Fig. 5. Functional analysis of hierarchical relationships between zygotic endoderm regulators. (A) At the two-cell stage, embryos were injected with either Cerb-S RNA to inhibit Nodal signaling (1 ng) or antisense morpholino oligos to Sox171+2+ß (Sox17-MO; 20 ng each). Some Cerb-S and Sox17-MO injected embryos were then injected with XtSox17ß RNA (from Xenopus tropicalis and resistant to the Sox17-MOs; 10-100 pg), Mixer RNA (50-500 pg) or Gata6 RNA (50-100 pg). A range of rescue RNA doses was used and the lowest does that gave a reproducible rescue is shown. At stage 11, RNA from the embryos was assayed by real time RT-PCR for expression of Bix1, Bix2, Bix4, Gata4, Gata5, Gata6, Edd, Foxa1, Xnr4, Sox17, Sox17ß, Mixer and Foxa2. Relative expression normalized to ODC is shown and the expression level in control gastrula was set to 1.0. This experiment was repeated three times and a representative example is shown. (B) These results, along with previously published reports, support the indicated regulatory relationships, which were previously not described in the existing models of endoderm development. Foxa2 (Ruiz i Altaba et al., 1993) is not present on the Affymetrix Xenopus chip and is distinct from Pintallavis (Ruiz i Altaba and Jessell, 1992) or XFKH1 (Dirksen and Jamrich, 1992), which are Foxa4a and Foxa4b, respectively (Kaestner et al., 2000).