DLG2 knockout reveals neurogenic transcriptional programs underlying neuropsychiatric disorders and cognition

Abstract

Genetic studies robustly implicate perturbation of DLG2-scaffolded mature postsynaptic signalling complexes in schizophrenia. Here we study in vitro cortical differentiation of DLG2 -/human embryonic stem cells via integrated phenotypic, gene expression and disease genetic analyses. This uncovers a developmental role for DLG2 in the regulation of neural stem cell proliferation and adhesion, and the activation of transcriptional programs during early excitatory corticoneurogenesis. Down-regulation of these programs in DLG2 -/lines delays expression of cell-type identity and causes marked deficits in neuronal migration, morphology and active properties. Genetic risk factors for neuropsychiatric and neurodevelopmental disorders converge on these neurogenic programs, each disorder displaying a distinct pattern of enrichment. These data unveil an intimate link between neurodevelopmental and mature signalling deficits contributing to disease -suggesting a dual role for known synaptic risk genes -and reveal a common pathophysiological framework for studying the neurodevelopmental origins of Mendelian and genetically complex mental disorders. Results Knockout generation and validationTwo DLG2 -/lines were created from H7 hESCs using the CRISPR/Cas9-D10A nickase system targeting the first PDZ domain ( Figure S1). Sequencing revealed no off-target mutations (see Methods, Figure S2 & Table S1). A significant decrease in DLG2 mRNA was observed for exons spanning the first PDZ domain, with a similar decrease inferred for PDZ-containing transcripts, indicating degradation of DLG2 -/transcripts via nonsense-mediated decay ( Figure S3A, B). Quantitative mass spectrometry-based proteomic analysis of peptide-affinity pulldowns using the NMDA receptor NR2 subunit PDZ peptide ligand (Husi and Grant, 2001) confirmed the presence of DLG2 in pulldowns from WT but not DLG2 -/lines ( Figure S3C-F & Table S2). Genotyping revealed no CNVs in either DLG2 -/line relative to WT ( Figure S4A). Both DLG2 -/lines expressed pluripotency markers OCT4, SOX2 and NANOG at 100% of WT levels ( Figure S4B-E). Cells were extensively characterised for their cortical identity using western blotting and immunocytochemistry from day 20 to 60 ( Figure S5). Over 90% of day 20 cells were positive for FOXG1, PAX6 and SOX2 confirming their dorsal forebrain fate ( Figure S5A-B). DLG2 regulates neural stem-cell proliferation and adhesionRNA was extracted in triplicate from each line at 4 timepoints spanning cortical excitatory neuron development (Figure 1A, B) and gene expression quantified. To robustly identify genes dysregulated by DLG2 knockout, expression data from the 2 DLG2 -/lines were pooled and compared to a WT sister line at each timepoint (see Methods). Disruption of DLG2 had a profound effect: of the >13,000 protein-coding genes expressed at each timepoint, ~7% were differentially expressed between DLG2 -/and WT at day 15, rising to 40-60% between days 20 and 30 then decreasing to ~25% by day 60 ( Figure 1C). Strikingly, the 3 genes with the strongest ...