Salic AN et al. (1997), Sizzled: a secreted Xwnt8 antagonist expressed ...

XB-ART-15617

Development 1997 Dec 01;12423:4739-48. doi: 10.1242/dev.124.23.4739.

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Sizzled: a secreted Xwnt8 antagonist expressed in the ventral marginal zone of Xenopus embryos.

Salic AN , Kroll KL , Evans LM , Kirschner MW .

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An expression cloning screen was used to isolate a novel gene homologous to the extracellular cysteine-rich domain of frizzled receptors. The gene (which we called sizzled for secreted frizzled) was shown to encode a soluble secreted protein, containing a functional signal sequence but no transmembrane domains. Sizzled (szl) is capable of inhibiting Xwnt8 as assayed by (1) dose-dependent inhibition of siamois induction by Xwnt8 in animal caps, (2) rescue of embryos ventralized by Xwnt8 DNA and (3) inhibition of XmyoD expression in the marginal zone. Szl can dorsalize Xenopus embryos if expressed after the midblastula transition, strengthening the idea that zygotic expression of wnts and in particular of Xwnt8 plays a role in antagonizing dorsal signals. It also suggests that inhibiting ventralizing wnts parallels the opposition of BMPs by noggin and chordin. szl expression is restricted to a narrow domain in the ventral marginal zone of gastrulating embryos. szl thus encodes a secreted antagonist of wnt signaling likely involved in inhibiting Xwnt8 and XmyoD ventrally and whose restricted expression represents a new element in the molecular pattern of the ventral marginal zone.

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Species referenced: Xenopus laevis
Genes referenced: acta4 actc1 actl6a bmp4 chrd ctnnb1 frzb gsc myc myod1 nog not post shh sia1 szl tbx2 tbxt tubb2b wnt8a

