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Deep single-cell RNAseq of microglia and brain myeloid cells from various brain regions and developmental stages

ABSTRACT: We generated single-cell RNAseq profiles of microglia and brain myeloid cells (1922 total; 1816 cells passed quality control) from different developmental stages (E14.5, P7 and P60) to study their heterogeneity. Cells were isolated from either the whole developing brain (for the E14.5 stage) or six separate regions (for P7 and P60 stages): Cortex (CTX), Cerebellum (CB), Hippocampus (HIP), Striatum (STR), Olfactory bulb (OB), Choroid plexus (CP). Single cells were FACS index sorted followed by Smart-seq2 library preparation and Illumina Nextseq (sequence depth > 1 million per cell). All 1816 cells were grouped into 15 clusters using Seurat package (Macosko, Basu, Satija et al. Cell. 2015), and manually annotated based on gene expression signatures and meta data. We found that the majority of adult microglia expressing homeostatic genes are remarkably similar in transcriptomes, regardless of brain region. By contrast, postnatal microglia represent a more heterogeneous population. We discovered a proliferative region-associated microglia (PAM) subset, mainly found in developing white matter, that share a characteristic gene signature with degenerative disease-associated microglia (DAM). Such PAM have amoeboid morphology, are metabolically active, and phagocytose newly formed oligodendrocytes. This scRNA-seq atlas will be a valuable resource for dissecting innate immune functions in health and disease.

ORGANISM(S): Mus musculus

PROVIDER: GSE123025 | GEO | 2018/12/31

REPOSITORIES: GEO

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Project description:Microglia, the resident immune cells of the central nervous system (CNS), have two distinct phenotypes in the developing brain: amoeboid form, known to be amoeboid microglial cells (AMC) and ramified form, known to be ramified microglial cells (RMC) alongside several intermediate forms. The AMC are characterized by being proliferative, phagocytic and migratory whereas the RMC are quiescent and exhibit a slow turnover rate. The AMC transform into RMC with advancing age, and this transformation is indicative of the gradual shift in the microglial functions. Both AMC and RMC respond to CNS inflammation, and they become hypertrophic when they are activated by trauma, infection or neurodegenerative stimuli. The molecular mechanisms and functional significance of morphological transformation of microglia during normal development and in disease conditions is not clear. It is hypothesized that AMC and RMC are functionally regulated by a specific set of genes encoding various signaling molecules and transcription factors. To address this, we carried out cDNA microarray analysis using lectin-labeled AMC and RMC isolated from frozen tissue sections of the corpus callosum of 5-day and 4-week old rat brain respectively, by laser capture microdissection (LCM). The global gene expression profiles of both microglial phenotypes were compared and the differentially expressed genes in AMC and RMC were clustered based on their functional annotations. This genome wide comparative analysis helps in identifying genes that are specific to AMC and RMC. The novel and specific molecules identified in both microglial phenotypes can be targeted for therapeutic purposes in developing and adult brain diseases. We used microarrays to identify the genes specific to amoeboid and ramified microglia. RNA was isolated from the laser-captured amoeboid and ramified microglia from the corpus callosum of 5-day and 4-week old rat brain. The RNA was hybridised onto Affymetrix Rat 230 2.0 array.

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Deep single-cell RNAseq of microglia and brain myeloid cells from various brain regions and developmental stages

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