not exhibit the above effects. Together, these results indicated that the molecular mechanism of PBDEs induced in ZFL cells is a chemically specific process that differs between members of the PBDE family. CYP1A induction derived by BDE-99 warrants further risk assessment as the humans, wildlife and environment are exposed to a complex mixture including dioxin-like compounds and carcinogenic compounds. - Highlights: • BDE-99 is an aryl hydrocarbon receptor (Ahr) agonist in zebrafish liver cell-line ZFL. • BDE-99 induced EROD activity, CYP1A and ugt1ab gene expression, in ZFL. • BDE-99 induced the pregnane X receptor (Pxr) luciferase reporter gene system in ZFL. • BDE-47 did not show any effects in ZFL to induce CYP1A, ugt1ab, and EROD. • BDE-47 and -99 showed no induction of Rxr and Pxr pathways in ZFL cells.« less

Development of a Convenient In Vivo Hepatotoxin Assay Using a Transgenic Zebrafish Line with Liver-Specific DsRed Expression

PubMed Central

Zhang, Xiaoyan; Li, Caixia; Gong, Zhiyuan

2014-01-01

Previously we have developed a transgenic zebrafish line (LiPan) with liver-specific red fluorescent protein (DsRed) expression under the fabp10a promoter. Since red fluorescence in the liver greatly facilitates the observation of liver in live LiPan fry, we envision that the LiPan zebrafish may provide a useful tool in analyses of hepatotoxicity based on changes of liver red fluorescence intensity and size. In this study, we first tested four well-established hepatotoxins (acetaminophen, aspirin, isoniazid and phenylbutazone) in LiPan fry and demonstrated that these hepatotoxins could significantly reduce both liver red fluorescence and liver size in a dosage-dependent manner, thus the two measurable parameters could be used as indicators of hepatotoxicity. We then tested the LiPan fry with nine other chemicals including environmental toxicants and human drugs. Three (mefenamic acid, lindane, and arsenate) behave like hepatotoxins in reduction of liver red fluorescence, while three others (17β-estradiol, TCDD [2,3,7,8-tetrachlorodibenzo-p-dioxin] and NDMA [N-nitrosodimethylamine]) caused increase of liver red fluorescence and the liver size. Ethanol and two other chemicals, amoxicillin (antibiotics) and chlorphenamine (pain killer) did not resulted in significant changes of liver red fluorescence and liver size. By quantitative RT-PCR analysis, we found that the changes of red fluorescence intensity caused by different chemicals correlated to the changes of endogenous fabp10a RNA expression, indicating that the measured hepatotoxicity was related to fatty acid transportation and metabolism. Finally we tested a mixture of four hepatotoxins and observed a significant reduction of red fluorescence in the liver at concentrations below the lowest effective concentrations of individual hepatotoxins, suggesting that the transgenic zebrafish assay is capable of reporting compound hepatotoxicity effect from chemical mixtures. Thus, the LiPan transgenic fry provide a rapid and convenient in vivo hepatotoxicity assay that should be applicable to high-throughput hepatotoxicity test in drug screening as well as in biomonitoring environmental toxicants. PMID:24626481

Magnetite-Based Magnetoreceptor Cells in the Olfactory Organ of Rainbow Trout and Zebrafish

NASA Astrophysics Data System (ADS)

Kirschvink, J. L.; Cadiou, H.; Dixson, A. D.; Eder, S.; Kobayashi, A.; McNaughton, P. A.; Muhamad, A. N.; Raub, T. D.; Walker, M. M.; Winklhofer, M.; Yuen, B. B.

2011-12-01

Many vertebrate and invertebrate animals have a geomagnetic sensory system, but the biophysics and anatomy of how magnetic stimuli are transduced to the nervous system is a challenging problem. Previous work in our laboratories identified single-domain magnetite chains in olfactory epithelium in cells proximal to the ros V nerve, which, in rainbow trout, responds to magnetic fields. Our objectives are to characterize these magnetite-containing cells and determine whether they form part of the mechanism of magnetic field transduction in teleost fishes, as a model for other Vertebrates. Using a combination of reflection mode confocal microscopy and a Prussian Blue technique modified to stain specifically for magnetite, our Auckland group estimated that both juvenile rainbow trout (ca. 7 cm total length) olfactory rosettes have ~200 magnetite-containing cells. The magnetite present in two types of cells within the olfactory epithelium appears to be arranged in intracellular chains. All of our groups (Munich, Auckland, Cambridge and Caltech) have obtained different types of structural evidence that magnetite chains closely associate with the plasma membrane in the cells, even in disaggregated tissues. In addition, our Cambridge group used Ca2+ imaging to demonstrate a clear response by individual magnetite-containing cells to a step change in the intensity of the external magnetic field and a slow change in Ca2+ activity when the external magnetic field was cancelled. In the teleost, zebrafish (Danio rerio), a small (~4 cm adult length in captivity) genetic and developmental biology model organism, our Caltech group detected ferromagnetic material throughout the body, but concentrated in the rostral trunk, using NRM and IRM scans of whole adults. Our analysis suggests greater than one million, 80-100 nm crystals, with Lowrie-Fuller curves strongly consistent with single-domain magnetite in 100-100,000 magnetocytes. Ferromagentic resonance (FMR) spectra show crystals with narrow particle size distribution concentrated in the trunk, similar to biogenic magnetofossils. In SQUID microscopy images numerous dipole spots are widely distributed throughout the flank, not correlated with skin pigments or the spinal cord and neural arches. We interpret this to indicate a lateral line location for trunk magnetite in zebrafish. In contrast to trout in which rock magnetic experiments and TEM suggest highly interacting bundled ropes of crystals, similar to those in the large magnetotactic bacterium, M. bavaricum, zebrafish magnetic aggregates apparently arrange in clumps or mixed chains and clumps. We report trout olfactory epithelium containing magnetite magnetoreceptors that transduce the external magnetic field, then encode and transmit it to the brain, while zebrafish contain magnetite in the lateral line region.

Zebrafish Health Conditions in the China Zebrafish Resource Center and 20 Major Chinese Zebrafish Laboratories.

PubMed

Liu, Liyue; Pan, Luyuan; Li, Kuoyu; Zhang, Yun; Zhu, Zuoyan; Sun, Yonghua

2016-07-01

In China, the use of zebrafish as an experimental animal in the past 15 years has widely expanded. The China Zebrafish Resource Center (CZRC), which was established in 2012, is becoming one of the major resource centers in the global zebrafish community. Large-scale use and regular exchange of zebrafish resources have put forward higher requirements on zebrafish health issues in China. This article reports the current aquatic infrastructure design, animal husbandry, and health-monitoring programs in the CZRC. Meanwhile, through a survey of 20 Chinese zebrafish laboratories, we also describe the current health status of major zebrafish facilities in China. We conclude that it is of great importance to establish a widely accepted health standard and health-monitoring strategy in the Chinese zebrafish research community.

Brain selective transgene expression in zebrafish using an NRSE derived motif

PubMed Central

Bergeron, Sadie A.; Hannan, Markus C.; Codore, Hiba; Fero, Kandice; Li, Grace H.; Moak, Zachary; Yokogawa, Tohei; Burgess, Harold A.

2012-01-01

Transgenic technologies enable the manipulation and observation of circuits controlling behavior by permitting expression of genetically encoded reporter genes in neurons. Frequently though, neuronal expression is accompanied by transgene expression in non-neuronal tissues, which may preclude key experimental manipulations, including assessment of the contribution of neurons to behavior by ablation. To better restrict transgene expression to the nervous system in zebrafish larvae, we have used DNA sequences derived from the neuron-restrictive silencing element (NRSE). We find that one such sequence, REx2, when used in conjunction with several basal promoters, robustly suppresses transgene expression in non-neuronal tissues. Both in transient transgenic experiments and in stable enhancer trap lines, suppression is achieved without compromising expression within the nervous system. Furthermore, in REx2 enhancer trap lines non-neuronal expression can be de-repressed by knocking down expression of the NRSE binding protein RE1-silencing transcription factor (Rest). In one line, we show that the resulting pattern of reporter gene expression coincides with that of the adjacent endogenous gene, hapln3. We demonstrate that three common basal promoters are susceptible to the effects of the REx2 element, suggesting that this method may be useful for confining expression from many other promoters to the nervous system. This technique enables neural specific targeting of reporter genes and thus will facilitate the use of transgenic methods to manipulate circuit function in freely behaving larvae. PMID:23293587

Anti-cancer Effect of Luminacin, a Marine Microbial Extract, in Head and Neck Squamous Cell Carcinoma Progression via Autophagic Cell Death.

PubMed

Shin, Yoo Seob; Cha, Hyun Young; Lee, Bok-Soon; Kang, Sung Un; Hwang, Hye Sook; Kwon, Hak Cheol; Kim, Chul-Ho; Choi, Eun Chang

2016-04-01

The purpose of this study is to determine whether luminacin, a marine microbial extract from the Streptomyces species, has anti-tumor effects on head and neck squamous cell carcinoma (HNSCC) cell lines via autophagic cell death. Inhibition of cell survival and increased cell death was measured using cell viability, colony forming, and apoptosis assays. Migration and invasion abilities of head and cancer cells were evaluated using wound healing, scattering, and invasion assays. Changes in the signal pathway related to autophagic cell death were investigated. Drug toxicity of luminacin was examined in in vitro HaCaT cells and an in vivo zebrafish model. Luminacin showed potent cytotoxicity in HNSCC cells in cell viability, colony forming, and fluorescence-activated cell sorting analysis. In vitro migration and invasion of HNSCC cells were attenuated by luminacin treatment. Combined with Beclin-1 and LC3B, Luminacin induced autophagic cell death in head and neck cancer cells. In addition, in a zebrafish model and human keratinocyte cell line used for toxicity testing, luminacin treatment with a cytotoxic concentration to HNSCC cells did not cause toxicity. Taken together, these results demonstrate that luminacin induces the inhibition of growth and cancer progression via autophagic cell death in HNSCC cell lines, indicating a possible alternative chemotherapeutic approach for treatment of HNSCC.

Intratumoral genetic heterogeneity in metastatic melanoma is accompanied by variation in malignant behaviors.

PubMed

Anaka, Matthew; Hudson, Christopher; Lo, Pu-Han; Do, Hongdo; Caballero, Otavia L; Davis, Ian D; Dobrovic, Alexander; Cebon, Jonathan; Behren, Andreas

2013-10-11

Intratumoral heterogeneity is a major obstacle for the treatment of cancer, as the presence of even minor populations that are insensitive to therapy can lead to disease relapse. Increased clonal diversity has been correlated with a poor prognosis for cancer patients, and we therefore examined genetic, transcriptional, and functional diversity in metastatic melanoma. Amplicon sequencing and SNP microarrays were used to profile somatic mutations and DNA copy number changes in multiple regions from metastatic lesions. Clonal genetic and transcriptional heterogeneity was also assessed in single cell clones from early passage cell lines, which were then subjected to clonogenicity and drug sensitivity assays. MAPK pathway and tumor suppressor mutations were identified in all regions of the melanoma metastases analyzed. In contrast, we identified copy number abnormalities present in only some regions in addition to homogeneously present changes, suggesting ongoing genetic evolution following metastatic spread. Copy number heterogeneity from a tumor was represented in matched cell line clones, which also varied in their clonogenicity and drug sensitivity. Minor clones were identified based on dissimilarity to the parental cell line, and these clones were the most clonogenic and least sensitive to drugs. Finally, treatment of a polyclonal cell line with paclitaxel to enrich for drug-resistant cells resulted in the adoption of a gene expression profile with features of one of the minor clones, supporting the idea that these populations can mediate disease relapse. Our results support the hypothesis that minor clones might have major consequences for patient outcomes in melanoma.

Genetic resistance to rhabdovirus infection in teleost fish is paralleled to the derived cell resistance status.

PubMed

Verrier, Eloi R; Langevin, Christelle; Tohry, Corinne; Houel, Armel; Ducrocq, Vincent; Benmansour, Abdenour; Quillet, Edwige; Boudinot, Pierre

2012-01-01

Genetic factors of resistance and predisposition to viral diseases explain a significant part of the clinical variability observed within host populations. Predisposition to viral diseases has been associated to MHC haplotypes and T cell immunity, but a growing repertoire of innate/intrinsic factors are implicated in the genetic determinism of the host susceptibility to viruses. In a long-term study of the genetics of host resistance to fish rhabdoviruses, we produced a collection of double-haploid rainbow trout clones showing a wide range of susceptibility to Viral Hemorrhagic Septicemia Virus (VHSV) waterborne infection. The susceptibility of fibroblastic cell lines derived from these clonal fish was fully consistent with the susceptibility of the parental fish clones. The mechanisms determining the host resistance therefore did not associate with specific host immunity, but rather with innate or intrinsic factors. One cell line was resistant to rhabdovirus infection due to the combination of an early interferon IFN induction--that was not observed in the susceptible cells--and of yet unknown factors that hamper the first steps of the viral cycle. The implication of IFN was well consistent with the wide range of resistance of this genetic background to VSHV and IHNV, to the birnavirus IPNV and the orthomyxovirus ISAV. Another cell line was even more refractory to the VHSV infection through different antiviral mechanisms. This collection of clonal fish and isogenic cell lines provides an interesting model to analyze the relative contribution of antiviral pathways to the resistance to different viruses.

Generation of human cortical neurons from a new immortal fetal neural stem cell line.

PubMed

Cacci, E; Villa, A; Parmar, M; Cavallaro, M; Mandahl, N; Lindvall, O; Martinez-Serrano, A; Kokaia, Z

2007-02-01

Isolation and expansion of neural stem cells (NSCs) of human origin are crucial for successful development of cell therapy approaches in neurodegenerative diseases. Different epigenetic and genetic immortalization strategies have been established for long-term maintenance and expansion of these cells in vitro. Here we report the generation of a new, clonal NSC (hc-NSC) line, derived from human fetal cortical tissue, based on v-myc immortalization. Using immunocytochemistry, we show that these cells retain the characteristics of NSCs after more than 50 passages. Under proliferation conditions, when supplemented with epidermal and basic fibroblast growth factors, the hc-NSCs expressed neural stem/progenitor cell markers like nestin, vimentin and Sox2. When growth factors were withdrawn, proliferation and expression of v-myc and telomerase were dramatically reduced, and the hc-NSCs differentiated into glia and neurons (mostly glutamatergic and GABAergic, as well as tyrosine hydroxylase-positive, presumably dopaminergic neurons). RT-PCR analysis showed that the hc-NSCs retained expression of Pax6, Emx2 and Neurogenin2, which are genes associated with regionalization and cell commitment in cortical precursors during brain development. Our data indicate that this hc-NSC line could be useful for exploring the potential of human NSCs to replace dead or damaged cortical cells in animal models of acute and chronic neurodegenerative diseases. Taking advantage of its clonality and homogeneity, this cell line will also be a valuable experimental tool to study the regulatory role of intrinsic and extrinsic factors in human NSC biology.

(Eco)toxicological effects of 2,4,7,9-tetramethyl-5-decyne-4,7-diol (TMDD) in zebrafish (Danio rerio) and permanent fish cell cultures.

PubMed

Vincze, Krisztina; Gehring, Martin; Braunbeck, Thomas

2014-01-01

2,4,7,9-tetramethyl-5-decyne-4,7-diol (TMDD) is a high-production volume chemical used in paper, ink, pesticide, and adhesive industries as a wetting and anti-foaming agent. The physicochemical properties and slow biodegradation rate of TMDD indicate a low bioaccumulation potential but a high prevalence in the environment. As a consequence, TMDD has been detected in several European rivers in the nanogram per liter and lower microgram per liter range; however, its environmental risk to aquatic organisms is considered low. Recent studies almost exclusively focused on acute effects by TMDD, little is known about cytotoxic and genotoxic effects, reproduction and developmental toxicity, endocrine disruption, and any kind of long-term toxicity and carcinogenicity so far. The present study aims to provide more specific baseline information on the ecotoxicological effects of TMDD in fish. For this end, cyto- and genotoxicity assays were carried out in vitro with the permanent fish cell line RTL-W1; in addition, in vivo studies were conducted with the early life stages of zebrafish (Danio rerio) in order to fill the data gaps in developmental toxicity and endocrine disruption. TMDD showed a cytotoxic and slight genotoxic potential in fish cell lines; moreover, various sublethal and lethal effects could be detected in developing zebrafish embryos. There was no evidence of endocrine-disrupting effects by TMDD; however, mortality following prolonged exposure to TMDD during fish sexual development test was clearly higher than mortality in the fish embryo test after 96-h exposure. Our results thus confirmed previous findings of laboratory screening tests, suggesting short-term toxic effects of TMDD in the intermediate, and long-term effects in the lower milligram per liter range.

The Importance of Stochastic Effects for Explaining Entrainment in the Zebrafish Circadian Clock.

PubMed

Heussen, Raphaela; Whitmore, David

2015-01-01

The circadian clock plays a pivotal role in modulating physiological processes and has been implicated, either directly or indirectly, in a range of pathological states including cancer. Here we investigate how the circadian clock is entrained by external cues such as light. Working with zebrafish cell lines and combining light pulse experiments with simulation efforts focused on the role of synchronization effects, we find that even very modest doses of light exposure are sufficient to trigger some entrainment, whereby a higher light intensity or duration correlates with strength of the circadian signal. Moreover, we observe in the simulations that stochastic effects may be considered an essential feature of the circadian clock in order to explain the circadian signal decay in prolonged darkness, as well as light initiated resynchronization as a strong component of entrainment.

Unregulated smooth-muscle myosin in human intestinal neoplasia.

PubMed

Alhopuro, Pia; Phichith, Denis; Tuupanen, Sari; Sammalkorpi, Heli; Nybondas, Miranda; Saharinen, Juha; Robinson, James P; Yang, Zhaohui; Chen, Li-Qiong; Orntoft, Torben; Mecklin, Jukka-Pekka; Järvinen, Heikki; Eng, Charis; Moeslein, Gabriela; Shibata, Darryl; Houlston, Richard S; Lucassen, Anneke; Tomlinson, Ian P M; Launonen, Virpi; Ristimäki, Ari; Arango, Diego; Karhu, Auli; Sweeney, H Lee; Aaltonen, Lauri A

2008-04-08

A recent study described a recessive ATPase activating germ-line mutation in smooth-muscle myosin (smmhc/myh11) underlying the zebrafish meltdown (mlt) phenotype. The mlt zebrafish develops intestinal abnormalities reminiscent of human Peutz-Jeghers syndrome (PJS) and juvenile polyposis (JP). To examine the role of MYH11 in human intestinal neoplasia, we searched for MYH11 mutations in patients with colorectal cancer (CRC), PJS and JP. We found somatic protein-elongating frameshift mutations in 55% of CRCs displaying microsatellite instability and in the germ-line of one individual with PJS. Additionally, two somatic missense mutations were found in one microsatellite stable CRC. These two missense mutations, R501L and K1044N, and the frameshift mutations were functionally evaluated. All mutations resulted in unregulated molecules displaying constitutive motor activity, similar to the mutant myosin underlying mlt. Thus, MYH11 mutations appear to contribute also to human intestinal neoplasia. Unregulated MYH11 may affect the cellular energy balance or disturb cell lineage decisions in tumor progenitor cells. These data challenge our view on MYH11 as a passive differentiation marker functioning in muscle contraction and add to our understanding of intestinal neoplasia.

Labelling and targeted ablation of specific bipolar cell types in the zebrafish retina

PubMed Central

2009-01-01

Background Development of a functional retina depends on regulated differentiation of several types of neurons and generation of a highly complex network between the different types of neurons. In addition, each type of retinal neuron includes several distinct morphological types. Very little is known about the mechanisms responsible for generating this diversity of retinal neurons, which may also display specific patterns of regional distribution. Results In a screen in zebrafish, using a trapping vector carrying an engineered yeast Gal4 transcription activator and a UAS:eGFP reporter cassette, we have identified two transgenic lines of zebrafish co-expressing eGFP and Gal4 in specific subsets of retinal bipolar cells. The eGFP-labelling facilitated analysis of axon terminals within the inner plexiform layer of the adult retina and showed that the fluorescent bipolar cells correspond to previously defined morphological types. Strong regional restriction of eGFP-positive bipolar cells to the central part of the retina surrounding the optic nerve was observed in adult zebrafish. Furthermore, we achieved specific ablation of the labelled bipolar cells in 5 days old larvae, using a bacterial nitroreductase gene under Gal4-UAS control in combination with the prodrug metronidazole. Following prodrug treatment, nitroreductase expressing bipolar cells were efficiently ablated without affecting surrounding retina architecture, and recovery occurred within a few days due to increased generation of new bipolar cells. Conclusion This report shows that enhancer trapping can be applied to label distinct morphological types of bipolar cells in the zebrafish retina. The genetic labelling of these cells yielded co-expression of a modified Gal4 transcription activator and the fluorescent marker eGFP. Our work also demonstrates the potential utility of the Gal4-UAS system for induction of other transgenes, including a bacterial nitroreductase fusion gene, which can facilitate analysis of bipolar cell differentiation and how the retina recovers from specific ablation of these cells. PMID:19712466

Genetic tagging of tumor cells with retrovirus vectors: Clonal analysis of tumor growth and metastasis in vivo

DOE Office of Scientific and Technical Information (OSTI.GOV)

Korczak; Robson, I.B.; Lamarche, C.

1988-08-01

Retrovirus vector infection was used to introduce large numbers of unique genetic markers into tumor cell populations for the purpose of analyzing comparative changes in the clonal composition of metastatic versus that of nonmetastatic tumors during their progressive growth in vivo. The cell lines were SP1, a nonmetastatic, aneuploid mouse mammary adenocarcinoma, and SP1HU9L, a metastatic variant of SP1. Cells were infected with ..delta..e..delta..rhoMoTn, a replication-defective retrovirus vector which possesses the dominant selectable neo gene and crippled long terminal repeats. G418/sup r/ colonies were obtained at a frequency of 4 x 10/sup -3/. Southern blot analysis of a number ofmore » clones provided evidence of random and heritable integration of one or two copies of the proviral DNA. Clonal equation of primary tumor growth and the nature of lineage relationships among spontaneous metastases and primary tumors were analyzed by subcutaneously injecting 10/sup 5/ cells from a pooled mixture of 3.6 x 10/sup 2/ G418/sup r/ SP1HU9L or 10/sup 4/ G418/sup r/ SP1 colonies into syngeneic CBA/J mice. The most striking finding was the relative clonal homogeneity of advanced primary tumors; they invariably consisted of a small number (less than 10) of distinct clones despite the fact that hundreds of thousands of uniquely marked clones had been injected.« less

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells (HSPCs) in Developing Zebrafish Embryos.

PubMed

Berrun, A C; Stachura, D L

2017-11-30

Hematopoiesis is an essential cellular process in which hematopoietic stem and progenitor cells (HSPCs) differentiate into the multitude of different cell lineages that comprise mature blood. Isolation and identification of these HSPCs is difficult because they are defined ex post facto; they can only be defined after their differentiation into specific cell lineages. Over the past few decades, the zebrafish (Danio rerio) has become a model organism to study hematopoiesis. Zebrafish embryos develop ex utero, and by 48 h post-fertilization (hpf) have generated definitive HSPCs. Assays to assess HSPC differentiation and proliferation capabilities have been developed, utilizing transplantation and subsequent reconstitution of the hematopoietic system in addition to visualizing specialized transgenic lines with confocal microscopy. However, these assays are cost prohibitive, technically difficult, and time consuming for many laboratories. Development of an in vitro model to assess HSPCs would be cost effective, quicker, and present fewer difficulties compared to previously described methods, allowing laboratories to quickly assess mutagenesis and drug screens that affect HSPC biology. This novel in vitro assay to assess HSPCs is performed by plating dissociated whole zebrafish embryos and adding exogenous factors that promote only HSPC differentiation and proliferation. Embryos are dissociated into single cells and plated with HSPC-supportive colony stimulating factors that cause them to generate colony forming units (CFUs) that arise from a single progenitor cell. These assays should allow more careful examination of the molecular pathways responsible for HSPC proliferation, differentiation, and regulation, which will allow researchers to understand the underpinnings of vertebrate hematopoiesis and its dysregulation during disease.

Characterization of a hypoxia-response element in the Epo locus of the pufferfish, Takifugu rubripes.

PubMed

Kulkarni, Rashmi P; Tohari, Sumanty; Ho, Adrian; Brenner, Sydney; Venkatesh, Byrappa

2010-06-01

Animals respond to hypoxia by increasing synthesis of the glycoprotein hormone erythropoietin (Epo) which in turn stimulates the production of red blood cells. The gene encoding Epo has been recently cloned in teleost fishes such as the pufferfish Takifugu rubripes (fugu) and zebrafish (Danio rerio). It has been shown that the transcription levels of Epo in teleost fishes increase in response to anemia or hypoxia in a manner similar to its human ortholog. However, the cis-regulatory element(s) mediating the hypoxia response of Epo gene in fishes has not been identified. In the present study, using the human hepatoma cell line (Hep3B), we have identified and characterized a hypoxia response element (HRE) in the fugu Epo locus. The sequence of the fugu HRE (ACGTGCTG) is identical to that of the HRE in the human EPO locus. However, unlike the HRE in the mammalian Epo locus, which is located in the 3' region of the gene, the fugu HRE is located in the 5' flanking region and on the opposite strand of DNA. This HRE is conserved in other teleosts such as Tetraodon and zebrafish in a similar location. A 365-bp fragment containing the fugu HRE was able to drive GFP expression in the liver of transgenic zebrafish. However, we could not ascertain if the expression of transgene is induced by hypoxia in vivo due to the low and variable levels of GFP expression in transgenic zebrafish. Our investigations also revealed that the Epo locus has experienced extensive rearrangements during vertebrate evolution. Copyright © 2010 Elsevier B.V. All rights reserved.

ngs (notochord granular surface) gene encodes a novel type of intermediate filament family protein essential for notochord maintenance in zebrafish.

PubMed

Tong, Xiangjun; Xia, Zhidan; Zu, Yao; Telfer, Helena; Hu, Jing; Yu, Jingyi; Liu, Huan; Zhang, Quan; Sodmergen; Lin, Shuo; Zhang, Bo

2013-01-25

The notochord is an important organ involved in embryonic patterning and locomotion. In zebrafish, the mature notochord consists of a single stack of fully differentiated, large vacuolated cells called chordocytes, surrounded by a single layer of less differentiated notochordal epithelial cells called chordoblasts. Through genetic analysis of zebrafish lines carrying pseudo-typed retroviral insertions, a mutant exhibiting a defective notochord with a granular appearance was isolated, and the corresponding gene was identified as ngs (notochord granular surface), which was specifically expressed in the notochord. In the mutants, the notochord started to degenerate from 32 hours post-fertilization, and the chordocytes were then gradually replaced by smaller cells derived from chordoblasts. The granular notochord phenotype was alleviated by anesthetizing the mutant embryos with tricaine to prevent muscle contraction and locomotion. Phylogenetic analysis showed that ngs encodes a new type of intermediate filament (IF) family protein, which we named chordostatin based on its function. Under the transmission electron microcopy, bundles of 10-nm-thick IF-like filaments were enriched in the chordocytes of wild-type zebrafish embryos, whereas the chordocytes in ngs mutants lacked IF-like structures. Furthermore, chordostatin-enhanced GFP (EGFP) fusion protein assembled into a filamentous network specifically in chordocytes. Taken together, our work demonstrates that ngs encodes a novel type of IF protein and functions to maintain notochord integrity for larval development and locomotion. Our work sheds light on the mechanisms of notochord structural maintenance, as well as the evolution and biological function of IF family proteins.

ngs (Notochord Granular Surface) Gene Encodes a Novel Type of Intermediate Filament Family Protein Essential for Notochord Maintenance in Zebrafish*

PubMed Central

Tong, Xiangjun; Xia, Zhidan; Zu, Yao; Telfer, Helena; Hu, Jing; Yu, Jingyi; Liu, Huan; Zhang, Quan; Sodmergen; Lin, Shuo; Zhang, Bo

2013-01-01

The notochord is an important organ involved in embryonic patterning and locomotion. In zebrafish, the mature notochord consists of a single stack of fully differentiated, large vacuolated cells called chordocytes, surrounded by a single layer of less differentiated notochordal epithelial cells called chordoblasts. Through genetic analysis of zebrafish lines carrying pseudo-typed retroviral insertions, a mutant exhibiting a defective notochord with a granular appearance was isolated, and the corresponding gene was identified as ngs (notochord granular surface), which was specifically expressed in the notochord. In the mutants, the notochord started to degenerate from 32 hours post-fertilization, and the chordocytes were then gradually replaced by smaller cells derived from chordoblasts. The granular notochord phenotype was alleviated by anesthetizing the mutant embryos with tricaine to prevent muscle contraction and locomotion. Phylogenetic analysis showed that ngs encodes a new type of intermediate filament (IF) family protein, which we named chordostatin based on its function. Under the transmission electron microcopy, bundles of 10-nm-thick IF-like filaments were enriched in the chordocytes of wild-type zebrafish embryos, whereas the chordocytes in ngs mutants lacked IF-like structures. Furthermore, chordostatin-enhanced GFP (EGFP) fusion protein assembled into a filamentous network specifically in chordocytes. Taken together, our work demonstrates that ngs encodes a novel type of IF protein and functions to maintain notochord integrity for larval development and locomotion. Our work sheds light on the mechanisms of notochord structural maintenance, as well as the evolution and biological function of IF family proteins. PMID:23132861

Zebrafish ( Danio rerio) as a model for investigating the safety of GM feed ingredients (soya and maize); performance, stress response and uptake of dietary DNA sequences.

PubMed

Sissener, Nini H; Johannessen, Lene E; Hevrøy, Ernst M; Wiik-Nielsen, Christer R; Berdal, Knut G; Nordgreen, Andreas; Hemre, Gro-Ingunn

2010-01-01

A 20-d zebrafish (Danio rerio) feeding trial, in which a near doubling of fish weight was achieved, was conducted with GM feed ingredients to evaluate feed intake, growth, stress response and uptake of dietary DNA. A partial aim of the study was to assess zebrafish as a model organism in GM safety assessments. Roundup Ready soya (RRS), YieldGard Bt maize (MON810) and their non-modified, maternal, near-isogenic lines were used in a 2 x 2 factorial design. Soya variety and maize variety were the main factors, both with two levels; non-GM and GM. Compared with fish fed non-GM maize, those fed GM maize exhibited significantly better growth, had lower mRNA transcription levels of superoxide dismutase (SOD)-1 and a tendency (non-significant) towards lower transcription of heat shock protein 70 in liver. Sex of the fish and soya variety had significant interaction effects on total RNA yield from the whole liver and transcription of SOD-1, suggesting that some diet component affecting males and females differently was present in different levels in the GM and the non-GM soya used in the present study. Dietary DNA sequences were detected in all of the organs analysed, but not all of the samples. Soya and maize rubisco (non-transgenic, multicopy genes) were most frequently detected, while MON810 transgenic DNA fragments were detected in some samples and RRS fragments were not detected. In conclusion, zebrafish shows promise as a model for this application.

Endotoxin molecule lipopolysaccharide-induced zebrafish inflammation model: a novel screening method for anti-inflammatory drugs.

