Injection of medaka embryos with Six3-PNAs. A wild-type embryo (no PNA injection) at 3 days is shown in (A). Embryos injected with 400 μM Ref16mix-PNA are indistinguishable from wild-type embryos; at 900 μM unspecific phenotypes were detected (both in A). The Six3mix-PNA injected embryos were evaluated 3 days after injection at stage 29. The phenotypes were divided into weak, moderate and strong (for criteria see text). Arrows indicate the anterior parts of the eyes pointing to the midline in weak phenotypes. The black arrowhead indicates remnants of the eye structures. All embryos in (A) are at stage 29 (34 somites). In (B) the expression of the bf1 gene in wild-type and strongly affected embryos injected with Six3mix-PNAs is shown (both at stage 22; 9 somites). Note the presence of bf1 expression in the otic vesicles, both in wild-type as well as in Six3mix-PNA injected embryos (marked by red arrowheads). Dorsal views of the anterior part of the embryos are shown. The quantitative evaluation of the six3 knock down experiments is shown in (C) and (D) for the indicated PNA molecules. The percentages of phenotypes for the diagrams were calculated from the surviving embryos. PNAs shown in (D) were injected at 400 μM. (E) Shows the mortality rate of embryos injected at the indicated concentration (survival was examined 24 hrs after injection, see also Additional file 5: Table S3).