January 1, 2018;
Mutations in Kinesin family member 6 reveal specific role in ependymal cell ciliogenesis and human neurological development.
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.
Disease Ontology terms:
primary ciliary dyskinesia
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Fig 7. Kif6-GFP localizes to the basal bodies and axonemes of Xenopus multi-ciliated cells.
Confocal imaging of the mucocilated Xenopus laevis epidermis demonstrating consistent Kif6-GFP localization within the axonemes (green; A, C) and at the basal bodies (green; D, F). Expression of pan-membrane-RFP marker (magenta; B, C) to co-label the axonemes and Centrin-BFP (blue; E, F) to co-label the basal bodies. Scale Bars: 10μm.
Fig 1. KIF6 mutation in a child with intellectual disability.(A) A low-set prominent anti-helical left pinna. (B) MRI of the brain at 7 months-old shows dolichocephaly with a normal brain structure. (C) and (D) CT of the brain at 8 years-old, sagittal and axial views, respectively show dolichocephalic shape of the cranium (cephalic index = 75) without demonstrable intracranial abnormality. (E) X-ray of the spine shows no scoliosis (F) Electropherograms of the patient, a control, the patient’s father, mother, and unaffected brother from top to bottom. The patient is homozygous for the c.1193delT mutantion while his father, mother, and unaffected brother are all heterozygous carriers. (G) Pedigree and RFLP, using MfeI restriction enzyme: Lane M = 100 bp marker. The arrow head indicates the 500 bp band. Lanes 1–5 are controls. Lanes 6 and 7 are the proband’s father and mother, respectively, showing that they are heterozygous. Lane 8 is the proband showing that he is homozygous for the c.1193delT. (H) Representative KIF6 structure. The arrow shows the position of the c.1193delT mutation.
Fig 2. Kif6
p.G555fs mutant mice display progressive hydrocephaly.(A) Schematic of the non-sense mutation in the patient (KIF6p.L398fsX2) and the mouse mutation (Kif6p.G555fs), both predicted to truncate the C-terminal domain of KIF6 protein. (B) qRT-PCR analyses of fold change of Kif6 expression using cDNA libraries derived from lateral ventricles from WT (black bars) and Kif6p.G555fs (gray bars) mutant mice. (C) Lateral X-rays of mouse cranium at P14 and P28 showing the progressive cranial expansion in Kif6p.G55 homozygous mutant mice. (D) Ventral view of whole mouse P28 brain to highlight hemorrhaging and slight enlargement of total brain size in Kif6p.G55 homozygous mutant mice. (E, F) H&E stained coronal sections of the mouse brain (P14), showing dilation of the lateral (LV) and third (3V) ventricles in Kif6p.G55 homozygous mutant mice (F). (G) Quantitation of ventricular area over total brain area in Kif6p.G55 homozygous mutant mice and heterozygous littermate controls (n = 7 mice per genotype; two-tailed t-test; p = 0.0173). Scale bars: 1cm in (C); 5mm in (D); and 300 μm in (F, F).
Fig 3. Kif6-LacZ expression is specific to the ependymal cells.(A-C', B”-C”) Representative LacZ staining in a variety of multiciliated tissues from P10 and P21 (A”) Kif6-LacZtm1b/+ transgenic mice. (A, A') Coronal section at the 4th ventricle showing specific LacZ expression in the ependymal cell (EC) layer and stark lack of expression in the choroid plexus (CP) or surrounding neuronal tissues. (B-C’) Sectioned oviduct and trachea tissue shows no LacZ expression, despite the presence of tufts of cilia observed under oblique lighting (red arrows B’, C’). Immunofluorescence shows acetylated tubulin labeling of ciliary axonemes in adjacent sections in oviduct which has supercoiled at this time point (B”) and in trachea (C”) at P10. LacZ staining labels the EC cells projecting tufts of cilia observed by oblique lighting (A”). Scale bars: 300μm in (A-C); 20μm in (A'-C'); and 10μm in (A”-C”).
