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First published online 13 June 2007
doi: 10.1242/dev.005611

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Cellular dynamics associated with the genome-wide epigenetic reprogramming in migrating primordial germ cells in mice

¹ Laboratory for Mammalian Germ Cell Biology, Center for Developmental Biology, RIKEN Kobe Institute, 2-2-3 Minatojima-Minamimachi, Chuo-ku, Kobe, 650-0047, Japan.
² Laboratory of Molecular Cell Biology and Development, Graduate School of Biostudies, Kyoto University, Oiwake-cho, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan.
³ Department of Biosystems Science, Graduate School of Science and Technology, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan.
⁴ Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University, 4-1 Seiryo-cho, Aoba-ku, Sendai 980-8575, Japan.
⁵ Department of Genetics and Division of Mammalian Development, National Institute of Genetics, SOKENDAI, 1111 Yata, Mishima, Shizuoka 411-8540, Japan.
⁶ Department of Cell Biology, Institute for Virus Research, Kyoto University, Shogoin Kawara-cho, Kyoto 606-8507, Japan.
⁷ Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, 4-1-8 Hon-cho, Kawaguchi, Saitama 332-0012, Japan.

View larger version (112K): [in this window] [in a new window]   Fig. 1. Epigenetic reprogramming proceeds progressively in migrating PGCs in mice. (A) H3K9me2 (red, left column) in migrating PGCs (green, middle) at E7.25, E7.75, E8.25 and E8.75 as indicated. The white bent arrow indicates a line used for the line-scan plot in C. (B) H3K27me3 (red, left column) in migrating PGCs (green, middle) at E7.25, E8.5, E8.75 and E9.5 as indicated. Images merged with Hoechst staining (blue) are shown on the right (A,B). The white bent arrow indicates a line used for the line-scan plot analysis in D. (C) Line-scan plot of relative intensity of H3K9me2 (red) and Hoechst (blue). E7.75 (top) and E8.75 (bottom) cells shown in A. (D) Line-scan plot of relative intensity of H3K27me3 (red) and Hoechst (blue). E8.75 (top) and E9.5 (bottom) cells shown in B. The areas in C and D shaded in green and pink represent PGC and somatic cell nuclei, respectively. (E) Ratio of PGCs strongly positive for H3K9me2 (top) and for H3K27me3 (bottom) during the course of development. All PGCs from each embryo were analyzed. Scale bars: 10 µm in A,B.

View larger version (97K): [in this window] [in a new window]   Fig. 2. Migrating PGCs exhibit transcriptional repression in mice. (A) (Top) Ser2 phosphorylation (red, left column) in PGCs (green, middle) at E8.75. (Bottom) Magnified images. (B) RNAP II expression (red, left) in PGCs (green, middle) at E8.75. (C) H3K4me2 (red, top, left) and H3K4me3 (red, bottom left) in migrating PGCs (green, middle) at E8.75. (D) Ser5 phosphorylation (red, left) in PGCs (green, middle) at E8.75. (E) BrUTP incorporation (red, left) in PGCs (green, middle) at E8.75. Percentages of BrUTP-weak, stella-positive cells are indicated with the number of cells analyzed in parenthesis (right). (F) Ser2 phosphorylation (red, left column) in PGCs (green, middle) at MS, E7.25, E8.0, E9.5 and E10.5 as indicated. An image for DAPI staining (F, MS) and images merged with Hoecsht staining (A-F, blue) are shown on the right. (G) Ratio of PGCs with strongly positive staining for Ser2 phosphorylation over the course of development. After E8.25 (dotted line), randomly chosen fragments were analyzed. (H) (Top) PGCs (green) stained by H5 (red), H3K9me2 (white) and Hoechst (blue) at E8.0. A white arrowhead denotes a PGC with reduced H3K9me2 but with Ser2 phosphorylation. (Bottom) PGCs (green) stained by H5 (red), H3K27me3 (white) and Hoechst (blue) at E8.75. Pink arrowheads denote PGCs with elevated H3K27me3 but without Ser2 phosphorylation. Scale bars: 100 µm in A, top row; 10 µm in A, bottom row; 10 µm in B-H.

View larger version (65K): [in this window] [in a new window]   Fig. 3. Cell cycle distribution of migrating PGCs. (A) PGC number with average and standard deviation (top), ratio of PGCs with BrdU (middle) and with cyclin B1-strong-positivity (bottom) over the course of development in mice. After E8.25 (dotted line), randomly chosen cells were analyzed for BrdU incorporation and cyclin B1 immunostaining. (B) Representative images of BrdU incorporation (red) into PGCs (green) at E7.25, E7.75, E8.5 and E10.5 as indicated. (C) Representative images of cyclin B1 immunoreactivity (red, left column) in PGCs (green, right) at E7.75, E8.25, E9.25 and E9.75 as indicated. (D) FACS analysis of the cell cycle distribution of PGCs (green) and embryonic somatic cells (pink) during the course of development. Red arrowheads denote the G1 and G2 peaks. Scale bars: 10 µm in B,C.

View larger version (50K): [in this window] [in a new window]   Fig. 4. Epigenetic reprogramming and transcriptional quiescence are independent of Nanos3 function in mice. H3K9me2 (A), H3K27me3 (B), H3K4me2 (C) and Ser2 phosphorylation (D) (red, middle columns) in wild-type (+/+) (top row) and Nanos3-null (-/-) (bottom row) PGCs (green, left columns). Merged images with Hoechst staining (blue) are shown in the right columns. Percentages of H3K9me2-reduced (A), H3K27me3-elevated (B), H3K4me2-normal (C) and H5-negative (D) PGCs are indicated with the number of cells analyzed in parenthesis. Scale bar: 10 µm.

View larger version (78K): [in this window] [in a new window]   Fig. 5. Heatmap representation of the expression of JmjC domain-containing genes in murine PGCs and their somatic neighbors. The expression levels relative to the average in all 24 cells are represented by a color code shown at the bottom, with corresponding fold difference values. Genes, the expression of which was not detected in all 24 cells, are shown in black.

View larger version (82K): [in this window] [in a new window]   Fig. 6. Specific repression of GLP in murine PGCs before their epigenetic reprogramming. (A) GLP expression (red, left) in PGCs (green, middle) in the extra-embryonic mesoderm at E7.75, and in the developing hindgut endoderm at E7.75 and E9.5 as indicated. (B) G9a expression (red, left) in PGCs (green, middle) in the developing hindgut endoderm at E7.75 (top) and at E9.5 (bottom). Images merged with Hoechst staining (blue) are shown on the right (A,B). (C) Triple staining of Blimp1-positive cells (green), GLP (red) and H3K9me2 (white), in addition to Hoechst (blue) at E7.5 (LB). Two representative fields are shown. Arrowheads indicate PGCs with repressed GLP but with high H3K9me2 (white), with high GLP with high H3K9me2 (pink), and with repressed GLP and reduced H3K9me2 (green). (D) Number and percentage (in parenthesis) of H3K9me2 weak-, GLP-negative and GLP-positive and H3K9me2 weak-cells among Blimp1-positive cells in two LB-stage embryos. Scale bars: 10 µm in A-C.

View larger version (34K): [in this window] [in a new window]   Fig. 7. Cellular dynamics and epigenetic reprogramming in migrating PGCs in mice. Events identified in both the current and previous (Seki et al., 2005) studies are shown.