Hikasa H , Ezan J , Itoh K , Li X , Klymkowsky MW , Sokol SY .

R01 HD031247-14 NICHD NIH HHS , R01 HD031247-15 NICHD NIH HHS , R01 HD031247-16A1 NICHD NIH HHS , R01 NS040972-08 NINDS NIH HHS , R01 NS040972-09 NINDS NIH HHS , R01 NS040972-10 NINDS NIH HHS , R01 HD031247 NICHD NIH HHS , R01 NS040972 NINDS NIH HHS

	Figure 3. Opposite Roles of TCF3 and HIPK2 in Dorsoanterior Development(A) CoMO (60 ng), HK2MO (60 ng), or TCF3MO (40 ng) were injected four times into the marginal zone of four-cell embryos as indicated. Anterior views are shown; dorsal is up. HK2MO enhances, whereas TCF3MO inhibits, the dorsoanterior fate, assessed by head and cement gland development at stage 28 (top). Effects of HIPK2 and TCF3 MOs on Vent2 (middle) and Otx2 (bottom) were analyzed by whole-mount in situ hybridization at stages 14 and 17, respectively. CoMO does not affect Vent2 and Otx2 gene expression (95%, n = 56, and 96%, n = 43, respectively). HK2MO suppresses (68%, n = 56), whereas TCF3MO expands (56%, n = 45), Vent2 expression in the anterior and ventral region. HK2MO expands (56%, n = 50), whereas TCF3MO suppresses (72%, n = 50), the Otx2 expression domain. Arrowheads demarcate cement gland (CG) boundaries (top), and the boundaries of the Vent2 (middle) and the Otx2 (bottom) expression domains.(B) Inhibition of anterior development by TCF3MO is rescued by TCF3 RNA. Embryos shown represent phenotype classification based on CG size at stage 28. Class I, CG is between one-third and one-half of normal size; class II, CG is less than one-third of normal size.(C) HK2MO enhances anterior development; this defect is rescued by RNA encoding HIPK1, a related protein kinase, and partially reversed by TCF3MO. Phenotype classes are based on cement gland size at stage 26. Class I, CG size is increased less than twice; class II, CG size is increased more than twice. RNAs or MOs were injected into the animal pole (B) or the marginal zone (C) of four-cell embryos at indicated doses.(D) HK2MO inhibits Vent2-Luc reporter activation via the TCF-binding site. MOs and reporter DNA were injected six times into the animal pole and the marginal zone for luciferase activity determination analysis at late gastrula stages. Data are shown as means ± SD.
	Figure 5. Wnt-Dependent TCF3 Phosphorylation Relieves Transcriptional Repression(A and B) Xenopus TCF3 phosphorylation mutants enhance the dorsoanterior fate (A) and inhibit Wnt8-dependent reporter activation of BREm-Luc (B). TCF3 RNAs were injected into four animal pole blastomeres at the eight-cell stage. Representative phenotype classes based on cement gland size at stage 28 are shown. Class I, more than double the size of a normal cement gland; class II, more than triple the size of a normal cement gland.(C) Mouse TCF3 P2/3/4 mutant inhibits Wnt-dependent reporter activation in HEK293 cells more efficiently than wild-type TCF3 (p < 0.05 for 1 ng and p < 0.01 for 10 ng, respectively). TOP/FOP is the ratio of STF reporter activity over STM reporter activity, presented as means ± SD.
	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.
	Figure S3. TCF3 is essential for anterior neural development. (A) TCF3MO blocks the anterior neural markers Xanf2 and Rx2a (95%, n=44, and 84%, n=45, respectively), but expands the midbrain-hindbrain boundary marker XHR1 (85%, n=41) and the posterior marker Meis3 (49%, n=89). Krox20 marks rhombomeres 3 and 5. TCF3MO (40 ng) was injected into animal blastomeres of 8 cell stage embryos. Injected embryos have been subjected to in situ hybridization analysis at stage 18. (B) Wnt8 depletion enhances anterior head development. Wnt8 MO (5 ng) was injected four times into the marginal zone of four-cell embryos. Anterior views are shown, dorsal is up. The panel for CoMO-injected embryo is the same image as shown in Fig. 3A.

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