Transcriptome and epigenome landscape of human cortical development modeled in organoids

Abstract

Genes implicated in neuropsychiatric disorders are active in human fetal brain, yet difficult to study in a longitudinal fashion. We demonstrate that organoids from human pluripotent cells model cerebral cortical development on the molecular level before 16 weeks postconception. A multiomics analysis revealed differentially active genes and enhancers, with the greatest changes occurring at the transition from stem cells to progenitors. Networks of converging gene and enhancer modules were assembled into six and four global patterns of expression and activity across time. A pattern with progressive down-regulation was enriched with human-gained enhancers, suggesting their importance in early human brain development. A few convergent gene and enhancer modules were enriched in autism-associated genes and genomic variants in autistic children. The organoid model helps identify functional elements that may drive disease onset.

Figure 1.. Comparison of transcriptome and epigenome of organoid and isogenic fetal brain.

(A) Dataset and sample annotation. Samples are from both our project (hiPSCs lines, organoids, fetal brain samples), other PsychENCODE projects, and the Roadmap epigenomics project. Colors correspond to datasets represented in B-D. (B-D) Hierarchical clustering dendrograms of samples by transcriptomes (B) and ChIP-seq peaks of H3K27ac (C) and H3K4me3 (D). (E) Hierarchical clustering of organoids and isogenic postmortem cortexes by transcriptomes and gene-associated enhancer elements. Organoid and brain samples used for clustering are shown on top. Colors and shapes correspond to the datasets represented in the panels below. (F) Transcriptome-based classification of organoids and isogenic cortexes by age (8) against the tissues from the PsychENCODE developmental dataset (PCW = post-conceptional week) from Li et al (9). For each sample, red shading indicates the average of correlation coefficients above the cut off as defined in (8) between the sample and those in Li et al. (9). White boxes indicate correlations below the cut-off. Correlations to brains older than 2 years of age where all below the cut-off, and thus were not displayed. (G) Overlap of differentially expressed genes (DEGs) and differentially active enhancers (DAEs) between organoids at each differentiation time point and isogenic fetal cortex (CTX). (H) tSNE scatterplot of 17,837 nuclei, colored by cluster. Clusters arising predominantly from fetal cortex are circled. RG = radial glia; MGE = medial ganglionic eminence; IPC = intermediate progenitor cells; OPC = oligodendrocyte precursor cells. Novel means no correspondence to previous annotations. (I) Counts of DEGs and DAEs between organoids at different stages of development.

Figure 2.. Modules of co-expressed genes and co-active enhancers during organoid differentiation.

(A) Unsupervised hierarchical clustering of gene modules (1 through 54) by expression eigengenes. Rows and columns represent gene modules and samples, respectively. (B) Unsupervised hierarchical clustering of enhancer modules (1 through 29) by activity eigengenes. Rows and columns represent samples and enhancer modules, respectively. (C,D) Mean module eigengenes (lines) across differentiation times grouped by gene (C) and enhancer (D) supermodules, respectively. Dots represent values of eigengenes for individual modules. (EH) Enrichment of gene (E,G) and enhancer (F,H) modules for DEGs/DAEs and for various enhancers/genes of interest from the literature, including HGE – human-gained enhancers (26), TF – genes encoding transcription factors during human fetal brain development (24), ASD – genes pertinent to autism spectrum disorder (22), and DBD – genes pertinent to developing brain disorder (23). (I) Correspondence between the gene and enhancer networks. The strongest A-reg (pink dots) and R-reg (cyan dots) for a subset of gene modules are overrepresented in a number of enhancer modules. Black circles emphasize converging genes and enhancer modules, both of which are ASD-associated (as shown in G and H). Panel (E-I) are aligned by gene and enhancer modules shown in panels A,B.

Figure 3.. ASD associated genes modules.

(A) Overlap of ASD gene modules MG4, MG5, and MG51 from this study with transcript modules associated with ASD from postmortem brain studies or enriched in ASD de novo mutations (DNM) (green, violet) (4, 25) and from an ASD patient-derived organoid study (brown) (6). Rows are modules from this study and columns are modules from other studies. Red shading represents the degree of enrichment between pairs of modules. Corrected p-values of significant overlaps (hypergeometric test) are numerically indicated as -log10(p-value). (B) Bar plots of the top scoring biological process terms for the ASD associated modules shown in (A). (C) Graphical representation of the strongest interacting hub genes in the MG4 module network. Circles: genes; lines: topological overlap above 0.95. Colors in circles annotate each gene as hub (red), DEG (green), SFARI gene (blue), and enhancer target (yellow). Enhancer target: genes targeted by enhancers in the ME9, ME29, ME13, and ME2 ASD-associated enhancer modules (Fig. 2I). (D) Frequency plots within the MG4 module showing that enhancer targets, DEGs, and SFARI genes have higher intramodular connectivity. X-axis shows the weighted gene connectivity, from low (peripheral genes) to high (central hub genes).

Figure 4.. Enrichment of variants in gene-associated enhancers.

A) Three subsets of enhancers were selected from all gene-associated enhancers. Early: enhancers active (denoted by +) in all organoid stages but inactive (denoted by −) in fetal brain (red), late: enhancers active in fetal brain but inactive in all organoid stages (blue), constant: enhancers active in all organoid stages and fetal brain (green). Variants in 540 families from the Simons Simplex Collection were analyzed for enrichment in these enhancer sets. B) Comparison of inherited personal SNPs between ASD probands and normal siblings from the SSC revealed significant enrichment in probands versus siblings (p-value ≤ 0.05 by one-sample t-test) of low allele frequency SNPs (MAF 0.1%-5%) in early enhancers (red) and enhancer modules ME2 and ME29 (black). Dashed line at value of 0 represents no difference between probands and siblings. * means p-value < 0.05. C) Fractions of DNMs in enhancers were compared in probands and siblings across the whole genome. P-values (shown above the bars) were calculated using the chi-square test. D) Count of motif-breaking DNMs in all gene-associated enhancers were compared between probands and siblings. Circles represent TFs with counts of broken motifs in probands and siblings plotted on X- and Y-axis. The size of the circles is proportional to the number of TFs. Circles away from diagonal represent TFs enriched with motif-breaking DNMs in probands or siblings. A few TFs in the probands (colored circles) but not in the siblings were significantly enriched (p-value < 0.05 by binomial test) with motif-breaking DNMs.