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First published online October 6, 2003
doi: 10.1242/10.1242/dev.00763

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The maternally expressed zebrafish T-box gene eomesodermin regulates organizer formation

Ashley E. E. Bruce^1,2,*,, Cristin Howley^2,*, Yi Zhou³, Sarah L. Vickers¹, Lee M. Silver², Mary Lou King³ and Robert K. Ho^1,2

¹ Department of Organismal Biology and Anatomy, University of Chicago, Chicago, IL 60637, USA
² Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA
³ Department of Cell Biology and Anatomy, Gautier Building, Room 517, 1011 NW 15th Street, University of Miami School of Medicine, Miami, FL 33136, USA

View larger version (102K): [in a new window]   Fig. 1. Localization of eomes in the early zebrafish. (A) Northern blot comparing eomes transcript levels at one-cell (0.3 hpf), eight-cell (1.25 hpf), 128-cell (2.25 hpf), 512-cell (2.75 hpf), 1000-cell (3 hpf), sphere (4 hpf) and dome (4.3 hpf) stages. Actin was used as a loading control. Exposure time, 7.5 hours. (B-D) Sections of adult ovaries: GV, germinal vesicle. (B) Stage I oocyte (20-140 µm), staining is uniform throughout cytoplasm (arrowhead). (C) Stage II oocyte (0.14-0.34 mm), eomes hybridization can be seen along the cortex of the oocyte (arrowhead). (D) Stage III oocyte (0.34-0.69 mm). eomes mRNA is detected cortically (arrowhead) and throughout the cytoplasm. (E,F,H,K) Whole-mount embryos, animal pole is toward the top. (G,J) Sections with the animal pole toward the top. (E) Activated egg, eomes is detected in the cytoplasmic streams in the yolk and in a gradient along the V/A axis. The arrow marks the region of most intense hybridization at the yolk/blastodisc junction. (F) The expression pattern in a four-cell-stage embryo is similar to that in E. (G) Section of a four-cell stage embryo showing the distribution of eomes mRNA. The arrowhead marks the most intense region of eomes expression at the yolk-blastomere junction. (H) Expression of eomes is maintained in vegetal to animal gradient in a 32-cell-stage embryo. (I) Nearly ubiquitous zygotic eomes expression at the 1000-cell stage. (J) Section of an oblong/sphere-stage embryo. eomes mRNA is detected in vegetally located cells and is absent from the YSL (arrow). (K) Sphere-stage embryo, eomes hybridization is reduced in the animal pole and is most intense in cells closest to the yolk.

View larger version (70K): [in a new window]   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.

View larger version (93K): [in a new window]   Fig. 3. Overexpression of eomes induces secondary axes. (A-C) Animal pole views. (D,F) Dorsal views with anterior to the left. (A) Embryo injected with myc-eomes. There is a thickened region (arrow) opposite the native shield (arrowhead). (B) Animal-pole view at shield stage of a live embryo injected with myc-eomes and GFP. Composite of white light and fluorescent images with GFP-expressing cells in green (arrow) opposite the native shield (arrowhead). (C) Live image of embryo in B at 1 dpf. Two heads are visible: the eyes from one axis are indicated by arrowheads and the eye from the second axis is indicated by arrow. (D) Embryo injected with myc-eomes and stained for fkd7 (black) has two axes side by side at 26 hpf. Arrows indicate the level of the section shown in E. (E) Section of embryo in D, dorsal is to the top. Two neural tubes are visible (arrowheads) and both stain with fkd7 (blue/purple staining). The eye is marked e. In addition to secondary axes, two other phenotypes were seen in eomes-overexpressing embryos. Embryos with bifurcated notochords in the trunk region were observed as well as embryos that resembled the dorsalized mutants previously described (Mullins et al., 1996). (F) Wild-type control embryo at 26 hpf stained for fkd7.

View larger version (115K): [in a new window]   Fig. 4. Analysis of gene expression in embryos injected with eomes mRNA. All embryos are at the shield stage (6 hpf) and all views are from the animal pole, except (C,) which is a lateral view. In A,B,D,E,G,H the shield is to the right. Bottom right corner indicates in situ probe used, top right corner indicates the gene construct, if injected. (A) Uninjected embryo stained for gsc expression in the shield region. (B) Injection of myc-eomes induced ectopic gsc expression at the margin (arrowheads). Arrow marks region shown in lateral view in C. (C) Embryo in B after anti-Myc antibody staining (brown). Myc-labeled cells and cells ectopically expressing gsc are in different focal planes. (D) Expression of chd in an uninjected control. (E) Ectopic chd expression in an embryo injected with myc-eomes (arrowheads). (F) High-magnification view of margin of an embryo injected with myc-eomes. White outline surrounds a cell that expresses chd and Myc. (arrow). Red outline demarcates a cell expressing chd but not Myc (arrowhead). (G) Expression of flh in an uninjected control. (H) Embryo injected with myc-eomes with ectopic flh expression (arrowhead). (I) High-magnification view of embryo injected with myc-eomes. The same cells stain for flh (arrowheads) and Myc (arrow).

