Differences found in expression of cell surface & ECM components during development of the rainbow trout ovary & testis.
ABSTRACT: A comparison of "maturing" and "prespawn" ovarian and testicular transciptomes was performed to determine the genes that are involved in regulating gametic and accessory cell function during maturation and development of the rainbow trout gonad. To identify some fo the genes involved in these processee, total RNA was compared between three-year-old normal vs two-year-old normal (maturing) and three-year-old normal vs two-year-old precocious (prespawn) gonadal tissue. Three biological replicates for both "maturing" and "prespawn" rainbow trout ovary and testis with corresponding dye flips.
Project description:Comparison of gene expression of exposed versus non-exposed Oncorhynchus mykiss hepatocytes to four model chemicals and a synthetic mixture. Hepatocytes were exposed for 24 hours to a single chemical and a synthetic mixture of 10 nM 17 alpha-ethinylestradiol (EE2), 0.75 nM 2,3,7,8-tetrachloro-di-benzodioxin (TCDD), 100 μM paraquat and 0.75 μM 4-nitroquinoline-1-oxide (NQO). Four biological replicates for both exposed and non-exposed Oncorhynchus mykiss hepatocytes with corresponding dye flips.
Project description:Hybridization of one kidney of cortisol treated fish vs. one kidney of control fish. Kidneys were collected from untreated juvenile sea bream (n=4) and from fish, which received for 72h a coconut-oil implant containing 10mg/Kg (fish wet weight) (n=4) cortisol. Experiments were carried out at the University of the Algarve, Portugal in accordance with National legislation for the welfare of animals. Experiments were conducted in two 125 l cylindriconical tanks supplied with a continuous through-flow of oxygenated seawater at 20+1 °C using juvenile sea bream (25 g + 3 g) adapted for 1 week to the experimental conditions. One tank contained 8 untreated fish (control) and the other tank 8 cortisol treated fish and the end of experiments fish were removed form tanks, decapitated and the kidneys rapidly removed and place in RNAlater (Qiagen) at –20 °C. No mortality occurred in the control tank but 2 fish died in the cortisol treated tank.
Project description:Five different developmental stages, neurula (1), hatching (48 h post-fertilization) (2), pectoral fin budding (96 h post-fertilization) (4), mouth opening (144 h post-fertilization) (6), and eye pigmentation (168 h post-fertilization) (7) were compared directly to each other in all possible combinations (Loop design).All experiments were perfomed by dye swap hybridizations.
Project description:Although studies have established that exogenous growth hormone (GH) treatment stimulates growth in fish, its effects on target tissue gene expression are not well characterized. We assessed the effects Posilac® (Monsanto Co., St. Louis, MO), a recombinant bovine somatotropin, on tissue transcript levels. Transcript abundance was measure in liver and muscle using the GRASP 16 K cDNA microarray. A selection of the genes identified as altered with the microarray, and also transcripts for insulin-like growth factors, growth hormone receptors (GHR) and myostatins were measured by realtime PCR in the liver, muscle, brain, kidney, intestine, stomach, gill and heart. In general, transcripts identified as differentially regulated in the muscle on the microarray showed similar direction of expression in the other non-hepatic tissues. Rainbow trout were selected from two high growth rate and two low growth rate families. A total of 113 and 67 transcripts were identified by microarray as differentially expressed with GH treatment across growth rate for muscle and liver respectively. The largest proportion of the transcripts represented novel transcripts, followed by immune and metabolism related genes. The immune related genes were primarily modulated in the liver and indicate activation of a non-specific immune response. The metabolic genes include lipid metabolism, oxidative phosphorylation and one carbon metabolism pathway transcripts. Most notable among the growth axis genes measured by realtime PCR were increases in GHR1 and-2 transcript in liver and muscle. Our results indicate that short-term GH treatment activates the immune system, shifts the metabolic sectors and modulates growth regulating genes. Keywords: Growth Hormone Injection Muscle and Liver Gene Expression Rainbow trout (hatched March 2005) selected for extreme growth rate were obtained from NCCCWA brood stock. Families were selected based on body weight at 7 months of age and thermal growth coefficient for the final month of growth. The two high growth families used in the study were in the top 2% in terms of growth rate, and the low growth families were in the lowest 10% for growth rate. Fish acclimated to the new tanks for two weeks prior to initiation of the treatments. Fish from each family were randomly selected to receive one of three treatments: 1) Posilac® injection (120 mg/kg BW, n = 4 per family); 2) vehicle injection (n = 4 per family); or 3) untouched controls (n = 2 per family). We had determined there was no effect to growth or the GH/IGF-I axis in the vehicle treated fish, and therefore, all of the microarray hybridizations were made between the GH and vehicle injected groups. This study included a total of 16 two-channel arrays designed for the direct comparison of GH treatment levels. That is, for each of the four groups, 1) High Growth Rate Liver; 2) Low Growth Rate Liver; 3) High Growth Rate Muscle; and 4) Low Growth Rate Muscle, four slide were hybridizes using individual RNA samples from individual fish. RNA isolation from each tissue/organ sample was handled separately (without pooling) with the purpose of using biological replications. We hybridized two slides with the GH cDNA labeled with Alexa 555 and vehicle cDNA labeled with Alexa 647; and two slides, using unique RNA samples, for the with GH cDNA labeled with Alexa 647 and tissue from the vehicle injected group labeled with Alexa 647 within each tissue and growth rate. Sixteen slides were used in the current study representing 32 individual tissue samples, meaning a total of four biological replicates for each treatment group.
