Extended Data Fig. 9. Characterization of GLI3 knock-out organoids.

a, Quantification of editing frequency as determined by the percentage and number of reads showing unmodified and modified alleles for the control and both KO cell lines. b, Frequency of frameshift of coding sequence reads as a result of the modifications seen in both KO lines. c, Western blot showing expression of Gli3-repressor (83kDA) in the control cell line. Catenin beta-1 and Ponceau were used as loading control. For western blot source data, see Supplementary Fig. 1 d, Sequences of the coding strand of the different indels of the different KO lines. The reference sequence is corresponding with the control line. The position of the gRNAs with the protospacer adjacent motif (PAM)-sequence is depicted above and underneath the sequence. Reference protein sequence with the protein sequences of each KO line of the altered protein sequences caused by the frame-shift. e, Brightfield images of brain organoid development with control and both KO cell lines. Images are representative for 16 imaged organoids per line. Scale bar is 2 mm. f, UMAP embedding showing trajectories from neural progenitor cells (NPCs) to neurons coloured by different clusters assigned to branches (dorsal, ventral, and non-telencephalon), with inset coloured by genetic condition and feature plots coloured by expression (log(transcript counts per 10k + 1)) of cell type markers. g, UMAP embedding of ventral telencephalic GLI3 KO neurons showing medial ganglionic eminence (MGE) and lateral/caudal ganglionic eminence (LGE/CGE) neuronal populations (top). Feature plots show selected marker gene expression on the UMAP embedding. The range of expression values is indicated for each feature plot. h, Volcano plot showing differential expression analysis in LGE neurons for GLI3 WT versus KO cells. i, Schematic of observed effect of GLI3 loss of function on dorsoventral telencephalic fate decisions.