The kinetochore protein, CENPF, is mutated in human ciliopathy and microcephaly phenotypes

Background Mutations in microtubule-regulating genes are associated with disorders of neuronal migration and microcephaly. Regulation of centriole length has been shown to underlie the pathogenesis of certain ciliopathy phenotypes. Using a next-generation sequencing approach, we identified mutations in a novel centriolar disease gene in a kindred with an embryonic lethal ciliopathy phenotype and in a patient with primary microcephaly. Methods and results Whole exome sequencing data from a non-consanguineous Caucasian kindred exhibiting mid-gestation lethality and ciliopathic malformations revealed two novel non-synonymous variants in CENPF, a microtubule-regulating gene. All four affected fetuses showed segregation for two mutated alleles [IVS5-2A>C, predicted to abolish the consensus splice-acceptor site from exon 6; c.1744G>T, p.E582X]. In a second unrelated patient exhibiting microcephaly, we identified two CENPF mutations [c.1744G>T, p.E582X; c.8692 C>T, p.R2898X] by whole exome sequencing. We found that CENP-F colocalised with Ninein at the subdistal appendages of the mother centriole in mouse inner medullary collecting duct cells. Intraflagellar transport protein-88 (IFT-88) colocalised with CENP-F along the ciliary axonemes of renal epithelial cells in age-matched control human fetuses but did not in truncated cilia of mutant CENPF kidneys. Pairwise co-immunoprecipitation assays of mitotic and serum-starved HEKT293 cells confirmed that IFT88 precipitates with endogenous CENP-F. Conclusions Our data identify CENPF as a new centriolar disease gene implicated in severe human ciliopathy and microcephaly related phenotypes. CENP-F has a novel putative function in ciliogenesis and cortical neurogenesis

Zebrafish as a model for kidney function and disease

Kidney disease is a global problem with around three million people diagnosed in the UK alone and the incidence is rising. Research is critical to develop better treatments. Animal models can help to better understand the pathophysiology behind the various kidney diseases and to screen for therapeutic compounds, but the use especially of mammalian models should be minimised in the interest of animal welfare. Zebrafish are increasingly used, as they are genetically tractable and have a basic renal anatomy comparable to mammalian kidneys with glomerular filtration and tubular filtration processing. Here, we discuss how zebrafish have advanced the study of nephrology and the mechanisms underlying kidney disease

The kinetochore protein, CENPF, is mutated in human ciliopathy and microcephaly phenotypes

Background: Mutations in microtubule-regulating genes are associated with disorders of neuronal migration and microcephaly. Regulation of centriole length has been shown to underlie the pathogenesis of certain ciliopathy phenotypes. Using a next-generation sequencing approach, we identified mutations in a novel centriolar disease gene in a kindred with an embryonic lethal ciliopathy phenotype and in a patient with primary microcephaly. Methods and results Whole exome sequencing data from a non-consanguineous Caucasian kindred exhibiting mid-gestation lethality and ciliopathic malformations revealed two novel non-synonymous variants in CENPF, a microtubule-regulating gene. All four affected fetuses showed segregation for two mutated alleles [IVS5-2A>C, predicted to abolish the consensus splice-acceptor site from exon 6; c.1744G>T, p.E582X]. In a second unrelated patient exhibiting microcephaly, we identified two CENPF mutations [c.1744G>T, p.E582X; c.8692 C>T, p.R2898X] by whole exome sequencing. We found that CENP-F colocalised with Ninein at the subdistal appendages of the mother centriole in mouse inner medullary collecting duct cells. Intraflagellar transport protein-88 (IFT-88) colocalised with CENP-F along the ciliary axonemes of renal epithelial cells in age-matched control human fetuses but did not in truncated cilia of mutant CENPF kidneys. Pairwise co-immunoprecipitation assays of mitotic and serum-starved HEKT293 cells confirmed that IFT88 precipitates with endogenous CENP-F. Conclusions: Our data identify CENPF as a new centriolar disease gene implicated in severe human ciliopathy and microcephaly related phenotypes. CENP-F has a novel putative function in ciliogenesis and cortical neurogenesis

Loss of Bardet-Biedl syndrome proteins causes synaptic aberrations in principal neurons

Bardet-Biedl syndrome (BBS), a ciliopathy, is a rare genetic condition characterised by retinal degeneration, obesity, kidney failure, and cognitive impairment. In spite of progress made in our general understanding of BBS aetiology, the molecular and cellular mechanisms underlying cognitive impairment in BBS remain elusive. Here, we report that the loss of BBS proteins causes synaptic dysfunction in principal neurons, providing a possible explanation for the cognitive impairment phenotype observed in BBS patients. Using synaptosomal proteomics and immunocytochemistry, we demonstrate the presence of Bbs proteins in the postsynaptic density (PSD) of hippocampal neurons. Loss of Bbs results in a significant reduction of dendritic spines in principal neurons of Bbs mouse models. Furthermore, we show that spine deficiency correlates with events that destabilise spine architecture, such as impaired spine membrane receptor signalling, known to be involved in the maintenance of dendritic spines. Our findings suggest a role for BBS proteins in dendritic spine homeostasis that may be linked to the cognitive phenotype observed in BBS

Loss of Bardet-Biedl syndrome proteins causes synaptic aberrations in principal neurons.

