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doi: 10.1242/10.1242/dev.00173

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A defect in a novel Nek-family kinase causes cystic kidney disease in the mouse and in zebrafish

¹ Genetics Division, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA
² Renal Unit, Massachusetts General Hospital, Boston, MA, USA
³ Whitehead Institute for Biomedical Research/MIT Center for Genome Research, Cambridge, MA, USA

View larger version (19K): [in a new window]   Fig. 1. Genetic and physical interval between D11Mit116 and D11Mit117 containing the jck mutation. The positions of STS markers, microsatellite markers and BAC clones are shown (not to scale). The brackets define the recombinant interval defined using a cross between B6 jck and Mus castaneus mice (see text). The boxed clones have been sequenced (67H12 is Accession Number AC025628; 199H17 is Accession Number AC022781; and 163A8 is Accession Number AC048361). Nek8 is located on clone 163A8.

View larger version (38K): [in a new window]   Fig. 2. The Nek8 gene is mutated in jck mice. (A) Protein domain structure of the murine NEK8 protein (analysis carried out using the PFAM search server: http://pfam.wustl.edu/index.html). (B) Sequence changes at bp 1346 and 1348 in the jck mutant mouse result in a Gly-to-Val substitution at amino acid 448. (C) Conservation of the region containing the jck mutation across species. Mouse and zebrafish sequence determined as described in the text, human sequence is from multiple ESTs (Accession Numbers BF795289, BG755480 and AA076459), Sus scrofa sequence is from an EST (Accession Number BF189288), Tetraodon sequence is from a genomic survey sequence (Accession Number AL324795), Xenopus sequence is from an EST (Accession Number BE132147). (D) Northern analysis of Nek8 and Actb (ß actin) control. Br, brain; He, heart; Ki, kidney; Li, liver; Lu, lung; Mu, muscle; Sk, skin; Sl, small intestine; Sp, spleen; St, stomach; Te, testes; Th, thymus.

View larger version (194K): [in a new window]   Fig. 3. Immunohistochemical localization of Nek8. Confocal immunofluorescence microscopy using affinity-purified anti-Nek8 revealed strong expression in the apical cytoplasm of two-week-old wild-type inner medullary collecting duct cells (A) shown in cross-section. Similar sections from jck-/- kidneys (B) show diffuse cytoplasmic Nek8 staining, often appearing as a perinuclear ring. Wild-type cortical collecting ducts also showed strong apical expression of Nek8 (C), while cystic collecting duct epithelia in jck-/- tissue (D) show a diffuse pattern of cytoplasmic expression.

View larger version (124K): [in a new window]   Fig. 4. Effect of transient expression of wild-type and mutant Nek8 in cultured cells. (Top) Images of Cos7 cells stained with anti-HA monoclonal antibody and DAPI after transfection with (A) wild-type, (B) jck or (C) kinase-deficient Nek8. Transfection with the mutant Nek8 kinases results in enlarged, multinucleated cells. (Bottom) Images of MDCK cells stained with anti-HA monoclonal antibody, phalloidin and DAPI after transfection with (A) wild-type, (B) jck or (C) kinase-deficient Nek8. Staining with phalloidin alone is shown in A', B' and C'. The Nek8 protein is expressed predominantly in the cytoplasm, and actin stress fibers are reduced in cells that express the mutant Nek8 kinases.

View larger version (162K): [in a new window]   Fig. 5. Epithelial cell adhesion and cytoplasmic organization are disrupted in jck-/- kidneys. Light micrographs of kidney sections from two-week-old wild-type (A) and jck-/- (B) mice from the kidney cortex. Tissue from jck mutants shows cyst formation in the connecting segments and cortical collecting ducts with evidence of cell detachment in the cyst lumens (arrowhead in B). Proximal tubules and glomeruli are not grossly affected in jck kidneys. Electron micrographs of wild type (C) and jck collecting ducts (D) reveal distension of the basal labyrinth in jck epithelia with large vacuolations (asterisk in D) and early signs of cell heightening and detachment (arrowhead in D) prior to overt cyst formation. Basal infoldings seen surrounding mitochondria in wild-type connecting segment cells (white arrowheads in E) are severely disrupted in jck connecting segments (arrowheads in F) and cellular organization is lost with large protrusions of cytoplasm extending into the forming cyst lumen (asterisk in F).

View larger version (34K): [in a new window]   Fig. 6. Pronephric cyst formation in Nek8 antisense oligonucleotide injected zebrafish embryos. (A) Splice donor blocking oligo (see Materials and Methods) disruption of Nek8 mRNA splicing. Sequence analysis of the resulting aberrant Nek8 mRNA in 24 hpf embryos by RT-PCR (B) showed that this oligo induced a 129 nucleotides (43 amino acid) in-frame deletion corresponding to the first RCC1 homology domain C-terminal to the Nek kinase domain. (C) Time course quantification of oligo efficacy showed complete absence of wild-type mRNA at 18 hpf and a gradual recovery of wild-type message by 24-60 hpf. (D) Histological analysis of embryos injected as in C show severe pronephric cysts (*) at 60 hpf. (E) Injection of control random oligo (500 nM cytoplasmic concentration) or lower doses of the splice donor blocking oligo (50 nM cytoplasmic concentration) produces normal pronephric kidney formation at 60 hpf.

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