Two independent ways of inhibiting BMP signaling result in massive ectopic expression of chordin. (A-E) BMP signaling detected by immunohistochemistry using an anti phospho-Smad1/5/8 antibody; green dots indicate nuclei containing the phosphorylated Smad. (F-J) In situ hybridization with chd probe. (K-O) Expression of the epidermal marker p63. Wild-type embryos (A,F,K) are shown with in lateral view with ventral side to the left. Untreated ich embryos (B,G,L), and ich embryos injected with bmp2bMO (C,H,M), or with the two βcatMOs (D,I,N), or with all three MOs (E,I,O) are shown in lateral views. All embryos are at ~70% epiboly.

Inhibition of BMP signaling in ich embryos does not result in organizer induction. In situ hybridization with boz (A,B,C), gsc (D,E,F) and chd (G,H,I) probes in uninjected ich and bmp2bMO-injected ich embryos demonstrate that while impaired BMP signaling results in the transcriptional derepression of chd at the margins of the embryo (H,I), other characteristic organizer genes such as boz and gsc are not induced (C and F). (In A-C dome staged embryos are shown from an animal pole view, D-F and G-I present lateral views of 50% epiboly and 70% epiboly staged embryos, respectively. For wild-type embryos, dorsal is on the left.)

Embryos inhibited in both BMP signaling and organizer function form neuroectoderm with correct AP pattern at the end of gastrulation and this patterning can be modulated by similar mechanisms as in wild-type embryos. Single in situ hybridization with cyp26 (A,D,G,J,M,P,S,V,Y) or hoxb1b (B,E,H,K,NQ,T,W,Z) probes or double in situ hybridization with both probes (C,F,I,L,O,R,U,X,A′) is shown for wild-type embryos (A-C), untreated ich embryos (D-F), or ich embryos treated with bmp2bMO (G-I), the two β-cat MOs (J-L), bmp2bMO plus the two β-cat MOs (M-O), or ich embryos injected with antivin mRNA (P-R), antivin mRNA plus bmp2bMO (S-U), SU5402 (V-X), or SU5402 plus bmp2bMO (Y-A′). Wild-type embryos [A-C] are shown in dorsal views, while ich embryos are shown in lateral views. The neuroectodermal and tailbud expression domains of cyp26 in wild-type embryos are marked with arrow and star, respectively. All embryos are at ~100% epiboly.

AP neurectodermal patterning is retained at 11 hpf in embryos lacking BMP signaling and an active organizer. Single in situ hybridization with rx3 (A,D,G,J,M) or fkd3 (B,E,H,K,N) probes or double in situ hybridization with both probes (C,F,I,L,O) is shown for wild-type embryos (A-C), untreated ich embryos (D-F), or ich embryos treated with bmp2bMO (G-I), the two β-cat MOs (J-L), or bmp2bMO plus the two β-cat MOs (M-O). In wild-type embryos, rx3 demarcates the eye-field (A,C) and fkd3 the diencephalon (arrows) and rhombomere 5 (arrowheads) of the hindbrain (B,C).

Germ layer segregation in ′ciuffo′ embryos. Sections through the posterior protrusions of 24hpf ′ciuffo′ embryos stained with characteristic germ layer markers show that neuroectoderm specific markers. islet1 (A) and krox20 (B) are expressed in the outermost layer of the protrusion. The endodermal marker gata5 (C) is expressed in the innermost layers, whereas the mesodermal marker myoD (B) can be observed in between. Sections were made of 24 hpf ich embryos that had been injected with βcatMO1 and βcatMO2, hybridized as whole mounts with the indicated probes.

Wnt-, TGFβ- and FGF-signaling have pivotal roles in the posteriorization of the neuroectoderm of embryos both inhibited in BMP signaling and devoid of organizer activity. Single in situ hybridization with otx1 (A,D,G,J,M,P,S,V,Y) or gbx1 (B,E,H,K,Q,T,W,Z) probes or double in situ hybridization with both probes (C,F,I,L,O,R,U,X,A′) is shown for wild-type embryos (A-C), untreated ich embryos (D-F), or ich embryos treated with bmp2bMO (G-I), the two β-cat MOs (J-L), bmp2bMO plus the two β-cat MOs (M-O), or ich embryos injected with antivin mRNA (P-R), antivin mRNA plus bmp2bMO (S-U), SU5402 (V-X), or SU5402 plus bmp2bMO (Y-A′). Wild-type embryos [A-C] are shown in dorsal views, while ich embryos are shown in lateral views. All embryos are at ~100% epiboly. Arrowheads in the antivin (atv) treated embryos point to the edge of the germ-ring.

