PMC:4157146 / 27055-29820 JSONTXT

{"project":"2_test","denotations":[{"id":"25192045-9007226-2045890","span":{"begin":311,"end":313},"obj":"9007226"},{"id":"25192045-24067530-2045891","span":{"begin":862,"end":864},"obj":"24067530"}],"text":"ccdc151 Is Mutated in the Zebrafish flanders Mutant, Leading to Ciliary Defects Including Laterality Defects\nThe evolutionarily conserved role of CCDC151 in vertebrate cilia was verified by examination of zebrafish flanders mutants. ccdc151ts272a (flanders) was generated in the Tübingen ENU mutagenesis screen.56 flanders mutants present morphologically with a ventral body curvature and kidney cysts (Figure 2A), characteristic of mutations that affect ciliary motility in zebrafish. We mapped the flanders mutation to a 2.4 Mb region on chromosome 6 and sequenced exons from candidate genes in this region. A c.631T\u003eA substitution was discovered in exon 6 of ccdc151 (RefSeq NM_001077369.2) that is predicted to introduce a premature stop codon at lysine 211 of the 545 amino acid protein (p.Lys211∗) (Figure 2B). Consistent with what was previously reported,54 whole mount in situ hybridization (WISH) analysis identified ccdc151 expression restricted to tissues that contain motile cilia in zebrafish including the left-right organizer (Kupffer’s vesicle [KV]), the otic vesicle, and the pronephric tubules (Figure 2C). Further support that ccdc151 is the gene mutated in flanders was provided by in situ hybridization showing evidence for nonsense-mediated decay of the transcript in embryos genotyped as mutant, which entirely lacked expression (Figure 2D). In addition, the flanders phenotype could be rescued by injection of ccdc151 RNA (Figure S2), and a phenocopy of the flanders mutant phenotype was generated by antisense morpholino injection (Figures S2 and S3).\nTo examine left-right patterning in flanders mutants and ccdc151 morphants, expression of the nodal gene southpaw (spaw) and the positioning of the visceral organs (heart, liver, and pancreas) were examined (Figure S3). Whereas wild-type siblings express spaw in the left lateral plate mesoderm and display situs solitus, flanders mutant embryos and ccdc151 morphants show randomization of spaw expression, situs inversus, and heterotaxic organ placement (Figures 2E, 2F, and S3). To explore the effect in flanders mutants and ccdc151 morphants on ciliary motility, cilia were imaged using high-speed videomicroscopy in the KV and developing kidney. In flanders mutants, cilia in the KV moved irregularly, occasionally switching direction, or were static (Movies S1 and S2). In the pronephric tubules, ciliary motility appeared less affected because cilia were able to bundle and beat regularly; however, the mutant cilia beat with significantly reduced beat frequency compared to those in unaffected sibling embryos (Figures 2G and S2; Movies S3 and S4). TEM ultrastructural analysis of pronephric cilia from flanders mutants revealed a loss of the ciliary outer dynein arms (Figure 2H)."}