Figure 5

Knockdown of VILL with a morpholino oligonucleotide (MO) interfered with the protein expression of ionocytes in the trunk epithelia of embryos incubated in FW for 6 dpf. The embryos injected with VILL-MO (A, C, and E) or standard control (SC) oligonucleotide (B, D, and F) were whole-mount double-immunofluorescence-stained with anti-VILL (green; A and B) and anti-NKA (red). The fixed regions for observation were indicated with white box on the lateral side of the posterior trunk. The merge images (C and D) revealed that VILL-IR signals were faint in the observed region of VILL-MO embryos compared to the signals of the SC group. The arrows indicated that the VILL-IR signals were found in the guts of both two groups of embryos. YS, yolk sac. (G) Average numbers of NKA-IR cells with VILL signals in the fixed region (the white boxes) of the trunk epithelia were quantified and compared between VILL-MO- and SC-injected embryos at 6 dpf (n=12 for all groups). The number of NKA-IR cells with VILL signals of the VILL-MO-injected embryos was significantly lower than in the SC-injected group. (H) Representative immunoblot of VILL protein detected with a specific polyclonal antibody in the embryos microinjected with VILL-MO or SC. The molecular mass of the single immunoreactive band was 100 kDa. β-actin was used as the loading control. Relative intensities of immunoreactive bands of VILL in embryos of the VILL-MO and SC groups (n=6 for both groups) were analyzed and compared to show that the amount of VILL in the VILL-MO group was significantly lower than in the SC group. The asterisk indicates significant differences (P <0.05, Student’s t-test). The values are means ± S.E.M.