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Fig. 7

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ZDB-IMAGE-190807-25
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Figures for Schulz et al., 2019
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Fig. 7

Functional assessment of the glomerular filtration barrier (GFB) after loss of transmembrane protein 63c (tmem63c) in zebrafish.

(A) Scheme of the GFB in healthy and albuminuric zebrafish embryos. Green fluorescent protein (gc-EGFP) represents an albumin surrogate and is filtrated after impairment of the GFB. fe, fenestrated endothelium; fp, foot processes of podocytes; GBM, glomerular basement membrane. (B) Schematic of tmem63c showing the target regions in Exon 2 (ex2) used for Morpholino (MO)- and CRISPR/Cas9-mediated somatic mutagenesis. (C–F) Bright field view of wildtype embryos at 48 hr post-fertilization (hpf) in uninjected controls (C), ATG-MO injected (D), 159.6 ng/µl ex2-sgRNA injected (E), and 250 ng/µl ex2-sgRNA injected embryos (F). Scale bar = 1 mm. (G–K) Fluorescence microscopy of Tg[fabp10a:gc-EGFP] embryos at 120 hpf. Uninjected control with clearly visible gc-EGFP fluorescence in the trunk vasculature (‘fluorescent’) (G). tmem63c ATG-MO-injected embryo with partial or a complete loss of trunk fluorescence (‘deficient-fluorescent’) (H) and tmem63c ATG-MO +Tmem63c mRNA (Rat) co-injected embryo (I) showing rescue of the phenotype. Tmem63c ex2-sgRNA-injected embryo with partial or a complete loss of trunk fluorescence (‘deficient-fluorescent’) (J) and tmem63c ex2-sgRNA + tmem63c mRNA (ZF) co-injected embryo (K) showing rescue of the phenotype. Scale bar = 1 mm. (L) Analysis of gc-EGFP in the trunk vasculature. Shown are embryos categorized as ‘deficient-fluorescent’ (df), see Materials and method section and Figure 7—figure supplement 2 for details. Experimental groups are normalized to the corresponding uninjected control group per experiment. Uninjected Control (n = 1198); Control-MO (n = 189); tmem63c ATG-MO (n = 227); tmem63c ATG-MO +Tmem63c mRNA (Rat) (n = 230); tmem63c ex2-sgRNA (n = 371); tmem63c ex2-sgRNA + tmem63c mRNA (ZF) (n = 126); One-way ANOVA with Bonferroni’s multiple comparisons test. Values plottet: mean ±SD, dashed line at y = 1 indicates the uninjected control level; *p=0.0002 vs. uninjected Control, #p<0.0001 vs. tmem63c ATG-MO. **p=0.0014 vs. uninjected Control, ##p<0.0001 vs. tmem63c ex2-sgRNA. Data points in the graph represent the ratio per independent experiment, %(Uninjected Control (df)) / %(experimental group (df)), N ≥ 3. (See Figure 7—figure supplement 1 and Figure 7—figure supplement 2).

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