Impairment of pronephros development upon indomethacin treatment.

<p>(A) Overview of pronephric alterations in zebrafish larvae (50 hpf) following indomethacin administration for 24 hours. Row 1 shows control embryos, rows 2-6 zebrafish embryos following indomethacin administration in increasing concentrations (row 2, 0.01 mM; row 3, 0.025 mM; row 4, 0.05 mM; row 5, 0.075 mM and row 6, 0.1 mM). (B-E) Comparison of (B-C) 50 hpf control larva and (D-E) indomethacin (0.1 mM) treated larva. (D) Brightfield image shows edema formation following indomethacin administration. (E) Fluorescence image showing nephron (glomerular and tubular) malformation. (F) Quantification of lethality rates and edema formation following indomethacin administration. (G) Concentration-dependent increases in glomerular malformation and decreases in glomerular fusion rates following indomethacin administration. (H) Widened tubular angles between neck segment and proximal convoluted tubule following indomethacin administration. Data are shown as mean ± SD. *p<0.05, **p<0.001.</p

Detection of cystic kidneys after Ift80 and Ift172 knockdown.

<p>(A) Automatically generated overview thumbnail image of 96 kidneys of morpholino injected larvae: standard ctr-MO (row 1-2), ift80-MO (row 3-5) and ift172-MO (row 6-8). (B-D) Phenotypic alterations of zebrafish larvae at 4 dpf after morpholino microinjection; (C) ift80-MO and (D) ift172-MO injected embryos exhibit glomerular cyst (arrow) and a ventrally curved tail compared to (B) standard ctr-MO injected embryos. (E-G) In-detail visualisation of glomerular cysts at 72 hpf in (F) ift80-MO and (G) ift172-MO injected embryos compared to standard ctr-MO injected embryos (E) using the Tg(wt1b:EGFP) transgenic line. </p

Overview of compound concentration-dependent pronephric phenotypes.

<p>Illustrative examples of pronephroi of a (A) non-treated embryo, and after treatment with (B) 20 mM penicillin, (C) 40 mM ampicillin, (D) 40 mM gentamicin, (E) 40 mM kanamycin, (F) 40 mM acetaminophen, (G) 40 mM captopril, (H) 10 mM losartan. For examples of phenotypes after Indomethacin treatment see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082137#pone-0082137-g004" target="_blank">Figure <b>4A-E</b></a>. Arrow and arrowheads in (A) indicate the different morphological parameters of the pronephros scored to evaluate compound effect on the developing kidney. Arrow: fused glomeruli; Arrowhead: angle between the neck segment and the proximal convoluted tubule. (I-M) Heatmaps showing (I) lethality rates, (J) edema rates and (K-M) changes in morphological parameters of the pronephros. In detail, (K) incomplete glomerular fusion, (L) glomerular malformation and (M) tubular angle. For further details see Materials and Methods and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082137#pone.0082137.s003" target="_blank">Tables <b>S1</b>-<b>S3</b></a>. Colour codes indicate the percentage of embryos (I-L) with particular phenotype, or the angle between neck segment and proximal convoluted tubule (M) as indicated by the colour coded legend. Grey squares indicate missing data points. Concentration ranges used are indicated above the heatmaps, or below for Indomethacin, respectively. Abbreviations: penicillin (Pen), ampicillin (Amp), gentamicin (Gen), kanamycin (Kan), acetaminophen (Ace), captopril (Cap), losartan (Los) and indomethacin (Ind). *p<0.05, **p<0.001.</p

Standardized orientation of zebrafish embryos.

<p>(A,B) Photographs of the brass tool for the simultaneous generation of agarose grooves within 96 well microtiter plates: (A) top view and (B) tilted view. For dimensions of the plate see Materials and Methods section. (C) Schematic depiction of a single well with a ventrally oriented embryo within an agarose cavity. Drawing is not to scale. (D) Illustrative example of aligned and oriented embryos. Shown are dorsal views of 48 hpf embryos acquired using a 2.5x objective on an inverted wide field screening microscope.</p

Overview of workflow for the automated imaging of the developing zebrafish pronephros.

