Fig. 5

Phase I expression requires a Nodal response by the margin enhancer and Zbtb4. A,B: In situ hybridizations for EGFP transcripts in shield stage Tg(-2.8) embryos, animal pole views, dorsal right. A: Control DMSO-treated embryo. B: SB431542-treated embryo. Note significant reduction in transcripts. Inset: Lateral view to show patchy expression in the margin. C,D: In situ hybridizations for ntla transcripts to confirm Nodal loss of function. C: Control DMSO-treated embryo. D: SB431542-treated embryo. Note loss of dorsal mesoderm indicative of strong reduction in Nodal signaling (arrows). E?I: In situ hybridizations on embryos injected with Taram-D mRNA at the 1-cell stage. E: EGFP transcripts in injected Tg(-2.8) embryo. Arrow indicates ectopic EGFP reporter expression. F: wnt8a.1 expression in injected Tg(-2.8) embryo. Note induction of ectopic expression, though not as robust as the transgene. G: ntla expression in injected Tg(-2.8) embryo. Note strong induction of ntla, arrow. H: EGFP expression in injected Tg(-1.8) embryo. Note ectopic expression, arrow. I: EGFP expression in injected embryo. Note absence of a reporter response to Taram-D when margin enhancer is deleted. J?L: Zbtb4 is required for Nodal-dependent phase I expression. EGFP in situ hybridizations. J: Embryo injected with Zbtb4 morpholino. Note absence of reporter expression in the margin. K: Embryos injected with Zbtb4 morpholino and Taram-D mRNA. Note absence of reporter expression. L: Embryo injected with Zbtb4 morpholino and Taram-D mRNA. Note that the Tg(-1.8) reporter lacks the DRR, and that reporter expression is activated prematurely (arrow, compare to Fig. 3B).