Fig. 4. Transcriptional profiling of 0-2 ss Wnt3a^-/- embryos identified the putative segment boundary determination gene Ripply2. (A) Hierarchical clustering of select differentially expressed genes revealed that several previously identified direct Wnt/β-catenin target genes such as Axin2, Tnfrsf9(Troy) and Nkd1 were downregulated (blue) in the Wnt3a^-/- mutants, as expected. The Riken EST C030002E08 (Ripply2) was identified in a class of genes that were upregulated (red) in embryos lacking Wnt3a. (B-E) Two-color WISH illustrating striped Ripply2 (purple) expression in the anterior PSM (arrows), overlapping with the Notch pathway components (orange) Dll1 (B) and Hes7 (C). A representative half-embryo culture experiment illustrates that at time 0, Ripply2 (purple) and Mesp2 (orange) are expressed as mutually exclusive domains in adjacent presumptive somites (S-I and S-II, respectively) in one half of the sagitally bisected embryo (D). The solid line represents segment border B0, which is out of the focal plane. After culturing the complementary half-embryo for 1 hour (E), Ripply2 was strongly co-expressed with Mesp2 in S-I, while disappearing in S0. The forming segment border B0 is represented by a dashed line. (F-I) Ripply2 expression in S-I (F) is posteriorized relative to the node (line) in Wnt3a^-/- embryos (G), and nearly absent from T-Cre;Ctnnb1^flLOF/ embryos (H). Ripply2 is not expressed in T-Cre;Ctnnb1^flGOF/+ embryos (I). Scale bars: 100 µm.