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Figure S1. Wnt/HIPK2-dependent TCF3 phosphorylation
(A) Wnt8 does not induce TCF3 phosphorylation before the midblastula stage. All blastomeres of 8-cell stage embryos were injected with Wn8 RNA (L: 40 pg, H: 200 pg) or ECD8 (2 ng).
(B) Wnt3a-containing medium stimulates TCF3 phosphorylation in dissociated ectoderm cells (stage 10) within 45 min.
(C-E) HIPK2 RNA during early development. (C) HIPK2 transcript levels have been assessed by RT-PCR at different developmental stages. Chordin is a zygotic gene, EF1a, a maternal and zygotic gene, is upregulated at late blastula stages. (D and E) Spatial distribution of HIPK2 RNA studied by whole mount in situ hybridization. HIPK2 is expressed strongly in the marginal zone and the animal pole at the late blastula stage (st. 9). At stage 14, HIPK2 RNA is abundant in the notochord and the anterior neural plate (left: dorsal view; right: anterior view).
(F-I) HIPK2 modulates TCF3 phosphorylation. (F) HIPK2 associates with TCF3. Four cell embryos were injected with mycHIPK2δPEST (δP), mycHIPK2KD (KD, kinase-dead), and HATCF3 RNAs (500 pg, 500 pg, and 80 pg, respectively) for the analysis at early gastrula stages. Anti-myc antibodies coprecipitate HATCF3 only in the presence of HIPK2 proteins. HK2δP is an active form of HIPK2 that lacks apoptosis-inducing activity (see Suppl. experimental procedures).
(G) HIPK2 does not alter -catenin stability in animal cap (AC) cells. AC lysates (stage 12.5) from embryos injected with HK2KD or HK2∆P RNA (400 pg each) were probed with anti-TCF3 (Zhang et al., 2003) and ABC (activated β-catenin) antibodies.
(H) HIPK2MO (HK2MO, 20 ng) specifically inhibits the translation of HIPK2 RNA (HK2) in injected embryos, but has no effect on HK2∆5NA lacking target sequence.
(I) HK2KD (400 pg) blocks the phosphorylation of endogenous TCF3 in ventral marginal zone (VMZ) explants. Embryos were dissected as described in Figure 1C.
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