title: FACS-Assisted Microarray Profiling Implicates Novel Genes and Pathways in Zebrafish Gastrointestinal Tract Development

status: Public on Jul 10 2009

city: Pittsburgh

first: Carsten

last: Stuckenholz

address: 3501 Fifth Avenue, Room 5065.5B

institute: University of Pittsburgh

summary:
Zebrafish (Danio rerio) gutGFP transgenic embryos [Tg(XlEef1a1:GFP)s854] were collected at 4 time points: 2 days post fertilization (dpf), 3, dpf, 4 dpf, 6 dpf. Embryos were dissociated into single cells and sorted by FACS based on GFP expression.
RNA was extracted from the different cell populations (Stratagene), amplified (NuGEN Ovation), and hybridized to Affymetrix Zebrafish GeneChips.

Keywords: Time course.

overall_design: Series of 24 samples: 4 time points, 3 replicates, 2 samples per replicate (GFP+ and GFP-).

first: Lili

last: Lu

first: Prakash

last: Thakur

first: Naftali

last: Kaminski

first: Nathan

last: Bahary


title: Gene profiling of Müller glia during early stages of zebrafish photoreceptor regeneration

status: Public on Feb 04 2009

city: Ann Arbor

first: Zhao

last: Qin

state: MI

address: 830 North University

department: Molecular, Cellular and Developmental Biology

country: USA

institute: University of Michigan

summary:
Photoreceptor damage in adult mammals results in permanent cell loss and glial scarring in the retina. In contrast, adult zebrafish can regenerate photoreceptors following injury. By using a stable transgenic line in which GFP is driven by the cis-regulatory sequences of a glial specific marker gfap, Tg(gfap:GFP)mi2002, previous studies showed that Müller glia, the radial glial cells in the retina, proliferate after photoreceptor loss and give rise to neuronal progenitors that eventually differentiate into regenerated photoreceptors. To identify the molecular mechanisms that initiate this regenerative response, Müller glia were isolated from Tg(gfap:GFP)mi2002 fish during the early stages of regeneration after light lesion and gene expression profiles were generated by microarray analyses.

Keywords: time course

overall_design: Retinas were dissected from Tg(gfap:GFP)mi2002 zebrafish at 8, 16, 24, 36 hour post-lesion (hpl) and non-light-treated controls (0 hpl) and were dissociated by enzymatic digestion. GFP+ Müller glia were isolated by fluorescence-activated cell sorting (FACS) for RNA extraction and hybridization on Affymetrix microarrays. Independent hybridization of three biological replicates were performed for each time point.

first: Matthew

last: Brooks

first: Ritu

last: Khanna

first: Pamela

middle: A

last: Raymond


title: Expression data from zebrafish pineal gland

status: Public on May 01 2009

city: Tel Aviv

first: Yoav

last: Gothilf

address: Sherman Building, Room 405

department: Neurobiology, Faculty of Life Sciences

country: Israel

institute: Tel Aviv University

summary:
Microarray data allowed detection of genes that are highly expressed in the pineal gland.

Keywords: tissue expression pattern

overall_design: Adult Tg(aanat2:EGFP)Y8 transgenic zebrafish in which EGFP marks the pineal gland were used for RNA extraction and hybridization on Affymetrix microarrays. Fish were anesthetized in 1.5 mM Tricane (Sigma) and sacrificed by decapitation, and pineal glands were removed under a fluorescent dissecting microscope. Since the pineal gland is a clock-containing organ and levels of certain transcripts may vary throughout the circadian cycle, glands were collected throughout the 24-hr cycle at 4 hr intervals. During the 24-hr cycle the fish were either maintained in a 12-h light/12-h dark cycle (LD) or kept in constant darkness (DD). 12 pineal glands were collected and pooled at each time point at each light condition, and total RNA was extracted (RNeasy, Qiagen).


title: Effects of the Aromatase Inhibitor Fadrozole on Gene Expression in the Zebrafish Telencephalon

status: Public on Feb 26 2009

city: Ottawa

first: Martin

last: Paquette

state: Ontario

address: 30 Marie Curie

department: Department of Biology

country: Canada

institute: University of Ottawa

summary: Cytochrome P450 aromatase is an essential enzyme for vertebrates.  It is responsible for the conversion of androgens to estrogens in the brain and gonadal tissues.  Aromatase converts testosterone into estradiol and therefore participates in the regulation of many processes which are controlled by estrogens such as development and fertility.  Teleosts have been characterized as having exceptionally high levels of brain estrogen biosynthesis when compared to the brains of the other vertebrates.  Little is known about the effects of estrogens on brain function in teleosts.  The main objective of this study was to assess the effects of fadrozole, a powerful aromatase inhibitor, on gene expression in the zebrafish brain.  To achieve this, male zebrafish were exposed to fadrozole for 10 days.  This exposure causes a decrease in estrogens along with an increase in androgens.  Plasma testosterone levels showed a 3-fold increase in response to the exposure.  In the telencephalon, 235 genes were identified by Affymetrix GeneChip analysis as being differentially regulated.  Real-time RT-PCR was used to validate the data obtained from the microarrays.  This technique was also used to evaluate the response in the hypothalamus, which appears to follow similar transcriptional regulation.  Gene ontology analysis of the microarray data revealed common biological processes and molecular functions such as sensory perception and detection of light stimulus, homeobox and transcription factor complexes.  This study has identified genes which are directly and/or indirectly controlled by estrogens and androgens which helped to demonstrate the pronounced effects of endocrine disrupting chemicals on gene expression in the brain of teleosts. Collectively, the results provide a better understanding of the effects of aromatase inhibitors on gene expression and also shed light on the underlying effects of sex hormone variation and their importance in the brain of teleosts.

overall_design:
Adult male zebrafish were exposed to fadrozole (200 ug/L) for 10 days.  Telencephalons were pooled; approximately 30 per array.
3 treated samples, 3 control samples

first: Vance

last: Trudeau

first: Marc

last: Ekker


title: Microcystin Genomic Effects on Zebrafish Larvae

status: Public on Jul 23 2009

city: Knoxville

first: Julia

last: Gouffon

state: TN

address: 1215 West Cumberland Ave, RM 229

department: Nutrition

institute: Univ. of TN

summary: Zebrafish (Danio rerio) were obtained from the Zebrafish Research Facility maintained in the Center for Environmental Biotechnology at the University of Tennessee.  Fish husbandry, spawning, and experimental procedures were conducted with approval from the University of Tennessee Institutional Animal Care and Use Committee (Protocol #1690-1007).  Water for holding fish and conducting experiments (hereafter referred to as fish water) consisted of MilliQ water (Millipore, Bedford, MA) with ions added: 19 mg/L NaHCO3, 1 mg/L sea salt (Instant Ocean Synthetic Sea Salt, Mentor, OH), 10 mg/L CaSO4, 10 mg/L MgSO4, 2 mg/L KCl.  Embryos were obtained by spawning adult fish with no history of contaminant exposure.  Fertilization of embryos took place at the same time (± 15 min.), such that larvae used in experiments were of similar age at the time of exposure.  All activities (maintenance of adult fish, spawning, and experiments) were conducted in an environmental chamber with a temperature of 27± 1 ºC and 14:10h light:dark photoperiod.

overall_design:
At 72 h post-fertilization, zebrafish larvae were exposed to lyophilized Microcystis and purified MC-LR at concentrations of 100 and 1,000 µg/L.  Controls consisted of zebrafish system water (negative control) and zebrafish system water containing 0.05% ethanol (vehicle control). Larvae from both control groups as well as 100 µg/L MC-LR, 1,000 µg/L MC-LR, and lyophilized Microcystis were exposed in groups of 50 with three replicates and were sacrificed after 96 hours for total RNA extraction and subsequent microarray analysis.  All larvae were exposed in beakers containing 100 ml of solution.
Water samples for microcystin analysis and water quality measurements were taken during the experiment, and mortality and behavioral observations were recorded at 24-hour intervals. Microcystin analysis was conducted by protein phosphatase inhibition assay.  Measured concentrations of microcystin-LR were 140 ± 12 SD (low concentration) and 1,703 ± 71 SD (high concentration).  The concentration of microcystin-LR in the lyophilized Microcystis treatment was 4.5 µg/L.  Water quality parameters measured included dissolved oxygen (6.7 mg/L), pH (6.9), total alkalinity (36 mg/L as CaCO3), total hardness (18 mg/L as CaCO3), and ammonia (<0.2 mg/L). No significant mortality or behavioral changes in larvae were observed during the exposure.

