Collective epithelial migration stimulates cell proliferation in the distal tubule.

<p>(A, B) Cell proliferation in the distal tubule (ET11-9 GFP domain) after anterior obstruction. Obstruction was induced at 30 hpf, BrdU incorporation was assessed between 2 and 3 dpf. (A) Total number of BrdU+ nuclei in the distal ET11-9 domain. White bar: control (n = 4), black bar: anterior obstruction (n = 4). P = 0.01. (B) Spatial distribution of the BrdU-positive nuclei per 100 µm length of the distal tubule (measured from the posterior border of the ET11-9 domain). Squares: control (n = 4), Circles: anterior obstruction (n = 4). (C,D) Cell proliferation in the distal nephron in <i>mindbomb</i> mutants. <i>mindbomb</i> (<i>mib</i>) heterozygotes were in-crossed and injected with <i>tnnt2</i> morpholino to control for vascular defects in <i>mib</i> mutants. BrdU incorporation was assessed between 2 and 3 dpf. Homozygous <i>mib</i> mutants were separated from their siblings based on their axis curvature phenotype. (E) Total amount of BrdU incorporation in pronephric duct (black bar: control, n = 8; dark-grey bar: <i>mindbomb</i>, n = 8; P>0.05) and in the posterior distal tubule (light grey bar: control, n = 8; white bar: <i>mindbomb</i>, n = 8; P<0.01). (D) Linear density of the BrdU+ nuclei. Squares: <i>tnnt2MO</i> only control (n = 4); circles: <i>mindbomb</i> +<i>tnnt2MO</i> (n = 4). The underlying bars (B,D) indicate the position of distal tubule/pronephric duct border. (E) 24 hour BrdU incorporation in the posterior proximal tubule (2–3 dpf). (F) BrdU incorporation during 24 hour post-obstruction (2–3 dpf). (G) total number of BrdU positive nuclei in the distal 600 µm of proximal tubule. Black bar: control (n = 4), white bar: obstructed nephrons (n = 8); P<0.01. (H) Proliferation profile in control ET11-9/<i>Tg(atp1a1a.4:GFP)</i> transgenic fish compared to LY294002 treated fish (2–3 dpf). Arrows point to the location of distal tubule/pronephric duct interface. (I) Up-regulation of BrdU incorporation in stretched proximal tubule between 12 and 36 hours post-obstruction. (J) BrdU incorporation was markedly reduced in LY294002 treated, obstructed tubules. (K) Total number of BrdU-positive nuclei in the anterior 500 µm of the proximal tubule. White bar: BrdU incorporation in obstructed nephron/−LY294002 (n = 3), black bar: BrdU incorporation in obstructed nephron/+LY294002 (n = 3); P<0.05. (E,F) Green: GFP (ET33d10 GFP); (I,J) Green – cadherin17; (E,F,I,J) Red: BrdU.</p

Pronephric epithelial proliferation.

<p>(A–C,E,F) Red: anti-BrdU staining, Green: anti-GFP staining. BrdU incorporation was measured for 24 h, between 48 and 72 hpf. (A) Proliferation in the proximal tubule is localized to the neck region (arrow). (B) Proliferation in the anterior part of the distal tubule is low. (C) Proliferation in the <i>ret1</i>-positive pronephric duct segment is high. (D) Cartoon depicting zebrafish pronephros. The proximal tubule (ET33d10 GFP positive domain) and the pronephric duct (<i>ret1</i>:GFP positive domain) are shaded black. The glomerulus and the distal tubule (ET11-9 GFP positive domain) are not shaded. (E) Proliferation in the posterior proximal tubule is low. (F) Proliferation in the posterior distal tubule is high. (G) Quantification of pronephric epithelial proliferation after 24 hour BrdU incorporation. The length of the tubule is plotted on the horizontal axis (measured from the cloaca). The linear density of BrdU positive nuclei (per 100 µm tubule length) is plotted on the vertical axis. Squares: pronephric proliferation between 3 and 4 dpf (n = 3). Circles: proliferation between 1 and 2 dpf (n = 3). See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039992#pone.0039992.s001" target="_blank">Figure S1</a>.</p

Distal proliferation supports prolonged epithelial migration.

