Fig. 2. Eomes protein expression. (A) Western blot of sphere-stage embryos. A 94 kDa band is recognized by the Eomes antibody but not by preimmune serum. (B) Embryo (1 dpf), dorsal side up and anterior to the top, stained with anti-Eomes antibody. Nuclear staining in the brain is light brown and indicated with arrowheads. (C) Embryo (1 dpf) in the same orientation as B incubated with preimmune serum. No staining is visible. (D) Eomes was not detected in early stage oocytes (arrowhead) but cytoplasmic staining (green) was detected in older oocytes (arrow, see text for details). (E) Lateral view of a one-cell stage embryo. Eomes is distributed throughout the blasoderm. (F) Lateral view of a sphere-stage embryo (4 hpf). Eomes is observed in nuclei on one side of the embryo (arrow). (G) Animal-pole view of sphere-stage embryo stained for Eomes (green) and flh transcript (red) demonstrates that Eomes is nuclear localized predominantly on the dorsal side of the embryo. (H) Lateral view of a dome-stage embryo (4.3 hpf) stained as in (G). Eomes and flh colocalize in some cells (yellow) as can be observed in the enlarged region, bottom right. (I) Animal-pole view of Eomes staining and flh in situ staining in a sphere-stage embryo. The white outline indicates a cell that co-expresses Eomes in the nucleus (brown) and flh in the cytoplasm (blue). (J) As in I, except gsc expression is in blue. The white oultine indicates a co-expressing cell and the black outline indicates a cell that expresses gsc but not Eomes. (K) Animal-pole view of sphere-stage embryo with Eomes expression visible in nuclei of the enveloping layer (arrowheads). F-H are images from Z-series taken on a confocal microscope.

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