first: Emily

middle: D

last: Rogers

first: Theodore

middle: B

last: Henry

middle: S

first: Jackson

last: McPherson

first: Gregory

middle: L

last: Boyer

first: Gary

last: Sayler

first: Steven

middle: W

last: Wilhelm


title: AHR Activation by TCDD Downregulates Sox9b Expression Producing Jaw Malformation in Zebrafish Embryos

status: Public on Nov 01 2008

city: Madison

first: Kong

middle: M

last: Xiong

state: WI

address: 777 Highland Ave, 5208 Rennebohm Hall

department: Biomolecular Chemistry

institute: University of Wisconsin at Madison

summary:
Exposure to environmental contaminants can disrupt normal development of the early vertebrate skeleton.  2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) impairs craniofacial skeletal development across many vertebrate species and its effects are especially prominent in early life stages of fish.  TCDD activates the aryl hydrocarbon receptor (AHR), a transcription factor that mediates most if not all TCDD responses.  We investigated the transcriptional response in the developing zebrafish jaw following TCDD exposure using DNA microarrays.  Zebrafish larvae were exposed to TCDD at 96 h postfertilization (hpf) and jaw cartilage tissue was harvested for microarray analysis at 1, 2, 4 and 12 h postexposure (hpe).  Numerous chondrogenic transcripts were misregulated by TCDD in the jaw.  Comparison of transcripts altered by TCDD in jaw with transcripts altered in embryonic heart showed that the transcriptional responses in the jaw and the heart were strikingly different.  Sox9b, a critical chondrogenic transcription factor, was the most significantly reduced transcript in the jaw.  We hypothesized that the TCDD reduction of sox9b expression plays an integral role in affecting formation of the embryonic jaw.  Morpholino knock down of sox9b expression demonstrated that partial reduction of sox9b expression alone was sufficient to produce a TCDD-like jaw phenotype.  Heterozygous sox9b deletion mutant embryos were sensitized to TCDD.  Lastly, embryos injected with sox9b mRNA and then exposed to TCDD blocked TCDD-induced jaw toxicity in approximately 14% of sox9b-injected embryos.  These results suggest that reduced sox9b expression in TCDD-exposed zebrafish embryos contributes to jaw malformation.

Keywords: Time course

overall_design: Three independent replicate microarray time course experiments were performed comparing transcript levels between TCDD-exposed and control zebrafish.  For each experiment, zebrafish were exposed to TCDD for 1 h starting at 96 hpf as described above.  For each time point (97, 98, 100 and 108 hpf) and treatment jaw samples were pooled from 10 dissections for RNA isolation and hybridization with Affymetrix zebrafish arrays (Affymetrix, Santa Clara, CA).  Each microarray contains roughly 14,900 probes corresponding to approximately 30% of the zebrafish genome.  For each array, total RNA (1 µg) was isolated from 10 jaw microdissections with the QIAGEN RNeasy Mini kit following the manufacturer’s protocol (QIAGEN, Valencia, CA).  The One-Cycle Target Labeling and Control Reagents kit was used to synthesize cDNA and biotinylated cRNA following the manufacturer’s protocol (Affymetrix, Santa Clara, CA).  Biotin-labeled cRNA (15 µg) was fragmented and hybridized onto Affymetrix Zebrafish Genechip Arrays following the protocol in the Affymetrix Genechip Expression Analysis Technical Manual.  Following hybdrization, the arrays were washed and stained with streptavidin-phycoerythrin on an Affymetrix Fluidics Station 400 using the protocol EukGE WS2v4.  Arrays were scanned with an Agilent Gene Array Scanner.

first: Richard

middle: E

last: Peterson

first: Warren

last: Heideman


title: Foggy mutant: brain (Guo-1R01NS042626-01A2)

status: Public on Oct 28 2008

city: Phoenix

first: Winnie

last: Liang

state: AZ

address: 445 N. Fifth Street

institute: Translational Genomics

summary:
The foggy mutant zebrafish show severe deficits in dopaminergic neurons, thus is an important  tool to understanding the development of these neurons, which undergo degeneration in Parkinson's disease.  Molecular study reveals that the foggy gene encodes a regulator of transcription elongation.  Biochemical study suggests that the repressive activity of the Foggy protein is lost in the foggy mutant.  The genes whose expression is normally repressed by Foggy are likely to play important roles in dopamine neuron development. However, their identity is unknown.  Finding out these target genes of Foggy will provide important insights into dopamine neuron development and regeneration.  They will also serve as an entry point to elucidate the role of transcription elongation in controlling neuronal development.
We will identify genes whose expression is altered in the foggy mutant. Their expression pattern and functional significance will be examined in zebrafish through in situ hybridization and morpholino antisense knockdowm methods.
We hypothesize that the genes whose transcription is regulated by Foggy play a critical role in the development of dopaminergic neurons.
The species noted above is Drosophila however we are using ZEBRAFISH. We will examine three stages: 24 hour post fertilization (hpf), 36 hpf, and 48 hpf. During these stages, the dopamine neuron deficits manifest in the foggy mutant. To identify genes whose expression is altered and important for dopamine neuron development, we shall dissect embryonic brain tissues from wildtype and the foggy mutant embryos at the stages mentioned above, and prepare RNA from them.  Since the foggy gene is expressed ubiquitously and also required for proper retinal and cardiovascular development, we would like to compare the gene expression between the whole embryos of wildtype and foggy mutant, to identify all possible downstream targets. We are currently applying for a supplement to support the entire microarray experiment.  As a pilot experiment, we would like to perform 6 array analysis as soon as possible.  Thus, we will examine the 48 hours post fertilzation time point in this pilot experiment and it will be performed in triplicate.

This dataset is composed of 36 hours post fertilization wild type and mutants and 12-somite stage Foggy knockdown.

first: Su

last: Guo


title: Gene expression in response to depletion of specific human histone H1 isoforms.

status: Public on Oct 20 2008

city: Badalona

first: Lauro

last: Sumoy

state: Barcelona

address: Ctra. Can Ruti, Camí de les escoles s/n

department: High Content Genomics and Bioinformatics

country: Spain

institute: IGTP

summary:
At least six histone H1 variants exist in mammalian somatic cells that bind to the linker DNA and stabilize the nucleosome particle contributing to higher order chromatin compaction. In addition, H1 seems to be involved in the active regulation of gene expression. It is not well known whether the different variants have specific roles or regulate specific promoters. We have explored this by inducible shRNA-mediated knock-down of each of the H1 variants in a human breast cancer cell line. A different subset of genes is altered in each H1 knock-down.

Keywords: cell type comparison; genetic modification; RNA interference

overall_design: Stable breast cancer-derived cell lines expressing an shRNA against one of each of the histone H1 isoforms in response to doxycycline (Dox) were grown for six days in the presence or absence of Dox, in duplicate, and RNA extracted for microarray hybridization. Cell lines used: inducible shRNA against H1.0, H1.2, H1.3, H1.4, H1.5, and control/empty shRNA-expression vector.

first: Albert

last: Jordan

first: Mónica

last: Sancho


title: The role of ERbeta2 in zebrafish neuromasts development

status: Public on Dec 02 2008

city: Dübendorf

first: Mirjam

last: Föhlicher

address: überlandstrasse 133

country: Switzerland

institute: eawag

summary: This SuperSeries is composed of the SubSeries listed below.

overall_design: Refer to individual Series


title: Isolation of single miRNA-expressing cells from zebrafish embryos

status: Public on Dec 25 2008

city: Cambridge

first: Alena

last: Shkumatava

state: MA

address: 9 Cambridge Center

department: Bartel Lab

institute: Whitehead Institute/MIT

summary:
The goal of the project was to isolate single miRNA-expressing cells labelled by GFP reporter genes under the control of endogenous miRNA promoters and analyze expression levels of miRNA target genes in these cells. GFP-positive miRNA-expressing cells and GFP-negative cells from the rest of the embryos were purified at the same developmental stage to the cellular resolution using fluorescent activated cell sorting (FACS).  Focus was on regulation by miR-206 and miR-133 in the developing somites and miR-124 in the developing central nervous system. Comparison of wild-type embryos and those lacking miRNAs revealed predicted
targets that responded to the miRNAs and distinguished miRNA-mediated mRNA
destabilization from other regulatory effects.
Keywords: cell type comparison

overall_design: Using FACS, GFP-negative and GFP-positive cells were isolated from two different miRNA reporter wild-type lines as well as from a Dicer-mutant zebrafish line at the same developmental stage (14-16 somites). Three biological replicates for each condition were used for microarray analyses.


title: Gene expression profiles in zebrafish brain after acute exposure to domoic acid at symptomatic and asymptomatic doses

status: Public on Jul 06 2009

city: Seattle

first: James

middle: William

last: MacDonald

state: WA

address: 4225 Roosevelt Way NE

department: Environmental and Occupational Health Sciences

institute: University of Washington

summary:
Domoic acid (DA) is a neuroexcitatory amino acid that is naturally produced by some marine diatom species of the genus Pseudo-nitzschia.  Ingestion of DA-contaminated seafood by humans results in a severe neurotoxic disease known as amnesic shellfish poisoning (ASP).  Clinical signs of ASP include seizures and neuronal damage from activation of AMPA and kainate receptors.    However, the impacts of DA exposure at levels below those known to induce outward signs of neurobehavioral exicitotoxicity have not been well characterized.  To further understand the mechanisms of neurotoxic injury associated with DA exposure, we examined the transcriptome of whole brains from zebrafish (Danio rerio) receiving intracoelomic (IC) DA at both symptomatic and asymptomatic doses.  A majority of zebrafish exposed to high-dose DA (1.2 µg DA/g) exhibited clinical signs of neuroexcitotoxicity (EC50 of 0.86 µg DA/g) within 5 to 20 minutes of IC injection.  All zebrafish receiving low-dose DA (0.47 µg DA/g) or vehicle only maintained normal behavior.  Microarray analysis of symptomatic and asymptomatic exposures collectively yielded 306 differentially expressed genes (1.5-fold, p = 0.05) predominately represented by signal transduction, ion transport, and transcription factor functional categories.  Transcriptional profiles were suggestive of neuronal apoptosis following an overwhelming of protective adaptive pathways.  Further, potential molecular biomarkers of neuropathic injury, including Nrdg4, were identified and may be relevant to DA exposure levels below that causing neurobehavioral injury.   Our results validate zebrafish as a vertebrate model to study mechanisms of DA neurotoxicity and provide a basis for identifying pathways of DA-induced injury as well as biomarkers of asymptomatic and symptomatic DA exposure levels.

