Sudou N , Yamamoto S , Ogino H , Taira M .

P40 OD010997 NIH HHS

	Fig. 2. Cis-element analysis of X. laevis and X. tropicalis cer promoter regions using transgenic reporter genes. (A) Deletion and point mutations of cer promoter-EGFP reporter constructs and their dorsal expression at the gastrula stage. Constructs of Xl_cer and Xt_cer promoter regions are schematically shown on the left, and numbers indicate positions from the transcription start site, whereas numbers in blue indicate position of Xt_cer when different from those of Xl_cer. A bent arrow indicates the transcription start site. Fixed embryos were bisected along the dorsal midline and whole-mount in situ hybridization was performed for EGFP mRNA. Dorsal expression of reporter genes was categorized into strong (orange) and partial or weak (pale orange) expression patterns (supplementary material Fig. S3A), and percentage incidences of each category are presented by bar graphs. The total number of embryos examined and those of experiments in the parentheses are indicated on the right. n.d., not determined. (B,C) Whole-mount in situ hybridization analysis of reporter expression. Typical expression patterns of Xl_cer (B) or Xt_cer (C) reporter constructs are shown. (D) Reporter gene expression of T-box mutant constructs.
	Fig. 3. Cooperation of VegT with Mix1, Siamois, Lim1 and Otx2 to activate the cer and gsc enhancers. (A) Dorsal-specific expression of –229Xl_cer and –229Xt_cer as assayed by DNA injection. Experiments were carried out as described in Fig. 1B. (B) Synergy of VegT with a mixture of Mix1, Sia, Lim1 and Otx2. Wild-type constructs of –229Xl_cer (white bar) and –229Xt_cer (diagonally striped bar) (left panel) or T-box mutant constructs of –229Xl_cer (right panel) were co-injected with mRNAs (25 pg mRNA each) for VegT and 4 mix (mix1, sia, lim1 and otx2), as indicated by a plus mark. Mutated T-box sites in –229Xl_cer-MT1, MT2, MT3 and MT123 are indicated by black diamonds, in which TNNCAC was mutated to CNNTAC. (C) Synergy of VegT with Sia and Lim1. The –229Xl_cer construct was co-injected with mRNA as indicated with or without mRNA for VegT. Amounts of injected mRNAs (pg/embryo): vegt, 25; lim1, 25; mix1, 25; sia, 12; otx2, 25. (D) Activation of gsc reporter constructs by VegT. –1500Xl_gsc or –492Xl_gsc was co-injected with vegt mRNA as indicated. The sequence of –1500Xl_gsc has been deposited in DDBJ/Genbank (AB698641).
	Fig. 5. Assignment of expression domains of organizer-expressing genes. (A-C) Blastula to gastrula stages. Expression domains at early blastula (A), mid-blastula (B for group 1; B′ for group 2 genes) and early gastrula (C) are surrounded by lines, as indicated on 32-cell blastomere fate maps reported by Bauer et al. (Bauer et al., 1994); brown, green, yellow, red, magenta and blue cells are derived from the B4, A4, A1, B1, C1 and D1 blastomeres, respectively. Group 1 genes, sia, gsc and chd; group 2 genes, lim1, otx2 and cer. Dotted lines indicate initially started or expanded expression. (D) Germ layers and the organizer region. The mesoderm (green line) is discriminated from the endoderm (black line) according to the size of cells (supplementary material Fig. S7). The superficial mesoderm (Shook et al., 2004) is indicated by a light magenta. A combined expression domain of chd, gsc, lim1 and otx2 is surrounded by a yellow line, and molecularly defined as the organizer region in this paper (supplementary material Fig. S8). (E) Neurula stage. Expression domains are illustrated on the diagram of bisected neurula embryos (stage 15) based on supplementary material Fig. S5N′ and Fig. S6C.
