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doi: 10.1242/10.1242/dev.00344

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Cell fate specification and competence by Coco, a maternal BMP, TGFß and Wnt inhibitor

Esther Bell*, Ignacio Muñoz-Sanjuán*, Curtis R. Altmann, Alin Vonica and Ali H. Brivanlou{dagger}

The Laboratory of Vertebrate Embryology, The Rockefeller University, NY 10021, New York, USA



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  		Fig. 1. Identification of Coco, a novel BMP inhibitor. (A) Nucleotide sequence of Xenopus Coco. ORF in blue and green. Green text indicates primers used for the RT-PCR. Translation is shown in red. RRK are putative cleavage sites similar to that found in Cerberus (Piccolo et al., 1999Go). (B) Alignment at the amino acid level of Xenopus, Fugu, human and mouse Coco and other family members, Cerberus and Caronte.

 


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  		Fig. 2. Expression pattern of Coco mRNA during Xenopus development. (A) Whole-mount in situ hybridisation of Coco, Bmp4 and VegT mRNA in the egg. Expression of Coco was also compared to Vg1 by RT-PCR. (B) Coco is expressed strongly in the animal pole at the 2-cell, 8-cell stages and at stage 8. (C) Coco is strongly expressed maternally and then is downregulated at the post-gastrula/pre-neurula stages. In contrast Cerberus is first expressed at stage 9 and then downregulated after the onset of neurulation. (D,E) At gastrula stage Coco is detected at high levels in the ectoderm and marginal zones by (D) whole-mount in situ hybridisation and (E) as seen by RT-PCR at much lower levels in the vegetal pole. ODC was used as a loading control for the RT-PCR. * indicates the organizer.

 


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  		Fig. 3. Phenotypes resulting from Coco overexpression in Xenopus embryos. Analysis of injected embryos at gastrula (A-D), neurula (H) and early tadpole stages (E-G,I-N). In A-D and H-N, the top panels are uninjected embryos, the lower panels are embryos injected with 1 ng Coco vegetally in 1 cell at the 2-cell stage (A-D) or ventrally in one cell at the 4-cell stage (G-N). (A) Xbra/brachyury (a marker of mesoderm); (B) Fgf8, also a mesoderm marker, (C) Otx2, (the organizer and anterior ectoderm); (D) gsc (organizer); (H) rx, (forebrain); (I) Emx1 (dorsal telencephalon); (J) Otx2, forebrain and midbrain; (K) En2 (midbrain/hindbrain boundary); (L) Hoxb9 (spinal cord); (M) Nkx2.5 (heart) (N) 12-101 Ab (muscle). (E-G) Phenotypes resulting from (E) no injection (control embryo); (F) dorsal animal injection at the 2-cell stage; (G) injection in one ventral vegetal cell at the 4-cell stage. *indicates the extra head structures. A,B,D are vegetal views; C, lateral with animal pole to the top; E-G,I-K, lateral views; H,L,N dorsal views; M, ventral view. In E-N, anterior is to the right.

 


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  		Fig. 4. Dorsalization effects of Coco in embryonic explants. (A) Animal caps injected with 1 ng Coco at the 2-cell stage and analyzed for the expression of epidermal, mesodermal and neural markers. (B) Animal caps analyzed for neural induction at early tadpole stages. Both the general neural markers Ncam and nrp1 have been induced as well as anterior markers Otx and XAG. (C) Morphology of the VMZ+Coco explants compared to control DMZ and VMZ. (D) RT-PCR analysis of VMZ explants injected with 1 ng Coco compared with DMZ and VMZ of uninjected explants at gastrula stages. The organizer markers chordin and goosecoid are induced in the VMZ+Coco explants. (E) Analysis at tadpole stages. The VMZ+Coco now expresses dorsal molecular markers. ODC was used as a loading control.

 


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  		Fig. 5. Inhibitory effects of Coco on BMP, TGFß and Wnt signaling. (A) Bmp4 and Coco RNAs were injected separately, and together, into embryos at the 2-cell stage. Animal caps were analyzed at gastrula stages for the presence of Xbra and epidermal keratin. Coco blocked the induction of both these markers by BMP4. (B) Wnt8 and Coco were injected into animal caps and the markers Xnr3 and Siamois analyzed. Coco blocked the induction of these markers by Wnt8. (C) Inhibition of nodal signaling by Coco. Coco blocked the induction of chordin, Xbra and Wnt8 by Xnr1. (D) Inhibition of activin signaling by Coco. (E,F) Direct binding of Coco to BMP4 and Xnr1. Flag-tagged Coco was co-injected with HA-BMP4 (E) and HA-Xnr1 (F). Coco inhibited the activation of both the Wnt8-responsive promoter TOP-FLASH (G) and the BMP response element (H). (I) Ectodermal competence assay. Ectodermal explants, either uninjected or Coco-injected, were exposed to activin-conditioned medium at different stages. Notice that in the presence of Coco, explants are unable to respond to activin from stage 10 onwards.

 




© The Company of Biologists Ltd 2003