Mutations in Kinesin family member 6 reveal specific role in ependymal cell ciliogenesis and human neurological development

Abstract

Cerebrospinal fluid flow is crucial for neurodevelopment and homeostasis of the ventricular system of the brain, with localized flow being established by the polarized beating of the ependymal cell (EC) cilia. Here, we report a homozygous one base-pair deletion, c.1193delT (p.Leu398Glnfs*2), in the Kinesin Family Member 6 (KIF6) gene in a child displaying neurodevelopmental defects and intellectual disability. To test the pathogenicity of this novel human KIF6 mutation we engineered an analogous C-terminal truncating mutation in mouse. These mutant mice display severe, postnatal-onset hydrocephalus. We generated a Kif6-LacZ transgenic mouse strain and report expression specifically and uniquely within the ependymal cells (ECs) of the brain, without labeling other multiciliated mouse tissues. Analysis of Kif6 mutant mice with scanning electron microscopy (SEM) and immunofluorescence (IF) revealed specific defects in the formation of EC cilia, without obvious effect of cilia of other multiciliated tissues. Dilation of the ventricular system and defects in the formation of EC cilia were also observed in adult kif6 mutant zebrafish. Finally, we report Kif6-GFP localization at the axoneme and basal bodies of multi-ciliated cells (MCCs) of the mucociliary Xenopus epidermis. Overall, this work describes the first clinically-defined KIF6 homozygous null mutation in human and defines KIF6 as a conserved mediator of neurological development with a specific role for EC ciliogenesis in vertebrates.

The Reissner Fiber is Highly Dynamic in vivo and Controls Morphogenesis of the Spine

“…Using high-speed imaging (10Hz) at higher magnification we also observed rapid movement of Scospondin-GFP-labeled puncta along the Reissner fiber in a rostro-caudal direction, which were occasionally observed to extend away from the fiber, towards the floor plate, and retract back into the bulk Reissner fiber ( Figure S4E As scospondin-GFP ut24 is an endogenous knock-in into a wild-type allele, we are currently precluded from dynamic imaging of the Reissner fiber in the hypomorphic scospondin mutants reported here, using this approach. However, there are obvious phenotypic similarities between scospondin hypomorphic mutants and other cilia-related scoliosis mutants described previously [1,2]. We hypothesized that the loss of the Reissner fiber is a common phenotype associated with the onset of scoliosis in zebrafish.…”

“…Spine morphogenesis requires the integration of multiple musculoskeletal tissues with the nervous system. Cerebrospinal fluid (CSF) physiology is important for development and homeostasis of the central nervous system and its disruption has been linked to scoliosis in zebrafish [1,2]. Suspended in the CSF is an enigmatic glycoprotein thread called the Reissner fiber, which is secreted from the subcomissural organ (SCO) in the brain and extends caudally through the central canal to where it terminates at the base of the spinal cord.…”

“…Recent efforts showed that AISassociated variants in the centrosomal protein gene POC5 lead to cilia defects in cell culture, and generate spine deformities when expressed in zebrafish [4,5]. Moreover, defects in motile ependymal cell cilia leading to disruption of cerebrospinal fluid (CSF) physiology in zebrafish is also associated with scoliosis, resembling AIS [1,2].…”

“…Mutations in the kinesin family member 6 (kif6) gene cause a loss of ependymal cell cilia and scoliosis, without vertebral malformations in zebrafish [1,21]. We assayed the Reissner fiber using AFRU immunostaining in kif6 sko/sko mutants which begin to display subtle axial curvatures at 3dpf [21].…”

“…

Highlights• Hypomorphic mutations in zebrafish scospondin result in progressive scoliosis • The disassembly of the Reissner fiber in scospondin hypomorphic mutants results in the strong upregulation of neuronal receptors and synaptic transport components • An endogenous fluorescent knock-in allele of scospondin reveals dynamic properties of the Reissner fiber during zebrafish development • Loss of the Reissner fiber during larval development is a common feature of zebrafish scoliosis models

SummarySpine morphogenesis requires the integration of multiple musculoskeletal tissues with the nervous system. Cerebrospinal fluid (CSF) physiology is important for development and homeostasis of the central nervous system and its disruption has been linked to scoliosis in zebrafish [1,2]. Suspended in the CSF is an enigmatic glycoprotein thread called the Reissner fiber, which is secreted from the subcomissural organ (SCO) in the brain and extends caudally through the central canal to where it terminates at the base of the spinal cord.

…”