Leyns L , Bouwmeester T , Kim SH , Piccolo S , De Robertis EM .

HD21502-11 NICHD NIH HHS , R37 HD021502-13 NICHD NIH HHS , R37 HD021502 NICHD NIH HHS , R01 HD021502 NICHD NIH HHS

	Figure 1. frzb-1 mRNA Expression in Xenopus, Mouse, and Human (A–D) Double-labeled in situ hybridization of Xenopus embryos showing frzb-1 (blue) and chordin expression (brown). (A) shows the vegetal view at stage 11; (B), lateral view of stage 13 embryo; (C), dorsal view of the same embryo; and (D), tailbud stage embryo. (E–J) Expression of frzb-1 in manipulated embryos. (E) shows the vegetal view of a Xenopus gastrula dorsalized by LiCl treatment; (F), Xenopus gastrula ventralized by UV irradiation; (G), vegetal view of an untreated embryo (stage 10 1/2); (H), Vegetal view of an embryo (stage 10 1/2) radially injected with siamois RNA; (I), frzb-1 expression in a stage 10 1/2 embryo radially injected with activated Xlim1 RNA; and (J), frzb-1 expression in a stage 10 1/2 embryo radially injected with goosecoid RNA. (K) Mouse gastrula (midstreak stage) showing frzb-1 expression in the primitive streak. (L) Mouse embryo (8.5 day) frzb-1 expression in the foregut diverticulum (arrowhead), nervous system, and posterior mesoderm. (M–N) Northern blot of adult mouse tissue (M) and human tissue (N) showing frzb-1 expression in heart (H), brain (B), spleen (S), skeletal muscle (M), kidney (K), testis (T), placenta (Pl), and pancreas (Pa). frzb-1 is detected at very low levels in lung (Lu) and liver (Li).
	Figure 2. Frzb-1 Is a Secreted Protein Containing a Frizzled-like CRD and a FUN Domain (A) Deduced amino acid sequences of the Xenopus, mouse, and human Frzb-1 proteins. Amino acid identity between frog and mouse or frog and human is indicated by an asterisk. The predicted signal peptide and the 10 cysteines typical of the extracellular cysteine-rich domain (CRD) of frizzled genes are shown in bold. The CRD is boxed, and the potential N-linked glycosylation site is indicated by an arrowhead. Outside the CRD, another region of Frzb-1 that shows homology to the Unc-6 and Netrin proteins, designated the FUN domain, is indicated by the dashed box. (B) Alignment of the FUN domains of mouse Frzb-1, C. elegans Unc-6, Drosophila Netrin-A, and chick Netrin-2. Residues identical in all four proteins and conserved acidic or basic residues are shown in bold; conserved residues in 3 of the 4 proteins are included in the consensus sequence. Dibasic sets of residues indicative of possible proteolytic cleavage sites are underlined. (C) [35S]Frzb-1 protein synthesized in vitro (rabbit reticulocyte, lane 1) or in vivo (culture supernatant of injected frog oocytes, lane 2) run on an SDS–PAGE gel. Treatment of the in vivo–synthesized protein with N-glycosidase A (lane 4) shows a shorter band (arrow), indicating signal peptide processing and possible carboxy-terminal cleavage of Frzb-1 (see text). Uninjected oocyte supernatant is shown in lane 3.
	Figure 3. frzb-1 mRNA Inhibits Skeletal Muscle Formation and Enhances Dorsoanterior Structures (A) Top, control embryo. Bottom embryo was injected into the vegetal pole with 200 pg of synthetic frzb-1 mRNA into each of the four blastomeres and allowed to develop to stage 36. Note the enlarged cement gland and shortening of the body in this dorsalized embryo. (B) Similar embryos after immunostaining with an antibody (MZ-15) labeling the notochord; the notochord is thickened in the frzb-1 dorsalized embryo. (C) Histological section through the eye of a normal embryo (top) and of an embryo injected as in (A) (bottom). The retina of the dorsalized embryo is folded multiple times, filling the enlarged eye. (D) Microinjection of frzb-1 mRNA into the marginal zone (400 pg/cell) leads to inhibition of trunk formation (bottom embryos). (E) Uninjected tadpole sectioned at the trunk level after staining with the skeletal muscle marker 12/101. (F) Sibling embryo injected with 250 pg/cell of frzb-1 mRNA into the marginal region of each of the four blastomeres. Note the decrease in the amount of 12/101 positive muscle and increase in notochord tissue ([So], somite; [No], notochord; [Ne], neural tube). (G) In situ hybridization at stage 103/4 showing expression of XMyoD in prospective skeletal muscle cells. (G′) frzb-1 mRNA inhibits XMyoD expression. (H and H′) Expression of XMyoD at late gastrula (stage 12 1/2) is inhibited by frzb-1 mRNA microinjection. (I and I′) Early expression (stage 10 1/2) of the organizer marker chordin is unaffected by frzb-1 ectopic expression. (J and J′) Late gastrula expression of chordin mRNA (stage 12 1/2) is expanded into the lateral marginal zone in frzb-1-injected embryos.