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	Fig. 1. Expression cloning of sizzled, a gene homologous to frizzled genes. (A) Sequence comparison between szl and frzb (Leyns et al., 1997; Wang et al., 1997). Conserved amino acid residues are boxed. The two proteins show 18.1% identity at the amino acid sequence level. (B) Phenotype displayed by stage 26 embryos injected with 200 pg of szl RNA into the ventral side at 4-cell stage. Top row of pictures shows lateral views of 4 embryos while the bottom row shows dorsal images of the same embryos. Arrows indicate the position of cement glands. Note the thick and stunted appearance and enlarged cement glands in szl-injected embryos as compared to the uninjected embryo. The neural plate is wide and fails to close properly (compare the area between arrowheads in a szl-injected embryo to that in an uninjected one â bottom row). In the embryo on the right, the phenotype caused by szl RNA injection has been rescued by coinjecting 100 pg of an Xwnt8 expression plasmid (CS2+Xwnt8). (C) Hematoxylin and eosin-stained frontal section through an embryo injected with 100 pg of szl RNA into one dorsal blastomere at 4-cell stage. The injected side is on the right. Note the increased cellularity and larger size of the paraxial mesoderm on the injected side. nt, neural tube; no, notochord; so, somite.
	Fig. 2. Sizzled encodes a secreted protein. (A,B) Confocal micrographs of Xenopus embryonic (stage 10.5) cells expressing myc-tagged sizzled. Two serial section planes located 15 mm apart are shown. The tagged protein appears red (a secondary antibody conjugated to Cy3 was used for immunofluorescent detection) while the nuclear DNA is blue (stained with DAPI). In A, the cell surfaces are roughly in the optical section plane, which corresponds to the surface of the embryo. Note the cell surface localization of szlMT in B, which shows an optical section plane 15 mm deeper into the embryo. (C) Immunoblot of myc-tagged szl secreted into the medium by 293T cells transfected with CMV-szlMT. Lane 1: cellular pellet; lane 2: tissue culture medium. Note the higher apparent MW of soluble szlMT protein in lane 2.
	Fig. 3. Szl has dorsalizing activity. (A) Stage 23 szl-injected embryos show increased muscle actin staining on the injected side compared to the uninjected one (top 2 embryos) or to uninjected embryos (bottom). Muscle actin is shown in blue; the localization of injected szl mRNA was followed using a lineage tracer RNA encoding GFP, which was detected using an antisense GFP probe and appears magenta. The top embryos are seen from their dorsal side and have their anterior ends to the left. The left embryo was injected in the right side and the right embryo in the left side (B) Sonic hedgehog (magenta) expression in a szl-injected stage 23 embryo, seen in anterolateral view. The lineage tracer is blue. Note the duplicated but short notochord, indicated by the top arrow. The bottom arrow points to the enlarged cement gland. (C) Inhibition of Xpo (blue), a posteroventral marker, by szl. Xpo expression is extinguished from szl-overexpressing cells (which stain magenta) on the left side of a stage 11.5 embryos. (D) Xnot expression (blue) in a normal stage 12 embryo and (E) in a szl-injected embryo of the same stage. Injected szl is magenta. Note the strong ectopic Xnot expression on the right side of the embryo (which overlaps in part with injected szl and thus appears dark blue) and the blocked elongation of the endogenous Xnot pattern. Also note the delayed blastopore closure in szlinjected embryos compared to the stage-matched uninjected embryo (which makes the embryo in E appear as if it were an earlier stage). (F) Stage 10.25 embryo stained for Xbra (blue) and the lineage tracer coinjected with szl (magenta). Xbra expression is unperturbed by overexpressed szl.
	Fig. 4. Expression pattern of sizzled transcripts in Xenopus embryos. In all embryos dorsal is up and ventral is down. Szl expression by in situ hybridization (ISH) in (A) a stage 10.25 embryo and (B) a stage 23 embryo. (C) Vegetal view of a stage 11 embryos dorsalized by lithium, a treatment which suppresses szl expression. (D)Vegetal view of a stage 11 UV-ventralized embryo showing szl expression around the entire blastopore. Double in situ hybridizations for szl and (E) Xwnt8 (blue) at stage 10.5, (F) Xvent1 (blue) at stage 10.5, (G) Xvent1 at stage 24, (I) Xnot (blue) at stage 11-11.5, (J) frzb (blue) at stage 11, (K) Xbra (magenta) at stage 10.5, (L) gsc (blue) at stage 11. (H) Section through a stage 11 embryo stained for szl. Note the expression of szl in both superficial and deep layers of the ventral blastopore lip (indicated by arrow). The unstained region above the szl-expressing cells is the yolk plug. Embryos in A, B and H were stained with NBT + BCIP. Embryos in C and D were stained with Magenta Phos. In E-G and I-L staining was done with BCIP (light blue) and Magenta Phos (magenta). The overlap between light blue and magenta appears dark blue.
	Fig. 5. Szl inhibits XmyoD expression. Double in situ hybridizations showing the relationship between szl and XmyoD expression in (A) stage 10.5 embryo, (B) stage 11.5 and (C) stage 13 normal embryos. sizzled is magenta, XmyoD is light blue. Note how XmyoD becomes gradually extinguished in the ventral sector occupied by sizzled. (D) szl-injected embryo, showing inhibition of XmyoD expression on the injected right side. XmyoD is light blue, the lineage tracer coinjected with szl is magenta.
	Fig. 6. (A) Szl blocks siamois induction by Xwnt8 in animal cap explants in a dose-dependent manner. RT-PCR analysis of siamois expression in stage 10.5 animal pole explants (caps) and embryos. Lane 1: no reverse transcriptase; lane 2: whole uninjected embryo; lane 3: uninjected caps; lane 4: caps injected with 250 pg Xwnt8 RNA; lane 5: caps injected with 250 pg Xwnt8 RNA + 250 pg szl RNA; lane 6: caps injected with 250 pg Xwnt8 + 2.5 ng szl RNA; lane 7: caps injected with 250 pg Xwnt8 and 2.5 ng antisense szl RNA. (B) Szl can block siamois induction in animal caps by Xwnt8 but not by b-catenin. Stage 10.5 animal pole explants were analyzed for siamois expression by RT-PCR. Lane 1: no reverse transcriptase; lane 2: stage 10.5 embryos; lane 3: uninjected caps; lane 4: caps injected with 250 pg Xwnt8 RNA; lane 5: caps injected with 250 pg Xwnt8 RNA + 2.5 ng szl RNA; lane 6: caps injected with 250 pg b- catenin RNA; lane 7: caps injected with 250 pg b-catenin RNA + 2.5 ng szl RNA.
	Fig. 7. Diagramme representing the expression patterns of Xwnt8, BMP4 and their secreted antagonists in the marginal zone of Xenopus gastrulae, seen from the vegetal pole. For clarity, the secreted components of the Xwnt8 and BMP4 pathways are shown as two non-overlapping concentric rings. In reality, BMP4 overlaps with Xwnt8, frzb overlaps with chordin and noggin while sizzled overlaps with both Xwnt8 and BMP4.