T-box gene eomesodermin and the homeobox-containing Mix/Bix gene mtx2 regulate epiboly movements in the zebrafish

Abstract

The T-box gene eomesodermin (eomes) has been implicated in mesoderm specification and patterning in both zebrafish and frog. Here, we describe an additional function for eomes in the control of morphogenesis. Epiboly, the spreading and thinning of an epithelial cell sheet, is a central component of gastrulation in many species; however, despite its importance, little is known about its molecular control. Here, we show that repression of eomes function in the zebrafish embryo dramatically inhibits epiboly movements. We also show that eomes regulates the expression of a zygotic homeobox transcription factor mtx2. Gene knockdown of mtx2 using antisense morpholino oligonucleotides, likewise, leads to an inhibition of epiboly; moreover, we show that knockdown of mtx2 function in the extraembryonic yolk syncytial layer only is sufficient to cause epiboly defects. Thus, we have identified two components in a molecular pathway controlling epiboly and show that interactions between deep layer cells of the embryo proper and extraembryonic tissues contribute in a coordinated manner to different aspects of epiboly movements.

Fig. 1

eomes-eng inhibits epiboly. A–O: All views lateral. J–O: anterior toward the left. Construct injected, if any, is indicated in the bottom left corner. A–C: Embryos at 30% epiboly (4.7 hours postfertilization [hpf]), (D–I) embryos at 60% epiboly (6.5 hpf). A: Uninjected control. B: Embryo injected with gfp and eomes-eng RNA. Arrowheads indicate the region of the blastoderm that has failed to thin, and the arrow indicates the normal region of the blastoderm. C: Same embryo as in B showing green fluorescent protein (GFP) fluorescence. The region indicated by the arrowheads in B is where most of the GFP expression is located. D: Control embryo injected with gfp and eng RNA. E: Higher power view of embryo in D. F: Same embryo as in E, showing that GFP fluorescence is distributed throughout the blastoderm. GFP-positive cells are intermingled with unlabeled cells. G: Embryo injected with gfp and eomes-eng RNA. H: Higher power view of embryo in G. Arrowheads indicate region of the blastoderm that has failed to thin, and the arrow indicates the normal region of the blastoderm. I: Same embryo as in H, showing GFP fluorescence. The region indicated by the arrowheads in H is where most of the GFP expression is located. J–O: Embryos at 1 day postfertilization. J: Control embryo injected with gfp and eng RNA. K: Higher magnification of J, showing the head region. L: Same embryo as in K, showing evenly distributed GFP fluorescence. M: Embryo injected with gfp and eomes-eng RNA. N: Higher magnification of M, showing abnormal head region. O: Same embryo as in N showing GFP fluorescence concentrated in the anterior portion of the head.

Fig. 2

Eomes is expressed throughout the blastoderm at early blastula stages. Images are single scans from a confocal z-series of embryos shown in lateral view and stained with the anti-Eomes antibody. A: Embryo at 512-cell stage (2.75 hours postfertilization [hpf]). Nuclear staining can be seen throughout the blastoderm, although not all nuclei are in the plane of view. B: Embryo at the high stage (3.3 hpf). Protein expression can be seen in nuclei throughout the blastoderm.

Fig. 3

eomes regulates mtx2 expression cell-autonomously. All views are lateral, and all embryos are at sphere stage (4 hours postfertilization [hpf]). Injected construct, if any, is indicated in lower left corner. A: In situ hybridization of mtx2 in an uninjected embryo, showing expression in the marginal cells of the blastoderm and the underlying yolk syncytial layer. B: eomes-VP–injected embryo with ectopic mtx2 expression (arrowhead). Inset shows a portion of the blastoderm of a myc-eomes–injected embryo with Eomes protein expression in the nucleus in brown and mtx2 expression in blue. White outline demarcates a group of cells that coexpress Eomes and ectopic mtx2, indicating a cell-autonomous induction of mtx2 by Eomes. C: Reduced mtx2 expression in an embryo injected with eomes-eng. D: ntl-VP–injected embryo with normal mtx2 expression.

Fig. 4

mtx2 morpholinos inhibit epiboly. All views are lateral with dorsal to the right; all embryos are at shield stage (6 hours postfertilization). Injected construct, if any, is indicated in lower left corner. A: Uninjected embryo. B: Embryo injected with Mtx2-MO1 into one cell at the two-cell stage; note that the blastoderm is thickened compared with control. C: Embryo injected with Mtx2-MO1 into the yolk syncytial layer (YSL); note that the blastoderm is thickened compared with control.

Fig. 5

Epiboly defect is Nodal-independent. All views are lateral; the mutant phenotype is indicated in upper right corner; the injected construct is indicated in lower left corner. A,B: At 50% epiboly (5.25 hours postfertilization [hpf]). C,D: At sphere stage (4 hpf). A: Embryo injected with eomes-eng; the blastoderm has failed to thin. B: Embryo injected with Mtx2-MO1; the blastoderm has failed to thin. C: mtx2 expression in an uninjected embryo. D: Ectopic mtx2 expression (arrowhead) in an eomes-VP–injected (into a single cell at the eight-cell stage) embryo.