	Figure 4. Frzb-1 Antagonizes the Early and Late Phenotypes of Xwnt-8 (A) Dorsal view of an embryo injected with Xwnt-8 RNA (4 pg) in a ventral-vegetal blastomere at the 32-cell stage. (B) Lateral view of an embryo coinjected with Xwnt-8 (4 pg) and frzb-1 (160 pg) mRNA. Note that the formation of a secondary axis is blocked. (C) Acephalous embryo resulting from the injection into the two dorsal blastomeres (four cell stage) of 80 pg of DNA construct expressing Xwnt-8 under the control of the cytoskeletal actin promoter. (D) Rescue of the ventralization by coinjection of Xwnt-8 DNA construct with 400 pg of frzb-1 mRNA into the dorsal blastomeres. Note that eye development is restored and that the cement gland is enlarged.
	Figure 5. Noncell-Autonomous Antagonism of Xwnt-8 by frzb-1 (A) Experimental design showing embryos injected with frzb-1 synthetic mRNA into the four animal blastomeres at the 8-cell stage. (B) Injection of Xwnt-8 synthetic mRNA into a single ventrovegetal blastomere at the 16–32 cell stage. (C) Dorsal view of uninjected siblings at neurula (stage 19). The anterior end is at the top, the neural tube is visible. (D) Sibling embryos injected with frzb-1 (200 pg/cell) mRNA as in (A). Note the single neural tube and the considerable enlargement of the cement gland at the anterior end (arrowhead), indicating dorsalization. (E) Embryos injected with Xwnt-8 (4 pg) synthetic mRNA as in B. The Y-shaped neural tube indicates the formation of a double axis. (F) Sibling embryos injected successively with frzb-1 mRNA in the animal cap and later with Xwnt-8 mRNA into a ventro-vegetal blastomere to reveal extracellular antagonism. Note that the embryos show a single axis; the Xwnt-8 phenotype was antagonized, but the dorsalization by frzb-1 (enlarged cement gland) is still present.
	Figure 6. Frzb-1 Protein Binds to Cells Expressing a Wnt-1/Transmembrane Chimera (A) Low magnification view of 293 cells transfected with a Wnt1CD8 construct expressing Wnt-1 tethered to the membrane. After incubation in conditioned medium containing 250 nM Frzb1–HA protein and staining with anti-HA, positive cells appear as red spots. (B) Cells transfected with the plasmid coding for truncated Chordin protein (pΔ-Chd), used here as a negative control. (C) The same field of view as in (A) in Nomarski optics. (D) Nomarski view of the same cells as in (B). (E) High power view of a culture co-transfected with Wnt1CD8 and LacZ in pcDNA3. (F) The same cell that binds Frzb1–HA (red in panel E) also expresses LacZ from the cotransfected plasmid (in green). (G) Nomarski view of the same field as in (E) and (F). (H) Cells cotransfected with the full-length CD8 control as well as LacZ plasmid and treated with the Frzb1–HA medium shows no specific staining. (I) The same cells as in panel (H) are shown after staining for β-galactosidase (in green). (J) The same field of cells as in (H) and (I) is shown using Nomarski optics. (K) Binding of Frzb1–HA (250 nM) performed at 4°C for 2.5 hr on Wnt1CD8-transfected cells stained with anti-HA (in red). (L) Incubation of cells expressing Wnt1CD8 chimera with Frzb1–HA diluted down to 125 pM. (M) Cell transfected with a mutant Wnt1CD8 (cysteine 369 mutated to tryptophan) that reduces its biological activity is able to bind diluted Frzb1–HA (125 pM). This experiment was carried out simultaneously to the one illustrated in (L) but exposure time was four times longer. Stained cells in (K)–(L) were GFP positive (data not shown). All pictures were taken on a Zeiss Axiophot microscope with 40X (A–D) or 100X objectives (E–M). Photos were taken with a Kodak Ektachrome P1600 film and printed photographically.
	Figure 7. Speculative Model for the Mechanism of Frzb-1 Function (A) Frzb-1 competes with frizzled receptors for the binding of Wnt factors. Frzb-1 and frizzled share a homologous cysteine-rich domain (PacMan shape) that is responsible for Wnt binding. (B) Diagram of the possible function of Frzb-1 during Xenopus gastrulation. Frzb-1 is secreted by the Spemann organizer (gray oval) and antagonizes Xwnt-8, which is involved in patterning mesoderm in the ventrolateral marginal zone. (C) Other organizer-specific inhibitory factors involved in patterning the mesoderm and neuroectoderm. BMP-4 is secreted by the ventral side of the embryo and is antagonized by chordin and noggin which directly bind to it in the extracellular space of ectoderm and mesoderm. In this view, the Spemann organizer would be a source of inhibitory secretory proteins.

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