Fig. 7

(A) Diagram of the exon and intron organization of zebrafish stx16. Thick red lines indicate the binding regions of two antisense morpholinos (MOs), one of which targets the start codon (AUG) to block translation of stx16, and the other the mRNA splice acceptor site of exon 4 (splicing) to block splicing of the stx16 mRNA. The two arrows indicate the locations of primers used in RT-PCR analysis in control and stx16 morphants. (B) Western blot analysis showing the expression of Stx16 in control and stx16-MO^AUG-injected embryos at 48 hpf; expression of alpha-actin served as a loading control. (C) RT-PCR showing 319 bp and 867 bp amplicons, which result, respectively, from amplification from exon 4 and exon 4 plus unspliced intron 3 in the control uninjected and stx16-MO^sp-injected embryos. (D) Expression of stx16 at 48 hrs, as detected by ISH. Left-hand panel: lateral view with anterior to the left. Right-hand panel: cross section of the region shown to left. Arrow: pronephric duct. (E) Normarski images (lateral view) of control and stx16 MO^sp-injected embryos at 48 hpf. Bar graph at right shows the percentage of embryos showing abnormalities (edema and curvature). Arrow: pericardial edema; arrowhead: body curvature. (F) pax2.1 expression at 48 hpf, as detected by ISH in the indicated embryos. Left-hand panels: Lateral view of the trunk region of indicated embryos, with anterior to the left. Right-hand panels: Transverse sections of embryos of the left-hand panel. Arrows: pronephric duct. (G) Top panel (i, ii): Representative epifluorescence images of 48-hpf Tg(cldnb:lynEGFP) uninjected control and stx16-MO^sp-injected embryos. Lateral view, anterior to the right, pronephric ducts. Middle panel (iii, iv): Confocal Z-stack images of pronephric ducts of Tg(cldnb:lynEGFP) taken at the regions shown in top panel. Bottom panel (v, vi): transverse sections of Tg(cldnb:lynEGFP) embryos showing the pronephric ducts (arrows). Nucleoli are stained with DAPI. Graph showing the percentages of embryos exhibiting pronephric-duct dilation. (H, I) Confocal (Z-stack) images of transverse sections of Tg(cldnb:lynEGFP) embryos at 48 hpf, showing the expression of PKCζ and pan-cadherin on one side of pronephric duct. Arrowheads: lumens.