Hagemann AI , Kurz J , Kauffeld S , Chen Q , Reeves PM , Weber S , Schindler S , Davidson G , Kirchhausen T , Scholpp S .

GM-075252 NIGMS NIH HHS , R01 GM075252 NIGMS NIH HHS

	Fig. 1. AP2μ2 has an essential function in Wnt/β-Catenin signaling. (A–F and H–S) In situ hybridization against lef1 or axin2 at shield stage. Embryos were injected with indicated mRNAs or morpholinos (MOs) and the lateral view is shown ( the dorsal side is on the left). (A–F) Expression of the Wnt target genes axin2 and lef1 is upregulated after Ap2μ2 protein synthesis, whereas the expression of both genes is downregulated when Ap2μ2 function is blocked. (A) n = 33; (B) n = 15/21; (C) n = 19/20; (D) n = 38; (E) 9/13; (F) 15/15. (G–L) Blockage of Ap2μ2 function by injection of 0.5 mM ap2μ2a/b morpholino solution rescues ectopic activation of indicated Wnt target gene expression by 1 ng mRNA of Wnt8, Ck1γ, and 2 ng Lrp6. (G) n = 29; (H) n = 20/28; (I) n = 13/21; (J) n = 45; (K) n = 21/22; (L) n = 17/22. (M–O) Overexpression of 2 ng Ap2μ2a mRNA counteracts the downregulation of lef1 after injection of 2 ng of axin1 mRNA. (P–R) Injection of ap2μ2a/b double morpholino oligomeres did not alter the expression of axin2 after Wnt-activation by 1 ng of β-Catenin mRNA. (M) n = 37; (N) n = 25/26; (O) n = 20/23; (P) n = 44; (Q) n = 23/28; (R) n = 13/16. (S) Luciferase assay with STF reporter construct in shield-stage zebrafish embryos. Error bars are given as +s.e. from four independent measurements. For each measurement ten embryos were pooled. **P<0.005, ***P<0.001. (T) Relative target gene expression analyzed by qRT-PCR with primers against axin2 (blue) and lef1 (red) on embryonic cDNA injected with the indicated constructs. Error bars are given as +s.e. of two independent experiments scanned each in duplicates.
	Fig. 2. Signalosome formation at the cytoplasmic membrane is dependent on Ap2μ2 function. Confocal microscopy analysis of live zebrafish embryos that express the indicated constructs at 30–50% epiboly stage; all constructs were used at 1 ng mRNA, except Axin1 and Dvl2 (used at 2 ng) and Ap2μ2 (used at 1,6 ng). Confocal images represent single z-sections. Constructs were expressed as indicated and are shown in the indicated colors together with 1,2 ng mRNA of the membrane marker CFP-GPI (blue in A,B,D,E–G,L,M). Magnified images in B-G and I represent boxed areas with split channels related to the indicated color code for injected constructs. Expression of Wnt8-GFP mRNA lead to the formation of signalosomes positive for Lrp6, CK1γ, β-Catenin, GSK3β, Axin1 and Dvl2 (A–D, J,L,M). Single channels for images B-G are shown in supplementary material Fig. S3 and control images of the indicated expression constructs in supplementary material Fig. S4. (H) Control of Dvl2 with CFPGPI (green). (I) Fz1 co-expression recruits Dvl2 to the plasma membrane. (J–L) Formation of the signalosome can be induced by expression of Wnt8 mRNA or Ap2μ2 mRNA (K). (L,M) Injection of 0.5 mM morpholino (MO) oligomers targeting Ap2μ2a and Ap2μ2b leads to strong reduction of Wnt8- and Dvl2-positive signalosomes at the plasma membrane. Yellow arrow, signalosome; green arrow, intracellular Wnt8 puncta; red arrow, intracellular Dvl2 puncta. (N,O) Manual quantification and measurment of signalosomes at the plasma membrane with Imaris software. Error bars are given as +s.e. of ten independent samples each. *P<0.05, ****P<0.001.
