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, 22 (2), 243-255

Pathological Priming Causes Developmental Gene Network Heterochronicity in Autistic Subject-Derived Neurons

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Pathological Priming Causes Developmental Gene Network Heterochronicity in Autistic Subject-Derived Neurons

Simon T Schafer et al. Nat Neurosci.

Abstract

Autism spectrum disorder (ASD) is thought to emerge during early cortical development. However, the exact developmental stages and associated molecular networks that prime disease propensity are elusive. To profile early neurodevelopmental alterations in ASD with macrocephaly, we monitored subject-derived induced pluripotent stem cells (iPSCs) throughout the recapitulation of cortical development. Our analysis revealed ASD-associated changes in the maturational sequence of early neuron development, involving temporal dysregulation of specific gene networks and morphological growth acceleration. The observed changes tracked back to a pathologically primed stage in neural stem cells (NSCs), reflected by altered chromatin accessibility. Concerted over-representation of network factors in control NSCs was sufficient to trigger ASD-like features, and circumventing the NSC stage by direct conversion of ASD iPSCs into induced neurons abolished ASD-associated phenotypes. Our findings identify heterochronic dynamics of a gene network that, while established earlier in development, contributes to subsequent neurodevelopmental aberrations in ASD.

Figures

Figure 1:
Figure 1:. Gene network analysis of time series-based RNA sequencing identifies dynamic developmental gene networks.
a, Schematic of FACS-based purification of NSCs and defined subpopulations of eGFP+/PSA-NCAM+ neurons over the time course of in vitro differentiation. b, WGCNA cluster dendrogram of all 65 time series samples groups genes into 44 distinct modules (top row). Bottom row shows strong differential expression relationships for developmental time (days of in vitro differentiation post lineage tracing). c, Heatmap showing relationships between modules, disease and developmental time. Relationship assignments are based on signed Pearson correlation coefficients indicating positive (red) or negative (blue) correlation of modules with trait and developmental time; n=65 time series samples; ASD: n=8 independent cell lines at 5 time points, control: n=5 independent cell lines at 5 time points. d, Time-based significance measure reveals three modules highly enriched for genes that change with developmental time. This module significance measure was defined as the absolute Pearson correlation between time (days of in vitro differentiation post lineage tracing) and module eigengenes; TM1: n=1,530 genes, TM2: n=294 genes, TM3: n=401 genes. Student p-values for associations with time were adjusted for multiple comparisons by using the false discovery rate (FDR) approach.
Figure 2:
Figure 2:. Gene network analysis and dynamic time warping identify ASD-specific heterochronicity in a time-related module.
a, Module eigengene (ME) dynamics of the three notable time-related modules averaged across developmental time. Upper panel: Heatmap showing average MEs across time. Blue, low expression: Red, high expression. Lower panel: Plot of TM1, TM2 and TM3 showing trajectories of module eigengenes across time. b, Three-way plot of the alignment for TM1, TM2 and TM3 between control (reference) and ASD (query). This plot allows display of alignments and places the query time series (ASD) in the small lower panel and the reference time series (control) vertically on the left; the large inner panel holds the warping curve. Left: Earlier time points in the query index (ASD time series) of TM1 correspond to later time points in the reference index (control time series), indicating an accelerated progression of TM1. c, Left: DTW alignment curves superimposed for all three TMs, showing the magnitude and onset of time shift. Right: Density plot showing alignment of TM1 between control (reference) and ASD (query). The average cost per step is displayed as a density distribution with contours superimposed. d, Module-specific enrichment for ASD-related risk factors based on the SFARI gene list (see Supplementary Table 5).
Figure 3:
Figure 3:. Characterization of the heterochronic TM1.
a, GO term analysis of the genes assigned to TM1 reveals specific neurodevelopmental signature. Point sizes correspond to the number of genes assigned to each category (Supplementary Table 6). b, Top gene-gene connections for TM1 are shown, highlighting their involvement in selected biological categories and ASD risk. Different point sizes correspond to the number of time points that show increased expression levels. c, Gene Significance (GS) values of the top 60 hub genes across developmental time and between conditions. GS measures are represented as mean ± s.d.; n=60 genes.
Figure 4:
Figure 4:. Aberrant neurodevelopmental growth dynamics in maturing ASD neurons.
