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Fig. 1. Nanos1-depleted Xenopus embryos are deficient in PGCs. (A) Nanos1-MO blocks the translation of endogenous nanos1. Confocal analysis showing the expression of Nanos1 (red) and Xiwi (germ plasm marker, green) at the eight-cell stage of embryos previously injected with Nanos1-Ctrl-MO or Nanos1-MO at the one-cell stage. Uninjected embryos served as controls. Arrows indicate Nanos1 signal. (B) Knockdown of Nanos1 results in loss of PGCs. WISH Xpat analysis of stage 37/38 wild-type (WT), Nanos1-Ctrl-MO- and Nanos1-MO-injected embryos. For the rescue, Nanos1-MO embryos were injected with nanos1-mut RNA. (C) Summary of PGC loss at different stages after Nanos1 knockdown. P-values by one-way ANOVA. Error bars indicate s.e. (D) Hematoxylin and Eosin-stained sections of gonads from WT (stage 54), Nanos1-Ctrl-MO (stage 55), Nanos1-MO (stage 54) and rescued (stage 54) embryos, Arrows indicate germ cells. Note that nanos1 morphants were completely rescued by nanos1-mut RNA injection. Scale bars: 130 μm in A; 100 μm in B; 20 μm in D. |
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Fig. 4. Nanos1-depleted PGCs exhibit CTD-PSer2 prematurely. (A) Confocal analysis of WT, Nanos1-Ctrl-MO- and Nanos1-MO-injected embryos at stage 11, showing double immunostaining with H5 monoclonal antibody (CTD-PSer2, green) and rabbit anti-Xiwi antibody (germ plasm, red). Merged images are shown at the top, with separate channels beneath. Images were taken from the endoderm core (see the diagram in Fig. 5). Scale bars: 50 μm. (B) PGCs immunopositive for H5 (which is specific for the CTD-PSer2 epitope) in Nanos1-MO (74%) as compared with WT (21%) and Nanos1-Ctrl-MO (16%) embryos. Differences between each group were highly significant by one-way ANOVA. The experiment was repeated three times. |
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Fig. 5. PGCs deficient in Nanos1 activity express endoderm-specific genes. (A,C) RT-PCR analysis of PGCs or endoderm cells isolated from the endoderm core at stage 11 (circled in red, surrounded by endomesoderm in blue) (A) and stage 15 (C). P, primordial germ cells; E, endoderm; WE, whole embryo. Xpat is a germ plasm marker, Xsox17α an endoderm marker, Bix4 an endomesoderm marker and Xbra a mesoderm marker. ODC provides an internal control. The asterisk marks endoderm cell contamination. (B) Confocal analysis of WT, Nanos1-Ctrl-MO- and Nanos1-MO-injected embryos at stage 11. Xsox17α RNA (red) was detected by WISH and PGCs (outlined by dashed line) were identified by Xiwi immunostaining (green). Merged images are shown at the top, with separate channels beneath. Images were taken from the endoderm core. Scale bars: 50 μm. |
