Project description:Purpose: The purpose of this study was to develop a framework for analyzing RPE expression profiles from zebrafish eye mutants. Methods: The fish model we used was smarca4 (SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 4), a retinal dystrophic mutant that the retinal phenotype and expression profiles were previously characterized. Histological and Affymetrix GeneChip analyses were conducted to define the RPE defects and underlying differential expression respectively. Results: Histological analysis indicates that smarca4 RPE was formed but its differentiation was abnormal. In particular, ultra-structural analysis of smarca4 RPE by transmission electron microscopy showed a number of defects in melanogenesis, suggesting that the cytoskeletal dynamics was impaired. To compare the expression profile of normal wild-type (WT) and smarca4 RPE, their retinas and RPE-attached retinas were microdissected and the gene expression values of these tissues measured by Affymetrix GeneChip analysis. The RPE expression values were then estimated from these samples using an approach previously established by us. A factorial analysis was conducted using the expression values of RPE, retinal as well as the whole-embryo samples. Specific rules (contrasts) were built using the coefficients of the resulting fitted model to select for three groups of genes: 1) Smarca4-regulated RPE genes, 2) Smarca4-regulated retinal genes, and 3) Smarca4-regulated RPE genes that are not differentially expressed in the retina. The latter group consists of 39 genes that are highly related to cytoskeletal dynamics, melanogenesis, paracrine and intracellular signal transduction. Conclusions: Our analytical framework can potentially identify genes in zebrafish mutants that both retina and RPE are affected by the underlying mutation. The gene expression levels of three independent replicates of WT whole embryos (WA52), WT retinas (WR52), WT RPE-attached retinas (WRR52), smarca4/yng retinas (YR52), smarca4 RPE-attached retinas (YRR52) and smarca4 whole embryos (YA52) were measured by Affymetrix Zebrafish Whole Genome Arrays. All samples were collected at 52 hpf. The probe-level data of these sample groups were background adjusted, normalized and summarized by a robust multiarray average (RMA) algorithm. RPE expression values were estimated from the comparison between the retinal and RPE-attached retinal samples of the same genotype based on a method we previously developed (Leung et al., Invest Ophthalmol Vis Sci. 2007; 48:881). Then, a 3x2 factorial design was used to analyze the expression values of three kinds of tissue (T): whole embryo, retina and RPE in two kinds of mutation (M) background: WT and smarca4. Using the coefficients from the fitted models, contrasts were built to identify candidate genes that fulfilled specific criteria. The false discovery rate (FDR) q-value cutoff for a contrast was 0.001. Several contrasts were ultimately combined to allow for selection of genes that were regulated by Smarca4 in the retina and RPE. These include 1) Smarca4-regulated RPE genes, 2) Smarca4-regulated retinal genes, and 3) Smarca4-regulated RPE genes that are not differentially expressed in the retina.

Project description:Purpose. XOPS-mCFP transgenic zebrafish experience a continual cycle of rod photoreceptor development and degeneration throughout life, making them a useful model to investigate the molecular determinants of rod photoreceptor regeneration. The purpose of this study was to compare the gene expression profiles of wild type and XOPS-mCFP retinas in order to identify genes that may contribute to the regeneration of the rods. Methods. Wild type and XOPS-mCFP retinal mRNA was subjected to microarray analysis using the Agilent platform. The Ingenuity Pathway Analysis program was used to identify biologically relevant processes that were significantly represented in the dataset. Expression changes were verified by RT-PCR. Selected genes were further examined during retinal development and in adult retinas by in situ hybridization, immunohistochemistry, and using a transgenic fluorescent reporter line. Results. Over 600 genes displayed significant expression changes in XOPS-mCFP retinas compared to wild type controls. Many of the downregulated genes were associated with phototransduction, whereas upregulated genes were associated with several biological functions, including cell cycle, DNA replication and repair, cell development and cell death. RT-PCR analysis of a subset of these genes confirmed the microarray results. Three transcription factors (sox11b, insm1a, and c-myb) displaying increased expression in XOPS-mCFP retinas were also expressed throughout retinal development and in the persistently neurogenic ciliary marginal zone. Conclusions. This study identified numerous gene expression changes in response to rod degeneration in zebrafish, and further suggests a role for the transcriptional regulators sox11b, insm1a, and c-myb in both retinal development and rod photoreceptor regeneration. Two-condition experiment: wild-type vs. XOPS-mCFP retinas. Four biological replicates for each condition. RNA was prepared from one retina for each sample. Each hybridization was accompanied by a dye-swap control, for a total of eight array hybridizations.

