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

Rescue of the pronephric uptake defect in OCRL1 deficient embryos by suppression of PIP5K.

<p>A. RT-PCR detection of PIP5Kαb and eIF1α in wild-type and <i>ocrl^-/-</i> embryos at the indicated developmental timepoints. B, left. RT-PCR of PIP5Kαb and eIF1α in 3 dpf zebrafish embryos injected with the indicated amount of PIP5Kαb splice morpholino. The asterisk indicates morpholino-induced abnormally spliced PIP5Kαb transcript. Right, mortality of PIP5Kαb morpholino-injected embryos at 24 hpf. C. PtdIns(4,5)P₂ levels in untreated wild-type or <i>ocrl^-/-</i> embryos or embryos injected with 2 ng PIP5Kαb morpholino. Data are presented as the mean ± SE (n = 6–13). Statistical analysis was performed using the one-way ANOVA with a post-hoc Dunnett’s multiple comparisons test. *p < 0.05. D. Images of pronephric uptake of Alexa 488-10 kDa dextran (green) in wild type (WT) or <i>ocrl^-/-</i> embryos or WT or <i>ocrl^-/-</i> embryos injected with 2 ng PIP5Kαb morpholino. The pronephric tubules are indicated with a green dashed line. E. Quantification of pronephric uptake of Alexa 488-10 kDa dextran in each of the indicated embryo types. F. Transverse confocal images showing megalin labelling in the proximal pronephric region of 72 hpf wild-type (WT), <i>ocrl^-/-</i> or <i>ocrl^-/-</i> embryos injected with 2 ng PIP5Kαb morpholino (top) and quantitation of megalin fluorescence (bottom). G. Transverse confocal images showing EEA1 labelling in the proximal pronephric region of 72 hpf wild-type (WT), <i>ocrl^-/-</i> or <i>ocrl^-/-</i> embryos injected with 2 ng PIP5Kαb morpholino. H. Block face scanning electron microscopy images of transverse sections through the proximal pronephric tubule of wild-type (WT), <i>ocrl^-/-</i> or <i>ocrl^-/-</i> embryos injected with 2 ng PIP5Kαb morpholino. The bottom row is a colour-coded version of the top row, with vacuaolar endosomes false coloured in green. I. Quantification of vacuolar endosome number, size and total area. Data in E, F and I 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. Scale bars represent 10 μm (F, G) and 2 μm (H).</p

Impairment of pronephric uptake in OCRL1 deficient zebrafish embryos.

<p>A. Confocal images of wild-type (WT), <i>ocrl^-/-</i> mutant, control morphant or OCRL1 morphant 72 hpf zebrafish embryos that were injected with Alexa 488-10 kDa dextran (white) and imaged after 2.5 h. The pronephric tubules are indicated with a green dashed line. B. Top: Quantification of pronephric uptake of 10 kDa (2.5 h) or 70 kDa dextran (4 h) in control, <i>ocrl^-/-</i> mutant and morphant embryos. Bottom: Representation of normal, low and no dextran uptake in injected. C. Wild-type (WT) and <i>ocrl^-/-</i> mutant embryos were injected with RAP-Cy3 (red) and pronephric accumulation after 60 min monitored by fluorescence microscopy. D. Quantification of pronephric uptake of RAP-Cy3 in control and <i>ocrl^-/-</i> mutant embryos. Data are presented as the mean ± SD. Statistical analysis was performed using the Pearson’s chi-squared test. ***p < 0.0001.</p

Reduced endosomal staining in OCRL1 deficient pronephros.

<p>A-C. Confocal transverse sections of the zebrafish proximal pronephric tubule of 72 hpf wild-type (WT) and <i>ocrl^-/-</i> mutant embryos labelled with antibodies to EEA1 or endofin (A), or to GFP (B and C) to detect expressed GFP-Rab11 (B) or GFP-Rab7 (C). White dashed lines indicate the outline of pronephric tubules. Scale bars represent 10 μm.</p

Elevated TGF-β signaling in <i>Gorab</i>-deficient skin, bone, and fibroblasts.

<p>(A) Quantitation of active and total TGF-β in skin lysates from E18.5 <i>Gorab</i>^Null mice (N = 3). (B) Enhanced expression of TGF-β regulated genes in the diaphysis of four week old <i>Gorab</i>^Prx1 mutants (N = 7–8). (C) Immunofluorescence staining for p-SMAD2 in <i>Gorab</i>^Prx1 mutants and controls at four weeks of age. Representative picture of N = 4 per group. Note stronger signals in periosteum in mutants. Scale bar = 50μm. (D) Western blot of p-SMAD2 in confluent fibroblasts from GO patients and healthy controls (N = 3) and quantitative evaluation. (E) Quantitative PCR to measure expression of TGF-β responsive genes in GO patient-derived fibroblasts (N = 4). P = periosteum, C = cortical bone. E = endosteum.</p

Golgi retention and reduced glycanation of decorin in <i>Gorab</i>-deficient fibroblasts.