	Fig. S3. Co-transgenic reporter analysis of cer regulatory regions and responsiveness of cer to VegT. (A) Co-transgenesis for searching cer regulatory regions. The PstI restriction site of the 5′ end of the cer EGFP reporter cassette −62Xt_cer and the 3′ end of PCR fragments (3′ primers have a PstI site) were digested by PstI. Sperm nuclei incubated with these fragments were injected into unfertilized eggs, and normal cleaved eggs were incubated until the gastrula stage. Fixed embryos were bisected along the dorsal midline and whole-mount in situ hybridization was performed for EGFP mRNA. Expression levels were divided into two groups as indicated. (B) Induction of the endogenous cer expression by VegT in the animal cap region. vegt mRNA (100 pg/embryo) was injected into the animal pole region of one blastomere at the two-cell stage. An arrowhead indicates the injected side. (C) Responsiveness of cer reporters to VegT. The reporter construct −229Xt_cer/EGFP with or without vegt mRNA (100 pg/embryo) was injected into the animal pole region at the two-cell stage. Expression of the cer (B) or reporter (C) gene was examined by whole-mount in situ hybridization with antisense probes as indicated.
	Fig. S6. Detailed comparison of expression domains between organizer-expressing genes in early X. laevis embryogenesis. mRNA localizations of organizer-expressing genes as well as other genes for comparison as indicated on the left were visualized by whole-mount in situ hybridization using bisected embryos from the early blastula to neural stages as indicted on the top. Arrows indicate mRNA localization in the BCNE region (green), the presumptive mesoderm or mesoderm (yellow), the presumptive endoderm or endoderm (magenta) and the prechordal plate (black). Arrowheads indicate the blastopore. (A) Stage 8 to 10.5. Maternal vegt mRNA was detected at the early blastula (stage 8) in the entire presumptive endoderm region, whereas zygotic vegt mRNA was seen in the presumptive mesoderm region at mid stage 9 thereafter, as has been reported (Stennard et al., 1999). sia and later gsc start to be expressed in the BCNE region. mix1 expression is already seen at mid-stage 9 in the presumptive endodermal region except for the central region, then expands to the central endoderm and mesodermal region during the late blastula (late stage 9) to the early gastrula stage (stage 10), and gradually decreased from the central region. Both chd and gsc are co-expressed at the late blastula (stage 9) and early gastrula stages (stages 10 and 10.5) in the region overlapping with the sia expression domain. At stage 10/10.5, both chd and gsc expression patterns expand to the dorsal endoderm based on the observation shown in supplementary material Fig. S7. cer expression starts in a central region of the dorsovegetal region (indicated by a magenta arrow), though very weakly, at stage 9 (mid blastula) and expands to the entire dorsal endoderm (indicated by a magenta arrow) and the anterior dorsal mesoderm at stages 10 and 10.5 (supplementary material Fig. S7). The lim1 and otx2 genes are expressed at a mid period of stage 9 in the marginal zone. It seems to start in an endodermal region and expand to the dorsal mesoderm (supplementary material Fig. S7). (B) Colocalization of gsc and vegt or cer and sia. Embryos were bisected along the dorsal-ventral axis. Each counterpart of embryo was treated with whole-mount in situ hybridization for the indicated probe. The data show that gsc expression corresponds to zvegt in the dorsal mesoderm region at mid-blastula stage, while cer and sia are co-expressed in the dorsal endoderm at the mid-blastula stage. (C) Expression pattern at the late gastrula to early neurula stage. Arrows indicate mRNA localization in the prechordal plate. mix1 expression ceased before the late gastrula stage. Note that lim1 mRNA disappears from the prechordal plate (indicated by an arrow at stage 15) and later the notochord, but the Lim1 protein persists at least to the tailbud stage (stage 28; supplementary material Fig. S5N).