Project description: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 (Â± 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 27Â± 1 ÂºC and 14:10h light:dark photoperiod. Experiment 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 Âµg/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 Âµg/L MC-LR, 1,000 Âµg/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. Experiment Overall Design: 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 Â± 12 SD (low concentration) and 1,703 Â± 71 SD (high concentration). The concentration of microcystin-LR in the lyophilized Microcystis treatment was 4.5 Âµg/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.

Project description:Pharmaceutical chemicals used in human medicine are released into surface waters via municipal effluents and pose a risk for aquatic organisms. Among these substances are selective serotonin reuptake inhibitors (SSRIs) which can affect aquatic organisms at sub ppb concentrations. To better understand biochemical pathways influenced by SSRIs, evaluate changes in the transcriptome, and identify gene transcripts with potential for biomarkers of exposure to SSRIs; larval zebrafish Danio rerio were exposed (96 h) to two concentrations (25 and 250 µg/L) of the SSRIs, fluoxetine and sertraline, and changes in global gene expression were evaluated (Affymetrix GeneChip® Zebrafish Array). Significant changes in gene expression (>=1.7 fold change, p<0.05) were determined with Partek® Genomics Suite Gene Expression Data Analysis System and ontology analysis was conducted using Molecular Annotation System 3. The number of genes differentially expressed after fluoxetine exposure was 288 at 25 µg/L and 131 at 250 µg/L; and after sertraline exposure was 33 at 25 µg/L and 52 at 250 µg/L. Five genes were differentially regulated in all treatments relative to control, suggesting that both SSRIs share some similar molecular pathways. Among them, expression of the gene coding for FK506 binding protein 5 (FKBP5), which is annotated to stress response regulation, was highly down-regulated in all treatments (results confirmed by qRT-PCR). Gene ontology analysis indicated that regulation of stress response and cholinesterase activity were critical functions influenced by these SSRIs, and suggested that changes in the transcription of FKBP5 or acetylcholinesterase could be useful biomarkers of SSRIs exposure in wild fish. Zebrafish (Danio rerio) were obtained from the Zebrafish Research Facility maintained at the Center for Environmental Biotechnology at the University of Tennessee. Fish husbandry, spawning, and experimental procedures were conducted with approval from the UT Insititutional 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. Embroyos were obtained by spawning adult fish with no history of contaminant exposure. Fertilization of embryos took place at the same time (<15 minutes), 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 C and 14:10h light:dark photoperiod. Larval zebrafish (72 hpf) were exposed for 96 h in 200ml fish water containing appropirate amount of SSRI stock (i.e. fluoxetine or sertraline). There were four SSRIs treatments (25 and 250 ug/L fluoxetine and 25 and 250 ug/L sertraline) and one control (no SSRIs) with triplicate beakers and each beaker contained about 100 larval fish. During exposure for 96 hours, beakers were kept covered to prevent water evaporation and fish were not fed (i.e., fish consumed their yolk sac).

