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Figure 1. In situ hybridization analysis showing the spatial expression pattern of Sp5.a at the indicated stages. A: Dorsal view, anterior to the top. B: Lateral view of the embryo in A, anterior to the left. C: Dorsal view, anterior to the top. D: Transverse section of the embryo in C at the level indicated by the dashed line. E: Magnified view of the boxed area in D. F, G: Anterior view, dorsal to the top. H: Arrowheads denote Sp5.a expression in the dorsal midline along the fin. ba, branchial arches; ov, otic vesicle.
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Figure 2. Knockdown of Sp5 leads to defective NC derivatives. A: Four-cell stage embryos were injected in the animal pole region with the indicated combination of Sp5.a-Myc (400 pg), Sp5.b-Myc (500 pg), Sp5 MO1 (60 ng), Sp5 MO2 (60 ng), and Co MO (60 ng), and the animal caps were dissected at stage 9 and cultured to stage 10.5 for western blotting. β-Actin serves as a loading control. B, C: Two blastomeres of 4-cell stage embryos were injected in the dorso-animal region with Sp5 MO1 (20 ng for B, 10 ng for C), Sp5 MO2 (40 ng), and Sp5.a (50 pg) as indicated and cultured to stage 38–40. An arrow indicates the defective dorsal fin. Control, uninjected control embryo. Sub., Suboptimal. D: One blastomere of 4-cell stage embryos was injected in the dorso-animal region with Sp5 MO1 (10 ng) or Co MO (10 ng) and then subjected to paraffin sectioning and eosin staining at stage 40. An arrow denotes the defective eye in the injected side. E: Alcian blue-stained cranial skeletal preparations from control and Sp5 MO-injected embryos at stage 45. One blastomere of 4-cell stage embryos was injected in the dorso-animal region as indicated with Sp5 MO1 (high, 10 ng; sub, 5 ng), Sp5 MO2 (20 ng), and Co MO (10 ng). Arrows indicate the severely disrupted ceratobranchial cartilages in the injected side. Control, uninjected control skeleton. cb, ceratobranchial cartilage; mc, Meckel's cartilage.
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Figure 3. Sp5 is involved in neural crest induction. A–C: Embryos were injected in the dorso-animal region of one blastomere at the 8-cell stage with Sp5.a (A, 300 pg), Sp5.a-Myc (B, 400 pg), Sp5.b-Myc (B, 400 pg) or Sp5.a-GR (500 pg) mRNA and the expression of Msx1, Sox9, Sox10, or Slug was analyzed at stages 16–17 by in situ hybridization. C: Injected embryos were treated or not with dexamethasone (DEX, 10 μM) from stage 10, 13 or 15 to 16. Embryos are shown in anterior view with dorsal to the top (A, B, and C, right panels) or in dorsal view with anterior to the top (C, left panels). Arrows in C denote the expansion of Msx1 or Slug expression.
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Figure 4. Sp5 is required for neural crest induction. A, B: Four-cell stage embryos were injected in the dorso-animal region of one blastomere as indicated with Sp5 MO1 (10 ng for A, 5 ng for B), Sp5 MO2 (20 ng), or Co MO (10 ng for A, 25 ng for B). Sox10, Sox9, Slug, or Zic1 expression was analyzed at stage 16–17 by in situ hybridization. Embryos are shown in dorso-anterior view with posterior to the top. C, D: Embryos were injected in the animal pole region at the 4-cell stage with the indicated combination of Noggin (100 pg), XWnt8-CSKA (100 pg), DN BR (400 pg), Sp5 MO1 (60 ng), Co MO (60 ng), and Sp5.a (50 pg for C, 400 pg for D) and then animal caps were excised at stage 8.5 and cultured to stage 16 (C) or 12 (D) for RT-PCR analysis. ODC serves as a loading control. WE, whole embryo; Con. AC, uninjected control animal cap; -RT, a control in the absence of reverse transcriptase.
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Figure 5. Depletion of Sp5 has no effect on neural development. A–N: Two- or four-cell stage embryos were injected in the dorso-animal region of one blastomere with Sp5 MO1 (10 ng) and Sp5 MO2 (30 ng) as indicated, and the expression of Sox2, N-tubulin, Otx2, Bf1, En2, Krox20, or HoxB9 was analyzed at stages 15–17. r3 and r5 denote the third and fifth rhombomeres, respectively. Embryos are shown in dorsal view with anterior to the top (A–D, M, N) or in anterior view with dorsal to the top (E–L).
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Figure 6. Sp5 acts upstream of NPB specifiers in NC induction. A, C: Four-cell stage embryos were injected in the animal pole region as indicated with a combination of Sp5.a (400 pg), dominant negative BMP4 receptor (DN BR, 1 ng), BMP4 (400 pg), Sp5 MO1 (40 ng), and Co MO (40 ng) and, subsequently, animal caps were excised at stage 8.5 and cultured to stage 11 for RT-PCR analysis. B: One blastomere of 8-cell stage embryos was injected in the dorso-animal region as indicated with GSK3 (500 pg), Sp5.a (200 pg), Sp5 MO1 (10 ng), and Co MO (10 ng) and then subjected to whole-mount in situ hybridization against Msx1 at stages 16–17. Arrows indicate inhibition of Msx1 expression in the injected side. Antero-dorsal view with posterior to the top. D: Four-cell stage embryos were injected in the animal pole region with Sp5.a-GR (30 pg) and then animal caps were dissected at the late blastula stage and immediately treated or not with cycloheximide (CHX, 10 μg/ml) as indicated. After 30 min, the tissue explants were treated with dexamethasone (DEX, 10 μM) and cultured for 5 hr for RT-PCR analysis. E: Embryos were injected in the dorso-animal region of one blastomere at the 8-cell stage with Sp5 MO1 (10 ng) alone or with Msx1 (100 pg) or Pax3 (70 pg) and then subjected to in situ hybridization against Sox10 at stage 16. Dorso-anterior view with posterior to the top.
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Figure 7. XWnt8 and FGF8a signals induce Sp5 expression. A, B: Four-cell stage embryos were injected in the animal pole region as indicated with FGF8a (1 ng), XWnt8 (400 pg), DN XWnt8 (500 pg), and DN Tcf3 (400 pg), and then animal caps were dissected stage 8.5 and cultured to stage 10.5 for qPCR analysis.
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Figure 8. Sp5 mediates the activity of FGF and Wnt signals in NC induction. A–N: Eight-cell stage embryos were injected in the dorso-animal region of one blastomere as indicated with FGF8a (40 pg), DN FGFR4 (350 pg), XWnt8-CSKA (100 pg), Sp5.a (100 pg for C, F and K, 200 pg for L–N), DN XWnt8 (400 pg), GSK3 (400 pg), DN Tcf3 (300 pg), and Sp5 MO1 (20 ng) and cultured to stage 16 for analysis of Sox9 or Sox10 expression. Embryos are shown in dorsal view with anterior to the top (A–C) or in dorso-anterior view with posterior to the top (D–N). O: Working model for NC specification by Sp5. See text for details.
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