PubMed

Yang, Li-Ling; Wang, Guo-Quan; Yang, Li-Mei; Huang, Zhi-Bing; Zhang, Wen-Qing; Yu, Lin-Zhong

2014-02-21

Lipopolysaccharide (LPS), an endotoxin molecule, has been used to induce inflammatory responses. In this study, LPS was used to establish an in vivo inflammation model in zebrafish for drug screening. We present an experimental method that conveniently and rapidly assesses the anti-inflammatory properties of drugs. The yolks of 3-day post-fertilization (dpf) larvae were injected with 0.5 mg/mL LPS to induce fatal inflammation. After LPS stimulation, macrophages were tracked by NR and SB staining and neutrophil migration was observed using the MPO:GFP line. Larval mortality was used as the primary end-point. Expression levels of key cytokines involved in the inflammatory response including IL-1β, IL-6, and TNF-α, were measured using quantitative reverse transcription polymerase chain reaction (RT-PCR). Macrophages and neutrophils were both recruited to the LPS-injected site during the inflammatory response. Mortality was increased by LPS in a dose-dependent manner within 48 h. Analyses of IL-1β, IL-6, and TNF-α expression levels revealed the upregulation of the inflammatory response in the LPS-injected larvae. Further, the anti-inflammatory activity of chlorogenic acid (CA) was evaluated in this zebrafish model to screen for anti-inflammatory drugs. A preliminary result showed that CA revealed a similar effect as the corticosteroid dexamethasone (DEX), which was used as a positive control, by inhibiting macrophage and neutrophil recruitment to the LPS site and improving survival. Our results suggest that this zebrafish screening model could be applied to study inflammation-mediated diseases. Moreover, the Traditional Chinese Medicine CA displays potential anti-inflammatory activity.

Fast Homozygosity Mapping and Identification of a Zebrafish ENU-Induced Mutation by Whole-Genome Sequencing

PubMed Central

Voz, Marianne L.; Coppieters, Wouter; Manfroid, Isabelle; Baudhuin, Ariane; Von Berg, Virginie; Charlier, Carole; Meyer, Dirk; Driever, Wolfgang; Martial, Joseph A.; Peers, Bernard

2012-01-01

Forward genetics using zebrafish is a powerful tool for studying vertebrate development through large-scale mutagenesis. Nonetheless, the identification of the molecular lesion is still laborious and involves time-consuming genetic mapping. Here, we show that high-throughput sequencing of the whole zebrafish genome can directly locate the interval carrying the causative mutation and at the same time pinpoint the molecular lesion. The feasibility of this approach was validated by sequencing the m1045 mutant line that displays a severe hypoplasia of the exocrine pancreas. We generated 13 Gb of sequence, equivalent to an eightfold genomic coverage, from a pool of 50 mutant embryos obtained from a map-cross between the AB mutant carrier and the WIK polymorphic strain. The chromosomal region carrying the causal mutation was localized based on its unique property to display high levels of homozygosity among sequence reads as it derives exclusively from the initial AB mutated allele. We developed an algorithm identifying such a region by calculating a homozygosity score along all chromosomes. This highlighted an 8-Mb window on chromosome 5 with a score close to 1 in the m1045 mutants. The sequence analysis of all genes within this interval revealed a nonsense mutation in the snapc4 gene. Knockdown experiments confirmed the assertion that snapc4 is the gene whose mutation leads to exocrine pancreas hypoplasia. In conclusion, this study constitutes a proof-of-concept that whole-genome sequencing is a fast and effective alternative to the classical positional cloning strategies in zebrafish. PMID:22496837

Establishment of two new cell lines derived from human breast carcinomas with HER-2/neu amplification.

PubMed Central

Meltzer, P.; Leibovitz, A.; Dalton, W.; Villar, H.; Kute, T.; Davis, J.; Nagle, R.; Trent, J.

1991-01-01

Two human cell lines (UACC-812 and 893), both containing significant amplification of the HER-2/neu gene, were established from biopsy specimens of breast carcinomas. One patient had Stage II breast carcinoma; the other had metastatic disease. Characterisation of these lines has revealed that both are highly aneuploid containing multiple clonal chromosome alterations, have doubling times near 100 h, and are oestrogen and progesterone receptor negative. Electron microscopy demonstrates that both lines contain numerous microvilli, cytoplasmic filaments, multivesicular bodies, and desmosomes. Immunoblot analysis for P-glycoprotein using the monoclonal antibody C219 was negative for both patient cell lines. These relatively rare cell lines may represent a useful model to investigate human breast carcinomas. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:1674877

Role of TGR-B1-Mediated Down Regulation of NF-kB/Rel Activity During Growth Arrest of Breast Cancer Cells

DTIC Science & Technology

2001-05-01

gallate ( EGCG ), which has been shown to inhibit the induction of NF-KB and growth of breast cancer cell lines in vitro. EGCG reduced NF-KB levels in the...demonstrated activation of NF-KB is induced upon over-expression of Her-2/neu. Thus, studies were initiated with green tea pholyphenol, epigallocatechin -3...NF639 cell line derived from an MMTV-Her-2/neu mouse tumor. NF639 clonal isolates resistant to EGCG appear to display elevated levels of NF-KB. Overall

Detection and isolation of rare cells by 2-step enrichment high-speed flow cytometry/cell sorting and single cell LEAP laser ablation

NASA Astrophysics Data System (ADS)

Zordan, M. D.; Leary, James F.

2011-02-01

The clonal isolation of rare cells, especially cancer and stem cells, in a population is important to the development of improved medical treatment. We have demonstrated that the Laser-Enabled Analysis and Processing (LEAP, Cyntellect Inc., San Diego, CA) instrument can be used to efficiently produce single cell clones by photoablative dilution. Additionally, we have also shown that cells present at low frequencies can be cloned by photoablative dilution after they are pre-enriched by flow cytometry based cell sorting. Circulating tumor cells were modeled by spiking isolated peripheral blood cells with cells from the lung carcinoma cell line A549. Flow cytometry based cell sorting was used to perform an enrichment sort of A549 cells directly into a 384 well plate. Photoablative dilution was performed with the LEAPTM instrument to remove any contaminating cells, and clonally isolate 1 side population cell per well. We were able to isolate and grow single clones of side population cells using this method at greater than 90% efficiency. We have developed a 2 step method that is able to perform the clonal isolation of rare cells based on a medically relevant functional phenotype.

Identification and removal of low-complexity sites in allele-specific analysis of ChIP-seq data.

PubMed

Waszak, Sebastian M; Kilpinen, Helena; Gschwind, Andreas R; Orioli, Andrea; Raghav, Sunil K; Witwicki, Robert M; Migliavacca, Eugenia; Yurovsky, Alisa; Lappalainen, Tuuli; Hernandez, Nouria; Reymond, Alexandre; Dermitzakis, Emmanouil T; Deplancke, Bart

2014-01-15

High-throughput sequencing technologies enable the genome-wide analysis of the impact of genetic variation on molecular phenotypes at unprecedented resolution. However, although powerful, these technologies can also introduce unexpected artifacts. We investigated the impact of library amplification bias on the identification of allele-specific (AS) molecular events from high-throughput sequencing data derived from chromatin immunoprecipitation assays (ChIP-seq). Putative AS DNA binding activity for RNA polymerase II was determined using ChIP-seq data derived from lymphoblastoid cell lines of two parent-daughter trios. We found that, at high-sequencing depth, many significant AS binding sites suffered from an amplification bias, as evidenced by a larger number of clonal reads representing one of the two alleles. To alleviate this bias, we devised an amplification bias detection strategy, which filters out sites with low read complexity and sites featuring a significant excess of clonal reads. This method will be useful for AS analyses involving ChIP-seq and other functional sequencing assays. The R package abs filter for library clonality simulations and detection of amplification-biased sites is available from http://updepla1srv1.epfl.ch/waszaks/absfilter

Viral Diseases in Zebrafish: What Is Known and Unknown

PubMed Central

Crim, Marcus J.; Riley, Lela K.

2013-01-01

Naturally occurring viral infections have the potential to introduce confounding variability that leads to invalid and misinterpreted data. Whereas the viral diseases of research rodents are well characterized and closely monitored, no naturally occurring viral infections have been characterized for the laboratory zebrafish (Danio rerio), an increasingly important biomedical research model. Despite the ignorance about naturally occurring zebrafish viruses, zebrafish models are rapidly expanding in areas of biomedical research where the confounding effects of unknown infectious agents present a serious concern. In addition, many zebrafish research colonies remain linked to the ornamental (pet) zebrafish trade, which can contribute to the introduction of new pathogens into research colonies, whereas mice used for research are purpose bred, with no introduction of new mice from the pet industry. Identification, characterization, and monitoring of naturally occurring viruses in zebrafish are crucial to the improvement of zebrafish health, the reduction of unwanted variability, and the continued development of the zebrafish as a model organism. This article addresses the importance of identifying and characterizing the viral diseases of zebrafish as the scope of zebrafish models expands into new research areas and also briefly addresses zebrafish susceptibility to experimental viral infection and the utility of the zebrafish as an infection and immunology model. PMID:23382345

Characterization of cancer stem-like cells derived from a side population of a human gallbladder carcinoma cell line, SGC-996

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Xin-xing; Wang, Jian, E-mail: dr_wangjian@yahoo.com.cn; Wang, Hao-lu

2012-03-23

Highlights: Black-Right-Pointing-Pointer We sorted SP cells from a human gallbladder carcinoma cell lines, SGC-996. Black-Right-Pointing-Pointer SP cells displayed higher proliferation and stronger clonal-generating capability. Black-Right-Pointing-Pointer SP cells showed more migratory and invasive abilities. Black-Right-Pointing-Pointer SP cells were more resistant and tumorigenic than non-SP counterparts. Black-Right-Pointing-Pointer ABCG2 might be a candidate as a marker for SP cells. -- Abstract: The cancer stem cell (CSC) hypothesis proposes that CSCs, which can renew themselves proliferate infinitely, and escape chemotherapy, become the root of recurrence and metastasis. Previous studies have verified that side population (SP) cells, characterized by their ability to efflux lipophilic substratemore » Hoechst 33342, to share many characteristics of CSCs in multiplying solid tumors. The purpose of this study was to sort SP cells from a human gallbladder carcinoma cell line, SGC-996 and to preliminarily identify the biological characteristics of SP cells from the cell line. Using flow cytometry we effectively sorted SP cells from the cell line SGC-996. SP cells not only displayed higher proliferative, stronger clonal-generating, more migratory and more invasive capacities, but showed stronger resistance. Furthermore, our experiments demonstrated that SP cells were more tumorigenic than non-SP counterparts in vivo. Real-time PCR analysis and immunocytochemistry showed that the expression of ATP-binding cassette subfamily G member 2 (ABCG2) was significantly higher in SP cells. Hence, these results collectively suggest that SP cells are progenitor/stem-like cells and ABCG2 might be a candidate marker for SP cells in human gallbladder cancer.« less

Use of the Zebrafish Larvae as a Model to Study Cigarette Smoke Condensate Toxicity

PubMed Central

Ellis, Lee D.; Soo, Evelyn C.; Achenbach, John C.; Morash, Michael G.; Soanes, Kelly H.

2014-01-01

The smoking of tobacco continues to be the leading cause of premature death worldwide and is linked to the development of a number of serious illnesses including heart disease, respiratory diseases, stroke and cancer. Currently, cell line based toxicity assays are typically used to gain information on the general toxicity of cigarettes and other tobacco products. However, they provide little information regarding the complex disease-related changes that have been linked to smoking. The ethical concerns and high cost associated with mammalian studies have limited their widespread use for in vivo toxicological studies of tobacco. The zebrafish has emerged as a low-cost, high-throughput, in vivo model in the study of toxicology. In this study, smoke condensates from 2 reference cigarettes and 6 Canadian brands of cigarettes with different design features were assessed for acute, developmental, cardiac, and behavioural toxicity (neurotoxicity) in zebrafish larvae. By making use of this multifaceted approach we have developed an in vivo model with which to compare the toxicity profiles of smoke condensates from cigarettes with different design features. This model system may provide insights into the development of smoking related disease and could provide a cost-effective, high-throughput platform for the future evaluation of tobacco products. PMID:25526262

A genetic screen for vascular mutants in zebrafish reveals dynamic roles for Vegf/Plcg1 signaling during artery development.

PubMed

Covassin, L D; Siekmann, A F; Kacergis, M C; Laver, E; Moore, J C; Villefranc, J A; Weinstein, B M; Lawson, N D

2009-05-15

In this work we describe a forward genetic approach to identify mutations that affect blood vessel development in the zebrafish. By applying a haploid screening strategy in a transgenic background that allows direct visualization of blood vessels, it was possible to identify several classes of mutant vascular phenotypes. Subsequent characterization of mutant lines revealed that defects in Vascular endothelial growth factor (Vegf) signaling specifically affected artery development. Comparison of phenotypes associated with different mutations within a functional zebrafish Vegf receptor-2 ortholog (referred to as kdr-like, kdrl) revealed surprisingly varied effects on vascular development. In parallel, we identified an allelic series of mutations in phospholipase c gamma 1 (plcg1). Together with in vivo structure-function analysis, our results suggest a requirement for Plcg1 catalytic activity downstream of receptor tyrosine kinases. We further find that embryos lacking both maternal and zygotic plcg1 display more severe defects in artery differentiation but are otherwise similar to zygotic mutants. Finally, we demonstrate through mosaic analysis that plcg1 functions autonomously in endothelial cells. Together our genetic analyses suggest that Vegf/Plcg1 signaling acts at multiple time points and in different signaling contexts to mediate distinct aspects of artery development.

Use of the zebrafish larvae as a model to study cigarette smoke condensate toxicity.

PubMed

Ellis, Lee D; Soo, Evelyn C; Achenbach, John C; Morash, Michael G; Soanes, Kelly H

2014-01-01

The smoking of tobacco continues to be the leading cause of premature death worldwide and is linked to the development of a number of serious illnesses including heart disease, respiratory diseases, stroke and cancer. Currently, cell line based toxicity assays are typically used to gain information on the general toxicity of cigarettes and other tobacco products. However, they provide little information regarding the complex disease-related changes that have been linked to smoking. The ethical concerns and high cost associated with mammalian studies have limited their widespread use for in vivo toxicological studies of tobacco. The zebrafish has emerged as a low-cost, high-throughput, in vivo model in the study of toxicology. In this study, smoke condensates from 2 reference cigarettes and 6 Canadian brands of cigarettes with different design features were assessed for acute, developmental, cardiac, and behavioural toxicity (neurotoxicity) in zebrafish larvae. By making use of this multifaceted approach we have developed an in vivo model with which to compare the toxicity profiles of smoke condensates from cigarettes with different design features. This model system may provide insights into the development of smoking related disease and could provide a cost-effective, high-throughput platform for the future evaluation of tobacco products.

A genetic screen for vascular mutants in zebrafish reveals dynamic roles for Vegf/Plcg1 signaling during artery development

PubMed Central

Covassin, L. D.; Siekmann, A. F.; Kacergis, M. C.; Laver, E.; Moore, J. C.; Villefranc, J. A.; Weinstein, B. M.; Lawson, N. D.

2009-01-01

In this work we describe a forward genetic approach to identify mutations that affect blood vessel development in the zebrafish. By applying a haploid screening strategy in a transgenic background that allows direct visualization of blood vessels, it was possible to identify several classes of mutant vascular phenotypes. Subsequent characterization of mutant lines revealed that defects in Vascular endothelial growth factor (Vegf) signaling specifically affected artery development. Comparison of phenotypes associated with different mutations within a functional zebrafish Vegf receptor-2 ortholog (referred to as kdr-like, kdrl) revealed surprisingly varied effects on vascular development. In parallel, we identified an allelic series of mutations in phospholipase c gamma 1 (plcg1). Together with in vivo structure-function analysis, our results suggest a requirement for Plcg1 catalytic activity downstream of receptor tyrosine kinases. We further find that embryos lacking both maternal and zygotic plcg1 display more severe defects in artery differentiation but are otherwise similar to zygotic mutants. Finally, we demonstrate through mosaic analysis that plcg1 functions autonomously in endothelial cells. Together our genetic analyses suggest that Vegf/Plcg1 signaling acts at multiple time points and in different signaling contexts to mediate distinct aspects of artery development. PMID:19269286

A novel subset of enteric neurons revealed by ptf1a:GFP in the developing zebrafish enteric nervous system.

PubMed

Uribe, Rosa A; Gu, Tiffany; Bronner, Marianne E

2016-03-01

The enteric nervous system, the largest division of the peripheral nervous system, is derived from vagal neural crest cells that invade and populate the entire length of the gut to form diverse neuronal subtypes. Here, we identify a novel population of neurons within the enteric nervous system of zebrafish larvae that express the transgenic marker ptf1a:GFP within the midgut. Genetic lineage analysis reveals that enteric ptf1a:GFP(+) cells are derived from the neural crest and that most ptf1a:GFP(+) neurons express the neurotransmitter 5HT, demonstrating that they are serotonergic. This transgenic line, Tg(ptf1a:GFP), provides a novel neuronal marker for a subpopulation of neurons within the enteric nervous system, and highlights the possibility that Ptf1a may act as an important transcription factor for enteric neuron development. © 2016 Wiley Periodicals, Inc.

Atomic Structure of the Cystic Fibrosis Transmembrane Conductance Regulator.

PubMed

Zhang, Zhe; Chen, Jue

2016-12-01

The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel evolved from the ATP-binding cassette (ABC) transporter family. In this study, we determined the structure of zebrafish CFTR in the absence of ATP by electron cryo-microscopy to 3.7 Å resolution. Human and zebrafish CFTR share 55% sequence identity, and 42 of the 46 cystic-fibrosis-causing missense mutational sites are identical. In CFTR, we observe a large anion conduction pathway lined by numerous positively charged residues. A single gate near the extracellular surface closes the channel. The regulatory domain, dephosphorylated, is located in the intracellular opening between the two nucleotide-binding domains (NBDs), preventing NBD dimerization and channel opening. The structure also reveals why many cystic-fibrosis-causing mutations would lead to defects either in folding, ion conduction, or gating and suggests new avenues for therapeutic intervention. Copyright © 2016 Elsevier Inc. All rights reserved.

Short-term developmental effects and potential mechanisms of azoxystrobin in larval and adult zebrafish (Danio rerio).

PubMed

Cao, Fangjie; Wu, Peizhuo; Huang, Lan; Li, Hui; Qian, Le; Pang, Sen; Qiu, Lihong

2018-05-01

Previous study indicated that azoxystrobin had high acute toxicity to zebrafish, and larval zebrafish were more sensitive to azoxystrobin than adult zebrafish. The objective of the present study was to investigate short-term developmental effects and potential mechanisms of azoxystrobin in larval and adult zebrafish. After zebrafish embryos and adults were exposed to 0.01, 0.05 and 0.20 mg/L azoxystrobin (equal to 25, 124 and 496 nM azoxystrobin, respectively) for 8 days, the lethal effect, physiological responses, liver histology, mitochondrial ultrastructure, and expression alteration of genes related to mitochondrial respiration, oxidative stress, cell apoptosis and innate immune response were determined. The results showed that there was no significant effect on larval and adult zebrafish after exposure to 0.01 mg/L azoxystrobin. However, increased ROS, MDA concentration and il1b in larval zebrafish, as well as increased il1b, il8 and cxcl-c1c in adult zebrafish were induced after exposure to 0.05 mg/L azoxystrobin. Reduced mitochondrial complex III activity and ATP concentration, increased SOD activity, ROS and MDA concentration, decreased cytb, as well as increased sod1, sod2, cat, il1b, il8 and cxcl-c1c were observed both in larval and adult zebrafish after exposure to 0.20 mg/L azoxystrobin; meanwhile, increased p53, bax, apaf1 and casp9, alteration of liver histology and mitochondrial ultrastructure in larval zebrafish, and alteration of mitochondrial ultrastructure in adult zebrafish were also induced. The results demonstrated that azoxytrobin induced short-term developmental effects on larval zebrafish and adult zebrafish, including mitochondrial dysfunction, oxidative stress, cell apoptosis and innate immune response. Statistical analysis indicated that azoxystrobin induced more negative effects on larval zebrafish, which might be the reason for the differences of developmental toxicity between larval and adult zebrafish caused by azoxystrobin. These results provided a new insight into potential mechanisms of azoxystrobin in larval zebrafish and adult zebrafish. Copyright © 2018 Elsevier B.V. All rights reserved.

19-nor vitamin-D analogs: a new class of potent inhibitors of proliferation and inducers of differentiation of human myeloid leukemia cell lines.

PubMed

Asou, H; Koike, M; Elstner, E; Cambell, M; Le, J; Uskokovic, M R; Kamada, N; Koeffler, H P

1998-10-01

We have studied the in vitro biological activities and mechanisms of action of 1,25-dihydroxyvitamin D3 (1,25D3) and nine potent 1,25D3 analogs on proliferation and differentiation of myeloid leukemia cell lines (HL-60, retinoic acid-resistant HL-60 [RA-res HL-60], NB4 and Kasumi-1). The common novel structural motiff for almost all the analogs included removal of C-19 (19-nor); each also had unsaturation of the side chain. All the compounds were potent; for example, the concentration of analogs producing a 50% clonal inhibition (ED50) ranged between 1 x 10(-9) to 4 x 10(-11) mol/L when using the HL-60 cell line. The most active compound [1, 25(OH)2-16,23E-diene-26-trifluoro-19-nor-cholecalciferol (Ro 25-9716)] had an ED50 of 4 x 10(-11) mol/L; in contrast, the 1,25D3 produced an ED50 of 10(-9) mol/L with the HL-60 target cells. Ro 25-9716 (10(-9) mol/L, 3 days) was a strong inducer of myeloid differentiation because it caused 92% of the HL-60 cells to express CD11b and 75% of these cells to reduce nitroblue tetrazolium (NBT). This compound (10(-8) mol/L, 4 days) also caused HL-60 cells to arrest in the G1 phase of the cell cycle (88% cells in G1 v 48% of the untreated control cells). The p27(kip-1), a cyclin-dependent kinase inhibitor which is important in blocking the cell cycle, was induced more quickly and potently by Ro 25-9716 (10(-7) mol/L, 0 to 5 days) than by 1,25D3, suggesting a possible mechanism by which these analogs inhibit proliferation of leukemic growth. The NB4 promyelocytic leukemia cells cultured with the Ro 25-9716 were also inhibited in their clonal proliferation (ED50, 5 x 10(-11) mol/L) and their expression of CD11b was enhanced (80% positive [10(-9) mol/L, 4 days] v 27% untreated NB4 cells). Moreover, the combination of Ro 25-9716 (10(-9) mol/L) and all-trans retinoic acid (ATRA, 10(-7) mol/L) induced 92% of the NB4 cells to reduce NBT, whereas only 26% of the cells became NBT positive after a similar exposure to the combination of 1,25D3 and ATRA. Surprisingly, Ro 25-9716 also inhibited the clonal growth of poorly differentiated leukemia cell lines (RA-res HL-60 [ED50, 4 x 10(-9) mol/L] and Kasumi-1 [ED50, 5 x 10(-10) mol/L]). For HL-60 cells, Ro 25-9716 markedly decreased the percent of the cells in S phase of the cell cycle and increased the expression of the cyclin-dependent kinase inhibitor, p27(kip-1). In summary, 19-nor vitamin D3 compounds strongly induced differentiation and inhibited clonal proliferation of various myeloid leukemia cell lines, suggesting a therapeutic niche for their use in myeloid leukemia.

Intratumoral genetic heterogeneity in metastatic melanoma is accompanied by variation in malignant behaviors

PubMed Central

2013-01-01

Background Intratumoral heterogeneity is a major obstacle for the treatment of cancer, as the presence of even minor populations that are insensitive to therapy can lead to disease relapse. Increased clonal diversity has been correlated with a poor prognosis for cancer patients, and we therefore examined genetic, transcriptional, and functional diversity in metastatic melanoma. Methods Amplicon sequencing and SNP microarrays were used to profile somatic mutations and DNA copy number changes in multiple regions from metastatic lesions. Clonal genetic and transcriptional heterogeneity was also assessed in single cell clones from early passage cell lines, which were then subjected to clonogenicity and drug sensitivity assays. Results MAPK pathway and tumor suppressor mutations were identified in all regions of the melanoma metastases analyzed. In contrast, we identified copy number abnormalities present in only some regions in addition to homogeneously present changes, suggesting ongoing genetic evolution following metastatic spread. Copy number heterogeneity from a tumor was represented in matched cell line clones, which also varied in their clonogenicity and drug sensitivity. Minor clones were identified based on dissimilarity to the parental cell line, and these clones were the most clonogenic and least sensitive to drugs. Finally, treatment of a polyclonal cell line with paclitaxel to enrich for drug-resistant cells resulted in the adoption of a gene expression profile with features of one of the minor clones, supporting the idea that these populations can mediate disease relapse. Conclusion Our results support the hypothesis that minor clones might have major consequences for patient outcomes in melanoma. PMID:24119551

Generation of human cortical neurons from a new immortal fetal neural stem cell line

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cacci, E.; Villa, A.; Parmar, M.

2007-02-01

Isolation and expansion of neural stem cells (NSCs) of human origin are crucial for successful development of cell therapy approaches in neurodegenerative diseases. Different epigenetic and genetic immortalization strategies have been established for long-term maintenance and expansion of these cells in vitro. Here we report the generation of a new, clonal NSC (hc-NSC) line, derived from human fetal cortical tissue, based on v-myc immortalization. Using immunocytochemistry, we show that these cells retain the characteristics of NSCs after more than 50 passages. Under proliferation conditions, when supplemented with epidermal and basic fibroblast growth factors, the hc-NSCs expressed neural stem/progenitor cell markersmore » like nestin, vimentin and Sox2. When growth factors were withdrawn, proliferation and expression of v-myc and telomerase were dramatically reduced, and the hc-NSCs differentiated into glia and neurons (mostly glutamatergic and GABAergic, as well as tyrosine hydroxylase-positive, presumably dopaminergic neurons). RT-PCR analysis showed that the hc-NSCs retained expression of Pax6, Emx2 and Neurogenin2, which are genes associated with regionalization and cell commitment in cortical precursors during brain development. Our data indicate that this hc-NSC line could be useful for exploring the potential of human NSCs to replace dead or damaged cortical cells in animal models of acute and chronic neurodegenerative diseases. Taking advantage of its clonality and homogeneity, this cell line will also be a valuable experimental tool to study the regulatory role of intrinsic and extrinsic factors in human NSC biology.« less

Genetic Resistance to Rhabdovirus Infection in Teleost Fish Is Paralleled to the Derived Cell Resistance Status

PubMed Central

Verrier, Eloi R.; Langevin, Christelle; Tohry, Corinne; Houel, Armel; Ducrocq, Vincent; Benmansour, Abdenour; Quillet, Edwige; Boudinot, Pierre

2012-01-01

Genetic factors of resistance and predisposition to viral diseases explain a significant part of the clinical variability observed within host populations. Predisposition to viral diseases has been associated to MHC haplotypes and T cell immunity, but a growing repertoire of innate/intrinsic factors are implicated in the genetic determinism of the host susceptibility to viruses. In a long-term study of the genetics of host resistance to fish rhabdoviruses, we produced a collection of double-haploid rainbow trout clones showing a wide range of susceptibility to Viral Hemorrhagic Septicemia Virus (VHSV) waterborne infection. The susceptibility of fibroblastic cell lines derived from these clonal fish was fully consistent with the susceptibility of the parental fish clones. The mechanisms determining the host resistance therefore did not associate with specific host immunity, but rather with innate or intrinsic factors. One cell line was resistant to rhabdovirus infection due to the combination of an early interferon IFN induction - that was not observed in the susceptible cells - and of yet unknown factors that hamper the first steps of the viral cycle. The implication of IFN was well consistent with the wide range of resistance of this genetic background to VSHV and IHNV, to the birnavirus IPNV and the orthomyxovirus ISAV. Another cell line was even more refractory to the VHSV infection through different antiviral mechanisms. This collection of clonal fish and isogenic cell lines provides an interesting model to analyze the relative contribution of antiviral pathways to the resistance to different viruses. PMID:22514610

Identification and characterization of the zebrafish glutathione S-transferase Pi-1.

PubMed

Abunnaja, Maryam S; Kurogi, Katsuhisa; Mohammed, Yasir I; Sakakibara, Yoichi; Suiko, Masahito; Hassoun, Ezdihar A; Liu, Ming-Cheh

2017-10-01

Zebrafish has in recent years emerged as a popular vertebrate model for use in pharmacological and toxicological studies. While there have been sporadic studies on the zebrafish glutathione S-transferases (GSTs), the zebrafish GST gene superfamily still awaits to be fully elucidated. We report here the identification of 15 zebrafish cytosolic GST genes in NCBI GenBank database and the expression, purification, and enzymatic characterization of the zebrafish cytosolic GST Pi-1 (GSTP1). The cDNA encoding the zebrafish GSTP1 was cloned from a 3-month-old female zebrafish, expressed in Eschelichia coli host cells, and purified. Purified GSTP1 displayed glutathione-conjugating activity toward 1-chloro-2,4-dinitrobenzene as a representative substrate. The enzymatic characteristics of the zebrafish GSTP1, including pH-dependency, effects of metal cations, and kinetic parameters, were studied. Moreover, the expression of zebrafish GSTP1 at different developmental stages during embryogenesis, throughout larval development, onto maturity was examined. © 2017 Wiley Periodicals, Inc.

Zebrafish neurobehavioral phenomics for aquatic neuropharmacology and toxicology research.

PubMed

Kalueff, Allan V; Echevarria, David J; Homechaudhuri, Sumit; Stewart, Adam Michael; Collier, Adam D; Kaluyeva, Aleksandra A; Li, Shaomin; Liu, Yingcong; Chen, Peirong; Wang, JiaJia; Yang, Lei; Mitra, Anisa; Pal, Subharthi; Chaudhuri, Adwitiya; Roy, Anwesha; Biswas, Missidona; Roy, Dola; Podder, Anupam; Poudel, Manoj K; Katare, Deepshikha P; Mani, Ruchi J; Kyzar, Evan J; Gaikwad, Siddharth; Nguyen, Michael; Song, Cai

2016-01-01

Zebrafish (Danio rerio) are rapidly emerging as an important model organism for aquatic neuropharmacology and toxicology research. The behavioral/phenotypic complexity of zebrafish allows for thorough dissection of complex human brain disorders and drug-evoked pathological states. As numerous zebrafish models become available with a wide spectrum of behavioral, genetic, and environmental methods to test novel drugs, here we discuss recent zebrafish phenomics methods to facilitate drug discovery, particularly in the field of biological psychiatry. Additionally, behavioral, neurological, and endocrine endpoints are becoming increasingly well-characterized in zebrafish, making them an inexpensive, robust and effective model for toxicology research and pharmacological screening. We also discuss zebrafish behavioral phenotypes, experimental considerations, pharmacological candidates and relevance of zebrafish neurophenomics to other 'omics' (e.g., genomic, proteomic) approaches. Finally, we critically evaluate the limitations of utilizing this model organism, and outline future strategies of research in the field of zebrafish phenomics. Copyright © 2015 Elsevier B.V. All rights reserved.

Zebrafish Transgenic Line huORFZ Is an Effective Living Bioindicator for Detecting Environmental Toxicants

PubMed Central

Chu, Chien; Li, Hong-Ping; Tsai, Huai-Jen

2014-01-01

Reliable animal models are invaluable for monitoring the extent of pollution in the aquatic environment. In this study, we demonstrated the potential of huORFZ, a novel transgenic zebrafish line that harbors a human upstream open reading frame of the chop gene fused with GFP reporter, as an animal model for monitoring environmental pollutants and stress-related cellular processes. When huORFZ embryos were kept under normal condition, no leaked GFP signal could be detected. When treated with hazardous chemicals, including heavy metals and endocrine-disrupting chemicals near their sublethal concentrations (LC50), huORFZ embryos exhibited different tissue-specific GFP expression patterns. For further analysis, copper (Cu2+), cadmium (Cd2+) and Chlorpyrifos were applied. Cu2+ triggered GFP responses in skin and muscle, whereas Cd2+ treatment triggered GFP responses in skin, olfactory epithelium and pronephric ducts. Moreover, fluorescence intensity, as exhibited by huORFZ embryos, was dose-dependent. After surviving treated embryos were returned to normal condition, survival rates, as well as TUNEL signals, returned to pretreatment levels with no significant morphological defects observed. Such results indicated the reversibility of treatment conditions used in this study, as long as embryos survived such conditions. Notably, GFP signals decreased along with recovery, suggesting that GFP signaling of huORFZ embryos likely reflected the overall physiological condition of the individual. To examine the performance of the huORFZ line under real-world conditions, we placed huORFZ embryos in different river water samples. We found that the huORFZ embryos correctly detected the presence of various kinds of pollutants. Based on these findings, we concluded that such uORFchop-based system can be integrated into a first-line water alarm system monitoring the discharge of hazardous pollutants. PMID:24594581

Clonal population of adult stem cells: life span and differentiation potential.