Project description:Ectothermic vertebrates are different from mammals that are sensitive to hypothermia and they have to maintain core temperature for survival. Why and how ectothermic animals can survive, grow and reproduce in low temperature have been for a long time a scientifically challenging and important inquiry to biologists. We used a microarray to profile the gill transcriptome in zebrafish (Danio rerio) after exposure to low temperature. Adult zebrafish were acclimated to a low temperature of 12 ï°C for 1 (1-d) and 30 d (30-d), and the gill transcriptome was compared to wild types by oligonucleotide microarray hybridization. Results showed 11 and 22 transcripts were found to be upregulated by low-temperature treatment for 1-d and 30-d respectively, while 56 and 70 transcrips were downregulated. The gill transcriptome profiles revealed that ionoregulation-related gene was highly upregulated in cold-acclimated zebrafish. This observation encouraged us to investigate the role of ionoregulatory genes in zebrafish gills during cold acclimation. Cold acclimation caused upregulation of genes that are essential for ionocyte specification, differentiation, ionoregulation, and acid/base balance, and also increased the numbers of cells expressing these genes. mRNA expression of epithelial Ca2+ channel (ECaC), one of these genes, was increased in parallel with the level of Ca2+ influx, revealing a functional compensation after long-term acclimation to cold. Phospho-histone H3 and TUNEL staining showed that the cell turnover rate was retarded in cold-acclimated gills. These results suggest that gills may sustain their functions by yielding mature ionocytes from preexisting undifferentiated progenitors in low-temperature environments. The AB strain of zebrafish (D. rerio) was originally obtained from the University of Oregon, and were kept in the zebrafish stock center at Academia Sinica, Taipei, Taiwan. Fish were reared in local tap water at 28 ï°C and a photoperiod regime of 14-h L/10-h D. Adult zebrafish were acclimated to 12 ï°C with a gradually reduced temperature at a gradient of 4 ï°C/h in order to prevent temperature shock and reduce mortality. After 30 d of acclimation, surviving (over an 80% survival rate) fish appeared to be feeding and behaving normally compared with control fish. The experimental protocols were approved by the Academia Sinica Institutional Animal Care and Utilization Committee (approval no. RFiZOOHP2006083). After the low-temperature treatment, gill tissues dissected from 6 individuals were pooled as a sample and then homogenized in 5 ml Trizol reagent (Invitrogen, Carlsbad, CA). Thirty six individuals (18 for low-temperature treatment and 18 for control) were sacrificed for each microarray hybridization experiment, and another 60 individuals were used for quantitative reverse-transcription polymerase chain reaction. After chloroform extraction, RNA precipitation and ethanol washing, the RNA samples were purified and treated with DNase1 to remove the genomic DNA by using RNeasy Mini Kit (Qiagen, Huntsville, Alabama). The quantity and quality of total RNA were assessed by spectrophotometry and agarose gel electrophoresis, respectively. The commercial zebrafish 14K oligonucleotides set (MWG Biotech AG, Ebersbach, Germany) were obtained and were printed on UltraGAPS Coated slide (Corning, New York, NY ) with use of the OmniGrid 100 microarrayer (Genomic Solutions, Ann Arbor, MI) according to the manufactureâs instructions. The 14,067 oligonucleotides represent 9666 genes (7009 singlet genes and 2657 redundant genes), and the redundancy of this chip is 31 %. The detailed description of the oligonucleotides information can be obtained on the Ocimun Biosolution website (http://www.ocimumbio.com/web/default.asp). cDNA probes were synthesized by reverse transcription of 20 Î¼g total RNA using a SuperScript indirect cDNA labeling system (Invitrogen) and were labeled with Cy5 (cold treatment groups) and Cy3 (control groups)(Amersham Bioscience, Buckinghamshire, UK) , respectively. The zebrafish 14K OciChip array chip was pretreated with 1% bovine serum albumin (BSA) (fraction V), 4x saline-sodium citrate (SSC), and 1% sodium dodecylsulfate (SDS) at 42 Â°C for 45 min, and then hybridized overnight in a cocktail containing 5x Denhardt's solution, 6x SSC, 0.5% SDS, 50% formamide, 50 mM sodium phosphate, and 2 Âµg/Âµl yeast tRNA. Slides were washed with 2x SSC and 0.1% SDS (5 min), 1x SSC and 0.1% SDS (5 min), 0.5x SSC (5 min), and twice with 0.1x SSC (2 min each). Scanning was performed with a Genepix scanner (Molecular Devices, Sunnyvale, CA). The acquired images were analyzed using ScanArray Express 3.0 (PerkinElmer, Waltham, MA) and Genespring (Aglient Technologies, Foster City, CA) software. The measurements of spots were filtered by flags, and the lowest normalization was performed after subtraction of the median background. To assess the differential expressions of genes, we identified the ratio of Cy5/Cy3 intensity > 2 or < 0.5. Each experiment contained 3 biological replicates (including 1 dye swap) with different samples, and the differentially expressed genes were selected from those with at least 2 of 3 significant signals (ratio > 2 or < 0.5).

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Transcriptional coupling between Nrf2 and aB-crystallin in the lens and heart of zebrafish under proteostatic stress

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