Agouti overexpression in a transgenic model regulates integrity, permeability and electrogenic amino acid transport in zebrafish intestine
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PROVIDER: PRJNA852246 | ENA |
REPOSITORIES: ENA
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Project description:Overexpression of asip1 in transgenic zebrafish disrupts dorsoventral pigment pattern in addition to increasing food intake levels and linear growth. A higher feed intake is unnecessary in transgenic fish to enable larger and heavier growth. A plausible explanation may rely on the enhanced feeding efficiency mediated by improved nutrient absorption in transgenic animals. To test this hypothesis, wide scope transcriptomic techniques were used to pipe the potential pathways involved in the enhanced nutrient absorption and intestinal epithelium permeability/integrity, in addition, the electrogenic capacity for amino acid transport was analysed. Transcriptomic results suggested that amino acid, monocarboxylates, ionic and vitamin transmembrane transport was substantially modified. Enrichment analysis also revealed an inhibition of intestinal lipid metabolism and down-regulation of KEGG pathways related to membrane integrity suggesting an augmented intestinal laxity that may result in enhanced paracellular transport. Electrophysiological experiments carried out in Ussing chambers show that asip1 overexpression decrease membrane tissue resistance (Rt), indicating a modification of the intestinal barrier function in ASIP1 transgenic animals. Similarly, paracellular permeability was higher in transgenic zebrafish. Both the decrease in Rt and the increase in permeability point to an ASIP1-dependent decrease in the tissue barrier function. Electrogenic amino acid transport was also enhanced in transgenic animals providing a strong indication that ASIP1 fish can extract more amino acids from their diet at similar feeding levels. Both transcriptomic and electrophysiological results suggest that asip1-overexpressing zebrafish display improved nutrient absorption and by extension a higher feed efficiency which explains enhanced growth in the absence of augmented food intake. The enhanced growth of ASIP1 zebrafish potentially mediated by improved nutrient uptake and feed efficiency suggests that the melanocortin system, specifically asip1 overexpression, is a potential target for the development of genetically engineered fish displaying improved performance and no differential lipid accumulation.
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Project description:The mouse agouti gene is normally expressed in the skin and regulates coat color pigmentation. Dominant regulatory mutations in the agouti gene, such as viable-yellow (Avy), cause ubiquitous over-expression of the wild-type gene product in every tissue in the body. As a result of this altered agouti expression, viable-yellow mice have solid yellow hair, they are obese and diabetic, and they have increased susceptibility to spontaneous and chemically induced tumors in a wide variety of tissues (e.g., skin, lung, liver, mammary gland and urinary bladder). Additionally, liver-specific expression of the agouti gene was shown to promote hepatocellular carcinogenesis, even in the absence of obesity and diabetes. Our lab recently extended these findings to show that the agouti gene also promotes skin cancer in the absence of obesity and diabetes. In K14-Agouti transgenic mice, the wild-type agouti gene is over-expressed in the skin under the regulatory control of the keratin 14 (K14) promoter. Over-expression of agouti in the skin results in yellow coat color without the obesity and diabetes. In two-stage skin carcinogenesis experiments the agouti protein was shown to act as a tumor promoter since it promotes the development of skin tumors in the K14-Agouti transgenic mice, but only after the skin was initiated with a single dose of 7, 12-dimethylbenz[a]anthrance (DMBA). These experiments set the stage for determining the mechanism of action of agouti protein in tumor promotion, which remains completely uncharacterized. As a first step in determining the role of agouti in skin cancer, we used cDNA microarray and quantitative real time polymerase chain reaction (QRT-PCR) analysis to identify and validate a set of differentially expressed genes in the skin of K14-Agouti transgenic mice at the promotion stage of carcinogenesis. Keywords: promotion stage of carcinogenesis, biological replicates,
