GSE133194
Single-cell transcriptomics of microglia isolated from the hypothalamic/thalamic region of E15.5 brains under control and stressed conditions
Microglia are the resident phagocytic immune cells of the central nervous system (CNS). In the embryo, microglia invade the CNS during early embryogenesis and continue to colonize alongside neural development. Microglia also mature across development, altering their morphology and expression signatures from embryonic to adult stages, suggesting differing roles for microglia in the developing brain than in the adult. While microglia were originally thought to be resting immune cells responsible for cleaning up cellular debris, studies are now showing that microglia are highly dynamic cells involved in proper postnatal development of the brain and adult neuronal plasticity. Although reports suggest that microglia in the embryonic brain are responsive to maternal stress, causing changes in neural development that lead to autistic- and schizophrenic-like behaviours later in life, the actual effects of microglia on embryonic developmental programs are just emerging. Our lab studies the hypothalamus, a brain region that controls a variety of physiologies including energy balance, reproduction, and even parenting behaviors, and it is also responsive to maternal stress. Interestingly, my previous work has shown that that the hypothalamus is uniquely comprised of at least two distinct populations of microglia. The first group of microglia are ramified, and appear to be passively surveying the hypothalamic mantle. The second cluster of microglia is amoeboid and activated, and are located adjacent to the ventricular zone (VZ) where hypothalamic neural progenitor cells (NPCs) reside. Given that this second group of microglia is in direct contact with NPCs, we hypothesize that microglia located in the VZ of the hypothalamus play an important role during embryonic development to organize the neurons and neuronal connections required for proper hypothalamic function. In order to identify the expression profiles of microglia in the VZ as compared to microglia present in the mantle of the hypothalamus, we extracted microglia from the brains of CD1 embryos and employed single-cell RNAseq. Specifically, we micro-dissected out the hypothalamic/thalamic region of E15.5 brains, created a single-cell suspension that was stained with Cd11b-PE / Cd45-FITC to mark microglia, and then used FACS to extract microglia from our mixed cell suspension. We then performed single-cell RNAseq on the microglia to determine the transcriptomic phenotype of these two populations either in a control condition or following maternal stress, where pregnant dams were exposed to a mild cold stress for 30 min a day over 5 days. Overall design: To isolate microglia from the hypothalamic/thalamic region of the E15.5 control and E15.5 stressed (pregnant dams were exposed to a mild cold stress over 5 days), we generated a single-cell suspensions and stained with Cd11b-PE / Cd45-FITC. We FACS collected viable PE / FITC double positive cells at E15.5 from the hypothalamic/thalamic region of the brain. The sample was processed according to 10X Genomics ChromiumTM Single Cell v3 10X Gene expression solution Chip B as per the manufacturer's protocol. In brief, single cells were sorted into 100% FBS and partitioned into Gel Bead-In-EMulsions (GEMs) using 10xTM GemCodeTM Technology. This process lysed cells and enabled barcoded reverse transcription of RNA, generating full-length cDNA from poly-adenylated mRNA. DynaBeads® MyOneTM Silane magnetic beads were used to remove leftover biochemical reagents, then cDNA was amplified by PCR over 10 cycles. Quality control size gating was used to select cDNA amplicon size prior to library construction. Read 1 primer sequences were added to cDNA during GEM incubation. P5 primers, P7 primers, i7 sample index, and Read 2 primer sequences were added during library construction. Quality control and cDNA quantification was performed using Agilent High Sensitivity DNA Kit. We recovered 1,156 cells from E15.5 control brains (hypothalamic/thalamic tissue pooled from 10 embryos) and 1,467 cells from E15.5 stressed brains (hypothalamic/thalamic tissue pooled from 5 embryos), with an estimated doublet rate of ~3-4%. Based on this, we determined lane distributions for sequencing using Illumina HiSeq 4000 PE (75 bp paired-end reads) with a targeted sequencing depth of ~100,000 reads/cell.