a, Schematic showing experimental design for lineage tracing-based morphometric assessment. b, Representative confocal images of developing neurons seven days post retroviral infection (dpi) expressing green fluorescent protein (GFP) and DCX and showing immunoreactivity for axonal filaments (Smi312). Scale bars 50 μm. Immunohistochemistry was performed in all 13 patient lines and 2 cell culture replicates per time point with similar results (also see Extended Data Fig. 5a). c, Reconstructions of retrovirus (RV)-labeled neurons derived from ASD and unaffected control individuals over time. Scale bar 50 μm. Experiments were performed in all 13 patient lines and 2 cell culture replicates per time point. Results are shown in d and e. d, ASD neurons showed accelerated growth properties and were significantly longer at 4, 7 and 14 dpi (two-way ANOVA with Sidak correction, 4 dpi: ***P=0.0002, 7 dpi: **P=0.006, 14 dpi: ****P<0.0001). Values show means ± s.d.; ASD (n=8; 320 technical tracing replicates total), control (n=5; 244 technical tracing replicates total); n refers to biologically independent patient lines. e, Sholl analysis of neurite length from ASD and control neurons at 14 dpi. Total sholl neurite length complexity was larger in the ASD group as compared with controls (two-way ANOVA with Sidak correction, *P<0.05, ***P<0.001, ****P<0.0001). Values show means ± s.e.m.; ASD (n=8; 320 technical tracing replicates total), control (n=5; 244 technical tracing replicates total); n refers to biologically independent patient lines. f, Schematic showing retroviral reporter construct designed to assess hDCX promoter activity and the flow cytometry-based gating strategy for the assessment of hDCX:GFP mean fluorescence intensities (MFIs). g, Representative histograms showing MFIs of the hDCX:GFP reporter through the time course of differentiation. Experiments were repeated in all 13 patient lines at 5 different time points; numerical values represent percentages. h, Relative hDCX promoter activities in differentiating neurons. ASD neurons showed premature activation of the inserted hDCX promoter with significantly elevated levels at the NSC stage and at 16 dpi (two-way ANOVA with Sidak correction, NSC stage: **P=0.0076, 16 dpi: **P=0.0059). The data are normalized to the maximum activity over time within each line. Values represent means ± s.e.m.; ASD (n=8), control (n=5); n refers to biologically independent patient lines. Also see Extended Data Figure 5.
Figure 5:
Figure 5:. Aberrant neurodevelopmental growth dynamics of early-born neurons in a three dimensional model of cortical development.
a, Schematic showing experimental design for lineage tracing-based morphometric assessment in forebrain organoids. b, Representative confocal images of retrovirally labeled radial glia-like (RGL) cells (upper panel). Over time, retrovirally labeled cells migrate into cortical plate (CP)-like regions and differentiate into neurons that integrate into the evolving cortical layer (bottom panel). Scale bars 20 μm. Experiments were repeated in 6 patient lines and 3 different organoid batches with similar results. c, Representative confocal images of retrovirally labeled neurons at 14 dpi in CP-like regions expressing TBR1. Arrowheads indicate GFP+ neurons co-expressing TBR1 (also see Extended Data Fig. 6k and l). Scale bar 20 μm. Immunohistochemistry was repeated in 6 patient lines and 3 different organoid batches with similar results. d, Representative confocal images of retrovirally labeled neurons in CP-like regions at 14 dpi between ASD and control forebrain organoids. Scale bar 20 μm. Immunohistochemistry was repeated in 6 patient lines and 3 different organoid batches with similar results. e, Developing cortical neurons of ASD patients showed significantly accelerated growth properties at 14 dpi (*P=0.0128, unpaired two-tailed Student’s t-test); Values represent mean ± s.e.m.; ASD: n=3 (9 organoids with 122 tracing replicates total); control: n=3 (9 organoids with 142 tracing replicates total); n refers to biologically independent patient lines. f, Sholl analysis of subtle branching patterns at 14 dpi between ASD and control cortical neurons. Sholl neurite length complexity (left) and intersections (right) were larger in the ASD group as compared with controls (two-way ANOVA with Sidak correction, *P<0.05, **P<0.001, ***P<0.001). Values represent mean ± s.e.m.; ASD: n=3 (12 organoids with 122 tracing replicates total); control: n=3 (12 organoids with 142 tracing replicates total); n refers to biologically independent patient lines.
Figure 6:
Figure 6:. Transgenic overexpression of proneural transcription factors in iPSCs (iPSC-iNs) circumvents proliferative NSC-like stages.
