February 1, 2010;
Repression of zygotic gene expression in the Xenopus germline.
Primordial germ cells (PGCs) in Xenopus are specified through the inheritance of germ plasm. During gastrulation, PGCs remain totipotent while surrounding cells in the vegetal mass become committed to endoderm
through the action of the vegetal localized maternal transcription factor VegT
. We find that although PGCs contain maternal VegT
RNA, they do not express its downstream targets at the mid-blastula
transition (MBT). Transcriptional repression in PGCs correlates with the failure to phosphorylate serine 2 in the carboxy-terminal domain (CTD) of the large subunit of RNA polymerase II (RNAPII). As serine 5 is phosphorylated, these results are consistent with a block after the initiation step but before the elongation step of RNAPII-based transcription. Repression of PGC
gene expression occurs despite an apparently permissive chromatin environment. Phosphorylation of CTD-serine 2 and expression of zygotic mRNAs in PGCs are first detected at neurula
, some 10 hours after MBT, indicating that transcription is significantly delayed in the germ cell lineage. Significantly, Oct-91, a POU subclass V transcription factor related to mammalian Oct3/4, is among the earliest zygotic transcripts detected in PGCs and is a likely mediator of pluripotency. Our findings suggest that PGCs are unable to respond to maternally inherited endoderm
determinants because RNAPII activity is transiently blocked while these determinants are present. Our results in a vertebrate system further support the concept that one strategy used repeatedly during evolution for preserving the germline is RNAPII repression.
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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).
Fig. 2. Pre-MBT PGCs and somatic cells express CTD-P-Ser5, but
not CTD-P-Ser2. Immunofluorescence of dissociated cells isolated from
pre-MBT stage 8 embryos previously stained with DiOC6 (A) to identify
PGCs. Monoclonal antibodies H5 and H14 were used to detect the
expression of CTD phospho-serines 2 (B, P-Ser2) and 5 (D, P-Ser5),
respectively, in PGCs (arrowheads) and somatic cells (arrows).
Monoclonal antibody 8WG16 recognizes the unphosphorylated RNA
Pol II large subunit (C, RNAPII) and served as a positive control.
Fig. 5. Pre-neurula PGCs display permissive chromatin and histone staining. (A)Fluorescence stereomicroscopy of PGCs (green) and somatic cells from dissociated embryos. Cells were stained with anti-hyperacetylated Histone H4 (Penta, red), a marker of transcriptionally active chromatin. Stage 8 and 10 represent merged images of Penta immunostaining and DiOC6 labeling (green). Stage 14: (i) Penta alone, (ii) DiOC6, (iii) merged image, (iv) merged confocal image showing Penta staining (green) and a PGC (outlined) identified by Xnos1 (red) immunostaining. (insets show separate images). Examples of PGCs (white arrows) and endoderm cells (green arrowheads) with nuclear staining. Both PGCs and somatic cells express Histone H4 Penta at every stage. (B)Cryostat sections from stages 10 and 14 showing the endodermal region immunostained for H4K20me3 or H3K4me3 (green). PGCs indicated by Xnos1 immunostaining (red); nuclei stained with Hoechst (blue). PGCs (white arrows), other nuclei from endoderm cells. PGC nuclei are outlined. No nuclear staining was detected for any of the histone methylated lysines tested (see Materials and methods). (C)Stage 10 isolated PGC and endoderm nuclei (blue) immunostained for 5mC (green). Both cell types were demethylated on cytosine residues. DiOC6-stained germ plasm remained associated with PGC nuclei (arrow). 5mC staining of ectoderm nuclei served as a
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