Complete repression of β-catenin signaling in ich embryos induces neuroectoderm with correct AP pattern. Untreated ich embryos do not express emx1, krox20, val, or hoxb6b (B,E,H), while their siblings coinjected with βcatMOs (C,C′,F,F′,I,I′) express these neuroectodermal markers in a correct order at 22 hpf, with the anterior to posterior direction corresponding to proximal to distal relative to the yolk (compare A with C,C′, D with F, F′, and G with I,I′). The following probe-pairs were used: emx1 (blue) and krox20 (red) (A-C′), krox20 (red) and val (blue) (D-F′), and krox20 (red) and hoxb6b (blue) (G-I′).

Posteriorizing TGFβ activity is only partly dependent on Nodal signals. In situ hybridization with cyp26 (A,C,E,G,I,K,M) or hoxb1b (B,D,F,H,J,L,N) probes is shown for wild-type embryos (A,B), untreated ich embryos (C,D), ich embryos treated with bmp2bMO (E,F), SB431542 (G,H), sqtMO and cycMO (K,L), or bmp2bMO in combination with SB431542 (I,J) or sqtMO and cyc MO (M,N). A small molecular inhibitor of the TGFβ pathway, SB431542, has no effect on untreated ich embryos (G,H), but when it is applied to bmp2bMO injected ich embryos, it anteriorizes the neuroectoderm (I,J). These embryos have gastrulation defects; the arrowheads point to the position of the stalled germ-ring. Coinjection of sqtMO and cycMO with bmp2bMO results in a slight expansion of the cyp26 domain (M), and a mild reduction of the hoxb1b domain (N), showing that the posteriorizing effects of TGFβ signaling are dependent to some extent on Nodal-independent signals. Injection of sqtMO along with cycMO into untreated ich embryos has no effect on cyp26 and hoxb1b expression (K,L). SB431542 was used at 2.4 mM concentration; sqtMO and cycMO were 3 mM each. Wild-type embryos (A,B) are shown in a dorsal view, ich embryos (C-N) from a lateral view. All embryos are at ~10 hpf.

Antagonists of the FGF pathway can anteriorize ich embryos with impaired BMP signaling. Single in situ hybridization with cyp26 (A,D,G,J,M,P,S) or hoxb1b (B,E,H,K,N,Q,T) probes or double in situ hybridization with both probes (C,F,I,L,O,R,U) is shown for wild-type embryos (A-C), untreated ich embryos (D-F), ich embryos treated with bmp2bMO (G-I), injected with XFD or mkp3 mRNAs alone (J-L and P-R), or in combination with bmp2bMO (M-O and S-U). The injection of mRNAs encoding a dominant negative FGF receptor, XFD, or a negative regulator of the MAPK pathway, mkp3, into bmp2bMO-injected embryos results in the posterior expansion of the anterior neurectodermal marker, cyp26 (M,O, and S,U, and a reduction of the posterior neurectodermal domain, marked by hoxb1b (N,O and T,U). Gastrulation movements seem to be impaired in such coinjected embryos. These antagonists of FGF signaling have no effect on untreated ich embryos (J-L and P-R). Wild-type embryos (A-C) are shown in a dorsal view, ich embryos (D-U) in lateral view. All embryos are at ~10 hpf.

Posterior movement of mesodermal cells only observed in ′ciuffo′ embryos. Lateral views of live, 12.5-13 hpf ich embryos untreated (A) or treated with bmp2bMO (B), βcat1MO (C) and bmp2bMO and βcat1MO (D). Note the clear vegetal migration of cells observable in ′ciuffo′ embryos (C), and the relatively symmetric distribution of tissues between the animal and vegetal poles of bmp2bMO (co-)injected embryos (B,D).

Acknowledgments
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