Sulfatases, in Particular Sulf1, Are Important for the Integrity of the Glomerular Filtration Barrier in Zebrafish
Figure 2
Morpholino induced knockdown of sulf1, sulf2a, and sulf2b leads to a reduction of circulating eGFP-DBP, pericardial effusion, and yolk sac edema. (a) Measurement of the maximum fluorescence intensity (AU) in the retinal vessel plexus at 96 hpf. Zebrafish embryos were injected in the yolk sac at the 1-2 cell stage with either a scrambled control (CTRL) or with sulf1 MO, sulf2a MO, or sulf2b MO. Additionally, a combined morpholino induced knockdown of Sulf1 and Sulf2a, Sulf1 and Sulf2b and Sulf1, Sulf2a and Sulf2b. In comparison with the CTRL larvae, , , and larvae display significant reduction of the maximum fluorescence indicating a loss of eGFP-DBP from the blood circulation. Neither combined and or and nor all of the three sulfatases were able to decrease the maximum fluorescence intensity below the maximum of a single knockdown of the respective sulfatases. n≥15 for all groups; each circle shows the maximum fluorescence for one individual fish; error bars are SEM; nonsignificant (ns), p≤0.0001; ANOVA and Tukey’s multiple comparison test. (b) Phenotype analysis of the CTRL shows no sign of pericardial effusion, yolk sac edema, or curvature of the tail. Knockdown of Sulf1, Sulf2a, and Sulf2b each lead to marked pericardial effusion, moderate to severe yolk sac edema, and mild curvature of the tail. Scale bar = 500 μm. (c) Real-time polymerase chain reaction quantification (qPCR) of mRNA levels of the sulfs after MO induced knockdown; mRNA levels are depicted as a fold change relative to the control-MO injection. mRNA expression of the sulfs is normalized to the housekeeping gene hprt. induces higher sulf2a and sulf2b expression and leads to an increase of sulf2a mRNA while sulf2b expression is highly variable. does not lead to a compensatory sulf1 or sulf2a mRNA expression. Error bars represent the SEM between three independent experiments (n = 3).