Oelgeschläger M , Tran U , Grubisic K , De Robertis EM .

R37 HD021502-16 NICHD NIH HHS , R37 HD21502-16 NICHD NIH HHS , R37 HD021502 NICHD NIH HHS

	Fig. 1. Analysis of the xTsg2 cDNA and protein sequence. (A) Nucleotide and amino acid sequence of xTsg-2. (B) Alignment of the two pseudoalleles of xTsg with xTsg-2. (C) Dendrogram showing the phylogenic relations between the Xenopus (xTsg, xTsgb, xTsg-2), mouse (mTsg), human (hTsg), zebrafish (zTsg, zTsg-2) and Drosophila Tsg proteins (dTsg, cv/dTsg2, dTsg3). (D) ClustalW alignment of the indicated protein sequences demonstrating the conservation of amino acids required for ventralizing activity or chordin binding (arrowheads) and the position of an in vivo glycosylation site (asterisk). The accession numbers of the sequences used for the alignments were for xTsg, AF245221; xTsgb, AF279246; xTsg-2, BJ075535; mTsg, AAG00605; hTsg, NP_065699; zTsg, AAK27324; zTsg-2, AAK13255; dTsg, A53836; cv, CG12410; dTsg3, CG11582.
	Fig. 2. Time course of xTsg and xTsg-2 gene expression. mRNA samples were analyzed by RT-PCR at the indicated developmental stages. Xenopus Tsg mRNA can be detected throughout development, whereas xTsg-2 expression starts at tailbud stages. Other markers shown are XNR-3, which responds specifically to the early ß-catenin signal in Xenopus; NCAM, which marks neural tissue; a-globin, which marks ventral mesoderm and blood differentiation; ODC (ornithine decarboxilase) serves as an mRNA loading control.
	Fig. 3. Whole-mount in situ hybridization analysis of xTsg-2 and xTsg expression. (A,C) xTsg-2 expression at stage 27 and (B,D) stage 33 embryos showing expression in the branchial arches (ba), neural crest (nc), lateral plate mesoderm (lpm) and head mesenchyme (hm). Embryos in C and D were made transparent in Murray�s Clearing Solution (2:1 benzyl benzoate: benzyl alcohol) to visualize the expression of xTsg-2 localized to the midbrain-hindbrain border, the isthmus (is). D is a close up of the embryo in B. (E) Stage 27 and (F) stage 33 embryos stained for xTsg and cleared in Murray�s solution reveal expression in the dorsal eye (ey), heart anlage (he) and proctodeum (pro), as well as in the posterior wall (pw) of the tailbud. cg, cement gland; fb, forebrain; hb, hindbrain; pn, pronephros.
	Fig. 4. xTsg-2 is expressed in the mesenchyme of the branchial arches, lateral plate and other tissues. Histological sections of whole-mount in situ hybridization. (A) Coronal section of an embryo at stage 33 showing expression of xTsg-2 in the branchial arches (ba), but not in the pharynx (ph). (B) Close up of (A) indicating the expression of xTsg-2 is restricted to the branchial arches (ba), but devoid of detectable expression in the pharyngeal pouches (pp) consisting of pharyngeal endoderm (pe) as well as the cement gland (cg). (C) xTsg-2 expression is observed at low amounts in the eye (ey), but strong in the surrounding head mesenchyme. The isthmus (is) is stained in this coronal section. (D) Transverse section of a stage 33 embryo at the level of the trunk showing xTsg-2 expression in the lateral plate mesoderm (lpm) and hindbrain (hb) and low levels in the pronephros (pn). The somites (so), endoderm (en) and notochord (no) are not stained. (E) Close up showing xTsg-2 expression in the ventricular (proliferative) zone (vz) of the hindbrain. (F) Transverse section showing xTsg-2 expression in the dorsal fin neural crest cells and dorsal spinal cord (sc).
	Fig. 5 Microinjection of xTsg-2 mRNA generates phenotypes similar to those of xTsg (A) Uninjected stage 42 embryo as control. (B) Embryo microinjected with 500 pg of xTsg mRNA or (C) 500 pg xTsg-2 mRNA ventrally at the 4-cell stage.
	twsg2 (twisted gastrulation homolog 2) gene expression in a Xenopus laevis embyro as assayed by in situ hybridization, NF stage 33. Lateral view: Anterior left, dorsal up

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