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. 2023 Apr 27;186(9):1930–1949.e31. doi: 10.1016/j.cell.2023.03.025

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© 2023 The Author(s)

This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by-nc/4.0/).

PMC Copyright notice

Perturbing autism-associated genes selectively in Rbp4-Cre neurons disrupts circuit organization and activity during embryonic development

(A) Expression (circles) of selected genes (Data S2) associated with autism spectrum disorder¹⁰⁷ in the three Rbp4-Cre neuron types and adult L5-PN types.⁸ Radius of circles: fraction of cells expressing the gene; color of circles: mean normalized transcripts per cell (log₂).

(B) Fraction of genes with a mean transcript count greater than the number of transcripts shown on the x axis, for all genes (black), and genes associated with autism spectrum disorder (magenta) in Rbp4-Cre neurons (top) and adult L5-PNs (bottom). Inset: Fold change of autism-associated gene expression compared to all genes in embryos and adult.

(C) Immunostaining of cortex of Rbp4-tdTomato-Chd8^+/− (top) and Rbp4-tdTomato-Grin2b^+/− (bottom) mice (Figure S8). Rbp4-Cre neurons (red), Bcl11b, (white), Hoechst (blue).

(D) Normalized depths of Rbp4-Cre neurons (as in Figure 2A) in Rbp4-tdTomato (WT) and the two mutant (top, Chd8^+/−; bottom, Grin2b^+/−) embryos (Figure S9). 125 neurons from each mouse line, sampled at random. χ² test.

(E) Mating strategy to generate Rbp4-GCaMP6s-tTA2-Chd8^+/− (Chd8^+/−) and Rbp4-GCaMP6s-tTA2-Grin2b^+/− (Grin2b^+/−) embryos.

(F) Example recordings from Rbp4-Cre neurons’ dendrites in Rbp4-GCaMP6s-tTA2 (WT), Grin2b^+/−, and Chd8^+/− embryos at E16.5 using 3D acousto-optic two-photon microscope.

(G) Distribution of activity in E16.5 embryos, shown in log-scale, for WT and two mutant genotypes. Circles: activity of each neurite; red line: median; shading: distribution. Wilcoxon rank-sum test. n = number of neurites.

(H) Immunostaining of local patches of cortical disorganization in Rbp4-tdTomato-Chd8^+/− and Rbp4-tdTomato-Grin2b^+/− mice, at E18.5. Rbp4-Cre neurons (red), Bcl11b, (white), Hoechst (blue).

(I) Fraction of mutant mice, of each genotype, showing at least one patch, summed across E16.5 to E18.5. Red line: Average across all four genotypes. Data from 14 (Rbp4-tdTomato-Chd8^+/−), 11 (Rbp4-tdTomato-Chd8^−/−), 9 (Rbp4-tdTomato-Grin2b^+/−), and 6 (Rbp4-tdTomato-Grin2b^−/−) embryos. Fisher’s exact test (p = 0.05, prior to Bonferroni correction).

(J) Fraction of neurons within the superficial layer that are located within patches of disorganization, in embryos with at least one patch. n = number of superficial layer neurons on each embryonic day.

Scale bars: 20 μm (C), 25s and 25 %ΔF/F (F), 50 μm (H).

See also Figures S8 and S9.