Project description:Perfluorooctanoic acid (PFOA) is a potent hepatocarcinogen and peroxisome proliferator (PP) in rodents. Humans are not susceptible to peroxisome proliferation and are thought to be refractory to carcinogenesis by PFOA and other PPs. However, previous studies with rainbow trout have shown that they are also insensitive to peroxisome proliferation by the PP, dehydroepiandrosterone (DHEA), but are still susceptible to enhanced hepatocarcinogenesis after chronic exposure. In this study, we determined whether PFOA is also a tumor promotor in trout and then examined hepatic gene expression profiles to further investigate possible mechanisms of action. Trout were initiated as fry to the hepatocarcinogen, aflatoxin B1, and then fed 200-1800 ppm PFOA in the diet for 30 weeks. Two structurally diverse PPs, clofibrate (CLOF) and DHEA, were included for comparison. Hepatic gene expression profiles were subsequently examined in animals exposed to similar doses of PFOA, DHEA and CLOF along with 5 ppm 17β-estradiol (E2; a known tumor promotor) in the diet. PFOA (1800 ppm) and DHEA treatments resulted in enhanced liver tumor incidence and multiplicity while CLOF showed no effect. Carcinogenesis seemed independent of peroxisome proliferation as no induction of peroxisomal β-oxidation and catalase activity were observed. Alternately, plasma VTG was elevated in fish fed PFOA and DHEA suggesting that estrogenic mechanisms may play a role. Both tumor promotors, PFOA and DHEA, resulted in strong correlation of transcriptional profiles with E2 by Pearson correlation (R=0.81 and 0.78, respectively). In comparison, CLOF regulated no genes in common with E2. Overall, these data suggest that the tumor promoting activities of DHEA and PFOA in trout are independent of peroxisome proliferation and may involve estrogenic mechanisms. Juvenile trout, 12-18 months old, were fed experimental diets containing 500 or 1800 ppm PFOA, 1800 ppm CLOF, 750 ppm DHEA, 5 ppm E2 or 0.15 % dimethyl sulfoxide vehicle control for 14 days. Liver samples were collected for microarray analysis. Hybridizations were performed using standard reference design with dye-swapping. For each sample, equal amounts of RNA (µg) were pooled from five fish per tank for every treatment (n=3 biological replicates per treatment). cDNA from two of the three biological replicates was dye-swapped and hybridized to two slides as technical replicates (5 arrays per treatment).
Project description:Vegetative phase change is the developmental transition from the juvenile phase to the adult phase during which a plant becomes competent for sexual reproduction. Gain of ability to flower is often accompanied by changes in patterns of differentiation in newly forming vegetative organs. In maize, juvenile leaves differ from adult leaves in morphology, anatomy, and cell wall composition. Whereas the normal sequence of juvenile followed by adult is repeated with every sexual generation, this sequence can be altered in maize by the isolation and culture of the shoot apex from an adult phase plant; an “adult” meristem so treated reverts to forming juvenile vegetative organs. To investigate the molecular differences between the juvenile and adult phases in maize comparisons among two juvenile samples, leaf 4 and culture-derived leaf 3 or 4, and an adult sample (leaf 9) were made using cDNA microarrays. All samples were leaf primordia at plastochron 6. A gene was scored as “phase specific” if it was up- (or down-) regulated in both juvenile samples compared to the adult sample with at least a twofold-change in gene expression at P-value less than or equal to 0.005. Some 221 ESTs up-regulated in juvenile and 28 ESTs up-regulated in adult were identified. Altered patterns of expression of selected ESTs in the phase change mutants Tp2, d1 and gl15 further confirmed these genes as being phase-specific and allowed us to position these genes in the known genetic hierarchy regulating phase change. Keywords: Transcript profiling among seed-derived juvenile leaf 4 and adult leaf 9 and culture-rejuvenated leaf 3 or 4 in maize To identify juvenile or adult specific ESTs, total RNA from leaf primordia at plastochron 6 (P6) was isolated from leaves 4 (L4) and 9 (L9) from seed-derived plants and leaf 3 or 4 (RL3/4) from culture-derived plants. For each of six biological replications, each of the three pairwise comparisons of P6-staged leaf primordia from L4, L9 and RL3/4 was made on one slide. With six biological replications and three slides per replication (L4 vs. L9, L9 vs. RL3/4, RL3/4 vs. L4), this replicated loop design used a total of 18 slides. To ensure dye balance, each of the 18 target samples was measured once with Cy3 labeling and once with Cy5 labeling.
