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

Arora, Altered mitotic domains reveal fate map changes in Drosophila embryos mutant for zygotic dorsoventral patterning genes. 1992, Pubmed

Arora, Altered mitotic domains reveal fate map changes in Drosophila embryos mutant for zygotic dorsoventral patterning genes. 1992, Pubmed
Beck, The role of BMP signaling in outgrowth and patterning of the Xenopus tail bud. 2001, Pubmed , Xenbase
Blitz, Twisted gastrulation loss-of-function analyses support its role as a BMP inhibitor during early Xenopus embryogenesis. 2003, Pubmed , Xenbase
Chang, Twisted gastrulation can function as a BMP antagonist. 2001, Pubmed , Xenbase
Conley, Crossveinless 2 contains cysteine-rich domains and is required for high levels of BMP-like activity during the formation of the cross veins in Drosophila. 2000, Pubmed
De Robertis, A common plan for dorsoventral patterning in Bilateria. 1996, Pubmed , Xenbase
De Robertis, The establishment of Spemann's organizer and patterning of the vertebrate embryo. 2000, Pubmed , Xenbase
Eldar, Robustness of the BMP morphogen gradient in Drosophila embryonic patterning. 2002, Pubmed
Ferguson, Localized enhancement and repression of the activity of the TGF-beta family member, decapentaplegic, is necessary for dorsal-ventral pattern formation in the Drosophila embryo. 1992, Pubmed
Garcia Abreu, Chordin-like CR domains and the regulation of evolutionarily conserved extracellular signaling systems. 2002, Pubmed
Gont, Tail formation as a continuation of gastrulation: the multiple cell populations of the Xenopus tailbud derive from the late blastopore lip. 1993, Pubmed , Xenbase
Goodman, BMP1-related metalloproteinases promote the development of ventral mesoderm in early Xenopus embryos. 1998, Pubmed , Xenbase
Graf, The developmentally regulated expression of Twisted gastrulation reveals a role for bone morphogenetic proteins in the control of T cell development. 2002, Pubmed
Graf, Evolutionary conservation, developmental expression, and genomic mapping of mammalian Twisted gastrulation. 2001, Pubmed , Xenbase
Larraín, Proteolytic cleavage of Chordin as a switch for the dual activities of Twisted gastrulation in BMP signaling. 2001, Pubmed , Xenbase
Nosaka, Mammalian twisted gastrulation is essential for skeleto-lymphogenesis. 2003, Pubmed
Oelgeschläger, The evolutionarily conserved BMP-binding protein Twisted gastrulation promotes BMP signalling. 2000, Pubmed , Xenbase
Oelgeschläger, The pro-BMP activity of Twisted gastrulation is independent of BMP binding. 2003, Pubmed , Xenbase
Oelgeschläger, Chordin is required for the Spemann organizer transplantation phenomenon in Xenopus embryos. 2003, Pubmed , Xenbase
Ross, Twisted gastrulation is a conserved extracellular BMP antagonist. 2001, Pubmed , Xenbase
Sasai, Xenopus chordin: a novel dorsalizing factor activated by organizer-specific homeobox genes. 1994, Pubmed , Xenbase
Scott, Homologues of Twisted gastrulation are extracellular cofactors in antagonism of BMP signalling. 2001, Pubmed , Xenbase
Vilmos, The Twisted gastrulation family of proteins, together with the IGFBP and CCN families, comprise the TIC superfamily of cysteine rich secreted factors. 2001, Pubmed , Xenbase
Zakin, Inactivation of mouse Twisted gastrulation reveals its role in promoting Bmp4 activity during forebrain development. 2004, Pubmed , Xenbase