<p>(A–D) 1.5 µm confocal slices of the distal tubule. Green: GFP (ET11-9 GFP transgenic in A–B, <i>Tg(atp1a1a.4:GFP)</i> transgenic in C&D). Red: BrdU. Magenta: DAPI. (A) BrdU incorporation in the distal tubule (2–3 dpf) in control fish. (B) Lack of BrdU incorporation in distal tubule between (2–3 dpf) when embryo is treated with 30 µM LY294002. Cells outside of the kidney continued to incorporate BrdU. (C) DAPI staining of distal tubule in control fish (4 dpf). (D) DAPI staining of distal tubule treated with 30 µM LY294002 (4 dpf). The distal tubule was markedly thinned in LY294002 condition (B, D). (E) estimated cross-sectional tubule area based on measured maximal diameter of the tubule in confocal stacks at 3 dpf. Circles: control (n = 5). Squares: LY294002 treated fish (n = 3). (F) Linear nuclear density (DAPI) in control fish (circles, n = 3) and LY294002 treated fish (squares, n = 3) confirmed linear stretching of the distal tubule in Ly294002 treated fish (4 dpf). (G, H) Kidney segment lengths in (G) control fish and (H) LY294002 treated fish at 4dpf. ET11-9 transgenic fish were used to estimate the segment lengths: ‘a’, ‘b’ and ‘c’ represent proximal tubule, distal tubule and the pronephric duct. (I–N) Individual frames of time lapse movies of the actively migrating pronephric epithelia in the presence of 30 µM LY294002. Arrowheads point to the individual traced cells. Time lapse immediately after addition of LY294002 (I, J), 24 hours after addition of LY294002 (K, L), and 48 hours after addition of LY294002 (M, N). Frame pairs in I and J, K and L, and M and N are separated by 6 hours. (O) Average epithelial migration rates after different durations of LY294002 exposure. Each bar represents average migration rate of 4 different individual cells in the proximal tubule (ET11-9:GFP). (P) Lengths of proximal tubule (designated ‘a’ in G), distal tubule (designated ‘b’ in G) and pronephric duct (designated ‘c’ in G) in control 96 hpf fish and fish treated with 30 µM LY294002 starting at 30 hpf. White bars: control (n = 9), black bars: LY294002 (n = 9). See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039992#pone.0039992.s002" target="_blank">Figure S2</a>.</p

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

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

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

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

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

Regenerating epithelia remain differentiated.

<p>Kidney epithelium is shown at 4–6 hours post injury (hpi), on the site of the laser ablation. (A, B): Anti-GFP (green), anti-acetylated-tubulin (red) and DAPI (magenta, A) staining of the injured tubule shows that cilia and cilia bundles (multiciliated cell in A) are present at the edge (white arrowheads, also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101304#pone.0101304.s004" target="_blank">Figure S4</a>) of surviving epithelium (white star in A and B). The aggregated cilia bundles in B (red star) are visible in the middle of the injured segment (between arrowheads). (C and D): Na/K ATPase (red) and Crumbs (green) expression in intact (C) and injured (D) epithelium (also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101304#pone.0101304.s004" target="_blank">Figure S4</a>). AKI does not affect the expression of Crumbs on the apical surface of surviving cells (D, arrow), while its distribution is randomized in the injured segment (right of the arrowhead in D, which marks the border between the intact and the injured segment. (E, F): Vimentin is not expressed in intact kidney epithelium (E) and is not up-regulated after acute injury (F, also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101304#pone.0101304.s004" target="_blank">Figure S4</a>). Vimentin staining can be seen outside of the kidney (arrow in (F)). Bar lengths in (A-F) are 30 µm. (G) Electron microscopy of a longitudinal section at the edge of surviving epithelium (the edge itself is not shown, to the left). Apical junctional complexes (arrow) are preserved. Bar length is 1 µm. One degenerated mitochondrion indicates partial injury to the cell at the edge of the injury (arrowhead). Different colors are used to delineate individual epithelial cells. The virtual slice thickness in (A-F): A- 1.4 µm; B-5.6 µm (7 slices); C,D- 1.4 µm; E- 7.0 µm (8 slices); F- 11.2 µm (15 slices).</p