Fig. S1. Syn4/PKC/Rac pathways involved in neural induction. Embryos were injected as indicated into the animal blastomeres of an 8-cell stage embryo or into A1 or A4 of a 32-cell embryo, and the expression of neural or epidermal markers analyzed at stage 19. (A) Control MO injected at the 8-cell stage. Analysis of Nrp1 (0% inhibition, n=18). (B) Syn4 MO injected at the 8-cell stage. Analysis of Nrp1 (96% inhibition, n=27). (C) PKCδ mRNA injected at the 8-cell stage. Analysis of Nrp1 (46% inhibition, n=23). (D) Constitutively active Rac mRNA injected at the 8-cell stage. Analysis of Nrp1 (39% inhibition, n=42). (E) PKCδ mRNA injected at the 32-cell stage. Analysis of NCAM (43% inhibition, n=7). (F) Constitutively active Rac mRNA injected at the 32-cell stage. Analysis of NCAM (32% inhibition, n=25). (G) Sox2 expression of embryo injected with PKCα and a dominant-negative form of FGF receptor (XFD-1). Note that the ectopic induction is not inhibited (58% induction, n=31). (H) Epidermal keratin (Epk) expression after injection of PKCα mRNA into A4 blastomeres. Note the inhibition of epidermis (63% inhibition, n=22).

Fig. S2. Syn4 has a neuralising activity that does not depend on BMP inhibition. (A,B) Formation of secondary axis. (A) Embryos were injected in ventral blastomeres with Syn4 (A) or chordin (B) mRNA and the lineage tracer β-galactosidase. Note the secondary axis induced by chordin injection; but no secondary axis was observed in Syn4-injected embryos. (C,D) Sox3 expression in stage 14 embryos, dorsal to the top. Inset shows the dorsal side. (C) Control expression of Sox3. (D) Embryo was injected with Syn4 mRNA into A4 blastomeres and the expression of Sox3 analyzed. Note the ectopic induction of Sox3 in ventral epidermis (89% induction, n=49). (E-G) Neuralisation by Syn4 does not require chordin. (E) Ventral view of an embryo injected with Syn4 mRNA. Note the ectopic Sox2 expression (91%, n=12). (F) Ventral view of embryo co-injected with Syn4 mRNA and chordin MO. There was no effect on the ectopic induction of Sox2 (89%, n=37). (G) Embryo injected with chordin MO to show the expected ventralised phenotype (100%, n=9). (H,I) Sox2 expression analysed in stage 20 embryos. Anterior view, dorsal to the top. (H) Control. (I) Embryo injected in A4 with Syn4 mRNA. Note the strong ectopic induction of Sox2. (J,K) Animal caps showing induction of Otx2 by Syn4 expression (K); no induction was observed in control animal caps (J).

Fig. S3. Analysis of neuralising activity of Syn4. (A) Deletion constructs of Syn4. 1, wild-type Syn4. 2, Syn4ΔGAG, with deletion of the glycosaminoglycan domain of Syn4. 3, Syn4ΔCyt-Cherry, with deletion of the intracellular domain of Syn4, which was replaced by the fluorescent protein Cherry. (B) Embryos were injected in A4 with the indicated mRNAs and the expression of Sox2 analyzed. The percentage of embryos showing normal (blue bars) or ectopic (red bars) Sox2 expression. Different concentrations of each construct were tested.