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Fig. 3. Location of FDL-labeled, pPGC (arrows) and somatic endoderm
cells derived from single FDL-injected GPBC of a stage 6 embryo. pPGC were identified by the criteria described in Materials and Method. ap, animal pole; b, blastocoel; e, epidermis; gc, gut cavity; no, notochord; nt, neural tube; s, somite; vp, vegetal pole.(A) A FDL-injected embryo at stage 7. A small cluster of FDL-labeled cells is situated in the vegetal surface of the embryo. Bar, 200 μm (A) A light micrograph of (A). Two pPGC are evident in the vegetal region of the embryo. The pPGC at the left (arrow) and right sides are found to have originated from the FDL injected and uninjected GPBC, respectively. Inset: a higher
magnification of the area of the pPGC. A yolk-free, granular cytoplasm
or the germ plasm is easily seen in the pPGC. Bar, 50 μm (B)
A FDL-injected embryo at stage 10. A cluster of the labeled cell is
extending from the vegetal surface nearly to the floor of the blastocoel
of the embryo. Bar, 100 μm (B) A light micrograph of (B). A pPGC (arrow) is located in the cluster. Inset: a higher magnification of the pPGC. A granular cytoplasm is observed in the perinuclear region of the pPGC. Bar, 50 μm (C) A FDL-injected embryo at stage 18. A cluster of FDL-labeled cells is located in the central part of the endodermal cell mass of the embryo. An edge of the cluster is seen in this figure because a
larger cluster, which was composed of more labeled cells, was
observed in the adjacent sections. Bar, 50 μm (C ) The embryo in (C)
is immunostained with the 2L-13 antibody. The cytoplasm of three
cells (arrows) is clearly stained with the antibody, indicating that
those cells are pPGC. (C) A merged image of (C) and (C). Two of the three pPGC (arrows) and the somatic endoderm cells are forming
a cluster. (D) A FDL-injected embryo at stage 24. A FDL-labeled
cell (arrow) in the centro-dorsal part (cf. Figure 2) of the endoderm cell mass is about to separate from the cluster of labeled
cells. Bar, 200 μm (D) The embryo in (D) stained with the 2L-13 antibody. The perinuclear cytoplasm of the cell (arrow) is strongly stained with the antibody, so that the cell is identified as a pPGC. The position of the nucleus seems to be empty. (D ) A merged image of (D) and (D'). (e) An FDL-injected embryo at stage 33/34. An FDL-labeled cell (arrow) is found as individual cells in the dorsal part (cf. Figure 2) of the endodermal cell mass, which is distinctly separated from a cluster of labeled somatic endoderm cells. Bar, 100 μm (E) The embryo in (E) is stained with the 2L-13 antibody. The FDL-labeled cell (arrow) is shown to be a pPGC by the positive stain in the perinuclear cytoplasm. The position of the nucleus seems to be empty. (E) A merged image of (E) and (E'). The top is the animal and bottom the vegetal side in (A) and (B), and in (C–E) the top is the dorsal and bottom the ventral side. |
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Fig. 5. The double staining with the anti-actin and 2-L-13 antibodies for transverse, polyester wax sections of stage-28 (A) and stage 40 (B) embryos. The position of the nucleus in (A–A’) and (B-B‘) seems to be empty. (A) A strong fluorescence representing actin is observed in the cytoplasm surrounding the nucleus of an endoderm cell (arrow) in the centro-dorsal part (cf. Figure 2) of the endoderm cell mass of the embryo. (A’) A similar part of the cell is also stained with the 2L-13antibody specific to the pPGC, indicating that the cell is a pPGC. (A”) A merged image of (A) and (A’) where coincidental staining is seen as yellow, revealing a partial overlap in the perinuclear cytoplasm.(B) The fluorescence for actin is not detected in the perinuclear cytoplasm of a cell or a pPGC (arrow) at the uppermost dorsal part of the endoderm cell mass (cf. Figure 1) of the embryo. (B’) The perinuclear cytoplasm of the pPGC is strongly stained with the 2L-13 antibody. (B”) A merged image of (B) and (B’). No overlap is seen in the pPGC, implying that the pPGC in the stage 40 embryo no longer has enough detectable actin. The top is the dorsal and bottom the ventral side. gc, gut cavity; no, notochord; s, somite. Bar, 50 μm.
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Fig. 7. (A,B) The double staining with the anti-CXCR4 and 2L-13 antibodies for a transverse section of embryos. (A) The molecule recognized with the anti-CXCR4 antibody, probably Xenopus CXCR4 (xCXCR4), is mostly detected throughout the cytoplasm of an endoderm cell (arrow) in the dorsal part (cf. Figure 2) of the endoderm cell mass of a stage 33/34 embryo. (A ) The perinuclear cytoplasm of the cell (arrow) in (A) is strongly stained with the 2L-13 antibody, indicating that the cell is a pPGC. (A ) A merged image of (A) and (A ). xCXCR4 is seen throughout the cytoplasm of the pPGC (arrow) while XVLG1 protein recognized with the 2 L-13 antibody is prominent in the perinuclear region. xCXCR4 is not always present in pPGC because it is not detected in a pPGC (arrowhead) situated more dorsally. The top is the dorsal and bottom the ventral side. Bar, 20 μm (B) No endoderm cell in the central part of the endoderm cell mass, including pPGC of a stage 23 embryo, is stained with the anti-CXCR4 antibody. (B ) The perinuclear cytoplasm of five cells (arrows) are stained with the 2L-13 antibody, indicating that they are pPGC. (B ) A merged image of (B) and (B ). xCXCR4 is never observed in the pPGC (arrows) at stages younger than 23. The top is the dorsal and bottom the ventral side. Bar, 50 μm. (C,D) In situ hybridization for polyester wax sections of a stage 28 embryo with riboprobes of xCXCR4. xCXCR4 RNA is prominent in the nucleus of three pPGC (arrows) in the lateral part (cf. Figure 2) of the endoderm cell mass with the antisense probe (C) while it is rarely detected in a pPGC (arrow) on the adjacent section of (C) with the sense probe (D). The pPGC are easily identified by the possession of a granular cytoplasm in the perinuclear region. Bar, 20 μm. |
