chordin expression is required for expression of neural markers when both β-catenins are inhibited. 24 hpf untreated ich embryos (A, E, I, and M), and ich embryos injected with chdMO alone (B, F, J, and N), βcat1MO and βcat2MO ([MO1 + MO2] C, G, K, and O), or all three MOs (D, H, L, and P) were photographed alive (A–D) or after in situ hybridization with probe for emx1 (E–H) krox20 (I–L), or myoD (M–P). ich embryos, which are highly ventralized if untreated (A), do not express emx1 (E), or krox20 (I), but do express myoD in the tail region (M). When depleted of β-catenin-1 and β-catenin-2 expression by MO treatment, embryos develop the ‘ciuffo’ phenotype (C, bracket indicates the protrusion that has very different morphology from the tail region of ich embryos, indicated by arrow in A) and express emx1 (G, arrow points to single band of expression), and krox20 (K, arrowheads point to two bands of expression), in addition to myoD (O). ich embryos injected with chdMO alone remain highly ventralized, with a broader but less protrusive tail (B, arrow) than in untreated ich embryos, and show no induction of emx1 (F) or krox20 (J). When ich embryos are injected with chdMO as well as the two β-catenin MOs, the posterior protrusion is no longer ‘ciuffo-like’, but rather is tail-like, and the anterior necrotic protrusion (at the end of the embryo opposite the arrow) seen in ich embryos (A) and embryos treated with chdMO alone (B) fails to form (D). The embryos treated with the three MOs are inhibited in both emx1 (H) and krox20 (L) expression but do express myoD in the tail region (P), indicating that the failure of emx1 and krox20 expression in the triple MO-injected embryos was not due to non-specific toxic effects of the MOs. Over 24 embryos were examined for each marker and each condition, with results identical to embryo shown in the panel (except for panel N in which 1 of 24 embryos did not express myoD and panel K in which 2 of 28 embryos did not express krox20). Concentrations of MOs injected were 3 mM for each of the β-catenin MOs and 0.6 mM chdMO.
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