© 2005 Nature Publishing Group The zebrafish dorsal axis is apparent at the four-cell stage Aniket V. Gore 1,2 , Shingo Maegawa 3 , Albert Cheong 1 , Patrick C. Gilligan 1 , Eric S. Weinberg 3 & Karuna Sampath 1,2 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 dorso- ventral axis requires signalling by proteins of the Transforming growth factor-b and Wnt families 1–3 . 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 morpho- lino 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 0 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 0 untranslated region of the human nodal gene can also localize exogenous sequences to dorsal cells, this mechanism may be evolutionarily conserved. Localized maternal factors specify the embryonic axes in several organisms 4 . In many vertebrates, specification of the dorsoventral (DV) axis is thought to require maternally encoded signals of the Transforming growth factor (TGF)-b and Wnt pathways 5,6 . In Xenopus, on the entry of sperm, cortical rotation results in the transport of dorsal determinants by means of microtubules 7 . In fish, removal of the vegetal yolk cell and disruption of microtubules by cold shock or by drugs disrupts the movement of dorsal determi- nants from the yolk to the blastoderm 8–10 . However, the nature of these dorsal determinants and the mechanism of movement are poorly understood. In zebrafish, transcripts of the diffusible morphogen Sqt are expressed in oocytes and embryos 11–14 . Although sqt RNA is present uniformly through oogenesis, on egg activation and fertilization it rapidly localizes to the blastoderm by a microtubule-dependent process 14 . Subsequently, during early gastrula stages, before the formation of the zebrafish organizer, the embryonic shield, sqt RNA is expressed in the dorsal side of the embryo 11–13 . We find, by in situ hybridization, that sqt RNA is localized asymmetrically in four-cell and eight-cell embryos (66%, n ¼ 72, and 67%, n ¼ 52, respectively; Fig. 1a–c, e–g, and Supplementary Table 1), and is detectable at least until the 32- and 64-cell stages (Fig. 1d, h). Real-time reverse-transcriptase-mediated polymerase chain reaction (RT–PCR) analysis (Supplementary Fig. S1) shows that sqt RNA levels are constant through the cleavage and blastula stages. To examine the dynamics of sqt RNA localization in living embryos, we injected synthetic Alexa-488-labelled sqt RNA into one- cell embryos and observed the movement of the fluorescent RNA by time-lapse videomicroscopy. Injected fluorescent sqt RNA is detected in the cells expressing endogenous sqt RNA (72%, n ¼ 19; Fig. 1m–o), LETTERS Figure 1 | Sqt RNA localizes in early embryos. a–h, In situ hybridization to detect endogenous sqt RNA (arrowheads) in embryos at the 4-cell (a, b, e, f), 8-cell (c, g), 32-cell (d) and 64-cell (h) stages. i–l, Stills from a movie of an embryo injected with Alexa-488-labelled sqt RNA (arrowheads) at the one- cell (i), two-cell (j), four-cell (k) and eight-cell (l) stages. Times are indicated as h:min. m–o, Four-cell embryo showing localization of endogenous sqt RNA (arrowhead, m), injected fluorescent sqt RNA (asterisk, n), and their overlap (o). p–r, t–v, First cleavage furrow labelled with FM4-64 (red; arrowhead) and Alexa-488-labelled sqt RNA (green; asterisks), in four-cell (p, t) and eight-cell (q, r, u, v) embryos. s, w, Immunodetection of g-tubulin (red), DNA (blue) Alexa-488-labelled sqt RNA (green; asterisks) in four-cell (s) and eight-cell (w) embryos. a–c, e–g, i–l, m–s, w, animal pole views; d, h, t–v, lateral views. Scale bar, 100 mm in h, l and v, for a–h, i–l and p–w, respectively. 1 Vertebrate Development Group, Temasek Life Sciences Laboratory, 1 Research Link, 117604 Singapore. 2 Department of Biological Sciences, National University of Singapore, 117543 Singapore. 3 Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA. Vol 438|15 December 2005|doi:10.1038/nature04184 1030