Harada Y , Yokota C , Habas R , Slusarski DC , He X .

R01 GM074241-03 NIGMS NIH HHS , R01 CA112369-01 NCI NIH HHS , R01 GM078172-06 NIGMS NIH HHS , R01 GM078172-07 NIGMS NIH HHS , R01 CA112369 NCI NIH HHS , R01 GM074241 NIGMS NIH HHS , R01 GM078172 NIGMS NIH HHS

	Fig. 1. Amino acid sequence of XRAIGs and the expression pattern of XRAIG mRNAs during embryogenesis. (A) Alignment of the predicted amino acid sequence of Xenopus RAIG1, RAIG2, RAIG3, RAIG4a, and RAIG4b. Amino acid residues conserved between all proteins are shaded in black, and putative transmembrane segments are overlined. Only partial sequences of XRAIG4a and XRAIG4b are available from EST databases. (B) Phylogenetic analysis of human and Xenopus RAIGs. The full-length hRAIGs and XRAIG1–3 and the transmembrane domains (I–IV) of XRAIG4a and XRAIG4b were aligned using CLUSTALW with the point accepted mutation (PAM) series substitution matrix. The tree was constructed using CLUSTALW with standard parameters. (C) RT-PCR analysis of XRAIGs during Xenopus development. −RT: without reverse transcriptase. EF-1α was used as an internal control. (D) A schematic diagram shows the dissection of blastula at stage 10.5 into six regions. (E) RT-PCR analysis of XRAIG2, 3, and 4. Xnr3 and Cerberus (Cer) are used as control markers for dissection.
	Fig. 2. Interaction between Frizzled and RAIG proteins. (A) HEK 293T cells were transiently transfected with plasmids expressing Flag-hRAIG1, HA-Smo, and HA-hFz5 as indicated. The cell lysate was subjected to immunoprecipitation with anti-HA antibody, and the immunocomplex was blotted with anti-Flag antibody (first panel) or anti-HA antibody (third panel). (B) Both blastomeres of two-cell embryos were injected with XRAIG2-Flag mRNA (2 ng), XRAIG2δC-Flag mRNA (2 ng), HA-SMO mRNA (2 ng), HA-hFz5 mRNA (2 ng), or HA-XFz7 mRNA (2 ng) in various combinations as indicated. Extracts were prepared form 20 embryos at stage 9, and subjected to immunoprecipitation with anti-HA antibody. The immunocomplex was blotted with anti-Flag antibody (top panel) or anti-HA antibody (bottom panel).
	Fig. 3. Functional analysis of overexpression of RAIG in Xenopus embryos. (A,B) Animal caps were excised from embryos injected with various combinations of mRNA as indicated. The explants were cultured until control sibling embryos reached stage 10.5, and total RNAs were isolated and subjected to RT-PCR analysis. (C) Normal un-injected tadpoles. (D) Both dorsal blastomeres of four-cell embryos were injected with hRAIG1 mRNA (1 ng). Embryos display severely reduced tail structures and kinked trunks. (E) Ventral injection of hRAIG1 mRNA (1 ng). (F–I) Activin-dependent animal cap elongation assay. Animal cap control explants from uninjected embryos were treated with (G) or without (F) activin. One nanogram of Xdd1 mRNA (H) or 200 pg of XRAIG2 mRNA (I) were injected into embryos, and the animal cap explants were treated with activin. (J–U) Expression of Xbra (J–O) and Gsc (P–U) in uninjected (J, M, P, and S), 1 ng of Xdd1-injected (K, N, Q, and T), and 1 ng of XRAIG2-injected (L, O, R, and U) embryos was examined at the indicated stage by whole-mount in situ hybridizastion. XRAIG2-injected embryos showed normal expression of mesodermal Xbra and dorsal Gsc at stage 10.5. At stage 12, the blastopore size of XRAIG2-injected embryos (O) was larger than that of uninjected (M), and the Xbra-positive cells at the involuting notochord (M) was missing in the XRAIG2-injected embryos (O). Gsc expression in control embryos at stage 13 is observed in anterior mesoendoderm far from the closed blastopore (S), but remained in a dorsal region close to the open blastopore in XRAIG2-injected embryos.
	Fig. 4. Signal transduction by XRAIGs. (A) Flag-Dvl2 or XRAIG2 was transfected to HEK 293T cells. GTP-bound RhoA in cell lysates was precipitated using GST-RBD and detected by an anti-RhoA antibody. GTP-bound Rac and Cdc42 were precipitated using GST-PBD and detected by an anti-Rac or an anti-Cdc42 antibody, respectively. (B–E) Activin-dependent animal cap elongation assay. Animal cap control explants from uninjected embryos were treated with (C) or without (B) activin. Injection of XRAIG2 mRNA (200 pg) blocked the activin-induced elongation of animal cap explants (D). Dominant-negative form of Cdc42 (200 pg) partially rescued the elongation inhibited by XRAIG2 injection (E). (F) The Myc-tagged c-Jun mRNA (200 pg) was co-injected with XFz7 mRNA (1 ng), XRAIG2 mRNA (500 pg), or XRAIG3 mRNA (500 pg). Myc-c-Jun was immunoprecipitated with anti-Myc antibody, and the immunoprecipitates were immunoblotted with anti-phospho-c-Jun antibody (top panel) or anti-c-Jun antibody (middle panel). (G–H) Phenotypes of wild-type (G,I) and XRAIG2-injected (H,J) zebrafish. (K,L) Ca2+ release dynamics in un-injected (K) and XRAIG2-injected (L) zebrafish embryos.

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