Bardet-Biedl syndrome (BBS), a ciliopathy, is a rare genetic condition characterised by retinal degeneration, obesity, kidney failure, and cognitive impairment. In spite of progress made in our general understanding of BBS aetiology, the molecular and cellular mechanisms underlying cognitive impairment in BBS remain elusive. Here, we report that the loss of BBS proteins causes synaptic dysfunction in principal neurons, providing a possible explanation for the cognitive impairment phenotype observed in BBS patients. Using synaptosomal proteomics and immunocytochemistry, we demonstrate the presence of Bbs proteins in the postsynaptic density (PSD) of hippocampal neurons. Loss of Bbs results in a significant reduction of dendritic spines in principal neurons of Bbs mouse models. Furthermore, we show that spine deficiency correlates with events that destabilise spine architecture, such as impaired spine membrane receptor signalling, known to be involved in the maintenance of dendritic spines. Our findings suggest a role for BBS proteins in dendritic spine homeostasis that may be linked to the cognitive phenotype observed in BBS

E15.5 Fto knockout mice embryos display tissue specific cilia defects.

<p>Paraffin sections from wild type and mutant animals showing immunolocalization of acetylated- α-tubulin (green) and γ-tubulin or IFT88 (red) in the choroid plexus (A,B); nasopharynx (C); cochlea (D); kidney (E). Loss of Fto results in shortened cilia in the choroid plexus, nasopharynx and kidney whilst cilia in the cochlea appear unperturbed. Scale bars: in A, B 50 µm; in C,D,E 20 µm.</p

Loss of <i>fto</i> results in a craniofacial zebrafish phenotype.

<p>(A,B) <i>fto</i> knockdown zebrafish display small eyes, shortened dorsal ventral axis (red brackets), reduced pharyngeal width and length (double headed blue arrows), mislocalised melanocytes (blue arrows and red asterisk) and a curved truncated body axis. Scale bar: 500 µm, n =  con 50/50, <i>fto</i>MO 48/50. (C) Hematoxylin and Eosin staining on paraffin sections highlight loss of lamination and reduced size of the eye in <i>fto</i> morphants at 78hpf (n =  con 10/10, <i>fto</i>MO 10/10. Scale bar: 100 µm. pl; photoreceptor layer, opl; outer plexiform layer, bcl; bipolar cell layer, acl; amacrine cell layer, ipl; inner plaxiform layer, gcl; ganglion cell layer, rpe; retinal pigmented epithelium. (D) Dorsal whole mount view of control uninjected and <i>fto</i> morphant embryos at 48 hpf, morphants have reduced optic spacing (blue brackets) indicative of microcephaly. (E) Hematoxlyin and Eosin staining on transverse paraffin sections through the brain at eye level showing microcephaly in morphant embryos at 78 hpf. Scale bar: 100 µm. ot; optic tectum, t; tegmentum, h; hypothalamus. (F) <i>Fto</i> knockdown was confirmed in 48 hpf morphant embryos by western analysis using an anti-human FTO antibody, note the missing band at approximately 65KDa (arrow head). Asterisks indicate non-specific bands. (G) Embryos treated with the <i>fto</i> MO fail to develop the majority of head cartilage at 5 dpf compared to untreated controls, a reduced basal plate remains intact between treatments (asterisks). <i>p53</i> MO was used to counteract off-target morpholino effects, <i>p53</i> MO failed to rescue the <i>fto</i>MO phenotype. Ventral and lateral views displayed in the top and bottom columns, respectively. Scale bar: 200 µm; n =  con 30/30, <i>fto</i>MO 36/41, <i>fto</i>MO;<i>p53</i>MO 28/32. (H) <i>In situ</i> hybridisation for <i>myod</i> and <i>krox20</i> in control and fto morphants at 14 hpf. Arrowheads indicate a reduction in somite size in morphants compared to controls. Scale bar 200 µm; n =  con 52/55, <i>fto</i>MO 45/48. (I) Aberrant migration of NCCs, visualised using <i>sox10</i> probe, was observed in the head (brackets) and trunk (arrows) of <i>fto</i> morphants. Scale bar: 500 µm; n =  con 33/33, <i>fto</i>MO 27/34.</p

Canonical Wnt signaling is downregulated in <i>fto</i> morphants zebrafish.

<p>(A) Dual luciferase assay using the β-catenin responsive TopFlash construct shows loss of reporter assay activity in <i>fto</i>MO embryos analysed at both 24 hpf (con: 1.000 SEM ±0.073, ftoMO: 0.491 SEM ±0.105) and 48 hpf (con:1.000 SEM ±0.103, ftoMO 0.580 SEM ±0.066) stages. (B) <i>Lef1</i> transcripts, a canonical Wnt target gene, were analysed by <i>in situ</i> hybridisation (ISH) at 48 hpf. <i>Fto</i> morphants showed marked loss of <i>lef1</i> expression in the optic-tectum (arrows). Scale bar: 500 µm. n =  con 56/66, <i>fto</i>MO 40/53. (C) Loss of β-Catenin was confirmed in <i>fto</i> morphants by western blotting at 48 hpf. β-Catenin protein levels were quantified relative to the loading control (Actin). (D) ISH analysis of <i>ctnnb1</i> (zebrafish <i>β-catenin 1</i>) at 48 hpf showed upregulation of transcripts specifically in areas of the lateral hindbrain (arrowheads). Scale bar: 500 µm. n =  con 70/70, <i>fto</i>MO 65/68 (E) <i>Fto</i> morphant <i>Tg(7xTCF-Xla.Siam:GFP)^ia4</i> display loss of GFP accumulation in both the Telen- and Diencephalic regions of the brain when compared to uninjected controls at 48 hpf, embryos viewed from a dorsal perspective. Scale bar: 100 µm; n =  con 20/20, <i>fto</i>MO 18/20.</p