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Fig. 1. Function of Hh and Wnt signals during neural induction, anteroposterior patterning and eye field development. Xenopus embryos were injected into the animal pole of a single blastomere at the 4-cell stage with the indicated constructs and nlacZ mRNA as a lineage tracer (red nuclei on injected right side). (A,A′) Control late gastrula in dorsal view depicting the neural plate marker Sox2 and the epidermal marker Cytokeratin. (B,B′,C,C′) XGli1 mRNA expands neural at the expense of epidermal tissue, while Gli3C′δClaI mRNA causes the opposite effect. (D,D′,E,E′) XWnt3a DNA promotes epidermal and dnXWnt8 mRNA neural development. (G–G′) Control embryos at neurula stage in anterior view (G,G′) and at tail bud stage in dorsal view (G′). Otx2 demarcates the developing cement gland, forebrain and midbrain (horizontal line), FoxG1 the telencephalon, En2 the posterior midbrain, and Krox20 the hindbrain rhombomeres 3 and 5 (bracket). Open arrowhead points to the anterior border of HoxC6 expression in the spinal cord. (H–H′,I–I′) XGli1 mRNA anteriorizes the brain, whereas Gli3C′δClaI causes very subtle posteriorization. (J–J′) XWnt3a DNA has posteriorizing activity. The arrow points to an expansion of Krox20 expression in neural crest cells. (K–K′) dnXWnt8 mRNA leads to anteriorization. (M) Control tail bud embryo in anterior view, showing Rx2A expression in the bilateral eyes. (N–O) XGli1 mRNAs diminish, whereas Gli3C′δClaI mRNAs expand the eye anlage. (P,Q) XWnt3a DNA leads to a smaller and dnXWnt8 mRNA to a larger eye anlage. (F,L,R) Summary of effects of Hh and zygotic Wnt signals on neural induction (F), anteroposterior patterning (L), and specification of the eye field (R). The indicated gene expression patterns were obtained in: B, 43/46; B′, 24/26; C, 48/49; C′, 41/52; D, 36/40; D′, 17/19; E, 57/57; E′, 64/68, H, 9/10; H′, 12/12 (FoxG1), 8/12 (En2); H′, 7/7 (Krox20), 11/11 (HoxC6); I, 11/13; I′, 18/20 (FoxG1), 15/20 (En2); I′, 17/26 (Krox20), 45/49 (HoxC6); J, 44/44; J′, 34/34 (FoxG1), 29/34 (En2); J′, 43/50 (Krox20), 12/17 (HoxC6); K, 42/45; K′, 37/37 (FoxG1), 29/37 (En2); K′, 15/15 (Krox20), 13/13 (HoxC6); N, 8/10; O, 17/20; P, 8/9; Q, 11/11 (Rx2A).
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Fig. 2. Gene expression of XSufu in Xenopus embryos. (A–C) RT-PCR of whole embryos (A) and embryonic explants (B,C). Histone H4 was used as RNA loading control. DMZ, dorsal marginal zone; VMZ, ventral marginal zone. (D–M) Whole-mount in situ hybridization of embryos shown in dorsal (D,E), anterior (F,I,J), and lateral views (K–M). Panels (G) and (H) are transversal sections of embryo in (I). anp, anterior neural plate; ba, branchial arch; ea, ear; ey, eye; fb, forebrain; mhb, mid–hindbrain boundary; nc, neural crest; ng, neural groove; np, neural plate; ppp, panplacodal primordium, tb, tail bud.
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Fig. 3. Microinjection of XSufu mRNA suppresses neural plate formation, stimulates anterior neural markers and promotes eye development. Embryos were animally injected at the 4-cell stage with nlacZ mRNA as control or XSufu mRNA. All specimens were injected into a single blastomere. Embryos are shown in dorsal (A,A′,B,B′,E–E′,F,F′,H), anterior (C–C′,D–D′,G,G′), lateral views (I,I′), or as transversal section (J,J′). (A,B,A′,B′) XSufu mRNA causes reduction of Sox2 and concomitant expansion of Cytokeratin expression on the injected right side (arrowhead). (C–E,C′–E′) XSufu mRNA causes posteriorward expansion of Otx2 expression and a posterior shift of En2 and Krox20 expression, while FoxG1 expression remains unaffected. (C′–E′) Co-injection of XSufu mRNA and Wnt3a DNA restores normal expression of Otx2, En2, and Krox20. (F,F′) XSufu mRNA has no effect on MyoD expression upon animal injection. (G,G′,H–J,I′,J′) XSufu mRNA leads to an enlargement of Rx2A expression, enlarged eye structures and expansion of the neural retina. Frequency of embryos with the indicated phenotype was: A′, 44/46; B′, 61/62; C′, 7/10; C′, 12/12; D′, 46/51; D′, 19/21; E′, 116/125; E′, 32/40; F′, 19/21; G′, 95/108; H, 56/115.
