Fig. 1. Sequence comparison and biological activity of Shisa proteins. (A) Comparison of the predicted amino acid sequences of the conserved amino-half of the Xenopus Shisa family. Conserved amino acids are shown in gray. Cysteine-rich domains (CRD1 and 2) are underlined. Sequence analysis was carried out using GeneWorks. (B) Schematic protein structures of Shisa family members. Total amino acid lengths are presented in parentheses. (C) Identities of amino acid sequences (%) in the conserved domain and non-conserved domain of Xenopus Shisa family members with Xenopus Shisa1. (D) XWnt8 (1 pg) RNA was injected either alone or together with Shisa2 or Shisa3 RNA (12.5 pg for lanes 4 and 7, 50 pg for lanes 5 and 8, 200 pg for lanes 6 and 9) into each animal blastomere at the eight-cell stage. RNAs were isolated from animal cap explants (AC) at late blastula and were analyzed by RT-PCR. (E) Shisa2 or Shisa3 RNA (100 pg for lanes 4 and 7, 200 pg for lanes 5 and 8, 400 pg for lanes 6 and 9) were injected into each animal blastomere at the four-cell stage. ACs were treated with or without bFGF for 3 hours and the expression of Xbra was analyzed as in D. (F-F'') Overexpression phenotype of Shisa genes. Radial injection of Shisa2 RNA (F') or Shisa3 (F'') RNAs (50 pg) into the animal side of each blastomere at the four-cell stage. Resulting embryos had open neural folds (arrow) and enlarged cement glands (arrowhead) (Shisa2: 50%, n=30; Shisa3: 77%, n=34). (G-H') Shisa2 inhibits the elongation of the dorsolateral marginal zone (DLMZ) explants. The DLMZ and ventral marginal zone (VMZ) explants (H'), were isolated at stage 10 and cultured until stage 12 (H) or stage 17 (G,G'). Explants from the normal embryos (G) or embryos injected with Shisa2 RNA (3 pg per blastomere) at the four-cell stage (G'). (H) RT-PCR analysis of the DLMZ and VMZ explants. (I,I') Whole-mount in situ hybridization of MyoD probe, dorsal view at neural stage. Shisa2 RNA (3 pg) was unilaterally injected into two right side blastomeres at the four-cell stage (I'). (I) Uninjected embryo. Note that injection of Shisa2 disturbed the convergence of the paraxial mesoderm on the injected side (double arrowhead) (n=16/28). CD, conserved domain; Chd: Chordin; Co, uninjected ACs; DLMZ, dorsolateral marginal zone; NCD, non-conserved domain; -RT, PCR with cDNAs synthesized without reverse transcriptase for H4; SP, signal peptide; VMZ, ventral marginal zone; WE, whole embryo.