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Project description:Twenty transgenic fish overexpressing agouti-signaling protein (ASIP, Ceinos et al., Pigment Cell and Melanoma Research 28:196-209, 2015) and wild type (WT) zebrafish were placed in 6-liter tanks and acclimated during 30 days prior to sampling to minimize physiological differences. Animals were sacrificed and the brain+pituitary and the skin dissected under the stereo microscope, placed in trizol and stored at -80C until RNA extraction. Six biological replicates (3 females and 3 males) of each tissue (skin and brain) of each group (ASIP vs WT) were analyzed in a total of 6 slides containing 4 microarrays in the 4 × 44 k format. Global gene expression analyses were performed to search for both sex-dependent and transgene-dependent effects as well as the interaction between both factors (sex x phenotype) on the brain+pituitary and skin transcriptome. Wild type (Tu) or transgenic zebrafish overexpression agouti-signaling protein (ASIP) were reared in similar conditions. Brain+pituitary and skin were sampled from 6 ASIP (3 males + 3 females) and WT (3 males + 3 females) zebrafish. Samples were individually hybridized in a total of 6 slides containing 4 microarrays in the 4 × 44 k format. Microarrays included 60-mer probes to the unique transcripts from Ensembl and Unigene which were annotated by functional categories of GO and pathways of KEGG with bioinformatics package STARS
2015-09-11 | E-GEOD-72908 | biostudies-arrayexpress
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Project description:The aim of this study was to examine the hippocampus, frontal cortex and dorsal raphe of Dark Agouti rats with gene expression arrays (Illumina RatRef bead arrays) looking for possible mechanisms and new candidates contributing to the effects of a single dose of MDMA (15 mg/kg) 3 weeks earlier or venlafaxine (40 mg/kg) administration for 3 weeks continously. We also examined the possible interaction between the single dose MDMA treatment and the continous venlafaxine treatment described above. With the present study we tried to evaluate the effects of a single, neurotoxic dose of 3,4-methylenedioxymethamphetamine (MDMA) and/or 3 weeks long venlafaxine (VLX) treatment in the dorsal raphe, frontal cortical or hippocampal neurons in Dark Agouti rats. For the analysis of MDMA long-term effects rats were treated once with 15 mg/kg MDMA i.p. in a volume of 1 ml/kg 3 weeks prior to sacrificing the animals. In the case of VLX, the animals were administered 40 mg/kg of the drug for 3 weeks via Alzet 2001 minipumps inserted under the skin of the rats. In the third group rats received the mentioned treatment protocols simultaneously. Vehicle and sham-operated controls were used for comparison. For microarray analysis all groups consisted of 8 animals. Every two randomly selected samples from the same brain regions were pooled resulting in 4 pooled samples per region and treatment. From the total 48 pooled samples 2 were excluded because of quality control reasons. For RNA extraction Trizol reagent was used and microarray analysis was done by Service XS (Netherlands) according to Illumina protocols (San Diego, CA, USA). Results were validated with Fluidigm GEx (San Francisco, CA, USA) arrays using TaqMan Gene Expression (Carlsbad, CA, USA) assays. In this study no replicates were used.
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2014-01-01 | E-GEOD-43786 | biostudies-arrayexpress
Project description:Vascular permeability is frequently associated with inflammation and it is triggered by chemokines and by a cohort of secreted permeability factors, such as VEGF. In contrast, here we showed that the physiological vascular permeability that precedes implantation is directly controlled by progesterone receptor (PR) and it is independent of VEGF. Both global and endothelial-specific deletion of PR block physiological vascular permeability in the uterus while misexpression of PR in the endothelium of other organs results in ectopic vascular leakage. Integration of genome-wide transcriptional profile of endothelium and ChIP-sequencing revealed that PR induces a NR4A1 (Nur77/TR3) specific transcriptional program that broadly regulates vascular permeability in response to progesterone. This program triggers concurrent suppression of several junctional proteins and leads to an effective, timely and venule-specific regulation of vascular barrier function. Silencing NR4A1 blocks PR-mediated permeability responses indicating a direct link between PR and NR4A1. These results reveal a previously unknown function for progesterone receptor on endothelial cell biology with consequences to physiological vascular permeability and implications to the clinical use of progestins and anti-progestins on blood vessel integrity. Examination of PR target genes in human umbilical vein endothelial cells (HUVECs) using RNA-seq (PR infected only -PR only and PR infected followed by ligand treatment-PR+P)
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