Fig 4. Kif6 mutant mice have defects in formation of ependymal cell cilia.(A-B') Scanning electron microscopy of the lateral ventricular wall (en face view) in Kif6p.G55 homozygous mutant mice and heterozygous littermate controls at P21, demonstrating a reduction in the number and density of EC cilia tufts in mutant ventricles (B, B’). (C-D‴) Immunofluorescence of the wild-type (C-C”‘) and Kif6p.G55 homozygous mutant (D-D”‘) mice at P21. (C-D) Three color merge of (C', D') αS100B (ependymal cell marker; magenta) channel; (C'', D'') α–γ-tubulin (basal bodies; cyan) channel; and (C‴, D‴) αCD133 (EC axoneme marker, Prominin-1; green). (C', D') αS100B staining showing no alterations of ependymal cell specification between Kif6p.G55 homozygous mutant and WT mice. (C'', D'') α–γ-tubulin staining showing typical basal body positioning at the apical surface of ECs in both Kif6p.G55 homozygous mutant and WT mice. (C‴, D‴) αCD133 staining reveals a marked of EC cilia projecting into the ventricular lumen in Kif6p.G555fs homozygous mutant mice compared to WT mice. (E) Quantitation of fluorescent intensity of the CD133 channel (EC axonemes). Scale bars: 20μM in (A, B); 2 μM in (A', B'); and 20 μM in (C-D‴).
Fig 5. kif6 mutant zebrafish display dilation of the ventricular system.(A-B’) 3D-reconstruction of representative iodine-contrasted μCT dataset from WT (A-A’) and kif6sko mutant zebrafish at 90dpf. (C-D’) 3D-reconstruction and segmentation of virtual endocasting of ventricular system in WT (blue; C-C’) and kif6sko mutant (red; D-D’) zebrafish from datasets in A-B’ demonstrated morphological alterations of the ventricular system including dilation of the central canal (red arrows; D-D’) and stenosis of small ventricles (asterisks, D-D’). (E) Schematic of adult zebrafish brain highlighting the relative transverse optical section of the zebrafish brain in WT (F, H, J) and kif6sko homozygous mutant (G, I, K) zebrafish brain at 90dpf. (F, G) The medial region of the TeO showing the medial TecV (yellow dashed line) which is dilated in kif6sko mutant fish (G) compared to age-matched WT (F). (H, I) Sectioning at the region of the medulla oblongata posterior to the lobus facialis showing dysmorphogenesis and deepening of the posterior RV (yellow dashed line) in kif6sko mutants (I) compared with WT (H) zebrafish. (J, K) Spinal cord sectioning showing dilation of the central canal in kif6sko mutant (K) compared to WT (J) zebrafish. (L) Quantitation of the areas (yellow dashed line) of the TecV and the RV posterior to the lobus facialis (pos. RV) in WT and kif6sko mutant zebrafish, highlighting a consistent dilation in kif6sko mutants (n = 11 sections/genotype; two-tailed t-test; ****, p<0.0001). Scale Bars: 1mm. DiV—diencephalic ventricle; TecV-tectal ventricle TeO-tectum opticum; CCe-corpus cerebelli; RV- rhombencephalic ventricle; Vam—medial division of valvula cerebelli; and Cc-central canal.
Fig 6. kif6 mutant zebrafish display defects in the formation of the ependymal cell cilia in the brain.(A-D) Immunofluorescence of Tg[foxj1a::GFP] (A, B) and Tg[foxj1a::Arl13bGFP] transgenic zebrafish in both heterozygous kif6sko/+ (A, C) and homozyogus kif6sko mutant (B, D) zebrafish backgrounds assayed with αGFP (green) and DAPI (nuclei, blue). The Tg[foxj1a::GFP] transgene demonstrates that GFP positive ECs are present in both genotypes. In contrast, kif6sko/+ heterozygous zebrafish display numerous apical tufts of cilia (red arrows; A) projecting into the ventricle lumen, which are markedly reduced in kif6sko mutant zebrafish (B). Similar results were observed with the Tg[foxj1a::Arl13bGFP] transgene, showing a obvious reduction in Arl13b-GFP positive EC axonemes in kif6sko mutant zebrafish (D), which are robustly labeled in kif6sko/+ heterozygous fish (C). Scale Bars: 20μm.
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