PubMed

Seruya, Mitchel; Shah, Anup; Pedrotty, Dawn; du Laney, Tracey; Melgiri, Ryan; McKee, J Andrew; Young, Henry E; Niklason, Laura E

2004-01-01

Adult stem cells derived from bone marrow, connective tissue, and solid organs can exhibit a range of differentiation potentials. Some controversy exists regarding the classification of mesenchymal stem cells as bona fide stem cells, which is in part derived from the limited ability to propagate true clonal populations of precursor cells. We isolated putative mesenchymal stem cells from the connective tissue of an adult rat (rMSC), and generated clonal populations via three rounds of dilutional cloning. The replicative potential of the clonal rMSC line far exceeded Hayflick's limit of 50-70 population doublings. The high capacity for self-renewal in vitro correlated with telomerase activity, as demonstrated by telomerase repeat amplification protocol (TRAP) assay. Exposure to nonspecific differentiation culture medium revealed multilineage differentiation potential of rMSC clones. Immunostaining confirmed the appearance of mesodermal phenotypes, including adipocytes possessing lipid-rich vacuoles, chondrocytes depositing pericellular type II collagen, and skeletal myoblasts expressing MyoD1. Importantly, the spectrum of differentiation capability was sustained through repeated passaging. Furthermore, serum-free conditions that led to high-efficiency smooth muscle differentiation were identified. rMSCs plated on collagen IV-coated surfaces and exposed to transforming growth factor-beta1 (TGF-beta1) differentiated into a homogeneous population expressing alpha-actin and calponin. Hence, clonogenic analysis confirmed the presence of a putative MSC population derived from the connective tissue of rat skeletal muscle. The ability to differentiate into a smooth muscle cell (SMC) phenotype, combined with a high proliferative capacity, make such a connective tissue-derived MSC population ideal for applications in vascular tissue construction.

Zebrafish vitamin K epoxide reductases: expression in vivo, along extracellular matrix mineralization and under phylloquinone and warfarin in vitro exposure.

PubMed

Fernández, Ignacio; Vijayakumar, Parameswaran; Marques, Carlos; Cancela, M Leonor; Gavaia, Paulo J; Laizé, Vincent

2015-06-01

Vitamin K (VK) acts as a cofactor driving the biological activation of VK-dependent proteins and conferring calcium-binding properties to them. As a result, VK is converted into VK epoxide, which must be recycled by VK epoxide reductases (Vkors) before it can be reused. Although VK has been shown to play a central role in fish development, particularly during skeletogenesis, pathways underlying VK actions are poorly understood, while good and reliable molecular markers for VK cycle/homeostasis are still lacking in fish. In the present work, expression of 2 zebrafish vkor genes was characterized along larval development and in adult tissues through qPCR analysis. Zebrafish cell line ZFB1 was used to evaluate in vitro regulation of vkors and other VK cycle-related genes during mineralization and upon 24 h exposure to 0.16 and 0.8 µM phylloquinone (VK1), 0.032 µM warfarin, or a combination of both molecules. Results showed that zebrafish vkors are differentially expressed during larval development, in adult tissues, and during cell differentiation/mineralization processes. Further, several VK cycle intermediates were differentially expressed in ZFB1 cells exposed to VK1 and/or warfarin. Present work provides data identifying different developmental stages and adult tissues where VK recycling is probably highly required, and shows how genes involved in VK cycle respond to VK nutritional status in skeletal cells. Expression of vkor genes can represent a reliable indicator to infer VK nutritional status in fish, while ZFB1 cells could represent a suitable in vitro tool to get insights into the mechanisms underlying VK action on fish bone.

Circadian Clock Synchronization of the Cell Cycle in Zebrafish Occurs through a Gating Mechanism Rather Than a Period-phase Locking Process.

PubMed

Laranjeiro, Ricardo; Tamai, T Katherine; Letton, William; Hamilton, Noémie; Whitmore, David

2018-04-01

Studies from a number of model systems have shown that the circadian clock controls expression of key cell cycle checkpoints, thus providing permissive or inhibitory windows in which specific cell cycle events can occur. However, a major question remains: Is the clock actually regulating the cell cycle through such a gating mechanism or, alternatively, is there a coupling process that controls the speed of cell cycle progression? Using our light-responsive zebrafish cell lines, we address this issue directly by synchronizing the cell cycle in culture simply by changing the entraining light-dark (LD) cycle in the incubator without the need for pharmacological intervention. Our results show that the cell cycle rapidly reentrains to a shifted LD cycle within 36 h, with changes in p21 expression and subsequent S phase timing occurring within the first few hours of resetting. Reentrainment of mitosis appears to lag S phase resetting by 1 circadian cycle. The range of entrainment of the zebrafish clock to differing LD cycles is large, from 16 to 32 hour periods. We exploited this feature to explore cell cycle entrainment at both the population and single cell levels. At the population level, cell cycle length is shortened or lengthened under corresponding T-cycles, suggesting that a 1:1 coupling mechanism is capable of either speeding up or slowing down the cell cycle. However, analysis at the single cell level reveals that this, in fact, is not true and that a gating mechanism is the fundamental method of timed cell cycle regulation in zebrafish. Cell cycle length at the single cell level is virtually unaltered with varying T-cycles.

Circadian Clock Synchronization of the Cell Cycle in Zebrafish Occurs through a Gating Mechanism Rather Than a Period-phase Locking Process

PubMed Central

Tamai, T. Katherine; Letton, William; Hamilton, Noémie; Whitmore, David

2018-01-01

Studies from a number of model systems have shown that the circadian clock controls expression of key cell cycle checkpoints, thus providing permissive or inhibitory windows in which specific cell cycle events can occur. However, a major question remains: Is the clock actually regulating the cell cycle through such a gating mechanism or, alternatively, is there a coupling process that controls the speed of cell cycle progression? Using our light-responsive zebrafish cell lines, we address this issue directly by synchronizing the cell cycle in culture simply by changing the entraining light-dark (LD) cycle in the incubator without the need for pharmacological intervention. Our results show that the cell cycle rapidly reentrains to a shifted LD cycle within 36 h, with changes in p21 expression and subsequent S phase timing occurring within the first few hours of resetting. Reentrainment of mitosis appears to lag S phase resetting by 1 circadian cycle. The range of entrainment of the zebrafish clock to differing LD cycles is large, from 16 to 32 hour periods. We exploited this feature to explore cell cycle entrainment at both the population and single cell levels. At the population level, cell cycle length is shortened or lengthened under corresponding T-cycles, suggesting that a 1:1 coupling mechanism is capable of either speeding up or slowing down the cell cycle. However, analysis at the single cell level reveals that this, in fact, is not true and that a gating mechanism is the fundamental method of timed cell cycle regulation in zebrafish. Cell cycle length at the single cell level is virtually unaltered with varying T-cycles. PMID:29444612

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

PubMed Central

Andersen, Erica; Asuri, Namrata; Clay, Matthew; Halloran, Mary

2010-01-01

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons. Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages. Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells1. Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this work. PMID:20130524

Early life stage trimethyltin exposure induces ADP-ribosylation factor expression and perturbs the vascular system in zebrafish.

PubMed

Chen, Jiangfei; Huang, Changjiang; Truong, Lisa; La Du, Jane; Tilton, Susan C; Waters, Katrina M; Lin, Kuanfei; Tanguay, Robert L; Dong, Qiaoxiang

2012-12-16

Trimethyltin chloride (TMT) is an organotin contaminant, widely detected in aqueous environments, posing potential human and environmental risks. In this study, we utilized the zebrafish model to investigate the impact of transient TMT exposure on developmental progression, angiogenesis, and cardiovascular development. Embryos were waterborne exposed to a wide TMT concentration range from 8 to 96 h post fertilization (hpf). The TMT concentration that led to mortality in 50% of the embryos (LC(50)) at 96 hpf was 8.2 μM; malformations in 50% of the embryos (EC(50)) was 2.8 μM. The predominant response observed in surviving embryos was pericardial edema. Additionally, using the Tg (fli1a: EGFP) y1 transgenic zebrafish line to non-invasively monitor vascular development, TMT exposure led to distinct disarrangements in the vascular system. The most susceptible developmental stage to TMT exposure was between 48 and 72 hpf. High density whole genome microarrays were used to identify the early transcriptional changes following TMT exposure from 48 to 60 hpf or 72 hpf. In total, 459 transcripts were differentially expressed at least 2-fold (P<0.05) by TMT compared to control. Using Ingenuity Pathway Analysis (IPA) tools, it was revealed that the transcripts misregulated by TMT exposure were clustered in numerous categories including metabolic and cardiovascular disease, cellular function, cell death, molecular transport, and physiological development. In situ localization of highly elevated transcripts revealed intense staining of ADP-ribosylation factors arf3 and arf5 in the head, trunk, and tail regions. When arf5 expression was blocked by morpholinos, the zebrafish did not display the prototypical TMT-induced vascular deficits, indicating that the induction of arf5 was necessary for TMT-induced vascular toxicity. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

The effects of rearing light level and duration differences on the optic nerve, brain, and associated structures in developing zebrafish larvae: a light and transmission electron microscope study.

PubMed

Chapman, George B; Tarboush, Rania; Connaughton, Victoria P

2012-03-01

The ultrastructure of the optic nerve, brain, and some associated structures of larval zebrafish, grown under three different light regimens were studied. Fish grown under cyclic light (control), constant dark (CD), and constant light (CL) were studied for 4 and 8 days postfertilization (dpf). We also studied the control and CD fish at 15 dpf. The brains of the control and CL fish were larger at 4 dpf than at 8 dpf. In all 4 dpf fish, the brain occupied the entire expanse between the two retinas and the optic nerve extended the shortest distance between the retina and the brain. The 15 dpf zebrafish had the smallest brain size. Groups of skeletal muscle cells associated with the optic nerves became visible in all older larvae. In the 15 dpf larvae, bulges and dilations in the optic nerve occurred as it reached the brain and optic chiasms occurred proximal to the brain. Electron microscopy yielded information about myelinated and unmyelinated axons in the optic nerve, the dimensions of neurotubules, neurofilaments, and myofilaments, including a unique variation in actin myofilaments, and a confirmation of reported myosin myofilament changes (but with dimensions). We also describe the ultrastructure of a sheath-like structure that is confluent over the optic nerve and the brain, which has not been described before in zebrafish. Also presented are images of associated fibroblasts, epithelial cells lining the mouth, cartilage plates, blood vessels, nerve bundles, and skeletal muscle cells, most of which have not been previously described in the literature. Copyright © 2012 Wiley Periodicals, Inc.

Neurochemical measurements in the zebrafish brain

PubMed Central

Jones, Lauren J.; McCutcheon, James E.; Young, Andrew M. J.; Norton, William H. J.

2015-01-01

The zebrafish is an ideal model organism for behavioral genetics and neuroscience. The high conservation of genes and neurotransmitter pathways between zebrafish and other vertebrates permits the translation of research between species. Zebrafish behavior can be studied at both larval and adult stages and recent research has begun to establish zebrafish models for human disease. Fast scan cyclic voltammetry (FSCV) is an electrochemical technique that permits the detection of neurotransmitter release and reuptake. In this study we have used in vitro FSCV to measure the release of analytes in the adult zebrafish telencephalon. We compare different stimulation methods and present a characterization of neurochemical changes in the wild-type zebrafish brain. This study represents the first FSCV recordings in zebrafish, thus paving the way for neurochemical analysis of the fish brain. PMID:26441575

Zebrafish as tools for drug discovery.

PubMed

MacRae, Calum A; Peterson, Randall T

2015-10-01

The zebrafish has become a prominent vertebrate model for disease and has already contributed to several examples of successful phenotype-based drug discovery. For the zebrafish to become useful in drug development more broadly, key hurdles must be overcome, including a more comprehensive elucidation of the similarities and differences between human and zebrafish biology. Recent studies have begun to establish the capabilities and limitations of zebrafish for disease modelling, drug screening, target identification, pharmacology, and toxicology. As our understanding increases and as the technologies for manipulating zebrafish improve, it is hoped that the zebrafish will have a key role in accelerating the emergence of precision medicine.

Evolutionary perspectives on clonal reproduction in vertebrate animals

PubMed Central

Avise, John C.

2015-01-01

A synopsis is provided of different expressions of whole-animal vertebrate clonality (asexual organismal-level reproduction), both in the laboratory and in nature. For vertebrate taxa, such clonal phenomena include the following: human-mediated cloning via artificial nuclear transfer; intergenerational clonality in nature via parthenogenesis and gynogenesis; intergenerational hemiclonality via hybridogenesis and kleptogenesis; intragenerational clonality via polyembryony; and what in effect qualifies as clonal replication via self-fertilization and intense inbreeding by simultaneous hermaphrodites. Each of these clonal or quasi-clonal mechanisms is described, and its evolutionary genetic ramifications are addressed. By affording an atypical vantage on standard vertebrate reproduction, clonality offers fresh perspectives on the evolutionary and ecological significance of recombination-derived genetic variety. PMID:26195735

Clonal Integration Enhances the Performance of a Clonal Plant Species under Soil Alkalinity Stress

PubMed Central

Sun, Juanjuan; Chen, Jishan; Zhang, Yingjun

2015-01-01

Clonal plants have been shown to successfully survive in stressful environments, including salinity stress, drought and depleted nutrients through clonal integration between original and subsequent ramets. However, relatively little is known about whether clonal integration can enhance the performance of clonal plants under alkalinity stress. We investigated the effect of clonal integration on the performance of a typical rhizomatous clonal plant, Leymus chinensis, using a factorial experimental design with four levels of alkalinity and two levels of rhizome connection treatments, connected (allowing integration) and severed (preventing integration). Clonal integration was estimated by comparing physiological and biomass features between the rhizome-connected and rhizome-severed treatments. We found that rhizome-connected treatment increased the biomass, height and leaf water potential of subsequent ramets at highly alkalinity treatments but did not affect them at low alkalinity treatments. However, rhizome-connected treatment decreased the root biomass of subsequent ramets and did not influence the photosynthetic rates of subsequent ramets. The biomass of original ramets was reduced by rhizome-connected treatment at the highest alkalinity level. These results suggest that clonal integration can increase the performance of clonal plants under alkalinity stress. Rhizome-connected plants showed dramatically increased survival of buds with negative effects on root weight, indicating that clonal integration influenced the resource allocation pattern of clonal plants. A cost-benefit analysis based on biomass measures showed that original and subsequent ramets significantly benefited from clonal integration in highly alkalinity stress, indicating that clonal integration is an important adaptive strategy by which clonal plants could survive in local alkalinity soil. PMID:25790352

Production of cloned embryos from caprine mammary epithelial cells expressing recombinant human β-defensin-3.

PubMed

Liu, Jun; Luo, Yan; Liu, Qingqing; Zheng, Liming; Yang, Zhongcai; Wang, Yongsheng; Su, Jianmin; Quan, Fusheng; Zhang, Yong

2013-03-01

Transgenic animals that express antimicrobial agents in their milk can inhibit bacterial pathogens that cause mastitis. Our objective was to produce human β-defensin-3 (HBD3) transgenic embryos by nuclear transfer using goat mammary epithelial cells (GMECs) as donor cells. Three GMEC lines (GMEC1, GMEC2, and GMEC3) were transfected with a HBD3 mammary-specific expression vector by electroporation. There was a difference (P < 0.05) in the rate of geneticin-resistant colony formation among cell lines GMEC1, GMEC2, and GMEC3 (39 and 47 vs. 19 colonies per 3 × 10(6) cells, respectively). After inducing expression, the mRNA and protein of HBD3 were detected by reverse transcription polymerase chain reaction and Western blot analysis in transgenic cells. Transgenic clonal cells expressing HBD3 were used as donor cells to investigate development of cloned embryos. There were no significant differences in rates of cleavage or blastocyst formation of cloned embryos from transgenic (GMEC1T2 and GMEC2T3) and nontransgenic (GMEC1 and GMEC2) GMECs (72.3 ± 5.0%, 69.5 ± 2.3%, 61.8 ± 4.8%, and 70.0 ± 2%; and 16.8 ± 0.5%, 17.5 ± 0.7%, 16.7 ± 0.9%, and 17.5 ± 0.6%, respectively). However, the fusion rate, cleavage rate, and blastocyst formation rate of cloned embryos from a transgenic clonal cell line (GMEC2T6, 50.7 ± 2.1%, 55.5 ± 2.0%, and 11.1 ± 0.6%) were lower than those of other groups (P < 0.05). We concluded that genetic modification of GMECs might not influence the in vitro development of cloned embryos, but that some of the transgenic clonal cells were not suitable for nuclear transfer to produce transgenic goats, because of low developmental rates. However, transgenic GMECs expressing HBD3 might be used as donor cells for producing transgenic goats that express increased concentrations of β-defensins in their milk. Copyright © 2013 Elsevier Inc. All rights reserved.

Germ line variants predispose to both JAK2 V617F clonal hematopoiesis and myeloproliferative neoplasms

PubMed Central

Hinds, David A.; Barnholt, Kimberly E.; Mesa, Ruben A.; Kiefer, Amy K.; Do, Chuong B.; Eriksson, Nicholas; Mountain, Joanna L.; Francke, Uta; Tung, Joyce Y.; Nguyen, Huong (Marie); Zhang, Haiyu; Gojenola, Linda; Zehnder, James L.

2016-01-01

We conducted a genome-wide association study (GWAS) to identify novel predisposition alleles associated with Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs) and JAK2 V617F clonal hematopoiesis in the general population. We recruited a web-based cohort of 726 individuals with polycythemia vera, essential thrombocythemia, and myelofibrosis and 252 637 population controls unselected for hematologic phenotypes. Using a single-nucleotide polymorphism (SNP) array platform with custom probes for the JAK2 V617F mutation (V617F), we identified 497 individuals (0.2%) among the population controls who were V617F carriers. We performed a combined GWAS of the MPN cases plus V617F carriers in the control population (n = 1223) vs the remaining controls who were noncarriers for V617F (n = 252 140). For these MPN cases plus V617F carriers, we replicated the germ line JAK2 46/1 haplotype (rs59384377: odds ratio [OR] = 2.4, P = 6.6 × 10−89), previously associated with V617F-positive MPN. We also identified genome-wide significant associations in the TERT gene (rs7705526: OR = 1.8, P = 1.1 × 10−32), in SH2B3 (rs7310615: OR = 1.4, P = 3.1 × 10−14), and upstream of TET2 (rs1548483: OR = 2.0, P = 2.0 × 10−9). These associations were confirmed in a separate replication cohort of 446 V617F carriers vs 169 021 noncarriers. In a joint analysis of the combined GWAS and replication results, we identified additional genome-wide significant predisposition alleles associated with CHEK2, ATM, PINT, and GFI1B. All SNP ORs were similar for MPN patients and controls who were V617F carriers. These data indicate that the same germ line variants endow individuals with a predisposition not only to MPN, but also to JAK2 V617F clonal hematopoiesis, a more common phenomenon that may foreshadow the development of an overt neoplasm. PMID:27365426

Sprouting Buds of Zebrafish Research in Malaysia: First Malaysia Zebrafish Disease Model Workshop.

PubMed

Okuda, Kazuhide Shaun; Tan, Pei Jean; Patel, Vyomesh

2016-04-01

Zebrafish is gaining prominence as an important vertebrate model for investigating various human diseases. Zebrafish provides unique advantages such as optical clarity of embryos, high fecundity rate, and low cost of maintenance, making it a perfect complement to the murine model equivalent in biomedical research. Due to these advantages, researchers in Malaysia are starting to take notice and incorporate the zebrafish model into their research activities. However, zebrafish research in Malaysia is still in its infancy stage and many researchers still remain unaware of the full potential of the zebrafish model or have limited access to related tools and techniques that are widely utilized in many zebrafish laboratories worldwide. To overcome this, we organized the First Malaysia Zebrafish Disease Model Workshop in Malaysia that took place on 11th and 12th of November 2015. In this workshop, we showcased how the zebrafish model is being utilized in the biomedical field in international settings as well as in Malaysia. For this, notable international speakers and those from local universities known to be carrying out impactful research using zebrafish were invited to share some of the cutting edge techniques that are used in their laboratories that may one day be incorporated in the Malaysian scientific community.

Construction and properties of a cell line constitutively expressing the herpes simplex virus glycoprotein B dependent on functional alpha 4 protein synthesis.

PubMed Central

Arsenakis, M; Hubenthal-Voss, J; Campadelli-Fiume, G; Pereira, L; Roizman, B

1986-01-01

We report the construction of a cell line constitutively expressing the glycoprotein B (gB) of herpes simplex virus (HSV) 1. The cell line was constructed in two steps. In the first, a baby hamster kidney cell line was transfected with the DNA of a plasmid containing the neomycin phosphotransferase gene that confers resistance to the antibiotic G418 and the gene specifying a temperature-sensitive (ts-) alpha 4 protein of HSV-1, the major viral regulatory protein. A clonal cell line, alpha 4/c113, selected for resistance to the antibiotic G418, expressed high levels of alpha 4 protein constitutively. Superinfection of these cells with HSV-2 resulted in twofold induction of the resident HSV-1 alpha 4 gene. In the second step, alpha 4/c113 cells were transfected with the DNA of a plasmid carrying the gB gene and the mouse methotrexate resistance dihydrofolate reductase gene. A clonal cell line, alpha 4/c113/gB, selected for methotrexate resistance expressed gB constitutively. Expression of both gB and alpha 4 continued unabated for at least 32 serial passages. Cells passaged serially in medium containing both methotrexate and G418 after passage 10 contained a higher copy number of the alpha 4 gene and produced larger amounts of both gB and alpha 4 proteins than did cells maintained in medium containing methotrexate alone. Expression of gB was dependent on the presence of functional alpha 4 protein inasmuch as expression of gB ceased on shift up to nonpermissive temperatures, when shifted to permissive temperatures, the cell line reinitiated expression of gB after a delay commensurate with the length of incubation at the nonpermissive temperature, and the cell-resident HSV-1 gB gene was expressed at the nonpermissive temperature in cells infected with a recombinant expressing a ts+ alpha 4 protein and an HSV-2 gB. The properties of the alpha 4/c113 cell line suggest that it may express other viral genes induced by alpha 4 protein constitutively, provided that the product is not toxic to the cells. Images PMID:3022001

Microglandular adenosis: a prime suspect in triple-negative breast cancer development.

PubMed

Tsang, Julia Ys; Tse, Gary Mk

2016-06-01

Microglandular adenosis (MGA) and atypical MGA (AMGA) are unusual lesions of the breast. They were once regarded as benign proliferative lesions and innocent bystanders. Several lines of evidence suggested that they could be neoplastic, clonal lesions and a non-obligate precursor for triple-negative breast cancers (TNBC). Recent work published in The Journal of Pathology by Guerini-Rocco and colleagues provided further evidence regarding the precursor-product relationship between MGA/AMGA and TNBC. Using a massively parallel sequencing approach, they demonstrated that MGA/AMGA, particularly those associated with TNBC, could be clonal neoplastic lesions showing clonal non-synonymous mutations, but none in pure MGA. Importantly, those alterations were observed in the associated TNBC. They were also able to identify recurrent alterations in TP53 in those MGA/AMGA cases as well as their associated TNBC. The findings, in conjunction with others, underscore the significance for MGA in clinical diagnosis. The potential of a benign lesion to progress into an aggressive malignant tumour implies that modification of the current management approach may be necessary. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Clonal variation in proliferation rate of cultures of GPK cells.

PubMed

Riley, P A; Hola, M

1981-09-01

Pedigrees of twenty-six clones of a line of keratocytes derived from guinea-pig ear epidermis (GPK cells) were analysed from time-lapse film. The mean interdivision time (IDT) for the culture was 1143 +/- 215 (SD) min. The mean generation rates (mean reciprocal interdivision times) of clones varied over a range of 3.93--10.2 x 10(-4)/min and the standard deviation of the clonal mean generation rates was 16.8% of the average value. Transient intraclonal variations in IDT due to mitoses in a plane perpendicular to the substratum were observed. The data were also analysed on the basis of cell location in sixteen equal zones (quadrats) of the filmed area. The mean generation rate of quadrats was 8.73 x 10(-4)/min (SD = 4.9%). The spatial distribution showed some clustering of cells. The mean local density of the clones (2.25 +/- 0.62 cells/10(-4) cm2) was significantly higher than the quadrat density (1.76 +/- 0.8 cells/10(-4) cm2). There was no significant correlation between clonal density and mean generation rates, whereas for quadrats a significant negative correlation was found (P = 2.7%). The results support the proposition that cell lineage is the major determinant of the proliferation rate of subconfluent cultures.

Rapid contemporary evolution and clonal food web dynamics

PubMed Central

Jones, Laura E.; Becks, Lutz; Ellner, Stephen P.; Hairston, Nelson G.; Yoshida, Takehito; Fussmann, Gregor F.

2009-01-01

Character evolution that affects ecological community interactions often occurs contemporaneously with temporal changes in population size, potentially altering the very nature of those dynamics. Such eco-evolutionary processes may be most readily explored in systems with short generations and simple genetics. Asexual and cyclically parthenogenetic organisms such as microalgae, cladocerans and rotifers, which frequently dominate freshwater plankton communities, meet these requirements. Multiple clonal lines can coexist within each species over extended periods, until either fixation occurs or a sexual phase reshuffles the genetic material. When clones differ in traits affecting interspecific interactions, within-species clonal dynamics can have major effects on the population dynamics. We first consider a simple predator–prey system with two prey genotypes, parametrized with data from a well-studied experimental system, and explore how the extent of differences in defence against predation within the prey population determine dynamic stability versus instability of the system. We then explore how increased potential for evolution affects the community dynamics in a more general community model with multiple predator and multiple prey genotypes. These examples illustrate how microevolutionary ‘details’ that enhance or limit the potential for heritable phenotypic change can have significant effects on contemporaneous community-level dynamics and the persistence and coexistence of species. PMID:19414472

Automatic multiple zebrafish larvae tracking in unconstrained microscopic video conditions.

PubMed

Wang, Xiaoying; Cheng, Eva; Burnett, Ian S; Huang, Yushi; Wlodkowic, Donald

2017-12-14

The accurate tracking of zebrafish larvae movement is fundamental to research in many biomedical, pharmaceutical, and behavioral science applications. However, the locomotive characteristics of zebrafish larvae are significantly different from adult zebrafish, where existing adult zebrafish tracking systems cannot reliably track zebrafish larvae. Further, the far smaller size differentiation between larvae and the container render the detection of water impurities inevitable, which further affects the tracking of zebrafish larvae or require very strict video imaging conditions that typically result in unreliable tracking results for realistic experimental conditions. This paper investigates the adaptation of advanced computer vision segmentation techniques and multiple object tracking algorithms to develop an accurate, efficient and reliable multiple zebrafish larvae tracking system. The proposed system has been tested on a set of single and multiple adult and larvae zebrafish videos in a wide variety of (complex) video conditions, including shadowing, labels, water bubbles and background artifacts. Compared with existing state-of-the-art and commercial multiple organism tracking systems, the proposed system improves the tracking accuracy by up to 31.57% in unconstrained video imaging conditions. To facilitate the evaluation on zebrafish segmentation and tracking research, a dataset with annotated ground truth is also presented. The software is also publicly accessible.

Comprehensive analysis of coding-lncRNA gene co-expression network uncovers conserved functional lncRNAs in zebrafish.

PubMed

Chen, Wen; Zhang, Xuan; Li, Jing; Huang, Shulan; Xiang, Shuanglin; Hu, Xiang; Liu, Changning

2018-05-09

Zebrafish is a full-developed model system for studying development processes and human disease. Recent studies of deep sequencing had discovered a large number of long non-coding RNAs (lncRNAs) in zebrafish. However, only few of them had been functionally characterized. Therefore, how to take advantage of the mature zebrafish system to deeply investigate the lncRNAs' function and conservation is really intriguing. We systematically collected and analyzed a series of zebrafish RNA-seq data, then combined them with resources from known database and literatures. As a result, we obtained by far the most complete dataset of zebrafish lncRNAs, containing 13,604 lncRNA genes (21,128 transcripts) in total. Based on that, a co-expression network upon zebrafish coding and lncRNA genes was constructed and analyzed, and used to predict the Gene Ontology (GO) and the KEGG annotation of lncRNA. Meanwhile, we made a conservation analysis on zebrafish lncRNA, identifying 1828 conserved zebrafish lncRNA genes (1890 transcripts) that have their putative mammalian orthologs. We also found that zebrafish lncRNAs play important roles in regulation of the development and function of nervous system; these conserved lncRNAs present a significant sequential and functional conservation, with their mammalian counterparts. By integrative data analysis and construction of coding-lncRNA gene co-expression network, we gained the most comprehensive dataset of zebrafish lncRNAs up to present, as well as their systematic annotations and comprehensive analyses on function and conservation. Our study provides a reliable zebrafish-based platform to deeply explore lncRNA function and mechanism, as well as the lncRNA commonality between zebrafish and human.

Integrated mechanism for the generation of the 5′ junctions of LINE inserts

PubMed Central

Yamaguchi, Katsumi; Kajikawa, Masaki; Okada, Norihiro

2014-01-01

To elucidate the molecular mechanism of the integration of long interspersed elements (LINEs), we characterized the 5′ ends of more than 200 LINE de novo retrotransposition events into chicken DT40 or human HeLa cells. Human L1 inserts produced 15-bp target-site duplications (TSDs) and zebrafish ZfL2-1 inserts produced 5-bp TSDs in DT40 cells, suggesting that TSD length depends on the LINE species. Further analysis of 5′ junctions revealed that the 5′-end-joining pathways of LINEs can be divided into two fundamental types—annealing or direct. We also found that the generation of 5′ inversions depends on host and LINE species. These results led us to propose a new model for 5′-end joining, the type of which is determined by the extent of exposure of 3′ overhangs generated after the second-strand cleavage and by the involvement of host factors. PMID:25378331

Integrated mechanism for the generation of the 5' junctions of LINE inserts.

PubMed

Yamaguchi, Katsumi; Kajikawa, Masaki; Okada, Norihiro

2014-12-01

To elucidate the molecular mechanism of the integration of long interspersed elements (LINEs), we characterized the 5' ends of more than 200 LINE de novo retrotransposition events into chicken DT40 or human HeLa cells. Human L1 inserts produced 15-bp target-site duplications (TSDs) and zebrafish ZfL2-1 inserts produced 5-bp TSDs in DT40 cells, suggesting that TSD length depends on the LINE species. Further analysis of 5' junctions revealed that the 5'-end-joining pathways of LINEs can be divided into two fundamental types-annealing or direct. We also found that the generation of 5' inversions depends on host and LINE species. These results led us to propose a new model for 5'-end joining, the type of which is determined by the extent of exposure of 3' overhangs generated after the second-strand cleavage and by the involvement of host factors. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Defining Clonal Color in Fluorescent Multi-Clonal Tracking

PubMed Central

Wu, Juwell W.; Turcotte, Raphaël; Alt, Clemens; Runnels, Judith M.; Tsao, Hensin; Lin, Charles P.

