Fig. 6: Experimental validation of Atoh1 spatial enhancer code in e11 mouse embryos.
From: Divergent combinations of enhancers encode spatial gene expression
Spatial mapping of Atoh1 gene activity scores (a) in the spinal cord and hindbrain in e11 mouse embryos (b), where spatial-ATAC-seq data were from a published study12. c Fluorescence in situ hybridization (FISH) images depicting Atoh1 expression at e11 mouse embryos. The experiment was repeated independently 3 times with similar results. Scale bars, 500 µm. d Heatmap showing the accessibility levels of Atoh1 enhancers. The spatial specificity scores of Atoh1 enhancers in the e11 spinal cord and hindbrain were labeled on the heatmap. e Genome tracks showing the chromatin accessibility, peak coordinates, gene activity sores (left), and gene tracks (right) for the Atoh1 locus in the spinal cord and hindbrain of e11 mouse. Spatial mapping of denoised chromatin accessibility of four Atoh1 enhancers (E0, E1, E2, and E3) in the e11 mouse spinal cord and hindbrain. f Whole-mount X-gal staining of E0 (top), E2 (middle), and E3 (bottom) reporter embryos at e11. The blue signal displays the β-gal activity of the LacZ reporter driven by the indicated enhancers, representing the activities of the Atoh1 enhancers. The experiment was repeated independently 3 times with similar results. Scale bars, 500 µm. g, h Schematic diagram represented sampled embryonic tissue (left), imaging perspectives (middle), and eSpatial prediction (right). The blue dot line indicates the section direction. The pink box denotes the RNAscope imaged region in the spinal cord (g). The pale green box denotes the RNAscope imaged region of the hindbrain (h). i–l RNAscope detection of Atoh1 mRNA in E0/E2/E3/E0 + 3 KO or WT mice in e11 spinal cord. Left: Representative images show staining for Atoh1 RNAscope probes (red) and DAPI (blue). Right: Quantification of Atoh1 RNAscope probe signal. Values shown are the mean ± standard error of the mean; n = 5 independent experiments. Statistical significance was determined by two-sided Student’s t-test: E0-WT vs. E0-KO (p < 0.0001), E2-WT vs. E2-KO (p = 0.8792), E3-WT vs. E3-KO (p = 0.0296), and E0 + 3-WT vs. E0 + 3-DKO (p < 0.0001). *p < 0.05, **p < 0.01, ***p < 0.001; n.s., not significant. Scale bar, 50 μm. WT wild type, KO knockout. m–p RNAscope detection of Atoh1 mRNA in E0/E2/E3/E0 + 3 KO or WT mice in e11 hindbrain. Left: Representative images show staining for Atoh1 RNAscope probes (red) and DAPI (blue). Right: Quantification of Atoh1 RNAscope probe signal. Values shown are the mean ± standard error of the mean; n = 5 independent experiments. Statistical significance was determined by two-sided Student’s t-test: E0-WT vs. E0-KO (p < 0.0001), E2-WT vs. E2-KO (p = 0.9910), E3-WT vs. E3-KO (p = 0.8914), and E0 + 3-WT vs. E0 + 3-DKO (p < 0.0001). *p < 0.05, **p < 0.01, ***p < 0.001; n.s., not significant. Scale bar, 50 μm. WT wild type, KO knockout. Source data are provided as a Source Data file.
