Characterization of a novel rat model of X-linked hydrocephalus by CRISPR-mediated mutation in L1cam

Abstract

OBJECTIVEEmergence of CRISPR/Cas9 genome editing provides a robust method for gene targeting in a variety of cell types, including fertilized rat embryos. The authors used this method to generate a transgenic rat L1cam knockout model of X-linked hydrocephalus (XLH) with human genetic etiology. The object of this study was to use diffusion tensor imaging (DTI) in studying perivascular white matter tract injury in the rat model and to characterize its pathological definition in histology.METHODSTwo guide RNAs designed to disrupt exon 4 of the L1cam gene on the X chromosome were injected into Sprague-Dawley rat embryos. Following embryo transfer into pseudopregnant females, rats were born and their DNA was sequenced for evidence of L1cam mutation. The mutant and control wild-type rats were monitored for growth and hydrocephalus phenotypes. Their macro- and microbrain structures were studied with T2-weighted MRI, DTI, immunohistochemistry, and transmission electron microscopy (TEM).RESULTSThe authors successfully obtained 2 independent L1cam knockout alleles and 1 missense mutant allele. Hemizygous male mutants from all 3 alleles developed hydrocephalus and delayed development. Significant reductions in fractional anisotropy and axial diffusivity were observed in the corpus callosum, external capsule, and internal capsule at 3 months of age. The mutant rats did not show reactive gliosis by then but exhibited hypomyelination and increased extracellular fluid in the corpus callosum.CONCLUSIONSThe CRISPR/Cas9-mediated genome editing system can be harnessed to efficiently disrupt the L1cam gene in rats for creation of a larger XLH animal model than previously available. This study provides evidence that the early pathology of the periventricular white matter tracts in hydrocephalus can be detected in DTI. Furthermore, TEM-based morphometric analysis of the corpus callosum elucidates the underlying cytopathological changes accompanying hydrocephalus-derived variations in DTI. The CRISPR/Cas9 system offers opportunities to explore novel surgical and imaging techniques on larger mammalian models.

Impaired neural differentiation and glymphatic CSF flow in the Ccdc39 rat model of neonatal hydrocephalus: genetic interaction with L1cam

“…To investigate the epistatic interactions between known hydrocephalus-causing gene mutations, we crossed the Ccdc39 prh allele with the L1cam − allele that we generated recently (Emmert et al, 2019). L1cam y/− single mutant rats show delayed growth and develop mild enlargement of the fourth and lateral ventricles by 3 and 6 weeks postnatally, respectively (Emmert et al, 2019), modeling a mild form of XLH.…”

“…To investigate the epistatic interactions between known hydrocephalus-causing gene mutations, we crossed the Ccdc39 prh allele with the L1cam − allele that we generated recently (Emmert et al, 2019). L1cam y/− single mutant rats show delayed growth and develop mild enlargement of the fourth and lateral ventricles by 3 and 6 weeks postnatally, respectively (Emmert et al, 2019), modeling a mild form of XLH. Survival rate and body weight analyses of male and female postnatal wild-type, L1cam heterozygous/ Ccdc39 heterozygous ( L1cam wt/− ;Ccdc39 wt/prh ), L1cam mutant/ Ccdc39 heterozygous ( L1cam y/− ;Ccdc39 wt/prh ), double-mutant ( L1cam y/− ;Ccdc39 prh/prh ), and L1cam heterozygous/ Ccdc39 mutant ( L1cam wt/− ;Ccdc39 prh/prh ) rats revealed that the stunted growth and premature mortality of Ccdc39 prh/prh rats were exaggerated in the presence of the L1cam -null allele, as evidenced by the decreased survival and growth of male L1cam y/− ;Ccdc39 prh/prh rats ( n =6 and n =2, respectively; P< 0.001 between wild-type and L1cam y/− ;Ccdc39 prh/prh and P< 0.01 between Ccdc39 prh/prh and L1cam y/− ;Ccdc39 prh/prh for survival; P< 0.05 between Ccdc39 prh/prh and L1cam y/− ;Ccdc39 prh/prh for body weight) (Fig.…”

“…The accessibility and affordability of CRISPR/Cas9 genome engineering have allowed multiple scientific groups to generate novel models of congenital hydrocephalus in a variety of organisms, including mice (Morimoto et al, 2019), rats (Emmert et al, 2019) and frogs (Date et al, 2019). The current study demonstrates our successful generation of a robust rat model of neonatal hydrocephalus via the use of CRISPR/Cas9 genome editing to introduce a homozygous splice-site mutation, Ccdc39 c.916+2T>A , in the Ccdc39 gene that we previously identified in the prh mouse mutant.…”

“…The emergence of CRISPR/Cas9 technology provides an accessible method for generating transgenic rat models of congenital hydrocephalus (Emmert et al, 2019) that were unfeasible with previous genetic techniques (Mashimo, 2014; Sander and Joung, 2014). Furthermore, CRISPR/Cas9 offers the opportunity to test genetic modifiers and possible genetic interactions that determine disease severity in congenital hydrocephalus.…”

“…Furthermore, CRISPR/Cas9 offers the opportunity to test genetic modifiers and possible genetic interactions that determine disease severity in congenital hydrocephalus. For instance, X-linked hydrocephalus (XLH), which can result from mutations in the L1 cell adhesion molecule ( L1cam ) gene in mice (Dahme et al, 1997; Demyanenko et al, 1999), rats (Emmert et al, 2019) and humans (Adle-Biassette et al, 2013; Dahme et al, 1997; Rosenthal et al, 1992), varies in severity from hydrocephalus with multiple structural abnormalities and prenatal death to a milder phenotype with cognitive impairment or isolated symptoms even within the same family (Fryns et al, 1991; Serville et al, 1992). CRISPR/Cas9-generated rodent models of congenital hydrocephalus resulting from mutations in different hydrocephalus-related genes, such as L1cam and Ccdc39 , can be interbred to investigate epistatic interactions previously believed to affect mutation penetrance and ventricular size in other models of hydrocephalus (Weller and Gärtner, 2001; Zhang et al, 2006).…”