	Fig. 3. Cell autonomous endocytosis of the signalosome. Confocal microscopy analysis of live zebrafish embryos that express 2 ng mRNA of the indicated constructs at 30–50% epiboly stage; all constructs were used at 2 ng, except Wnt8 (1 ng), CFP-GPI (1,2 ng) and Ap2μ2 (1,6 ng). Confocal images represent single z-sections. Constructs were expressed as indicated and are shown in the indicated colors together with membrane markers CFP-GPI (blue). (A) Still pictures of a time-lapse analysis (from supplementary material Movie 2) of endocytosis of Wnt8 (green) together with Dvl2 (red) from a signalosome. (B–E) Dvl2 does neither colocalize with Wnt8 nor with Lrp6, unless in the plasma membrane after induction by Wnt8 (B,C). (F–I) Embryos treated with 150 nM bafilomycin A1 for 1 hour before scanning show colocalization of Wnt8 with Lrp6 (F,G) or β-Catenin (H,I). Arrow in A indicates a membranous cluster of Wnt8 and Dvl2 that is going to be internalized. Magnified images represent boxed areas with split channels related to the indicated color code for injected constructs.
	Fig. 4. Clonal analysis of signalosome internalization. Confocal microscopy analysis of live zebrafish embryos injected with a set of mRNAs of indicated constructs (2 ng Dvl2, 1,2 ng of CFP-GPI, 1,6 ng Ap2μ2 or Fz1) at one-cell stage and later at eight-cell stage injected with 0.25 ng Wnt8 and 0.1 ng CFP-Histone H2B into one cell only. For convenience, Wnt8-producing clones were marked by nuclear CFP (indicated by dashed lines). Confocal images represent single z-sections. (A,C) Controls. (E) Co-injection of Ap2μ2 morpholinos (MOs) at one-cell stage. (B,D,F) Embryos were treated with bafilomycin A1 for 1 hour before scanning. Magnified images represent boxed areas with split channels related to the indicated color code for injected constructs. (G) Manual quantification of intracellular Dvl2-Cherry vesicles in cells next to Wnt8 clone from (C–F). Error bars are given as +s.e. of ten independent samples each. *P<0.05; *** P<0.005; n.s., not significant (P≥0.05). (H) Automatic quantification after surface application (using Imaris software) of intracellular Wnt8-GFP total volume in 15 responding cells of three different embryos. Represented in volume per cell normalized to volume in control cells (1 = 25,6 µm3). (I) Still pictures of a time-lapse analysis of endocytosis of Wnt8 (green) together with Dvl2 (red) from a signalosome (supplementary material Movie 3). (J) Experimental procedure to generate clones displayed in (A–F and (I).
	Fig. 5. Colocalization of Wnt8 and Dvl2 with endosomal markers. Confocal microscopy analysis of Rab5-GFP or Rab7-GFP transgenic zebrafish embryos that express mRNA of indicated constructs at 30–50% epiboly stages. (A–D) Expression of Dvl2 in Rab5-GFP (A,B) or Rab7-GFP (C,D) zebrafish transgenic line. Dashed lines in E–H indicate the border between Wnt8-positive cell clone (left) and host tissue (right). Confocal images represent single z-sections. Constructs were expressed as indicated and are shown in the indicated colors together with membrane marker CFP-GPI (blue). (E–H) Clonal expression of Wnt8 together with nuclear marker His-CFP in Rab5-GFP (A,B) or Rab7-GFP (C,D) zebrafish transgenic line transiently expressing CFP-GPI. (I) Quantification of colocalization of Dvl2 with Rab5 and Wnt8 with Rab7. Analysis was performed using the Imaris Coloc software module function. Thresholds were set manually and Manders Coefficient for either Dvl2 or Wnt8 was calculated automatically. Error bars represent +s.e. of five independent samples each. (J) Control embryos treated with 0.3% DMSO, (K) Embryos treated with Vacuolin-1 in 0.3% DMSO for 1.5 hours before scanning.
	Fig. 6. Dvl2 stability and activity depends on Ap2μ2 function. (A–C,L,M) Confocal microscopy analysis of live zebrafish embryos that express synthetic mRNA of the indicated constructs and morpholino (MO) oligomers against ap2μ2a/b at 40% epiboly stage. (A–C) Intracellular clusters of Dvl2 are hardly detectable in Ap2μ2 double-morphant embryos (B) and in embryos that express dominant-negative (DN) Dyn (C) compared to control. (D) Western blot analysis of Ap2m2 morphant embryos and Ap2m2-mRNA-expressing embryos shows a reduction of endogenous Dvl2 in double-morphant embryos. PCNA was used as loading control. (E–K) In situ hybridization with axin2 and lef1 probed in embryos injected with indicated constructs at blastula-stage. Embryos shown are representative of 30–40 stained embryos; the lateral view is shown (the dorsal side is on the right). (L,M) Ectopic expression of Ap2μ2 mRNA leads to increased size of Dvl2 aggregate. (N) Quantification of data shown in L and M. Error bars are given as +s.e. of five independent samples each. ****P<0.001.
