Extended Data Fig. 4: Comparing the effects of FGF8 versus SHH and BMP4 versus BMP7 treatment.

Results shown correspond to the lines HES3 (single-cell RNA-seq analysis) and WTC (HCR image). a, Effects of different FGF8 treatment windows (left) and concentration steps (right) at early (day 3-14) and late (day 12-20) time windows on the abundance of telencephalic progenitor populations at 21 days. Bottom left: Spatial representation of the clusters enriched by FGF8 and SHH in a schematic representation of a ~ 4 GW human embryo. b, Effects of different SHH treatment windows (left) and concentration steps (right) on the abundance of retinal, telencephalic and hypothalamic progenitor populations in 21-day-old neural organoids. P-values were derived using two-sided Fisher’s exact test with Bonferroni correction. For panels a and b, *** p-value < 0.001, ** 0.001< p-value < 0.01, * 0.01 < p-value < 0.05. Exact p-values can be found in Supplementary Table 3. c, HCR in situ hybridization for PTCH1 (hypothalamic marker gene and target gene of SHH signaling) comparing SHH treatment conditions c1 and c5. Cell line = WTC. Images are representative of 3 organoids per condition. d, Dorso-ventral score values of forebrain single cells for different concentration steps of SHH and FGF8 (both given on days 3-14) with their corresponding controls showing higher ventralization in SHH-treated organoids, which reach the hypothalamic compartment. Horizontal bars depict the median dorso-ventral score across all cells categorized as telencephalic and hypothalamic progenitors for each condition. n = 12 for Ctrl (SHH experiment), n = 424 for SHH c1, n = 271 for SHH c2, n = 379 for SHH c3, n = 380 for SHH c4, n = 361 for SHH c5, n = 17 for Ctrl (FGF-8 experiment), n = 446 for FGF-8 c1, n = 239 for FGF-8 c2, n = 526 for FGF-8 c3, n = 407 for FGF-8 c4 and n = 472 for FGF-8 c5. e, Anteroposterior scores for telencephalic progenitors treated with SHH or FGF8 concentration steps on days 3-14. Vertical bars depict the median dorso-ventral score across all cells categorized as telencephalic progenitors for each condition. n = 12 for Ctrl (SHH experiment), n = 1165 for SHH c1, n = 269 for SHH c2, n = 274 for SHH c3, n = 304 for SHH c4, n = 356 for SHH c5, n = 17 for Ctrl (FGF-8 experiment), n = 439 for FGF-8 c1, n = 238 for FGF-8 c2, n = 523 for FGF-8 c3, n = 406 for FGF-8 c4, and n = 470 for FGF-8 c5. f, Volcano Plot showing the differentially expressed genes between the telencephalic progenitors of FGF-8 and SHH conditions, promoting the greatest increase in telencephalic progenitors (c3 on days 3-14 for FGF-8, and c2 on days 3-14 for SHH). Differentially expressed genes were obtained by applying the Wilcoxon rank sum test for differential expression analysis. P-values were then subjected to Bonferroni correction for multiple testing. g, Endogenous FGF8 expression in telencephalic progenitors in response to SHH or FGF8 concentration steps applied at the same time window. SHH expression is not shown, as it consistently yielded zero values. h, UMAP representation of cell fateshifts under BMP4 and BMP7 treatment in comparison to control. Only cells from the BMP concentration steps experiment are shown and are coloured by cluster. Legend with relevant clusters is shown below. i, Heatmap showing differentially expressed genes in ectodermal cells from control, BMP4 or BMP7 conditions.