	Fig. S6. Detailed comparison of expression domains between organizer-expressing genes in early X. laevis embryogenesis. mRNA localizations of organizer-expressing genes as well as other genes for comparison as indicated on the left were visualized by whole-mount in situ hybridization using bisected embryos from the early blastula to neural stages as indicted on the top. Arrows indicate mRNA localization in the BCNE region (green), the presumptive mesoderm or mesoderm (yellow), the presumptive endoderm or endoderm (magenta) and the prechordal plate (black). Arrowheads indicate the blastopore. (A) Stage 8 to 10.5. Maternal vegt mRNA was detected at the early blastula (stage 8) in the entire presumptive endoderm region, whereas zygotic vegt mRNA was seen in the presumptive mesoderm region at mid stage 9 thereafter, as has been reported (Stennard et al., 1999). sia and later gsc start to be expressed in the BCNE region. mix1 expression is already seen at mid-stage 9 in the presumptive endodermal region except for the central region, then expands to the central endoderm and mesodermal region during the late blastula (late stage 9) to the early gastrula stage (stage 10), and gradually decreased from the central region. Both chd and gsc are co-expressed at the late blastula (stage 9) and early gastrula stages (stages 10 and 10.5) in the region overlapping with the sia expression domain. At stage 10/10.5, both chd and gsc expression patterns expand to the dorsal endoderm based on the observation shown in supplementary material Fig. S7. cer expression starts in a central region of the dorsovegetal region (indicated by a magenta arrow), though very weakly, at stage 9 (mid blastula) and expands to the entire dorsal endoderm (indicated by a magenta arrow) and the anterior dorsal mesoderm at stages 10 and 10.5 (supplementary material Fig. S7). The lim1 and otx2 genes are expressed at a mid period of stage 9 in the marginal zone. It seems to start in an endodermal region and expand to the dorsal mesoderm (supplementary material Fig. S7). (B) Colocalization of gsc and vegt or cer and sia. Embryos were bisected along the dorsal-ventral axis. Each counterpart of embryo was treated with whole-mount in situ hybridization for the indicated probe. The data show that gsc expression corresponds to zvegt in the dorsal mesoderm region at mid-blastula stage, while cer and sia are co-expressed in the dorsal endoderm at the mid-blastula stage. (C) Expression pattern at the late gastrula to early neurula stage. Arrows indicate mRNA localization in the prechordal plate. mix1 expression ceased before the late gastrula stage. Note that lim1 mRNA disappears from the prechordal plate (indicated by an arrow at stage 15) and later the notochord, but the Lim1 protein persists at least to the tailbud stage (stage 28; supplementary material Fig. S5N).
	Fig. S6. Detailed comparison of expression domains between organizer-expressing genes in early X. laevis embryogenesis. mRNA localizations of organizer-expressing genes as well as other genes for comparison as indicated on the left were visualized by whole-mount in situ hybridization using bisected embryos from the early blastula to neural stages as indicted on the top. Arrows indicate mRNA localization in the BCNE region (green), the presumptive mesoderm or mesoderm (yellow), the presumptive endoderm or endoderm (magenta) and the prechordal plate (black). Arrowheads indicate the blastopore. (A) Stage 8 to 10.5. Maternal vegt mRNA was detected at the early blastula (stage 8) in the entire presumptive endoderm region, whereas zygotic vegt mRNA was seen in the presumptive mesoderm region at mid stage 9 thereafter, as has been reported (Stennard et al., 1999). sia and later gsc start to be expressed in the BCNE region. mix1 expression is already seen at mid-stage 9 in the presumptive endodermal region except for the central region, then expands to the central endoderm and mesodermal region during the late blastula (late stage 9) to the early gastrula stage (stage 10), and gradually decreased from the central region. Both chd and gsc are co-expressed at the late blastula (stage 9) and early gastrula stages (stages 10 and 10.5) in the region overlapping with the sia expression domain. At stage 10/10.5, both chd and gsc expression patterns expand to the dorsal endoderm based on the observation shown in supplementary material Fig. S7. cer expression starts in a central region of the dorsovegetal region (indicated by a magenta arrow), though very weakly, at stage 9 (mid blastula) and expands to the entire dorsal endoderm (indicated by a magenta arrow) and the anterior dorsal mesoderm at stages 10 and 10.5 (supplementary material Fig. S7). The lim1 and otx2 genes are expressed at a mid period of stage 9 in the marginal zone. It seems to start in an endodermal region and expand to the dorsal mesoderm (supplementary material Fig. S7). (B) Colocalization of gsc and vegt or cer and sia. Embryos were bisected along the dorsal-ventral axis. Each counterpart of embryo was treated with whole-mount in situ hybridization for the indicated probe. The data show that gsc expression corresponds to zvegt in the dorsal mesoderm region at mid-blastula stage, while cer and sia are co-expressed in the dorsal endoderm at the mid-blastula stage. (C) Expression pattern at the late gastrula to early neurula stage. Arrows indicate mRNA localization in the prechordal plate. mix1 expression ceased before the late gastrula stage. Note that lim1 mRNA disappears from the prechordal plate (indicated by an arrow at stage 15) and later the notochord, but the Lim1 protein persists at least to the tailbud stage (stage 28; supplementary material Fig. S5N).
