Yasuoka Y , Suzuki Y , Takahashi S , Someya H , Sudou N , Haramoto Y , Cho KW , Asashima M , Sugano S , Taira M .

R01 GM054704 NIGMS NIH HHS , R01 HD073179 NICHD NIH HHS

             head development
             anterior head development

Xla Wt + otx2 + ssbp3 + ldb1 + lhx1 (Fig. 1. C)
Xla Wt + otx2 + ssbp3 + ldb1 + lhx1 mRNA (Fig. S3 A)
Xla Wt + tle1 + gsc + otx2 + ssbp3 + ldb1 + lhx1 mRNA (Fig. 1. C)
Xla Wt + tle1 + gsc + otx2 + ssbp3 + ldb1 + lhx1 mRNA (Fig. S3 A)
Xla Wt + tle1 + gsc + otx2 + ssbp3 + ldb1 + lhx1 mRNA (Fig. S3 B)
Xla Wt + tle1 + otx2 + gsc mRNA (Fig. S3 A)
Xtr Wt + crx MO (Fig. S2 C (graph))
Xtr Wt + crx MO + otx2 MO (Fig. S2 C (graph))
Xtr Wt + crx MO + otx2 MO + lhx1 MO (Fig. S2 C (graph))
Xtr Wt + gsc MO (Fig. S2 C (graph))
Xtr Wt + gsc MO + crx MO + otx2 MO (Fig. S2 C (graph))
Xtr Wt + lhx1 MO (Fig S2 C (graph))
Xtr Wt + lhx1 MO + gsc MO (Fig. S2 C (graph))
Xtr wt + otx2 MO (Fig. S2 C (graph))
Xtr Wt + otx2 MO + crx MO + gsc MO + lhx1 MO (Fig. 1 b row2)
Xtr Wt + otx2 MO + crx MO + gsc MO + lhx1 MO (Fig. S2 C (graph))
Xtr Wt + otx2 MO + crx MO + gsc MO + lhx1 MO (Fig. S2 C (graph))
Xtr Wt + ssbp3 + ldb1 + lhx1 mRNA (Fig. S3 A)