2016-01-01

Clonal heterogeneity and selection underpin many biological processes including development and tumor progression. Combinatorial fluorescent protein expression in germline cells has proven its utility for tracking the formation and regeneration of different organ systems. Such cell populations encoded by combinatorial fluorescent proteins are also attractive tools for understanding clonal expansion and clonal competition in cancer. However, the assignment of clonal identity requires an analytical framework in which clonal markings can be parameterized and validated. Here we present a systematic and quantitative method for RGB analysis of fluorescent melanoma cancer clones. We then demonstrate refined clonal trackability of melanoma cells using this scheme. PMID:27073117

A comparative genomic hybridization approach to study gene copy number variations among Chinese hamster cell lines.

PubMed

Vishwanathan, Nandita; Bandyopadhyay, Arpan; Fu, Hsu-Yuan; Johnson, Kathryn C; Springer, Nathan M; Hu, Wei-Shou

2017-08-01

Chinese Hamster Ovary (CHO) cells are aneuploid in nature. The genome of recombinant protein producing CHO cell lines continuously undergoes changes in its structure and organization. We analyzed nine cell lines, including parental cell lines, using a comparative genomic hybridization (CGH) array focused on gene-containing regions. The comparison of CGH with copy-number estimates from sequencing data showed good correlation. Hierarchical clustering of the gene copy number variation data from CGH data revealed the lineage relationships between the cell lines. On analyzing the clones of a clonal population, some regions with altered genomic copy number status were identified indicating genomic changes during passaging. A CGH array is thus an effective tool in quantifying genomic alterations in industrial cell lines and can provide insights into the changes in the genomic structure during cell line derivation and long term culture. Biotechnol. Bioeng. 2017;114: 1903-1908. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Invasive alien plants benefit more from clonal integration in heterogeneous environments than natives.

PubMed

Wang, Yong-Jian; Müller-Schärer, Heinz; van Kleunen, Mark; Cai, Ai-Ming; Zhang, Ping; Yan, Rong; Dong, Bi-Cheng; Yu, Fei-Hai

2017-12-01

What confers invasive alien plants a competitive advantage over native plants remains open to debate. Many of the world's worst invasive alien plants are clonal and able to share resources within clones (clonal integration), particularly in heterogeneous environments. Here, we tested the hypothesis that clonal integration benefits invasive clonal plants more than natives and thus confers invasives a competitive advantage. We selected five congeneric and naturally co-occurring pairs of invasive alien and native clonal plants in China, and grew pairs of connected and disconnected ramets under heterogeneous light, soil nutrient and water conditions that are commonly encountered by alien plants during their invasion into new areas. Clonal integration increased biomass of all plants in all three heterogeneous resource environments. However, invasive plants benefited more from clonal integration than natives. Consequently, invasive plants produced more biomass than natives. Our results indicate that clonal integration may confer invasive alien clonal plants a competitive advantage over natives. Therefore, differences in the ability of clonal integration could potentially explain, at least partly, the invasion success of alien clonal plants in areas where resources are heterogeneously distributed. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Selection of early-occurring mutations dictates hormone-independent progression in mouse mammary tumor lines.

PubMed

Gattelli, Albana; Zimberlin, María N; Meiss, Roberto P; Castilla, Lucio H; Kordon, Edith C

2006-11-01

Mice harboring three mouse mammary tumor virus (MMTV) variants develop pregnancy-dependent (PD) tumors that progress to pregnancy-independent (PI) behavior through successive passages. Herein, we identified 10 predominant insertions in PI transplants from 8 independent tumor lines. These mutations were also detected in small cell populations in the early PD passages. In addition, we identified a new viral insertion upstream of the gene Rspo3, which is overexpressed in three of the eight independent tumor lines and codes for a protein very similar to the recently described protein encoded by Int7. This study suggests that during progression towards hormone independence, clonal expansion of cells with specific mutations might be more relevant than the occurrence of new MMTV insertions.

An invasive clonal plant benefits from clonal integration more than a co-occurring native plant in nutrient-patchy and competitive environments.

PubMed

You, Wenhua; Fan, Shufeng; Yu, Dan; Xie, Dong; Liu, Chunhua

2014-01-01

Many notorious invasive plants are clonal, however, little is known about the different roles of clonal integration effects between invasive and native plants. Here, we hypothesize that clonal integration affect growth, photosynthetic performance, biomass allocation and thus competitive ability of invasive and native clonal plants, and invasive clonal plants benefit from clonal integration more than co-occurring native plants in heterogeneous habitats. To test these hypotheses, two stoloniferous clonal plants, Alternanthera philoxeroides (invasive), Jussiaea repens (native) were studied in China. The apical parts of both species were grown either with or without neighboring vegetation and the basal parts without competitors were in nutrient- rich or -poor habitats, with stolon connections were either severed or kept intact. Competition significantly reduced growth and photosynthetic performance of the apical ramets in both species, but not the biomass of neighboring vegetation. Without competition, clonal integration greatly improved the growth and photosynthetic performance of both species, especially when the basal parts were in nutrient-rich habitats. When grown with neighboring vegetation, growth of J. repens and photosynthetic performance of both species were significantly enhanced by clonal integration with the basal parts in both nutrient-rich and -poor habitats, while growth and relative neighbor effect (RNE) of A. philoxeroides were greatly improved by clonal integration only when the basal parts were in nutrient-rich habitats. Moreover, clonal integration increased A. philoxeroides's biomass allocation to roots without competition, but decreased it with competition, especially when the basal ramets were in nutrient-rich sections. Effects of clonal integration on biomass allocation of J. repens was similar to that of A. philoxeroides but with less significance. These results supported our hypothesis that invasive clonal plants A. philoxeroides benefits from clonal integration more than co-occurring native J. repens, suggesting that the invasiveness of A. philoxeroides may be closely related to clonal integration in heterogeneous environments.

An Invasive Clonal Plant Benefits from Clonal Integration More than a Co-Occurring Native Plant in Nutrient-Patchy and Competitive Environments

PubMed Central

You, Wenhua; Fan, Shufeng; Yu, Dan; Xie, Dong; Liu, Chunhua

2014-01-01

Many notorious invasive plants are clonal, however, little is known about the different roles of clonal integration effects between invasive and native plants. Here, we hypothesize that clonal integration affect growth, photosynthetic performance, biomass allocation and thus competitive ability of invasive and native clonal plants, and invasive clonal plants benefit from clonal integration more than co-occurring native plants in heterogeneous habitats. To test these hypotheses, two stoloniferous clonal plants, Alternanthera philoxeroides (invasive), Jussiaea repens (native) were studied in China. The apical parts of both species were grown either with or without neighboring vegetation and the basal parts without competitors were in nutrient- rich or -poor habitats, with stolon connections were either severed or kept intact. Competition significantly reduced growth and photosynthetic performance of the apical ramets in both species, but not the biomass of neighboring vegetation. Without competition, clonal integration greatly improved the growth and photosynthetic performance of both species, especially when the basal parts were in nutrient-rich habitats. When grown with neighboring vegetation, growth of J. repens and photosynthetic performance of both species were significantly enhanced by clonal integration with the basal parts in both nutrient-rich and -poor habitats, while growth and relative neighbor effect (RNE) of A. philoxeroides were greatly improved by clonal integration only when the basal parts were in nutrient-rich habitats. Moreover, clonal integration increased A. philoxeroides's biomass allocation to roots without competition, but decreased it with competition, especially when the basal ramets were in nutrient-rich sections. Effects of clonal integration on biomass allocation of J. repens was similar to that of A. philoxeroides but with less significance. These results supported our hypothesis that invasive clonal plants A. philoxeroides benefits from clonal integration more than co-occurring native J. repens, suggesting that the invasiveness of A. philoxeroides may be closely related to clonal integration in heterogeneous environments. PMID:24816849

Synthesis and toxicity evaluation of hydrophobic ionic liquids for volatile organic compounds biodegradation in a two-phase partitioning bioreactor.

PubMed

Rodriguez Castillo, Alfredo Santiago; Guihéneuf, Solène; Le Guével, Rémy; Biard, Pierre-François; Paquin, Ludovic; Amrane, Abdeltif; Couvert, Annabelle

2016-04-15

Synthesis of several hydrophobic ionic liquids (ILs), which might be selected as good candidates for degradation of hydrophobic volatile organic compounds in a two-phase partitioning bioreactor (TPPB), were carried out. Several bioassays were also realized, such as toxicity evaluation on activated sludge and zebrafish, cytotoxicity, fluoride release in aqueous phase and biodegradability in order to verify their possible effects in case of discharge in the aquatic environment and/or human contact during industrial manipulation. The synthesized compounds consist of alkylimidazoliums, functionalized imidazoliums, isoqinoliniums, triazoliums, sulfoniums, pyrrolidiniums and morpholiniums and various counter-ions such as: PF6(-), NTf2(-) and NfO(-). Toxicity evaluation on activated sludge of each compound (5% v/v of IL) was assessed by using a glucose uptake inhibition test. Toxicity against zebrafish and cytotoxicity were evaluated by the ImPACCell platform of Rennes (France). Fluoride release in water was estimated by regular measurements using ion chromatography equipment. IL biodegradability was determined by measuring BOD28 of aqueous samples (compound concentration,1mM). All ILs tested were not biodegradable; while some of them were toxic toward activated sludge. Isoquinolinium ILs were toxic to human cancerous cell lines. Nevertheless no toxicity was found against zebrafish Danio rerio. Only one IL released fluoride after long-time agitation. Copyright © 2016 Elsevier B.V. All rights reserved.

LSD1 is Required for Hair Cell Regeneration in Zebrafish.

PubMed

He, Yingzi; Tang, Dongmei; Cai, Chengfu; Chai, Renjie; Li, Huawei

2016-05-01

Lysine-specific demethylase 1 (LSD1/KDM1A) plays an important role in complex cellular processes such as differentiation, proliferation, apoptosis, and cell cycle progression. It has recently been demonstrated that during development, downregulation of LSD1 inhibits cell proliferation, modulates the expression of cell cycle regulators, and reduces hair cell formation in the zebrafish lateral line, which suggests that LSD1-mediated epigenetic regulation plays a key role in the development of hair cells. However, the role of LSD1 in hair cell regeneration after hair cell loss remains poorly understood. Here, we demonstrate the effect of LSD1 on hair cell regeneration following neomycin-induced hair cell loss. We show that the LSD1 inhibitor trans-2-phenylcyclopropylamine (2-PCPA) significantly decreases the regeneration of hair cells in zebrafish after neomycin damage. In addition, immunofluorescent staining demonstrates that 2-PCPA administration suppresses supporting cell proliferation and alters cell cycle progression. Finally, in situ hybridization shows that 2-PCPA significantly downregulates the expression of genes related to Wnt/β-catenin and Fgf activation. Altogether, our data suggest that downregulation of LSD1 significantly decreases hair cell regeneration after neomycin-induced hair cell loss through inactivation of the Wnt/β-catenin and Fgf signaling pathways. Thus, LSD1 plays a critical role in hair cell regeneration and might represent a novel biomarker and potential therapeutic approach for the treatment of hearing loss.

Kinase-activating and kinase-impaired cardio-facio-cutaneous syndrome alleles have activity during zebrafish development and are sensitive to small molecule inhibitors.

PubMed

Anastasaki, Corina; Estep, Anne L; Marais, Richard; Rauen, Katherine A; Patton, E Elizabeth

2009-07-15

The Ras/MAPK pathway is critical for human development and plays a central role in the formation and progression of most cancers. Children born with germ-line mutations in BRAF, MEK1 or MEK2 develop cardio-facio-cutaneous (CFC) syndrome, an autosomal dominant syndrome characterized by a distinctive facial appearance, heart defects, skin and hair abnormalities and mental retardation. CFC syndrome mutations in BRAF promote both kinase-activating and kinase-impaired variants. CFC syndrome has a progressive phenotype, and the availability of clinically active inhibitors of the MAPK pathway prompts the important question as to whether such inhibitors might be therapeutically effective in the treatment of CFC syndrome. To study the developmental effects of CFC mutant alleles in vivo, we have expressed a panel of 28 BRAF and MEK alleles in zebrafish embryos to assess the function of human disease alleles and available chemical inhibitors of this pathway. We find that both kinase-activating and kinase-impaired CFC mutant alleles promote the equivalent developmental outcome when expressed during early development and that treatment of CFC-zebrafish embryos with inhibitors of the FGF-MAPK pathway can restore normal early development. Importantly, we find a developmental window in which treatment with a MEK inhibitor can restore the normal early development of the embryo, without the additional, unwanted developmental effects of the drug.

Fetal Origins of Life Stage Disease: A Zebrafish Model for the ...

EPA Pesticide Factsheets

In the U.S., childhood obesity has more than doubled in children and quadrupled in adolescents in the past 30 years, affects 35% of adults, and costs the U.S. healthcare industry >$200 billion annually. The chemical environment in the womb may cause susceptibility to different life-stage and life-long metabolic diseases including obesity. The challenge is to understand if exposures during developmentally sensitive windows impact life-stage disease, such as obesity, by increasing adipose tissue mass. In vitro models lack the integrated systems approach needed to assess adipose development, while mammalian models are impractical in a screen of thousands of chemicals. Therefore, an obesogen screening method was developed to interrogate bioactivity using a full systems approach, in a vertebrate zebrafish model with complete metabolic activity, at a time when the full signaling repertoire is expressed and active, to optimally examine how chemical dose and duration impact life-stage adipose mass. A time-line for adipose depot formation was mapped in zebrafish 6−14 days post fertilization (dpf) using the lipophilic dye, Nile Red, in combination with fluorescent microscopy. Those time points were then used to investigate the impact of embryonic tributyltin chloride (TBT, a known obesogen) exposure (10nM daily renewal, 0−5dpf) on adipose mass. Fluorescent microscopy revealed adipose depots that were larger and appeared 2 days earlier in TBT treated compared to contro

Phenotype-based Discovery of 2-[(E)-2-(Quinolin-2-yl)vinyl]phenol as a Novel Regulator of Ocular Angiogenesis*

PubMed Central

Reynolds, Alison L.; Alvarez, Yolanda; Sasore, Temitope; Waghorne, Nora; Butler, Clare T.; Kilty, Claire; Smith, Andrew J.; McVicar, Carmel; Wong, Vickie H. Y.; Galvin, Orla; Merrigan, Stephanie; Osman, Janina; Grebnev, Gleb; Sjölander, Anita; Stitt, Alan W.; Kennedy, Breandán N.

2016-01-01

Retinal angiogenesis is tightly regulated to meet oxygenation and nutritional requirements. In diseases such as proliferative diabetic retinopathy and neovascular age-related macular degeneration, uncontrolled angiogenesis can lead to blindness. Our goal is to better understand the molecular processes controlling retinal angiogenesis and discover novel drugs that inhibit retinal neovascularization. Phenotype-based chemical screens were performed using the ChemBridge DiversetTM library and inhibition of hyaloid vessel angiogenesis in Tg(fli1:EGFP) zebrafish. 2-[(E)-2-(Quinolin-2-yl)vinyl]phenol, (quininib) robustly inhibits developmental angiogenesis at 4–10 μm in zebrafish and significantly inhibits angiogenic tubule formation in HMEC-1 cells, angiogenic sprouting in aortic ring explants, and retinal revascularization in oxygen-induced retinopathy mice. Quininib is well tolerated in zebrafish, human cell lines, and murine eyes. Profiling screens of 153 angiogenic and inflammatory targets revealed that quininib does not directly target VEGF receptors but antagonizes cysteinyl leukotriene receptors 1 and 2 (CysLT1–2) at micromolar IC50 values. In summary, quininib is a novel anti-angiogenic small-molecule CysLT receptor antagonist. Quininib inhibits angiogenesis in a range of cell and tissue systems, revealing novel physiological roles for CysLT signaling. Quininib has potential as a novel therapeutic agent to treat ocular neovascular pathologies and may complement current anti-VEGF biological agents. PMID:26846851

Therapeutic Role of Bmi-1 Inhibitors in Eliminating Prostate Tumor Stem Cells

DTIC Science & Technology

2015-10-01

antitumor activity in mouse xenografts did not exert toxic effects on normal tissues. BMI-1 targeted therapy when combined with taxotere resulted in...utilizing zebrafish xenografts (Sabaawy Lab) and prostate cancer cell lines (Bertino Lab), and 3) Confirmation of the antitumor activity of C-209...in mouse xenografts alone and upon combination with taxotere (Bertino Lab). The following tasks from the approved SOW were performed to achieve the

Characterization of spaC-type Erysipelothrix sp. isolates causing systemic disease in ornamental fish.

PubMed

Pomaranski, E K; Reichley, S R; Yanong, R; Shelley, J; Pouder, D B; Wolf, J C; Kenelty, K V; Van Bonn, B; Oliaro, F; Byrne, B; Clothier, K A; Griffin, M J; Camus, A C; Soto, E

2018-01-01

Since 2012, low-to-moderate mortality associated with an Erysipelothrix sp. bacterium has been reported in ornamental fish. Histological findings have included facial cellulitis, necrotizing dermatitis and myositis, and disseminated coelomitis with abundant intralesional Gram-positive bacterial colonies. Sixteen Erysipelothrix sp. isolates identified phenotypically as E. rhusiopathiae were recovered from diseased cyprinid and characid fish. Similar clinical and histological changes were also observed in zebrafish, Danio rerio, challenged by intracoelomic injection. The Erysipelothrix sp. isolates from ornamental fish were compared phenotypically and genetically to E. rhusiopathiae and E. tonsillarum isolates recovered from aquatic and terrestrial animals from multiple facilities. Results demonstrated that isolates from diseased fish were largely clonal and divergent from E. rhusiopathiae and E. tonsillarum isolates from normal fish skin, marine mammals and terrestrial animals. All ornamental fish isolates were PCR positive for spaC, with marked genetic divergence (<92% similarity at gyrB, <60% similarity by rep-PCR) between the ornamental fish isolates and other Erysipelothrix spp. isolates. This study supports previous work citing the genetic variability of Erysipelothrix spp. spa types and suggests isolates from diseased ornamental fish may represent a genetically distinct species. © 2017 John Wiley & Sons Ltd.

Mixtures, Metabolites, and Mechanisms: Understanding Toxicology Using Zebrafish.

PubMed

Gamse, Joshua T; Gorelick, Daniel A

2016-10-01

For more than 60 years, zebrafish have been used in toxicological studies. Due to their transparency, genetic tractability, and compatibility with high-throughput screens, zebrafish embryos are uniquely suited to study the effects of pharmaceuticals and environmental insults on embryonic development, organ formation and function, and reproductive success. This special issue of Zebrafish highlights the ways zebrafish are used to investigate the toxic effects of endocrine disruptors, pesticides, and heavy metals.

Open-RAC: Open-Design, Recirculating and Auto-Cleaning Zebrafish Maintenance System.

PubMed

Nema, Shubham; Bhargava, Yogesh

2017-08-01

Zebrafish is a vertebrate animal model. Their maintenance in large number under laboratory conditions is a daunting task. Commercially available recirculating zebrafish maintenance systems are used to efficiently handle the tasks of automatic sediment cleaning from zebrafish tanks with minimal waste of water. Due to their compact nature, they also ensure the maximal use of available lab space. However, the high costs of commercial systems present a limitation to researchers with limited funds. A cost-effective zebrafish maintenance system with major features offered by commercially available systems is highly desirable. Here, we describe a compact and recirculating zebrafish maintenance system. Our system is composed of cost-effective components, which are available in local markets and/or can be procured via online vendors. Depending on the expertise of end users, the system can be assembled in 2 days. The system is completely customizable as it offers geometry independent zebrafish tanks that are capable of auto-cleaning the sediments. Due to these features, we called our setup as Open-RAC (Open-design, Recirculating and Auto-Cleaning zebrafish maintenance system). Open-RAC is a cost-effective and viable alternative to the currently available zebrafish maintenance systems. Thus, we believe that the use of Open-RAC could promote the zebrafish research by removing the cost barrier for researchers.

A Zebrafish Heart Failure Model for Assessing Therapeutic Agents.

PubMed

Zhu, Xiao-Yu; Wu, Si-Qi; Guo, Sheng-Ya; Yang, Hua; Xia, Bo; Li, Ping; Li, Chun-Qi

2018-03-20

Heart failure is a leading cause of death and the development of effective and safe therapeutic agents for heart failure has been proven challenging. In this study, taking advantage of larval zebrafish, we developed a zebrafish heart failure model for drug screening and efficacy assessment. Zebrafish at 2 dpf (days postfertilization) were treated with verapamil at a concentration of 200 μM for 30 min, which were determined as optimum conditions for model development. Tested drugs were administered into zebrafish either by direct soaking or circulation microinjection. After treatment, zebrafish were randomly selected and subjected to either visual observation and image acquisition or record videos under a Zebralab Blood Flow System. The therapeutic effects of drugs on zebrafish heart failure were quantified by calculating the efficiency of heart dilatation, venous congestion, cardiac output, and blood flow dynamics. All 8 human heart failure therapeutic drugs (LCZ696, digoxin, irbesartan, metoprolol, qiliqiangxin capsule, enalapril, shenmai injection, and hydrochlorothiazide) showed significant preventive and therapeutic effects on zebrafish heart failure (p < 0.05, p < 0.01, and p < 0.001) in the zebrafish model. The larval zebrafish heart failure model developed and validated in this study could be used for in vivo heart failure studies and for rapid screening and efficacy assessment of preventive and therapeutic drugs.

Chemical-Induced Erythrocytosis in Wistar Rats: Assessment as a Model for Human Polycythemia.

DTIC Science & Technology

1985-05-01

polycythemic condition are unusual features that are more typically found in polycythemia vera, an autonomous myeloproliferative disorder in man that results...polycythemia vera, an autonomous myeloproliferative disorder in man that results from clonal neoplasia of bone marrow stem cells. However, the data described...5 and stroma cell lines [55]. These diseases are commonly termed ’ myeloproliferative disorders’, or better, ’myelodysplastic disease’ which emphasizes

Comparison of stem morphology and anatomy of two alfalfa clonal lines exhibiting divergent cell wall composition.

PubMed

Gronwald, John W; Bucciarelli, Bruna

2013-08-30

In previous research, two alfalfa clonal lines (252 and 1283) were identified that exhibited environmentally stable differences in stem cell walls. Compared with stems of 1283, stems of 252 have a higher cell wall concentration and greater amounts of lignin and cellulose but reduced levels of pectic sugar residues. These results suggest greater deposition of secondary xylem and a reduction in pith in stems of 252 compared with 1283. The stem morphology and anatomy of first-cut and second-cut harvests of field-grown 1283 and 252 were examined. For both harvests, stems of 1283 were thicker and had a higher leaf/stem ratio compared with stems of 252. Stem cross-sections of both genotypes were stained for lignin, and the proportions of stem area that were pith and secondary xylem were measured using ImageJ. Stems of 252 exhibited greater deposition of secondary xylem and a reduction in pith proportion compared with stems of 1283 for the first-cut harvest, but this difference was not statistically significant for the second-cut harvest. The results indicate that the proportions of secondary xylem and pith are not environmentally stable in these two genotypes and hence cannot be the sole basis for the differences in cell wall concentration/composition. © 2012 Society of Chemical Industry.

Clonal analysis of human embryonic stem cell differentiation into teratomas.

PubMed

Blum, Barak; Benvenisty, Nissim

2007-08-01

Differentiation of human embryonic stem cells (HESCs) can be studied in vivo through the induction of teratomas in immune-deficient mice. Cells within the teratomas differentiate into all three embryonic germ layers. However, the exact nature of the proliferation and differentiation of HESCs within the teratoma is not fully characterized, and it is not clear whether the differentiation is cell autonomous or affected by neighboring cells. Here, we establish a genetic approach to study the clonality of differentiation in teratomas using a mixture of HESC lines. We first demonstrate, by means of 5-bromo-2'-deoxyuridine incorporation, that cell proliferation occurs throughout the teratoma, and that there are no clusters of undifferentiated-proliferating cells. Using a combination of laser capture microdissection and DNA fingerprinting analysis, we show that different cell lines contribute mutually to the same distinctive tissue structures. Further support for the nonclonal differentiation within the teratoma was achieved by fluorescence in situ hybridization analysis of sex chromosomes. We therefore suggest that in vivo differentiation of HESCs is polyclonal and, thus, may not be cell autonomous, stressing the need for a three-dimensional growth in order to achieve complex differentiation of HESCs. Disclosure of potential conflicts of interest is found at the end of this article.

Adipose cell differentiation: evidence for a two-step process in the polyamine-dependent Ob1754 clonal line.

PubMed Central

Amri, E Z; Dani, C; Doglio, A; Etienne, J; Grimaldi, P; Ailhaud, G

1986-01-01

A subclone of preadipocyte Ob17 cells has been isolated (Ob1754 clonal line). Confluent Ob1754 cells treated with an inhibitor of spermidine and spermine synthesis, methylglyoxal bis(guanylhydrazone), were totally dependent upon putrescine addition for the expression of glycerol-3-phosphate dehydrogenase which behaved as a late marker of adipose conversion. Under these conditions, the early expression of lipoprotein lipase during growth arrest remained unchanged. Studies at the mRNA level showed that the expression of unidentified pOb24 and pGH3 mRNAs, which was parallel to that of lipoprotein lipase, is independent of polyamine addition whereas the late emergence of glycerol-3-phosphate dehydrogenase mRNA was putrescine-dependent and co-ordinated with the expression of pAL422 mRNA encoding for a myelin-P2 homologue [Bernlohr, Angus, Lane, Bolanowski & Kelly (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 5468-5472]. The appearance of lipoprotein lipase preceded DNA synthesis and post-confluent mitoses which were both putrescine-dependent and which took place before the appearance of glycerol-3-phosphate dehydrogenase. Thus the adipose conversion of Ob1754 cells involves the expression of at least two separate sets of markers which are differently regulated. Images Fig. 3. Fig. 6. PMID:3800927

Effect of aspirin on tumour cell colony formation and evolution.

PubMed

Wodarz, Dominik; Goel, Ajay; Boland, C Richard; Komarova, Natalia L

2017-09-01

Aspirin is known to reduce the risk of colorectal cancer (CRC) incidence, but the underlying mechanisms are not fully understood. In a previous study, we quantified the in vitro growth kinetics of different CRC tumour cell lines treated with varying doses of aspirin, measuring the rate of cell division and cell death. Here, we use these measured parameters to calculate the chances of successful clonal expansion and to determine the evolutionary potential of the tumour cell lines in the presence and absence of aspirin. The calculations indicate that aspirin increases the probability that a single tumour cell fails to clonally expand. Further, calculations suggest that aspirin increases the evolutionary potential of an expanding tumour cell colony. An aspirin-treated tumour cell population is predicted to result in the accumulation of more mutations (and is thus more virulent and more difficult to treat) than a cell population of the same size that grew without aspirin. This indicates a potential trade-off between delaying the onset of cancer and increasing its evolutionary potential through chemoprevention. Further work needs to investigate to what extent these findings apply to in vivo settings, and to what degree they contribute to the epidemiologically documented aspirin-mediated protection. © 2017 The Author(s).

Clonal evolution and heterogeneity in metastatic head and neck cancer-An analysis of the Austrian Study Group of Medical Tumour Therapy study group.

PubMed

Melchardt, Thomas; Magnes, Teresa; Hufnagl, Clemens; Thorner, Aaron R; Ducar, Matthew; Neureiter, Daniel; Tränkenschuh, Wolfgang; Klieser, Eckhard; Gaggl, Alexander; Rösch, Sebastian; Rasp, Gerd; Hartmann, Tanja N; Pleyer, Lisa; Rinnerthaler, Gabriel; Weiss, Lukas; Greil, Richard; Egle, Alexander

2018-04-01

Tumour heterogeneity and clonal evolution within a cancer patient are deemed responsible for relapse in malignancies and present challenges to the principles of targeted therapy, for which treatment modality is often decided based on the molecular pathology of the primary tumour. Nevertheless, the clonal architecture in distant relapse of head and neck cancer is fairly unknown. For this project, we analysed a cohort of 386 patients within the Austrian Registry of head and neck cancer. We identified 26 patients with material from the primary tumour, the distant metastasis after curative first-line treatment and a germline sample for analysis of clonal evolution. After pathological analyses, these samples were analysed using a targeted massively parallel sequencing (MPS) panel of 257 genes known to be recurrently mutated in head and neck cancer plus a genome-wide SNP-set. Despite histological diagnosis of distant metastasis, no corresponding mutation in the supposed metastases was found in two of 23 (8.6%) evaluable patients suggesting a primary tumour of the lung instead of a distant metastasis of head and neck cancer. We observed a branched pattern of evolution in 31.6% of the analysed patients. This pattern was associated with a shorter time to distant metastasis, compared with a pattern of punctuated evolution. Structural genomic changes over time were also present in 7 of 12 (60%) evaluable patients with metachronous metastases. Targeted MPS demonstrated substantial heterogeneity at the time of diagnosis and a complex pattern of evolution during disease progression in head and neck cancer. Copy number analyses revealed additional changes that were not detected by mutational analyses. Mutational and structural changes contribute to tumour heterogeneity at diagnosis and progression. Copyright © 2018 Elsevier Ltd. All rights reserved.

Mixtures, Metabolites, and Mechanisms: Understanding Toxicology Using Zebrafish

PubMed Central

Gamse, Joshua T.

2016-01-01

Abstract For more than 60 years, zebrafish have been used in toxicological studies. Due to their transparency, genetic tractability, and compatibility with high-throughput screens, zebrafish embryos are uniquely suited to study the effects of pharmaceuticals and environmental insults on embryonic development, organ formation and function, and reproductive success. This special issue of Zebrafish highlights the ways zebrafish are used to investigate the toxic effects of endocrine disruptors, pesticides, and heavy metals. PMID:27618129

Molecular epidemiology of clonal diploids: a quick overview and a short DIY (do it yourself) notice.

PubMed

De Meeûs, Thierry; Lehmann, Laurent; Balloux, François

2006-03-01

In this short review we report the basic notions needed for understanding the population genetics of clonal diploids. We focus on the consequences of clonality on the distribution of genetic diversity within individuals, between individuals and between populations. We then summarise how to detect clonality in mainly sexual populations, conversely, how to detect sexuality in mainly clonal populations and also how genetic differentiation between populations is affected by clonality in diploids. This information is then used for building recipes on how to analyse and interpret genetic polymorphism data in molecular epidemiology studies of clonal diploids.

Preparing for a Semiannual IACUC Inspection of a Satellite Zebrafish (Danio rerio) Facility

PubMed Central

Koerber, Amy S; Kalishman, Jennifer

2009-01-01

Institutions worldwide have experienced a rapid growth in the use of zebrafish as a research model for a variety of molecular and genetic studies of vertebrate development. This expansion in zebrafish research essentially has outpaced the establishment of specific recommendations for the care and use of fish in research. In some cases, this situation has created a dilemma where an Institutional Animal Care and Use Committee, which is responsible for oversight of vertebrate animal research, is not fully prepared to undertake this role for a decentralized zebrafish facility. IACUC inspectors will be more equipped to ask pertinent questions by understanding the basic principles of zebrafish health and facility management. Concurrently, zebrafish facility managers can contribute to the progress of a semiannual facility inspection by maintaining fully accessible operating records. In the context of presenting a well-established and useful model of zebrafish management and recordkeeping to the zebrafish facility operator, the information we present here also prepares a potential IACUC inspector to conduct a constructive and positive inspection. PMID:19245754

Making Waves: New Developments in Toxicology With the Zebrafish.