Keywords: neurotoxic disease

overall_design: To further understand the mechanisms of neurotoxic injury associated with DA exposure, we examined the transcriptome of whole brains from zebrafish (Danio rerio) receiving intracoelomic (IC) DA at both symptomatic and asymptomatic doses.  A majority of zebrafish exposed to high-dose DA (1.2 µg DA/g) exhibited clinical signs of neuroexcitotoxicity (EC50 of 0.86 µg DA/g) within 5 to 20 minutes of IC injection.  All zebrafish receiving low-dose DA (0.47 µg DA/g) or vehicle only maintained normal behavior.  Microarray analysis of symptomatic and asymptomatic exposures collectively yielded 306 differentially expressed genes (1.5-fold, p = 0.05) predominately represented by signal transduction, ion transport, and transcription factor functional categories.  All animal studies were carried out under approved IACUC protocols at the University of Washington.

middle: P

last: Beyer


title: miRNAs in activated mouse CD4 T cells by Bcl-6 overexpression

status: Public on Jul 24 2009

city: Sydney

first: Di

last: Yu

address: 384 Victoria Street

department: Immunology and inflammation

country: Australia

institute: The Garvan Institute of Medical Research

summary: Compare miRNA expression profiles in mouse CD4 T cells overexpression of Bcl-6 vs empty vector

overall_design: Mouse naïve CD4+ T cells were transduced with a Bcl-6-expressing retrovirus or the control vector and cultured in Th0 condition for 2 days. Cells expressing medium to high levels of GFP were sorted and total RNAs were isolated using mirVana miRNA Isolation Kit (Applied Biosystems). MiRNA expression was profiled using Agilent 8x15K mouse miRNA microarray miRNA-v1_95_May07 (miRBase Release 9.2) by Ramaciotti Centre for Gene Function Analysis (Sydney). Each sample was run in duplicate.

first: Louis

last: Tsai

first: Charles

middle: R

last: Mackay

first: Carola

middle: G

last: Vinesa


title: The cellular expression of midkine-a and midkine-b during retinal development and photoreceptor regeneration

status: Public on Dec 17 2008

first: Peter

middle: F.

last: Hitchcock

address: 1000 Wall Street

department: Ophthy and Vis Sci

summary: In the retina of adult teleosts, stem cells are sustained in two specialized niches: the ciliary marginal zone (CMZ) and the microenvironment surrounding adult Müller glia. Recently, Müller glia were identified as the regenerative stem cells in the teleost retina. Secreted signaling molecules that regulate neuronal regeneration in the retina are largely unknown. In a microarray screen to discover such factors, we identified midkine-b (mdkb). Midkine is a highly conserved heparin-binding growth factor with numerous biological functions. The zebrafish genome encodes two distinct midkine genes: mdka and mdkb. Here, we describe the cellular expression of mdka and mdkb during retinal development and the initial, proliferative phase of photoreceptor regeneration. The results show that in the embryonic and larval retina mdka and mdkb are expressed in stem cells, retinal progenitors and neurons in distinct patterns that suggest different functions for the two molecules. Following the selective death of photoreceptors in the adult, mdka and mdkb are co-expressed in horizontal cells and proliferating Müller glia and their neurogenic progeny. These data reveal that Mdka and Mdkb are signaling factors present in the retinal stem cell niches in both embryonic and mature retinas, and that their cellular expression is actively modulated during retinal development and regeneration.

overall_design: As a means to identify genes necessary for photoreceptor regeneration, we evaluated transcriptional in the retina of the zebrafish as photoreceptors are regenerated. Albino zebrafish were dark adapted, then half were exposed to bright constant light and half were returned to normal lighting. After 72hrs of light exposure, retinal RNA was isolated and analysis of gene expression was performed using Affymetriz zebrafish gene arrays.

middle: F

first: David

last: Hyde

first: Thomas

last: Vihtelic


title: The role of ERbeta2 in zebrafish neuromasts development 15uM

status: Public on Oct 16 2008

summary:
The role of ERbeta2 in zebrafish larvae was investigated by injection of a Morpholino against ERbeta2. After 72hpf, the morphants showed a strong disruption in their sensory systems. ERbeta2 has been shown to be needed for the normal functioning of the sensory system organs, the neuromasts. The mechanisms involved in the neuromast disruption in ERbeta2 morphants was identified by microarrays gene screening. After comparison of two screening with low and high concentration of Morpholinos, genes that were present in the two microarrays screening were selected. The genes were then chosen by relevance for the mechanisms involved in the role of ERbeta2 in neuromast development. The ngn1 transcription factor, Notch3 and Notch1a showed to be up-regulated, also confirmed by in situ hybridization. The Notch signaling is known to be involved in cell fate in developing neuromasts. The overall conclusion is that ERbeta2 by interacting with the notch signaling pathways is critical for normal development of the neuromast of the lateral line in zebrafish.

Keywords: knock down experiment

overall_design: Zebrafish eggs until 4 cell stage were injected either with Morpholino blocking the translation ERbeta2 (ERbeta2 MO), either with a standard control Morpholino (coMO) at a concentration of 15uM. Two biological replicates for ERbeta2 MO, two replicates for coMO and two replicates for uninjected embryos (blank). A total of 6 samples were performed.

last: Fröhlicher


title: Identification of the molecular signatures integral to regenerating photoreceptors in the retina of the zebrafish

status: Public on Oct 08 2008

summary: Investigating neuronal and photoreceptor regeneration in the retina of zebrafish has begun to yield insights into both the cellular and molecular means by which this lower vertebrate is able to repair its central nervous system. However, knowledge about the signaling molecules in the local microenvironment of a retinal injury and the transcriptional events they activate during neuronal death and regeneration is still lacking.  To identify genes involved in photoreceptor regeneration, we combined light-induced photoreceptor lesions, laser-capture microdissection (LCM) of the outer nuclear layer (ONL) and analysis of gene expression to characterize transcriptional changes for cells in the ONL as photoreceptors die and are regenerated. Using this approach, we were able to characterize aspects of the molecular signature of injured and dying photoreceptors, cone photoreceptor progenitors and microglia within the ONL. We validated changes in gene expression and characterized the cellular expression for three novel, extracellular signaling molecules that we hypothesize are involved in regulating regenerative events in the retina.

overall_design: As a means to identify genes necessary for photoreceptor regeneration, we evaluated transcriptional changes for cells in the outer nuclear layer (ONL) as photoreceptors die and are regenerated. To accomplish this, we combined light-induced photoreceptor lesions, laser-capture microdissection (LCM) of the ONL and analysis of gene expression using oligonucleotide arrays.

first: Sonya

last: Craig

first: Anda-Alexandra

last: Calinescu


title: The effect of cardiac troponin T2 knockdown on gene expression in zebrafish embryos

status: Public on Jun 24 2009

city: Sheffield

first: Marta

last: Milo

state: South Yorkshire

address: Western Bank

department: Biomedical Science

country: United Kingdom

institute: University of Sheffield

summary:
We examined the transcriptional effect of preventing cardiac contraction in zebrafish embryos which can be deprived of circulation without experiencing hypoxia since the fish obtain sufficient oxygen via diffusion. Morpholino antisense knockdown of cardiac troponin T2 (tnnt2) prevented cardiac contraction without affecting vascular development. We concluded that absence of hemodynamic force induces endothelial CXCR4a up-regulation and promotes recovery of blood flow.

We used microarrays to define the genetic signatures of the development  of collateral vessels in zebrafish.