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Fig. 4. Depletion of XSufu induces expansion of the neural plate, stimulates anterior neural markers and suppresses eye development. (A) Targeting sequence of the XSufu morpholino oligonucleotide (XSufu-MO). The non-targeted XSufu* mRNA construct lacks the 5′ untranslated region. Blue letters indicate start codon. (B) In vitro transcription–translation assay. The XSufu-MO, but not an unspecific control-MO, inhibits XSufu protein synthesis. XSufu-MO does not reduce translation of XSufu* mRNA. (C,C′) At the tadpole stage, XSufu-MO-injected embryos have reduced head and tail structures. (D–D′) Dorsal view of early neurula embryos. Injection was performed animally into one blastomere at the 2-cell stage together with nlacZ mRNA as tracer. XSufu-MO, but not control-MO, causes expansion of Sox2 expression on the injected side (arrowhead); this effect is reverted by co-injection with XSufu* mRNA. (E,E′) XSufu-MO leads to a reduction of Cytokeratin expression. (F–H,F′–H′,G′,H'') Anterior view of neurula (F,F′,G–G′) and dorsal view of tailbud embryo (H–H′). XSufu-MO causes expansion of Otx2, reduced intensity of En2, and a posterior shift of En2 and Krox20 expression, while FoxG1 expression remains unaffected. Co-injection of XSufu-MO and XSufu* mRNA rescues normal En2 and Krox20 expression. (I,I′) Dorsal view of early neurula. XSufu-MO induces reduction of MyoD expression (arrowhead). (J,J′) Anterior view of tail bud embryo. XSufu-MO leads to a reduction of Rx2A expression. (K–M,K′–M′) A single injection of XSufu-MO causes reduction of eye structures (arrowheads), which is reverted by co-injection of XSufu* mRNA. The indicated phenotypes were observed in: C, 70/83; C′, 21/43; J, 22/24; D, 50/50; D′, 71/77; D′, 82/87; E, 24/27; E′, 39/40; F, 23/24; F′, 20/20; G, 39/39 (FoxG1), 55/55 (En2); G′, 42/42 (FoxG1), 54/54 (En2); G′, 31/31; H, 65/67; H′, 79/79; H′, 34/40; I, 24/26; I′, 27/28; J, 40/42; J′, 67/73; L,L′, 25/25; M,M′, 21/43.
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Fig. 5. XSufu is important for cement gland, neural crest, neuronal and paraxial mesoderm development. Embryos were injected together with nlacZ mRNA as tracer animally into one blastomere at the 2- or 4-cell stage. (A,A′,B,B′) Whole-mount in situ hybridization of early neurula embryos in anterior view. XSufu mRNA and XSufu-MO lead to a reduction of the cement gland marker XAG while a non-specific control-MO has no effect. (C–F,C′–F′,D′,F′) Late gastrulae in dorsal and tail bud embryo in lateral view. XSufu mRNA and XSufu-MO cause a reduction of the neural crest markers Slug and Snail. Co-injection of XSufu-MO and XSufu* mRNA reverts to normal Slug and Snail expression. (G,G′,H–H′) Early neurulae in dorsal view. XSufu mRNA causes slight expansion of the neuronal marker N-tubulin. XSufu-MO reduces N-tubulin expression. Normal N-tubulin expression is seen after co-injection of XSufu-MO with XSufu* mRNA. (I–I′) Tail bud embryos in dorsal view. XSufu-MO inhibits formation of MyoD-positive segmented somites (see also magnification in inset). Indicated effects were observed in: A′, 9/16; B, 40/45; B′, 7/7; C′, 70/70; D, 65/68; D′, 14/15; D′, 62/64; E′, 62/65; F, 13/13; F′, 19/19; F′, 30/33; G′, 66/81; H, 102/105; H′, 78/84; H′, 31/39; I, 15/20; I′, 17/17.