PubMed

Horzmann, Katharine A; Freeman, Jennifer L

2018-05-01

The laboratory zebrafish (Danio rerio) is now an accepted model in toxicologic research. The zebrafish model fills a niche between in vitro models and mammalian biomedical models. The developmental characteristics of the small fish are strategically being used by scientists to study topics ranging from high-throughput toxicity screens to toxicity in multi- and transgenerational studies. High-throughput technology has increased the utility of zebrafish embryonic toxicity assays in screening of chemicals and drugs for toxicity or effect. Additionally, advances in behavioral characterization and experimental methodology allow for observation of recognizable phenotypic changes after xenobiotic exposure. Future directions in zebrafish research are predicted to take advantage of CRISPR-Cas9 genome editing methods in creating models of disease and interrogating mechanisms of action with fluorescent reporters or tagged proteins. Zebrafish can also model developmental origins of health and disease and multi- and transgenerational toxicity. The zebrafish has many advantages as a toxicologic model and new methodologies and areas of study continue to expand the usefulness and application of the zebrafish.

A zebrafish (Danio rerio) model of infectious spleen and kidney necrosis virus (ISKNV) infection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu Xiaopeng; Zhang Lichun; Weng Shaoping

2008-06-20

Zebrafish is a model animal for studies of genetics, development, toxicology, oncology, and immunology. In this study, infectious spleen and kidney necrosis virus (ISKNV) was used to establish an infection in zebrafish, and the experimental conditions were established and characterized. Mortality of adult zebrafish infected with ISKNV by intraperitoneal (i.p.) injection exceeded 60%. ISKNV can be passed stably in zebrafish for over ten passages. The ailing zebrafish displayed petechial hemorrhaging and scale protrusion. Histological analysis of moribund fish revealed necrosis of tissue and enlarged cells in kidney and spleen. The real-time RT-PCR analysis of mRNA level confirmed that ISKNV wasmore » replicated in zebrafish. Immunohistochemistry and immunofluorescence analyses further confirmed the presence of ISKNV-infected cells in almost all organs of the infected fish. Electron microscope analyses showed that the ISKNV particle was present in the infected tissues. The establishment of zebrafish infection model of ISKNV can offer a valuable tool for studying the interactions between ISKNV and its host.« less

Zebrafish: an animal model for research in veterinary medicine.

PubMed

Nowik, N; Podlasz, P; Jakimiuk, A; Kasica, N; Sienkiewicz, W; Kaleczyc, J

2015-01-01

The zebrafish (Danio rerio) has become known as an excellent model organism for studies of vertebrate biology, vertebrate genetics, embryonal development, diseases and drug screening. Nevertheless, there is still lack of detailed reports about usage of the zebrafish as a model in veterinary medicine. Comparing to other vertebrates, they can lay hundreds of eggs at weekly intervals, externally fertilized zebrafish embryos are accessible to observation and manipulation at all stages of their development, which makes possible to simplify the research techniques such as fate mapping, fluorescent tracer time-lapse lineage analysis and single cell transplantation. Although zebrafish are only 2.5 cm long, they are easy to maintain. Intraperitoneal and intracerebroventricular injections, blood sampling and measurement of food intake are possible to be carry out in adult zebrafish. Danio rerio is a useful animal model for neurobiology, developmental biology, drug research, virology, microbiology and genetics. A lot of diseases, for which the zebrafish is a perfect model organism, affect aquatic animals. For a part of them, like those caused by Mycobacterium marinum or Pseudoloma neutrophila, Danio rerio is a natural host, but the zebrafish is also susceptible to the most of fish diseases including Itch, Spring viraemia of carp and Infectious spleen and kidney necrosis. The zebrafish is commonly used in research of bacterial virulence. The zebrafish embryo allows for rapid, non-invasive and real time analysis of bacterial infections in a vertebrate host. Plenty of common pathogens can be examined using zebrafish model: Streptococcus iniae, Vibrio anguillarum or Listeria monocytogenes. The steps are taken to use the zebrafish also in fungal research, especially that dealing with Candida albicans and Cryptococcus neoformans. Although, the zebrafish is used commonly as an animal model to study diseases caused by external agents, it is also useful in studies of metabolic disorders including fatty liver disease and diabetes. The zebrafish is also a valuable tool as a model in behavioral studies connected with feeding, predator evasion, habituation and memory or lateralized control of behavior. The aim of the present article is to familiarize the reader with the possibilities of Danio rerio as an experimental model for veterinary medicine.

Acute toxicity effects of perfluorooctane sulfonate on sperm vitality, kinematics and fertilization success in zebrafish

NASA Astrophysics Data System (ADS)

Xia, Jigang; Niu, Cuijuan

2017-07-01

Perfluorooctane sulfonate (PFOS) has emerged as one of the most concerning contaminants in recent years. This study aimed to investigate the acute toxicity effect of PFOS on sperm viability, kinematics and fertilization success in zebrafish ( Danio rerio). Sperm were activated in aqueous media containing a range of PFOS concentrations (0, 0.09, 0.9 and 9 mg/L). Viabilities and kinematics of the sperm exposed to different PFOS treatments were assessed via computer-assisted sperm analysis (CASA) at 20, 40, 60, and 80 s after activation. PFOS exposure decreased the percentage of motile sperm, the curvilinear velocity (VCL), and the mean angular displacement (MAD) of spermatozoa, but showed no influence on the straight-line velocity (VSL) or the angular path velocity (VAP). Furthermore, a significant decrease in fertilization success was observed in spermatozoa that were exposed to 0.9 mg/L PFOS or more. These findings indicate that PFOS pollution in natural aquatic environment may be a potential threaten to successful reproduction of fish.

Automation of Technology for Cancer Research.

PubMed

van der Ent, Wietske; Veneman, Wouter J; Groenewoud, Arwin; Chen, Lanpeng; Tulotta, Claudia; Hogendoorn, Pancras C W; Spaink, Herman P; Snaar-Jagalska, B Ewa

2016-01-01

Zebrafish embryos can be obtained for research purposes in large numbers at low cost and embryos develop externally in limited space, making them highly suitable for high-throughput cancer studies and drug screens. Non-invasive live imaging of various processes within the larvae is possible due to their transparency during development, and a multitude of available fluorescent transgenic reporter lines.To perform high-throughput studies, handling large amounts of embryos and larvae is required. With such high number of individuals, even minute tasks may become time-consuming and arduous. In this chapter, an overview is given of the developments in the automation of various steps of large scale zebrafish cancer research for discovering important cancer pathways and drugs for the treatment of human disease. The focus lies on various tools developed for cancer cell implantation, embryo handling and sorting, microfluidic systems for imaging and drug treatment, and image acquisition and analysis. Examples will be given of employment of these technologies within the fields of toxicology research and cancer research.

Effects of Double Transgenesis of Somatotrophic Axis (GH/GHR) on Skeletal Muscle Growth of Zebrafish (Danio rerio).

PubMed

Silva, Ana Cecilia Gomes; Almeida, Daniela Volcan; Nornberg, Bruna Felix; Figueiredo, Marcio Azevedo; Romano, Luis Alberto; Marins, Luis Fernando

2015-12-01

Transgenic fish for growth hormone (GH) has been considered as a potential technological improvement in aquaculture. In this study, a double-transgenic zebrafish was used to evaluate the effect of GH and its receptor (GHR) on muscle growth. Double transgenics reached the same length of GH transgenic, but with significantly less weight, featuring an unbalanced growth. The condition factor of GH/GHR-transgenic fish was lower than the other genotypes. Histological analysis showed a decrease in the percentage of thick muscle fibers in GH/GHR genotype of ∼ 80% in comparison to GH-transgenic line. The analysis of gene expression showed a significant decrease in genes related to muscle growth in GH/GHR genotype. It seems that concomitant overexpression of GH and GHR resulted in a strong decrease of the somatotrophic axis intracellular signaling by diminishing its principal transcription factor signal transducer and activator of transcription 5.1 (STAT5.1).

Genetic analysis of vertebrate sensory hair cell mechanosensation: the zebrafish circler mutants.

PubMed

Nicolson, T; Rüsch, A; Friedrich, R W; Granato, M; Ruppersberg, J P; Nüsslein-Volhard, C

1998-02-01

The molecular basis of sensory hair cell mechanotransduction is largely unknown. In order to identify genes that are essential for mechanosensory hair cell function, we characterized a group of recently isolated zebrafish motility mutants. These mutants are defective in balance and swim in circles but have no obvious morphological defects. We examined the mutants using calcium imaging of acoustic-vibrational and tactile escape responses, high resolution microscopy of sensory neuroepithelia in live larvae, and recordings of extracellular hair cell potentials (microphonics). Based on the analyses, we have identified several classes of genes. Mutations in sputnik and mariner affect hair bundle integrity. Mutant astronaut and cosmonaut hair cells have relatively normal microphonics and thus appear to affect events downstream of mechanotransduction. Mutant orbiter, mercury, and gemini larvae have normal hair cell morphology and yet do not respond to acoustic-vibrational stimuli. The microphonics of lateral line hair cells of orbiter, mercury, and gemini larvae are absent or strongly reduced. Therefore, these genes may encode components of the transduction apparatus.

Osteosarcoma Models: From Cell Lines to Zebrafish

PubMed Central

Mohseny, Alexander B.; Hogendoorn, Pancras C. W.; Cleton-Jansen, Anne-Marie

2012-01-01

High-grade osteosarcoma is an aggressive tumor most commonly affecting adolescents. The early age of onset might suggest genetic predisposition; however, the vast majority of the tumors are sporadic. Early onset, most often lack of a predisposing condition or lesion, only infrequent (<2%) prevalence of inheritance, extensive genomic instability, and a wide histological heterogeneity are just few factors to mention that make osteosarcoma difficult to study. Therefore, it is sensible to design and use models representative of the human disease. Here we summarize multiple osteosarcoma models established in vitro and in vivo, comment on their utilities, and highlight newest achievements, such as the use of zebrafish embryos. We conclude that to gain a better understanding of osteosarcoma, simplification of this extremely complex tumor is needed. Therefore, we parse the osteosarcoma problem into parts and propose adequate models to study them each separately. A better understanding of osteosarcoma provides opportunities for discovering and assaying novel effective treatment strategies. “Sometimes the model is more interesting than the original disease” PJ Hoedemaeker (1937–2007). PMID:22566751

Control of metazoan heme homeostasis by a conserved multidrug resistance protein

PubMed Central

Korolnek, Tamara; Zhang, Jianbing; Beardsley, Simon; Scheffer, George L; Hamza, Iqbal

2014-01-01

Several lines of evidence predict that specific pathways must exist in metazoans for the escorted movement of heme, an essential but cytotoxic iron-containing organic ring, within and between cells and tissues, but these pathways remain obscure. In Caenorhabditis elegans, embryonic development is inextricably dependent on both maternally-derived heme and environmentally-acquired heme. Here, we show that the multidrug resistance protein, MRP-5/ABCC5, likely acts as a heme exporter and targeted depletion of mrp-5 in the intestine causes embryonic lethality. Transient knockdown of mrp5 in zebrafish leads to morphological defects and failure to hemoglobinize red blood cells. MRP5 resides on the plasma membrane and endosomal compartments and regulates export of cytosolic heme. Together, our genetic studies in worms, yeast, zebrafish, and mammalian cells identify a conserved, physiological role for a multidrug resistance protein in regulating systemic heme homeostasis. We envision other MRP family members may play similar unanticipated physiological roles in animal development. PMID:24836561

Application of Zebrafish Model to Environmental Toxicology.

PubMed

Komoike, Yuta; Matsuoka, Masato

2016-01-01

Recently, a tropical freshwater fish, the zebrafish, has been generally used as a useful model organism in various fields of life science worldwide. The zebrafish model has also been applied to environmental toxicology; however, in Japan, it has not yet become widely used. In this review, we will introduce the biological and historical backgrounds of zebrafish as an animal model and their breeding. We then present the current status of toxicological experiments using zebrafish that were treated with some important environmental contaminants, including cadmium, organic mercury, 2,3,7,8-tetrachlorodibenzo-p-dioxin, and tributyltin. Finally, the future possible application of genetically modified zebrafish to the study of environmental toxicology is discussed.

Strategies to Mitigate a Mycobacterium marinum Outbreak in a Zebrafish Research Facility

PubMed Central

Snell, Kathy; Mittge, Erika; Melancon, Ellie; Montgomery, Rebecca; McFadden, Marcie; Camoriano, Javier; Kent, Michael L.; Whipps, Christopher M.; Peirce, Judy

2016-01-01

Abstract In 2011, the zebrafish research facility at the University of Oregon experienced an outbreak of Mycobacterium marinum that affected both research fish and facility staff. A thorough review of risks to personnel, the zebrafish veterinary care program, and zebrafish husbandry procedures at the research facility followed. In the years since 2011, changes have been implemented throughout the research facility to protect the personnel, the fish colony, and ultimately the continued success of the zebrafish model research program. In this study, we present the history of the outbreak, the changes we implemented, and recommendations to mitigate pathogen outbreaks in zebrafish research facilities. PMID:27351618

The zebrafish genome: a review and msx gene case study.

PubMed

Postlethwait, J H

2006-01-01

Zebrafish is one of several important teleost models for understanding principles of vertebrate developmental, molecular, organismal, genetic, evolutionary, and genomic biology. Efficient investigation of the molecular genetic basis of induced mutations depends on knowledge of the zebrafish genome. Principles of zebrafish genomic analysis, including gene mapping, ortholog identification, conservation of syntenies, genome duplication, and evolution of duplicate gene function are discussed here using as a case study the zebrafish msxa, msxb, msxc, msxd, and msxe genes, which together constitute zebrafish orthologs of tetrapod Msx1, Msx2, and Msx3. Genomic analysis suggests orthologs for this difficult to understand group of paralogs.

The ontogeny of sleep-wake cycles in zebrafish: a comparison to humans

PubMed Central

Sorribes, Amanda; Þorsteinsson, Haraldur; Arnardóttir, Hrönn; Jóhannesdóttir, Ingibjörg Þ.; Sigurgeirsson, Benjamín; de Polavieja, Gonzalo G.; Karlsson, Karl Æ.

2013-01-01

Zebrafish (Danio rerio) are used extensively in sleep research; both to further understanding of sleep in general and also as a model of human sleep. To date, sleep studies have been performed in larval and adult zebrafish but no efforts have been made to document the ontogeny of zebrafish sleep–wake cycles. Because sleep differs across phylogeny and ontogeny it is important to validate the use of zebrafish in elucidating the neural substrates of sleep. Here we describe the development of sleep and wake across the zebrafish lifespan and how it compares to humans. We find power-law distributions to best fit wake bout data but demonstrate that exponential distributions, previously used to describe sleep bout distributions, fail to adequately account for the data in either species. Regardless, the data reveal remarkable similarities in the ontogeny of sleep cycles in zebrafish and humans. Moreover, as seen in other organisms, zebrafish sleep levels are highest early in ontogeny and sleep and wake bouts gradually consolidate to form the adult sleep pattern. Finally, sleep percentage, bout duration, bout number, and sleep fragmentation are shown to allow for meaningful comparisons between zebrafish and human sleep. PMID:24312015

Preliminary Evaluation on the Effects of Feeds on the Growth and Early Reproductive Performance of Zebrafish (Danio rerio)

PubMed Central

2012-01-01

This study evaluated the effects of several commercially available feeds and different feeding regimes on the growth and early reproductive performance of zebrafish (Danio rerio). Juvenile zebrafish (n= 20; 5.06 ± 0.69 mg) were stocked into each of 24 tanks (volume, 2 L); 3 tanks were assigned to each of 8 feeding combinations for a period of 60 d. At the end of 60 d, 2 male and 2 female fish from each tank were pooled by dietary treatment (n = 6) and used to evaluate the effects of feeding combinations on early reproductive performance. Zebrafish fed dietary treatments 3 and 7 had significantly greater weight gain than zebrafish fed diet 5. Mean spawning success was significantly greater in zebrafish fed the control diet (Artemiaonly) than in those fed diet 1. Mean hatch rates were greater in zebrafish fed the control feed and diets 1, 2, 3, 5, and 6 than zebrafish fed diet 4. Additional results suggest that female zebrafish are sexually mature after 90 d post fertilization and that fertilization rates are the limiting factor in early reproduction. PMID:23043806

In vitro and in vivo characterization of the anticancer activity of Thai stingless bee (Tetragonula laeviceps) cerumen

PubMed Central

Nugitrangson, Pongvit; Puthong, Songchan; Iempridee, Tawin; Pimtong, Wittaya; Chanchao, Chanpen

2015-01-01

Tetragonula laeviceps cerumen was sequentially extracted with 80% (v/v) methanol, dichloromethane, and hexane and also in the reverse order. By the MTT assay and the respective 50% inhibition concentration value, the most active fraction was further purified to apparent homogeneity by bioassay-guided silica gel column chromatography. α-Mangostin was identified by high-resolution electrospray ionization mass spectrometry and nuclear magnetic resonance analyses. It had a potent cytotoxicity against the BT474, Chago, Hep-G2, KATO-III, and SW620 cell lines (IC50 values of 1.22 ± 0.03, 2.25 ± 0.20, 0.94 ± 0.01, 0.88 ± 0.16, and 1.50 ± 0.39 µmol/L, respectively). The in vitro cytotoxicity of α-mangostin against the five human cancer cell lines and primary fibroblasts was further characterized by real-time impedance-based analysis. Interestingly, α-mangostin was more cytotoxic against the cancer-derived cell lines than against the primary fibroblasts. Later, the migration assay was performed by continuously measuring the attachment of cells to the plate electrodes at the bottom of the transwell membrane. The combined caspase-3 and -7 activities were assayed by the Caspase-Glo® 3/7 kit. It showed that the cytotoxic mechanism involved caspase-independent apoptosis, while at low (non-toxic) concentrations α-mangostin did not significantly alter cell migration. Furthermore, the in vivo cytotoxicity and angiogenesis were determined by alkaline phosphatase staining in zebrafish embryos along with monitoring changes in the transcript expression level of two genes involved in angiogenesis (vegfaa and vegfr2) by quantitative real-time reverse transcriptase- polymerase chain reaction. It was found that the in vivo cytotoxicity of α-mangostin against zebrafish embryos had a 50% lethal concentration of 9.4 µM, but no anti-angiogenic properties were observed in zebrafish embryos at 9 and 12 µM even though it downregulated the expression of vegfaa and vegfr2 transcripts. Thus, α-mangostin is a major active compound with a potential anticancer activity in T. laeviceps cerumen in Thailand. PMID:26290139

In vitro and in vivo characterization of the anticancer activity of Thai stingless bee (Tetragonula laeviceps) cerumen.

PubMed

Nugitrangson, Pongvit; Puthong, Songchan; Iempridee, Tawin; Pimtong, Wittaya; Pornpakakul, Surachai; Chanchao, Chanpen

2016-01-01

Tetragonula laeviceps cerumen was sequentially extracted with 80% (v/v) methanol, dichloromethane, and hexane and also in the reverse order. By the MTT assay and the respective 50% inhibition concentration value, the most active fraction was further purified to apparent homogeneity by bioassay-guided silica gel column chromatography. α-Mangostin was identified by high-resolution electrospray ionization mass spectrometry and nuclear magnetic resonance analyses. It had a potent cytotoxicity against the BT474, Chago, Hep-G2, KATO-III, and SW620 cell lines (IC50 values of 1.22 ± 0.03, 2.25 ± 0.20, 0.94 ± 0.01, 0.88 ± 0.16, and 1.50 ± 0.39 µmol/L, respectively). The in vitro cytotoxicity of α-mangostin against the five human cancer cell lines and primary fibroblasts was further characterized by real-time impedance-based analysis. Interestingly, α-mangostin was more cytotoxic against the cancer-derived cell lines than against the primary fibroblasts. Later, the migration assay was performed by continuously measuring the attachment of cells to the plate electrodes at the bottom of the transwell membrane. The combined caspase-3 and -7 activities were assayed by the Caspase-Glo® 3/7 kit. It showed that the cytotoxic mechanism involved caspase-independent apoptosis, while at low (non-toxic) concentrations α-mangostin did not significantly alter cell migration. Furthermore, the in vivo cytotoxicity and angiogenesis were determined by alkaline phosphatase staining in zebrafish embryos along with monitoring changes in the transcript expression level of two genes involved in angiogenesis (vegfaa and vegfr2) by quantitative real-time reverse transcriptase- polymerase chain reaction. It was found that the in vivo cytotoxicity of α-mangostin against zebrafish embryos had a 50% lethal concentration of 9.4 µM, but no anti-angiogenic properties were observed in zebrafish embryos at 9 and 12 µM even though it downregulated the expression of vegfaa and vegfr2 transcripts. Thus, α-mangostin is a major active compound with a potential anticancer activity in T. laeviceps cerumen in Thailand. © 2015 by the Society for Experimental Biology and Medicine.

Clonal growth: invasion or stability? A comparative study of clonal architecture and diversity in native and introduced lineages of Phragmites australis (Poaceae).

PubMed

Douhovnikoff, Vladimir; Hazelton, Eric L G

2014-09-01

• The characteristics of clonal growth that are advantageous in invasive plants can also result in native plants' ability to resist invasion. In Maine, we compared the clonal architecture and diversity of an invasive lineage (introduced Phragmites) and a noninvasive lineage (native Phragmites) present in much of North America. This study is the first on stand-scale diversity using a sample size and systematic spatial-sampling scheme adequate for characterizing clonal structure in Phragmites. Our questions included: (1) Does the structure and extent of clonal growth suggest that the potential for clonal growth contributes to the invasiveness of the introduced lineage? (2) Is clonal growth common in the native lineage, acting as a possible source of ecological resistance and resilience?• Microsatellite markers were used to measure clonal sizes, architecture, and diversity within each lineage in stands within four marshes in Maine.• Clonal diversity measures indicated that clonal growth was significantly greater in stands of the native lineage than in the introduced. While lineage was a consistent predictor of clonal diversity relative ranking, the marsh location was a much stronger predictor of the absolute range of these values.• Our results indicate an important role for clonal growth in the space consolidation of native Phragmites and could explain why the introduced lineage, with stronger competitive traits, has not replaced the native where they co-occur. These results with regard to clone size, size distributions, singleton occurrence, and clonal architecture provide some evidence for stand development that follows a genotypic initial floristics model. © 2014 Botanical Society of America, Inc.

Establishment and Characterization of Novel Human Primary and Metastatic Anaplastic Thyroid Cancer Cell Lines and Their Genomic Evolution Over a Year as a Primagraft

PubMed Central

Okamoto, Ryoko; Nagata, Yasunobu; Kanojia, Deepika; Venkatesan, Subhashree; M. T., Anand; Braunstein, Glenn D.; Said, Jonathan W.; Doan, Ngan B.; Ho, Quoc; Akagi, Tadayuki; Gery, Sigal; Liu, Li-zhen; Tan, Kar Tong; Chng, Wee Joo; Yang, Henry; Ogawa, Seishi; Koeffler, H. Phillip

2015-01-01

Context: Anaplastic thyroid cancer (ATC) has no effective treatment, resulting in a high rate of mortality. We established cell lines from a primary ATC and its lymph node metastasis, and investigated the molecular factors and genomic changes associated with tumor growth. Objective: The aim of the study was to understand the molecular and genomic changes of highly aggressive ATC and its clonal evolution to develop rational therapies. Design: We established unique cell lines from primary (OGK-P) and metastatic (OGK-M) ATC specimen, as well as primagraft from the metastatic ATC, which was serially xeno-transplanted for more than 1 year in NOD scid gamma mice were established. These cell lines and primagraft were used as tools to examine gene expression, copy number changes, and somatic mutations using RNA array, SNP Chip, and whole exome sequencing. Results: Mice carrying sc (OGK-P and OGK-M) tumors developed splenomegaly and neutrophilia with high expression of cytokines including CSF1, CSF2, CSF3, IL-1β, and IL-6. Levels of HIF-1α and its targeted genes were also elevated in these tumors. The treatment of tumor carrying mice with Bevacizumab effectively decreased tumor growth, macrophage infiltration, and peripheral WBCs. SNP chip analysis showed homozygous deletion of exons 3–22 of the PARD3 gene in the cells. Forced expression of PARD3 decreased cell proliferation, motility, and invasiveness, restores cell-cell contacts and enhanced cell adhesion. Next generation exome sequencing identified the somatic changes present in the primary, metastatic, and primagraft tumors demonstrating evolution of the mutational signature over the year of passage in vivo. Conclusion: To our knowledge, we established the first paired human primary and metastatic ATC cell lines offering unique possibilities for comparative functional investigations in vitro and in vivo. Our exome sequencing also identified novel mutations, as well as clonal evolution in both the metastasis and primagraft. PMID:25365311

A strategy to accelerate protein production from a pool of clones in Chinese hamster ovary cells for toxicology studies.

PubMed

Hu, Zhilan; Hsu, Wendy; Pynn, Abby; Ng, Domingos; Quicho, Donna; Adem, Yilma; Kwong, Zephie; Mauger, Brad; Joly, John; Snedecor, Bradley; Laird, Michael W; Andersen, Dana C; Shen, Amy

2017-11-01

In the biopharmaceutical industry, a clonally derived cell line is typically used to generate material for investigational new drug (IND)-enabling toxicology studies. The same cell line is then used to generate material for clinical studies. If a pool of clones can be used to produce material for IND-enabling toxicology studies (Pool for Tox (PFT) strategy) during the time a lead clone is being selected for clinical material production, the toxicology studies can be accelerated significantly (approximately 4 months at Genentech), leading to a potential acceleration of 4 months for the IND submission. We explored the feasibility of the PFT strategy with three antibodies-mAb1, mAb2, and mAb3-at the 2 L scale. For each antibody, two lead cell lines were identified that generated material with similar product quality to the material generated from the associated pool. For two antibody molecules, mAb1 and mAb2, the material generated by the lead cell lines from 2 L bioreactors was tested in an accelerated stability study and was shown to have stability comparable to the material generated by the associated pool. Additionally, we used this approach for two antibody molecules, mAb4 and mAb5, at Tox and GMP production. The materials from the Tox batch at 400 L scale and three GMP batches at 2000 L scale have comparable product quality attributes for both molecules. Our results demonstrate the feasibility of using a pool of clonally derived cell lines to generate material of similar product quality and stability for use in IND-enabling toxicology studies as was derived from the final production clone, which enabled significant acceleration of timelines into clinical development. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1449-1455, 2017. © 2017 American Institute of Chemical Engineers.

Zebrafish as model organisms for studying drug-induced liver injury

PubMed Central

Vliegenthart, A D Bastiaan; Tucker, Carl S; Del Pozo, Jorge; Dear, James W

2014-01-01

Drug-induced liver injury (DILI) is a major challenge in clinical medicine and drug development. New models are needed for predicting which potential therapeutic compounds will cause DILI in humans, and new markers and mediators of DILI still need to be identified. This review highlights the strengths and weaknesses of using zebrafish as a high-throughput in vivo model for studying DILI. Although the zebrafish liver architecture is different from that of the mammalian liver, the main physiological processes remain similar. Zebrafish metabolize drugs using similar pathways to those in humans; they possess a wide range of cytochrome P450 enzymes that enable metabolic reactions including hydroxylation, conjugation, oxidation, demethylation and de-ethylation. Following exposure to a range of hepatotoxic drugs, the zebrafish liver develops histological patterns of injury comparable to those of mammalian liver, and biomarkers for liver injury can be quantified in the zebrafish circulation. The zebrafish immune system is similar to that of mammals, but the zebrafish inflammatory response to DILI is not yet defined. In order to quantify DILI in zebrafish, a wide variety of methods can be used, including visual assessment, quantification of serum enzymes and experimental serum biomarkers and scoring of histopathology. With further development, the zebrafish may be a model that complements rodents and may have value for the discovery of new disease pathways and translational biomarkers. PMID:24773296

Quantification of birefringence readily measures the level of muscle damage in zebrafish

DOE Office of Scientific and Technical Information (OSTI.GOV)

Berger, Joachim, E-mail: Joachim.Berger@Monash.edu; Sztal, Tamar; Currie, Peter D.

2012-07-13

Highlights: Black-Right-Pointing-Pointer Report of an unbiased quantification of the birefringence of muscle of fish larvae. Black-Right-Pointing-Pointer Quantification method readily identifies level of overall muscle damage. Black-Right-Pointing-Pointer Compare zebrafish muscle mutants for level of phenotype severity. Black-Right-Pointing-Pointer Proposed tool to survey treatments that aim to ameliorate muscular dystrophy. -- Abstract: Muscular dystrophies are a group of genetic disorders that progressively weaken and degenerate muscle. Many zebrafish models for human muscular dystrophies have been generated and analysed, including dystrophin-deficient zebrafish mutants dmd that model Duchenne Muscular Dystrophy. Under polarised light the zebrafish muscle can be detected as a bright area in anmore » otherwise dark background. This light effect, called birefringence, results from the diffraction of polarised light through the pseudo-crystalline array of the muscle sarcomeres. Muscle damage, as seen in zebrafish models for muscular dystrophies, can readily be detected by a reduction in the birefringence. Therefore, birefringence is a very sensitive indicator of overall muscle integrity within larval zebrafish. Unbiased documentation of the birefringence followed by densitometric measurement enables the quantification of the birefringence of zebrafish larvae. Thereby, the overall level of muscle integrity can be detected, allowing the identification and categorisation of zebrafish muscle mutants. In addition, we propose that the establish protocol can be used to analyse treatments aimed at ameliorating dystrophic zebrafish models.« less

The HDAC Inhibitor TSA Ameliorates a Zebrafish Model of Duchenne Muscular Dystrophy.

PubMed

Johnson, Nathan M; Farr, Gist H; Maves, Lisa

2013-09-17

Zebrafish are an excellent model for Duchenne muscular dystrophy. In particular, zebrafish provide a system for rapid, easy, and low-cost screening of small molecules that can ameliorate muscle damage in dystrophic larvae. Here we identify an optimal anti-sense morpholino cocktail that robustly knocks down zebrafish Dystrophin (dmd-MO). We use two approaches, muscle birefringence and muscle actin expression, to quantify muscle damage and show that the dmd-MO dystrophic phenotype closely resembles the zebrafish dmd mutant phenotype. We then show that the histone deacetylase (HDAC) inhibitor TSA, which has been shown to ameliorate the mdx mouse Duchenne model, can rescue muscle fiber damage in both dmd-MO and dmd mutant larvae. Our study identifies optimal morpholino and phenotypic scoring approaches for dystrophic zebrafish, further enhancing the zebrafish dmd model for rapid and cost-effective small molecule screening.