Keywords: Time course comparison of wild type zebrafish embryos and morpholino antisense against troponin t2 treated embryos.

overall_design: One cell zebrafish embryos were injected with morpholino antisense against control or cardiac troponin t2, which prevents cardiac development. Whole embryo RNA was extracted at 36, 48, and 60h post fertilization and transcriptionally profiled.

first: Ian

last: Packham

first: Caroline

last: Gray

first: Tim

last: Chico


title: T47D_MTVL_H1.2sh or T47D_MTVL_controlsh

city: barcelona

first: BELEN

last: MINANA

state: barcelona

address: dr aiguader 88

department: GENE REGULATION, STEM CELLS AND CANCER

institute: Center for genomic regulation

summary:
H1.2 (and control) knock-down cell lines were established from T47D-MTVL cells: Human breast cancer cell line T47D modified to contain one stably integrated copy of Luciferase reporter gene driven by the Mouse Mammary Tumor Virus promoter, named T47D-MTVL (Truss, M., J. Bartsch, A. Schelbert, R. J. Hache, and M. Beato. 1995).
Initially, a cell line expressing the Dox-responsive KRAB repressor and RedFP (ptTR-KRAB-Red) was generated. Then, this cell line was infected with viruses for expression of the different H1 variants shRNAs (pLVTHM). The inducible knocked-down cell lines were sorted in a FACSvantageSE (Becton Dickinson) for RedFP-positive and GFP-positive fluorescence after 3 days of Dox treatment. Then cells were amplified in the absence of Dox until an experiment was performed. Plasmids for the lentivirus vector-mediated drug-inducible RNA interference system (pLVTHM, ptTR-KRAB-Red, pCMC-R8.91 and pMD.G) were provided by D. Trono (University of Geneva) (Wiznerowicz M & Trono D (2003) Conditional suppression of cellular genes: lentivirus vector-mediated drug-inducible RNA interference. J Virol 77: 8957-8961).
Keywords: Gene expression - T47D-MTVL-control sh/sh H1.2 +/- Dox induction

overall_design:
4 biological replicates are included of each sample:
Control sh samples are treated with/without Dox
H1.2 sh samples are treated with/without Dox
Cell line sample was labeled with Cy5 and hybridized against Universal Human Reference RNA (Stratagene) labeled with Cy3.

first: Albert

last: Jordan

first: Monica

last: Sancho

first: Belen

last: Minana

first: Lauro

last: Sumoy

first: Cecilia

last: Ballare

first: Miguel

last: Beato


title: Zebrafish 24hpf mutant sk8 gene expression

status: Public on Sep 18 2008

city: Cincinnati

first: Nathan

last: Salomonis

state: OH

address: 3333 Burnet Avenue

department: Biomedical Informatics

institute: Cincinnati Children's Hospital

summary:
The production of functional mRNA involves multiple steps including transcription initiation, elongation, and termination.  spt5 encodes a conserved essential transcription elongation factor that controls RNAPII processivity in vitro and co-localizes with RNAPII in vivo.
We used microarrays to detail the global expression of gene expression at 24 hours post fertilization in wild-type and Spt5 mutant (fog-sk8) to identify Spt5 transcriptionally dependent genes and affected pathways. Here we report that a mutation in zebrafish spt5, sk8, deregulates <5% of ~10,000 genes examined.  These findings reveal a small but diverse set of developmentally regulated genes, whose expression is critically dependent on Spt5-regulated RNAPII stalling in vertebrate development.

Keywords: comparison

overall_design: RNA from zebrafish embryos were harvested at 24 hours post fertilization for hybridization to Affymetrix gene arrays.

first: Guo

last: Su

first: Krishnan

last: Keerthi

first: Salomonis

last: Nathan


title: Gene expression profile of zebrafish kidney side population (SP) cells

status: Public on Oct 10 2008

city: Shinjuku-ku

first: Isao

last: Kobayashi

state: Tokyo

address: 35 Shinanoma-chi

department: Cell Differentiation

country: Japan

institute: Keio University

summary: Side population (SP) cells are identified based on their capacity to efflux of the fluorescent dye Hoechst 33342, and are enriched for hematopoietic stem cells (HSCs) in mammalian bone marrow. We recently demonstrated that SP cells were present in the teleost kidney, the main hematopoietic organ in teleosts, and were enriched for HSCs. In this analysis, to identify the regulated genes in teleost HSCs, gene expression analysis of zebrafish kidney SP cells were performed using the GeneChip Zebrafish Genome Array.

overall_design: Based on their Hoechst fluorescence intensity, lymphoid cells (FS-low, SS-low) from zebrafish kidney were subdivided into SP and MP populations (referred to as “zSP” and “zMP”). To minimize the biological variability, 20 zebrafish were used for 3 independent cell sorting experiments and lysates from these 3 experiments were pooled by cell type. Each RNA sample was split into two aliquots and used for amplification, labeling, and hybridization to independent arrays.

first: Hiromasa

last: Ono

first: Tadaaki

last: Moritomo

first: Koichiro

last: Kano

first: Teruyuki

last: Nakanishi


title: The role of ERbeta2 in zebrafish neuromasts development 50uM

first: Mirjam

last: Föhlicher

summary:
The role of ERbeta2 in zebrafish larvae was investigated by injection of a Morpholino against ERbeta2. After 72hpf, the morphants showed a strong disruption in their sensory systems. ERbeta2 has been shown to be needed for the normal functioning of the sensory system organs, the neuromasts. The mechanisms  involved in the neuromast disruption in ERbeta2 morphants was identified by microarrays gene screening. After comparison of two screening with low and hign concentration of Morpholinos, genes that were present in the two microarrays screening were selected. The genes were then chosen by relevance for the mechanisms involved in the role of ERbeta2 in neuromast development. The ngn1 transcription factor, Notch3 and Notch1a showed to be up-regulated, also confirmed by in situ hybridization. The Notch signaling is known to be involved in cell fate in developing neuromasts. The overall conclusion is that ERbeta2 by interacting with the notch signaling pathways is critical for normal development of the neuromast of the lateral line in zebrafish.

Keywords: knock down experiment

overall_design: Zebrafish eggs until 4 cell stage were injected either with Morpholino blocking the translation ERbeta2 (ERbeta2 MO), either with a standard control Morpholino (coMO) at a concentration of 50uM. Two biological replicates for ERbeta2 MO, two replicates for coMO and two replicates for uninjected embryos (blank). A total of 6 samples were performed.

last: Fröhlicher


title: Regulation of zebrafish endothelial gene expression by miR-126

status: Public on Aug 12 2008

city: San Francisco

first: Jason

middle: E.

last: Fish

state: CA

address: 1650 Owens Street

department: Gladstone Institute of Cardiovascular Disease

institute: J. David Gladstone Institute

summary:
Fish, JE, Santoro, MM, Morton, SU, Yu, S, Yeh, RF, Wythe, JD, Ivey, KI, Bruneau, BG, Stainier, DYR, and Srivastava, D. (2008). miR-126 Regulates Angiogenic Signaling and Vascular Integrity. Developmental Cell 15, 272-284.

Precise regulation of the formation, maintenance, and remodeling of the vasculature is required for normal development, tissue response to injury, and tumor progression. How specific microRNAs intersect with and modulate angiogenic signaling cascades is unknown. Here, we identified microRNAs that were enriched in endothelial cells derived from mouse embryonic stem (ES) cells and in developing mouse embryos. We found that miR-126 regulated the response of endothelial cells to VEGF. Additionally, knockdown of miR-126 in zebrafish resulted in loss of vascular integrity and hemorrhage during embryonic development. miR-126 functioned in part by directly repressing negative regulators of the VEGF pathway, including the Sprouty-related protein SPRED1 and phosphoinositol-3 kinase regulatory subunit 2 (PIK3R2/p85-β). Increased expression of Spred1 or inhibition of VEGF signaling in zebrafish resulted in defects similar to miR-126 knockdown. These findings illustrate that a single miRNA can regulate vascular integrity and angiogenesis, providing a new target for modulating vascular formation and function.

overall_design: To identify the genes regulated by miR-126 in vivo, flk1:GFP transgenic zebrafish embryos (which express GFP in the endothelium) were injected at the one-cell stage with 4 ng of two independent antisense morpholinos that block the processing of the miR-126 pri-cursor. At 48 hours post-fertilization, endothelial cells were isolated by fluorescence-activated cell sorting from flk1:GFP transgenic fish, and RNA was extracted. Arrays were performed on four biological replicates of control and two independent miR-126 morpholinos.

first: Ru-Fang

last: Yeh

first: Deepak

last: Srivastava


title: Sexual dimorphism in the zebrafish hepatic transcriptome and response to dietary carbohydrate

status: Public on Mar 31 2008

city: Davis

first: Matt

last: Settles

address: 451 Health Sciences Sr

department: Genome Center

institute: University of California, Davis

summary:
The liver plays a central role in vertebrate glucose homeostasis, and is also one of the most sexually dimorphic organs in terms of gene expression.  While the extent of hepatic sexual dimorphism has been well described in mammals, little is known regarding this phenomenon in non-mammalian species, particularly fish.  In this study, we examined hepatic gene expression and physiological phenotypes (growth, proximate body composition, retention efficiencies) to determine whether male and female zebrafish respond differently to diets comprised of 0, 15, 25, or 35 % carbohydrate.  Using both Affymetrix microarrays and qRTPCR, we observed substantial sexual dimorphism in the hepatic transcriptome, and the response of some genes to dietary carbohydrate manipulation also varied by sex.  Males upregulated genes associated with oxidative metabolism, carbohydrate metabolism, energy production, and amelioration of oxidative stress, while females had higher expression levels of genes associated with translation.  Males also expressed elevated levels of hnf4a, a gene thought to be involved in regulating hepatic sexual dimorphism in the rodent.  Dietary carbohydrate affected hepatic gene expression, growth performance, retention efficiencies of protein and energy, and percentage of moisture, lipid, and ash.  Significant diet effects reflected differences between the 0% carbohydrate diet and the other diets, consistent with previous work on other cyprinids showing a high tolerance for dietary carbohydrate.  Our data support the use of the zebrafish as a model for the study of both normal and disease states associated with carbohydrate metabolism, and highlight the importance of accounting for both sex and diet
Keywords: Nutritional manipulation, sex comparison

overall_design: 17 samples are analyzed from a factorial combination of sex (male or female) and dietary treatment (0% or 25% dietary carbohydrate).  5 biological replicates for females fed each diet, and 3 or 4 biological replicates for males fed the 0% and 25% diets, respectively.

first: Barrie

last: Robison

first: Matthew


title: Comparison of transcripts in zebrafish tumors

status: Public on May 19 2008

first: Charles

middle: Arthur

last: Whittaker

institute: Koch Institute

summary:
The goal of this experiment was to compare the genes expressed in malignant peripheral nerve sheath tumors (MPNSTs) that arise in zebrafish as a result of homozygous mutation of the p53 gene or heterozygous mutation of several different ribosomal protein (rp) mutations.  Since MPNSTs arise very rarely in wild type zebrafish, it seemed a possibility that p53 and rps may in fact be functioning in similar pathways.  The tumors arising from the different mutations had been previously classified as similar by histology, thus the goal of the array experiments was to establish if any molecular signatures could be found that could delineate the p53 from the rp MPNSTs.

