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Multiple oxygen tension environments reveal diverse patterns of transcriptional regulation in primary astrocytes.

ABSTRACT: The central nervous system normally functions at O(2) levels which would be regarded as hypoxic by most other tissues. However, most in vitro studies of neurons and astrocytes are conducted under hyperoxic conditions without consideration of O(2)-dependent cellular adaptation. We analyzed the reactivity of astrocytes to 1, 4 and 9% O(2) tensions compared to the cell culture standard of 20% O(2), to investigate their ability to sense and translate this O(2) information to transcriptional activity. Variance of ambient O(2) tension for rat astrocytes resulted in profound changes in ribosomal activity, cytoskeletal and energy-regulatory mechanisms and cytokine-related signaling. Clustering of transcriptional regulation patterns revealed four distinct response pattern groups that directionally pivoted around the 4% O(2) tension, or demonstrated coherent ascending/decreasing gene expression patterns in response to diverse oxygen tensions. Immune response and cell cycle/cancer-related signaling pathway transcriptomic subsets were significantly activated with increasing hypoxia, whilst hemostatic and cardiovascular signaling mechanisms were attenuated with increasing hypoxia. Our data indicate that variant O(2) tensions induce specific and physiologically-focused transcript regulation patterns that may underpin important physiological mechanisms that connect higher neurological activity to astrocytic function and ambient oxygen environments. These strongly defined patterns demonstrate a strong bias for physiological transcript programs to pivot around the 4% O(2) tension, while uni-modal programs that do not, appear more related to pathological actions. The functional interaction of these transcriptional 'programs' may serve to regulate the dynamic vascular responsivity of the central nervous system during periods of stress or heightened activity.

SUBMITTER: Chadwick W

PROVIDER: S-EPMC3124552 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Multiple oxygen tension environments reveal diverse patterns of transcriptional regulation in primary astrocytes.

Chadwick Wayne W Boyle John P JP Zhou Yu Y Wang Liyun L Park Sung-Soo SS Martin Bronwen B Wang Rui R Becker Kevin G KG Wood William H WH Zhang Yongqing Y Peers Chris C Maudsley Stuart S

PloS one 20110627 6

The central nervous system normally functions at O(2) levels which would be regarded as hypoxic by most other tissues. However, most in vitro studies of neurons and astrocytes are conducted under hyperoxic conditions without consideration of O(2)-dependent cellular adaptation. We analyzed the reactivity of astrocytes to 1, 4 and 9% O(2) tensions compared to the cell culture standard of 20% O(2), to investigate their ability to sense and translate this O(2) information to transcriptional activity ...[more]

PMID: 21738745

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Project description:The central nervous system normally functions at O2 levels which would be regarded as hypoxic by most other tissues. However, most in vitro studies of neurons and astrocytes are conducted under hyperoxic conditions without consideration of O2-dependent cellular adaptation. We analyzed the reactivity of astrocytes to 1, 4 and 9% O2 tensions compared to the cell culture standard of 20% O2, to investigate their ability to sense and translate this O2 information to transcriptional activity. Variance of ambient O2 tension for rat astrocytes resulted in profound changes in ribosomal activity, cytoskeletal and energy-regulatory mechanisms and cytokine-related signaling. Clustering of transcriptional regulation patterns revealed four distinct response pattern groups that directionally pivoted around the 4% O2 tension, or demonstrated coherent ascending/decreasing gene expression patterns in response to diverse oxygen tensions. Immune response and cell cycle/cancer-related signaling pathway transcriptomic subsets were significantly activated with increasing hypoxia, whilst hemostatic and cardiovascular signaling mechanisms were attenuated with increasing hypoxia. Our data indicate that variant O2 tensions induce specific and physiologically-focused transcript regulation patterns that may underpin important physiological mechanisms that connect higher neurological activity to astrocytic function and ambient oxygen environments. These strongly defined patterns demonstrate a strong bias for physiological transcript programs to pivot around the 4% O2 tension, while uni-modal programs that do not, appear more related to pathological actions. The functional interaction of these transcriptional ‘programs’ may serve to regulate the dynamic vascular responsivity of the central nervous system during periods of stress or heightened activity. Cerebral cortices were removed from 6-8-day-old Wistar rats, cortical astrocytes were isolated and these cells were maintained in a humidified incubator at 37°C (95% air; 5% CO2). This resulted in a culture of cortical astrocytes, as confirmed by visual inspection the following day and later by glial fibrillary acidic protein immunohistochemistry. Any cortical astrocyte culture that was not homogenous was disposed of and not used in this study. Culture medium was exchanged every 7 days and cells were grown in culture for up to 14 days. 24hr prior to experimentation cells were transferred to an hypoxic workstation equilibrated with either 1%, 4%, 9% or 21% O2 and 5% CO2, with the remaining percentage gas being N2. Once cortical astrocytes had reached approximately 90% confluence (75cm2 flask) they were subjected to hypoxia as above, washed with PBS, removed from the flask base with 0.05% trypsin-EDTA (Gibco) and then gently centrifuged (500xg). The cell pellet was re-suspended in PBS, centrifuged twice more to remove any traces of media, then triturated in 8-10 volumes of RNAlater (Applied Biosystems), frozen and stored at -80oC. Messenger RNA was amplified and labeled with biotin using the standard Illumina protocol (Illumina TotalPrep RNA Amplification Kit Ambion; Austin, TX, cat # IL1791), and hybridized to Illumina's Sentrix Rat Ref-12,v1 Expression BeadChips (Illumina, San Diego, CA). Three replicates from each treatment group (1%, 21%, 4%, and 9% O2) were hybridized and the data was extracted using the Illumina BeadStudio software(v3.4).

Project description:A broad array of endosymbionts radiate through host populations via vertical transmission, yet much remains unknown concerning the cellular basis, diversity, and routes underlying this transmission strategy. Here, we address these issues, by examining the cellular distributions of Wolbachia strains that diverged up to 50 million years ago in the oocytes of 18 divergent Drosophila species. This analysis revealed 3 Wolbachia distribution patterns: (1) a tight clustering at the posterior pole plasm (the site of germline formation); (2) a concentration at the posterior pole plasm, but with a significant bacteria population distributed throughout the oocyte; and (3) a distribution throughout the oocyte, with none or very few located at the posterior pole plasm. Examination of this latter class indicates Wolbachia accesses the posterior pole plasm during the interval between late oogenesis and the blastoderm formation. We also find that 1 Wolbachia strain in this class concentrates in the posterior somatic follicle cells that encompass the pole plasm of the developing oocyte. In contrast, strains in which Wolbachia concentrate at the posterior pole plasm generally exhibit no or few Wolbachia in the follicle cells associated with the pole plasm. Taken together, these studies suggest that for some Drosophila species, Wolbachia invade the germline from neighboring somatic follicle cells. Phylogenomic analysis indicates that closely related Wolbachia strains tend to exhibit similar patterns of posterior localization, suggesting that specific localization strategies are a function of Wolbachia-associated factors. Previous studies revealed that endosymbionts rely on 1 of 2 distinct routes of vertical transmission: continuous maintenance in the germline (germline-to-germline) or a more circuitous route via the soma (germline-to-soma-to-germline). Here, we provide compelling evidence that Wolbachia strains infecting Drosophila species maintain the diverse arrays of cellular mechanisms necessary for both of these distinct transmission routes. This characteristic may account for its ability to infect and spread globally through a vast range of host insect species.

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Multiple oxygen tension environments reveal diverse patterns of transcriptional regulation in primary astrocytes.

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Multiple oxygen tension environments reveal diverse patterns of transcriptional regulation in primary astrocytes.

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