Astrocytes and neurons share region-specific transcriptional signatures that confer regional identity to neuronal reprogramming

Abstract

Neural cell diversity is essential to endow distinct brain regions with specific functions. During development, progenitors within these regions are characterized by specific gene expression programs, contributing to the generation of diversity in postmitotic neurons and astrocytes. While the region-specific molecular diversity of neurons and astrocytes is increasingly understood, whether these cells share region-specific programs remains unknown. Here, we show that in the neocortex and thalamus, neurons and astrocytes express shared region-specific transcriptional and epigenetic signatures. These signatures not only distinguish cells across these two brain regions but are also detected across substructures within regions, such as distinct thalamic nuclei, where clonal analysis reveals the existence of common nucleus-specific progenitors for neurons and astrocytes. Consistent with their shared molecular signature, regional specificity is maintained following astrocyte-to-neuron reprogramming. A detailed understanding of these regional-specific signatures may thus inform strategies for future cell-based brain repair.

Fig. 1. Astrocytes show region-specific transcriptomic profiles.

(A) Schematic of the RNA-seq experiments for cortical and thalamic astrocytes. Astrocytes from P7 Gfap::Gfp mice were fluorescence-activated cell sorting (FACS)–purified and sequenced. Right, images showing the thalamus and cortex of a Gfap::Gfp mouse at P7. (B) Principal components analysis (PCA) of the transcriptomes of astrocytes (As) from the thalamus (Th), including dLG (n = 5 samples), VPM (n = 4), and MGv (n = 4), and cortex (Ctx, n = 4) at P7. (C) Heatmap of z score normalized regularized logarithm (Rlog) expression and unbiased clustering of significantly DEGs between thalamic (As-Th) and cortical astrocytes (As-Ctx). Each row represents a gene, the columns are biological replicates, and the color code represents the normalized expression for up-regulated genes in yellow versus down-regulated genes in purple. (D) MA plot displaying DEGs. Blue and light gray dots represent thalamic and cortical DEGs with their mean normalized counts, respectively. Dark gray dots represent genes that failed to give a significant result. (E) Enrichment plots from the GSEA of two specific GO terms related to the thalamic and cortical formation. (F) GO biological process (BP) enrichment analysis of significantly DEGs in thalamic and cortical astrocytes and associated gene networks. The size of every node (enriched term) represents the number of genes enriched and the color code (yellow, high expression; purple, low expression) corresponds to the log₂FC in DE analysis. In (A), scale bars, 400 μm.

Fig. 2. Astrocytes and neurons share region-specific transcriptome profiles.

(A) Schematic of the RNA-seq experiments for comparing thalamic and cortical neurons. Thalamic neurons were obtained from Gbx2-Cre::Tomato-floxed P0 mice and cortical neurons from publicly available datasets (20). Ns-Th included dLG (n = 4), VPM (n = 4), and MGv (n = 3), and Ns-Ctx (n = 6). (B) Venn diagram showing the genes that overlap between astrocytes (As) and neurons (Ns) in both the thalamus and cortex. Bar plots represent the percentage of the enriched genes shared between populations. (C) Heatmap showing overlapping genes between As and Ns in the thalamus and cortex. (D) Box plots showing expression levels of selected region-specific genes shared between neurons and astrocytes of the thalamus (top) or the cortex (bottom). TPM, transcripts per million. (E) Heatmap of the z score of average expression levels of DEGs at the single-cell level, identified by comparing cell types among different regions of origin (As-Th versus As-Ctx; Ns-Th versus Ns-Ctx) from publicly available data (23). (F) Comparison matrix of the number of shared specific gene lists between As and Ns datasets of every specific region. Color code according to significance of overlap. (G) Schematic of the main conclusion of the experiments. Data are plotted with box-and-whisker plots, which give the median, 25th and 75th percentiles, and range. Dots in (D) represent every single value.

Fig. 3. Sensory-modality thalamic astrocytes and neurons express common specific genes for every nucleus.

(A) Schematic of the RNA-seq experiments for comparing neurons and astrocytes from the sensory thalamic nuclei (dLG, VPM, and MGv) and main conclusion obtained. (B) PCA of transcriptomes from astrocytes (As) isolated from the distinct sensory-modality thalamic nuclei [dLG (n = 5), VPM (n = 4), and MGv (n = 4)] at P7. (C) PCA of transcriptomes of neurons (Ns) from the distinct sensory-modality thalamic nuclei [dLG (n = 4), VPM (n = 4), and MGv (n = 3)] at P0. (D) Left, comparison matrix of the number of shared specific gene lists between neurons and astrocytes datasets of every thalamic nuclei. Color code according to significance of overlap. Right, bar plots representing the percentage of gene overlap between As and Ns from each thalamic nucleus. (E) Heatmap showing the overlapping DEGs between As and Ns in each nucleus. Each column represents a biological replicate and the color code represents the z score normalized expression (up-regulated genes in yellow, down-regulated genes in purple). (F) Box plots showing expression levels of nuclei-specific shared genes between astrocytes and neurons in the distinct sensory-modality thalamic nuclei. TMP, transcripts per million. Data are plotted with box-and-whisker plots, which give the median, 25th and 75th percentiles, and range. Dots in (F) represent every single value. ***P < 0.0005.

