Fig. 3

Fig. 3 The forerunner cell cluster forms a wedge-shaped cap at the posterior limit of the embryonic axis during late-epiboly. Scale bars, 50 μm. (A–E) The forerunner cells form a wedge-shaped cap at the dorsal margin of the blastoderm. EVL cells, which derive from the original NEM cell cluster, overlie the forerunner cells. Forerunner cells are derived from deep NEM cells. The embryo is viewed from its dorsal side. The embryo was stained with SYTO-11 and red-emitting BODIPY 564/591 at 30%-epiboly and then imaged at 65%-epiboly. Scale bar, 50 μm. (A) SYTO-11-labeled cells include both EVL – NEM cells (thin white arrows) and deep (forerunner) cells (thick arrow). A group of brightly labeled nuclei (black arrow) in the YSL is located just vegetal to the NEM/forerunner cluster margin. This surface-view image was taken by using a dual excitation with 488- and 568-nm laser light, while fluorescence emission was collected >585 nm. Since SYTO-11 exhibits a red fluorescence, dual excitation of the specimen allows cell nuclei and endosomes, labeled with SYTO-11, and cellular cytoplasm, labeled with BODIPY 564/591, to be imaged simultaneously. The animal-vegetal (An-Vg) axis is shown. (B-E) A focus-through of the NEM/forerunner cell cluster in the same embryo as in F but rotated slightly counterclockwise and shifted into an oblique orientation. B-E were only collected with 488-nm excitation so that only SYTO-11 emission is seen. (B) This is a surface focal plane of the NEM/forerunner cell region showing the same EVL (white arrows) and YSL nuclei (black arrow) shown in A. (C) 15 μm deeper than B. Brightly labeled YSL nuclei (arrows are visible at this plane of focus. (D) 23 μm deeper into the embryo. Brightly labeled, multi-nucleate EVL-NEM cells (arrow) lie directly above the forerunner cell cluster. (E) 23 μm deeper into the embryo. SYTO-11-labeled deep cells (arrow) of the forerunner cell cluster are closely associated to overlying EVL-NEM cells. Panels F-J show a focus-through (15-μm steps) of a 80%-epiboly-stage embryo labeled with SYTO-11, starting from the posterior edge of the NEM/forerunner cell cluster and progressing toward the anterior end of the cell cluster. The embryo is viewed from the vegetal pole. The dorsal-ventral (D-V) axis is shown. (F) The focus-through starts at the level of the EVL-YSL boundary. (G) Labeled NEM/forerunner cells and YSL cytoplasm are prominent. (H) A distinct layer of forerunner cells lies directly above the internal YSL which contains numerous SYTO-11-filled endosomes. (I, J) Edges of the germ ring converge toward the embryonic midline which is defined by the dorsal apex (arrow) of the forerunner cell cluster. The NEM cell cluster in J also has an apex (arrow) pointing toward the anterior end of the embryo. The anterior apex of the forerunner cluster lies in a more ventral plane than the dorsal apex seen in H.