Signaling Pathways in Pathogenesis of Diamond Blackfan Anemia

DTIC Science & Technology

2014-10-01

specificity of innate immune changes that we observed in rps19 morphants is that we saw analogous changes in rpl11 mutants and in human cells with RPS19...a TGFβ family member activin in RP-deficient zebrafish and in RPS19-deficient human cells , which include a lymphoid cell line from a DBA patient... cells with varying conclusions. Therefore, understanding the precise role of not only adaptive immunity but also innate immunity in regulation of

Tcf3 and cell cycle factors contribute to butyrate resistance in colorectal cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chiaro, Christopher, E-mail: cchiaro@tcmedc.org; Lazarova, Darina L., E-mail: dlazarova@tcmedc.org; Bordonaro, Michael, E-mail: mbordonaro@tcmedc.org

2012-11-09

Highlights: Black-Right-Pointing-Pointer We investigate mechanisms responsible for butyrate resistance in colon cancer cells. Black-Right-Pointing-Pointer Tcf3 modulates butyrate's effects on Wnt activity and cell growth in resistant cells. Black-Right-Pointing-Pointer Tcf3 modulation of butyrate's effects differ by cell context. Black-Right-Pointing-Pointer Cell cycle factors are overexpressed in the resistant cells. Black-Right-Pointing-Pointer Reversal of altered gene expression can enhance the anti-cancer effects of butyrate. -- Abstract: Butyrate, a fermentation product of dietary fiber, inhibits clonal growth in colorectal cancer (CRC) cells dependent upon the fold induction of Wnt activity. We have developed a CRC cell line (HCT-R) that, unlike its parental cell line, HCT-116,more » does not respond to butyrate exposure with hyperactivation of Wnt signaling and suppressed clonal growth. PCR array analyses revealed Wnt pathway-related genes, the expression of which differs between butyrate-sensitive HCT-116 CRC cells and their butyrate-resistant HCT-R cell counterparts. We identified overexpression of Tcf3 as being partially responsible for the butyrate-resistant phenotype, as this DNA-binding protein suppresses the hyperinduction of Wnt activity by butyrate. Consequently, Tcf3 knockdown in HCT-R cells restores their sensitivity to the effects of butyrate on Wnt activity and clonal cell growth. Interestingly, the effects of overexpressed Tcf3 differ between HCT-116 and HCT-R cells; thus, in HCT-116 cells Tcf3 suppresses proliferation without rendering the cells resistant to butyrate. In HCT-R cells, however, the overexpression of Tcf3 inhibits Wnt activity, and the cells are still able to proliferate due to the higher expression levels of cell cycle factors, particularly those driving the G{sub 1} to S transition. Knowledge of the molecular mechanisms determining the variable sensitivity of CRC cells to butyrate may assist in developing approaches that prevent or reverse butyrate resistance.« less

Ecological Consequences of Clonal Integration in Plants

PubMed Central

Liu, Fenghong; Liu, Jian; Dong, Ming

2016-01-01

Clonal plants are widespread throughout the plant kingdom and dominate in diverse habitats. Spatiotemporal heterogeneity of environment is pervasive at multiple scales, even at scales relevant to individual plants. Clonal integration refers to resource translocation and information communication among the ramets of clonal plants. Due to clonal integration, clonal plant species possess a series of peculiar attributes: plasticity in response to local and non-local conditions, labor division with organ specialization for acquiring locally abundant resources, foraging behavior by selective placement of ramets in resource-rich microhabitats, and avoidance of intraclonal competition. Clonal integration has very profound ecological consequences for clonal plants. It allows them to efficiently cope with environmental heterogeneity, by alleviating local resource shortages, buffering environmental stresses and disturbances, influencing competitive ability, increasing invasiveness, and altering species composition and invasibility at the community level. In this paper, we present a comprehensive review of research on the ecological consequences of plant clonal integration based on a large body of literature. We also attempt to propose perspectives for future research. PMID:27446093

An individual-based model of zebrafish population dynamics accounting for energy dynamics.

PubMed

Beaudouin, Rémy; Goussen, Benoit; Piccini, Benjamin; Augustine, Starrlight; Devillers, James; Brion, François; Péry, Alexandre R R

2015-01-01

Developing population dynamics models for zebrafish is crucial in order to extrapolate from toxicity data measured at the organism level to biological levels relevant to support and enhance ecological risk assessment. To achieve this, a dynamic energy budget for individual zebrafish (DEB model) was coupled to an individual based model of zebrafish population dynamics (IBM model). Next, we fitted the DEB model to new experimental data on zebrafish growth and reproduction thus improving existing models. We further analysed the DEB-model and DEB-IBM using a sensitivity analysis. Finally, the predictions of the DEB-IBM were compared to existing observations on natural zebrafish populations and the predicted population dynamics are realistic. While our zebrafish DEB-IBM model can still be improved by acquiring new experimental data on the most uncertain processes (e.g. survival or feeding), it can already serve to predict the impact of compounds at the population level.

An Individual-Based Model of Zebrafish Population Dynamics Accounting for Energy Dynamics

PubMed Central

Beaudouin, Rémy; Goussen, Benoit; Piccini, Benjamin; Augustine, Starrlight; Devillers, James; Brion, François; Péry, Alexandre R. R.

2015-01-01

Developing population dynamics models for zebrafish is crucial in order to extrapolate from toxicity data measured at the organism level to biological levels relevant to support and enhance ecological risk assessment. To achieve this, a dynamic energy budget for individual zebrafish (DEB model) was coupled to an individual based model of zebrafish population dynamics (IBM model). Next, we fitted the DEB model to new experimental data on zebrafish growth and reproduction thus improving existing models. We further analysed the DEB-model and DEB-IBM using a sensitivity analysis. Finally, the predictions of the DEB-IBM were compared to existing observations on natural zebrafish populations and the predicted population dynamics are realistic. While our zebrafish DEB-IBM model can still be improved by acquiring new experimental data on the most uncertain processes (e.g. survival or feeding), it can already serve to predict the impact of compounds at the population level. PMID:25938409

Differential expression of neuroligin genes in the nervous system of zebrafish.

PubMed

Davey, Crystal; Tallafuss, Alexandra; Washbourne, Philip

2010-02-01

The establishment and maturation of appropriate synaptic connections is crucial in the development of neuronal circuits. Cellular adhesion is believed to play a central role in this process. Neuroligins are neuronal cell adhesion molecules that are hypothesized to act in the initial formation and maturation of synaptic connections. In order to establish the zebrafish as a model to investigate the in vivo role of Neuroligin proteins in nervous system development, we identified the zebrafish orthologs of neuroligin family members and characterized their expression. Zebrafish possess seven neuroligin genes. Synteny analysis and sequence comparisons show that NLGN2, NLGN3, and NLGN4X are duplicated in zebrafish, but NLGN1 has a single zebrafish ortholog. All seven zebrafish neuroligins are expressed in complex patterns in the developing nervous system and in the adult brain. The spatial and temporal expression patterns of these genes suggest that they occupy a role in nervous system development and maintenance.

Mutagenesis and phenotyping resources in zebrafish for studying development and human disease

PubMed Central

Varshney, Gaurav Kumar

2014-01-01

The zebrafish (Danio rerio) is an important model organism for studying development and human disease. The zebrafish has an excellent reference genome and the functions of hundreds of genes have been tested using both forward and reverse genetic approaches. Recent years have seen an increasing number of large-scale mutagenesis projects and the number of mutants or gene knockouts in zebrafish has increased rapidly, including for the first time conditional knockout technologies. In addition, targeted mutagenesis techniques such as zinc finger nucleases, transcription activator-like effector nucleases and clustered regularly interspaced short sequences (CRISPR) or CRISPR-associated (Cas), have all been shown to effectively target zebrafish genes as well as the first reported germline homologous recombination, further expanding the utility and power of zebrafish genetics. Given this explosion of mutagenesis resources, it is now possible to perform systematic, high-throughput phenotype analysis of all zebrafish gene knockouts. PMID:24162064

United we stand, divided we fall: a meta-analysis of experiments on clonal integration and its relationship to invasiveness.

PubMed

Song, Yao-Bin; Yu, Fei-Hai; Keser, Lidewij H; Dawson, Wayne; Fischer, Markus; Dong, Ming; van Kleunen, Mark

2013-02-01

Many ecosystems are dominated by clonal plants. Among the most distinctive characteristics of clonal plants is their potential for clonal integration (i.e. the translocation of resources between interconnected ramets), suggesting that integration may play a role in their success. However, a general synthesis of effects of clonal integration on plant performance is lacking. We conducted a meta-analysis on the effects of clonal integration on biomass production and asexual reproduction of the whole clone, the recipient part (i.e. the part of a clone that imports resources) and the donor part (i.e. the part of a clone that exports resources). The final dataset contained 389 effect sizes from 84 studies covering 57 taxa. Overall, clonal integration increased performance of recipient parts without decreasing that of donor parts, and thus increased performance of whole clones. Among the studies and taxa considered, the benefits of clonal integration did not differ between two types of experimental approaches, between stoloniferous and rhizomatous growth forms, between directions of resource translocation (from younger to older ramet or vice versa), or among types of translocated resources (water, nutrients and carbohydrates). Clonal taxa with larger benefits of integration on whole-clone performance were not more invasive globally, but taxa in which recipient parts in unfavorable patches benefited more from integration were. Our results demonstrate general performance benefits of clonal integration, at least in the short term, and suggest that clonal integration contributes to the success of clonal plants.

Modeling Leukemogenesis in the Zebrafish Using Genetic and Xenograft Models.

PubMed

Rajan, Vinothkumar; Dellaire, Graham; Berman, Jason N

2016-01-01

The zebrafish is a widely accepted model to study leukemia. The major advantage of studying leukemogenesis in zebrafish is attributed to its short life cycle and superior imaging capacity. This chapter highlights using transgenic- and xenograft-based models in zebrafish to study a specific leukemogenic mutation and analyze therapeutic responses in vivo.

Kidney organogenesis in the zebrafish: insights into vertebrate nephrogenesis and regeneration

PubMed Central

Gerlach, Gary F.; Wingert, Rebecca A.

2012-01-01

Vertebrates form a progressive series of up to three kidney organs during development—the pronephros, mesonephros, and metanephros. Each kidney derives from the intermediate mesoderm and is comprised of conserved excretory units called nephrons. The zebrafish is a powerful model for vertebrate developmental genetics, and recent studies have illustrated that zebrafish and mammals share numerous similarities in nephron composition and physiology. The zebrafish embryo forms an architecturally simple pronephros that has two nephrons, and these eventually become a scaffold onto which a mesonephros of several hundred nephrons is constructed during larval stages. In adult zebrafish, the mesonephros exhibits ongoing nephrogenesis, generating new nephrons from a local pool of renal progenitors during periods of growth or following kidney injury. The characteristics of the zebrafish pronephros and mesonephros make them genetically tractable kidney systems in which to study the functions of renal genes and address outstanding questions about the mechanisms of nephrogenesis. Here, we provide an overview of the formation and composition of these zebrafish kidney organs, and discuss how various zebrafish mutants, gene knockdowns, and transgenic models have created frameworks in which to further delineate nephrogenesis pathways. PMID:24014448

Cardiac Ca2+ signalling in zebrafish: Translation of findings to man.

PubMed

van Opbergen, Chantal J M; van der Voorn, Stephanie M; Vos, Marc A; de Boer, Teun P; van Veen, Toon A B

2018-05-07

Sudden cardiac death is a leading cause of death worldwide, mainly caused by highly disturbed electrical activation patterns in the heart. Currently, murine models are the most popular model to study underlying molecular mechanisms of inherited or acquired cardiac electrical abnormalities, although the numerous electrophysiological discrepancies between mouse and human raise the question whether mice are the optimal model to study cardiac rhythm disorders. Recently it has been uncovered that the zebrafish cardiac electrophysiology seems surprisingly similar to the human heart, mainly because the zebrafish AP contains a clear plateau phase and ECG characteristics show alignment with the human ECG. Although, before using zebrafish as a model to study cardiac arrhythmogenesis, however, it is very important to gain a better insight into the electrophysiological characteristics of the zebrafish heart. In this review we outline the electrophysiological machinery of the zebrafish cardiomyocytes, with a special focus on the intracellular Ca 2+ dynamics and excitation-contraction coupling. We debate the potential of zebrafish as a model to study human cardiovascular diseases and postulate steps to employ zebrafish into a more 'humanized' model. Copyright © 2018 Elsevier Ltd. All rights reserved.

Understanding behavioral and physiological phenotypes of stress and anxiety in zebrafish.

PubMed

Egan, Rupert J; Bergner, Carisa L; Hart, Peter C; Cachat, Jonathan M; Canavello, Peter R; Elegante, Marco F; Elkhayat, Salem I; Bartels, Brett K; Tien, Anna K; Tien, David H; Mohnot, Sopan; Beeson, Esther; Glasgow, Eric; Amri, Hakima; Zukowska, Zofia; Kalueff, Allan V

2009-12-14

The zebrafish (Danio rerio) is emerging as a promising model organism for experimental studies of stress and anxiety. Here we further validate zebrafish models of stress by analyzing how environmental and pharmacological manipulations affect their behavioral and physiological phenotypes. Experimental manipulations included exposure to alarm pheromone, chronic exposure to fluoxetine, acute exposure to caffeine, as well as acute and chronic exposure to ethanol. Acute (but not chronic) alarm pheromone and acute caffeine produced robust anxiogenic effects, including reduced exploration, increased erratic movements and freezing behavior in zebrafish tested in the novel tank diving test. In contrast, ethanol and fluoxetine had robust anxiolytic effects, including increased exploration and reduced erratic movements. The behavior of several zebrafish strains was also quantified to ascertain differences in their behavioral profiles, revealing high-anxiety (leopard, albino) and low-anxiety (wild type) strains. We also used LocoScan (CleverSys Inc.) video-tracking tool to quantify anxiety-related behaviors in zebrafish, and dissect anxiety-related phenotypes from locomotor activity. Finally, we developed a simple and effective method of measuring zebrafish physiological stress responses (based on a human salivary cortisol assay), and showed that alterations in whole-body cortisol levels in zebrafish parallel behavioral indices of anxiety. Collectively, our results confirm zebrafish as a valid, reliable, and high-throughput model of stress and affective disorders.

[Effect of rhynchophylline on behaviors of methamphetamine-dependent zebrafish and the mechanism].

PubMed

Chen, Yi-Fei; Peng, Ju; Fang, Miao; Liu, Yi; Nie, Ling-Hui; Mo, Zhi-Xian; Zhu, Ling-Ling

2016-11-20

To observe the effect of rhynchophylline on methamphetamine-dependent zebrafish and explore the possible mechanism. Zebrafish were divided into control group, amphetamine group, low- (50 mg/kg) and high (100 mg/kg)-dose rhynchophylline groups, and ketamine (150 mg/kg) group. Conditioned place preference (CPP) was induced in zebrafish with methamphetamine, and the staying time in the drug box and the tracking map of the zebrafish were observed with Noldus Ethovision XT system. The protein expressions of TH, NR2B and GLUR2 in the brain of zebrafish with CPP were detected with Western blotting. Compared with the control group, zebrafish in methamphetamine group showed significant variations in the staying time and swimming distance in the drug box after conditioning (P<0.05) with obvious alterations of NR2B, TH and GLUR2 expressions in the brain (P<0.05). Treatment of methamphetamine-dependent zebrafish with high-dose rhynchophylline significantly reduced the variations in the staying time and swimming distance in the drug box (P<0.05) and in the expressions of NR2B, TH and GLUR2 in the brain (P<0.05). Rhynchophylline can inhibit methamphetamine dependence in zebrafish, the mechanism of which may involve the expressions of TH, NR2B and GLUR2 proteins in the brain.

Glutathione S-Transferase Protein Expression in Different Life Stages of Zebrafish (Danio rerio)

PubMed Central

Tierbach, Alena; Groh, Ksenia J; Schönenberger, René; Schirmer, Kristin

2018-01-01

Abstract Zebrafish is a widely used animal model in biomedical sciences and toxicology. Although evidence for the presence of phases I and II xenobiotic defense mechanisms in zebrafish exists on the transcriptional and enzyme activity level, little is known about the protein expression of xenobiotic metabolizing enzymes. Given the important role of glutathione S-transferases (GSTs) in phase II biotransformation, we analyzed cytosolic GST proteins in zebrafish early life stages and different organs of adult male and female fish, using a targeted proteomics approach. The established multiple reaction monitoring-based assays enable the measurement of the relative abundance of specific GST isoenzymes and GST classes in zebrafish through a combination of proteotypic peptides and peptides shared within the same class. GSTs of the classes alpha, mu, pi and rho are expressed in zebrafish embryo as early as 4 h postfertilization (hpf). The majority of GST enzymes are present at 72 hpf followed by a continuous increase in expression thereafter. In adult zebrafish, GST expression is organ dependent, with most of the GST classes showing the highest expression in the liver. The expression of a wide range of cytosolic GST isoenzymes and classes in zebrafish early life stages and adulthood supports the use of zebrafish as a model organism in chemical-related investigations. PMID:29361160

Characterization of behavioral and endocrine effects of LSD on zebrafish.

PubMed

Grossman, Leah; Utterback, Eli; Stewart, Adam; Gaikwad, Siddharth; Chung, Kyung Min; Suciu, Christopher; Wong, Keith; Elegante, Marco; Elkhayat, Salem; Tan, Julia; Gilder, Thomas; Wu, Nadine; Dileo, John; Cachat, Jonathan; Kalueff, Allan V

2010-12-25

Lysergic acid diethylamide (LSD) is a potent hallucinogenic drug that strongly affects animal and human behavior. Although adult zebrafish (Danio rerio) are emerging as a promising neurobehavioral model, the effects of LSD on zebrafish have not been investigated previously. Several behavioral paradigms (the novel tank, observation cylinder, light-dark box, open field, T-maze, social preference and shoaling tests), as well as modern video-tracking tools and whole-body cortisol assay were used to characterize the effects of acute LSD in zebrafish. While lower doses (5-100 microg/L) did not affect zebrafish behavior, 250 microg/L LSD increased top dwelling and reduced freezing in the novel tank and observation cylinder tests, also affecting spatiotemporal patterns of activity (as assessed by 3D reconstruction of zebrafish traces and ethograms). LSD evoked mild thigmotaxis in the open field test, increased light behavior in the light-dark test, reduced the number of arm entries and freezing in the T-maze and social preference test, without affecting social preference. In contrast, LSD affected zebrafish shoaling (increasing the inter-fish distance in a group), and elevated whole-body cortisol levels. Overall, our findings show sensitivity of zebrafish to LSD action, and support the use of zebrafish models to study hallucinogenic drugs of abuse. Copyright (c) 2010 Elsevier B.V. All rights reserved.

Mapping the zebrafish brain methylome using reduced representation bisulfite sequencing

PubMed Central

Chatterjee, Aniruddha; Ozaki, Yuichi; Stockwell, Peter A; Horsfield, Julia A; Morison, Ian M; Nakagawa, Shinichi

2013-01-01

Reduced representation bisulfite sequencing (RRBS) has been used to profile DNA methylation patterns in mammalian genomes such as human, mouse and rat. The methylome of the zebrafish, an important animal model, has not yet been characterized at base-pair resolution using RRBS. Therefore, we evaluated the technique of RRBS in this model organism by generating four single-nucleotide resolution DNA methylomes of adult zebrafish brain. We performed several simulations to show the distribution of fragments and enrichment of CpGs in different in silico reduced representation genomes of zebrafish. Four RRBS brain libraries generated 98 million sequenced reads and had higher frequencies of multiple mapping than equivalent human RRBS libraries. The zebrafish methylome indicates there is higher global DNA methylation in the zebrafish genome compared with its equivalent human methylome. This observation was confirmed by RRBS of zebrafish liver. High coverage CpG dinucleotides are enriched in CpG island shores more than in the CpG island core. We found that 45% of the mapped CpGs reside in gene bodies, and 7% in gene promoters. This analysis provides a roadmap for generating reproducible base-pair level methylomes for zebrafish using RRBS and our results provide the first evidence that RRBS is a suitable technique for global methylation analysis in zebrafish. PMID:23975027

Short- and long-term effects of nicotine and the histone deacetylase inhibitor phenylbutyrate on novel object recognition in zebrafish.

PubMed

Faillace, M P; Pisera-Fuster, A; Medrano, M P; Bejarano, A C; Bernabeu, R O

2017-03-01

Zebrafish have a sophisticated color- and shape-sensitive visual system, so we examined color cue-based novel object recognition in zebrafish. We evaluated preference in the absence or presence of drugs that affect attention and memory retention in rodents: nicotine and the histone deacetylase inhibitor (HDACi) phenylbutyrate (PhB). The objective of this study was to evaluate whether nicotine and PhB affect innate preferences of zebrafish for familiar and novel objects after short- and long-retention intervals. We developed modified object recognition (OR) tasks using neutral novel and familiar objects in different colors. We also tested objects which differed with respect to the exploratory behavior they elicited from naïve zebrafish. Zebrafish showed an innate preference for exploring red or green objects rather than yellow or blue objects. Zebrafish were better at discriminating color changes than changes in object shape or size. Nicotine significantly enhanced or changed short-term innate novel object preference whereas PhB had similar effects when preference was assessed 24 h after training. Analysis of other zebrafish behaviors corroborated these results. Zebrafish were innately reluctant or prone to explore colored novel objects, so drug effects on innate preference for objects can be evaluated changing the color of objects with a simple geometry. Zebrafish exhibited recognition memory for novel objects with similar innate significance. Interestingly, nicotine and PhB significantly modified innate object preference.

Hospital clonal dissemination of Enterobacter aerogenes producing carbapenemase KPC-2 in a Chinese teaching hospital.

PubMed

Qin, Xiaohua; Yang, Yang; Hu, Fupin; Zhu, Demei

2014-02-01

Carbapenems are first-line agents for the treatment of serious nosocomial infections caused by multidrug-resistant Enterobacteriaceae. However, resistance to carbapenems has increased dramatically among Enterobacteriaceae in our hospital. In this study, we report clonal dissemination caused by carbapenem-resistant Enterobacter aerogenes (CREA). In 2011, CREA was identified from 12 patients admitted to the neurosurgical ward. All 12 clinical isolates were non-susceptible to cefotaxime, ceftazidime, cefoxitin, ertapenem, imipenem or meropenem. All isolates carried the gene encoding Klebsiella pneumoniae carbapenemase-2 (KPC-2), except for the isolate E4. However, a remarkably lower expression level of the porin OmpF was detected in the non-KPC-2-producing isolate E4 on SDS-PAGE compared with the carbapenem-susceptible isolate. Epidemiological and molecular investigations showed that a single E. aerogenes strain (PFGE type A), including seven KPC-2-producing clinical isolates, was primarily responsible for the first isolation and subsequent dissemination. In a case-control study, we identified risk factors for infection/colonization with CREA. Mechanical ventilation, the changing of sickbeds and previous use of broad-spectrum antibiotics were identified as potential risk factors. Our findings suggest that further studies should focus on judicious use of available antibiotics, implementation of active antibiotic resistance surveillance and strict implementation of infection-control measures to avoid the rapid spread or clonal dissemination caused by carbapenem-resistant Enterobacteriaceae in healthcare facilities.

Invasive clonal plant species have a greater root-foraging plasticity than non-invasive ones.

PubMed

Keser, Lidewij H; Dawson, Wayne; Song, Yao-Bin; Yu, Fei-Hai; Fischer, Markus; Dong, Ming; van Kleunen, Mark

2014-03-01

Clonality is frequently positively correlated with plant invasiveness, but which aspects of clonality make some clonal species more invasive than others is not known. Due to their spreading growth form, clonal plants are likely to experience spatial heterogeneity in nutrient availability. Plasticity in allocation of biomass to clonal growth organs and roots may allow these plants to forage for high-nutrient patches. We investigated whether this foraging response is stronger in species that have become invasive than in species that have not. We used six confamilial pairs of native European clonal plant species differing in invasion success in the USA. We grew all species in large pots under homogeneous or heterogeneous nutrient conditions in a greenhouse, and compared their nutrient-foraging response and performance. Neither invasive nor non-invasive species showed significant foraging responses to heterogeneity in clonal growth organ biomass or in aboveground biomass of clonal offspring. Invasive species had, however, a greater positive foraging response in terms of root and belowground biomass than non-invasive species. Invasive species also produced more total biomass. Our results suggest that the ability for strong root foraging is among the characteristics promoting invasiveness in clonal plants.

Effect of clonal integration on nitrogen cycling in rhizosphere of rhizomatous clonal plant, Phyllostachys bissetii, under heterogeneous light.

PubMed

Li, Yang; Chen, Jing-Song; Xue, Ge; Peng, Yuanying; Song, Hui-Xing

2018-07-01

Clonal integration plays an important role in clonal plant adapting to heterogeneous habitats. It was postulated that clonal integration could exhibit positive effects on nitrogen cycling in the rhizosphere of clonal plant subjected to heterogeneous light conditions. An in-situ experiment was conducted using clonal fragments of Phyllostachys bissetii with two successive ramets. Shading treatments were applied to offspring or mother ramets, respectively, whereas counterparts were treated to full sunlight. Rhizomes between two successive ramets were either severed or connected. Extracellular enzyme activities and nitrogen turnover were measured, as well as soil properties. Abundance of functional genes (archaeal or bacterial amoA, nifH) in the rhizosphere of shaded, offspring or mother ramets were determined using quantitative polymerase chain reaction. Carbon or nitrogen availabilities were significantly influenced by clonal integration in the rhizosphere of shaded ramets. Clonal integration significantly increased extracellular enzyme activities and abundance of functional genes in the rhizosphere of shaded ramets. When rhizomes were connected, higher nitrogen turnover (nitrogen mineralization or nitrification rates) was exhibited in the rhizosphere of shaded offspring ramets. However, nitrogen turnover was significantly decreased by clonal integration in the rhizosphere of shaded mother ramets. Path analysis indicated that nitrogen turnover in the rhizosphere of shaded, offspring or mother ramets were primarily driven by the response of soil microorganisms to dissolved organic carbon or nitrogen. This unique in-situ experiment provided insights into the mechanism of nutrient recycling mediated by clonal integration. It was suggested that effects of clonal integration on the rhizosphere microbial processes were dependent on direction of photosynthates transport in clonal plant subjected to heterogeneous light conditions. Copyright © 2018 Elsevier B.V. All rights reserved.

Disturbance Is an Important Driver of Clonal Richness in Tropical Seagrasses

PubMed Central

McMahon, Kathryn M.; Evans, Richard D.; van Dijk, Kor-jent; Hernawan, Udhi; Kendrick, Gary A.; Lavery, Paul S.; Lowe, Ryan; Puotinen, Marji; Waycott, Michelle

2017-01-01

Clonality is common in many aquatic plant species, including seagrasses, where populations are maintained through a combination of asexual and sexual reproduction. One common measure used to describe the clonal structure of populations is clonal richness. Clonal richness is strongly dependent on the biological characteristics of the species, and how these interact with the environment but can also reflect evolutionary scale processes especially at the edge of species ranges. However, little is known about the spatial patterns and drivers of clonal richness in tropical seagrasses. This study assessed the spatial patterns of clonal richness in meadows of three tropical seagrass species, Thalassia hemprichii, Halodule uninervis, and Halophila ovalis, spanning a range of life-history strategies and spatial scales (2.5–4,711 km) in Indonesia and NW Australia. We further investigated the drivers of clonal richness using general additive mixed models for two of the species, H. uninervis and H. ovalis, over 8° latitude. No significant patterns were observed in clonal richness with latitude, yet disturbance combined with sea surface temperature strongly predicted spatial patterns of clonal richness. Sites with a high probability of cyclone disturbance had low clonal richness, whereas an intermediate probability of cyclone disturbance and the presence of dugong grazing combined with higher sea surface temperatures resulted in higher levels of clonal richness. We propose potential mechanisms for these patterns related to the recruitment and mortality rates of individuals as well as reproductive effort. Under a changing climate, increased severity of tropical cyclones and the decline in populations of mega-grazers have the potential to reduce clonal richness leading to less genetically diverse populations. PMID:29259609

Automatic zebrafish heartbeat detection and analysis for zebrafish embryos.

PubMed

Pylatiuk, Christian; Sanchez, Daniela; Mikut, Ralf; Alshut, Rüdiger; Reischl, Markus; Hirth, Sofia; Rottbauer, Wolfgang; Just, Steffen

2014-08-01

A fully automatic detection and analysis method of heartbeats in videos of nonfixed and nonanesthetized zebrafish embryos is presented. This method reduces the manual workload and time needed for preparation and imaging of the zebrafish embryos, as well as for evaluating heartbeat parameters such as frequency, beat-to-beat intervals, and arrhythmicity. The method is validated by a comparison of the results from automatic and manual detection of the heart rates of wild-type zebrafish embryos 36-120 h postfertilization and of embryonic hearts with bradycardia and pauses in the cardiac contraction.

The Visual System of Zebrafish and its Use to Model Human Ocular Diseases

PubMed Central

Gestri, Gaia; Link, Brian A; Neuhauss, Stephan CF

2011-01-01

Free swimming zebrafish larvae depend mainly on their sense of vision to evade predation and to catch prey. Hence there is strong selective pressure on the fast maturation of visual function and indeed the visual system already supports a number of visually-driven behaviors in the newly hatched larvae. The ability to exploit the genetic and embryonic accessibility of the zebrafish in combination with a behavioral assessment of visual system function has made the zebrafish a popular model to study vision and its diseases. Here, we review the anatomy, physiology and development of the zebrafish eye as the basis to relate the contributions of the zebrafish to our understanding of human ocular diseases. PMID:21595048

Heart Repair and Regeneration: Recent Insights from Zebrafish Studies

PubMed Central

Lien, Ching-Ling; Harrison, Michael R.; Tuan, Tai-Lan; Starnes, Vaughn A

2012-01-01

Cardiovascular disease is the leading cause of death in United States and worldwide. Failure to properly repair or regenerate damaged cardiac tissues after myocardial infarction is a major cause of heart failure. In contrast to humans and other mammals, zebrafish hearts regenerate after substantial injury or tissue damage. Here, we review recent progress in studying zebrafish heart regeneration, addressing the molecular and cellular responses in the three tissue layers of the heart: myocardium, epicardium, and endocardium. We also compare different injury models utilized to study zebrafish heart regeneration, and discuss the differences in responses to injury between mammalian and zebrafish hearts. By learning how zebrafish hearts regenerate naturally, we can better design therapeutic strategies for repairing human hearts after myocardial infarction. PMID:22818295

Zebrafish models for translational neuroscience research: from tank to bedside

PubMed Central

Stewart, Adam Michael; Braubach, Oliver; Spitsbergen, Jan; Gerlai, Robert; Kalueff, Allan V.

2014-01-01

The zebrafish (Danio rerio) is emerging as a new important species for studying mechanisms of brain function and dysfunction. Focusing on selected central nervous system (CNS) disorders (brain cancer, epilepsy, and anxiety) and using them as examples, we discuss the value of zebrafish models in translational neuroscience. We further evaluate the contribution of zebrafish to neuroimaging, circuit level, and drug discovery research. Outlining the role of zebrafish in modeling a wide range of human brain disorders, we also summarize recent applications and existing challenges in this field. Finally, we emphasize the potential of zebrafish models in behavioral phenomics and high-throughput genetic/small molecule screening, which is critical for CNS drug discovery and identifying novel candidate genes. PMID:24726051

Genomic, cDNA and embryonic expression analysis of zebrafish IRF6, the gene mutated in the human oral clefting disorders Van der Woude and popliteal pterygium syndromes.

PubMed

Ben, Jin; Jabs, Ethylin Wang; Chong, Samuel S

2005-06-01

Van der Woude syndrome (VWS) and popliteal pterygium syndrome (PPS) are autosomal dominant clefting disorders recently discovered to be caused by mutations in the IRF6 (Interferon Regulatory Factor 6) gene. The IRF gene family consists of nine members encoding transcription factors that share a highly conserved helix-turn-helix DNA-binding domain and a less conserved protein-binding domain. Most IRFs regulate the expression of interferon-alpha and -beta after viral infection, but the function of IRF6 remains unknown. We have isolated a full-length zebrafish irf6 cDNA, which encodes a 492 amino acid protein that contains a Smad-IRF interaction motif and a DNA-binding domain. The zebrafish irf6 gene consists of eight exons and maps to linkage group 22 closest to marker unp1375. By in situ hybridization analysis of embryo whole-mounts and cryosections, we demonstrate that irf6 is first expressed as a maternal transcript. During gastrulation, irf6 expression was concentrated in the forerunner cells. From the bud stage to the 3-somite stage, irf6 expression was observed in the Kupffer's vesicle. No expression could be detected at the 6-somite and 10-somite stages. At the 14-somite stage, expression was detected in the otic placode. At the 17-somite stage, strong expression was also observed in the cloaca. During the pharyngula, hatch and larva periods up to 5 days post-fertilization, irf6 was expressed in the pharyngeal arches, olfactory and otic placodes, and in the epithelial cells of endoderm derived tissues. The latter tissues include the mouth, pharynx, esophagus, endodermal lining of swim bladder, liver, exocrine pancreas, and associated ducts. Overall, the zebrafish expression data are consistent with the observations of lip pits in VWS patients, as well as more recent reports of alae nasi, otitis media and sensorineural hearing loss documented in some patients.

