Gene expression profile after Spring Viraemia Carpae Virus (SVCV) infection in zebrafish mutants rag1 -/- versus wt
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ABSTRACT: Immune responses in higher vertebrates are classically separated into innate and adaptive (or specific) immunity. However, important gaps of knowledge about how adaptive responses are generated in lower vertebrates still remain unsolved. In order to explore the relative importance of adaptive and innate immune responses, we have studied zebrafish transcriptional responses to an infection with the Spring Viraemia of Carp virus (SVCV) in rag1-/- zebrafish mutants compared to wild type zebrafish by using both genome wide and immunological-targeted gene expression microarrays. Both wild type (wt) and mutant (rag1) zebrafish were divided in two groups with 3 individuals per group. First group was infected with 10^5 pfu per ml of SVCV, second group was mock-infected. Two days after challenge zebrafish were sampled, head kidney and spleen of each fish were extracted and pooled between each group. The experiment was repeated once to obtain two biological replicates.
Project description:Vertebrates are protected from pathogens by an adaptive and an innate immune system. In mammals, loss of the adaptive immune system severely compromises viability. Zebrafish, in contrast, can survive for relatively long without adaptive immunity, as demonstrated by the immuno-deficient Rag1 mutant. Here, we examine whether the innate immune system is activated in the mutant, and ask whether this has any effect on the nervous system, given the finding that inflammation is linked to neurodegeneration in higher vertebrates. Using microarray analysis of the olfactory epithelium, we show that innate immune responses are upregulated. Neuronal genes are down-regulated, and immunofluorescence for the neural marker HuC shows a progressive loss of olfactory sensory neurons in mutants. Propodium iodide uptake indicates a corresponding increase in cell death. Rag1 mutant fish progressively lose their sensitivity to an olfactory cue, the alarm pheromone Schreckstoff, indicating that neuro-degeneration has a behavioral significance. Taken together, these data establish that immuno-deficiency in the zebrafish is accompanied by neuro-inflammation and loss of neurons. The microarray data document transcriptional changes associated with activation of innate immunity in the context of immunodeficiency and also identify novel genes that are potentially related to function of the olfactory system. Keywords: analysis of mutant A total of 6 RNA samples were used for hybridization. 3 were from the olfactory rosettes of wild type adult zebrafish and 3 were from Rag1 mutants.
Project description:We describe here transcripts induced after infection of zebrafish with Spring Viremia Carp Virus (SVCV). Two days after infection, differentially expressed transcript levels from selected immune-related zebrafish genes were studied in internal organs (pooled spleen, head kidney). Also, transcripts from resistant fishes to viral infection one month after inoculation were studied. Three different experiments were performed to get three biological replicates. Fishes were divided into two groups in each experiment. First group was infected by immersion with SVCV 10^7 pfu/ml, second group was used as a control of non-infected fishes. 6 fishes per group were sacrificed two days post infection, whereas the rest of the infected fishes from the three experiments were maintained for 30 days in the aquariums and then survivors (six for experiment) were sacrificed. This submission includes three biological replicate groups for the non-infected fish and the two days post-infected fish, and two biological replicate groups for the 30 days post-infected fish.
Project description:In order to explore the relative importance of adult zebrafish innate and adaptive immune responses to different kinds of infections, we compared the gene expression profile of VHSV-challenge-survivors, bacterial infection survivors and control non infected zebrafish. VHSV -infection-survivors zebrafish were divided in two groups of 12 individuals, first group was infected with VHSV (samples named A) and second group was mock-infected (samples named B). Two days after challenge, zebrafish of each group were divided in four subgroups (3 fishes per group) and head kidney and spleen of each individual were sampled. For each group, internal organs of three fishes were pooled, so four biological replicates were generated . Zebrafish surviving a natural infection caused by Aeromonas hydrophila and Vibrio fluvialis (samples named C) were divided in four groups (3 fishes per group) and head kidney and spleen were extracted. Finally, 12 healthy zebrafish were used as control group (samples named D) and processed in the same way as group C.
