Novel role of the synaptic scaffold protein Dlgap4 in ventricular surface integrity and neuronal migration during cortical development

Subcortical heterotopias are malformations associated with epilepsy and intellectual disability, characterized by the presence of ectopic neurons in the white matter. Mouse and human heterotopia mutations were identified in the microtubule-binding protein Echinoderm microtubule-associated protein-like 1, EML1. Further exploring pathological mechanisms, we identified a patient with an EML1-like phenotype and a novel genetic variation in DLGAP4. The protein belongs to a membrane-associated guanylate kinase family known to function in glutamate synapses. We showed that DLGAP4 is strongly expressed in the mouse ventricular zone (VZ) from early corticogenesis, and interacts with key VZ proteins including EML1. In utero electroporation of Dlgap4 knockdown (KD) and overexpression constructs revealed a ventricular surface phenotype including changes in progenitor cell dynamics, morphology, proliferation and neuronal migration defects. The Dlgap4 KD phenotype was rescued by wild-type but not mutant DLGAP4. Dlgap4 is required for the organization of radial glial cell adherens junction components and actin cytoskeleton dynamics at the apical domain, as well as during neuronal migration. Finally, Dlgap4 heterozygous knockout (KO) mice also show developmental defects in the dorsal telencephalon. We hence identify a synapse-related scaffold protein with pleiotropic functions, influencing the integrity of the developing cerebral cortex.

resulting Dlgap4 tm1a(KOMP)Wtsi mice were then exposed to Cre recombinase to create a nonconditional lacZ-tagged null allele without exon 8 removing the promoter-driven neo cassette.
This generated the Dlgap4 tm1b(KOMP)Wtsi mice that were then phenotyped (Suppl. Fig. 10a). At weaning age, mouse survival was assessed from 55 successfully genotyped mice originating from several different litters and derived from a heterozygous-by-heterozygous breeding scheme. We obtained the expected number of wild type and heterozygous mice, but no homozygous, suggesting that the tm1b allele of Dlgap4 is not compatible with life. To determine the window of death, we carried out a recessive lethality screen at mouse embryonic day 14.5 (E14.5) and found 6 homozygous embryos amongst 35 collected (17%). The reason underlying lethality is unknown and will require further studies; however, these preliminary data suggest that death mainly occurs late during development between E14.5 and weaning age.
Male and female heterozygous mice were studied independently and developed properly.
Mouse weight did not significantly differ from their local matched controls.

Supplementary methods for Dlgap4 mutant mice
Mice on the C57BL/6N background were maintained by the Mouse Genetics Project (MGP) Select pipeline at the Wellcome Sanger Institute (UK) and were given a breeders chow diet (Mouse Breeder Diet 5021, 9% crude fat content, 21% kcal as fat, 0.276ppm cholesterol, Labdiet, London,UK) from weaning. After weaning, animals were housed three to four mice per cage with WT controls housed separately, in specific-pathogen-free environment in individually ventilated cages under 12/12 light/dark cycle with temperature-controlled conditions and free access to food and water with hardwood bedding. All animals were regularly monitored for health and welfare concerns and were additionally checked prior to and after procedures.
We use a statistical model validated for comparison of a small number of knockout mice. More specifically, in inbred mutant mice, we are able to detect neuroanatomical defects with an effect size of 10% or more with 80% detection power using 3 mice per assessed groups (calculated using Gpower) (Collins et al. 2019).

Neuroanatomical studies
Neuroanatomical studies were carried out using 3 heterozygous knock-out Dlgap4 +/and 3 littermate WT mice at 16 weeks old as previously described (Collins et al., 2018). Mice were anaesthetized with Ketamine (100 mg/kg, intraperitoneally) and Xylazine (10 mg/kg, i.p.), blood collected via the retro-orbital route and death confirmed before the brains were dissected and fixed in 4% buffered formalin for 48 hours, then transferred to 70% ethanol. Samples were embedded in paraffin using an automated embedding machine (Sakura Tissue-Tek VIP) and cut at a thickness of 5µm with a microtome in order to obtain sagittal brain section at Lateral +0.60 mm. The sections were then stained with 0.1% Luxol Fast Blue (Solvent Blue 38; Sigma-Aldrich) and 0.1% Cresyl violet acetate (Sigma-Aldrich) and scanned using Nanozoomer 2.0HT, C9600 series at 20× resolution. 40 brain parameters, made of area and length measurements as well as cell level features, were taken blind to the genotype across the sagittal section. Data were analyzed using a linear mixed model to determine whether a brain region was associated with neuroanatomical defect or not.

Ethical considerations in animal use
The care and use of mice in the Wellcome Sanger Institute study was carried out in accordance with UK Home Office regulations, UK Animals (Scientific Procedures) Act of 1986 under UK