FIGURE 10.

PCSK7 regulates TGFβ1a, which affects the zebrafish larva development and otolith formation. A, FURIN-deficient RPE.40 cells were transiently transfected with zebrafish tgfβ1a-myc or human TGFβ1-myc together with zffurinA, zffurinB, zfpcsk7, huFURIN, or huPCSK7. Pro-TGFβ1 (45 kDa) and mature TGFβ1 (16 kDa in zebrafish and 14 kDa in human) expressions were detected with Western blotting (WB). Mature/pro-TGFβ1 ratios were quantified using NIH ImageJ software, and ratios in cells transfected only with tgfβ1 cDNAs were given an arbitrary value of 1. Equal loading of cell lysates and supernatants was verified by Ponceau S staining (data not shown). The experiment was repeated twice with similar results. B, tgfβ1a mRNA expression was measured by QRT-PCR from pcsk7 e3 + p53 and RC morphant embryos at 48 hpf (**, p = 0.0052, two-tailed Student's t test). C and D depict the phenotypes (4 dpf) of zebrafish injected with tgfβ1a + p53 MOs (tgfβ1a MO, 1.0 pmol; p53 MO, 1.5 pmol). tgfβ1a morphants had incorrect otolith numbers: tgfβ1a MO, 65 fish with two otoliths per ear, 21 fish with three otoliths per ear; RC MO, 120 fish with two otoliths per ear, 0 fish with three otoliths per ear (p = 2.16e−9 in Fisher 2 × 2 test).