Fig. 4. Clonally related astrocytes and neurons remain within the same nuclear boundaries.

(A) Experimental design for the analysis of astrocytic clones in the sensory nuclei (dLG, VPM, and MGv). A cocktail of integrative plasmids encoding six different fluorescent proteins under the Gfap promoter (GFAP-StarTrack) was electroporated in the third ventricle at E11.5. (B) Thalamic astrocytes labeled with the GFAP-StarTrack constructs at P8. Insets show the expression of each fluorescent reporter in a dLG astrocyte clone (white square). (C) Quantification of the dispersion of the clonally related astrocytes (n = 320 clones from five electroporated animals). (D) Experimental design for the study of neuronal and nonneuronal clonal cells with the UbC-StarTrack constructs. (E) Example of a neuron (white arrows) and two astrocytes (purple arrows) from the VPM coming from the same progenitor, thus sharing the same color code. (F) Three types of clones were analyzed clones based on their cell-type composition: mixed clones (containing neurons and nonneuronal cells), clones with neurons only, or clones with nonneuronal cells only (n = 4 electroporated animals). (G) Quantification of the dispersion of clonally related neuronal and nonneuronal cells from mixed clones, in the different thalamic sensory nuclei (n = 130 clones from four electroporated animals). (H) Schema representing the specificity in the nuclei-dependent localization of clonal cells. Cells coming from the same progenitor are colored with the same color. Note that most clonally related cells respect the nuclei segregation and only few cells are dispersed. Data are plotted with box-and-whisker plots, which give the median, 25th and 75th percentiles, and range. Scale bars, 100 μm. ns, not significant; *P < 0.05, **P < 0.005, and ***P < 0.0005.

Fig. 5. Astrocytes are reprogrammed into region-specific neurons.

(A) Experimental design for the in vivo reprogramming. Retrovirus encoding Neurog2 and Bcl2 or only Bcl2 were injected in the thalamus and cortex of P3 animals. (B) Immunofluorescence for thalamic and cortical markers in iNs reprogrammed from cortical or thalamic astrocytes in vivo. (C) Percentage of iNs expressing thalamic or cortical markers after reprogramming in vivo (n = 4 to 6 injected mice). (D) Experimental design for assessing the influence of the environment on the induced neurons identity. Isolated cortical or thalamic astrocytes were infected and then cocultured with thalamic or cortical astrocytes or neurons. (E) Immunostaining for the thalamic marker Rorα in cortical or thalamic iNs (RFP⁺/Tuj1⁺) in the different conditions. (F) Quantification of the percentage of iNs generated from cortical or thalamic astrocytes that express vGlut2, Rorα, Tbr1, or Ctip2 in control conditions or when mixed with astrocytes or neurons from the thalamus or the cortex, respectively (n = 6 to 14 independent cultures per condition). (G) Left, experimental design. Astrocytes from dLG, VPM, and MGv were isolated, cultured, and infected with Neurog2 retrovirus. Right, image of an iN from dLG astrocytes at 10 days post infection (dpi). (H) Reverse transcription (RT)–qPCR showing the expression of specific neuronal genes in the iNs after 10 dpi (n = 10 to 14 independent cultures per condition). Data are plotted with box-and-whisker plots, which give the median, 25th and 75th percentiles, and range. Dots in (C) represent every single value. Scale bars, 100 μm in (B) (insets, 25 μm) and 25 μm in (E) and (G). *P < 0.05, **P < 0.005, and ***P < 0.0005.

Fig. 6. Poised epigenetic state of region-specific gene expression in astrocytes.

(A) Experimental design. Astrocytes from the thalamus and cortex were cultured and infected with either Neurog2-i-Gfp retrovirus alone or both Neurog2-i-Gfp and Gbx2-i-DsRed. After 2 days, astrocytes were FACS-purified based on the presence of the reporter protein in three groups (noninfected, infected only with Neurog2, or infected with Neurog2 and Gbx2). (B) Quantification of specific gene expression by RT-qPCR in astrocytes in basal conditions and 2 days after the overexpression of Neurog2 alone or with a thalamic-specific gene (Gbx2) (n = 6 to 14 independent cultures per condition). Data are means ± SEM. (C) Schematic conclusion of the experiment. (D) Astrocytes from the thalamus and cortex were isolated, and the expression levels of some region-specific genes were assessed by RT-qPCR or ChIP-qPCR. Box-and-whisker plots represent the basal expression levels of the studied genes in thalamic and cortical astrocytes (left axis), and dots show the means ± SEM of the epigenetic state of the promoter of those genes, in terms of the presence of two histone marks, H3K4me3 and H3K27me3 (right axis) (n = 12 to 23 independent ChIP samples per condition). The red dashed line indicates the point where H3K4me3 and H3K27me3 marks are present at the same level. (E) Box-and-whisker plots showing the H3K4me3 and H3K27me3 ratio in vitro (left axis) (n = 14 to 18 independent ChIP samples per condition) and the basal in vivo expression of neuronal specific genes in thalamic astrocytes from each nucleus (right axis). ns, not significant; *P < 0.05, **P < 0.005, and ***P < 0.0005.