Fig. 1

SYTO-11 labeling of mid-blastula-stage zebrafish embryos. Scale bars, 50 μm. Embryos at 1 k-cell stage to high-stage embryos were labeled with SYTO-11 and observed at later developmental stages. All panels are confocal micrographs. (A) SYTO-11 labeling is seen in the enveloping layer cells of a sphere-stage zebrafish embryo. SYTO-11 has been internalized by endocytosis into numerous fluorescent endosomes. Few EVL nuclei exhibit SYTO-11 labeling in this particular embryo. Animal pole view. (B) 15 μm deeper into the same embryo. Deep cells, which have delaminated from the EVL, contain numerous fluorescent endosomes in their cytoplasm. (C) 23 μm deeper into the blastoderm than B. No deep cells containing SYTO-11 are seen in the center of the blastoderm. Those deep cells that exhibit SYTO-11 labeling are in close proximity to the EVL epithelium. (D) SYTO-11 is also internalized by endocytosis into the cytoplasm of the yolk syncytial layer (YSL) and yolk cytoplasmic layer (YCL). Endosomes stream along linear tracks (white arrows) toward nuclei in the external YSL in an oblong-stage embryo. Black arrows point to the EVL – YSL margin. The animal-vegetal (An-Vg) axis is shown. (E) Nuclei in the YSL exhibit SYTO-11 labeling (black arrows). In this 30%-epiboly-stage embryo, a large fraction of EVL cells exhibit nuclear staining by SYTO-11. Vesicular labeling is observed throughout the cytoplasm of EVL cells and the YSL. Numerous fluorescent endosomes accumulate at the EVL-YSL margin. (F) Fixation releases SYTO-11 that has been internalized by endocytosis. Embryos were first labeled as above and then fixed with 4% paraformaldehyde at dome stage. The vesicular labeling pattern within the cytoplasm of EVL and deep cells is no longer observed. Instead, distinct nuclear labeling is observed in both cell populations. At this plane of focus, EVL nuclei (white arrows) are seen at the periphery of the image, and deep cell nuclei (dark arrows) are seen in the center. Animal pole view.