Impaired neural development in a zebrafish model for Lowe syndrome

Lowe syndrome, which is characterized by defects in the central nervous system, eyes and kidneys, is caused by mutation of the phosphoinositide 5-phosphatase OCRL1. The mechanisms by which loss of OCRL1 leads to the phenotypic manifestations of Lowe syndrome are currently unclear, in part, owing to the lack of an animal model that recapitulates the disease phenotype. Here, we describe a zebrafish model for Lowe syndrome using stable and transient suppression of OCRL1 expression. Deficiency of OCRL1, which is enriched in the brain, leads to neurological defects similar to those reported in Lowe syndrome patients, namely increased susceptibility to heat-induced seizures and cystic brain lesions. In OCRL1-deficient embryos, Akt signalling is reduced and there is both increased apoptosis and reduced proliferation, most strikingly in the neural tissue. Rescue experiments indicate that catalytic activity and binding to the vesicle coat protein clathrin are essential for OCRL1 function in these processes. Our results indicate a novel role for OCRL1 in neural development, and support a model whereby dysregulation of phosphoinositide metabolism and clathrin-mediated membrane traffic leads to the neurological symptoms of Lowe syndrome

TbG63, a golgin involved in Golgi architecture in Trypanosoma brucei

10.1242/jcs.014324Journal of Cell Science12191538-1546JNCS

Measurement of phosphoinositides in the zebrafish Danio rerio

Phosphoinositides represent a minor fraction of the total glycerolipids in cells. Despite the fact that phosphoinositides are present in small quantities, they have crucial roles during cell signaling and in regulating numerous intracellular processes. Measuring changes in the levels of different phosphoinositides in animals is difficult, but it is essential in order to define the important functions of specific members of the phosphoinositide family. Here we detail procedures for measuring phosphoinositides in 2-days-postfertilization (2-d.p.f.) embryos in zebrafish (Danio rerio). Both in vivo radiolabeling (using [(32)P]orthophosphate) followed by thin-layer or high-performance liquid chromatography (TLC or HPLC) analysis and specific in vitro phosphorylation assays (using [(32)P]γATP) permit the quantitative measurement of phosphoinositides. Normalization of both measurements can be achieved by the determination of total lipid phosphate in embryos. All the techniques described are relatively inexpensive and accessible to most laboratories with an interest in studying the effect of gene manipulation on phosphoinositide metabolism in zebrafish. All the procedures described herein will take up to 10 working days.</p

The lowe syndrome protein OCRL1 is involved in primary cilia assembly

Lowe syndrome (LS) is a devastating, X-linked genetic disease characterized by the presence of congenital cataracts, profound learning disabilities and renal dysfunction. Unfortunately, children affected with LS often die early of health complications including renal failure. Although this syndrome was first described in the early 1950s and the affected gene, OCRL1, was identified more than 17 years ago, the mechanism by which Ocrl1 defects lead to LS's symptoms remains unknown. Here we show that LS display characteristics of a ciliopathy. Specifically, we found that patients' cells have defects in the assembly of primary cilia and this phenotype was reproduced in cell lines by knock-down of Ocrl1. Importantly, this defect could be rescued by re-introduction of WT Ocrl1 in both patient and Ocrl1 knock-down cells. In addition, a zebrafish animal model of LS exhibited cilia defects and multiple morphological and anatomical abnormalities typically seen in ciliopathies. Mechanistically, we show that Ocrl1 is involved in protein trafficking to the primary cilia in an Rab8-and IPIP27/Ses-dependent manner. Taking into consideration the relevance of the signaling pathways hosted by the primary cilium, our results suggest hitherto unrecognized mechanisms by which Ocrl1 deficiency may contribute to the phenotypic characteristics of LS. This conceptual change in our understanding of the disease etiology may provide an alternative avenue for the development of therapies

Electron microscopy analysis of endocytic compartments in OCRL1 deficient pronephros.