Project description:To identify mechanisms that regulate V(D)J recombination, we used proximity-dependent biotin identification to analyze the interactomes of full length and truncated forms of RAG1 in pre-B cells. This revealed an association of RAG1 with numerous nucleolar proteins in a manner dependent on amino acids 216-383 and allowed identification of a motif required for nucleolar localization. Experiments in transformed pre-B cell lines and cultured primary pre-B cells reveal a strong correlation between disruption of nucleoli, reduced association of RAG1 with a nucleolar marker, and increases in V(D)J recombination activity. Mutation of the RAG1 nucleolar localization motif boosts recombination while removal of the first 215 amino acids of RAG1, which are required for efficient egress from nucleoli, reduces recombination activity. Our findings indicate that nucleolar sequestration of RAG1 is a negative regulatory mechanism in V(D)J recombination and identify regions of the RAG1 N-terminal region that control nucleolar association and egress.
Project description:Domesticated animal populations often show profound reductions in predator avoidance and fear-related behavior compared to wild populations. These reductions are remarkably consistent and have been observed in a diverse array of taxa including fish, birds, and mammals. Experiments conducted in common environments indicate that these behavioral differences have a genetic basis. In this study, we quantified differences in fear-related behavior between wild and domesticated zebrafish strains and used microarray analysis to identify genes that may be associated with this variation. Compared to wild zebrafish, domesticated zebrafish spent more time near the water surface and were more likely to occupy the front of the aquarium nearest a human observer. Microarray analysis of the brain transcriptome identified high levels of population variation in gene expression, with 1,749 genes significantly differentially expressed among populations. Genes that varied among populations belonged to functional categories that included DNA repair, DNA photolyase activity, response to light stimulus, neuron development and axon guidance, cell death, iron-binding, chromatin reorganization, and homeobox genes. Comparatively fewer genes (112) differed between domesticated and wild strains with notable genes including gpr177 (wntless), selenoprotein P1a, synaptophysin and synaptoporin, and acyl-CoA binding domain containing proteins (acbd3 and acbd4). Microarray analysis identified a large number of genes that differed among zebrafish populations and may underlie behavioral domestication. Comparisons with similar microarray studies of domestication in rainbow trout and canids identified sixteen evolutionarily or functionally related genes that may represent components of shared molecular mechanisms underlying convergent behavioral evolution during vertebrate domestication. However, this conclusion must be tempered by limitations associated with comparisons among microarray studies and the low level of population-level replication inherent to these studies. RNA was extracted from the brains of fish from four behaviorally distinct strains of zebrafish and hybridized on Affymetrix microarrays. Brains from 2-5 individual fish of the same sex were pooled and homogenized together, for a total of two biological replicate pools per sex per strain (16 microarrays total).
Project description:We performed microarray-based expression profiling on liver of male zebrafish exposed to 7 mg/L of nitrophenol for 8-96 h, to identify global transcriptional programs and biological pathways involved in chloroaniline-induced adaptive responses under in vivo environment. We analyzed 12 arrays of nitrophenol-treated adult male zebrafish liver and 12 arrays of control fish.
Project description:We performed microarray-based expression profiling on liver of male zebrafish exposed to 7 mg/L of nitrophenol for 8-96 h, to identify global transcriptional programs and biological pathways involved in chloroaniline-induced adaptive responses under in vivo environment. We analyzed 12 arrays of nitrophenol-treated adult female zebrafish liver and 12 arrays of control fish.
Project description:We performed microarray-based expression profiling on liver of male zebrafish exposed to 15ppm (~ 192 µM) of arsenic [As(V)] for 8-96 h, to identify global transcriptional programs and biological pathways involved in chloroaniline-induced adaptive responses under in vivo environment. We analyzed 12 arrays of chloroaniline-treated adult female zebrafish liver and 12 arrays of control fish.
Project description:We performed microarray-based expression profiling on liver of female zebrafish exposed to 30 µg/L of cadmium (II) chloride for 8-96 h, to identify global transcriptional programs and biological pathways involved in cadmium-induced adaptive responses under in vivo environment. We analyzed 10 arrays of Cadmium-treated adult female zebrafish liver and 12 arrays of control fish.
Project description:We performed microarray-based expression profiling on liver of male zebrafish exposed to 30 µg/L of cadmium (II) chloride for 8-96 h, to identify global transcriptional programs and biological pathways involved in cadmium-induced adaptive responses under in vivo environment. We analyzed 10 arrays of Cadmium-treated adult male zebrafish liver and 12 arrays of control fish.