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Zebrafish one-eyed pinhead (oep) is essential for embryonic axis and dorsal midline formation by promoting Nodal signalling and is thought to act as a permissive factor. Here we describe that oep elicits profound phenotypic effects when overexpressed in Xenopus and zebrafish. In Xenopus, wild-type oep inhibits mesoderm induction, disrupts axis formation and neuralizes animal caps. A secreted Oep dorsoanteriorizes and neuralizes Xenopus embryos indicative of BMP inhibition. In zebrafish, misexpression of smad1 in oep mutant embryos also reveals an interaction of oep with BMP signalling. Furthermore, the phenotypic effect of nodal overexpression can be rescued by coexpression of oep both in Xenopus and zebrafish. Taken together, our results support an interaction between oep and nodal but they suggest also (1) that the role of oep in Nodal signalling may include negative as well as positive regulation, (2) that oep is able to function in an active fashion and (3) that oep exerts a regulatory effect on the BMP signalling pathway.
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10842057
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Fig. 1. Overexpression of oep leads to anteriorization and axis disruption in whole embryos and neuralization of animal caps. Embryos were injected at the 4-cell-stage into all blastomeres with 2.5 ng/blastomere control (ppl) (A,C), oep (D) or s-oep RNA (B,E). (A,B) Stage-22 embryos, ventral view, anterior to the top. (C–E) Stage-35 embryos, lateral view, anterior to the left, dorsal to the top. Note the anteriorization of s-Oep-injected embryos. (F–H) Embryos were injected animally at the 4-cell stage into all four blastomeres with 1.25 ng/blastomere control (ppl), s-oep or oep RNA. Animal caps were cut at stages 8–8.5 and cultured until control embryos reached stage 36. Note induction of cement glands by s-oep and oep (arrowheads). (I) RT–PCR analysis of animal caps. Four-cell embryos were injected animally with 0.375 or 1.25 ng/blastomere of the indicated RNAs into all four blastomeres, cut at stages 8–8.5 and cultured until control embryos reached stage 11. (J) RT–PCR analysis of animal caps. Four-cell embryos were injected and animal caps were cut as described above and cultured until control embryos reached stage 36. H4, Histone 4; −RT, negative control without reverse transcriptase; we, whole embryo positive control; ni, animal caps cut from noninjected embryos as negative control.
Fig. 2. S-oep inhibits mesoderm induction. (A) RT–PCR analysis of stage-11 whole embryos (left side) and animal caps (right side). For the analysis of animal caps, 4-cell-stage embryos were injected animally into all four blastomeres with the following mRNAs: 0.0125 ng/blastomere Xnr1, 0.0625 ng/blastomere Activin, 0.25 ng/blastomere BMP-4, 0.625 ng/blastomere s-Oep. All RNA samples were adjusted to 0.875 ng/blastomere with PPL as carrier RNA where required. Two batches of both PPL-injected and s-Oep-injected caps were treated with basic fibroblast growth factor protein (bFGF) or γ-globulins (control) as described in Section 4. For RT–PCR analysis of whole embryos, 4-cell-stage embryos were radially injected with 2.5 ng/blastomere control (ppl) or s-oep RNA. H4, Histone 4; −RT, negative control without reverse transcriptase; we, whole embryo positive control; ni, caps from noninjected embryos as negative control. (B) Embryos were injected at the 4-cell stage into all four blastomeres with the following RNAs in ng/blastomere: 1.25 PPL (left), 0.025 PPL (middle) or 0.025 s-Oep (right). Note the axial rescue of Xnr1-injected embryos (0% complete or partial axis, n=45) by s-Oep (96% complete or partial axis, n=45).
Fig. 3. Overexpression of oep causes posterior truncations (A) and inhibits hyperdorsalization by cyc in zebrafish embryos (B,C). (A) Uninjected control (left) and oep mRNA-injected embryos (right) stained with an antisense shh probe. Truncation of the posterior body axis is depicted by arrowheads. (B) Representative examples of phenotypic groups observed after injection of cyc mRNA alone or together with oep mRNA. Embryos were hybridized to shh mRNA at 24 h of development and are shown anterior to the left and dorsal side up except for group III embryos which are shown animal pole up. Ectopic shh expression is labelled with arrows. (C) Bar diagram depicting the frequency of group I–III phenotypes caused by injection of cyc mRNA (n=323) or the combination of cyc and oep mRNA (n=238).
Fig. 4. Oep controls the activity of smad1 in zebrafish embryos. (A,B) Control and smad1 mRNA-injected wild-type embryos. (C,D) Control and smad1 mRNA-injected oep mutant embryos. (E,F) Control and smad1 mRNA-injected cyc mutant embryos. Embryos were hybridized to an antisense shh probe (blue/black, A–F) and an antisense hgg1 probe (red, A–D), which marks hatching gland tissue. Arrowhead and arrows point out rescued hatching glands and shh expression in smad1-injected oep mutant embryos, respectively. Embryos are 24 h old and are oriented anterior to the left and dorsal side up. The yolk was removed from the embryos in panels E,F.
Fig. 5. Soluble Oep protein does not bind to 293T cells expressing Xnr1. (A) Western blot analysis of HA-Xnr1, Flag-s-Oep and Cer-Fc proteins expressed in 293T cells. co, conditioned medium from mock-transfected 293T cells. (B) 293T cells mock-transfected or (B′) transfected with pCS2+HA-Xnr1 were stained with anti-HA antibody. (C) 293T cells mock-transfected or (C′) transfected with pCS2+HA-Xnr1 treated with Cer-Fc-conditioned medium were stained with anti-Fc antibody. (D) pCS2+HA-Xnr1-transfected cells treated with Flag-s-Oep-conditioned medium and stained with anti-Flag antibody.
