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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
positive control. |
