XB-ART-15902Dev Growth Differ October 1, 1997; 39 (5): 625-33.
Involvement of the protein of Xenopus vasa homolog (Xenopus vasa-like gene 1, XVLG1) in the differentiation of primordial germ cells.
In order to understand the role of the protein of Xenopus vasa homolog (Xenopus vasa-like gene 1, XVLG1) in germ line cells, an attempt was made to perturb the function of the protein with the anti-vasa antibody 2L-13. The 2L-13 or the control antibody was microinjected with a lineage tracer (FITC-dextran-lysine, FDL) into single vegetal blastomeres containing the germ plasm of Xenopus 32-cell embryos, the descendants of which were destined to differentiate into a small number of primordial germ cells (PGC) and a large number of somatic cells, mostly of endodermal tissues at the tadpole stage. No significant effect of the injection of the antibodies on FDL-labeled, presumptive PGC (pPGC) was observed in embryos until stage 37/38. However, FDL-labeled PGC were not observed in almost all the 2L-13 antibody-injected tadpoles, although a similar number of labeled somatic cells were always present. As 2L-13 antibody specifically reacts with XVLG1 protein in the embryos by immunoblotting, the present results suggest that the antibody perturbed the function of XVLG1 protein in the pPGC, resulting in failure of PGC differentiation at the tadpole stage.
PubMed ID: 9338598
Article link: Dev Growth Differ
Species referenced: Xenopus laevis
Genes referenced: ddx4 ednra myh3 pgc
Antibodies: Ddx4 Ab1
Article Images: [+] show captions
|Fig. 1. A schematic representation of a perturbation experiment with an antibody. 2L-13 antibody specific to XVLGI protein, or the control, antibody or modified Steinberg's saline was microinjected with FITC-dextran-lysine (FOL) into single vegetal blastomeres with a 'dark area' (lkenishi & Nakazato 1986) or those containing the germ plasm of 32-cell embryos. FDL-Iabeled, pPGC and PGC derived from the blastomeres were examined in experimental embryos and tadpoles. See text for details.|
|Fig. 2. A transverse, paraplast section of a stage-45 tadpole developed from a 32-cell embryo with an injection of FDL. About 2 nL of FDL (at a concentration of 100 mg/ml in Steinberg's saline) was injected into the vegetal blastomere containing the germ plasm of the embryo. (A) A fluorescent micrograph showing that FDL-Iabeled cells are mainly observed in the endodermal tissues such as intestine (i), stomach (st) and lung (I) of the tadpole. The labeled PGC (arrowheads) are also recognized in the genital ridges. Top is dorsal and bottom, ventral. e, epidermis. Bar, 100 ~m. (B) A phase-contrast micrograph of a higher magnification of the genital ridge region in A. Four PGC (arrowheads) are situated in the genital ridges. dm, dorsal mesentery; m, mesonephrogenic blastema; w, Wolffian duct. Bar, 20 ~m. (C) A fluorescent micrograph of B with FITC. Two of the four PGC (arrowheads), and some cells in the lung and the mesonephrogenic blastema are labeled with FDL. The other two, unlabeled PGC are thought to be originated from uninjected blastomeres containing the germ plasm. Bar, 20 ~m.|
|Fig. 3. A transverse. polyester wax section of a 2L-13 tadpole at stage 45. Top is dorsal and bottom, ventral. Bar 100 ~m. (A) A fluorescent micrograph with FITC. FDL-Iabeled cells derived from the injected blastomere are seen in the midgut (m). h, hindgut. (B) A fluorescent micrograph with Cy3. FDL-Iabeled cells in A are certainly stained with Cy3- conjugated anti-mouse lgG, recognizing the injected antibody. This means that the descendant, somatic cells from the injected blastomere have differentiated into intestinal cells, without suffering from any influence by the injected antibody. e, epidermis; s, somite; w, Wolffian duct.|
|Fig. 4. lmmunoblotting to Xenopus embryonic proteins with 2L-13 (lane 1) and the commercially purchased, control antibodies (lanes 2-5). Proteins from one quarter of an embryo were loaded onto each lane. 2L-13 antibody specifically reacts with ca 80 kDa band (lane 1, arrowhead) or XVLG1 protein, while the control antibody does not at all. Lanes 1 and 2, stage 6; lane 3, stage 12; lane 4, stage 28; lane 5, stage 37/38.|
|Fig. 5. A frontal, paraplast section just below the archenteron floor of a 2L-13 antibody-injected embryo at stage 18. Only a somewhat centro-lateral part of the endodermal cell mass is shown. Top is anterior and bottom, posterior. Bar, 50 !Jm. (A) A phase-contrast micrograph. Three cells (arrows), the pPGC, having a granular cytoplasm or the germ plasm adjacent to the nucleus (n), are seen in the endodermal cell mass, forming a cluster. In this photo, the nucleus is seen only in two of the three pPGC but the nucleus of the rest was in the vicinity of the granular cytoplasm in the adjacent sections. (B) A fluorescent micrograph with FITC. The nucleus and granular cytoplasm of the pPGC in A (arrows) are heavily stained with FDL. The somatic cells next to the pPGC on the right side are also stained.|