Differential gene expression in mouse retina related to regional differences in vulnerability to hyperoxia
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ABSTRACT: Purpose: In the C57BL/6J mouse retina, hyperoxia-induced degeneration of photoreceptors shows strong regional variation, beginning at a locus ~0.5mm inferior to the optic disc. To identify gene expression differences that might underlie this variability in vulnerability, we have used microarray techniques to describe regional (superior-inferior) variations in gene expression in the retina. Methods: Young adult C57BL/6J mice raised in dim cyclic illumination (12h 5lux, 12h darkness) were exposed to hyperoxia (75% oxygen for 2w). Retinas were collected from hyperoxia-exposed and control animals without fixation, and divided into superior and inferior halves. RNA was extracted from each sample, purified and hybridised to mouse 1.0 ST arrays (Affymetrix). The consistency of the microarray results was assessed with quantitative PCR for selected genes. Expression data were analysed to identify genes and ncRNAs whose differential expression between superior and inferior retina could be associated with relative vulnerability to hyperoxia.
Project description:Purpose: In the C57BL/6J mouse retina, hyperoxia-induced degeneration of photoreceptors shows strong regional variation, beginning at a locus ~0.5mm inferior to the optic disc. To identify gene expression differences that might underlie this variability in vulnerability, we have used microarray techniques to describe regional (superior-inferior) variations in gene expression in the retina.
Project description:PURPOSE: Hyperoxia is toxic to photoreceptors, and this toxicity may be important in the progress of retinal dystrophies. This microarray study examines gene expression induced in the C57BL/6J mouse retina by hyperoxia over the 14-day period during which photoreceptors first resist, then succumb to, hyperoxia. METHODS: Young adult C57BL/6J mice were exposed to hyperoxia (75% oxygen) for up to 14 days. On day 0 (control), day 3, day 7, and day 14, retinal RNA was extracted and processed on Affymetrix GeneChip Mouse Genome 430 2.0 arrays. Microarray data were analyzed using GCOS Version 1.4 and GeneSpring Version 7.3.1. RESULTS: The overall numbers of hyperoxia-regulated genes increased monotonically with exposure. Within that increase, however, a distinctive temporal pattern was apparent. At 3 days exposure, there was prominent upregulation of genes associated with neuroprotection. By day 14, these early-responsive genes were downregulated, and genes related to cell death were strongly expressed. At day 7, the regulation of these genes was mixed, indicating a possible transition period from stability at day 3 to degeneration at day 14. CONCLUSIONS: Microarray analysis of the response of the retina to prolonged hyperoxia demonstrated a temporal pattern involving early neuroprotection and later cell death, and provided insight into the mechanisms involved in the two phases of response. As hyperoxia is a consistent feature of the late stages of photoreceptor degenerations, understanding the mechanisms of oxygen toxicity may be important therapeutically. 4 timepoints in total, 0d (control), 3d, 7d and 14d and a replicate per timepoint. Total of 8 chips.
Project description:<p>Proper spatial differentiation of retinal cell types is necessary for normal human vision. Many retinal diseases, such as Best disease and male germ cell associated kinase (MAK)-associated retinitis pigmentosa, preferentially affect distinct topographic regions of the retina. While much is known about the distribution of cell types in the retina, the distribution of molecular components across the posterior pole of the eye has not been well-studied. To investigate regional difference in molecular composition of ocular tissues, we assessed differential gene expression across the temporal, macular, and nasal retina and retinal pigment epithelium (RPE)/choroid of human eyes using RNA-Seq. RNA from temporal, macular, and nasal retina and RPE/choroid from four human donor eyes was extracted, poly-A selected, fragmented, and sequenced as 100 bp read pairs. Digital read files were mapped to the human genome and analyzed for differential expression using the Tuxedo software suite. Retina and RPE/choroid samples were clearly distinguishable at the transcriptome level. Numerous transcription factors were differentially expressed between regions of the retina and RPE/choroid. Photoreceptor-specific genes were enriched in the peripheral samples, while ganglion cell and amacrine cell genes were enriched in the macula. Within the RPE/choroid, RPE-specific genes were upregulated at the periphery while endothelium associated genes were upregulated in the macula. Consistent with previous studies, BEST1 expression was lower in macular than extramacular regions. The MAK gene was expressed at lower levels in macula than in extramacular regions, but did not exhibit a significant difference between nasal and temporal retina. The regional molecular distinction is greatest between macula and periphery and decreases between different peripheral regions within a tissue. Datasets such as these can be used to prioritize candidate genes for possible involvement in retinal diseases with regional phenotypes. </p> <p>Reprinted from <a href="https://www.ncbi.nlm.nih.gov/pubmed/?term=25446321">Whitmore, S.S., Wagner, A.H., DeLuca, A.P., Drack, A.V., Stone, E.M., Tucker, B.A., Zeng, S., Braun, T.A., Mullins, R.F., Scheetz, T.E., 2014. Transcriptomic analysis across nasal, temporal, and macular regions of human neural retina and RPE/choroid by RNA-Seq. Experimental Eye Research</a>. Used under <a href="http://creativecommons.org/licenses/by-nc-sa/3.0/">Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported </a>license.