View larger version (61K): [in a new window]   Fig. 5. Dominant-negative eomes inhibits dorsal expression of gsc and flh. (A-D) The dorsal blastoderm margin of embryos at 50% epiboly was dissected and flat-mounted. Bottom right corner indicates in situ probe used, top right corner indicates gene construct, if injected. (A) Expression of gsc in an uninjected embryo. (B) Injection of eomes-eng inhibits gsc expression. Eomes-eng expressing cells shown in brown by anti-GFP antibody staining. (C) Expression of flh in an uninjected embryo. (D) Injection of eomes-eng inhibits flh expression. Eomes-eng expressing cells shown in brown by anti-GFP staining.

View larger version (83K): [in a new window]   Fig. 6. Eomes-MO2 reduces Eomes protein levels and expression of gsc and flh. (A) Western blot of in vitro transcription and translation of eomes-pCS2+ probed with the anti-Eomes antibody. Lane 1, protein produced in the presence of 5 ng of cyclops morpholino. Lane 2, protein produced without morpholino present. Lane 3, protein produced in the presence of Eomes-MO2. The amount of Eomes protein produced is not affected by the presence of cyclops morpholino but is dramatically reduced in the presence of Eomes-MO2. (B) Embryo (1 dpf), dorsal side up and anterior to the top, stained with anti-Eomes antibody. Nuclear staining in the brain is in green (arrowheads). (C) Embryo (1 dpf) in the same orientation as B that has been injected with Eomes-MO2 and stained with the anti-Eomes antibody. No staining is visible. (D) Sphere-stage embryo stained with the anti-Eomes antibody. Staining is visible in nuclei on the dorsal side of the embryo. (E) Sphere-stage embryo injected with Eomes-MO2 and stained with the anti-Eomes antibody. Staining is reduced compared to D, but nuclear-localized protein is visible on the dorsal side of the embryo (arrowhead). (F-I) Animal-pole view of shield-stage embryos. Bottom right corner indicates in situ probe used, top right corner indicates gene construct, if injected. (F) Uninjected embryo stained for gsc. (G) Eomes-MO2 injected embryo, expression of gsc is reduced. (H) Uninjected embryo stained for flh. (I) Eomes-MO2 injected embryo expression of flh is reduced.

View larger version (57K): [in a new window]   Fig. 7. eomes induces its own expression. Lateral views at 50% epiboly. (A) Uninjected embryo with no detectable eomes expression at shield stage. (B) Injection of eomes-VP leads to ectopic expression of the endogenous eomes gene at shield stage (arrowhead, see text for details).

View larger version (77K): [in a new window]   Fig. 8. Overexpression of eomes in mutant embryos. All embryos are at 6 hpf (equivalent to the shield stage) with dorsal to the right and are animal-pole views, except K,L which are lateral views. (A-F) boz-mutant embryos. (G-L) MZoep-mutant embryos. (A,C,E,G,I,K) Uninjected embryos. (B,D,F,J,L) Embryos injected with myc-eomes. (H) An eomes-VP-injected embryo. Expression of gsc is shown in A,B,G,H, chd in C,D,I,J and flh in E,F,K,L. Arrowheads indicate regions of ectopic expression. (J) Two cells of an eight-cell-stage embryo were injected and stained with the anti-Myc antibody. Regions of Myc staining are indicated with an arrow and arrowhead. Expanded chd expression is only observed on the dorsal side (purple stain, arrowhead).

View larger version (82K): [in a new window]   Fig. 9. eomes modulates Sqt signaling. Animal pole views at 50% epiboly (5.3 hpf). Bottom right corner indicates probe, top right corner indicates gene construct, if injected. (A) Expression of gsc in embryo injected with sqt at the animal pole. (B) Expression of gsc in embryo injected with sqt and myc-eomes at the animal pole. gsc is induced in a ring-like pattern. (C) Embryo in B after Myc-antibody staining. Note that the region of high Myc staining corresponds to the region of reduced gsc expression in B. (D) Expression of a ring of flh in embryo injected with sqt. (E) Expression of a solid domain of flh in an embryo injected with sqt and myc-eomes. (F) Embryo injected with sqt and myc-eomes, and stained with the Myc antibody.

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