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Figure 1. Structure and Expression Pattern of XsalF(A) Percent identities of amino acid residues among XsalF and Sal family proteins. Highly conserved Zn finger domains are indicated by solid box. The structure of the dominant-negative XsalF is shown on the top. Amino acid residue numbers are indicated above.(B–I) Temporal and spatial expression of XsalF analyzed by whole-mount in situ hybridization. (B) Stage 13. (C) Stage 15. (D–I) Double-labeled in situ hybridization. (D) Slug (blue) and XsalF (indigo) probes. (E) XAG1 (blue) and XsalF (indigo) probes. (F) Otx2 (blue) and XsalF (indigo) probes. (G) Tcf3 (blue) and XsalF (indigo) probes. (H) Pax2 (indigo) and XsalF (blue) probes. (I) Gbx2 (blue) and XsalF (indigo) probes. (B, C, H, and I) Dorsal views with the anterior side up, (D–G) anterior view with the dorsal side up. (F and G) Arrows indicate the posterior borders.
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Figure 2. Microinjection of XsalF mRNA Induces Expansion of Forebrain/Midbrain Markers and Suppression of MHB/Hindbrain MarkersXsalF mRNA (400 pg) was injected into two left animal blastomeres of 8-cell embryos. Embryos were harvested at the neurula stage. (A–L) Analyzed by whole-mount in situ hybridization with the following probes. (A and B) Otx2 probe, anterior view with the dorsal side up; (C and D) Pax6, anterior view; (E and F) Pax2, anterior view; (G and H) Gbx2, anterior view; (I and J) MafB, dorsal view with the anterior side up; (K and L) MyoD, dorsal view. (A–D) Arrowheads indicate the midline.
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Figure 3. Attenuation of XsalF Function Results in Suppression of Forebrain/Midbrain Marker Genes and Upregulation of MHB/Anterior Hindbrain Genes(A) External phenotypes caused by injecting the dominant-negative XsalF (XsalF-delC). Top, control sibling. (B–N) Effects of XsalF-delC and XsalF-delC-GR injection. XsalF-delC (50 pg; [C, D, F, H, J, L]) and XsalF (400 pg; [D]) mRNA were injected into all animal blastomeres in 8-cell embryos. (M and N) XsalF-delC-GR (50 pg) was injected into two unilateral blastomeres and Dex was added at stage 13 (N). Embryos were harvested at late neurula stage and analyzed by whole-mount in situ hybridization with the following probes (anterior views): (B, C, D, M, N) Pax6 probe, (E and F) Otx2, (G and H) Bf1, (I and J) En2, (K and L) Pax2.(O) External phenotypes caused by injecting XsalF-MO. Top, control sibling. (P–Z) XsalF-MO injection experiments. XsalF-MO (10 ng; [Q, R, T, V, X, Z]), five base-mispaired control MO (10 ng; [P]) and XsalF mRNA (400 pg; [R]) were injected.Embryos were harvested at the neurula stage and analyzed by whole-mount in situ hybridization with the following probes (anterior views with the dorsal side up). (P, Q, R) Pax6 probes, (S and T) Otx2, (U and V) Bf1, (W and X) En2, (Y and Z) Pax2.
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Figure 4. XsalF Antagonizes the Wnt Activity and Is Required for Anterior-Specific Expression of GSK3β and Tcf3(A–C) Effects of overexpression of Wnt1 and XsalF on Pax6 expression (anterior view). (B and C) pCS2-Wnt1 (5 pg) and XsalF (C) mRNA (400 pg) were injected into all animal blastomere of 8-cell embryos. Embryos were analyzed at the neurula stage.(D) Animal caps were prepared from stage 10 embryos injected with Chd (50 pg), Chd (50 pg)+XWnt3a(50 pg), or Chd+XWnt3a+XsalF (400 pg) mRNA, cultured until siblings reached at late neurula stage, and analyzed by RT-PCR.(E–G and I–K) Transcriptional regulations of GSK3β and Tcf3. XsalF-MO ([F, G, J, K]; 10 ng) and XsalF mRNA ([G and K]; 400 pg) were injected into all animal blastomeres of 8-cell embryos and embryos were analyzed by whole-mount in situ hybridization with GSK3-β ([E–G]; dorsal view with the anterior side up; cleared embryos) and Tcf3 ([I–K]; anterior view) probes at the mid-neurula stage.(H) RT-PCR analysis of GSK3-β expression in the whole embryo (lane 1), anterior and posterior halves of the stage 15 embryo (lanes 2 and 3), XsalF-MO-injected and XsalF-MO+XsalF mRNA-injected embryos (lanes 4 and 5).(L) Animal caps were prepared from stage 10 embryos injected with Chd mRNA (50 pg), Chd mRNA+XsalF-MO (10 ng), Chd mRNA+XsalF-MO+XsalF mRNA (400 pg), cultured until siblings reached at late neurula stage, and analyzed by RT-PCR.
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Figure 5. Effects of GSK3β and GSK3β-MO on A-P Patterning(A–D) Gain-of-function of GSK3β. GSK3β mRNA (200 pg) was injected into two left animal blastomeres of 8-cell embryos.(E–L) GSK3β-MO ([F–H, J, and L]; 10 ng), GSK3β mRNA ([G]; 200 pg), and XsalF mRNA ([H]; 400 pg) were injected into all animal blastomeres of 8-cell embryos.(M–O) Injection of XsalF-delC mRNA ([N and O]; 50 pg) and pCS2-GSK3β DNA ([O]; 200 pg). Embryos were harvested at the neurula stage and analyzed by whole-mount in situ hybridization with the following probes (anterior views): (A, E–H) Otx2 probe, (B, I, J, M–O) Pax6, (C) Pax2, (D) Gbx2, (K and L) Six3.
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Figure 6. Effects of Tcf3 and Tcf3-delC on A-P PatterningTcf3 mRNA ([A–D]; 10 pg) was injected into two left animal blastomeres of 8-cell embryos. Tcf3-delC mRNA ([F, G, I, K]; 10 pg), XsalF-delC ([M, N, P, Q]; 50 pg), and Tcf3 mRNA ([G, N, Q]; 10 pg) were injected into all animal blastomeres of 8-cell stage embryos.Embryos were harvested at the neurula stage and analyzed by whole-mount in situ hybridization with the following probes (anterior view): (A, E–G, and O–Q) Otx2 probe, (B, H, I, L–N) Pax6, (C, J, K) Pax2, (D) Gbx2.
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