</p> <p>Additional RNA sequencing was performed on temporal, macular, nasal, inferior, and superior retina from a fifth subject and is included in this dataset. See original publication for details.</p>
Project description:To detect sex-specific differences in gene expression in a model of hyperoxic lung injury in adult C56BL/6J mice. In this dataset, we include the expression data obtained from lungs from 8-10 week old male and female WT C57BL/6J mice exposed to hyperoxia for 48 hours amd room air controls.These data were used to determine differences in transcriptome between male and female mice after hyperoxia exposure 12 total samples were analyzed. We generated the following pairwise comparisons: 5 comparisons (M/F: male/female, A/O: air/oxygen): 1) M_O - M_A, 2) F_O - F_A, 3) M_O - F_O, 4) M_A - F_A, 5) (M_O - M_A) - (F_O - F_A). Genes with an FDR≤5% and a fold-change ≥1.4 were selected.
Project description:Background: Nrf2 is an essential cytoprotective transcription factor. However, association of Nrf2 in organ development and neonatal disease is rarely examined. Hyperoxia exposure to newborn rodents generates pulmonary phenotypes which resemble bronchopulmonary dysplasia (BPD) of prematurity. Methods: To investigate the role of Nrf2 in lung maturation and BPD pathogenesis, Nrf2-deficient (Nrf2-/-) and wild-type (Nrf2+/+) neonates were exposed to air or hyperoxia (O2). Transcriptome analysis determined Nrf2-directed mechanisms in premature lung. Lung injury was assessed by bronchoalveolar lavage analysis and histopathology. Results: In Nrf2-/- neonates, basal expression of cell cycle machinery, redox balance, and lipid/carbohydrate metabolism genes were suppressed while immunity genes were overexpressed compared to Nrf2+/+ pups. O2-induced mortality and pulmonary inflammation/injury were significantly higher in Nrf2-/- than in Nrf2+/+. Lung DNA lesion and oxidation were greater in Nrf2-/- than in Nrf2+/+, constitutively and after O2. Nrf2-dependent genes modulated cellular growth/proliferation, defense, immunity, and lipid metabolism against hyperoxia. Bioinformatic elucidation of Nrf2 binding motifs and augmented O2-induced inflammation in genetically deficient neonates validated Gpx2 and Marco as Nrf2 effectors. Conclusion: Overall, Nrf2 in underdeveloped lungs orchestrated cell cycle, morphogenesis, and immunity as well as cellular defense constitutively and under oxidant stress. Results provide putative molecular mechanisms of Nrf2-directed lung alveolarization and BPD of prematurity. PARALLEL study design with 42 samples comparing 14 groups of age (P1 to P4 corresponding to day 0 to day 3 animals), gene, and exposure: (4 groups Nrf+/+ wild type P1-P4 air exposure) (4 groups Nrf -/- knockout P1-P4 air exposure), (3 groups Nrf+/+ wild type P2-P4 with 100 percent O2 (hyperoxia exposure) and 3 groupsNrf -/- knockout P2-P4 with 100 percent O2 (hyperoxia exposure)) Biological replicates: 3 per group
Project description:Hyperoxia has a potential to alter DNA methylation status. We assessed the effect of long term hyperoxia in mouse lung tissue. A total of 24 mice were randomized to hyperoxia (85% O2; 12 animals) or normoxia (21% O2; 12 animals) for 14 days continued with normoxia conditions for all animals for the subsequent 14 days. All mice had free access to food and water and were kept under standard conditions in A-Chambers (O2 – monitor ProOX110, CO2 – monitor ProCO2 P120, BioSpherix). The animals were sacrificed on day 28. Lung tissue was harvested on day 28 after euthanasia with a zolazepam/tiletamine/xylazine/fentanyl cocktail. Tissues samples were snap-frozen in liquid nitrogen immediately after cessation of circulation for the subsequent analysis. Subsequently, DNA methylation profiles in lung tissue were compared by means of methylation microarrays between both groups. We used the Genomic Workbench software (Agilent) to assess the mean methylation status of each DNA fragment (array probe) in each group. We first calculated the normalised, combined Z-scores, representing summation of the left and right Gaussian Z-scores and reflecting the location of a probe log-ratio value in relation to the Gaussian distribution of probes. A strong positive value of the combined Z-score means that a given probe is methylated and strong negative value means that it is unmethylated. Next, we compared the mean combined Z-scores (average methylation patterns) of the probes between hyperoxia and normoxia groups.
