Extended Data Fig. 8: Lack of FMRP causes altered neural differentiation and aberrant developmental trajectory in forebrain organoids.

a, A heat map of expression of annotation reference genes in 14 cell type specific clusters present during human forebrain organoids shows the differential expression of various marker genes for specific cell types in each cluster. (C1: fate determining stage neurons toward excitatory neuron, C2: excitatory neuron, C3: neural stem cell /radial glia2, C4: immature neuron, C5: neural stem cell /radial glia1 cell, C6: glial progenitor, C7: inhibitory neuron, C8: astrocyte, C9: radial glia, C10: astrocyte, C11: immature neuron very early stage, C12: oligodendrocyte, C13: ectodermal origin non-neuronal cells, C14: non-neuronal cells) b, The expression of neural stem cell/progenitor marker, SOX2 (red) and differentiated cortical plate neuron marker, BCL11B (CTIP2, green) were presented simultaneously in the UMAP plot. Compared to control, cells in FXS organoids expressing BCL11B/CTIP2 at low level were increased and widely distributed spanning various cell types regardless of differentiation status and cell function. Many of these are accompanied by the expression of SOX2. Significantly high co-expression rate of the NPC marker, SOX2, and cortical plate marker, BCL11B in the C7, young inhibitory neuron cluster (19% in FXS forebrain organoids compared to 0% in control forebrain organoids), suggest that the spatiotemporal regulation of SOX2 and BCL11B expression critical for proper specification and lamination of neurons is severely perturbed in FXS organoids. Data are presented as mean ± s.e.m. (n=3 single cell RNAseq of 3 independent culture sets, **P=0.0025, two-tailed unpaired t test) (c) Among the 14 clusters, the highest number of DEGs were detected in the young inhibitory neuron cluster, C7. PANTHER analyses show high relevance to regulation of synapse organization, learning and memory, and forebrain development with down-regulated DEGs and protein targeting. mRNA stability and regulation of cell cycle. Yellow represents up-regulated genes and blue represents down-regulated genes. The numbers on the bars are the associated two-sided p-values by Fisher’s Exact test. The p values have been adjusted for multiple testing using Bonferroni correction. d, Transcriptional features of the cluster 6 at the developmental break point between FXS and control (arrow in red) in the time trajectory was assessed. The Monocle cluster 4, one of the major break point in the time trajectory, has marker genes associated with cell proliferation and regulation of DNA methylation, (for example, KMT2A), neuron migration and regulation of neuron projection development (ACAP3), synapse organization and axon guidance (NFASC).