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Goosecoid promotes head organizer activity by direct repression of Xwnt8 in Spemann’s organizer

Jie Yao and Daniel S. Kessler*

Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6058, USA



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  		Fig. 1. Transcriptional repression by Gsc regulates anterior development. (A) Schematic of the Gsc fusion constructs. A C-terminal region of Gsc (residues 128-244), containing the homeodomain (HD), was fused to the Engrailed repressor (residues 1-298) (Eng-Gsc) or the VP16 activator (residues 410-490) (VP16-Gsc). At the four-cell stage, one ventral (C,E,G) or two dorsal (B,D,F,J,K) blastomeres were injected with 150 pg of Gsc (B,C), 150 pg of Eng-Gsc (D,E) or 500 pg of VP16-Gsc (F,G,J,K) mRNA. See Table 1 for quantitation. In situ hybridization for Otx2 (H,J) or En2 (I,K) and immunocytochemistry with the muscle antibody 12/101 (H,J) or the notochord antibody Tor 70 (I,K) were performed to assess the axial and neural development of VP16-Gsc-injected or uninjected (Control) embryos. White arrowheads indicate partial secondary axis (C,E) or cement gland (B,D). Black arrowheads indicate muscle (H,J) or notochord staining (I,K). Black arrows indicate Otx2 (H) or En2 (I,K) staining. Scale bar: 0.5 mm.

 


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  		Fig. 2. VP16-Gsc is a specific inhibitor of Gsc function. One ventral (A,B) or two dorsal (C,D) blastomeres were injected at the four-cell stage with 150 pg of Gsc (A), 500 pg of VP16-Gsc (C) or a combination of Gsc and VP16-Gsc (B,D). An uninjected embryo is also shown (E). Arrowheads indicate partial secondary axis (A) or cement gland (D). Scale bar: 0.5 mm.

 


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  		Fig. 3. Ectopic activation of Wnt signaling by VP16-Gsc inhibits head formation. At the four-cell stage two dorsal blastomeres were injected with 500 pg of VP16-Gsc (B), 100 pg of Frzb (C), 150 pg of tBMPR (E), VP16-Gsc and Frzb (D) or VP16-Gsc and tBMPR (F). An uninjected embryo is also shown (A). Quantitation is shown in G. Each bar represents the percentage of embryos with anterior truncations resulting from five independent experiments. n, total number of injected embryos. Scale bar: 0.5 mm.

 


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  		Fig. 4. VP16-Gsc activates Xwnt8 expression in animal explants. Animal explants were isolated from uninjected (A,B) or VP16-Gsc-injected embryos (C,D), and were cultured in the absence (A,C) or presence (B,D) of Activin protein. The morphology of explants was assessed at the tailbud stage. The expression of Xbra, Xwnt8 and BMP4 in response to Gsc or VP16-Gsc was examined by RT-PCR analysis at the gastrula stage (E). EF1{alpha} is a control for RNA recovery and loading. Intact embryos (embryo) served as a positive control and an identical reaction without reverse transcriptase controlled for PCR contamination (embryo-RT). Scale bar: 0.2 mm.

 


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  		Fig. 5. Cell autonomous activation of Xwnt8 by VP16-Gsc. At the 16-cell stage, one dorsal marginal zone blastomere was injected with 750 pg of ß-galactosidase (ß-gal) mRNA (A) or a combination of ß-gal and 500 pg of VP16-Gsc (B). Descendents of the injected cell were identified at the gastrula stage by the presence of ß-gal, using the Rose-gal substrate. Activation of Xwnt8 by VP16-Gsc in the dorsal marginal zone and endogenous ventrolateral expression of Xwnt8 was detected by in situ hybridization with BM-purple substrate (A,B). Dorsal views of stage 10.25 embryos with endogenous Xwnt8 expression visible in lateral regions. Insets show higher magnification views of boxed regions. Black arrowheads indicate ß-gal-positive nuclei (red) and the white arrowhead indicates cytoplasmic Xwnt8 in situ stain (purple). Scale bar: 0.25 mm; 80 µm in the insets.