Keywords: Zebrafish malignant peripheral nerve sheath tumors from different genetic backgrounds

overall_design: The mRNAs from 6 different MPNSTs arising from rp mutations (3 from rpL35, 1 from rpL14, 1 from rpS5, 1 from rpS11) were compared to mRNA from 3 MPNSTs arising from the p53 M214K/M214K  mutation.  These were also compared to the mRNA from 2 spontaneously forming seminoma tumors from random genetic backgrounds.

first: Alyson

last: MacInnes

first: Adam

last: Amsterdam

first: Jacqueline

last: Lees

first: Nancy

last: Hopkins


title: Comparative genomic analysis between adult and larval fin regeneration

status: Public on Feb 29 2008

city: Corvallis

first: Robert

last: Tanguay

state: OR

department: EMT

institute: Oregon State University

summary:
Zebrafish have the remarkable ability to regenerate body parts including the heart, spinal cord and fins by a process referred to as epimorphic regeneration. Recent studies have illustrated that similar to adult zebrafish, early life stage-larvae also possess the ability to regenerate the caudal fin. A comparative genomic analysis was used to determine the degree of conservation in gene expression among the regenerating adult caudal fin, adult heart and larval fin. Results indicate that these tissues respond to amputation/injury with strikingly similar genomic responses. Comparative analysis revealed raldh2, a rate-limiting enzyme for the synthesis of Retinoic acid (RA), as one of the highly induced genes across the three regeneration platforms.
Keywords: comparative genomics

overall_design: The caudal fin of zebrafish larvae at 2days post fertilization were amputated. Caudal fin tissue at 2dpf and regenerating fins were isolated at 1, 2and 3 days post amputation. Three replicates were collected at each time point. 150 fins were pooled to comprise one replicate.

first: Lijoy

middle: K

last: Mathew

first: Sumitra

last: Sengupta

first: Jessica

last: Perry

first: Jane

last: LaDu

first: Eric

last: Andreasen


title: Gene Expression Profile of Hematopoietic Stem Cells during Zebrafish Development

status: Public on Apr 26 2008

city: Boston

first: Gerhard

middle: J

last: Weber

address: 1 Blackfan Circle

department: Hematology / Oncology

institute: Children's Hospital Boston, Harvard Medical School

summary:
In order to detect transcriptional differences between primitive and definitive hematopoietic stem and progenitor cells during regular development in the zebrafish embryo, gata1-GFP+/+(18 somites),  lmo2-GFP+/+ (18 somites and 35 hpf)1 and cd41-GFP+/+ (35 hpf) cells from transgenic embryos were individually separated from GFP-/- cells by flow cytometry at the indicated stages. For each individual population, pools of 600 - 1500 transgenic embryos were collected.  After RNA extraction, labelled cRNA was hybridized onto Affymetrix microarrays. Individual experiments were performed with 2 or 3 biological replicates.
Keywords: cell type comparison

overall_design:
gata1-GFP+/+(18 somites),  lmo2-GFP+/+ (18 somites and 35 hpf)1 and cd41-GFP+/+ (35 hpf)2 cells were separated from GFP-/- cells
by flow cytometry at the indicated stages.  Sorting was based on propidium iodide exclusion, forward scatter, and GFP
fluorescence, using a FACSVantage flow cytometer (Beckton Dickinson).  Sorted cell populations were run twice to optimize cell
purity.  Total RNA from cell-sorted populations was extracted with TRIzol reagent (Invitrogen) and purified on RNeasy resins
(Qiagen) according to the manufacturer’s recommendations.  Total RNA was subjected to two rounds of linear amplification
(Ambion) and hybridized to Affymetrix zebrafish Gene Chips, according to Affymetrix guidelines. After staining with a
streptavidin-phycoerythrin conjugate (Molecular Probes), the fluorescence of bound RNA was quantitated by using a Gene Chip
scanner (Affymetrix). The raw expression data were calculated and, after pairing of GFP+/+ and GFP-/- samples, statistically
analyzed using methods implemented in Bioconductor’s “affy” package and available custom scripts 3,4.
references:
1. Zhu, H. et al. Regulation of the lmo2 promoter during hematopoietic and vascular development in zebrafish. Dev Biol 281,
256-69 (2005).
2. Lin, H. F. et al. Analysis of thrombocyte development in CD41-GFP transgenic zebrafish. Blood 106, 3803-10 (2005).
3. Choe, S. E., Boutros, M., Michelson, A. M., Church, G. M. & Halfon, M. S. Preferred analysis methods for Affymetrix
GeneChips revealed by a wholly defined control dataset. Genome Biol 6, R16 (2005).
4. Weber, G. J. et al. Mutant-specific gene programs in the zebrafish. Blood 106, 521-30 (2005).

first: Hsiao-Chun

last: Huang

first: Elizabeth

middle: A

last: Mayhall

first: Yi

last: Zhou

first: Leonard

middle: I

last: Zon


title: Expression data from embryos injected with DrC1q-like morpholino and control morpholino

status: Public on Dec 29 2007

city: Wuhan

first: Jie

last: Mei

state: Hubei

address: 7# Donghu South Road

department: Chinese Academy of Sciences

country: China

institute: Institute of Hydrobiology

summary:
Except complement C1q, the immunological functions of other C1q family members have remained unclear. In vitro studies showed that DrC1q-like could inhibit the apoptosis induced by ActD and CHX in EPC cells, through repressing the activation of caspase 3/9. Moreover, its physiological roles were studied by morpholino-mediated knockdown in zebrafish embryogenesis
We used microarrays to compare the global programme of gene expression of DrC1q-like deficient embryos and control embryos. And identified distinct classes of up-regulated and down-regulated genes in DrC1q-like deficient embryos.
Keywords: time course

overall_design: Zebrafish embryos were selected at 24hpf for RNA extraction and hybridization on Affymetrix microarrays. We sought to obtain expression profiles of DrC1q-like deficient embryos compared to the control. And we could find out which genes and pathways were affected by DrC1q-like

first: Jianfang

last: Gui


title: Comparative Genomics Identifies Gene Targets for Retinoic Acid in the Embryonic Zebrafish Hearts

status: Public on Sep 12 2007

first: Jing

last: Chen

address: 777 Highland Ave

department: pharmaceutical Sciences

institute: University of Wisconsin - Madison

summary:
Retinoic acid (RA) and 2,3,7,8-tetrachlorodibenzo-p-dioxin activate distinct ligand-dependent transcription factors, and both cause cardiac malformation and heart failure in zebrafish embryos.  We hypothesized that they cause this response by hyperactivating a common set of genes critical for heart development.  To test this, we used microarrays to measure transcripts changes in hearts isolated from zebrafish embryos 1,2,4 and 12 h after exposure to 1μM RA.  We used hierarchical clustering to compare the transcriptional responses produced in the embryonic heart by RA and TCDD.  We could identify no early responses in common between the two agents.  However, at 12 h both treatments produced a dramatic downregulation of a common cluster of cell cycle progression genes, which we term the Cell Cycle Gene Cluster (CCGC).  This was associated with a halt in heart growth.  These results suggest that RA and TCDD ultimately trigger a common transcriptional response associated with heart failure, but not through the direct activation of a common set of genes.  Among the genes rapidly induced by RA was Nr2F5, a member of the COUP-TF family of transcription repressors.  We found that induction of Nr2F5 was both necessary and sufficient for the cardiotoxic response to RA.
Keywords: Comparative Genomics, Timecourse

overall_design: For RA study, total of 24 samples were collected and analyzed by microarray.   Samples of zebrafish embryonic hearts were collected at four timepoints: 1, 2, 4, 12 hours post RA dosing.  For each timepoint, there are three replicates of microarray studies.  Each replicate includes microarray studies of one sample of RA treated heats and one sample of vehicle control (DMSO) treated hearts.

