A central question in the development of multicellular organisms pertains to the timing and mechanisms of specification of the embryonic axes. In many organisms, specification of the dorsoventral axis requires signalling by proteins of the Transforming growth factor-β and Wnt families1,2,3. Here we show that maternal transcripts of the zebrafish Nodal-related morphogen, Squint (Sqt), can localize to two blastomeres at the four-cell stage and predict the dorsal axis. Removal of cells containing sqt transcripts from four-to-eight-cell embryos or injection of antisense morpholino oligonucleotides targeting sqt into oocytes can cause a loss of dorsal structures. Localization of sqt transcripts is independent of maternal Wnt pathway function and requires a highly conserved sequence in the 3′ untranslated region. Thus, the dorsoventral axis is apparent by early cleavage stages and may require the maternally encoded morphogen Sqt and its associated factors. Because the 3′ untranslated region of the human nodal gene can also localize exogenous sequences to dorsal cells, this mechanism may be evolutionarily conserved.
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We thank M. Balasubramanian, R. Dosch, R. Dunn, S. Jesuthasan, E. Raz, S. Roy, A. Schier, and members of the Sampath laboratory for discussions and suggestions; S. Koshida, E. Yamaha and H. Takeda for embryo operation techniques; A. Chang and the Qian Hu fish farm for cyprinids; H. Ngoc Bao Quach for technical assistance; M. Hidayat for sequencing; and J. Bradner, C. Hu and C. Goh and the TLL fish facility for fish care. This work was supported by NIH grants (E.W.) and by TLL, Singapore (K.S.).
The cyprinid sqt sequences have been deposited in GenBank (accession numbers DQ080241, DQ080242 and DQ080243). Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests.
Real-Time PCR analysis of sqt transcripts (a) and ef1-α (b) at various stages of development. (PDF 354 kb)
Colocalization of β-catenin with β-Gal human nodal 3’UTR. (PDF 76 kb)
Percentage embryos expressing localized sqt through cleavage, blastula and early gastrula stages. (DOC 20 kb)
Marker gene expression in operated embryos. (DOC 24 kb)
Marker gene expression in morpholino injected oocytes or fertilized embryos (DOC 28 kb)
Dynamic localization of fluorescent sqt RNA in zebrafish embryos at early cleavage stages. (MOV 2570 kb)
Dynamic localization of fluorescent sqt RNA in zebrafish embryos at early cleavage stages. (AVI 517 kb)
Uniform distribution of fluorescent lacZ RNA with the β-globin 3’ UTR. (MOV 976 kb)
Localization of fluorescent sqt RNA is inhibited by the microtubule poison, nocodazole. (MOV 3388 kb)
The 3’UTR of sqt RNA is required for localization. The embryo was injected with sqt RNA lacking its 3’UTR. (AVI 3076 kb)
Exogenous 3’UTRs do not localize sqt RNA. The embryo was injected with labelled sqt coding region RNA fused with the β-globin 3’UTR. (MOV 1176 kb)
Fluorescent lacZ RNA fused to sqt 3’UTR is localized by early cleavage stages. (MOV 3337 kb)
Fluorescent lacZ RNA fused to sqt 3’UTR is localized by early cleavage stages. (MOV 2176 kb)
Fluorescent sqt RNA with the first 50 nucleotides of its 3’UTR is localized by the 4-cell stage (MOV 2363 kb)
Fluorescent sqt RNA with the first 50 nucleotides of its 3’UTR is localized by the 4-cell stage. (MOV 3411 kb)
Fluorescent lacZ RNA with the human nodal 3’UTR is localized by the 4-cell stage. (MOV 2774 kb)
Fluorescent lacZ RNA with the human nodal 3’UTR is localized by the 4-cell stage. Lateral view shown. (AVI 656 kb)
Text to accompany the above Supplementary Figures and Supplementary videos. (DOC 24 kb)
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Gore, A., Maegawa, S., Cheong, A. et al. The zebrafish dorsal axis is apparent at the four-cell stage. Nature 438, 1030–1035 (2005). https://doi.org/10.1038/nature04184
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