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Fig. 1. PGC isolation and gene expression analyses in Xenopus embryos. (A) PGCs can be identified based on the distinctive fluorescence staining of the germ plasm with DiOC6. (i) Eight-cell embryo and (ii) corresponding image after stereofluorescence microscopy showing DiOC6 staining of vegetal pole germ plasm. (iii) PGCs near the floor of the blastocoel just after removal of the animal cap (arrows). (iv) Stereofluorescence microscopy of PGCs (arrows) and unstained somatic cells (arrowheads) from embryos dissociated at gastrula (stage 10). (B) VegT RNA is found in PGCs. WISH with anti-VegT probe of (i) early embryo and (ii) isolated PGC from a blastula-stage embryo. (C) Zygotic genes are not expressed in PGCs at MBT. Gene expression in PGCs isolated before (stage 8) and after (stages 10 and 14) MBT was compared with that in whole embryos (WE) by semi-quantitative RT-PCR with gene-specific primers. Xpat, PGC-specific marker; Xbra, mesoderm marker; Bix4, Xnr1 and Xsox17, endodermal-specific and downstream targets of maternal VegT; Xsurv (survivin), a maternal gene. Presence of Xpat but not Xbra or zygotic VegT at stage 10 and 14 indicates that PGCs were not contaminated with mesoderm. Zygotic VegT, a spliced variant of maternal VegT, is expressed at post-MBT stages in the mesoderm. Ornithine decarboxylase (ODC) served as a control. PGCs did not express endoderm markers (Bix4, Xnr1, Xsox17), but did contain maternal VegT RNA (asterisk). |