first: Sara

last: Carney

first: Richard

last: Peterson

first: Warren

last: Heideman


title: 2 or 3 Day Post Amputation Vehicle or TCDD Exposed

first: Robert

last: Tanguay

summary:
Exposures to dioxin, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) cause a wide array of toxicities in vertebrates and is mostly considered to be mediated through the inappropriate activation of the aryl hydrocarbon receptor (Ahr) signaling pathway. Although transcriptional regulation by Ahr is widely studied, the molecular mechanisms responsible for the adverse outcomes after Ahr activation are largely unknown. To identify the important events downstream of AHR activation that play an actual role in the toxic responses, we employed the zebrafish caudal fin regeneration models since Ahr activation blocks the regenerative process. Zebrafish regenerate their caudal fins by an orchestrated progression of cell migration, differentiation and proliferation controlled by a multitude of signaling pathways. This complex process was exploited as an in vivo platform to identify cross talk between Ahr and other signaling pathways. Global genomic analysis was performed in the larval regenerating fin tissue after exposure to TCDD in order to identify genes differentially regulated after Ahr activation. Comparative toxicogenomic analysis revealed that both adult and larval fins respond to TCDD during regeneration with mis-expression of Wnt signaling pathway members and Wnt target genes.
Keywords: comparative toxico-genomics

overall_design: The caudal fin of zebrafish larvae at 2days post fertilization were amputated and exposed to vehicle control alone or TCDD. Regenerating fins were isolated at 2and 3 days post amputation. Three replicates were collected at each time point. 150 fins were pooled to comprise one replicate.

middle: k


title: The expression profiles of control embryos and pbx2-MO;pbx4-MO embryos at 10 somites and at 18 somites.

status: Public on Jul 11 2007

city: Seatttle

last: Cao

address: 1100 Fairview Ave. N

department: Human Biology

institute: Fred Hutchinson CRC

summary:
Pbx homeodomain proteins have been implicated in the regulation of gene expression during muscle development.  Whether Pbx proteins are required broadly for the regulation of muscle gene expression or are required for the expression of a specific subset of muscle gene expression is not known.  We employed microarrays to determine the requirements for Pbx proteins during zebrafish development.
Keywords: developmental time course analysis

overall_design: Total RNA samples from control and pbx2-MO;pbx4-MO embryos were collected at two developmental stages: 10 somites and 18 somites. Their expression profiles were analyzed using Affymetrix zebrafish genome arrays, and the differentially hybridized genes were determined to be significant according to a FDR<0.05.

first: Lisa

last: Maves

first: Andrew

last: Waskiewicz

first: Biswajit

last: Paul

first: Ashlee

last: Tyler

first: Cecilia

middle: B

last: Moens

first: Stephen

last: Tapscott


title: Zebrafish response to Mycobacterium marinum infection (Affymetrix series)

status: Public on Jan 01 2008

city: Chapel Hill

first: John

last: Rawls

state: NC

address: 111 Mason Farm Rd.

department: Department of Cell and Molecular Physiology

institute: University of North Carolina at Chapel Hill

summary:
Mycobacteria infect macrophages that aggregate with additional macrophages and lymphocytes to form granulomas. We have used a functional genomics approach to identify immune response genes expressed during granuloma formation in Mycobacterium marinum-infected transparent zebrafish larvae where individual infection steps can be viewed in real time. We assessed RNA expression profiles from zebrafish larvae that were either infected with Mycobacterium marinum, mock-infected, or uninfected. Zebrafish infections were performed at 1 day post-fertilization (dpf), and samples were derived from pools of 6dpf zebrafish larvae.
Keywords: host response to infection

overall_design: Total RNA was purified from pooled intact zebrafish larvae (31-82 larvae/pool, 3 biological replicate pools/condition) using Trizol reagent (Invitrogen) followed by DNase I digestion (DNA-Free, Ambion) according to manufacturers' protocols. Total RNA from each replicate pool (12ug RNA/replicate) was used as template for independent cDNA synthesis and in vitro transcription reactions (BioArray HighYield RNA Transcript Labeling Kit; Enzo Life Sciences) to generate biotinylated cRNA targets. cRNA targets (30ug/replicate) were fragmented using standard methods. Hybridization and scanning were performed using standard Affymetrix protocol. Raw expression values were normalized (Invariant set method) and modeled (PM-MM model), and present/absent calls were generated using dChip software (build date Dec.11, 2005).

first: Tamara

middle: C

last: Pozos

first: Hannah

last: Volkman

first: Lalita

last: Ramakrishnan


title: Comparison of gene expression profiles between GnRH-treated and control rat pituitary organ cultures

status: Public on Aug 31 2006

city: San Diego

last: Roth

address: 12790 El Camino Real

department: Molecular Medicine

institute: Neurocrine Biosciences, Inc.

summary:
Rat pituitary organ cultures were treated with either GnRH at 10nM for 6 hours, or treated with control for 6 h, both groups in triplicate; samples were processed to generate total RNA, which were subsequently analyzed for gene expression using Affymetrix rat 230 2.0 arrays
Keywords: rat pituitary organ culture treated vs control


title: Expression data from egy wildtype and mutant embryo comparison

status: Public on Jan 31 2007

first: Gerhard

last: Weber

summary:
U4/U6 di-snRNPs were disrupted and singular U4 and U6 snRNPs accumulated in egy mutant embryos, establishing the recycling function of p110 in vivo. Based on microarray analyses, a subset of spliceosome components and splicing-related factors was coordinately upregulated in the egy mutant. This revealed an extensive network of coregulated components of the spliceosome cycle, compensating – albeit inefficiently – for the recycling defect. In contrast, another set of genes, many of them eye- and pancreas-specific, was downregulated in the egy mutant embryos.
Keywords: mut / wt comparison

overall_design: Zebrafish earl grey (egy) mutant embryos carry an autosomal recessive defect in the p110-orthologous gene which leads to microcephaly, microphthalmia, underdevelopment of the pharyngeal arches, and thymus hypoplasia by day 8 of development. To characterize the defect on the transcriptional level, egy whole embryos (n>100) were collected and morphologically separated into pools of mutant (mut) and wildtype (wt) sibling embryos. Pools of embryos were collected at 2 time points (3 and 4 days post fertilization) with 2 and 3 biological replicates, resp.. After RNA extraction, labelled cRNA was hybridized onto Affymetrix microarrays.

first: Nikolaus

last: Trede

first: Yi

last: Zhou

first: Leonard

last: Zon


title: Gene expression analysis in the zebrafish embryos silenced by zfPGRP-SC

status: Public on Aug 13 2007

first: M

middle: X

last: Chang

address: Luojiaashan

institute: Institute of Hydrobiology, Chinese Academy of Sciences

summary:
Peptidoglycan recognition proteins (PGRPs) are a family of pattern recognition receptors (PRRs) that serve as a strong link between invertebrate and vertebrate immunity.It has been demonstrated that PGRPs in insects function as bacterial recognition molecules which trigger proteolytic or signal transduction pathways, being thus important in the antimicrobial innate immunity.
To explore the effect of zebrafish short-form PGRP (zfPGRP-SC) on proteolytic or signal transduction pathway, RNA interference (siRNA) and high-density oligonucleotide microarray methods were used to identify differentially expressed genes regulated by zfPGRP-SC.
Keywords: RNA interference

overall_design: The control siRNA, zfPGRP-SC siRNA were introduced into zebrafish zygotes at the concentration of 300 ng / μl under a normal microscope.The injection volume was 1 nL per embryo. To insure the isochronous development, the embryos of one experimental and two different control samples (the group injected with the control siRNA and the uninjected group) were from the artificial fertilized embryos collected within 5 minutes. To eliminate the difference from the different parents, all embryos were randomly mixed. The microinjection was conducted within 30 minutes. Twenty-four hours later, 400 embryos for each group were collected.

first: MingXian

first: Pin

last: Nie


title: The expression of Spt5 targeted genes

status: Public on Oct 25 2007

city: Yokohama

first: Tadashi

last: Wada

state: Kanagawa

address: 4259 Nagatsuta-cho, Midori-ku

department: Bioscience and Biotechnology

institute: Tokyo Institute of Technology

summary:
The Spt4-Spt5 complex, and its human homolog DSIF (DRB sensitivity-inducing factor), is unique in its ability to regulate Pol II processivity. Previous studies have shown that Spt5 has the characteristics of a general transcription-elongation factor. However, mutagenesis of Spt5 showed specific phenotypes during development, which were far less severe than those of Pol II defects or TBP deficient embryos. It seems paradoxical that a mutation which alters a general elongation factor can cause rather specific developmental defects. By using Spt5 knockdown zebrafish embryos and microarrays, here we showed that transcript abundance for only a small subset of genes is altered by loss of Spt5. Further investigation of the down-regulated genes showed that the genes most intensely repressed by the knockdown were strongly activated during early development in untreated embryos. Thus, this study shows that gene activation levels may create different requirements for Pol II processivity. Active transcription requires Spt5 for efficient elongation through its stimulatory activity on Pol II processivity.
Keywords: Spt5, microarray analysis, development, transcription, transcription elongation

overall_design: The expression pattern of Spt5 knockdown embryos was compared to that of the control mopholino oligo injected embryos, both at 21 hours post-fertilization, to show the targets of Spt5. The activation levels of the Spt5-knockdown affected genes were measured by compareing the abundance of transcripts in 21 hours post-fertilization untreated embryos to that in 5 hours post-fertilization untreated embryos.

