Nishiumi F et al. (2005), The mode and molecular mechanisms of the migrat...

XB-ART-2249

Dev Growth Differ 2005 Jan 01;471:37-48. doi: 10.1111/j.1440-169x.2004.00777.x.

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The mode and molecular mechanisms of the migration of presumptive PGC in the endoderm cell mass of Xenopus embryos.

Nishiumi F , Komiya T , Ikenishi K .

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We investigated the mode of migration of presumptive primordial germ cells (pPGC) in the endoderm cell mass of Xenopus embryos at stages 7-40. The molecules underlying the migration were also studied cytochemically and immunocytologically. By examining the relative positions of pPGC and somatic cells derived from the single, fluorescein-dextran lysine (FDL)-injected, germ plasm-bearing cells of stage 6 embryos, pPGC in embryos at stages 7-23 and those at stages later than 24 were assumed to passively and actively migrate in the endoderm cell mass, respectively. This assumption was supported by the observation that F-actin, essential for active cell migration, was recognized on pPGC of the latter stages, but never on those of the former ones. In addition, the molecule like CXC chemokine receptor 4 (CXCR4) found on directionally migrating PGC in mouse and zebrafish, probably Xenopus CXCR4 (xCXCR4), was detected on pPGC only at latter stages. Accordingly, F-actin and xCXCR4, and probably beta1-integrin and collagen type IV, which are indispensable for the formation of F-actin, are thought to be involved in the active migration of pPGC in the endoderm cell mass.

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Species referenced: Xenopus
Genes referenced: actb cxcl12 cxcr4 ddx4 fn1 itgb1 pgc
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	Fig. 1. An outline of the experiment showing the behavior of descendant cells of fluoresceindextran lysine (FDL)-injected, single germ plasm-bearing cells (GPBC) of stage 6 embryos. The relative positions of FDL-labeled, presumptive primordial germ cells (pPGC) and somatic cells in the endoderm cell mass were examined in embryos at stages 7–40. The germline cells derived from GPBC were designated as pPGC in embryos at stages 7–40, and as PGC in embryos from stage 41 onward. Approximate positions of pPGC and PGC are shown in red circles on the schematic drawing of embryos.
	Fig. 2. A diagrammatic transverse section of an embryo showing five different parts of the endoderm cell mass. As for the locations of pPGC in the endoderm cell mass of embryos at stages 24–40 in Figs 3–9, refer to this drawing. The top is the dorsal and bottom the ventral side. d, dorsal part; l, lateral part; c-d, centro-dorsal part; c-v, centro-ventral part; v, ventral part.
	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.
	Fig. 4. A transverse section of a stage 33/34 embryo stained with rhodamine-phalloidin (R-P). R-P positive cells containing F-actin (arrows), which are usually spherical in shape, are found in the dorsal part (cf. Figure 2) of the endoderm cell mass of the embryo. The epidermal cells (e) are also stained with R-P. The top is the dorsal and bottom the ventral side. Bar, 50 μm.
	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.
	Fig. 6. The double staining with the anti-Beta1 integrin and antiactin antibodies for a transverse section of a stage 26 embryo. (A) The integrin is faintly detected in the cell membrane and prominently in the cytoplasm surrounding the nucleus of an endoderm cell (arrow), possibly a pPGC in the centro-dorsal part (cf. Figure 2) of the endoderm cell mass. It is also prominent in cells of the neural tube (nt), notochord (no) and somite (s). gc, gut cavity. (B) A higher magnification of the area of the cell in (A). Integrin is faintly localized in the cell membrane, and moderately in the perinuclear cytoplasm of the pPGC. (B‘) Nearly the same region of the cell is stained with the anti-actin antibody, so that it must be the pPGC as described in Fig. 5. (B”) A merged image of (B) and (B’). An overlap is clearly seen in the perinuclear cytoplasm of the pPGC. The position of the nucleus seems to be empty. The top is the dorsal and bottom the ventral side. Bar, 200 μm in (A), and 50 μm in (B).
	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.
	Fig. 8. (A) CBB stain (lane 1) and immunoblotting for a fusion protein of Xenopus collagen type IV. The antiserum from a rat immunized with the fusion protein reacts with a 37 kDa protein band (lane 2), which corresponds to the predicted size of the fusion protein (151 amino acids of thioredoxin and 192 amino acids of the collagen type IV), while a pre-immune serum of the same rat does not react at all (lane 3). The epitope was confirmed to reside on the collagen, but not on the thioredoxin of the fusion protein because of the positive stain for Xenopus embryos, as shown in this study. (B) The double staining with the antiserum and 2L-13 antibody for a transverse section of a stage 23 embryo. Xenopus collagen type IV is detected in the perinuclear region and in the vicinity of the cell membrane of the cells (arrows) in the ventral part of the endoderm cell mass of the embryo. The perinuclear region of those cells is stained with the 2L-13 antibody, confirming that they are pPGC (B). (C) The double staining with the antiserum and 2L-13 antibody for a transverse section of a stage 26 embryo. The collagen is usually recognized in the perinuclear cytoplasm of a pPGC (arrow) in the centro-dorsal part (cf. Figure 2) of the embryo. The perinuclear cytoplasm of the pPGC is stained with 2L-13 antibody (C). The position of the nucleus seems to be empty. The top is the dorsal and bottom the ventral side. Bar, 50 μm.
	Fig. 9. The double staining with the anti-fibronectin and anti-actin antibodies for transverse sections of stage 26 (A) and stage 40 (B) embryos. (A) Fibronectin is first detected in the cytoplasm of many cells (arrows) or the pPGC in the centro-dorsal part (cf. Figure 2) of the endoderm cell mass of the embryo. (A) Those pPGC are also stained strongly with the anti-actin antibody. (A) A merged image of (A) and (A). A partial overlap is observed of pPGC. (B) Fibronectin is abundant in the perinuclear cytoplasm of a pPGC (arrow) in the dorsal part (cf. Figure 2) of the endoderm cell mass of the embryo. (B) The cytoplasm in the pPGC is exceptionally stained with the anti-actin antibody, though the pPGC in the uppermost dorsal part at stage 40 were never stained with the antibody, as shown in Fig. 5 (B). (B) A merged image of (B) and (B). The distribution of fibronectin is somewhat similar to that of actin. The position of the nucleus seems to be empty. The top is the dorsal and bottom the ventral side. e, epidermis; no, notochord; s, somite. Bar, 100 μm in (A) and (B).