Fig. 4: Spatial transcriptomic analysis reveals reversal of disease-associated genetic changes following microglial replacement in SD mice.
a Image of WT, Hexb-/-, bone marrow transplant (BMT)-treated Hexb-/-, and BMT + colony-stimulating factor 1 receptor inhibitor (CSF1Ri)-treated Hexb-/- groups (n = 3/group) imaged for cell segmentation markers histone (green), DAPI (gray), and GFAP (magenta). b Uniform Manifold Approximation and Projection (UMAP) of 389,585 cells across 12 brains. c Violin plot of Hexb transcript counts in broad cell types demonstrating myeloid-specific Hexb expression. d Comparison matrix scatterplot of average difference in significant (padj < 0.05) differentially expressed genes (DEGs) in inhibitory neurons, excitatory neurons, oligodendrocytes, astrocytes, and myeloid cells between Hexb-/- vs. WT and Hexb-/- BMT + CSF1Ri vs. Hexb-/-. The plot shows inversely correlated genes (yellow), directly correlated genes (blue), a linear regression line, and error bands (gray) representing a 95% confidence interval. Differential gene expression analysis per cell type between groups was performed on scaled expression data using Model-based Analysis of Single-cell Transcriptomics (MAST) to calculate average difference, defined as the difference in log-scaled average expression between two groups for each broad cell type. e The monocyte/macrophage subcluster (black) in Hexb-/- BMT and Hexb-/- BMT + CSF1Ri brains plotted in XY space. f Hexb-expressing cells (blue) plotted in XY space in WT, Hexb-/-, Hexb-/- BMT, and Hexb-/- BMT + CSF1Ri brains. Points sized by Hexb expression: cells with 0 transcripts not plotted, cells with 1 transcript plotted at point size=0.001, cells with 2 transcripts plotted at point size = 0.15, and cells with 3 + transcripts plotted at point size = 0.3. g Subclusters in XY space colored by DEG score calculated in comparison to WT controls in representative Hexb-/- BMT and Hexb-/- BMT + CSF1Ri brains. DEG score was calculated using the DEGs from treatment condition pairs in each subcluster by summing the absolute value of the log2 fold change values for all significant (padj< 0.05) DEGs identified between WT control and Hexb-/- BMT or Hexb-/- BMT + CSF1Ri. Cell clusters with higher DEG scores colored with darker shades to visually represent the degree of difference in XY space. h Dot plot representing pseudo-bulked expression values across groups in genes related to monocyte/macrophage identity, myeloid cell activation, and apoptosis and/or cellular stress in excitatory neurons, inhibitory neurons, and oligodendrocytes.