	Figure 1 | Phenotypes of loss- and gain-of-function of head organizer TFs in frogs. (a) Expression patterns of otx2, otx5, lim1 and gsc in Xenopus gastrula embryos. Schematic representation shows co-expression of lim1, otx2, otx5 and gsc in the head organizer4–9,16,22,23,56 and lim1 expression in the trunk organizers16,56. A double-headed arrow indicates the dorsal (D) and ventral (V) axis. Co-expression of these genes in X. tropicalis early gastrula embryos was shown with in situ hybridization using hemisections. In anterior neuroectoderm, otx2 is weakly expressed, while otx5 is more strongly expressed. Arrowhead, dorsal blastopore; open arrowhead, expression in the anterior neuroectoderm; and dashed line, boundary of ectoderm and mesoderm. (b) Loss- of-function analysis in X. tropicalis. Phenotypes of normal embryos (control morphants) and head-reduced embryos (lim1/otx2/otx5/gsc morphants) are shown. Sagittal section and whole mount in situ hybridization of the telencephalon marker (eomes) and the midbrain–hindbrain boundary marker (en2) showed that forebrain, midbrain and foregut were shrunk in head-reduced embryos. Note that the notochord reached the anterior-most region in the morphant. Tailbud embryos are shown in lateral (anterior to the left; three left panels) or dorsoanterior (right-most panels) views. fb, forebrain; fg, foregut; no, notochord. (c) Gain-of-function analysis in X. laevis. Left panels show lateral views of a secondary head with one eye generated after ventral injection (inj.) of a cocktail of mRNAs as indicated. Right panels show immunostaining of head organizer cocktail-injected embryos against notochord by MZ15 (dorsal view of the same embryo as shown in the left panel) and somites by 12/101 (lateral view), respectively. Scale bar, 250 mm.
	Supplementary Figure 2. Loss of function analysis using Xenopus tropicalis embryos (A) Sequence alignment of MOs and mRNAs. WT, wild type; mut, MO resistant mRNAs. Start codons are underlined. (B) Specificity of MOs. Translation of WT mRNA, but not mut mRNA for lim1, otx2, or HA-tagged otx5 was inhibited by the corresponding MO. Western blotting was performed using lysates from X. laevis embryos, which were first injected with MO (5 pmol/embryo), followed by a second injection of lim1, otx2 or HA-tagged otx5 mRNA (200 pg/embryo) to avoid interactions between MO and mRNA prior to injection. AntiYLim1, antiYOtx2, and anti-HA antibodies were used for detection. (C) Morphant phenotypes in X. tropicalis. Five phenotypes were defined as indicated, (+++) being the most severe phenotypes. Tailbud embryos were shown in lateral views. Combinations of injected MOs demonstrated synergisms among lim1, otx2, otx5, and gsc. Phenotypes are summarized as bar graphs. Scale bar is 250 Pm. n, the number of embryos; exp, the number of independent experiments. (D) Rescuing morphant phenotypes in X. tropicalis. mRNAs were injected into the dorsal equatorial region of two blastomeres at 4Ycell stage embryos which had been injected with MOs at 2Ycell stage. Head defects were partially rescued by injection of MO-resistant mRNAs for lim1, otx2, and otx5 in a dose-dependent manner. Significance was examined by chi-square test. (E) Rescue experiments for morphant gene expression. Reduction of gsc and chordin expression by triple MOs was recovered by injection of MO-resistant mRNAs for lim1, otx2, and otx5 in a dose dependent manner. RNA samples 1 to 4 were prepared from embryos corresponding to (D). RTYqPCR was performed to quantify changes in mRNA levels. Relative expression changes compared to control morphants are shown. Normalization was done with the expression level of ef1D. k, P<0.05 (t-test, two-tailed).
	Supplementary Figure 1. Summary of this paper (A) Schematic presentation of gene expression patterns in Xenopus mid-gastrula embryos. Expression patterns of head organizer genes (1, 2) and non-head organizer genes (3Y6) are shown in head, notochord, and posterior mesoderm (indicated by dotted lines) or neural genes (7) in dorsal ectoderm. Some expression patterns were referred to Xenbase (http://www.xenbase.org/common/). (B) Model for Otx2 functions as a molecular landmark of the head. Otx2 is involved in activation of anterior genes (head organizer genes) while repressing the expression of the posterior/ventral genes (trunk genes) within the same cells. Otx2 and Lim1 together with indicated transcriptional factors bind to cis-regulatory modules (CRMs) containing Lim1Ybinding motifs (type I CRMs), which are found among head organizer genes, such as gsc, cerberus, chordin and crescent, as suggested by reporter gene assays 1,2,3, animal cap assays4,5 and this study. Otx2 binding to chordin and crescent CRMs is probably indirect because there are no Otx2 binding motifs (bicoid/P3C) in these CRMs. The co-activator, p300, binds to the CRMs, but the factor directly binding to p300 is unknown. Otx2, Gsc, and TLE bind to CRMs containing multiple bicoid/P3C binding motifs (type II CRMs) located within the trunk genes. Otx2 and Gsc possibly form a heterodimer on P3C sites,6 and bind directly to the co-repressor, TLE/Groucho.7,8 Thus, Otx2 influences expression of a battery of genes, each of which interprets the input for activation or repression via various CRMs to provide head identify. HD, homeodomain; RD, transrepression domain; AD, transactivation domain.
	Supplementary figure 3. Gain of Function analysis using Xenopus laevis embryos. A) head phenotypes. combinations of injected mRNAs demonstrated synergisms among lim1/ldb1/ssbp3, otx2, and gsc/tle1. Phenotypes were classified as indicated and summarized as bar graphs. Statistical significance was examined by chi-squared test. B). Animal cap Rt-qPCR assays. Animal caps injected with control mRNA ( GFP) or the head organizer cocktail ( head. org) or uninfected caps (union.) were used. bars represent relative expression levels +/- s.d. of head organizers gene s ( chrodin, gsc, cerebrus) , a truck organizer gene ( brachyura) , and a muscle marker gene ( actc1) at indicated stages. *, P<0.05; **, P<0.01 ( t-test, two tailed) .

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