Extended Data Fig. 6: Intersection of ASD DEGs with cluster markers of EN and fetal brain derived cortical area-specific markers (Bhaduri et al.⁴⁵) (related to Fig. 3).

a Dot plot showing log2FC differential expression results in the IPC/nN cell type (left) for genes identified as specific cluster markers of EN-PP and EN-DCP clusters as indicated by dots on the right side (clusters are referred by numbers as shown in the initial clustering in Main Fig. 1a; pct.exp= percentage of cells expressing the gene in the corresponding cluster. Specific cluster markers were defined as cluster markers with average log-fold change > 0.25, BH adjusted-pvalue < 0.01 (Wilcoxon rank sum test) and pct.1/pct.2 > 1.2 in no more than 2 clusters; see full cluster marker list in Supplementary Table 3). The panel show that IPC/nN cells are showing a differential expression in cluster markers compatible with a shift in fate preference in ASD probands (increase in EN-DCP in macro and in EN-PP in normo-ASD). b Dot plots showing enrichment of cortical area-specific markers in upregulated (upDEG) or downregulated (downDEG) ASD DEGs at TD30/60 separated by cohort and cell types. Cortical area-specific markers from fetal brain major cell type were selected and matched to corresponding organoid cell type (‘RG’ for RG-related cell type, ‘IPC” for IPC/nN and”Neuron” for neuronal cell types, as reported in Table S8 of Bhaduri et al for ‘mid’ fetal stage). Overall the there is a stronger downregulation of area-specific genes at TD30/TD60 in both cohorts. However, upregulated DEGs in macro-ASD are notably more enriched in genes specific to V1 area, which, put together with an enrichment of genes marking PFC in downDEGs (notably in RG) could point to a differential area-specification in macro-ASD. c To further investigate the upregulation of V1 area markers, the most significant areal markers differentiating PFC and V1 cortical areas in fetal brain (y axis: ‘PFC-enriched’, or ‘V1-enriched’) were selected (main figure in Bhaduri et al.) and ASD DEG results from our study were plotted as a differential expression heatmap (as in main Fig. 3c). When considering this limited list of important genes, both important V1-enriched and PFC-enriched genes are found upregulated (for example LHX2/TENM4/BCL11A for V1 and NEUROG1/2/HOPX/PAX6 for PFC) and downregulated (NR2F1/WNT7B/NPY for V1 or FOS/CTNNB1 for PFC), which suggest that area misspecification alone do not account for the full phenotype. Note that most of those cortical area marker genes have several other canonical functions in neurodevelopment (see for instance alternative annotations in known marker list in Supplementary Table 3 in our study).