Project description:We aimed to identify differential expression of microRNAs between superior and inferior spikelets by using a deep sequencing approach developed by Solexa (Illumina). Two small RNA libraries were constructed from superior and inferior spikelets at 18 days after fertilization, and more than nine million small RNA sequence reads were generated for each library. Totals of 351 and 312 known miRNAs were obtained from the superior and inferior spikelets, respectively. Analysis of the expression profiles of these miRNAs showed that 189 miRNAs were differentially expressed between superior spikelets and inferior spikelets. In addition, 43 novel miRNAs were identified mostly by the accumulations of miRNA*s were also expressed differentially. Further analysis shows that these miRNAs may individually participate in regulating hormone metabolism, carbohydrate metabolic pathways, and cell division during rice grain development. These results indicate that slow grain filling and low grain weight of rice inferior spikelets probably relation to the expression and function differences between superior and inferior spikelet miRNAs. Examination of 2 different small RNA expression profilings in superior and inferior spikelets at 18 days after fertilization.
Project description:Gene expression changes in response to aging, hyperoxia, hydrogen peroxide, ionizing radiation, and heat stress were compared using microarrays. While aging shared features with each stress, aging was more similar to the stresses most associated with oxidative stress (hydrogen peroxide, hyperoxia, ionizing radiation) than to heat stress. Aging is associated with down-regulation of numerous mitochondrial genes, including electron-transport-chain (ETC) genes and mitochondrial metabolism genes, and a sub-set of these changes was also observed upon hydrogen peroxide stress and ionizing radiation stress. Aging shared the largest number of gene expression changes with hyperoxia. The extensive down-regulation of mitochondrial and ETC genes during aging is consistent with an aging-associated failure in mitochondrial maintenance, which may underlie the oxidative stress-like and proteotoxic stress-like responses observed during aging. Thirty five sample of RNA including Stress Controls (4 replicates), Heat Stress (3 replicates), Ionizing Radiation (4 replicates), Sugar (4 replicates), Hydrogen Peroxide (4 replicates), Young Controls (6 replicates) , Hyperoxia (6 replicates) and Old (4 replicates) adult Drosophila were analysed by Affymetrix microarrays. Stress Controls were used as controls for Heat Stress, Ionizing Radiation, Sugar and Hydrogen Peroxide samples. Young Controls were used as controls for Hyperoxia and Old samples. All flies were male progeny of a cross between Oregon-R wildtype and transgenic strain w[1118];P{w+ rtTA}(3)[E2]/ TM3. Flies lacking the balancer but bearing the transgene were used.
Project description:This experiment aimed to investigate the differences in the transcriptional profile of the neurogenic niche regions of mouse pups that were raised in room air verses mouse pups that were exposed to hyperoxia during the neonatal period. Pups were housed in room air or hyperoxia (85% O2) from P0 to P14. Brain tissue (the subventricular zone and the hippocampus) was collected at P14 and 12 months of age. RNA was extracted from the brain tissue and the microarray labelling, hybridization, and scanning was conducted by the Génome Québec Innovation Centre (Montréal, Canada).