<p>(A) Western blot analysis of decorin in control and <i>Gorab</i>^Null MEF cell lysates. The size ranges for the fully glycanated form of decorin (DCN) and of its core protein are indicated. (B) Left, western blot analysis of decorin in lysates of the extracellular matrix produced by cultured control and GO fibroblasts. Right, levels of glycanated decorin were quantified against total decorin levels (N = 3). (C) Analysis of intra-Golgi levels of decorin in co-cultured control and GO human skin fibroblasts. Immunofluorescence labeling was performed with anti-decorin, anti-GORAB and anti-GM130 antibodies. GORAB staining was used to distinguish control (yellow arrows) and GO (red arrows) cells. Decorin fluorescence intensity in both cell types was normalized against that of the Golgi marker GM130 (N = 3, >500 cells analyzed per cell line). Scale bar = 10 μm. (D) Analysis of intra-Golgi levels of dermatan sulfate (DS)-modified proteins in co-cultured control (yellow arrows) and GO (red arrows) human skin fibroblasts. Cells were labeled with anti-DS (GD3A12), anti-GORAB and anti-TGN46 antibodies. The intensity of the GD3A12 fluorescence signals were measured relative to that of the Golgi marker TGN46 (N = 3, >500 cells analyzed per cell line). Scale bar = 10 μm.</p

Loss of <i>Gorab</i> resulted in underglycanation of proteoglycans.

<p>(A) Quantitation of dermatan sulfate and chondroitin sulfate in skin, cartilage, and lung samples from E18.5 <i>Gorab</i>^Null mice (N = 3–4). (B) Total amount of GAGs in the cortical bone of femora from four week old <i>Gorab</i>^<i>Prx1</i> mutants and littermate controls (N = 3–4). (C) Percentage of dermatan sulfate in the total amount of glycosaminoglycans (GAGs). (D) Immunoblotting for decorin in skin samples from E18.5 <i>Gorab</i>^Null mice. Loss of the 100kDa band, corresponding to the fully glycanated decorin, and higher intensity the core protein band in mutant lysate indicate a glycanation defect. (E) Immunoblotting for decorin in cortical bone lysates from four week old <i>Gorab</i>^Prx1 mice and littermate controls also showing higher intensity of lower bands in mutant. (F) Immunoblot of decorin in cortical bone lysates from wildtype (WT) mice at different ages: newborn (P0), 5 weeks (5W), 26 weeks (26W) and 2 years (2Y). Note reduction in glycanation with increasing age. (G) Immunofluorescence detection of decorin and (H) biglycan in tibia of four week old <i>Gorab</i>^Prx1 mice. Sections were not pretreated with chondroitinase. Higher staining intensities therefore indicate lower glycanation of the core proteins in the periosteum. P = periosteum, C = cortical bone. Scale bar = 50μm. Experiments (D) to (H) were repeated at least three times with independent biological samples, representative results are shown.</p

Cortical porosity and osteoblast dysfunction in the <i>Gorab</i>^Prx1 model recapitulate the gerodermia osteodysplastica bone phenotype.

<p>(A) Von Kossa / hematoxylin stained cortical bone of twelve week old <i>Gorab</i>^Prx1 mouse. Inset showing magnified view of large pores in the mutant diaphysis. Scale bar = 200 μm. (B) Sections of metaphyseal cortical bone of the tibia of four week old <i>Gorab</i>^Prx1 mice stained with Masson Goldner trichrome. Scale bar = 100μm. (C) μCT reconstructed image of a spontaneously fractured humerus from a four week old <i>Gorab</i>^Prx1 mouse. Arrowhead = fracture site. Scale bar = 1mm. (D) Histomorphometric quantitation number of osteoblast per bone perimeter (N.Ob/B.Pm), (E) number of osteocytes per cortical bone perimeter (N.Ot/cort B.Pm). (F) Mineral apposition rate (MAR) at the endosteum of tibia midshaft in four week old <i>Gorab</i>^<i>Prx1</i> (N = 4). Inset showing calcein double labeling, C = control, M = mutant. (G) osteoid volume (OV/TV) in secondary spongiosa of the proximal tibia in <i>Gorab</i>^Prx1 mutants vs. controls at four weeks of age (N = 4–6). (H) Altered expression of osteoblast lineage marker genes in femoral cortical bone of four week old <i>Gorab</i>^Prx1 mutants (N = 6–8). (I) Immunohistochemical detection of Sp7/osterix, Spp1/osteopontin and Dmp1 expression in cortical bone. Note higher number of osterix + cells in <i>Gorab</i>^Prx1 mutants. Scale bar = 50μm. (J) Number of osteoclasts per bone perimeter (N.Oc/B.Pm). (K) Opg to Rankl expression ratio in four week old <i>Gorab</i>^<i>Prx1</i> mutants (N = 6). P = periosteum, C = cortical bone, E = endosteum.</p