<p>(A) Overview of the workflow for screening larval kidneys. The flowchart illustrates the different steps carried out to obtain overview images of kidneys. (B) Initial compound treatment or microinjection of embryos prior to sample preparation and imaging. (C) Schematic illustrating the transfer of embryos into agarose coated microtiter plates, and alignment and orientation of embryos. (D-G) Acquisition and processing of image Data. D to F show data of the same embryo. (D) Automated acquisition of z-stacks (33 z-slices, dZ=15µm) on an inverted widefield screening microscope. (E) Deblurring of images using deconvolution. Shown are maximum projections of z-stacks of raw data (left panel) and deconvolved data (right panel). (F) Automated detection and cropping of the kidney region. The red square indicates the position and dimensions of the cropped region. (G) Automated generation of overview images for quick assessment of overall morphological changes. Indomethacin skeletal formula in (A) taken from (<a href="http://en.wikipedia.org/wiki/file:indometacin_skeletal.svg" target="_blank"><u>http://en.wikipedia.org/wiki/File:Indometacin_skeletal.svg</u></a>).</p

Additional file 1 of Compound heterozygous IFT140 variants in two Polish families with Sensenbrenner syndrome and early onset end-stage renal disease

Additional file 1. List of genes and SNPs included in the NGS gene panel

Additional file 2 of Compound heterozygous IFT140 variants in two Polish families with Sensenbrenner syndrome and early onset end-stage renal disease

Additional file 2. List of primer sequences used for PCR, Sanger sequencing and qPCR

MOESM1 of Cellular ciliary phenotyping indicates pathogenicity of novel variants in IFT140 and confirms a Mainzer–Saldino syndrome diagnosis

Additional file 1: Figure S1. Schematic overview of the workflow described in this study. Figure S2. Sequence validation of CRISPR/Cas9-derived Ift140 knockout cells

Scribble localizes to the cell-cell contacts and the basolateral membrane of podocytes.

<p>(<b>A</b>) Immunogold electron microscopy of P0 rat kidney sections displays localization of Scribble (arrows) at immature podocyte cell-cell contacts. (<b>B</b>) In adult rat kidney sections Scribble localizes predominantly at the basolateral side of podocyte foot processes and partially at the slit-diaphragm. Scale bars: 200 nm.</p

Migration of apical and basolateral polarity proteins during podocyte differentiation.

<p>Frozen kidney sections of newborn Wistar rat (P0) were stained using antibodies against the apical membrane protein Podocalyxin, the apical polarity protein Par3 and the basolateral polarity protein Scribble and were subjected to confocal laser microscopy. Since glomerular development is asynchronous, kidneys of newborn rats display various glomerular developmental stages. Each panel displays the expression pattern of the accordant proteins during glomerular development (from left to right): Developmental stages ranging from comma-shaped body (I), s-shaped body (II), capillary loop stage (III to IV), to a maturing glomerulus (V). (<b>A</b>) Whereas Par3 is expressed during comma-shaped body stage and localizes to the apical sited cell-cell junctions, expression of Podocalyxin starts during s-shaped body stage, when Par3 and the cell-cell contacts translocate along the lateral side of immature podocytes to basal. During this translocation the apical membrane area, marked by Podocalyxin, increases while the basolateral membrane area shrinks relatively. Arrows indicate translocation of Par3 from the apical cell-cell contacts in I to the developing foot processes in V. (<b>B</b>) Scribble localizes basal of Par3 at the cell-cell junctions and at the basolateral membrane during comma-shaped body stage (I) and translocates like Par3 during podocyte differentation to the developing foot processes in V. (<b>C</b>) While Podocalyxin and Par3 as well as Par3 and Scribble display an partial overlap of their localization (yellow in A and B), no overlap of Podocalyxin and Scribble can be detected indicating a localization to completely distinct membrane areas with Podocalyxin as an apical membrane marker and Scribble as a basolateral marker protein. Scale bars: 5 µm.</p