Project description:Tadpoles of the anuran species Rana pirica can undergo predator-specific morphological responses. Exposure to a predation threat by larvae of the salamander Hynobius retardatus results in formation of a bulgy body (bulgy morph) with a higher tail. The tadpoles revert to a normal phenotype upon removal of the larval salamander threat. The objective of the present study was to use our own fabricated tadpole Rana pirica cDNA microarray to profile gene expression patterns during the predation threat. Experimental design used to produce control, bulgy morph and reversion type tadpoles for the microarray analysis was as follows. One group of tadpoles was placed with a larval salamander for 4 days to induce formation of the bulgy morph phenotype; the predator was then removed and the tadpoles were allowed to revert to the normal phenotype for 4 days. This group is termed “8 day out tadpoles”. A second group of tadpoles was placed with the predator for the full 8 days. The control group was not exposed to a predator. Tadpoles from the predator induced groups were sampled at 6 hours, 4 days and 8 days, those from the control group at 0 hour, 4 days, and 8 days. The comparative design of the microarray analysis was performed as (Exp 6 hours VS Cont 0 hours), (Exp 4 days VS Cont 4 days), (Exp 8 days VS Cont 8 days), and (Exp 8 days-Out VS Exp 8 days), respectively. These analyses were performed in triplicate with a dye swap experiment. Samples 0hr~8days were as follows. Sample plate (1~3A):Channel 1-Cy5 Experiment,Channel 2-Cy3 Control. Sample plate (1~3B):Channel 1-Cy5 Control, Channel 2-Cy3 Experiment. In case of 8day-out samples were as follows. Sample plate (1~3A):Channel 1-Cy5 8day-out,Channel 2-Cy3 full 8day. Sample plate (1~3B):Channel 1-Cy5 full 8day,Channel 2-Cy3 8day-out.
Project description:A large-scale functional genomics study revealed shifting energy generating processes in white muscle during the final 1,300 km migration of wild sockeye salmon to their spawning grounds in the Fraser River, British Columbia. In 2006, Lower Adams stock sockeye salmon ceased feeding after passing the Queen Charlotte Islands, 850 km from the Fraser River. Enhanced protein turnover and reduced transcription of actin, muscle contractile and heme-related proteins were early starvation responses in saltwater. Arrival to the estuarine environment triggered massive protein turnover through induction of proteasoma and lysosomal proteolysis and protein biosynthesis, and a shift from anaerobic glycolysis to oxidative phosphorylation. Response to entry into freshwater was modest, with up-regulation of heat shock proteins and nitric oxide biosynthesis. High river temperatures resulted in a strong defense/immune response and high mortalities in 50% of fish. Arrival to the spawning grounds triggered further up-regulation of oxidative phosphorylation and proteolysis, down-regulation of protein biosynthesis and helicase activity, and continued down-regulation of muscle proteins and most glycolytic enzymes. However, sharp up-regulation of PFK-I indicated induction of glycolytic potential at the spawning grounds. The identification of potential environmental cues triggering genome-wide transcriptional shifts in white muscle associated with migration and the strong activation of proteasomal proteolysis were both novel findings. Keywords: Functional genomics study on wild-caught fish The experiment was based on expression profiles of white muscle tissue collected from wild migrating adult sockeye salmon during their return spawning migration back to the Fraser River. Fish were collected from seven sites along the final 1,300 km migration path, and white muscle samples were quickly frozen in liquid nitrogen upon capture. Marine sampling sites included (from north to south) the Queen Charlotte Islands (QCI), Johnstone Strait (JS), Juan de Fuca Strait (JDFS), and the Strait of Georgia (SOG). Freshwater sampling sites included Whonnock (W), Savona (SV) and the Lower Adams Spawning Grounds. Genetically-based stock ID was used to identify the natal sites of fish collected from the wild. The experiment was designed to profile the transcriptional shifts associated with migration of the Adams River stock complex. The total experiment included 80 microarray slides, with a minimum biological replicate size per site of 6 (SV), and maximum of 18 (JS) (see supplemental table for details). Additional intra-site variables, which could only be addressed in some sites, included sex (female biased) and river entry timing (for JS, JDFS and W sites; identified through radio-tracking of marine collected fish). Total RNA was amplified (1 round) with MessageAmpTMII-96 kit (Ambion, TX, USA), and reverse transcribed to cDNA before labelling with ALEXA dyes using the Invitrogen Indirect Labelling Kit. The experiment was based on a reference design, with the reference containing the combined aRNA of all individuals used in the experiment. Individual samples were labelled with Alexa 555 and the reference control with Alexa 647, with no dye flips included. A single technical replicate of one SV fish (replicate 5) was included in the experimental design. This experiment is part of a larger white muscle experiment containing additional sockeye salmon stocks.
