Abstract
Abstract INTRODUCTION 70% of all oligodendrogliomas (ODG) harbor hemizygous loss-of-function Capicua (Cic) mutations. Cic is a transcriptional repressor downstream of MAPK signaling. Loss of Cic in neural precursors has been linked to increased proliferation and developmental bias towards differentiation-incompetent oligodendrocyte precursor cells (OPCs). This study aims to elucidate the consequences of OPC-specific Cic loss for oligodendroglia development and assess its contribution to ODG formation. METHODS Pdgfra-CreERT2;Cicfl/+;Rosa26EYFP (heterozygote control, Cic-HET) and Pdgfra-CreERT2;Cicfl/fl;Rosa26EYFP (homozygous knockout, Cic-KO) mice were used to induce Cic deletion and EYFP expression in OPCs during the postnatal wave of oligodendrocyte production (P3+P5). Double labeling with BrdU at P6 and EdU 6 hours prior to experimental endpoint was used to birthdate and assess active proliferation, respectively. Cic knockout was confirmed on the genomic level by qPCR. Formalin-fixed mouse brains were analyzed at 7-, 14- and 28-days post injection (dpi) for expression of proliferation and oligodendrocyte stage-specific markers by multiplex immunofluorescent staining. FACS-sorting was used to enrich for recombined cells based on EYFP expression. The 10x Genomics platform was used to analyze the single cell transcriptome (scRNAseq) of Cic-KO vs Cic-HET brains at designated time points. RESULTS 7dpi Cic-KO brains displayed an increased percentage of Pdgfra+ OPCs (25.76% KO vs. 15.95% HET) and a decreased percentage of CC1+ mature oligodendrocytes (27.69% KO vs. 33.93% HET) compared to heterozygote control. Cic-KO OPCs showed an increased proliferation rate as demonstrated by higher frequency of Ki67+ (3.16% HET vs 7.26%KO) and EdU+ (22.47% HET vs 44.44% KO) cells. scRNAseq revealed upregulation of genes involved in “nervous system development” and downregulation of “myelination” program upon Cic knockout. CONCLUSION We showed that OPC-specific Cic knockout results in increased cellular proliferation and maturation defects in the developing mouse brain. Cic loss may contribute to ODG formation by promoting developmental programs and inhibiting oligodendrocyte maturation in postnatal OPCs.
Published Version
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