Journal: bioRxiv
Article Title: Intrinsic coordination of dynamic molecular signatures shape the human prefrontal cortex
doi: 10.64898/2026.05.13.724991
Figure Lengend Snippet: A) Schematic of the YBX1 knock-down (KD) workflow in hCOs. Three stem cell lines (UCLA1 hESC, UCLA6 hESC, KOLF iPSC) were infected with a YBX1-targeting shRNA (EGFP-labeled). Cortical organoids were generated from each cell line, which, after 18 days, were dissociated and re-aggregated together in the presence of YBX1 shRNA lentivirus (Anton-Bolanos, et al., 2024; Nano et al., 2025). The resulting chimeroids were grown for 8 weeks in culture, at which time EGFP+ (YBX1-KD) and EGFP– (unperturbed) cells were isolated by FACS and captured for single-cell multi-omic profiling (simultaneous scRNA-seq and scATAC-seq). Representative immunofluorescence images of 5-week-old chimeroids show YBX1 shRNA (EGFP, green), SOX2 (DAPI), and CTIP2 (DAPI), confirming cortical identity. Scale bar = 300 μm. B) YBX1 expression is significantly and consistently depleted in YBX1-KD cells across all three cell lines. (Top) UMAP projection from both RNA and ATAC modalities (UMAP WNN) of YBX1-KD hCO cells colored by perturbation condition. (Bottom) YBX1 expression in unperturbed versus YBX1-KD cells for each cell line, summarized with boxplots (two-sided Wilcoxon test). C) Loss of YBX1 favors deep layer fate over upper layer fate. (Top) UMAP (WNN) projection of cells colored by cell type, with the Deep Layer Excitatory Neuron cluster highlighted. (Bottom) Percent change in cell type proportion in YBX1-KD versus unperturbed cells for each cell type. Individual dots represent each cell line, data summarized by boxplot. Dashed line at 0 indicates no change. D) Loss of YBX1 activates neuronal gene programs in both radial glia and deep layer neurons. Dot plots show the percent change in activity of developmental meta-modules (Nano et al., 2025) in YBX1-KD versus unperturbed cells in radial glia (top) and deep layer excitatory neurons (bottom), colored by biological process. Module 20, associated with deep layer fate (Nano et al., 2025), is among the most elevated modules in both cell types. Only statistically significant effects are shown (p < 0.05, two-sided Wilcoxon test). E) YBX1 depletion attenuates most PFC signatures across the excitatory neuronal lineage. Bar and error bars show the average percent change in PFC signature activity in YBX1-KD versus unperturbed cells across radial glia, IPC, deep layer excitatory neurons, and upper layer excitatory neurons. Values for each individual cell line shown as dots. Only statistically significant effects are shown (p < 0.05, two-sided Wilcoxon test). F) Across the glutamatergic lineage, YBX1 is required for the majority of cell type-specific PFC signatures. Pie charts summarizing the number of cell type-specific PFC signatures that are dependent (pink) versus independent (teal) of YBX1 in each cell type. White indicates signatures that are not enriched in the PFC within the indicated cell type. G) YBX1 depletion induces broad shifts in chromatin accessibility, with effects significantly amplifying from radial glia to deep layer excitatory neurons (two-sided Wilcoxon test). Dots represent significantly differentially accessible chromatin peaks between unperturbed and YBX1-KD cells within radial glia and deep layer excitatory neurons, plotted based on fold-change in accessibility (p < 0.05, logistic regression test). Data are colored by YBX1-dependent regions (less accessible in YBX1-KD), YBX1-repressed regions (more accessible in YBX1-KD), and relatively unchanged regions (less than 25% change in accessibility, grey dots). Data summarized by boxplot. H) Loss of YBX1 modestly decreases the chromatin accessibility of PFC signatures. The average promoter region accessibility in each PFC signature was calculated per cell. Data show the percent change in this “PFC signature chromatin accessibility” in YBX1-KD vs unperturbed cells. Comparisons were conducted within radial glia and deep layer neurons, focusing solely on the PFC signatures relevant to each cell type. Dots indicate the percent-change in average chromatin accessibility for each cell type-specific signature, summarized by boxplots. P-value calculated by two-sided Wilcoxon test. I) YBX1 regulates PFC signatures at both the chromatin and transcriptional level in radial glia, but acts predominantly as a transcriptional regulator in deep layer neurons. Dot plot displays the percent change induced by YBX1-KD in each cell type-specific PFC signature, both in terms of average chromatin accessibility (blue) and gene expression (green). Dashed line at 0 indicates no change. J–K) In YBX1-sensitive PFC signatures shared between radial glia and deep layer neurons (J), YBX1 is required to open chromatin in radial glia but shifts to a predominantly transcriptional role in deep layer neurons – a cascade not observed in non-YBX1-sensitive signatures (K). Bar and error bars show, for the indicated PFC signatures, the average percent change in chromatin accessibility (blue) and gene expression (green) induced by YBX1-KD. Effects in radial glia and deep layer neurons are shown, with the values from individual cell lines shown as dots.
Article Snippet: Multiomic libraries were generated using the Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Kit (10X Genomics, CG000338 Rev F) according to manufacturer instructions, using 7-8 cycles for ATAC library amplification, 6-7 cycles for cDNA amplification, and 10-14 cycles for gene expression library amplification depending on input DNA amount.
Techniques: Knockdown, Infection, shRNA, Labeling, Generated, Isolation, Single Cell, Immunofluorescence, Expressing, Activity Assay, Gene Expression
