The role of ASD-linked histone lysine demethylase KDM5B in cortical development

Abstract

Epigenetic mechanisms are of great importance in the temporal and spatial regulation of the formation of the cortex. Disruption can result in neurodevelopmental disorders, such as autism spectrum disorder (ASD). Multiple mutations in the human KDM5B gene, which is a histone demethylase that removes histon-3-lysin-4 (H3K4) methyl modifications, have been linked to ASD. KDM5B and H3K4me3 have been associated with both differentiation of NSCs and self-renewal of NSCs. A KDM5B KO has been generated before to study the effect of KDM5B, but the role of KDM5B in late embryonic and early postnatal development, as well as the downstream targets and mechanisms of KDM5B, remains poorly understood. The aim of this project is to investigate the function of KDM5B in cortical development, which I will do by generating a KDM5B KO with a CRISPR/Cas9 pipeline that was optimised in the Basak Lab. To disrupt the function of KDM5B, I designed, amplified and cloned four different gRNAs that are part of a pCAG-Cre-U6-4xgRNA construct. Next, I introduced plasmids containing this construct in NSCs with an in utero electroporation (IUE) at embryonic day 13 (E13) and isolated the embryos at E17. To validate the efficiency of the CRISPR/Cas9 complex, I analysed the loss of GFP fluorescence in the control group during FACS. Immunohistochemistry shows a lower expression of KDM5B in the mutant, indicating the successful generation of a KDM5B KO. This KDM5B KO results in a relatively lower number of cells below cortical layers V and VI. A lower percentage of KDM5B KO cells among the live cells during FACS suggests a possible indispensable effect of KDM5B on the proliferation of NSCs. This is supported by the observation that KDM5B is highly expressed in the ventricular zone. By generating a KDM5B KO, I thus show that KDM5B potentially is involved in the proliferation of NSCs. These are the first steps in determining the function of KDM5B in corticogenesis, which can help in unravelling how bivalent chromatin modifications affect the development of the cortex, and how this might be altered in ASD patients. This will contribute to our overall understanding of the role of epigenetic regulation in ASD