Phenotype and nephrin expression in nephrin depleted zebrafish.

(A) Three categories of phenotypes were defined in nephrin morpholino injected embryos: embryos without edema (normal), embryos with visible pericardial edema (white arrow) and dysmorphic or dead embryos. Pictures were taken at 5 dpf. (B) 80 embryos per condition were live-screened at 5 dpf and assigned to a phenotype category. Increasing concentrations of the injected nephrin morpholino were associated with an increasing percentage of embryos with pericardial edema, but also with increased numbers of severely dysmorphic or dead embryos. (C) RT-PCR was performed using total RNA extracted from nephrin morpholino (100 μM) and control morpholino injected embryos at 24, 48 and 72 hpf. Reduced expression of normal nephrin (389 bp) was found in the nephrin depleted versus control embryos. Moreover, injection of nephrin morpholino induced alternative splicing resulted in 272 and 1070 bp fragments, resulting from an exon deletion and a retained intron, respectively. (D) Quantitation of nphs1 RNA expression in control versus nephrin morpholino injected larvae after 24, 48 and 72 h of injection. M, marker; Mo, nephrin morpholino injected; Co, control morpholino injected.

Quantification of thrombocytes in nephrin depleted Tg(cd41:EGFP) transgenic zebrafish.

(A) LEFT: GFP-labeled thrombocytes and thrombocyte precursors are formed in the zebrafish caudal hematopoietic tissue (CHT) region (white arrows) at 3 dpf. Representative pictures of the CHT region of a control and a nephrin depleted (100 μM nephrin morpholino) embryo at 3 dpf are shown. No obvious differences in thrombocyte numbers were observed. RIGHT: Pixel intensity was measured using ImageJ software. The graph represents means ± SD from measurements in three embryos per condition. (B) LEFT: Western blot for GFP and β-actin (loading control) was performed using total zebrafish lysates at 3 dpf. A representative blot is shown. RIGHT: Signal intensity was measured using ImageJ software. The graph represents means ± SD from measurements in two repeated experiments. (C) LEFT: Fluorescence-activated cell sorter (FACS) analysis of control zebrafish lysates for CD41 positive cells was performed at 3 dpf. MIDDLE: FACS analysis of morphant zebrafish lysates for CD41 positive cells was performed at 3 dpf. RIGHT: A diagrammatic representation of the number of GFP-positive cells per 100,000 counted cells. For each zebrafish lysate, 500,000 cells were counted and analyzed. Graphs represent means ± SD from three repeated experiments performed in duplicate. Mo, nephrin morpholino injected; Co, control morpholino injected; SCC, side scatter; GFP, green fluorescent protein.

PACAP morpholino suppression and PACAP-38 rescue in nephrin depleted embryos.

(A) Representative larvae of the phenotypes observed in different groups with or without PACAP (adcyap1a and adcyap1b) morpholinos injections (100 μM each). (B) Approximately 100 injected embryos per condition were live-screened at 4 dpf and assigned to a phenotype category. Compared to the control morpholino injection alone, PACAP morpholinos produced a harmful effect when injected together with the control morpholino and a devastating effect with the nphs1 morpholino. (C) Representative larvae of the phenotypes observed in different groups with human PACAP-38 injection (5μM). (D) Approximately 100 injected embryos per condition were live-screened at 4 dpf and assigned to a phenotype category. Human PACAP-38 could rescue to a great extent the PACAP morpholino injected embryos; however, they produced no beneficial effect on the nphs1 morpholino injected embryos.

Phenotype analysis, nephrin expression and evaluation of glomerular function in adriamycin exposed zebrafish.

(A) Different categories of phenotype were defined in the adriamycin exposed embryos at 4 dpf: embryos with a normal phenotype, embryos with visible pericardial edema (white arrow) and dysmorphic or dead embryos. (B) 100 embryos per condition were live-screened at 4 dpf and assigned to a phenotype category. Increasing adriamycin concentrations in the culture medium were associated with an increasing percentage of embryos with pericardial edema. (C) qPCR was performed using total RNA from control and adriamycin exposed embryos at 4 dpf. A significantly decreased expression of nephrin (corrected for housekeeping gene elfa) was observed in the adriamycin exposed embryos compared to the control fish. The experiment was performed twice in triplicate. Bars represent means ± SD. * P<0.05 in comparison to condition without the addition of adriamycin. (D) Rhodamine-labeled 70 kDa dextran was injected in the cardiac venous sinus of 75 hpf old embryos. LEFT: A representative immunofluorescence picture of a control embryo immediately after injection shows the distribution of fluorescence through the vascular system of the zebrafish larva. A dose-dependent diminishing effect of adriamycin on fluorescence recorded in the fish eye 5 hours after injection was observed. Representative images of the eye from 0, 10 and 30 μM adriamycin treated embryos 5 hours after injection are shown. RIGHT: A diagrammatic representation shows the quantification of the mean fluorescence intensity ± SD recorded in the retinal vascular bed. * P < 0.05 in comparison to condition without the addition of adriamycin.

Quantification of thrombocytes in adriamycin treated zebrafish.

(A) LEFT: GFP-labeled thrombocytes and thrombocyte precursors are formed in the zebrafish caudal hematopoietic tissue (CHT). Representative pictures of the CHT region at 4 dpf of embryos exposed to adriamycin 0, 10 and 30 μM are shown. No obvious difference in thrombocytes was observed between the controls and the adriamycin exposed fish. RIGHT: Pixel intensity was measured using ImageJ software. The graph represents means ± SD from measurements in three embryos per condition. No significant difference was observed between adriamycin exposed and control embryos. (B) LEFT: Western blot for GFP and β-actin (loading control) was performed using total zebrafish lysates at 4 dpf. A representative blot is shown. RIGHT: Signal intensity was measured using ImageJ software. The graph represents means ± SD from measurements in two repeated experiments. No significant difference was observed between adriamycin exposed and control embryos.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.