Microglia are the resident immune cells of the central nervous system (CNS) and are derived from yolk sac progenitors. In the embryo, microglia invade the CNS starting at embryonic day 9.5 (E9.5) 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 recent reports have demonstrated 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 uniquely is 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 as we would expect. 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 used Cx3cr1-Cre; Rosa26tdTomato mice to mark microglia. We then micro-dissected the hypothalamic/thalamic region of the E15.5 brains and used flow cytometry to extract tdTomato+ microglia. We then performed single-cell RNAseq on the microglia to determine the transcriptomic phenotype of these two populations.
Overall design: To isolate microglia from the hypothalamic/thalamic region of the E15.5, we generated Cx3cr1Cre:tdTomato+ve dams that were treated with tamoxifen while pregnant (E10.5-E11.5). We FACS collected viable Cx3cr1Cre:tdTomato+ve cells at E15.5 from the hypothalamic/thalamic region of the brain. The sample was processed according to 10X Genomics ChromiumTM Single Cell 3’ Reagent Guidelines v2 Chemistry as per the manufacturer’s protocol. In brief, single cells were sorted into 0.1% BSA–PBS 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. Sequencing was performed first using Illumina MiSeq SR50 to approximate the number of recovered cells in each sample. We recovered 375 cells, with an estimated doublet rate of ~0.4%. 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 ~115,000 reads/cell.
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