PMC

Representative images of embryos hatching through the embryonic or abembryonic pole. a, b Embryos escaping the zona pellucida through the embryonic pole. c, d Embryos hatching through the abembryonic pole completely opposite to the ICM (c, d). The area encircled with the dotted line represents the inner cell mass. Scale bar = 50 μM

(A) Expression of a mitotic marker PH3 was examined in pole cells of embryos derived from y w, nos, impα2-nos3’UTR, and pgc impα2-nos3’UTR females mated with y w males. Stage-7–9 embryos were double-stained with anti-PH3 (green) and anti-Vasa (a marker for pole cells: magenta) antibodies. Left: representative images of pole cells negative (top) and positive (bottom) for PH3 in stage-8 embryos. DIC images merged with PH3- and Vasa-signals are also shown. Right: percentages of embryos carrying 0 (white), 1 (gray), 2 (pale green), or ≥3 (green) pole cells with PH3 signal are shown. The numbers of embryos examined are shown in parentheses. Significance was calculated using Fisher’s exact test (*: P < 0.05, n.s.: P > 0.5). (B) Expression of an apoptotic marker, cleaved Caspase-3, was examined in pole cells of embryos derived from y w, impα2-nos3’UTR, pgc/pgc (pgc), and pgc impα2-nos3’UTR females mated with y w males. Stage-10–16 embryos were double-stained with anti–cleaved Caspase-3 (green) and anti-Vasa (magenta) antibodies. Left: representative images of pole cells negative (top) and positive (bottom) for cleaved Caspase-3 in stage-12 embryos. DIC images merged with cleaved Caspase-3- and Vasa-signals are also shown. Right: percentages of pole cells expressing cleaved Caspase-3 are shown. The numbers of pole cells examined are shown in parentheses. For each genotype, 10–26 embryos were examined. Significance was calculated using Fisher’s exact test (*: P < 0.01, n.s.: P > 0.1). (C) Stage-14–16 embryos derived from y w, impα2-nos3’UTR, pgc/Df (pgc), and pgc impα2-nos3’UTR females mated with y w males were stained with anti-Vasa antibody (magenta). Left: representative images of pole cells within (top) and outside of (bottom) gonads in stage-14 embryos. DIC images merged with Vasa signal are also shown. Dotted line shows contour of embryonic gonads. Right: percentages of pole cells incorporated in the embryonic gonads. Total numbers of pole cells examined are shown in parentheses. For each genotype, 10–20 embryos were examined. Significance was calculated using Fisher’s exact test (*: P < 0.01, n.s.: P > 0.1). Scale bars, 10 μm (A–C).

nos-H99 pole cells have the ability to adopt somatic cell fate and to enter the embryonic gonads. Pole cells from nos (B), nos-H99 (C–F), and control (A) embryos were transplanted into hosts, and the hosts were developed until stages 15–17. The transplanted pole cells were identified as blue cells after heat treatment and staining for β-gal (arrowheads). The transplanted control (A) and nos-H99 (C) pole cells were observed within the gonads, whereas nos pole cells remained outside the gonads (B). Some nos-H99 pole cells were integrated within somatic tissues (D–F). (D) nos-H99 pole cell within midgut epithelium is shown. Note that nos-H99 pole cell and the neighboring midgut epithelial cells were columnar in shape at stage 17. nos-H99 pole cells within gastric ceca (E) and tracheal (F) epithelium are also shown. The lower cell indicated by an arrowhead in E was out of focus. (G–I) Drawings tracing the integrated nos-H99 pole cells (blue) and the surrounding somatic cells in D–F, respectively. (Scale bar = 10 μm.)

A:: Stage 6 lateral view, B: Stage 8 animal pole view; Circle, the area of fortilin expression, C: Stage 10 lateral view; an, animal pole; ve, vegetal pole. D: Stage 10 sagittal section; an, animal pole; ve, vegetal pole. E: Stage 12 dorsal view; a, anterior; p, posterior; arrowheads, the area of fortilin expression. F: Stage 15 dorsal view; a, anterior; p, posterior; arrowheads, the area of fortilin expression.