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Fig. 3
PI4K2β/AP-1 Complex Regulates Wnt-Dependent Fin Development
(A) Zebrafish embryos injected at the single-cell stage with a morpholino (MO) targeting pi4k2β (MOpi4k2β). Detailed head and tail views of zebrafish noninjected (WT), injected with MOpi4k2β, or injected with MOpi4k2β and MO-insensitive rescue mRNA (MOpi4k2β +rescue) at 24 and 48 hr postfertilization (hpf) are shown.
(B) At 24 hpf (lateral view), pi4k2β morphants display shortened body axes and head defects comparable to Tg[hsp70:dkk1-GFP]w32 fish heat-shocked at the one- to three-somite stage (hs:dkk1). Ventral views of 72 hpf ap-1 and pi4k2β morphants and hs:dkk1 embryos heat shocked at 24 hpf with defective pectoral fin development (arrows) are shown. pi4k2β morphant phenotypes are partially rescued by coinjection of MO-insensitive zebrafish pi4k2β mRNA (+ rescue).
(C) Quantification of MO phenotypes shown in (B) at 72 hpf (>130 embryos per condition; chi-square test: p < 0.001).
(D) Zebrafish embryos injected at the single-cell stage with a MO targeting pi4k2β lose pectoral fins. Coinjection of MO-insensitive pi4k2β mRNA encoding WT, but not dileucine (LLAA) or kinase-inactive (D325A) mutant zPI4K2β, rescued defective pectoral fin development in pi4k2β morphants at 48 hpf (see also Figure S3E).
(E) Dorsal view of in situ hybridization at 28 hpf for Wnt target genes lef1 and tcf7 in the developing pectoral fin bud (arrow). pi4k2β morphants have reduced expression of lef1 (33 out of 36) and tcf7 (28 out of 28) in the developing pectoral fin bud. 100% of Tg[hsp70:dkk1-GFP]w32 embryos heat shocked at 24 hpf show downregulation of lef1 (n = 21) and tcf7 (n = 26) expression.
(F) In situ hybridization shows downregulation of lef1 (23 out of 27), tcf7 (34 out of 36), and tcf4 (27 out of 30) at 38 hpf upon MOpi4k2β injection. 100% of Tg[hsp70:dkk1-GFP]w32 transgenic fish heat shocked at 24 hpf (hs:dkk1) show downregulation of lef1 (n = 32), tcf7 (n = 43), and tcf4 (n = 23). Arrows indicate developing pectoral fin.
(G) pi4k2β expression in pectoral fin buds (boxed area, inset) at 24 hpf visualized by in situ hybridization.