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Fig. 6. XSufu inhibits Hh/Gli signaling in the Xenopus embryo. (A) Molecular analysis by RT-PCR of animal cap (AC) explants cultured until stage 16. Animal injection of 500 pg XBhh mRNA elevates the expression of XPtc2, XPtc1, and XGli1, and this effect is reverted by co-injection of 16 ng XSufu mRNA. XSufu-MO, but not control-MO, increases the expression of these Hh target genes. H4 was used for normalization. (B,B′,C,C′) Whole-mount in situ hybridization of early neurula embryos in dorsal view. Animal injection of 15 ng XSufu mRNA reduces, while XSufu-MO, but not control-MO, expands the expression of XPtc1 expression. Indicated effects were observed in: B, 61/61; B′, 29/38; C, 58/58; C′, 32/37.
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Fig. 7. XSufu inhibits Wnt/β-catenin signaling in the Xenopus embryo. (A) Molecular analysis by RT-PCR of animal cap (AC) explants cultured until stage 10. Embryos were animally injected with 25 pg XWnt8 mRNA either alone or in combination with the indicated amount of XSufu mRNA. H4 was used for normalization. XSufu mRNA downregulates XWnt8-induced transcription of Siamois and Xnr3 in a dose-dependent manner. (B) XSufu-MO, but not control-MO, induced expression of the Wnt target genes Siamois, Xnr3, and Chordin in injected AC explants at stage 10. (C–F,C′–F′) Whole-mount in situ hybridization of early gastrula embryos in dorsal view. A single marginal injection of XSufu mRNA at the 4-cell stage reduces, while a single animal injection of XSufu-MO, but not of control-MO, at the 2-cell stage expands endogenous expression of Xnr3 and Chordin. (G–G′) Animal view of early gastrulae. A single animal injection of 12 pg XWnt8 mRNA at the 4-cell stage induces ectopic Xnr3 expression (G′) that is reduced by co-injection of XWnt8 and XSufu mRNA (G′). Injected nlacZ-positive cells are highlighted with a strippled line. (H) Dorsal view of uninjected neurula embryo. Condensed natural pigmentation at the site of neural tube closure delineates the main body axis. (I–M) A single ventral injection of 4 pg XWnt8 mRNA induces a complete secondary body axis (arrowhead in I) that is blocked by co-injection with 4 ng XSufu mRNA (J) or by co-injection with a combination of XSufu and 150 pg XGli1 mRNA (K). Note that XWnt8-induced secondary axis formation is partly reversed by co-injection of 150 pg XGli1 mRNA (arrowhead in L) and inhibited by co-injection of 450 pg XGli1 mRNA (M). (N) Quantification of secondary axis formation. Indicated effects were observed in: C, 14/14; C′, 8/10; D, 15/18; D′, 24/36; E, 13/13; E′, 7/8; F, 24/26; F′, 21/28; G, 47/47; G′, 14/14; G′, 55/55.
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sufu (suppressor of fused homolog) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 12, blastoporal view, dorsal up.
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sufu (suppressor of fused homolog) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 13, dorsal view, anterior up.
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sufu (suppressor of fused homolog) gene expression in Xenopus laevis embryo via in situ hybridization, NF stage 15, anterior view, dorsal up.
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sufu (suppressor of fused homolog) gene expression in Xenopus laevis embryo via in situ hybridization, NF stage 18, anterior view, dorsal up.
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sufu (suppressor of fused homolog) gene expression in Xenopus laevis embryo via in situ hybridization, NF stage 23, anterior view, dorsal up.
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sufu (suppressor of fused homolog) gene expression in Xenopus laevis embryo via in situ hybridization, NF stage 26, later view, dorsal up, head region only.
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sufu (suppressor of fused homolog) gene expression in Xenopus laevis embryo via in situ hybridization, NF stage 32, later view, dorsal up.
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