Fig. 1: PACS1 p.R203W variant does not impact cytoarchitectural organization of cortical organoids.

a Experimental overview. Control, patient, and isogenic pluripotent cell lines were generated using CRISPR/Cas9 and differentiated to dorsal cortical organoids through exposure to a series of growth factors. Patient and control organoids were then assessed using immunohistochemistry, single-cell RNA sequencing, and calcium imaging. Additional downstream functional analyses were also performed with rapidly directed glutamatergic neurons. b Expression of telencephalic protein FOXG1, dorsal neuronal transcription factor TBR1, and upper layer marker SATB2 in day 40 and 80 organoids. Scale bar = 100 µm. DAPI is in greyscale. c Expression of neural precursor protein SOX2 and projection neuron marker CTIP2 in day 40 and 80 organoids. Scale bar = 100 µm. DAPI is in greyscale. d A quantification of (b). Mean ± SEM fraction of FOXG1⁺, TBR1⁺, or SATB2⁺ cells contributing to the total number of positive cells per field of view. Stains are different by day (FOXG1 F_1,9 = 24.004, p_adj = 8.43E-04; TBR1 F_1,9 = 9.459, p_adj = 0.013; SATB2 H₁ = 9.466, p_adj = 0.004) but not genotype (FOXG1 F_1,9 = 0.061, p_adj = 0.811; TBR1 F_1,9 = 1.811, p_adj = 0.211; SATB2 H₁ = 0.183, p_adj = 0.669) using a two-way ANOVA unless p < 0.05 in a Shapiro test of normality, in which case a one-sided Kruskal-Wallis test was performed between conditions and adjusted using a Benjamini Hochberg correction (see Methods). n = 3 organoids for each condition (Day 40: three C1, one CRISPR R203W, and two A1 organoids from four independent differentiations; Day 80: two C2, one CRISPR A1, two CRISPR R203W, and one A1 organoids from three independent differentiations. See Table S1). Data from PACS1^(+/R203W) lines is indicated by the dot pattern. Results per organoid are shown in Fig. S4d. e A quantification of (c). Mean ± SEM fraction of CTIP2⁺ or SOX2⁺ cells contributing to the total number of positive cells per field of view was significant between time points (H₁ = 12.808, p_adj = 6.90E-04) but not genotypes (H₁ = 3.360, p_adj = 0.067) using a one-sided Kruskal-Wallis test (p_Shapiro = 0.003) and Benjamini Hochberg correction. n = 7+ organoids per condition (Day 40: five C1, one C3, one CRISPR A1, one CRISPR A2, one CRISPR R203W, five A1, and one A2 organoids from four independent differentiations; Day 80: two C1, three C2, one C3, one CRISPR A1, three CRISPR R203W, and four A1 organoids from three independent differentiations. See Table S1). Data from PACS1^(+/R203W) lines is indicated by the dot pattern. Results per organoid are shown in Fig. S4e. f Western blot for the PACS1 protein abundance in day 40 organoid samples. An uncropped version can be found in the Source Data file. g Quantification of the ratio of PACS1 to α-tubulin signal in day 40 organoids shown in (f). The bar represents mean ± SEM. U = 26, p = 0.867 using a two-sided Mann–Whitney test. n = 7 PACS1^(+/+) organoids (two C1, three C2, and two CRISPR A1) and n = 8 PACS1^(+/R203W) organoids (four CRISPR R203W and four A1) from two independent differentiations. See Table S1. Source data for (d–g) are provided in the Source Data file.