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| Status |
Public on Mar 27, 2017 |
| Title |
Microglia self-renewal comprises context-dependent random or clonal expansion. |
| Organism |
Mus musculus |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
Microglia constitute a highly specialized network of tissue resident immune cells that is important for the control of tissue homeostasis and the resolution of virtually all diseases of the central nervous system (CNS). However, how this dissemination is established and maintained in vivo and its kinetics of these processes are poorly understood. Here we established a new multicolor fluorescence fate mapping system to monitor microglia dynamics during steady state and disease. Our findings that microglia establish a stable network over time albeit with regional differences and remarkably high turnover rates challenge their postulated longevity. Microglia self-renewal constitutes a random process without evidence of defined progenitor cells. Under pathological conditions this randomness shifts to clonal microglia expansion that is finally resolved by both cell apoptosis and egression for re-establishment of the stable microglia network. Our data reveal new insights on how microglia ensure their complex distribution throughout the healthy and diseased CNS .
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| Overall design |
Total RNA was column purified using the RNeasy Mini Kit (QIAGEN) from the ipsilateral and contralateral ventral pons (containing the facial nucleus, and parts of the gigantocellular reticular nucleus, intermediate reticular nucleus, parvicellular reticular nucleus, alpha, and spinal trigeminal nucleus, oral) of CX3CR1GFP/wt mice that underwent unilateral facial nerve axotomy at 8 weeks of age. A rodent brain matrix (RBM-2000C, ASI Instruments) was used to ensure consistent cutting of brain tissue. Total RNA from the contralateral ventral pons of un-operated 8-week-old CX3CR1GFP/wt was used as baseline control (Day 0 post nerve transection) for the analysis. Three replicates were used per time point (Day 0, 7, 14 and 30 post axotomy). Illumina HiSeq deep sequencing was carried out at a genomics core facility (Center of Excellence for Fluorescent Bioanalytics, KFB, University of Regensburg, Germany) for 50 bases single-read sequencing that generate about 30 million reads per sample. Data analysis was performed using Bioconductor (Huber et al., 2015). Sequences were aligned using the Rsubread aligner (Liao et al., 2013) with default settings using the Gencode mouse assembly (version M7) as reference. Gene counts were generated using the featureCounts program included in the Rsubread library after sequence alignment (Liao et al., 2013). Counts were filtered for subsequent analysis. The sum of all counts over all samples was calculated. Genes with a sum of counts of 0 were removed from the data. All genes at or below the 32nd percentile of the sum of gene counts were also excluded from the remaining genes. Differential gene expression was tested for using limma (Ritchie et al., 2015), which utilizes parts of the edgeR (Robinson et al., 2010) code for RNAseq analysis, as described in the limma documentation (Ritchie et al., 2015). Genes were recognized as differentially expressed when having a BH (Benjamini-Hochberg) adjusted P < 0.05. The most significantly differentially expressed genes were identified based on a log2 (fold change) ≥ 1.7 as suggested by the SEQC consortium (Consortium 2014). Heat maps were generated with the heatmap3 Bioconductor package (Zhao et al., 2014). Pathway and Functional analysis was performed using Ingenuity Pathway Analysis (IPA, Qiagen). Subclustering of gene expression profiling utilized the Short Time-series Expression Miner (STEM) (Ernst et al., 2005; Ernst and Bar-Joseph 2006).
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| Contributor(s) |
Tay T, Staszewski O |
| Citation(s) |
28414331 |
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| Submission date |
Mar 15, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Ori Staszewski |
| E-mail(s) |
ori.staszewski@uniklinik-freiburg.de
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| Organization name |
University Medical Center Freiburg
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| Department |
Institute of Neuropathology
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| Street address |
Breisacher Str. 64
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| City |
Freiburg |
| ZIP/Postal code |
D-79106 |
| Country |
Germany |
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| Platforms (1) |
| GPL15103 |
Illumina HiSeq 1000 (Mus musculus) |
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| Samples (21)
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| Relations |
| BioProject |
PRJNA315281 |
| SRA |
SRP071802 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE79252_fC_counts.txt.gz |
1.1 Mb |
(ftp)(http) |
TXT |
| GSE79252_voom_matrix.txt.gz |
2.5 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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