a, Schematic of lentiviral system for inducible overexpression of Ngn2-2A-eGFP (top). Direct conversion of iPSCs into induced neurons (iNs) by forced expression of an inducible Ngn2 transgene may bypass early NSC-like stages (bottom). b, Representative images of patient-derived iPSC-iNs at different stages in the course of neuronal differentiation. Scale bar 200 μm. Experiments were repeated in all 13 patient lines at 4 different time points. c, Immunocytochemical characterization of iPSC-iNs after two weeks of conversion. Scale bar represents 25 μm. Immunohistochemistry was performed in all 13 patient lines at least once with similar results d, BrdU-labeling experiment with unselected iPSC-iNs during conversion (arrowheads highlight GFP-expressing iPSC-iNs). Scale bar 50 μm. Values represent mean ± s.d.; n=4 independent control cell lines. e, Representative reconstructions of patient-derived iPSC-iNs at the stages indicated in b. Scale bar 100 μm. Experiments were repeated in all 13 patient lines at 4 different time points. Also see Extended Data Figures 8 and 9.
Figure 7:
Figure 7:. Bypassing the NSC state restores early neurodevelopmental aberrations.
a, A significant enrichment of PSA-NCAM+ cells was present in ASD NSC-derived neurons (NSC-N) at 4 dpi as compared to their respective controls (**P=0.0016, Mann-Whitney U-test), whereas no difference was observed in iPSC-iNs (P=0.805, Mann-Whitney U-test, not significant). Box plots show median (center line), mean (‘+’) and interquartile range (IQR), with whiskers representing the minimum and maximum of data points; ASD: n=8 biologically independent patient lines (42 technical replicates total), control n=5 biologically independent patient lines (22 technical replicates total). b, Sholl analysis of subtle branching patterns in patient-derived iPSC-iNs at 14 days post conversion. Values represent mean ± s.e.m.; ASD: n=8 biologically independent patient lines, control: n=5 biologically independent patient lines. c, Left: Total neurite growth assessment of converting iPSC-iNs. Values represent mean ± s.d.; ASD: n=8 biologically independent patient lines (482 tracing replicates total), control: n=5 biologically independent patient lines (264 tracing replicates total). Right: ASD NSC-Ns had significantly longer neurites at 14 dpi as compared with their respective controls (control NSC-N: 421.8 ± 14.14 μm, ASD NSC-N: 552.32 ± 24.86 μm, **P=0.0062, Mann-Whitney U-test), whereas no difference was observed in iPSC-iPSC-iNs at 14 days post conversion (control iPSC-INs: 442.39 ± 23.93 μm, ASD iPSC-iNs: 439.07 ± 14.51 μm, P=0.9433, Mann-Whitney U-test, not significant). Values represent mean ± s.e.m.; ASD: n=8 biologically independent patient lines, control: n=5 biologically independent patient lines. d, WGCNA cluster dendrogram of all 52 iPSC-iN time series samples groups genes into distinct modules (top row). The middle row shows strong differential expression relationships for developmental time (days of iPSC-iN conversion). TM1 genes (bottom row; Fig. 2) show high module preservation in the iPSC-iN gene network (also see Extended Data Fig. 10c-e). e, Density plot showing the temporal ME alignment for the TM1-equivalent iPSC-iN module blue between control (reference) and ASD (query) during iPSC-iN maturation. The average cost per step is displayed as a density distribution with contours superimposed. f, GS values of the top 60 TM1 hub genes for ASD and control individuals in NSC-derived neurons (left) or when bypassing the NSC stage (iPSC-iN technology, right) after 14 days. Violin plots show median (center line), IQR (box), 95% confidence interval and the kernel probability density at different values; n=60 genes. Also see Extended Data Figures 8, 9 and 10.
Figure 8:
Figure 8:. Aberrant gene network dynamics at early neuronal stages are associated with changes in chromatin accessibility at preceding NSC stages.
a, Coverage maps of normalized ATAC-seq signals from ASD and control NSCs showing a differentially accessible (DA) peak (highlighted in red) near the DNMBP gene on chromosome 10. Group-wise sample coverages are displayed and annotated chromatin states are based on the ENCODE 25-state model (see Supplementary Methods). b, Binding affinity heatmap showing normalized accessibilities for DA peaks. c, Correlation heatmap showing hierarchical clustering based on DA peaks (Pearson correlations of peak scores); ASD: n=8 biologically independent patient lines, control: n=5 biologically independent patient lines. d, Log2 fold enrichment of DA peaks in epigenetically annotated regions of the genome (ENCODE 25-state model) shows significant enrichment in promoter and enhancer regions (GAT randomization test; n=1,593 DA elements; see Supplementary Methods). e, Significance calculations for the enrichment of DA peaks within gene-distal regions (50kbp windows) of TM-associated genes (GAT randomization test; ASD enriched: n=721 gene-distal DA peaks, ASD depleted: n=755 gene-distal DA peaks; see Supplementary Methods). f, Metagene profiles of normalized ATAC-seq signals at promoters around ±1kb from the transcription start sited (TSS) in NSCs from ASD patients (red) or controls (black). Left: Metagene profiles of promoter regions from TM1 hub genes (kME > 0.8) show higher accessibility in ASD NSCs (P=6.717×10−4, Hotelling’s t test; n=273 elements). Right: Metagene profiles of promoter regions from randomly accessible background genes (P=0.7761, Hotelling’s t test, n.s. – not significant; n=273 elements). Numbers of plotted elements (genes) are similar.

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