Fig. S1. RT-PCR analyses of the expression and distribution of Xdscr6 transcripts during early development.

Transcription

1 Fig. S1. RT-PCR analyses of the expression and distribution of Xdscr6 transcripts during early development. (A) Temporal expression of Xdscr6 at various stages (numbers on the top) and its distribution at stage 35 in the head (He), trunk (Tr) and posterior (P) regions. (B) Schema showing the dissection of different early gastrula tissues including ectoderm (ect), mesoderm, endoderm, dorsal endoderm (DE) and ventral endoderm (VE). (C) RT-PCR analyses of the distribution of Xdscr6 transcripts in different regions of the early gastrula. (D) Induction of Xdscr6 expression in early gastrula ectoderm following overexpression of squint and Wnt8. ODC was used as an input control. Fig. S2. XDSCR6 differentially induces the expression of nodal-related genes. Embryos at the 2-cell stage were injected at the animal pole region with Xdscr6 mrna (0.5 ng), RT-PCR analyses were performed on control embryo and dissected ectoderm explants at the early gastrula stage.

2 Fig. S3. Comparison of sequence and activity between Xenopus and human RIPPLY2 proteins. (A) Sequence alignment. Conserved residues are indicated in red. The amino- and carboxyl-terminal halves of XDSCR6 used for functional analyses are indicated. (B) RT-PCR analyses of the activity of XDSCR6 and human RIPPLY2 in mesoderm gene expression at the early gastrula stage. (C) A control embryo at the stage 33. (D) Induction of a partial secondary axis by human RIPPLY2. The frequency of this induction is similar as XDSCR6 (not shown). Fig. S4. Uniform expression of XDSCR6 suppresses trunk and posterior development. Embryos at the 8-cell stage were radially injected with Xdscr6 mrna (0.4 ng) at the equatorial region and live images of control (A) and injected (B) embryos were taken at larval stage 41. Note that embryos radially injected with Xdscr6 lacks trunk and posterior regions.

3 Fig. S5. XDSCR6 induces chordin and Xbra expression independently of nodal signalling. Embryos at the 2-cell stage were injected at the animal pole region with low amount of Xdscr6 mrna alone or coinjected with VegT-EnR mrna or antivin mrna. They were also injected with VegT mrna (40 pg) or coinjected with VegT-EnR mrna or antivin mrna. RT-PCR analyses on control embryo and dissected ectoderm explants were performed at the early gastrula stage. VegT-EnR and antivin does not block the activity of XDSCR6, but inhibits the activity of VegT. Fig. S6. RT-PCR analyses of Otx2, chordin and Xbra expression in ectoderm explants injected with wild-type Xdscr6 or its mutants, as indicated. Fig. S7. BMI1 inhibits ectopic chordin expression induced by XDSCR6. Embryos at the 4-cell stage were ventrally injected with Xdscr6 mrna alone, BMI1 mrna alone or coinjected with Xdscr6 and BMI1 mrna. In situ hybridization was performed at the early gastrula stage.

4 Table S1. Primers for semi-quantitative PCR. PCR primer pair VegT Krox20 Xdscr6 Xnr1 Xnr2 Xnr4 Xnr5 Xnr6 Goosecoid (promoter) Sequence F: 5'-CAAGTAAATGTGAGAAACCG-3' R: 5'-CAAATACACACACATTTCCC-3' F: 5'-AACCGCCCCAGTAAGACC-3' R: 5'-GTGTACGCCTGTCCTGTTAG-3' F: 5'-GTTCTACTATGGAGCCGAATC-3' R: 5'-CTGTGCAAGATTTAATTTTCAT-3' F: 5'-TAAAAGCAAAGGAAAGGGCAGAG-3' R : 5'-GATGCTTCCTATTGATAAGTGATG-3' F : 5'-ATCTGATGCCGT TCTAAGCC-3' R: 5'-GACCTTCTTCAACCTCAGCC-3' F: 5'-GAAATGGAGGTGATGGTAGAC-3' R: 5'-GACCATCATCACTATCCTGCTG-3' F: 5'-TCACAATCCTTTCACTAGGGC-3' R : 5'-GGAACCTCTGAAAGGAAGGC-3' F : 5'-TCCAGTATGATCCATCTGTTGC-3' R : 5'-GTCAAAGTCAACAATCATGTCGA-3' F: 5'-CCAGAGAAACAAAACAGTCATTCC-3' R: 5'-GCAGACTCTCCCTGTAGTTATTCAC-3'

5 Table S2. Primers for real-time PCR. PCR primer pair Chordin Xbra Wnt8 Sox17α EF1α ODC Goosecoid (promoter) Sequence F: 5'-AGCTGTCAGAAGCGTACCGTG-3' R: 5'-CAGAGGTGGGCACACAATAGG-3' F: 5'-CACCAAGAATGGAAGACGAATG-3' R: 5'-CCCGACATGCTCACCTTCA-3' F: 5'-GCTACCCACAATGGACTTCGA-3' R: 5'-AGCTAATGGCATGCACAAAGG-3' F : 5'-ACAGAACCCCGACCTGCA-3' R : 5'-GGATTTCCATGATTTACCAAGCAT-3' F: 5'-CTTCTCAGGCCGACTGTGC-3' R : 5'-ATTCACCAACACCAGCAGCA-3' F : 5'-GGGCAAAGGAGCTTAATGTGG-3' R: 5'-TGCCAACATGGAAACTCACAC-3' F: 5'-CCAGAGAAACAAAACAGTCATTCC-3' R: 5'-GGTGCAATTTCCTTGCTCTC-3'

6 Table S3. Induction of a secondary embryonic axis by overexpression of XDSCR6. Injection Single axis Secondary axis with head Partial secondary axis Uninjected n Xdscr6 mrna (0.4 ng) (Ventro-vegetal) Xdscr6 mrna (0.4 ng) (Ventro-animal) Xdscr6 DNA (0.4 ng) (Ventro-vegetal) Embryos were injected at the 4-cell stage and the phenotypes were scored when uninjected embryos reached stage 35. The result is presented as percentage, except n, which refers to total embryos scored from three independent experiments.

7 Table S4. XDSCR6 rescues anterior defects produced by dorsal overexpression of BMI1. Injection Normal Anterior defects n Uninjected BMI1 (0.5 ng) Xdscr6 (0.2 ng) BMI1/Xdscr6 (0.5/0.2 ng) Embryos at 4-cell stage were dorsally injected with indicated mrna. They were cultured until stage 32 to score phenotypes. Anterior defects include phenotypes with small head, absence of eye or complete absence of head region as shown in Figure 9C. The results are expressed as percentage, except n, which refers to total embryos scored from three independent experiments.

8 Table S5. BMI1 inhibits secondary axis formation induced by XDSCR6. Injection Normal Secondary axis n Uninjected Xdscr6 (0.1 ng) BMI1 (0.5 ng) Xdscr6/BMI1 (0.1/0.5 ng) Embryos at 4-cell stage were ventrally injected with indicated mrna. They were cultured until stage 35 to score the presence or absence of axis duplication. The results are presented as percentage except n, which refers to total embryos scored from three independent experiments.

9 Table S6. Ectopic chordin expression following overexpression of XDSCR6 or XDSCR6/BMI1. Injection Strong Weak Absence n Uninjected Xdscr6 (0.1 ng) Xdscr6/BMI1 (0.1/0.5 ng) Embryos at 4-cell stage were ventrally injected with indicated mrna and were fixed at early gastrula stage for in situ hybridization. The intensity of ectopic chordin expression was as shown in Figure S7. The results are presented as percentage except n, which refers to total embryos scored from three independent experiments.