Repression of BIM mediates survival signaling by MYC and AKT in high-risk T-cell acute lymphoblastic leukemia.

PubMed

Reynolds, C; Roderick, J E; LaBelle, J L; Bird, G; Mathieu, R; Bodaar, K; Colon, D; Pyati, U; Stevenson, K E; Qi, J; Harris, M; Silverman, L B; Sallan, S E; Bradner, J E; Neuberg, D S; Look, A T; Walensky, L D; Kelliher, M A; Gutierrez, A

2014-09-01

Treatment resistance in T-cell acute lymphoblastic leukemia (T-ALL) is associated with phosphatase and tensin homolog (PTEN) deletions and resultant phosphatidylinositol 3'-kinase (PI3K)-AKT pathway activation, as well as MYC overexpression, and these pathways repress mitochondrial apoptosis in established T-lymphoblasts through poorly defined mechanisms. Normal T-cell progenitors are hypersensitive to mitochondrial apoptosis, a phenotype that is dependent on the expression of proapoptotic BIM. In a conditional zebrafish model, MYC downregulation induced BIM expression in T-lymphoblasts, an effect that was blunted by expression of constitutively active AKT. In human T-ALL cell lines and treatment-resistant patient samples, treatment with MYC or PI3K-AKT pathway inhibitors each induced BIM upregulation and apoptosis, indicating that BIM is repressed downstream of MYC and PI3K-AKT in high-risk T-ALL. Restoring BIM function in human T-ALL cells using a stapled peptide mimetic of the BIM BH3 domain had therapeutic activity, indicating that BIM repression is required for T-ALL viability. In the zebrafish model, where MYC downregulation induces T-ALL regression via mitochondrial apoptosis, T-ALL persisted despite MYC downregulation in 10% of bim wild-type zebrafish, 18% of bim heterozygotes and in 33% of bim homozygous mutants (P=0.017). We conclude that downregulation of BIM represents a key survival signal downstream of oncogenic MYC and PI3K-AKT signaling in treatment-resistant T-ALL.

Depletion of Aurora-A in zebrafish causes growth retardation due to mitotic delay and p53-dependent cell death.

PubMed

Jeon, Hee-Yeon; Lee, Hyunsook

2013-03-01

Aurora-A is a serine/threonine mitotic kinase that is required for centrosome maturation. Many cancer cells over-express Aurora-A, and several reports have suggested that Aurora-A has prognostic value in the clinical treatment of cancer. Therefore, inhibitors for Aurora-A kinase have been developed. However, studies on Aurora-A are largely performed in cancer cell lines and are sometimes controversial. For effective evaluation of Aurora-A inhibitors in cancer treatment, it is essential to understand its function at the organism level. Here, we report the crucial functions of Aurora-A in homeostasis of spindle organization in mitosis using zebrafish embryogenesis as a model system. Using morpholino technology, we show that depletion of Aurora-A in zebrafish embryogenesis results in short bent trunks, accompanied by growth retardation and eventual cell death. Live-imaging and immunofluorescence analyses of the embryos revealed that the developmental defects are due to problems in mitosis, manifested through monopolar and disorganized spindle formation. Aurora-A-depleted cells exhibited mitotic arrest with congression failure, leading to activation of the spindle assembly checkpoint. Cell death in the absence of Aurora-A was partially rescued by co-injection of the p53 morpholino, suggesting that apoptosis after Aurora-A depletion is p53-dependent. The clinical implications of these results relate to the indication that Aurora-A inhibitors may be effective towards cancers with intact p53. © 2013 The Authors Journal compilation © 2013 FEBS.

Hypoxia-induced retinal neovascularization in zebrafish embryos: a potential model of retinopathy of prematurity.

PubMed

Wu, Yu-Ching; Chang, Chao-Yuan; Kao, Alex; Hsi, Brian; Lee, Shwu-Huey; Chen, Yau-Hung; Wang, I-Jong

2015-01-01

Retinopathy of prematurity, formerly known as a retrolental fibroplasia, is a leading cause of infantile blindness worldwide. Retinopathy of prematurity is caused by the failure of central retinal vessels to reach the retinal periphery, creating a nonperfused peripheral retina, resulting in retinal hypoxia, neovascularization, vitreous hemorrhage, vitreoretinal fibrosis, and loss of vision. We established a potential retinopathy of prematurity model by using a green fluorescent vascular endothelium zebrafish transgenic line treated with cobalt chloride (a hypoxia-inducing agent), followed by GS4012 (a vascular endothelial growth factor inducer) at 24 hours postfertilization, and observed that the number of vascular branches and sprouts significantly increased in the central retinal vascular trunks 2-4 days after treatment. We created an angiography method by using tetramethylrhodamine dextran, which exhibited severe vascular leakage through the vessel wall into the surrounding retinal tissues. The quantification of mRNA extracted from the heads of the larvae by using real-time quantitative polymerase chain reaction revealed a twofold increase in vegfaa and vegfr2 expression compared with the control group, indicating increased vascular endothelial growth factor signaling in the hypoxic condition. In addition, we demonstrated that the hypoxic insult could be effectively rescued by several antivascular endothelial growth factor agents such as SU5416, bevacizumab, and ranibizumab. In conclusion, we provide a simple, highly reproducible, and clinically relevant retinopathy of prematurity model based on zebrafish embryos; this model may serve as a useful platform for clarifying the mechanisms of human retinopathy of prematurity and its progression.

Actin binding GFP allows 4D in vivo imaging of myofilament dynamics in the zebrafish heart and the identification of Erbb2 signaling as a remodeling factor of myofibril architecture.

PubMed

Reischauer, Sven; Arnaout, Rima; Ramadass, Radhan; Stainier, Didier Y R

2014-10-24

Dilated cardiomyopathy is a leading cause of congestive heart failure and a debilitating complication of antineoplastic therapies. Despite disparate causes for dilated cardiomyopathy, maladaptive cardiac remodeling and decreased systolic function are common clinical consequences, begging an investigation of in vivo contractile dynamics in development and disease, one that has been impossible to date. To image myocardial contractile filament dynamics in vivo and to assess potential causes of dilated cardiomyopathy in antineoplastic therapies targeting the epidermal growth factor receptor Erbb2. We generated a transgenic zebrafish line expressing an actin-binding green fluorescent protein in cardiomyocytes, allowing an in vivo imaging of myofilaments. Analysis of this line revealed architectural differences in myofibrils of the distinct cardiomyocyte subtypes. We used this model to investigate the effects of Erbb2 signaling on myofibrillar organization because drugs targeting ERBB2 (HER2/NEU) signaling, a mainstay of breast cancer chemotherapy, cause dilated cardiomyopathy in many patients. High-resolution in vivo imaging revealed that Erbb2 signaling regulates a switch between a dense apical network of filamentous myofibrils and the assembly of basally localized myofibrils in ventricular cardiomyocytes. Using this novel line, we compiled a reference for myofibrillar microarchitecture among myocardial subtypes in vivo and at different developmental stages, establishing this model as a tool to analyze in vivo cardiomyocyte contractility and remodeling for a broad range of cardiovascular questions. Furthermore, we applied this model to study Erbb2 signaling in cardiomyopathy. We show a direct link between Erbb2 activity and remodeling of myofibrils, revealing an unexpected mechanism with potentially important implications for prevention and treatment of cardiomyopathy. © 2014 American Heart Association, Inc.

Long interspersed element-1 protein expression is a hallmark of many human cancers.

PubMed

Rodić, Nemanja; Sharma, Reema; Sharma, Rajni; Zampella, John; Dai, Lixin; Taylor, Martin S; Hruban, Ralph H; Iacobuzio-Donahue, Christine A; Maitra, Anirban; Torbenson, Michael S; Goggins, Michael; Shih, Ie-Ming; Duffield, Amy S; Montgomery, Elizabeth A; Gabrielson, Edward; Netto, George J; Lotan, Tamara L; De Marzo, Angelo M; Westra, William; Binder, Zev A; Orr, Brent A; Gallia, Gary L; Eberhart, Charles G; Boeke, Jef D; Harris, Chris R; Burns, Kathleen H

2014-05-01

Cancers comprise a heterogeneous group of human diseases. Unifying characteristics include unchecked abilities of tumor cells to proliferate and spread anatomically, and the presence of clonal advantageous genetic changes. However, universal and highly specific tumor markers are unknown. Herein, we report widespread long interspersed element-1 (LINE-1) repeat expression in human cancers. We show that nearly half of all human cancers are immunoreactive for a LINE-1-encoded protein. LINE-1 protein expression is a common feature of many types of high-grade malignant cancers, is rarely detected in early stages of tumorigenesis, and is absent from normal somatic tissues. Studies have shown that LINE-1 contributes to genetic changes in cancers, with somatic LINE-1 insertions seen in selected types of human cancers, particularly colon cancer. We sought to correlate this observation with expression of the LINE-1-encoded protein, open reading frame 1 protein, and found that LINE-1 open reading frame 1 protein is a surprisingly broad, yet highly tumor-specific, antigen. Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Clonal Evolution and Blast Crisis Correlate with Enhanced Proteolytic Activity of Separase in BCR-ABL b3a2 Fusion Type CML under Imatinib Therapy.

PubMed

Haaß, Wiltrud; Kleiner, Helga; Weiß, Christel; Haferlach, Claudia; Schlegelberger, Brigitte; Müller, Martin C; Hehlmann, Rüdiger; Hofmann, Wolf-Karsten; Fabarius, Alice; Seifarth, Wolfgang

2015-01-01

Unbalanced (major route) additional cytogenetic aberrations (ACA) at diagnosis of chronic myeloid leukemia (CML) indicate an increased risk of progression and shorter survival. Moreover, newly arising ACA under imatinib treatment and clonal evolution are considered features of acceleration and define failure of therapy according to the European LeukemiaNet (ELN) recommendations. On the basis of 1151 Philadelphia chromosome positive chronic phase patients of the randomized CML-study IV, we examined the incidence of newly arising ACA under imatinib treatment with regard to the p210BCR-ABL breakpoint variants b2a2 and b3a2. We found a preferential acquisition of unbalanced ACA in patients with b3a2 vs. b2a2 fusion type (ratio: 6.3 vs. 1.6, p = 0.0246) concurring with a faster progress to blast crisis for b3a2 patients (p = 0.0124). ESPL1/Separase, a cysteine endopeptidase, is a key player in chromosomal segregation during mitosis. Separase overexpression and/or hyperactivity has been reported from a wide range of cancers and cause defective mitotic spindles, chromosome missegregation and aneuploidy. We investigated the influence of p210BCR-ABL breakpoint variants and imatinib treatment on expression and proteolytic activity of Separase as measured with a specific fluorogenic assay on CML cell lines (b2a2: KCL-22, BV-173; b3a2: K562, LAMA-84). Despite a drop in Separase protein levels an up to 5.4-fold increase of Separase activity under imatinib treatment was observed exclusively in b3a2 but not in b2a2 cell lines. Mimicking the influence of imatinib on BV-173 and LAMA-84 cells by ESPL1 silencing stimulated Separase proteolytic activity in both b3a2 and b2a2 cell lines. Our data suggest the existence of a fusion type-related feedback mechanism that posttranslationally stimulates Separase proteolytic activity after therapy-induced decreases in Separase protein levels. This could render b3a2 CML cells more prone to aneuploidy and clonal evolution than b2a2 progenitors and may therefore explain the cytogenetic results of CML patients.

CHARACTERIZATION OF AN EQUINE MACROPHAGE CELL LINE: APPLICATION TO STUDIES OF EIAV INFECTION

PubMed Central

Fidalgo-Carvalho, Isabel; Craigo, Jodi K.; Barnes, Shannon; Costa-Ramos, Carolina; Montelaro, Ronald C.

2009-01-01

EIAV is a monocyte/macrophage tropic virus. To date, even though EIAV has been under investigation for numerous years, very few details have been elucidated about EIAV/macrophage interactions. This is largely due to the absence of an equine macrophage cell line that would support viral replication. Herein we describe the spontaneous immortalization and generation of a clonal equine macrophage-like (EML) cell line with the functional and immunophenotype characteristics of differentiated equine monocyte derived macrophage(s) (eMDM(s)). These cells possess strong non-specific esterase (NSE) activity, are able to phagocytose fluorescent bioparticles, and produce nitrites in response to LPS. The EML-3C cell line expresses the EIAV receptor for cellular entry (ELR1) and supports replication of the virulent EIAVPV biological clone. Thus, EML-3C cells provide a useful cell line possessing equine macrophage related properties for the growth and study of EIAV infection as well as of other equine macrophage tropic viruses. PMID:19038510

Ion transport in the zebrafish kidney from a human disease angle: possibilities, considerations, and future perspectives.

PubMed

Kersten, Simone; Arjona, Francisco J

2017-01-01

Unique experimental advantages, such as its embryonic/larval transparency, high-throughput nature, and ease of genetic modification, underpin the rapid emergence of the zebrafish (Danio rerio) as a preeminent model in biomedical research. Particularly in the field of nephrology, the zebrafish provides a promising model for studying the physiological implications of human solute transport processes along consecutive nephron segments. However, although the zebrafish might be considered a valuable model for numerous renal ion transport diseases and functional studies of many channels and transporters, not all human renal electrolyte transport mechanisms and human diseases can be modeled in the zebrafish. With this review, we explore the ontogeny of zebrafish renal ion transport, its nephron structure and function, and thereby demonstrate the clinical translational value of this model. By critical assessment of genomic and amino acid conservation of human proteins involved in renal ion handling (channels, transporters, and claudins), kidney and nephron segment conservation, and renal electrolyte transport physiology in the zebrafish, we provide researchers and nephrologists with an indication of the possibilities and considerations of the zebrafish as a model for human renal ion transport. Combined with advanced techniques envisioned for the future, implementation of the zebrafish might expand beyond unraveling pathophysiological mechanisms that underlie distinct genetic or environmentally, i.e., pharmacological and lifestyle, induced renal transport deficits. Specifically, the ease of drug administration and the exploitation of improved genetic approaches might argue for the adoption of the zebrafish as a model for preclinical personalized medicine for distinct renal diseases and renal electrolyte transport proteins. Copyright © 2017 the American Physiological Society.

Animal Models of Human Granulocyte Diseases

PubMed Central

Schäffer, Alejandro A.; Klein, Christoph

2012-01-01

In vivo animal models have proven very useful to understand basic biological pathways of the immune system, a prerequisite for the development of innovate therapies. This manuscript addresses currently available models for defined human monogenetic defects of neutrophil granulocytes, including murine, zebrafish and larger mammalian species. Strengths and weaknesses of each system are summarized, and clinical investigators may thus be inspired to develop further lines of research to improve diagnosis and therapy by use of the appropriate animal model system. PMID:23351993

Teratogenic responses of zebrafish embryos to decabromodiphenyl ether (BDE-209) in the presence of nano-SiO2 particles.

PubMed

Chao, Shu-Ju; Huang, Chin Pao; Chen, Pei-Chung; Huang, Chihpin

2017-07-01

This study investigated the influence of nano-SiO 2 particles (nSiO 2 ) on the teratogenic responses of zebrafish embryos to decabromodiphenyl ether (BDE-209). Zebrafish embryos were exposed to BDE-209 in the absence and presence of nSiO 2 for 96 h post fertilization (hpf). Results showed that formation of nSiO 2 -BDE-209 associates promoted both extracellular and intracellular uptake of BDE-209 by zebrafish embryos, thereby increasing the bioconcentration of BDE-209 on the chorion surface and the embryos. Results also showed embryos delay hatching temporarily when co-exposure to BDE-209 and nSiO 2 at 60 hpf. Furthermore, there was heartbeat decline (28.3 beats/10s) and increase in irregular heartbeat (45.8%) in zebrafish larvae at 96 hpf, compared to the sole exposure to BDE-209 (32.7 beats/10s and 0%). Malformation in terms of spinal curvature (SC), pericardial edema (PE) and yolk sac edema (YSE) were observed on zebrafish larvae at 33.9, 23.4, and 18%, respectively. Overall, abnormal development of zebrafish was apparent when co-exposure to BDE-209 and nSiO 2 . All relevant evidence considered, nSiO 2 could facilitate the transport of BDE-209 towards zebrafish embryos and negatively impact the development of zebrafish. Copyright © 2017 Elsevier Ltd. All rights reserved.

Dietary Strontium Increases Bone Mineral Density in Intact Zebrafish (Danio rerio): A Potential Model System for Bone Research

PubMed Central

Padgett-Vasquez, Steve; Garris, Heath W.; Nagy, Tim R.; D'Abramo, Louis R.; Watts, Stephen A.

2010-01-01

Abstract Zebrafish (Danio rerio) skeletal bone possesses properties similar to human bone, which suggests that they may be used as a model to study mineralization characteristics of the human Haversian system, as well as human bone diseases. One prerequisite for the use of zebrafish as an alternative osteoporotic bone model is to determine whether their bone displays functional plasticity similar to that observed in other bone models. Strontium citrate was supplemented into a laboratory-prepared diet (45% crude protein) to produce dietary strontium levels of 0%, 0.63%, 1.26%, 1.89%, and 2.43% and fed ad libitum twice daily for 12 weeks to 28-day-old intact zebrafish. Length was determined at 4-week intervals, and both weight and length were recorded at 12 weeks. At 12 weeks, seven zebrafish from each dietary level were analyzed for total bone mineral density by microcomputed tomography. Dietary strontium citrate supplementation significantly (p < 0.05) increased zebrafish whole-body and spinal column bone mineral density. In addition, trace amounts of strontium were incorporated into the scale matrix in those zebrafish that consumed strontium-supplemented diets. These findings suggest that zebrafish bone displays plasticity similar to that reported for other bone models (i.e., rat, mouse, and monkey) that received supplements of strontium compounds and zebrafish should be viewed as an increasingly valuable bone model. PMID:20874492

Husbandry stress exacerbates mycobacterial infections in adult zebrafish, Danio rerio (Hamilton)

USGS Publications Warehouse

Ramsay, J.M.; Watral, Virginia G.; Schreck, C.B.; Kent, M.L.

2009-01-01

Mycobacteria are significant pathogens of laboratory zebrafish, Danio rerio (Hamilton). Stress is often implicated in clinical disease and morbidity associated with mycobacterial infections but has yet to be examined with zebrafish. The aim of this study was to examine the effects of husbandry stressors on zebrafish infected with mycobacteria. Adult zebrafish were exposed to Mycobacterium marinum or Mycobacterium chelonae, two species that have been associated with disease in zebrafish. Infected fish and controls were then subjected to chronic crowding and handling stressors and examined over an 8-week period. Whole-body cortisol was significantly elevated in stressed fish compared to non-stressed fish. Fish infected with M. marinum ATCC 927 and subjected to husbandry stressors had 14% cumulative mortality while no mortality occurred among infected fish not subjected to husbandry stressors. Stressed fish, infected with M. chelonae H1E2 from zebrafish, were 15-fold more likely to be infected than non-stressed fish at week 8 post-injection. Sub-acute, diffuse infections were more common among stressed fish infected with M. marinum or M. chelonae than non-stressed fish. This is the first study to demonstrate an effect of stress and elevated cortisol on the morbidity, prevalence, clinical disease and histological presentation associated with mycobacterial infections in zebrafish. Minimizing husbandry stress may be effective at reducing the severity of outbreaks of clinical mycobacteriosis in zebrafish facilities. ?? 2009 Blackwell Publishing Ltd.

A two-scale model for correlation between B cell VDJ usage in zebrafish

NASA Astrophysics Data System (ADS)

Pan, Keyao; Deem, Michael

2011-03-01

The zebrafish (Danio rerio) is one of the model animals for study of immunology. The dynamics of the adaptive immune system in zebrafish is similar to that in higher animals. In this work, we built a two-scale model to simulate the dynamics of B cells in primary and secondary immune reactions in zebrafish and to explain the reported correlation between VDJ usage of B cell repertoires in distinct zebrafish. The first scale of the model consists of a generalized NK model to simulate the B cell maturation process in the 10-day primary immune response. The second scale uses a delay ordinary differential equation system to model the immune responses in the 6-month lifespan of zebrafish. The generalized NK model shows that mature B cells specific to one antigen mostly possess a single VDJ recombination. The probability that mature B cells in two zebrafish have the same VDJ recombination increases with the B cell population size or the B cell selection intensity and decreases with the B cell hypermutation rate. The ODE model shows a distribution of correlation in the VDJ usage of the B cell repertoires in two six-month-old zebrafish that is highly similar to that from experiment. This work presents a simple theory to explain the experimentally observed correlation in VDJ usage of distinct zebrafish B cell repertoires after an immune response.

The role of idiotypic interactions in the adaptive immune system: a belief-propagation approach

NASA Astrophysics Data System (ADS)

Bartolucci, Silvia; Mozeika, Alexander; Annibale, Alessia

2016-08-01

In this work we use belief-propagation techniques to study the equilibrium behaviour of a minimal model for the immune system comprising interacting T and B clones. We investigate the effect of the so-called idiotypic interactions among complementary B clones on the system’s activation. Our results show that B-B interactions increase the system’s resilience to noise, making clonal activation more stable, while increasing the cross-talk between different clones. We derive analytically the noise level at which a B clone gets activated, in the absence of cross-talk, and find that this increases with the strength of idiotypic interactions and with the number of T cells sending signals to the B clones. We also derive, analytically and numerically, via population dynamics, the critical line where clonal cross-talk arises. Our approach allows us to derive the B clone size distribution, which can be experimentally measured and gives important information about the adaptive immune system response to antigens and vaccination.

Single Cell Proteolytic Assays to Investigate Cancer Clonal Heterogeneity and Cell Dynamics Using an Efficient Cell Loading Scheme

NASA Astrophysics Data System (ADS)

Chen, Yu-Chih; Cheng, Yu-Heng; Ingram, Patrick; Yoon, Euisik

2016-06-01

Proteolytic degradation of the extracellular matrix (ECM) is critical in cancer invasion, and recent work suggests that heterogeneous cancer populations cooperate in this process. Despite the importance of cell heterogeneity, conventional proteolytic assays measure average activity, requiring thousands of cells and providing limited information about heterogeneity and dynamics. Here, we developed a microfluidic platform that provides high-efficiency cell loading and simple valveless isolation, so the proteolytic activity of a small sample (10-100 cells) can be easily characterized. Combined with a single cell derived (clonal) sphere formation platform, we have successfully demonstrated the importance of microenvironmental cues for proteolytic activity and also investigated the difference between clones. Furthermore, the platform allows monitoring single cells at multiple time points, unveiling different cancer cell line dynamics in proteolytic activity. The presented tool facilitates single cell proteolytic analysis using small samples, and our findings illuminate the heterogeneous and dynamic nature of proteolytic activity.

Biochemical and pharmacological characterization of the thyrotropin releasing hormone (TRH) receptor from clonal GH sub 4 C sub 1 pituitary cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Phillips, W.J.

1987-01-01

The effect of drugs with anesthetic properties on the activity of the pituitary thyrotropin-releasing hormone (TRH) receptor was determined in the clonal GH{sub 4}C{sub 1} somatomammotropic cell line. Classic local anesthetics and other drugs with anesthetic activity inhibited binding of ({sup 3}H)methyl-TRH to cell receptors at concentrations known to produce anesthetic effects on the membrane. The inhibition of TRH receptor binding by tetracaine was competitive and temperature and pH dependent. Verapamil and tetracaine inhibited TRH-stimulated prolactin secretion at concentrations that inhibited peptide binding. TRH-stimulated prolactin secretion was equivalent with or without Ca{sup 2+} channel activity. Verapamil and tetracaine also inhibitedmore » basal prolactin and secretion stimulated by drugs that bypass membrane receptors, db-cAMP and TPA. These results indicate that inhibition of TRH binding and responses by diverse drugs results from an anesthetic effect on the cell membrane.« less

Zebrafish: A Versatile Animal Model for Fertility Research.

PubMed

Hoo, Jing Ying; Kumari, Yatinesh; Shaikh, Mohd Farooq; Hue, Seow Mun; Goh, Bey Hing

2016-01-01

The utilization of zebrafish in biomedical research is very common in the research world nowadays. Today, it has emerged as a favored vertebrate organism for the research in science of reproduction. There is a significant growth in amount numbers of scientific literature pertaining to research discoveries in reproductive sciences in zebrafish. It has implied the importance of zebrafish in this particular field of research. In essence, the current available literature has covered from the very specific brain region or neurons of zebrafish, which are responsible for reproductive regulation, until the gonadal level of the animal. The discoveries and findings have proven that this small animal is sharing a very close/similar reproductive system with mammals. More interestingly, the behavioral characteristics and along with the establishment of animal courtship behavior categorization in zebrafish have laid an even stronger foundation and firmer reason on the suitability of zebrafish utilization in research of reproductive sciences. In view of the immense importance of this small animal for the development of reproductive sciences, this review aimed at compiling and describing the proximate close similarity of reproductive regulation on zebrafish and human along with factors contributing to the infertility, showing its versatility and its potential usage for fertility research.

The essential role of endogenous ghrelin in growth hormone expression during zebrafish adenohypophysis development.

PubMed

Li, Xi; He, Jiangyan; Hu, Wei; Yin, Zhan

2009-06-01

Ghrelin, a multifunctional hormone, including potent GH stimulation activity, has been suggested to be important during embryonic development. Expression of ghrelin has been confirmed in the zebrafish pancreas during embryonic stages. Interfering with ghrelin function using two specific antisense morpholino oligonucleotides causes defects during zebrafish embryonic development. In ghrelin morphants the expression of GH was abolished in zebrafish somatotropes, whereas the expression patterns of the other key molecules involved in hypothalamic-pituitary development and distinct pituitary hormones genes remain largely intact at the appropriate time during zebrafish adenohypophysis development. Effective rescue of the ghrelin morphants with exogenous ghrelin mRNA showed that the correct gene had been targeted. Moreover, by analyzing the efficiencies of the ghrelin morphants rescue experiments with various forms of exogenous mutant ghrelin mRNAs, we also demonstrated the essentiality of the form acyl-ghrelin on GH stimulation during zebrafish adenohypophysis development. Our in vivo experiments, for the first time, also provided evidence of the existence of functional obestatin in the C-terminal part of zebrafish proghrelin peptides. Our research here has demonstrated that zebrafish is a unique model for functional studies of endogenous ghrelin, especially during embryonic development.

Translating Discovery in Zebrafish Pancreatic Development to Human Pancreatic Cancer: Biomarkers, Targets, Pathogenesis, and Therapeutics

PubMed Central

Kazi, Abid A.; Yee, Rosemary K.

2013-01-01

Abstract Experimental studies in the zebrafish have greatly facilitated understanding of genetic regulation of the early developmental events in the pancreas. Various approaches using forward and reverse genetics, chemical genetics, and transgenesis in zebrafish have demonstrated generally conserved regulatory roles of mammalian genes and discovered novel genetic pathways in exocrine pancreatic development. Accumulating evidence has supported the use of zebrafish as a model of human malignant diseases, including pancreatic cancer. Studies have shown that the genetic regulators of exocrine pancreatic development in zebrafish can be translated into potential clinical biomarkers and therapeutic targets in human pancreatic adenocarcinoma. Transgenic zebrafish expressing oncogenic K-ras and zebrafish tumor xenograft model have emerged as valuable tools for dissecting the pathogenetic mechanisms of pancreatic cancer and for drug discovery and toxicology. Future analysis of the pancreas in zebrafish will continue to advance understanding of the genetic regulation and biological mechanisms during organogenesis. Results of those studies are expected to provide new insights into how aberrant developmental pathways contribute to formation and growth of pancreatic neoplasia, and hopefully generate valid biomarkers and targets as well as effective and safe therapeutics in pancreatic cancer. PMID:23682805

Translating discovery in zebrafish pancreatic development to human pancreatic cancer: biomarkers, targets, pathogenesis, and therapeutics.

PubMed

Yee, Nelson S; Kazi, Abid A; Yee, Rosemary K

2013-06-01

Abstract Experimental studies in the zebrafish have greatly facilitated understanding of genetic regulation of the early developmental events in the pancreas. Various approaches using forward and reverse genetics, chemical genetics, and transgenesis in zebrafish have demonstrated generally conserved regulatory roles of mammalian genes and discovered novel genetic pathways in exocrine pancreatic development. Accumulating evidence has supported the use of zebrafish as a model of human malignant diseases, including pancreatic cancer. Studies have shown that the genetic regulators of exocrine pancreatic development in zebrafish can be translated into potential clinical biomarkers and therapeutic targets in human pancreatic adenocarcinoma. Transgenic zebrafish expressing oncogenic K-ras and zebrafish tumor xenograft model have emerged as valuable tools for dissecting the pathogenetic mechanisms of pancreatic cancer and for drug discovery and toxicology. Future analysis of the pancreas in zebrafish will continue to advance understanding of the genetic regulation and biological mechanisms during organogenesis. Results of those studies are expected to provide new insights into how aberrant developmental pathways contribute to formation and growth of pancreatic neoplasia, and hopefully generate valid biomarkers and targets as well as effective and safe therapeutics in pancreatic cancer.

BMP signaling modulates hepcidin expression in zebrafish embryos independent of hemojuvelin.

PubMed

Gibert, Yann; Lattanzi, Victoria J; Zhen, Aileen W; Vedder, Lea; Brunet, Frédéric; Faasse, Sarah A; Babitt, Jodie L; Lin, Herbert Y; Hammerschmidt, Matthias; Fraenkel, Paula G

2011-01-21

Hemojuvelin (Hjv), a member of the repulsive-guidance molecule (RGM) family, upregulates transcription of the iron regulatory hormone hepcidin by activating the bone morphogenetic protein (BMP) signaling pathway in mammalian cells. Mammalian models have identified furin, neogenin, and matriptase-2 as modifiers of Hjv's function. Using the zebrafish model, we evaluated the effects of hjv and its interacting proteins on hepcidin expression during embryonic development. We found that hjv is strongly expressed in the notochord and somites of the zebrafish embryo and that morpholino knockdown of hjv impaired the development of these structures. Knockdown of hjv or other hjv-related genes, including zebrafish orthologs of furin or neogenin, however, failed to decrease hepcidin expression relative to liver size. In contrast, overexpression of bmp2b or knockdown of matriptase-2 enhanced the intensity and extent of hepcidin expression in zebrafish embryos, but this occurred in an hjv-independent manner. Furthermore, we demonstrated that zebrafish hjv can activate the human hepcidin promoter and enhance BMP responsive gene expression in vitro, but is expressed at low levels in the zebrafish embryonic liver. Taken together, these data support an alternative mechanism for hepcidin regulation during zebrafish embryonic development, which is independent of hjv.

Computerized image analysis for quantitative neuronal phenotyping in zebrafish.

PubMed

Liu, Tianming; Lu, Jianfeng; Wang, Ye; Campbell, William A; Huang, Ling; Zhu, Jinmin; Xia, Weiming; Wong, Stephen T C

2006-06-15

An integrated microscope image analysis pipeline is developed for automatic analysis and quantification of phenotypes in zebrafish with altered expression of Alzheimer's disease (AD)-linked genes. We hypothesize that a slight impairment of neuronal integrity in a large number of zebrafish carrying the mutant genotype can be detected through the computerized image analysis method. Key functionalities of our zebrafish image processing pipeline include quantification of neuron loss in zebrafish embryos due to knockdown of AD-linked genes, automatic detection of defective somites, and quantitative measurement of gene expression levels in zebrafish with altered expression of AD-linked genes or treatment with a chemical compound. These quantitative measurements enable the archival of analyzed results and relevant meta-data. The structured database is organized for statistical analysis and data modeling to better understand neuronal integrity and phenotypic changes of zebrafish under different perturbations. Our results show that the computerized analysis is comparable to manual counting with equivalent accuracy and improved efficacy and consistency. Development of such an automated data analysis pipeline represents a significant step forward to achieve accurate and reproducible quantification of neuronal phenotypes in large scale or high-throughput zebrafish imaging studies.

