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First published online 21 July 2004
doi: 10.1242/dev.01277

Development 131, 3991-4000 (2004)
Published by The Company of Biologists 2004
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Repression of the vertebrate organizer by Wnt8 is mediated by Vent and Vox

Marie-Christine Ramel and Arne C. Lekven*

Department of Biology, Texas A&M University, College Station, TX 77843-3258, USA



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  		Fig. 1. The wnt8– phenotype is similar to the vent–;vox– and swr phenotypes. (A,B,D) Double in situ hybridization for eve1 and gsc. (C) eve1 expression, inset shows gsc. Note strongly reduced eve1 in wnt8 and swr mutants but only slightly reduced eve1 in vent;vox mutants. Arrowheads indicate the width of gsc expression (note circumferential gsc in C, inset). (E-H) In situ hybridization for chd (domain width indicated by arrowheads). Note expansion in both wnt8 and vent;vox mutants, but not swr mutants. All embryos are at shield stage. Animal view, dorsal right.

 


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  		Fig. 2. vent and vox mRNA levels are reduced in wnt8 mutants. In situ hybridization for vent (A-F), vox (G-L) or chd (M-R). Embryo genotypes are indicated above each column; stages are also indicated. At 30% epiboly, vent expression is reduced in wnt8 mutants/morphants (arrows in B,C). vox is reduced at 40% epiboly (arrows in H,I), corresponding to increased chd expression (arrowheads in N,O). Both vent (E,F) and vox (K,L) expression are reduced in shield stage wnt8 mutants/morphants. Animal view, dorsal right.

 


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  		Fig. 3. Wnt8 ORF1 and ORF2 expression in vent;vox mutants. In situ hybridization for wnt8 ORF1 (A-F) and wnt8 ORF1+ORF2 (G-L). Genotypes are indicated above each column; stages are also indicated. Arrowheads indicate the dorsal limit of wnt8 expression. Note the slight decrease in ORF1 dorsally in shield stage vent;vox mutants (C,D), and the broadened dorsal clearing of wnt8 ORF1 expression at 75% epiboly (F). wnt8 ORF2 expression is not affected. (A-D,G-J) Animal view, dorsal right. (E,F,K,L) Vegetal view, dorsal right.

 


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  		Fig. 4. vent and vox are direct transcriptional targets of Wnt8/ß-catenin signaling. (A) vent and vox expression in control (a,c) or treated (b,d) embryos. Arrows in panels b and d indicate ectopic expression upon induction of GR-LEF{Delta}N-ßCTA with DEX. (B) Percentage of embryos displaying ectopic vent or vox domains (y-axis) upon treatment with CHX alone, DEX alone, or CHX+DEX (x-axis). The control bar represents embryos injected with GR-LEF{Delta}N-ßCTA and treated with ethanol (n=109 for vent, n=177 for vox). Error bars represent s.e.m. When performing the {chi}2 test on DEX versus DEX+CHX means, P>0.05 for both vent and vox, meaning that the difference between the means is not statistically significant.

 


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  		Fig. 5. The wnt8– expanded organizer phenotype is due to reduced vent and vox expression. (A) Rescue of wnt8 mutants by Vox. gsc expression (bracket) in wild-type (a,c) or wnt8– (b,d) embryos, uninjected (a,b) or injected (c,d) with a vox expression plasmid. Some isolated lateral cells still express gsc in injected wnt8– embryos because of the mosaic expression of Vox (panel d, arrow). Embryos shown are at 70% epiboly, dorsal view. (B) Reduction of Vent/Vox enhances the wnt8– organizer phenotype. Graph shows the percentage of embryos belonging to a specific phenotypic class. Class I, wild-type chd expression; class II to IV, increasingly expanded chd expression. 100% of wild-type and wnt8– embryos belong to class I and class II, respectively. Upon injection of vent+vox MOs, most wild-type embryos belong to class II (96.5%, n=85), whereas wnt8– embryos belong to both classes III and IV (60.7% and 39.3%, respectively, n=28). Embryos shown at the bottom of the graph are at shield stage, animal view, dorsal right.

 


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  		Fig. 6. Wnt8 requires Vent and Vox to repress dorsal genes. (A-D) GFP in situ hybridization to embryos homozygous for the TOPdGFP transgene. (E-H) opl, pax2a and tbx6 in situ hybridization. Genotype/treatment is indicated above each panel. (A) TOPdGFP is expressed in the mesoderm. In wnt8 morphants (B), TOPdGFP is barely detectable (arrow). vent+vox MO-injected embryos display mostly wild-type TOPdGFP expression (C), but some display somewhat reduced expression (D, arrows). Arrowheads in A-D indicate the AP extent of the TOPdGFP positive domain. (E) In wild type, opl and pax2a expression domains in relation to tbx6 indicate normal neural posteriorization. In wnt8 morphants, opl is expanded posteriorly, pax2a is delayed and tbx6 is reduced (F). vent;vox mutants (G) do not display a strong AP defect, and ventral tbx6 staining is as strong as in wild-type embryos. Reducing Wnt8 in vent;vox mutants (H) results in decreased tbx6 and pax2a expression. The distance between the arrowheads in F, G and H show the degree of posteriorization. Embryos shown are at ~100% epiboly, lateral view, dorsal right.

 


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  		Fig. 7. Wnt8 and zygotic BMP both regulate vent and vox, but do so differently. In situ hybridization for gsc (A,B,F,G), vent (C-E), vox (H-J), wnt8 (K,L) and bmp2b (M-O). Genotypes/treatments are indicated above each panel. Note circumferential gsc in vent;vox (A) and wnt8;swr (B,G) double mutants/morphants, and the strong reduction of vent (E) and vox (J) in wnt8–;swr–. wnt8 is still expressed in swr mutants (L), and bmp2b is still expressed in wnt8 mutants/morphants (N,O). Arrowheads in M-O indicate the dorsal limits of mesodermal bmp2b, which is shifted slightly ventrally in wnt8 mutants/morphants (N,O). Embryos shown are at shield stage, animal view, dorsal right.

 


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  		Fig. 8. Regulation of vent and vox by Wnt8 and zygotic BMP. (A) vent and vox are induced around MBT by an unknown factor. (B) At 40% epiboly, Wnt8 is required to maintain high levels of vent and mesodermal vox expression. (C) At 70% epiboly, in addition to Wnt8, zygotic BMP is required to maintain vent expression. BMP is also required for ectodermal vox expression. Thicker arrows represent stronger regulatory connections, as vent and ectodermal vox expression is absent in zygotic BMP mutants at this stage, whereas vent and mesodermal vox expression are only reduced in wnt8 mutants.

 

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© The Company of Biologists Ltd 2004