 


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  		Fig. 6. Gsc inhibition of trunk formation. At the two-cell stage, both blastomeres were injected in the equatorial region with 100 pg of Gsc (B), Eng-Gsc (C) or Frzb (D). The presence of axial structures was examined by immunostaining with a muscle-specific antibody (12/101). Quantitation is shown in E. Each bar represents the percentage of embryos with reduction or absence of trunk resulting from three independent experiments. n, total number of injected embryos. Scale bar: 0.5 mm.

 


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  		Fig. 7. Complete axis induction by co-expression of Gsc and a truncated BMP receptor. At the four-cell stage, two ventral blastomeres were injected with 100 pg of Gsc (B), 250 pg of a truncated BMPR (tBMPR) (C) or a combination of both (D), and axis induction was assessed at the late tadpole stage. Arrowhead indicates partial axis (C) or ectopic eye in a complete secondary axis (D). Quantitation is shown in E. Each bar represents the percentage of embryos with ectopic axes resulting from four independent experiments. Dark bar, complete secondary axes; light bar, partial secondary axes; n, total number of injected embryos. Scale bar: 1.0 mm.

 


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  		Fig. 8. VP16-Gsc alters gene expression at the gastrula and neurula stages. At the four-cell stage, two dorsal blastomeres were injected with 500 pg of VP16-Gsc. Embryos were fixed at stage 10.5 (early gastrula), 12.5 (late gastrula), 15 (mid-neurula) or 20 (late neurula). Gene expression was detected by in situ hybridization of uninjected (Control) and VP16-Gsc-injected embryos. VP16-Gsc caused an expansion of Xwnt8 (B) and MyoD (F) into the dorsal marginal zone at stage 10.5, and MyoD-positive cells were present at the midline (white arrow) at stage 15 (H). Frzb and Chordin expression in the organizer was not affected at stage 10.5 (D,J) and Chordin expression in the chordamesoderm was normal at stage 15 (L). Otx2 was reduced or absent at all stages examined (N,P,R,T). Opl expression in the prospective neural plate was not affected by VP16-Gsc at stage 10.5 (V), but was absent from the most anterior domain of the neural plate (bracket) at stage 12.5 (X). Black arrowheads indicate the dorsal blastopore lip. The black arrows indicate the primordia of the forebrain (fb), eyes (e) and cement gland (cg) (S). Vegetal views are shown in A-F,I,J,M,N, dorsal view is shown in G,H,K,L,U,V with anterior on the left in G,H,K,L and anterior view is shown in O-T,W,X. Scale bar: 0.5 mm.

 


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  		Fig. 9. Direct regulation of Xwnt8 transcription by Gsc. (A) Xwnt8 promoter-luciferase reporter plasmids containing 628 bp of upstream sequence (–628Xwnt8/Luc), 628 bp with site-directed mutations at each P3 site (–628Xwnt8-4Xmut/Luc) or 202 bp (–202Xwnt8/Luc). Four Paired-type homeodomain binding sites (P3 consensus sequences) present in the –628Xwnt8/Luc reporter are indicated by dark gray boxes, and the TATA element (–36) and transcriptional start site (+1) are also indicated. (B) The animal pole was injected at the one-cell stage with 200 pg of the indicated reporter plasmid and 10 pg of the internal control (pRLCMV), in combination with 500 pg of Gsc or 1000 pg of VP16-Gsc mRNA and animal explants were analyzed for luciferase activity at the gastrula stage. Mean luciferase activity was determined by normalizing to the activity of each reporter plasmid in the absence of co-injected RNA (control), and the standard error is shown for each sample. Statistical analysis confirmed the significance of the response of –628Xwnt8/Luc to Gsc and VP16-Gsc (**, P<0.01; *, P<0.05). The data presented are the average of three independent experiments. (C) Binding of Gsc to the Xwnt8 promoter was examined by electrophoretic mobility shift assay. In vitro translated Gsc protein was incubated with a radiolabeled probe encoding a single P3 site (–458). Gsc bound strongly to the P3 site (lane 3) and formation of this Gsc-DNA complex (bound) was competed by an excess of unlabeled P3 site (lane 4). Complex formation was not observed with the addition of a control in vitro translation reaction (cont) lacking Gsc protein (lane 2) or in the absence of any added protein (lane 1). Unbound P3 site is also indicated (free).

 

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