Project description:Natural Antisense Transcripts (NATs) can regulate gene expression by virtue of their ability to form double-stranded RNA duplexes. To investigate NATs in the maize transcriptome, cDNAs from seedling of two inbred lines (B73 and Mo17) were hybridized to an oligonucleotide microarray designed to validate the expression of in silico detected NATs and to screen for NATs that can anneal to a random set of 3’ UTRs and selected repeats found in 3’ UTRs regions. Quantitative Real-Time PCR experiments were conducted to determine the minimum detection threshold of microarray experiment and to thereby identify genes for which both sense and antisense transcripts accumulate to detectable levels. Two independent approaches, strand-specific RT-PCR and S1 nuclease assays were conducted to validate the results of the microarray experiment. Based on these conservative assays, NATs accumulate in seedlings that can anneal to over 70% of a random set of maize genes. In addition, both sense and antisense transcripts anneal to more than 80% of a set of maize repeats. Significantly, sense and antisense transcripts exhibit significant different expression patterns between the two genotypes. Based on these findings we hypothesize that interactions between sense and antisense transcripts may contribute to the differential patterns of gene expression in maize hybrids and to heterosis. Keywords: Global antisense transcripts profiling between two maize inbreds To systematically identify NATs in maize, we employed multiple strategies to computationally identify putative antisense transcripts from our partial genome assembly (MAGIs) and a collection of ESTs we sequenced with known sequence orientation. Additionally, we randomly sampled and surveyed maize UTRs which often harbor transposons. Strand-specific oligonucleotides which can hybridize to the antisense strand were designed and spotted with together with the oligonucleotides hybridizing sense strands on a custom, strand-specific oligoarray, providing the first global expression profiling study of NATs in maize UTRs. In total, 10 biological replication were conducted to compare the global gene expression profiles between two maize genotypes (B73 and Mo17).
Project description:Heterosis is the phenomenon whereby the progeny of particular inbred lines have enhanced agronomic performance relative to both parents. Although several hypotheses have been proposed to explain this fundamental biological phenomenon, the responsible molecular mechanisms have not been determined. The maize inbred lines B73 and Mo17 produce a heterotic F1 hybrid. Global patterns of gene expression were compared in seedlings of these three genotypes using a microarray that contains 13,999 cDNAs. Using an estimated 15% false discovery rate as a cut-off, 1,367 ESTs (9.8%) were identified as being significantly differentially expressed among genotypes. All possible modes of gene action were observed, including additivity, high- and low-parent dominance, under-dominance, and over-dominance. The largest proportion of the ESTs (78%, 1,062/1,367) exhibited expression patterns that are not statistically distinguishable from additivity. Even so, 22% of the differentially regulated genes exhibited non-additive modes of gene expression. Classified on the basis of significant pair-wise comparisons of genotype means, 181 of these 305 genes exhibited high-parent dominance and 23 exhibited low-parent dominance. In addition, 44 genes exhibited under- or over-dominant gene action. These findings are consistent with the hypothesis that multiple molecular mechanisms contribute to heterosis, including over-dominance Keywords: Genotype Comparison The inbreds B73 (Schnable Lab Accession #660) and Mo17 (Schnable Lab Accession #2618) used in this study were derived by self-pollination from stocks originally obtained from Don Robertson (Iowa State University) and Mike Lee (Iowa State University), respectively. Mo17 was crossed as a female by B73 to generate the F1. Kernels from three different seed sources (ears) per genotype were used in the experimental design. Prior to microarray analyses genotypes were confirmed by genotyping DNA extracted from each seed source using co-dominant IDP genetic markers that distinguish B73 from Mo17 (Fu et al., in preparation). Ten biological replications of B73, Mo17, and their F1 (Mo17xB73) were grown under highly controlled conditions in a randomized complete block design. For each replication, the B73, Mo17, and F1 samples were hybridized to three two-color cDNA microarrays using a loop design such that each loop included all pairwise comparisons between genotypes. RNA pools for each genotype were alternately labeled providing dye balance within each loop. After hybridization, one biological replicate was removed due to poor quality. The final analysis incorporated 27 microarray slides (3 slides for each of nine high-quality biological replicates).