first: Wenyan

last: Zhu

first: Sachiko

last: Okabe

first: Takuya

last: Taneda

first: Yoichi

last: Kohara

first: Hajime

last: Watanabe

first: Yuki

last: Yamaguchi

first: Hiroshi

last: Handa


title: Gene Expression Profiling of Zebrafish Embryonic Retinal Pigment Epithelium in vivo.

status: Public on Jan 25 2007

city: West Lafayette

first: Yuk Fai

last: Leung

state: IN

address: 915 W. State Street

department: Biological Sciences

institute: Purdue University

summary:
Eye development and photoreceptor maintenance requires the retinal pigment epithelium (RPE), a thin layer of cells that underlies the neural retina. Despite its importance, RPE development has not been studied by a genomic approach. A microarray expression profiling methodology was established in this study for studying RPE development. The intact retina with RPE attached was dissected from developing embryos, and differentially expressed genes in RPE were inferred by comparing the dissected tissues with retinas without RPE using microarray and statistical analyses. We found 8810 probesets to be significantly expressed in RPE at 52 hours post-fertilization (hpf), of which 1443 might have biologically meaningful expression levels. Further, 78 and 988 probesets were found to be significantly over- or under-expressed in RPE respectively compared to retina. Also, 79.2% (38/48) of the known over-expressed probesets have been independently validated as RPE-related transcripts. The results strongly suggest that this methodology can obtain in vivo RPE specific gene expression from the zebrafish embryos and identify novel RPE markers.
Keywords: Development, retina, retinal pigment epithelium

overall_design: The gene expression levels of three independent replicates of retina with RPE attached consisting of ten samples each at 52hpf (WRR52) were compared with three independent pure retinal samples consisting of ten retinas each at 52hpf (WR52). The yield-adjusted WR52 expression values were assumed to be equivalent to the retinal contribution in WRR52 samples and deducted from WRR52 expression values to obtain estimations of RPE gene expression at 52hpf (RPE52). Differential gene expressions between RPE and retina were inferred by comparing the RPE52 estimates and WR52 expression values.

first: Yuk

first: Ping

last: Ma

first: John

middle: E

last: Dowling


title: The zebrafish runzel mutant results in a novel muscular dystrophy phenotype due to a titin mutation

status: Public on Jul 30 2006

first: Leta

middle: S

last: Steffen

address: 320 Longwood Ave

department: Program in Genomics

institute: Harvard Medical School, Children's Hospital

summary:
The zebrafish runzel (ruz) carries a mutation in the titin gene resulting in muscle degeneration and embryonic lethality.  At the onset of the visible phenotype (3-3.5 days post fertilization), RNA expression of the tail and trunk (primarily skeletal muscle) was compared between ruz mutants and wildtype siblings using the Affymetrix Zebrafish Gene Chip.  Mutant RNA was matched with RNA of wildtype siblings from the same clutch.  3 separate clutches were used (n=3).  RNA was labeled, sheered, and hybridized as in Shephard et al. (Shepard et al., 2005) and the data analyzed as in Choe et al. and Weber et al. (Choe et al., 2005; Weber et al., 2005).  Results suggest a novel mechanism of titin muscular dystrophy pathogenesis including upregulation of heat shock proteins.
Keywords: disease state analysis

overall_design: At the onset of the visible phenotype (3-3.5 days post fertilization), RNA expression of the tail and trunk (primarily skeletal muscle) was compared between ruz mutants and wildtype siblings using the Affymetrix Zebrafish Gene Chip.  RNA of mutants was matched with RNA of wildtype siblings from the same clutch.  3 separate clutches were used (6 arrays total).

first: Jeffrey

last: Guyon

first: Emily

middle: D

last: Vogel

first: Gerhard

last: Weber

first: Yi

first: Louis

middle: M

last: Kunkel


title: Molecular targets of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) within the zebrafish ovary

status: Public on Sep 01 2007

city: Milwaukee

first: Craig

last: Struble

department: Math, Stat, and Comp Sci

institute: Marquette University

summary:
2,3,7,8-TCDD (TCDD) is a reproductive toxicant and endocrine disruptor in nearly all vertebrates, yet the mechanisms by which TCDD induces these reproductive alterations have not been fully characterized.  Fish are among the most sensitive vertebrates to the toxic effects of TCDD, and even subtle physiologic changes induced by TCDD can impair reproduction. Previously, we have shown that chronic, sub-lethal exposure to TCDD decreased reproductive capacity, reduced serum estradiol and vitellogenin concentrations, and altered follicular development. Here we investigate the transcriptional changes in zebrafish ovary as they relate to observed attenuated estradiol concentrations and ovarian development.  We used quantitative RT-PCR to assess TCDD’s effects on the expression of several candidate genes important in the regulation of follicular development and steroidogenesis. Additionally, global changes in gene expression in the ovary caused by TCDD exposure were identified using Affymetrix Gene Chip Analysis.  Our data suggest that TCDD may inhibit follicle maturation via attenuated gonadotropin responsiveness and/or depressed estradiol biosynthesis. Additionally, genes involved in glucose and lipid metabolism, regulation of transcription, and immune function were dysregulated by at least 2-fold suggesting that TCDD alters various integrated networks of signaling pathways.  Approximately 89% of dysregulated transcripts contain putative AHR response elements (AHRE) within 5kb upstream of the predicted transcriptional start site suggesting ovarian toxicity is AHRE driven.  Furthermore, approximately 49% of dysregulated transcripts contain putative estrogen response elements (ERE) suggesting that dysregulation of estrogen-responsive genes may also contribute to TCDD-induced attenuated follicular development.  Patterns in gene expression were correlated with putative EREs and AHREs, and suggest that impacts on the regulation of transcription may play a large role in TCDD’s ovarian toxicity.  Taken together, these data illustrate the complexity of TCDD’s ovarian toxicity.
Keywords: dose response

overall_design: A total of eight samples were analyzed including three TCDD doses (10, 40, 100 ppb, dietary exposure) and vehicle control samples. cRNA targets were synthesized from pooled total RNA isolated from five ovaries from different individuals in each treatment group. Each individual contributed equally to a given pool. Two technical replicates were run for each treatment group. Several genes of interest arising from this microarray study were validated by Quantitative RT-PCR with individual ovary samples to assess biological variability.

first: Tisha

last: King Heiden

first: Matthew

middle: L

last: Rise

first: Martin

last: Hessner

first: Reinhold

last: Hutz

first: Michael

last: Carvan III


title: Gene Expression Profiles of Intact and Regenerating Zebrafish Retina

status: Public on Sep 16 2005

city: Syracuse

first: David

last: Cameron

state: NY

address: 750 E Adams St

department: Neuroscience & Physiology

institute: SUNY Upstate

summary:
Purpose: Investigate the molecular determinants of retinal regeneration in adult vertebrates by analyzing the gene expression profiles of control and post-lesion retina of adult zebrafish, a system that regenerates following injury.
Methods: Gene expression profiles of zebrafish retina and brain were determined with DNA microarray, RT-PCR, and real-time quantitative PCR analyses.  Damaged retinas and their corresponding controls were analyzed 2-5 days post-lesion (acute injury condition) or 14 d post-lesion (cell regeneration condition).
Results: Expected similarities and differences in the gene expression profile of zebrafish retina and brain were observed, confirming the applicability of the gene expression techniques.  Mechanical lesion of retina triggered significant, time-dependent changes in retinal gene expression.  The induced transcriptional changes were consistent with cellular phenomena known to occur, in a time-dependent manner, subsequent to retinal lesion, including cell cycle progression, axonal regeneration, and regenerative cytogenesis.
Conclusions: The results indicate that retinal regeneration in adult zebrafish involves a complex set of induced, targeted changes in gene transcription, and suggest that these molecular changes underlie the ability of the adult vertebrate retina to regenerate.
Keywords: time course; injury response; cellular correlation

overall_design:
Control brain and retina (unlesioned);
Control and lesioned retina (matched animals,
at least n = 8 for each condition).

