Fig. 4: Transcriptomic analysis of Gad1 expression in NG2 glia.
From: NG2 glia-derived GABA release tunes inhibitory synapses and contributes to stress-induced anxiety
a The gene expression levels with Log2 (FPKM) reveal a transcriptomic profile related to GABA synthetic processes and degradation, clathrin-sculpted GABA transport vesicle membrane, GABA transport, secretion, and GABA signaling pathway. Note that Gad1 gene (GAD67) is relatively highly expressed in NG2 glia isolated from 3 Pdgfrα-creERTM;mGFP mice brains at postnatal 3–4 weeks. b Representative images of RNAscope with post-immunohistochemistry reveal a colocalization of Gad1 mRNA but not Slc17a7 mRNA with an individual GFP-labeled NG2 glial cell. The colocalization image in the white dotted box is magnified as indicated with arrows. The image data are repeated from 3 mice at postnatal 4–6 weeks. Scale bars, 20 μm. c Representative images and bar graph summary showing single-cell RT-PCR analysis of Gad1 and Gad2 mRNA expression in NG2 glia with interneuron as its control from hippocampal CA1 region of Pdgfrα-creERTM;mGFP mice at postnatal 3–4 weeks. Note 12 out of 19 GFP (+) cells showed distinct Gad1 but not Gad2 expression in NG2 glia. d Representative images of western blot and bar graph summary showing GAD67 protein expression in the purified NG2 glia by FACS from 3 Pdgfrα-creERTM;mGFP mice brain at postnatal 3–4 weeks. GAD67 expression in NG2 glia is 17.81 ± 4.76% (n = 3) relative to hippocampal tissue (n = 4). MOLT-4 cells as a negative control (n = 3). e Single-cell RNA-sequencing analysis shows a t-distributed stochastic neighbor embedding (t-SNE) plot depicting the cell-type identity of total 4014 EGFP (+) cells by FACS from a Pdgfrα-creERTM;mGFP mouse hippocampus at postnatal 3 weeks. Six clusters identified on the basis of the expression of well-established marker genes are shown in the right graph. f t-SNE maps visualizing single-cell gene expression of Gad1 and Gad2 within six clusters. Note that Gad1 but not Gad2 mainly distributes in the OPC subclusters 1 and 2. Bar graph summarizes the percentage of Gad1 expression within each cluster cells. Data are presented as mean values ± SEM and error bar represents SEM.
