Fig. 3
RNAscope reveals Gfap mRNA within neurons of the mPFC (A, B) Images of sections from the MC (A) and mPFC (B) following processing for RNAscope for Gfap mRNA (GFAPRNA, red) and immunohistochemistry for NeuN (green) and DAPI (blue) staining. Arrowheads indicate the presence of Gfap mRNA in NeuN + cells. (C) Quantification of the percentage of neurons expressing NeuN + cells reveal significantly more neurons in the mPFC are expressing Gfap mRNA. Unpaired t-test used for statistical analysis. n = 3 mice per group. Data represents mean ± SEM. (D) Quantification of the number of Gfap mRNA puncta per Gfap mRNA-expressing cell, revealing significantly more Gfap mRNA puncta in Gfap + Neurons of the mPFC. Mann–Whitney, nonparametric test was performed, n = 60 cells per group distributed over n = 3 mice per group. (E) Cortical and hippocampal single-cell RNAseq data was used to analyze the expression of 131 transcription factors with binding sites in the hGFAP promoter. Thirteen transcription factors are expressed in numerous cortical and/or hippocampal cell types in the mouse brain. Image was adapted from the Allan Brain Atlas Transcriptomics Explorer (https://celltypes.brain-map.org/rnaseq/mouse_ctx-hpf_10x). Examples of layer 2—layer 6 cortical neurons are highlighted with a dashed white line. (F) Transcription factor binding sites (green) of the thirteen cortically and/or hippocampally expressed transcription factors were mapped onto 15 TFBS within the hGFAP promoter (blue), revealing clustering of twelve of the fifteen TFBS at two loci in the promoter (160–216 bp, and 531–568 bp). CGE—Caudal ganglionic eminence neighborhood, MGE—medial ganglionic eminence neighborhood, Sst—Somatostatin subclass, NP—Cortex layer 5/6 near projecting (NP)/ Layer 6 corticothalamic (CT)/layer 6b (L6b)(NP/CT/L6b) neighborhood, CA3—Cornu Ammonis 3 subclass, V—Vascular and immune cells.