first: Karen

last: Gentile

first: Frank

last: Middleton

first: Patrick

last: Yurco


title: Zebrafish Hypoxia study Heart array

status: Public on Jul 06 2006

city: Leiden

first: Christoph

middle: Peter

last: Bagowski

country: Netherlands

institute: Leiden University

summary:
Gene expression profile of the response to chronic constant hypoxia in the heart of adult zebrafish
Keywords: Adaptive response to chronic constant hypoxia in the heart

overall_design: 10 arrays (5 for hypoxia and 5 normoxia); in total 8 biological samples plus 2 technical replicates

first: Jelani

middle: T

last: Leito

first: Ines

last: Marques

middle: P


title: Gene expression in foggy mutant. Guo-1R01NS042626-01A2

status: Public on Nov 24 2004

city: Phoenix

first: Winnie

last: Liang

address: 445 N. Fifth Street

institute: Translational Genomics

summary:
The foggy mutant zebrafish show severe deficits in dopaminergic neurons, thus is an important  tool to understanding the development of these neurons, which undergo degeneration in Parkinson's disease.  Molecular study reveals that the foggy gene encodes a regulator of transcription elongation.  Biochemical study suggests that the repressive activity of the Foggy protein is lost in the foggy mutant.  The genes whose expression is normally repressed by Foggy are likely to play important roles in dopamine neuron development. However, their identity is unknown.  Finding out these target genes of Foggy will provide important insights into dopamine neuron development and regeneration.  They will also serve as an entry point to elucidate the role of transcription elongation in controlling neuronal development.
We will identify genes whose expression is altered in the foggy mutant. Their expression pattern and functional significance will be examined in zebrafish through in situ hybridization and morpholino antisense knockdowm methods.
We hypothesize that the genes whose transcription is regulated by Foggy play a critical role in the development of dopaminergic neurons.
The species noted above is Drosophila however we are using ZEBRAFISH. We will examine three stages: 24 hour post fertilization (hpf), 36 hpf, and 48 hpf. During these stages, the dopamine neuron deficits manifest in the foggy mutant. To identify genes whose expression is altered and important for dopamine neuron development, we shall dissect embryonic brain tissues from wildtype and the foggy mutant embryos at the stages mentioned above, and prepare RNA from them.  Since the foggy gene is expressed ubiquitously and also required for proper retinal and cardiovascular development, we would like to compare the gene expression between the whole embryos of wildtype and foggy mutant, to identify all possible downstream targets. We are currently applying for a supplement to support the entire microarray experiment.  As a pilot experiment, we would like to perform 6 array analysis as soon as possible.  Thus, we will examine the 48 hours post fertilzation time point in this pilot experiment and it will be performed in triplicate.
Keywords: other

first: Su

last: Guo


title: nck2 vs. wt morpholino

status: Public on Oct 30 2004

city: Berlin

first: Henning

last: Witt

address: Ihnestrasse 65

department: Hans Lehrach

country: Germany

institute: Max Planck Institute for Molecular Genetics

summary:
pooled individuals of nck2 and control morpholinos are compared in duplicated (A and B) hybridisations
Keywords: repeat sample


title: Factorial Microarray Analysis of Zebrafish Retinal Development

status: Public on Aug 04 2008

first: Yuk Fai

last: Leung

summary:
Retinal cells are specified in a zebrafish recessive mutant called young (yng) but they fail to terminally differentiate; i.e. extend neurites and make synaptic contacts. A point mutation in a brahma-related gene 1 (brg1) is responsible for this phenotype. In this microarray study, a three-factor factorial design was utilized to investigate the effects of 1) mutation, 2) change in time (36 vs. 52hpf), and 3) change in tissue (retina vs. whole embryos), and their interactions on gene expression. Significant probesets were inferred by using both specific contrasts of the fitted Analysis of Variance (ANOVA) models and a corresponding 2-fold expression cutoff. The probesets were grouped into three broad categories: 1) Brg1-regulated retinal differentiation genes (731 probsets), 2) Retinal specific genes but independent of Brg1 regulation (3038 probesets) and 3) Genes regulated by Brg1 but outside the retina (107 probesets). Four gene groups/pathways including neurite outgrowth regulators, Delta-Notch signalling molecules, Irx family members and specific cell cycle regulators were identified in the first group, and their relevance for retinal differentiation functionally validated. This study demonstrates that an approach such as ours can identify relevant genes and pathways involved in retinal development as well as the development of other tissues at the same time.
Keywords: Development, retina, terminal differentiation, chromatin remodeling, factorial anlaysis

overall_design: The gene expression levels of three independent replicates of retina consisting of ten samples each at 36 and 52hpf retinas from WT (WR36 & WR52) and yng (YR36 & YR52) larvae, and stage-matched whole embryos: WT at 36hpf (WA36), WT at 52hpf (WA52), yng at 36hpf (YA36), and yng at 52hpf (YA52) were measured by Affymetrix Zebrafish Whole Genome Arrays. We analyzed the effects of these factors on gene expression levels: 1) mutation (M), 2) tissue (R), and 3) time (T). Each of these factors have two levels: mutant and WT (M); retinas and whole body (R); and 36 and 52 hpf (T). As a result, there are totally eight conditions for all the comparisons and the design is called a 2x2x2 factorial design. The aim of a factorial analysis is to delineate the effects of each factor and their combinations (interaction) on gene expression. The overall analysis strategy is to first fit a full Analysis of Variance (ANOVA) model with all possible main effects (T, M, R) and their interactions (T*M, M*R, R*M, T*M*R) to the gene expression data for each probeset. The estimates of the model are obtained through a maximum likelihood estimation method. Then a backward elimination strategy is used to remove insignificant main effects and interactions to get the most parsimonious models. Finally, a group of contrasts and their corresponding fold changes are used to infer whether that probeset is significantly associated with a particular biological process.

first: Yuk

middle: F

first: Ping

last: Ma

first: Brian

last: Link

first: John

last: Dowling


title: 1, 3, or 5 Day Post Amputation Vehicle or TCDD Exposed

status: Public on Mar 20 2006

first: Robert

last: Tanguay

summary:
Adult zebrafish can completely regenerate their caudal fin following amputation. This complex process is initiated by the formation of an epithelial would cap over the amputation site by 12 hours post amputation (hpa). Once the cap is formed, mesenchymal cells proliferate and migrate from sites distal to the wound plane and accumulate under the epithelial cap forming the blastemal structure within 48 hpa. Blastemal cells proliferate and differentiate, replacing the amputated tissues, which are populated with angiogenic vessels and innervating nerves during the regenerative outgrowth phase which is completed around 14 days post amputation (dpa). Regenerative outgrowth does not occur in TCDD-exposed zebrafish. To identify the molecular pathways that are perturbed by TCDD exposure, male zebrafish were i.p. injected with 50 ng/g TCDD or vehicle and caudal fins were amputated. Regenerating fin tissue was collected at 1, 3 and 5 dpa for mRNA abundance analysis. Microarray analysis and quantitative real time PCR revealed that wound healing and regeneration alone altered the expression of nearly 900 genes by at least two fold between 1 and 5 dpa. TCDD altered the abundance of 370 genes at least two fold. Among these, several known aryl hydrocarbon responsive genes were identified in addition to several genes involved in extracellular matrix composition and metabolism. The profile of misexpressed genes is suggestive of impaired cellular differentiation and extracellular matrix composition potentially regulated by Sox9b.
Keywords: Time course and TCDD exposure response

overall_design:
Regenerating fins were isolated at 1, 3 and 5 days post amputation. Three replicates were collected at each time point.  10 fins were pooled to comprise one replicate.  Fish were dosed at 0 days post amputation with vehicle control alone or 50 ng/g TCDD.

1 Day Post Amputation Vehicle Exposed: GSM85187, GSM85188, and GSM85189

1 Day Post Amputation TCDD Exposed: GSM85190, GSM85191, and GSM85192

3 Days Post Amputation Vehicle Exposed: GSM85193, GSM85194, and GSM85195

3 Day Post Amputation TCDD Exposed: GSM85196, GSM85197, and GSM85198

5 Days Post Amputation Vehicle Exposed: GSM85199, GSM85200, and GSM85201

5 Days Post Amputation TCDD Exposed: GSM85202, GSM85203, and GSM85204

first: Eric

last: Andreasen

first: Lijoy

middle: K

last: Mathew


title: Zebrafish microRNA miR-430 promotes deadenylation and clearance of maternal mRNAs

status: Public on Feb 17 2006

city: New Haven

first: Antonio

middle: J.

last: Giraldez

state: CT

address: 333 Cedar Street

department: Genetics

institute: Yale University

summary:
MicroRNAs comprise 1-3% of all vertebrate genes, but their in vivo functions and mechanisms of action remain largely unknown. Zebrafish miR-430 is expressed at the onset of zygotic transcription and regulates morphogenesis during early development. Using a microarray approach and in vivo target validation, we find that miR-430 directly regulates several hundred target mRNAs. Targets are highly enriched for maternal mRNAs that accumulate in the absence of miR-430. We also show that miR-430 accelerates the deadenylation of target mRNAs. These results suggest that miR-430 facilitates the deadenylation and clearance of maternal mRNAs during early embryogenesis.
Keywords: Dicer, MZdicer, miR-430, miRNA target, maternal, zygotic

overall_design:
As a first step towards the identification of miR-430 targets, we compared the rate of mRNA degradation in wild type and maternal-zygotic (MZ) dicer mutant embryos, which are deficient in miRNA processing. We found that the rate of degradation of a target mRNA with partial complementarity to miR-430 is significantly reduced in MZdicer mutants. Providing miR-430 duplexes to MZdicer mutants restores the rate of target decay. These results indicate that miR-430 enhances the decay of target mRNAs.

To identify miR-430 in vivo targets, we compared the mRNA expression profile of embryos that were wild-type, MZdicer, or MZdicer rescued with miR-430 (MZdicer+miR-430). This analysis identified ~600 genes that were upregulated in MZdicer, but not in MZdicer+miR-430.

We have also analyzed the expression profile of the genes in the array at three different time points, one maternal stage(16 cell), and two Zygotic stages 5 hours and 9 hours

middle: J

first: Yuichiro

last: Mishima

first: Jason

last: Rihel

first: Russel

last: Grocock

first: Stijn

last: Van Dongen

first: Kuino

last: Inoue

first: Anton

last: Enright

first: Alexander

last: Schier