Fig. 6. Mesoderm induction by FoxD3 is non-cell-autonomous and dependent on the Nodal pathway. (A) At the one-cell stage the animal pole was injected with 100 pg of FoxD3 RNA and animal explants prepared at the early blastula (stage 7) were cultured intact or dissociated into individual cells in the absence of calcium (Disso.). The expression of Brachyury (Xbra), and MyoD was examined in uninjected (Control) and injected explants by RT-PCR at the gastrula stage (stage 11). (B) At the 32-cell stage a single animal pole blastomere was injected with 100 pg of FoxD3 RNA and explants prepared and fixed at the early gastrula stage (stage 10.5) were sequentially examined for Brachyury (Xbra) expression by in situ hybridization and FoxD3 protein expression by immunocytochemistry. To assess the dependence of FoxD3 function on Smad2 and Nodal, FoxD3 (100 pg) was injected alone, or in combination with 1 ng of the Smad2-interaction domain of Fast1 (SID) (C) or 1 ng of a truncated form of Cerberus (CerS) (D). Animal explants prepared at the midblastula stage (stage 9) were collected for RT-PCR analysis of Brachyury (Xbra) at the gastrula stage (stage 11) and Muscle Actin (M. Actin) at the tailbud stage (stage 25). Xnr1 (50 pg) was used as a positive control for the inhibitory activity of SID and CerS. PCR controls are as described in Fig. 2.