	Fig. 7. Dvl2 internalization and activity is dependent on Ap2μ2-binding sites. (A–E) In situ hybridization shows decreased activity of the Wnt/β-catenin signaling pathway reflected by the induction of axin2 expression in blastula-stage embryos at 30% epiboly. Embryos were injected with indicated constructs; the lateral view is shown (the dorsal side is on the right). (A) n = 72; (B) 33/39; (C) n = 27/29; (D) n = 23/29; (E) n = 33/35. (F) Relative target gene expression analyzed by real-time PCR with primers against Axin2 (blue) and Lef1 (red) on embryonic cDNA injected with the indicated constructs. Error bars are given as +s.e. of two independent experiments scanned in duplicates. (G,H) Blockage of Dynamin-dependent endocytosis reduces the Axin1-GFP signal in the cytoplasm. Confocal images show 12 µm z-stacks of live embryos at 40% epiboly stage. Indicated constructs were injected at 1 ng as synthetic mRNA, except Axin1 (2 ng), at one-cell stage. (I–M) Axin1 is blocked at the cytoplasmic membrane and its stability reduced when interaction between Dvl2 and AP2μ2 is impaired. (I) Control. Axin1 aggregates in the cytoplasm. (J–L) Co-expression of Dvl2 single- (J,K) and double-mutants (L) together with Axin1. (M) Colocalization of Axin1 with wild-type Dvl2 in cytoplasmic punctae. Confocal images represent single z-sections of live embryos at 40% epiboly stage. Indicated constructs were injected as synthetic mRNA (2 ng each) at one-cell stage.
	Fig. 8. Members of the transducer complex cluster intracellularly with Dvl2. Confocal Images represent single z-sections of live embryos at 40% epiboly stage. Indicated constructs were injected as synthetic mRNA (2 ng each, 1,6 ng of Ap2μ2, 1,2 ng CFP-GPI) at one-cell stage and displayed with indicated colors. Subcellular distribution of Axin1 (A–D), GSK3β (D–H) and β-catenin (I–L) in the context of Dvl2 co-expression. (A,B) Axin1 is distributed as intracellular punctae that colocalize with Dvl2 (A) even in the presence of Ap2μ2 (B). (C) Axin1 is found predominantly in plasma membrane clusters after co-expression of Ap2μ2 and DvlK340M/AHEA double mutant mRNA. (D) Automatic quantification of punctae size in (A,B) as volume after surface application using Imaris software. GSK3β (D–H) clusters in Dvl2-positive aggregates and colocalization can be improved by co-expression of Ap2μ2 mRNA. (G) GSK3β distribution is unaltered (compare with supplementary material Fig. S3E) after co-expression of Ap2μ2 and DvlK340M/AHEA double mutant mRNA. (H) Quantification of punctae size in (E,F). (I) β-Catenin clusters are less apparent in Dvl2-positive aggregates, but colocalization can be improved by co-expression of Ap2μ2 mRNA (J). (G) β-Catenin distribution is unaltered (compare with supplementary material Fig. S3G) after co-expression of Ap2μ2 and DvlK340M/AHEA double mutant mRNA. (L) Quantification of punctae size in (E,F). Error bars are given as +s.e. of ten independent samples each. ****P<0.001; n.s., not significant (P≥0.05).
	Fig. 9. Model for Ap2μ2 function. Schematic drawing of Ap2μ2 action during canonical Wnt/β-Catenin-signaling. (A) In the Wnt-off state, the β-Catenin destruction complex is active and degrades β-Catenin. (B) In the Wnt-on situation, ligand is bound to Fz receptor and Lrp6 co-receptor, thereby inducing Ap2-dependent recruitment of Dvl2 to Fz and signalosome formation at the cytoplasmic membrane. (C) Subsequent internalization is mediated by the interaction of Dvl2 with Ap2μ2 and leads to the sequestration of Axin, CK1γ, GSK3β and β-Catenin in cytoplasmic aggregates. The ligand-receptor complex is routed to late endosomes for degradation or recycling (E), whereas the signal-transducer complex (D) maintains signaling activity by stabilizing Dvl2 and by, for example, keeping GSK3β inactive. Subsequently, the destruction complex becomes activated again by, for example, phosphorylation of Axin1, leading to the initial Wnt-off state (A).

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