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 + 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)
Xtr wt + otx2 MO (Fig. S2 C (graph))

	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 organizer 49,16,22,23,56 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 midbrainhindbrain 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) .

Acampora, Forebrain and midbrain regions are deleted in Otx2-/- mutants due to a defective anterior neuroectoderm specification during gastrulation. 1995, Pubmed

Acampora, Forebrain and midbrain regions are deleted in Otx2-/- mutants due to a defective anterior neuroectoderm specification during gastrulation. 1995, Pubmed
Agulnick, Interactions of the LIM-domain-binding factor Ldb1 with LIM homeodomain proteins. 1996, Pubmed , Xenbase
Akkers, A hierarchy of H3K4me3 and H3K27me3 acquisition in spatial gene regulation in Xenopus embryos. 2009, Pubmed , Xenbase
Ang, A targeted mouse Otx2 mutation leads to severe defects in gastrulation and formation of axial mesoderm and to deletion of rostral brain. 1996, Pubmed
Arendt, Common ground plans in early brain development in mice and flies. 1996, Pubmed
Artinger, Interaction of goosecoid and brachyury in Xenopus mesoderm patterning. 1997, Pubmed , Xenbase
Blitz, Anterior neurectoderm is progressively induced during gastrulation: the role of the Xenopus homeobox gene orthodenticle. 1995, Pubmed , Xenbase
Chen, Integration of external signaling pathways with the core transcriptional network in embryonic stem cells. 2008, Pubmed
Chen, Ssdp proteins interact with the LIM-domain-binding protein Ldb1 to regulate development. 2002, Pubmed , Xenbase
Choo, Genome-wide analysis of the binding of the Hox protein Ultrabithorax and the Hox cofactor Homothorax in Drosophila. 2011, Pubmed
Ferreiro, Antimorphic goosecoids. 1998, Pubmed , Xenbase
Harland, In situ hybridization: an improved whole-mount method for Xenopus embryos. 1991, Pubmed , Xenbase
Heasman, Patterning the early Xenopus embryo. 2006, Pubmed , Xenbase
Hiratani, Functional domains of the LIM homeodomain protein Xlim-1 involved in negative regulation, transactivation, and axis formation in Xenopus embryos. 2001, Pubmed , Xenbase
Izzi, Foxh1 recruits Gsc to negatively regulate Mixl1 expression during early mouse development. 2007, Pubmed
Jiménez, A conserved motif in goosecoid mediates groucho-dependent repression in Drosophila embryos. 1999, Pubmed
Johnson, Genome-wide mapping of in vivo protein-DNA interactions. 2007, Pubmed
Kato, A component of the ARC/Mediator complex required for TGF beta/Nodal signalling. 2002, Pubmed , Xenbase
Koide, Xenopus as a model system to study transcriptional regulatory networks. 2005, Pubmed , Xenbase
Kuroda, Cloning a novel developmental regulating gene, Xotx5: its potential role in anterior formation in Xenopus laevis. 2000, Pubmed , Xenbase
Latinkić, The Xenopus Brachyury promoter is activated by FGF and low concentrations of activin and suppressed by high concentrations of activin and by paired-type homeodomain proteins. 1997, Pubmed , Xenbase
Lemaire, The vertebrate organizer: structure and molecules. 1996, Pubmed , Xenbase
Machanick, MEME-ChIP: motif analysis of large DNA datasets. 2011, Pubmed
Matsuo, Mouse Otx2 functions in the formation and patterning of rostral head. 1995, Pubmed
Mochizuki, Xlim-1 and LIM domain binding protein 1 cooperate with various transcription factors in the regulation of the goosecoid promoter. 2000, Pubmed , Xenbase
Mortazavi, Mapping and quantifying mammalian transcriptomes by RNA-Seq. 2008, Pubmed
Noyes, Analysis of homeodomain specificities allows the family-wide prediction of preferred recognition sites. 2008, Pubmed
Pannese, The Xenopus homologue of Otx2 is a maternal homeobox gene that demarcates and specifies anterior body regions. 1995, Pubmed , Xenbase
Pavesi, Weeder Web: discovery of transcription factor binding sites in a set of sequences from co-regulated genes. 2004, Pubmed
Puelles, Otx2 regulates the extent, identity and fate of neuronal progenitor domains in the ventral midbrain. 2004, Pubmed
Reichert, Developmental genetic evidence for a monophyletic origin of the bilaterian brain. 2001, Pubmed
Rivera-Pérez, Goosecoid is not an essential component of the mouse gastrula organizer but is required for craniofacial and rib development. 1995, Pubmed , Xenbase
Sander, The opposing homeobox genes Goosecoid and Vent1/2 self-regulate Xenopus patterning. 2007, Pubmed , Xenbase
Sandmann, A core transcriptional network for early mesoderm development in Drosophila melanogaster. 2007, Pubmed
Shanmugam, PBX and MEIS as non-DNA-binding partners in trimeric complexes with HOX proteins. 1999, Pubmed
Shawlot, Requirement for Lim1 in head-organizer function. 1995, Pubmed
Slattery, Genome-wide tissue-specific occupancy of the Hox protein Ultrabithorax and Hox cofactor Homothorax in Drosophila. 2011, Pubmed
Sudou, Dynamic in vivo binding of transcription factors to cis-regulatory modules of cer and gsc in the stepwise formation of the Spemann-Mangold organizer. 2012, Pubmed , Xenbase
Suga, Xenopus ADAMTS1 negatively modulates FGF signaling independent of its metalloprotease activity. 2006, Pubmed , Xenbase
Taira, Expression of the LIM class homeobox gene Xlim-1 in pronephros and CNS cell lineages of Xenopus embryos is affected by retinoic acid and exogastrulation. 1994, Pubmed , Xenbase
Vignali, Xotx5b, a new member of the Otx gene family, may be involved in anterior and eye development in Xenopus laevis. 2000, Pubmed , Xenbase
Visel, ChIP-seq accurately predicts tissue-specific activity of enhancers. 2009, Pubmed
Whyte, Master transcription factors and mediator establish super-enhancers at key cell identity genes. 2013, Pubmed
Wilson, Cooperative dimerization of paired class homeo domains on DNA. 1993, Pubmed
Yamamoto, Molecular link in the sequential induction of the Spemann organizer: direct activation of the cerberus gene by Xlim-1, Xotx2, Mix.1, and Siamois, immediately downstream from Nodal and Wnt signaling. 2003, Pubmed , Xenbase
Yamashita, Genome-wide characterization of transcriptional start sites in humans by integrative transcriptome analysis. 2011, Pubmed
Yao, Goosecoid promotes head organizer activity by direct repression of Xwnt8 in Spemann's organizer. 2001, Pubmed , Xenbase
Yasuoka, Evolutionary origins of blastoporal expression and organizer activity of the vertebrate gastrula organizer gene lhx1 and its ancient metazoan paralog lhx3. 2009, Pubmed , Xenbase
Ye, seqMINER: an integrated ChIP-seq data interpretation platform. 2011, Pubmed
Yoon, HEB and E2A function as SMAD/FOXH1 cofactors. 2011, Pubmed , Xenbase
Zeitlinger, Whole-genome ChIP-chip analysis of Dorsal, Twist, and Snail suggests integration of diverse patterning processes in the Drosophila embryo. 2007, Pubmed
van Heeringen, Principles of nucleation of H3K27 methylation during embryonic development. 2014, Pubmed , Xenbase