<p>A. Block face scanning electron microscopy (SEM) images of transverse sections through the zebrafish proximal pronephric tubule of wild-type and <i>ocrl^-/-</i> mutant 72 hpf embryos. The apical membrane, identified by numerous microvilli, lines the central lumen of the pronephric tubule. Vacuolar endosomes are false coloured in green. B and D. Block face SEM showing apical endocytic vesicles at the apical pole of pronephric proximal tubule cells (false coloured in orange in top row) (B) and vacuolar endosomes (false coloured in green in top row) (D). C and E. Quantification of endocytic compartments. Numbers of apical endocytic vesicles were counted per region of interest (C), and vacuolar endosome number, size and total area were counted per entire section (E). Data are presented as the mean ± SD. Statistical analysis was performed using the unpaired t-test. ***p < 0.0001. Scale bars represent 5 μm (A), 2 μm (D) or 1 μm (B).</p

Megalin transcript and protein analysis in OCRL1-deficient zebrafish embryos.

<p>A. Transverse confocal images of the proximal pronephric region of wild-type (WT) and <i>ocrl^-/-</i> mutant 72 hpf embryos labelled with anti-megalin antibodies. The white dashed lines indicate the outline of pronephric tubules. Arrowheads indicate sub-apical punctate and vacuolar megalin staining. B. Transverse confocal images of the proximal pronephric region of 72 hpf <i>ocrl^-/-</i> embryos labelled with antibodies to megalin (green in left panel, red in right panel) and EEA1 (red) or GFP (gfp-, green) to detect ectopically expressed Rab5 or Rab7. mApple (a-) tagged Rab11 is in red. Arrowheads indicate colocalisation. C. Quantification of the relative fluorescence levels of megalin in confocal transverse sections of the indicated embryo types. D. Western blot of 72 hpf wild-type (WT) or <i>ocrl^-/-</i> embryos with antibodies to megalin and tubulin. Three equivalent samples for genotype are analyzed. E. In situ hybridisation of megalin transcript in 48 hpf (top) and 72 hpf (bottom) wild-type (WT) or <i>ocrl^-/-</i> embryos. F. Quantitative RT-PCR (qPCR) of megalin transcript levels in wild type and <i>ocrl^-/-</i> embryos at 72 hpf. Data are presented as the mean ± SD. Statistical analysis was performed using the unpaired t-test. ***p < 0.0001. Scale bars in A, B and E represent 10, 2 and 20 μm respectively.</p

Pronephric cilia in <i>ocrl^-/-</i> zebrafish.

<p>A. Confocal images of pronephric cilia, detected using anti-acetylated tubulin antibody, in wild-type, <i>ocrl^-/-</i> mutant, control morphant or OCRL1 morphant zebrafish embryos (26hpf). B. Fluorescence dissecting microscope image of excretion of Alexa 488-10 kDa dextran from the cloacae of zebrafish embryos (72hpf). Bottom panels show cloacae immediately after injection (left) and excreting dextran 30–60s after injection (wild-type middle, <i>ocrl^-/-</i> right). Dextran excretion was identical in control and <i>ocrl^-/-</i> embryos (20 embryos of each genotype, 2 independent experiments). C. Brightfield images of wild-type (WT), <i>ocrl^-/-</i> mutant or IFT88/polaris morphant (MO) embryos. The morphants were injected with different concentrations of morpholino as indicated. Embryos were imaged using brightfield microscopy. Bottom panel shows <i>ocrl^-/-</i> mutant and polaris morphant (injected with 4 ng MO) and zoom of boxed area. The arrowhead indicates a pronephric cyst in the polaris morphant. D. Confocal images of pronephric cilia, detected using anti-acetylated tubulin antibody, in wild-type (WT), <i>ocrl^-/-</i> mutant or IFT88/polaris morphant (MO) embryos. E. Wild-type (WT), <i>ocrl^-/-</i> mutant and IFT88/polaris morphant embryos were injected with Alexa 488-10 kDa dextran (green) and pronephric accumulation after 2.5 h monitored by fluorescence microscopy. The pronephric tubules are indicated with a dashed line. Uptake was quantitated as indicated. Data are presented as the mean ± SEM. Statistical analysis was performed using the Pearson’s chi-squared test. ***p < 0.0001, **p < 0.001, *p < 0.01. F. Confocal transverse sections of the zebrafish proximal pronephric tubule of 72 hpf wild type and <i>double bubble (dbb</i>) cilia mutant showing 10 kDa-FD uptake into endocytic compartments in pronephric cells 2h after injection. Scale bars represent 10 μm (A and D).</p