Induction of primary glomerular disease in larval zebrafish: Zebrafish embryos were injected with 2 nl 100 µM translation-blocking anti wt1a, splice-blocking anti-nphs1 or non-binding control MOs. (A) Total of n = 564 zebrafish embryos were injected and phenotyped at 120 hpf. Phenotype distribution graded in 0:no edema to 3: severe edema with bent body axis and dead larvae. As shown in the stacked boxplots in (B), the phenotype of the wt1a knockdown larvae was generally more severe compared to nphs1 knockdown while the general proportion of larvae with edema was similar in both groups (C). No overt edema was seen in control MO injected larvae and the percentage of larvae with edema of any kind was statistically significant higher compared to paired control groups (paired t-test, p = 0.01). Shown in panel (D) are the results of the zebrafish proteinuria assay. While control MO-injected Tg(fabp10a:gc-eGFP) larvae showed intravascular localization of gc-eGFP, wt1a and nphs1 MO-injected larvae showed significantly less eGFP-fluorescence indicating clearance of the 78 kDa fusion protein from the blood plasma. (E) shows the exon–intron structure of exons 24–26 in the nphs1 gene with respective PCR-primer sites and the SBM MO binding site. Binding of the nphs1 SBM should lead to integration of a 679 bp intron. (F) shows agarose gel-resolved RT-PCR products for the nphs1 region described in (D) for three independent control and nphs1 SBM injected groups. Arrowheads show the size of the respective wildtype-bands at 210 and the shifted PCR product after intronic integration.

Acute and chronic zebrafish podocyte depletion models: (A) Two models of podocyte depletion were used: First, 80 µM MTZ was applied over 48 h. Edema developed in the proportions shown in (A). As shown in (B), mortality increased significantly 48 h after MTZ washout.

Abundant expression of candidate as endogenous control miRs. All miR candidates were detectable in the whole sample set. In general, they exhibit a high homogeneity as indicated by low standard deviations (SD). MiR-92b-3p shows most homogenous expression levels in mixed values (A), strains only (B), MTZ-treatment group (C) and in MO-treated group (D). Data is presented as inter-run calibrator (ICR) corrected expression. Box-Whisker plots with upper and lower quartiles and outliers from technical triplicates. (A) 45 samplings of 20 pooled larvae; (B) 21 samplings of 20 pooled larvae; (C) 15 samplings of 20 pooled larvae; (D) 9 samplings of 20 pooled larvae.

Ranking of potential endogenous control miRs by 4 different normalization determination softwares. Data was ranked by DeltaCt, Normfinder, Genorm and Bestkeeper with respect to mixed values, strains only, MTZ only and morpholinos only as well as the mean rank.

Average ranking of candidate miRs by 4 different normalization determination algorithms. Data was ranked by DeltaCt, Normfinder, Genorm and Bestkeeper. SSS = summarized stability score.

Differences in candidate miR expression between different subgroups. There are no significant differences between the presented subgroups. Data is presented as inter-run calibrator (ICR) corrected expression. All values are shown as mean from technical triplicates. Error bars = SD.

Differences in candidate miR expression between different developmental stages. There are no significant differences between the presented developmental stages in the expression of dre-miR-92a-3p, dre-miR-92b-3p and dre-miR-126a-5p. Data is presented as inter-run calibrator (ICR) corrected expression. All values are shown as mean from technical triplicates. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. Error bars = SD.