	Fig. S8. Assignment of expression domains of organizer genes. The mesoderm (green line) is discriminated from the endoderm according to the size of cells, and from the ectoderm based on the fate map of yellow and red cells, as shown by Bauer et al. (Bauer et al., 1994). Arrowhead indicates dorsal blastopore; bc, blastocoel.
	Fig. S5. Immunodetection of the endogenous Lim1, Otx2, Sia, Mix1 and VegT proteins in X. laevis embryos. (A) Schematic diagrams of antigen regions. A green region of each protein is a part of Lim1 amino acids (aa) 275-403; Accession Number NP_001084128, Sia (aa 1-141; NP_001079305), Mix1 (aa 156−377; NP_001081294), Otx2 (aa 99-288; NP_001084160) and VegT (aa 237-363; NP_001081665), which was used for raising antibodies in rabbits. (B,D,F) Amino acid sequence comparisons. Epitope sequences are indicated by bold and arrows. (C,E,G) Western blotting. Sequence comparison between Lim1 and Lim5/Lhx5 (NP_001084038.1) shows that there is a single stretch of consecutive six amino acids at most in the epitope region (B) and anti-Lim1 antibodies did not crossreact with Lim5 (C). Sequence comparison between Mix1 and Mixer (NP_001081760.1) shows that there is a single stretch of seven identical amino acids at most in the epitope region (D), and anti-Mix1 antibodies did not crossreact with Flag-Mixer. Synthesis of Flag-Mixer was confirmed by western blotting with anti-Flag antibody. Sequence comparison between Otx2 and Otx5 (NP_001081916.1) shows that there are several stretches with more than seven identical amino acids, and anti-Otx2 antibodies crossreacted, though weakly, Flag-Otx5 at the same position as anti-Flag antibody detected. As otx5 is expressed in the organizer in the same manner as otx2, our ChIP-qPCR data for Otx2 included Otx5, but this did not seem to affect the conclusion because Otx5 and Otx2 have supposedly the same function (Kuroda et al., 2000). (H,I) Detection of the endogenous Lim1 and VegT proteins by western blotting. Lysates obtained from 250 gastrula embryos were treated with anti-Lim1 or anti-VegT antibodies, and immunoprecipitates were subjected to western blotting with the same antibodies. One embryo equivalent of lysates from embryos, which had been injected with lim1 or vegt mRNA (100 pg/embryo) was used as positive control. (J-N) Immunostaining for Lim1, Otx2, Sia and Mix1. Bisected gastrula embryos (J-L), the eye at the tailbud stage (M) or tailbud embryos (N) were immunostained with antibodies as indicated. (J′-M′) Higher magnifications of boxed regions indicated in J-M. Staining of animal pole region with anti-Sia antibodies was probably nonspecific, because sia mRNA was not detected by whole-mount in situ hybridization (supplementary material Fig. S6A). The Lim1 protein was detected in the brain (br), spinal cord (sc), notochord (not), prechordal plate (pc) and pronephros (pn) at the tailbud stage (N). (N′,N′′) Higher magnification of boxed regions in N.

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