PI4K2β/AP-1 Complex Regulates Wnt-Dependent Fin Development

(A) Zebrafish embryos injected at the single-cell stage with a morpholino (MO) targeting pi4k2β (MO^pi4k2β). Detailed head and tail views of zebrafish noninjected (WT), injected with MO^pi4k2β, or injected with MO^pi4k2β and MO-insensitive rescue mRNA (MO^pi4k2β +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 MO^pi4k2β 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.

Role of pi4k2β-­AP-­1 in pectoral fin development and Wnt signaling in zebrafish (A) Expression of EGFP from MO target sequence reporters at 24hpf. a, Injections of AP-­ 1(γ1)_EGFP or b, of zebrafish pi4k2β (zpi4k2β)_EGFP reporter plasmids cause high levels of EGFP expression in WT embryos. c, Similar to non-­injected WT embryos, d, WT embryos co-­ injected with the AP-­1γ1_EGFP reporter plasmid and ap-­1(γ1) MO (250µM) or e, with zpi4k2β_EGFP reporter plasmid and the pi4k2β MO (250µM) completely lack EGFP expression. f, Western blot with quantifications of EGFP levels from embryos as shown in a-­f. Both MOs completely abolish EGFP expression from their respective EGFP reporter plasmids. (B) Quantification of loss-­of-­fin phenotype at 72 hpf in Tg[hsp70:dkk1-­GFP]^w32 embryos. Control (WT) or Tg[hsp70:dkk1-­GFP]^w32 (hs:dkk1) embryos were heat-­shocked at either 24 hpf (n=238) or 48 hpf (n=178). (C) Cos7 cells expressing WT, dileucine (LLAA), kinase-­inactive (D325A) zpi4k2β were analyzed by immunoblotting. All zpi4k2β variants are expressed at similar levels. (D) Localization of myc-­tagged WT, dileucine mutated (LLAA), or kinase-­ inactive (D325A) zpi4k2β (anti-­Myc, green) in Cos7 cells. Nuclei stained with DAPI. Scale bar, 10 µm. (E) Zebrafish embryos injected at single cell stage with MO targeting pi4k2β lose pectoral fins (arrows). 65 ng of MO-­insensitive pi4k2β mRNA encoding WT, dileucine (LLAA) or kinase inactive (D325A) zpi4k2β variants was co-­injected and embryos were analyzed at 48 hpf. WT mRNA partially rescued the morphant phenotype while mRNA encoding D325A and LLAA-­mutant proteins could not. For quantification see figure 3C. (F) Dorsal views of tbx5.1 in situ hybridization at 20 and 24 hpf of control, pi4k2β morphants, heat-­shocked Tg[hsp70:dkk1-­GFP]^w32 (at 17 hpf, hs:dkk1) and SU5402 treated embryos. At 24 hpf, pi4k2β morphants have less tbx5.1 expression. In SU5402 treated embryos, tbx5.1 expression is not affected at 20hpf. At 24 hpf, SU5402 treated embryos show elongation of tbx5.1 expression domain. (G, H, I) Quantification of F: n>40 embryos per condition.