Knockdown of Shroom4 leads to glomerular cysts, eye abnormalities and PE. (A) Western blot analysis demonstrates a protein decrease in shroom4 MO-injected zfl for Shroom4 (184 kDa, F1Q6C1), derived from transcript shroom4-201, in comparison with Ctrl MO-injected zfl. The protein product for transcript shroom4-202 is too small to be detected by Western blot analysis (11 kDa, B3DGK9). β-Actin serves as Ctrl and shows an equal amount of protein in both samples. (B) Quantification of survival (n=5); zfl injected with shroom4 MO show a significant reduction of survival rate at 5 dpf compared with Ctrl MO. Survival of shroom4 MO is significantly rescued by coinjection of human WT SHROOM4 RNA. No alterations in survival have been observed by solely injecting WT SHROOM4 RNA. (C,D) Quantification of PE (n=3) shows significant occurrence in shroom4 morphants, a phenotype, which could be rescued by co-njection of WT SHROOM4 RNA. Black arrowhead highlights PE observed in a zfl injected with shroom4 MO at 4 dpf. (E,F) Eye-to-head ratio of injected zfl at 4 dpf (n=4). Measurement of the eye (red line) and head (black line, distance between the snout to the otic vesicle) was performed as visualised (F). Injection of shroom4 MO significantly reduced eye-to-head ratio, while WT mRNA co-injection in shroom4 MO-injected zfl significantly rescues the phenotypical effect. (G,H) Zfl injected with shroom4 MO develop glomerular cysts (white arrowheads in H) and dilatation of the pronephric ducts (white asterisks in H) that could not be rescued by human WT RNA (n=4). Images from in vivo observation through fluorescence microscopy (dorsal view) in Tg(wt1b:GFP) were taken at 2 dpf. Scale bars represent 100 µm. Ctrl, control; MO, morpholino oligonucleotide; ns, nonsignificant; PE, pericardial effusion; wt, wild type; zfl, zebrafish larvae. p <0.05 (*), p <0.01 (**), p <0.001 (***)
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