From Omics to Drug Metabolism and High Content Screen of Natural Product in Zebrafish: A New Model for Discovery of Neuroactive Compound

PubMed Central

Hung, Ming Wai; Zhang, Zai Jun; Li, Shang; Lei, Benson; Yuan, Shuai; Cui, Guo Zhen; Man Hoi, Pui; Chan, Kelvin; Lee, Simon Ming Yuen

2012-01-01

The zebrafish (Danio rerio) has recently become a common model in the fields of genetics, environmental science, toxicology, and especially drug screening. Zebrafish has emerged as a biomedically relevant model for in vivo high content drug screening and the simultaneous determination of multiple efficacy parameters, including behaviour, selectivity, and toxicity in the content of the whole organism. A zebrafish behavioural assay has been demonstrated as a novel, rapid, and high-throughput approach to the discovery of neuroactive, psychoactive, and memory-modulating compounds. Recent studies found a functional similarity of drug metabolism systems in zebrafish and mammals, providing a clue with why some compounds are active in zebrafish in vivo but not in vitro, as well as providing grounds for the rationales supporting the use of a zebrafish screen to identify prodrugs. Here, we discuss the advantages of the zebrafish model for evaluating drug metabolism and the mode of pharmacological action with the emerging omics approaches. Why this model is suitable for identifying lead compounds from natural products for therapy of disorders with multifactorial etiopathogenesis and imbalance of angiogenesis, such as Parkinson's disease, epilepsy, cardiotoxicity, cerebral hemorrhage, dyslipidemia, and hyperlipidemia, is addressed. PMID:22919414

Understanding spatio-temporal strategies of adult zebrafish exploration in the open field test.

PubMed

Stewart, Adam Michael; Gaikwad, Siddharth; Kyzar, Evan; Kalueff, Allan V

2012-04-27

Zebrafish (Danio rerio) are emerging as a useful model organism for neuroscience research. Mounting evidence suggests that various traditional rodent paradigms may be adapted for testing zebrafish behavior. The open field test is a popular rodent test of novelty exploration, recently applied to zebrafish research. To better understand fish novelty behavior, we exposed adult zebrafish to two different open field arenas for 30 min, assessing the amount and temporal patterning of their exploration. While (similar to rodents) zebrafish scale their locomotory activity depending on the size of the tank, the temporal patterning of their activity was independent of arena size. These observations strikingly parallel similar rodent behaviors, suggesting that spatio-temporal strategies of animal exploration may be evolutionarily conserved across vertebrate species. In addition, we found interesting oscillations in zebrafish exploration, with the per-minute distribution of their horizontal activity demonstrating sinusoidal-like patterns. While such patterning is not reported for rodents and other higher vertebrates, a nonlinear regression analysis confirmed the oscillation patterning of all assessed zebrafish behavioral endpoints in both open field arenas, revealing a potentially important aspect of novelty exploration in lower vertebrates. Copyright © 2012 Elsevier B.V. All rights reserved.

The importance of Zebrafish in biomedical research.

PubMed

Tavares, Bárbara; Santos Lopes, Susana

2013-01-01

Zebrafish (Danio rerio) is an ideal model organism for the study of vertebrate development. This is due to the large clutches that each couple produces, with up to 200 embryos every 7 days, and to the fact that the embryos and larvae are small, transparent and undergo rapid external development. Using scientific literature research tools available online and the keywords Zebrafish, biomedical research, human disease, and drug screening, we reviewed original studies and reviews indexed in PubMed. In this review we summarized work conducted with this model for the advancement of our knowledge related to several human diseases. We also focused on the biomedical research being performed in Portugal with the zebrafish model. Powerful live imaging and genetic tools are currently available for zebrafish making it a valuable model in biomedical research. The combination of these properties with the optimization of automated systems for drug screening has transformed the zebrafish into "a top model" in biomedical research, drug discovery and toxicity testing. Furthermore, with the optimization of xenografts technology it will be possible to use zebrafish to aide in the choice of the best therapy for each patient. Zebrafish is an excellent model organism in biomedical research, drug development and in clinical therapy.

The zebrafish world of colors and shapes: preference and discrimination.

PubMed

Oliveira, Jessica; Silveira, Mayara; Chacon, Diana; Luchiari, Ana

2015-04-01

Natural environment imposes many challenges to animals, which have to use cognitive abilities to cope with and exploit it to enhance their fitness. Since zebrafish is a well-established model for cognitive studies and high-throughput screening for drugs and diseases that affect cognition, we tested their ability for ambient color preference and 3D objects discrimination to establish a protocol for memory evaluation. For the color preference test, zebrafish were observed in a multiple-chamber tank with different environmental color options. Zebrafish showed preference for blue and green, and avoided yellow and red. For the 3D objects discrimination, zebrafish were allowed to explore two equal objects and then observed in a one-trial test in which a new color, size, or shape of the object was presented. Zebrafish showed discrimination for color, shape, and color+shape combined, but not size. These results imply that zebrafish seem to use some categorical system to discriminate items, and distracters affect their ability for discrimination. The type of variables available (color and shape) may favor zebrafish objects perception and facilitate discrimination processing. We suggest that this easy and simple memory test could serve as a useful screening tool for cognitive dysfunction and neurotoxicological studies.

Designing and Testing of Self-Cleaning Recirculating Zebrafish Tanks.

PubMed

Nema, Shubham; Bhargava, Yogesh

2016-08-01

Maintenance of large number of zebrafish in captive conditions is a daunting task. This can be eased by the use of recirculating racks with self-cleaning zebrafish tanks. Commercially available systems are costly, and compatibility of intercompany products has never been investigated. Although various cost-effective designs and methods of construction of custom-made recirculating zebrafish racks are available in literature, the design of self-cleaning zebrafish tanks is still not available. In this study, we report the design and method of construction of the self-cleaning unit, which can be fitted in any zebrafish tank. We validated the design by investigating sediment cleaning process in rectangular and cylindrical tank geometries using time lapse imaging. Our results suggest that for both tank geometries, the tanks fitted with self-cleaning unit provided superior sediment cleaning than the tanks fitted with overflow-drain unit. Although the self-cleaning unit could clean the sediment completely from both geometries over prolonged period, the cleaning of sediments was faster in the cylindrical tank than the rectangular tank. In conclusion, cost and efforts of zebrafish maintenance could be significantly reduced through the installation of our self-cleaning unit in any custom-made zebrafish tank.

Zebrafish: A Versatile Animal Model for Fertility Research

PubMed Central

Hoo, Jing Ying; Kumari, Yatinesh; Shaikh, Mohd Farooq; Hue, Seow Mun

2016-01-01

The utilization of zebrafish in biomedical research is very common in the research world nowadays. Today, it has emerged as a favored vertebrate organism for the research in science of reproduction. There is a significant growth in amount numbers of scientific literature pertaining to research discoveries in reproductive sciences in zebrafish. It has implied the importance of zebrafish in this particular field of research. In essence, the current available literature has covered from the very specific brain region or neurons of zebrafish, which are responsible for reproductive regulation, until the gonadal level of the animal. The discoveries and findings have proven that this small animal is sharing a very close/similar reproductive system with mammals. More interestingly, the behavioral characteristics and along with the establishment of animal courtship behavior categorization in zebrafish have laid an even stronger foundation and firmer reason on the suitability of zebrafish utilization in research of reproductive sciences. In view of the immense importance of this small animal for the development of reproductive sciences, this review aimed at compiling and describing the proximate close similarity of reproductive regulation on zebrafish and human along with factors contributing to the infertility, showing its versatility and its potential usage for fertility research. PMID:27556045

Differences in Acute Alcohol-Induced Behavioral Responses Among Zebrafish Populations

PubMed Central

Gerlai, Robert; Ahmad, Fahad; Prajapati, Sonal

2009-01-01

Background With the arsenal of genetic tools available for zebrafish, this species has been successfully used to investigate the genetic aspects of human diseases from developmental disorders to cancer. Interest in the behavior and brain function of zebrafish is also increasing as CNS disorders may be modeled and studied with this species. Alcoholism and alcohol abuse are among the most devastating and costliest diseases. However, the mechanisms of these diseases are not fully understood. Zebrafish has been proposed as a model organism to study such mechanisms. Characterization of alcohol’s effects on zebrafish is a necessary step in this research. Methods Here, we compare the effects of acute alcohol (EtOH) administration on the behavior of zebrafish from 4 distinct laboratory-bred populations using automated as well as observation based behavioral quantification methods. Results Alcohol treatment resulted in significant dose-dependent behavioral changes but the dose–response trajectories differed among zebrafish populations. Conclusions The results demonstrate for the first time a genetic component in alcohol responses in adult zebrafish and also show the feasibility of high throughput behavioral screening. We discuss the exploration and exploitation of the genetic differences found. PMID:18652595

d-Tubocurarine and Berbamine: Alkaloids That Are Permeant Blockers of the Hair Cell's Mechano-Electrical Transducer Channel and Protect from Aminoglycoside Toxicity

PubMed Central

Kirkwood, Nerissa K.; O'Reilly, Molly; Derudas, Marco; Kenyon, Emma J.; Huckvale, Rosemary; van Netten, Sietse M.; Ward, Simon E.; Richardson, Guy P.; Kros, Corné J.

2017-01-01

Aminoglycoside antibiotics are widely used for the treatment of life-threatening bacterial infections, but cause permanent hearing loss in a substantial proportion of treated patients. The sensory hair cells of the inner ear are damaged following entry of these antibiotics via the mechano-electrical transducer (MET) channels located at the tips of the hair cell's stereocilia. d-Tubocurarine (dTC) is a MET channel blocker that reduces the loading of gentamicin-Texas Red (GTTR) into rat cochlear hair cells and protects them from gentamicin treatment. Berbamine is a structurally related alkaloid that reduces GTTR labeling of zebrafish lateral-line hair cells and protects them from aminoglycoside-induced cell death. Both compounds are thought to reduce aminoglycoside entry into hair cells through the MET channels. Here we show that dTC (≥6.25 μM) or berbamine (≥1.55 μM) protect zebrafish hair cells in vivo from neomycin (6.25 μM, 1 h). Protection of zebrafish hair cells against gentamicin (10 μM, 6 h) was provided by ≥25 μM dTC or ≥12.5 μM berbamine. Hair cells in mouse cochlear cultures are protected from longer-term exposure to gentamicin (5 μM, 48 h) by 20 μM berbamine or 25 μM dTC. Berbamine is, however, highly toxic to mouse cochlear hair cells at higher concentrations (≥30 μM) whilst dTC is not. The absence of toxicity in the zebrafish assays prompts caution in extrapolating results from zebrafish neuromasts to mammalian cochlear hair cells. MET current recordings from mouse outer hair cells (OHCs) show that both compounds are permeant open-channel blockers, rapidly and reversibly blocking the MET channel with half-blocking concentrations of 2.2 μM (dTC) and 2.8 μM (berbamine) in the presence of 1.3 mM Ca2+ at −104 mV. Berbamine, but not dTC, also blocks the hair cell's basolateral K+ current, IK,neo, and modeling studies indicate that berbamine permeates the MET channel more readily than dTC. These studies reveal key properties of MET-channel blockers required for the future design of successful otoprotectants. PMID:28928635

Analysis of Lethality and Malformations During Zebrafish (Danio rerio) Development.

PubMed

Raghunath, Azhwar; Perumal, Ekambaram

2018-01-01

The versatility offered by zebrafish (Danio rerio) makes it a powerful and an attractive vertebrate model in developmental toxicity and teratogenicity assays. Apart from the newly introduced chemicals as drugs, xenobiotics also induce abnormal developmental abnormalities and congenital malformations in living organisms. Over the recent decades, zebrafish embryo/larva has emerged as a potential tool to test teratogenicity potential of these chemicals. Zebrafish responds to compounds as mammals do as they share similarities in their development, metabolism, physiology, and signaling pathways with that of mammals. The methodology used by the different scientists varies enormously in the zebrafish embryotoxicity test. In this chapter, we present methods to assess lethality and malformations during zebrafish development. We propose two major malformations scoring systems: binomial and relative morphological scoring systems to assess the malformations in zebrafish embryos/larvae. Based on the scoring of the malformations, the test compound can be classified as a teratogen or a nonteratogen and its teratogenic potential is evaluated.

Conserved gene regulation during acute inflammation between zebrafish and mammals

PubMed Central

Forn-Cuní, G.; Varela, M.; Pereiro, P.; Novoa, B.; Figueras, A.

2017-01-01

Zebrafish (Danio rerio), largely used as a model for studying developmental processes, has also emerged as a valuable system for modelling human inflammatory diseases. However, in a context where even mice have been questioned as a valid model for these analysis, a systematic study evaluating the reproducibility of human and mammalian inflammatory diseases in zebrafish is still lacking. In this report, we characterize the transcriptomic regulation to lipopolysaccharide in adult zebrafish kidney, liver, and muscle tissues using microarrays and demonstrate how the zebrafish genomic responses can effectively reproduce the mammalian inflammatory process induced by acute endotoxin stress. We provide evidence that immune signaling pathways and single gene expression is well conserved throughout evolution and that the zebrafish and mammal acute genomic responses after lipopolysaccharide stimulation are highly correlated despite the differential susceptibility between species to that compound. Therefore, we formally confirm that zebrafish inflammatory models are suited to study the basic mechanisms of inflammation in human inflammatory diseases, with great translational impact potential. PMID:28157230

Genotoxicity and oxidative stress induced by the fungicide azoxystrobin in zebrafish (Danio rerio) livers.

PubMed

Han, Yingnan; Liu, Tong; Wang, Jinhua; Wang, Jun; Zhang, Cheng; Zhu, Lusheng

2016-10-01

Azoxystrobin is a frequently used fungicide in agriculture. Its toxicological effects on non-target organisms have aroused attention. In the present work, the toxic effects of azoxystrobin on zebrafish (Danio rerio) were investigated. Male and female zebrafish were separately exposed to a control solution and three azoxystrobin treatments (1, 10, and 100μg/L) and were sampled on days 7, 14, 21, and 28. Reactive oxygen species (ROS) were accumulated in excess in the zebrafish livers. Superoxide dismutase (SOD) activity was significantly inhibited in the male zebrafish. Moreover, a notable decrease was also observed after day 21 in the female zebrafish. Catalase (CAT) activity was induced by the azoxystrobin treatments with the exception of the 1μg/L treatment. A significant increase in glutathione-S-transferase (GST) activity was observed after day 21. Lipid peroxidation (LPO) was generated, and DNA damage was enhanced in a concentration-dependent manner. In conclusion, azoxystrobin induced oxidative stress and genotoxicity in zebrafish livers. Copyright © 2016 Elsevier Inc. All rights reserved.

Developmental social isolation affects adult behavior, social interaction, and dopamine metabolite levels in zebrafish.

PubMed

Shams, Soaleha; Amlani, Shahid; Buske, Christine; Chatterjee, Diptendu; Gerlai, Robert

2018-01-01

The zebrafish is a social vertebrate and an excellent translational model for a variety of human disorders. Abnormal social behavior is a hallmark of several human brain disorders. Social behavioral problems can arise as a result of adverse early social environment. Little is known about the effects of early social isolation in adult zebrafish. We compared zebrafish that were isolated for either short (7 days) or long duration (180 days) to socially housed zebrafish, testing their behavior across ontogenesis (ages 10, 30, 60, 90, 120, 180 days), and shoal cohesion and whole-brain monoamines and their metabolites in adulthood. Long social isolation increased locomotion and decreased shoal cohesion and anxiety in the open-field in adult. Additionally, both short and long social isolation reduced dopamine metabolite levels in response to social stimuli. Thus, early social isolation has lasting effects in zebrafish, and may be employed to generate zebrafish models of human neuropsychiatric conditions. © 2017 Wiley Periodicals, Inc.

Physical exercise improves learning in zebrafish, Danio rerio.

PubMed

Luchiari, Ana Carolina; Chacon, Diana Marques Martins

2013-11-01

Zebrafish is an ideal vertebrate model for neuroscience studies focusing on learning and memory. Although genetic manipulation of zebrafish is available, behavioral protocols are often lacking. In this study we tested whether physical activity can facilitate zebrafish's learning process in an associative conditioning task. Learning was inferred by the approach of the feeding area just after the conditioned stimulus (light). Unexercised zebrafish showed conditioning response from the 5th testing day while fish previously submitted to swim against the water current showed learning by the 3rd day of testing. It seems that physical activity may accelerate associative learning response in zebrafish, indicating the benefits of exercise for cognitive processes. We suggest that this preliminary work could be useful for high throughput screening. Copyright © 2013 Elsevier B.V. All rights reserved.

Zebrafish models of cardiovascular diseases and their applications in herbal medicine research.

PubMed

Seto, Sai-Wang; Kiat, Hosen; Lee, Simon M Y; Bensoussan, Alan; Sun, Yu-Ting; Hoi, Maggie P M; Chang, Dennis

2015-12-05

The zebrafish (Danio rerio) has recently become a powerful animal model for cardiovascular research and drug discovery due to its ease of maintenance, genetic manipulability and ability for high-throughput screening. Recent advances in imaging techniques and generation of transgenic zebrafish have greatly facilitated in vivo analysis of cellular events of cardiovascular development and pathogenesis. More importantly, recent studies have demonstrated the functional similarity of drug metabolism systems between zebrafish and humans, highlighting the clinical relevance of employing zebrafish in identifying lead compounds in Chinese herbal medicine with potential beneficial cardiovascular effects. This paper seeks to summarise the scope of zebrafish models employed in cardiovascular studies and the application of these research models in Chinese herbal medicine to date. Crown Copyright © 2015. Published by Elsevier B.V. All rights reserved.

Identification of a role for the nuclear receptor EAR-2 in the maintenance of clonogenic status within the leukemia cell hierarchy

PubMed Central

Ichim, CV; Atkins, HL; Iscove, NN; Wells, RA

2016-01-01

Identification of genes that regulate clonogenicity of acute myelogenous leukemia (AML) cells is hindered by the difficulty of isolating pure populations of cells with defined proliferative abilities. By analyzing the growth of clonal siblings in low passage cultures of the cell line OCI/AML4 we resolved this heterogeneous population into strata of distinct clonogenic potential, permitting analysis of the transcriptional signature of single cells with defined proliferative abilities. By microarray analysis we showed that the expression of the orphan nuclear receptor EAR-2 (NR2F6) is greater in leukemia cells with extensive proliferative capacity than in those that have lost proliferative ability. EAR-2 is expressed highly in long-term hematopoietic stem cells, relative to short-term hematopoietic stem and progenitor cells, and is downregulated in AML cells after induction of differentiation. Exogenous expression of EAR-2 increased the growth of U937 cells and prevented the proliferative arrest associated with terminal differentiation, and blocked differentiation of U937 and 32Dcl3 cells. Conversely, silencing of EAR-2 by short-hairpin RNA initiated terminal differentiation of these cell lines. These data identify EAR-2 as an important factor in the regulation of clonogenicity and differentiation, and establish that analysis of clonal siblings allows the elucidation of differences in gene expression within the AML hierarchy. PMID:21637284

TALEN mediated targeted editing of GM2/GD2-synthase gene modulates anchorage independent growth by reducing anoikis resistance in mouse tumor cells

PubMed Central

Mahata, Barun; Banerjee, Avisek; Kundu, Manjari; Bandyopadhyay, Uday; Biswas, Kaushik

2015-01-01

Complex ganglioside expression is highly deregulated in several tumors which is further dependent on specific ganglioside synthase genes. Here, we designed and constructed a pair of highly specific transcription-activator like effector endonuclease (TALENs) to disrupt a particular genomic locus of mouse GM2-synthase, a region conserved in coding sequence of all four transcript variants of mouse GM2-synthase. Our designed TALENs effectively work in different mouse cell lines and TALEN induced mutation rate is over 45%. Clonal selection strategy is undertaken to generate stable GM2-synthase knockout cell line. We have also demonstrated non-homologous end joining (NHEJ) mediated integration of neomycin cassette into the TALEN targeted GM2-synthase locus. Functionally, clonally selected GM2-synthase knockout clones show reduced anchorage-independent growth (AIG), reduction in tumor growth and higher cellular adhesion as compared to wild type Renca-v cells. Insight into the mechanism shows that, reduced AIG is due to loss in anoikis resistance, as both knockout clones show increased sensitivity to detachment induced apoptosis. Therefore, TALEN mediated precise genome editing at GM2-synthase locus not only helps us in understanding the function of GM2-synthase gene and complex gangliosides in tumorigenicity but also holds tremendous potential to use TALENs in translational cancer research and therapeutics. PMID:25762467

TALEN mediated targeted editing of GM2/GD2-synthase gene modulates anchorage independent growth by reducing anoikis resistance in mouse tumor cells.

PubMed

Mahata, Barun; Banerjee, Avisek; Kundu, Manjari; Bandyopadhyay, Uday; Biswas, Kaushik

2015-03-12

Complex ganglioside expression is highly deregulated in several tumors which is further dependent on specific ganglioside synthase genes. Here, we designed and constructed a pair of highly specific transcription-activator like effector endonuclease (TALENs) to disrupt a particular genomic locus of mouse GM2-synthase, a region conserved in coding sequence of all four transcript variants of mouse GM2-synthase. Our designed TALENs effectively work in different mouse cell lines and TALEN induced mutation rate is over 45%. Clonal selection strategy is undertaken to generate stable GM2-synthase knockout cell line. We have also demonstrated non-homologous end joining (NHEJ) mediated integration of neomycin cassette into the TALEN targeted GM2-synthase locus. Functionally, clonally selected GM2-synthase knockout clones show reduced anchorage-independent growth (AIG), reduction in tumor growth and higher cellular adhesion as compared to wild type Renca-v cells. Insight into the mechanism shows that, reduced AIG is due to loss in anoikis resistance, as both knockout clones show increased sensitivity to detachment induced apoptosis. Therefore, TALEN mediated precise genome editing at GM2-synthase locus not only helps us in understanding the function of GM2-synthase gene and complex gangliosides in tumorigenicity but also holds tremendous potential to use TALENs in translational cancer research and therapeutics.

Characterization of the OmyY1 region on the rainbow trout Y chromosome

USGS Publications Warehouse

Phillips, Ruth B.; DeKoning, Jenefer J.; Brunelli, Joseph P.; Faber-Hammond, Joshua J.; Hansen, John D.; Christensen, Kris A.; Renn, Suzy C.P.; Thorgaard, Gary H.

2013-01-01

We characterized the male-specific region on the Y chromosome of rainbow trout, which contains both sdY (the sex-determining gene) and the male-specific genetic marker, OmyY1. Several clones containing the OmyY1 marker were screened from a BAC library from a YY clonal line and found to be part of an 800 kb BAC contig. Using fluorescence in situ hybridization (FISH), these clones were localized to the end of the short arm of the Y chromosome in rainbow trout, with an additional signal on the end of the X chromosome in many cells. We sequenced a minimum tiling path of these clones using Illumina and 454 pyrosequencing. The region is rich in transposons and rDNA, but also appears to contain several single-copy protein-coding genes. Most of these genes are also found on the X chromosome; and in several cases sex-specific SNPs in these genes were identified between the male (YY) and female (XX) homozygous clonal lines. Additional genes were identified by hybridization of the BACs to the cGRASP salmonid 4x44K oligo microarray. By BLASTn evaluations using hypothetical transcripts of OmyY1-linked candidate genes as query against several EST databases, we conclude at least 12 of these candidate genes are likely functional, and expressed.

Biotechnology to harness the benefits of dietary phenolics; focus on Lamiaceae.

PubMed

Shetty, K

1997-09-01

Phytochemicals from herbs and fermented legumes are excellent dietary sources of phenolic metabolites. These phenolics have importance not only as food preservatives but increasingly have therapeutic and pharmaceutical applications. The long-term research objecitves of the food biotechnology program at the University of Massachusetts are to elucidate the molecular and physiological mechanisms associated with synthesis of important health-related, therapeutic phenolic metabolites in food-related plants and fermented plant foods. Current efforts focus on elucidation of the role of the proline-linked pentose phosphate pathway in regulating the synthesis of anti-inflammatory compound, rosmarinic acid (RA). Specific aims of the current research efforts are: (i) To develop novel tissue culture-based selection techniques to isolate high RA-producing, shoot-based clonal lines from genetically heterogeneous, cross-pollinating species in the family Lamiaceae; (ii) To target genetically uniform, regenerated shoot-based clonal lines for: (a) preliminary characterization of key enzymes associated with the pentose phosphate pathway and linked to RA synthesis; (b) development of genetic transformation techniques for subsequent engineering of metabolic pathways associated with RA synthesis. These research objectives have substantial implications for harnessing the genetic and biochemical potential of genetically heterogeneous, food-related medicinal plant species. The success of this research also provides novel methods and strategies to gain access to metabolic pathways of pharmaceutically important metabolites from ginger, curcuma, chili peppers, melon or other food-related species with novel phenolics.

Time-lapse imaging of neural development: zebrafish lead the way into the fourth dimension.

PubMed

Rieger, Sandra; Wang, Fang; Sagasti, Alvaro

2011-07-01

Time-lapse imaging is often the only way to appreciate fully the many dynamic cell movements critical to neural development. Zebrafish possess many advantages that make them the best vertebrate model organism for live imaging of dynamic development events. This review will discuss technical considerations of time-lapse imaging experiments in zebrafish, describe selected examples of imaging studies in zebrafish that revealed new features or principles of neural development, and consider the promise and challenges of future time-lapse studies of neural development in zebrafish embryos and adults. Copyright © 2011 Wiley-Liss, Inc.

Intraspecific competition and light effect on reproduction of Ligularia virgaurea, an invasive native alpine grassland clonal herb

PubMed Central

Xie, Tian-peng; Zhang, Ge-fei; Zhao, Zhi-gang; Du, Guo-zhen; He, Gui-yong

2014-01-01

The relationship between sexual reproduction and clonal growth in clonal plants often shows up at the ramet level. However, only a few studies focus on the relationship at the genet level, which could finally account for evolution. The sexual reproduction and clonal growth of Ligularia virgaurea, a perennial herb widely distributed in the alpine grasslands of the Qinghai-Tibetan Plateau of China, were studied under different competition intensities and light conditions at the genet level through a potted experiment. The results showed that: (1) sexual reproduction did not depend on density or light, and increasing clonal growth with decreasing density and increasing light intensity indicated that intraspecific competition and light intensity may affect the clonal life history of L. virgaurea; (2) both sexual reproduction and clonal growth show a positive linear relationship with genet size under different densities and light conditions; (3) a threshold size is required for sexual reproduction and no evidence of a threshold size for clonal growth under different densities and light conditions; (4) light level affected the allocation of total biomass to clonal and sexual structures, with less allocation to clonal structures and more allocation to sexual structures in full sunlight than in shade; (5) light determined the onset of sexual reproduction, and the genets in the shade required a smaller threshold size for sexual reproduction to occur than the plants in full sunlight; and (6) no evidence was found of trade-offs between clonal growth and sexual reproduction under different densities and light conditions at the genet level, and the positive correlation between two reproductive modes indicated that these are two integrated processes. Clonal growth in this species may be viewed as a growth strategy that tends to maximize genet fitness. PMID:24683463

Microsatellites within the feline androgen receptor are suitable for X chromosome-linked clonality testing in archival material.

PubMed

Farwick, Nadine M; Klopfleisch, Robert; Gruber, Achim D; Weiss, Alexander Th A

2017-04-01

Objectives A hallmark of neoplasms is their origin from a single cell; that is, clonality. Many techniques have been developed in human medicine to utilise this feature of tumours for diagnostic purposes. One approach is X chromosome-linked clonality testing using polymorphisms of genes encoded by genes on the X chromosome. The aim of this study was to determine if the feline androgen receptor gene was suitable for X chromosome-linked clonality testing. Methods The feline androgen receptor gene was characterised and used to test clonality of feline lymphomas by PCR and polyacrylamide gel electrophoresis, using archival formalin-fixed, paraffin-embedded material. Results Clonality of the feline lymphomas under study was confirmed and the gene locus was shown to represent a suitable target in clonality testing. Conclusions and relevance Because there are some pitfalls of using X chromosome-linked clonality testing, further studies are necessary to establish this technique in the cat.

Aging, clonal hematopoiesis and preleukemia: not just bad luck?

PubMed

Shlush, Liran I; Zandi, Sasan; Itzkovitz, Shalev; Schuh, Andre C

2015-11-01

Chronological human aging is associated with a number of changes in the hematopoietic system, occurring at many levels from stem to mature cells, and the marrow microenvironment as well. This review will focus mainly on the aging of hematopoietic stem and progenitor cells (HSPCs), and on the associated increases in the incidence of hematological malignancies. HSPCs manifest reduced function and acquire molecular changes with chronological aging. Furthermore, while for many years it has been known that the human hematopoietic system becomes increasingly clonal with chronological aging (clonal hematopoiesis), only in the last few years has it become clear that clonal hematopoiesis may result from the accumulation of preleukemic mutations in HSPCs. Such mutations confer a selective advantage that leads to clonal hematopoiesis, and that may occasionally result in the development of leukemia, and define the existence of both preleukemic stem cells, and of 'preleukemia' as a clinical entity. While it is well appreciated that clonal hematopoiesis is very common in the elderly, several questions remain unanswered: why and how does clonal hematopoiesis develop? How is clonal hematopoiesis related to the age-related changes observed in the hematopoietic system? And why do only some individuals with clonal hematopoiesis develop leukemia?

Glucocorticoid activity detected by in vivo zebrafish assay and in vitro glucocorticoid receptor bioassay at environmental relevant concentrations.

PubMed

Chen, Qiyu; Jia, Ai; Snyder, Shane A; Gong, Zhiyuan; Lam, Siew Hong

2016-02-01

Glucocorticoids are pharmaceutical contaminants of emerging concern due to their incomplete removal during wastewater treatment, increased presence in aquatic environment and their biological potency. The zebrafish is a popular model for aquatic toxicology and environmental risk assessment. This study aimed to determine if glucocorticoids at environmental concentrations would perturb expression of selected glucocorticoid-responsive genes in zebrafish and to investigate their potentials as an in vivo zebrafish assay in complementing in vitro glucocorticoid receptor bioassay. The relative expression of eleven glucocorticoid-responsive genes in zebrafish larvae and liver of adult male zebrafish exposed to three representative glucocorticoids (dexamethasone, prednisolone and triamcinolone) was determined. The expression of pepck, baiap2 and pxr was up-regulated in zebrafish larvae and the expression of baiap2, pxr and mmp-2 was up-regulated in adult zebrafish exposed to glucocorticoids at concentrations equivalent to total glucocorticoids reported in environmental samples. The responsiveness of the specific genes were sufficiently robust in zebrafish larvae exposed to a complex environmental sample detected with in vitro glucocorticoid activity equivalent to 478 pM dexamethasone (DEX-EQ) and confirmed to contain low concentration (0.2 ng/L or less) of the targeted glucocorticoids, and possibly other glucocorticoid-active compounds. The findings provided in vivo relevance to the in vitro glucocorticoid activity and suggested that the environmental sample can perturb glucocorticoid-responsive genes in its original, or half the diluted, concentration as may be found in the environment. The study demonstrated the important complementary roles of in vivo zebrafish and in vitro bioassays coupled with analytical chemistry in monitoring environmental glucocorticoid contaminants. Copyright © 2015 Elsevier Ltd. All rights reserved.