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Whether all descendants of germline founder cells inheriting the germ plasm can migrate correctly to the genital ridges and differentiate into primordial germ cells (PGCs) at tadpole stage has not been elucidated in Xenopus. We investigated precisely the location of descendant cells, presumptive primordial germ cells (pPGCs) and PGCs, in embryos at stages 23-48 by whole-mount in situ hybridization with the antisense probe for Xpat RNA specific to pPGCs and whole-mount immunostaining with the 2L-13 antibody specific to Xenopus Vasa protein in PGCs. Small numbers of pPGCs and PGCs, which were positively stained with the probe and the antibody, respectively, were observed in ectopic locations in a significant number of embryos at those stages. A few of the ectopic PGCs in tadpoles at stages 44-47 were positive in TdT-mediated dUTP digoxigenin nick end labeling (TUNEL) staining. By contrast, pPGCs in the embryos until stage 40, irrespective of their location and PGCs in the genital ridges of the tadpoles at stages 43-48 were negative in TUNEL staining. Therefore, it is evident that a portion of the descendants of germline founder cells cannot migrate correctly to the genital ridges, and that a few ectopic PGCs are eliminated by apoptosis or necrosis at tadpole stages.
Fig. 1. Whole-mount immunostaining for tadpoles with the 2L-13 antibody against XVLG1 (Xenopus Vasa) protein. Positive cells with the
antibody or primordial germ cells (PGCs) were stained dark brown with diaminobenzidine (DAB). The top of all figures is dorsal and
the bottom is ventral. e, esophagus; ep, epidermis; i, intestine; l, liver; s, somite; Wd, Wolffian duct. (A) A transverse section of a stage-
47 tadpole. PGCs in the genital ridges (arrowheads) or a proper location are heavily stained. Bar, 50 μM. (B1) A transverse section of
the same tadpole in A at a more anterior level than the genital ridges (corresponding to region ‘a’ in Fig. 2C). A positively stained
cell, probably an ectopic PGC (arrow), is seen in the intestine at the ventral side of the tadpole. Bar, 50 μM. (B2) A higher magnification
of the cell (arrow) in B1. It is strongly stained with the antibody like PGCs in the genital ridges, indicating it is an ectopic PGC. Bar,
25 μM. (C) Lateral view of a stage-46 tadpole. Heavily stained PGCs lining the genital ridges (bracket) are externally visible because
of the bleaching and clearing agent. Ecotopic PGCs in region ‘a’ was defined as ‘anterior’ and those in region ‘b’ as ‘genital ridges’
in Series IV of Table 1. Bar, 200 μM. (D1) A transverse section of a stage-47 tadpole at the level of the genital ridges (corresponding to
region ‘b’ in Fig. 2C). Positively stained PGCs are seen in the genital ridges (arrowheads) and the intestine at the ventral side (arrow)
of the tadpole. Bar, 100 μM. (D2) A higher magnification of the ectopic PGC in the intestine (arrow). Bar, 25 μM.
Fig. 2. Distribution of presumptive
primordial germ cells (pPGCs) in
the endoderm cell mass (a part
stained black) of embryos at
stages 23 (A), 28 (B), 33/34 (C)
and 40 (D) in Series III. Location
of pPGCs, being positive by
whole-mount in situ hybridization
with the antisense probe for Xpat
RNA (arrows in A2) (216, 404, 563
and 131 in total at stages 23 [A3],
28 [B2], 33/34 [C2] and 40 [D2],
respectively), was plotted on
schematic drawings of the lateral
profile of the endoderm cell mass
of the embryos. It was difficult to
distinguish accurately ectopic
pPGCs from pPGCs in their proper
location in embryos at stages 23â
40. Therefore, pPGCs forming a
cluster (blue dots) are defined as
pPGCs in a âproperâ location and
those distinctly apart from the
cluster (red dots) as pPGC in an
âectopicâ location for convenience.
Each dot represents the location
of a single pPGC. 0 and 1 on the
horizontal line represent the most
anterior and posterior ends of the
mass, respectively, to normalize the
location of pPGCs in the anteroposterior
direction of all embryos
at each stage. The values on the
vertical line (μM) represent the
distance of each pPGC (d1 in A2)
from the bottom of the mass. Lateral
profile of the mass at each stage
was drawn based on the average
values of the top of the endoderm
cell mass (d2 in A2) at every 10
sections from the first section of
all embryos examined, in which the
anterior end of the endoderm cell
mass appeared. See Methods for
details. The top and the bottom of
A2 are dorsal and ventral sides,
respectively. Bar in A2, 100 μM.
Fig. 3. Immunostaining with the
2L-13 antibody and TdT-mediated
dUTP digoxigenin nick end labeling
(TUNEL) staining in combination.
The cytoplasm of primordial germ
cells (PGCs) reacted with the
antibody was stained red with
Cy3, and the cytoplasm and the
nucleus of PGCs in apoptosis or
necrosis were stained red with
Cy3 and green with Alexa 488,
respectively. Cells, except for
PGCs, in apoptosis or necrosis
were stained green only. The top
of all figures is dorsal and the
bottom is ventral. e, esophagus;
ep, epidermis; i, intestine; l, liver;
n, neural tube; s, somite; Wd,
Wolffian duct. (A) A merged image
of transmission and Alexa 488 of
a transverse section of a stage-
33/34 embryo at the level of the
tail. Many epidermal cells and a
cell in the neural tube are stained
with Alexa 488 (arrowheads),
indicating that they are apoptotic
or necrotic cells. Bar, 50 μM. (B1)
A transverse section of a stage-46
tadpole. An ectopic PGC (arrow)
which is clarified in B3 is present
in the intestine situated in the
ventral side of the tadpole. Bar,
100 μM. (B2) A higher magnification
of the cell in B1 (arrow). Bar,
25 μM. (B3) The same view of B2
with Cy3. The fluorescence of Cy3
for the marker protein or XVLG1
protein in PGCs is detected on
the cell (arrow), indicating that the
cell is an ectopic PGC. (B4) The same view of B2 with Alexa 488. The fluorescence of Alexa 488 seems to be detected on the nucleus of the cell (arrow). (B5) A
merged image of B3 and B4. This clearly shows the PGC being in apoptosis or necrosis. (C1) A transverse section of a stage-47
tadpole. PGCs (arrowheads) are seen in the genital ridges. Bar, 50 μM. (C2) A merged image of the same section in C1 with Cy3 and
Alexa 488. PGCs (arrowheads) are stained with Cy3 but not with Alexa 488. Likewise, PGCs in the genital ridges of tadpoles at stages
44–48 were negative in TUNEL staining.
