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Fig. 1. dkk1 but not frzb induces complete secondary heads. Coinjections of tBR mRNAs with dkk1 (A) or frzb (B) into ventral blastomeres of four-cell embryos results in the development of secondary heads with two eyes or one eye, respectively. Embryos shown are 4 days postfertilisation (pf). (C-G) dkk1 rescues the cyclopic phenotype elicited by anti15 Ab, while frzb and bhh are unable to rescue cyclopia. Embryos are day 6 pf, shown from the dorsal side, anterior up. (C) Uninjected control embryo. (D) Embryo injected with anti15 Ab into the blastocoel. (E) Embryo injected radially with dkk1 mRNA and anti15 Ab. (F) Embryo injected radially with frzb mRNA and anti15 Ab. (G) Embryo injected radially with bhh mRNA and anti15 Ab. (H) Embryo injected radially with bhh mRNA. (I) Uninjected control embryo.
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Fig. 2. dkk1 but not frzb induces complete prechordal plates. (A,B) In situ hybridization with sonic hedgehog (Shh) in embryos coinjected at the four-cell stage ventrally with tBR and either dkk1 (A) or frzb (B). Stage-30 embryos are shown from the anterior. Arrowhead indicates the anterior limit of Shh expression. (C-D) in situ hybridization for Shh in embryos injected at the four-cell stage ventrally with nuclear lacZ lineage tracer, tBR and either dkk1 (C) or frzb (D). Stage-16 embryos were cut sagittally; secondary axes are marked by light blue β-gal staining. Arrowheads indicate the anterior limit of secondary Shh expression. Note that the expression in the prechordal plate in (D) is lacking. (E,F) In situ hybridization for goosecoid (gsc) in embryos injected as in A and B. Sagittal sections of stage-14 embryos are shown. Arrowheads indicate the mesodermal zone of gsc expression in the anterior mesoderm of the induced secondary axis (2. Note that mesodermal gsc expression extends anterior to the neuroectodermal in the primary axis (1 and secondary axis induced by dkk1, but is in register with the neural expression in frzb-injected embryos (F). a, anterior; p, posterior.
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Fig. 3. dkk1 is required for prechordal plate formation. (A,D,G) Uninjected controls, (B,E,H) Embryos injected with anti15 Ab into the blastocoel at blastula stage and (C,F,I) embryos injected radially at the four-cell stage with dkk1 mRNA. In situ hybridization with probes to XHex1 (A-C), XBlimp1 (D-F) and XNot2 + gsc (G-I). (A-F) Embryos from early neurula stage (stage 13) were cut sagittally; dorsal side up, anterior to the right. White arrowheads indicate the anterior and posterior limits of expression. (G-I) embryos from stage 13 are shown from the dorsal side, anterior side up. (J-L) The prechordal plates of control (J), anti15 Ab- injected (K), or dkk1 mRNA-injected embryos (L) are shown from gastrocoel, anterior side is up. Red arrows indicate the lateral limit of Shh expression. PCP, prechordal plate, NOT, notochord.
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Fig. 4. dkk1 regulates anteroposterior patterning of neurectoderm. (A,E,I) control embryos, (B,F,J) embryos injected radially at the four-cell stage with dkk1 (C,G,K) embryos injected with anti15 Ab into the blastocoel and (D,H,L) embryos treated with 10−7 M retinoic acid (RA). In situ hybridization for BF1 and En2 (A-D), Xanf1 (E-H), and XOtx2 (I-L). Midgastrula-stage embryos (stage 15) are shown from anterior, dorsal side up.
(M) Schematic diagram of the BF1 (dark green), Xanf1 (blue), XOtx2 (violet) and En2 (yellow) expression under the treatments indicated above. Note the changes in size of the head anlage and change in the proportions between its anterior and posterior regions. (N-U) dkk1 affects forebrain at the expense of midbrain, whereas RA affects both regions. (N-U) In situ hybridization for BF1 and En2 under the treatments indicated above. Brains of stained embryos were excised from 4-day embryos and are shown from the dorsal (N-Q) and lateral (R-U) sides, anterior side up. Note the expansion of tel- and diencephalon and reduction of mesencephalon in O and S and the opposite changes in P and T. di, diencephalon; mes, mesencephalon; rh, rhomencephalon; tel, telencephalon.
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Fig. 5. dkk1 regulates dorsoventral patterning of neuroectoderm. (A,E) Control embryo, (B,F) embryos radially injected at the four-cell stage with dkk1, (C,G) injected with anti15 Ab into the blastocoel and (D,H) treated with 10−7 M RA. In situ hybridisation for XNot2 (A-D) and Shh (E-H).
(A-D) Embryos are shown from the anterior, dorsal side up, (E-H) brains excised from day 4 pf. Brains are shown from the lateral side. zli, zona limitans intrathalamica, mhb, midbrain-hindbrain boundary.
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Fig. 6. Timing of dkk1 action. (A,F) Schematic drawings of experiments. The anterior halve of the gastrocoel roof containing the head anlage was excised from stage 12.5 or 13.5 embryos and cultured in the presence of anti14 (control, D,E) or anti15 Ab (B,C) until stage 20, then transferred to 0.5Barth solution and cultured for an additional 24 hours. Note cyclopia in (B). (F-J) Anterior halves of gastrocoel roof excized from stage-12.5 or -13.5 embryos were conjugated with animal caps, injected with dkk1 mRNA (G,H) or control animal caps (I,J) and cultured for 2 days. Note that the head in G is strongly anteriorised.
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Fig. 7. dkk1 rescues embryos posteriorized by bFGF, BMP4, XSmad1 and Wnt3A, but not by RA. Where indicated, embryos were injected radially at the four-cell stage with dkk1 or frzb mRNA. (A,B) Embryos treated from stages 8-13 with 10−7M retinoic acid (RA). (D-F) Embryos were injected radially at the four- cell stage with BMP4 or (G-I) XSmad1 mRNA. (J-L) Embryos were injected radially at the four-cell stage with dkk1 or frzb mRNA and at the blastula stage with bFGF protein into the blastocoel. (M-O) Embryos were injected animally at the four-cell stage with mWnt3A plasmid DNA. (C) Uninjected control.
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