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First published online 3 August 2006
doi: 10.1242/dev.02500

Development 133, 3411-3418 (2006)
Published by The Company of Biologists 2006
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DNA methylation is a primary mechanism for silencing postmigratory primordial germ cell genes in both germ cell and somatic cell lineages

Danielle M. Maatouk1, Lori D. Kellam1, Mellissa R. W. Mann2, Hong Lei3, En Li3, Marisa S. Bartolomei2 and James L. Resnick1,*

1 Department of Molecular Genetics and Microbiology, PO Box 100266, University of Florida, Gainesville, FL 32610-0266, USA.
2 Howard Hughes Medical Institute and Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.
3 Epigenetics Program, Models of Disease Center, Novartis Institute for Biomedical Research, 250 Massachusetts Avenue, Cambridge, MA 02139, USA.


Figure 1
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  		Fig. 1. CpG island location in several germ cell-specific genes. The genomic structures from -2 kb to +2 kb relative to the transcription start site are depicted for Dazl, Mvh, Scp3 and Tnap. For each gene, exon 1 (exon 1a for Tnap) is surrounded by a CpG island defined as previously described (Gardiner-Garden and Frommer, 1987Go). Black boxes represent exons, gray bars represent CpG islands. The regions amplified for bisulfite analysis are indicated by black bars below each CpG island.

 

Figure 2
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  		Fig. 2. DNA methylation analysis of postmigratory germ cell-specific genes. (A-C) Bisulfite sequence analysis of (A) Mvh, (B) Scp3 and (C) Dazl was performed on immunomagnetically purified germ cell and depleted somatic cell fractions from 10.5 and 13.5 dpc embryos. Each line represents an individually sequenced clone and circles represent CpG residues. White circles indicate unmethylated CpG sites; black circles represent methylated CpG sites.

 

Figure 3
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  		Fig. 3. DNA methylation analysis of Tnap. Bisulfite sequence analysis of Tnap was performed on immunomagnetically purified germ cell and somatic cell fractions from 10.5, 13.5 and 14.5 dpc embryos. Each line represents an individually sequenced clone. Numbers indicate the frequency of each observed clone. White circles indicate unmethylated CpG sites; black circles indicate methylated CpG sites.

 

Figure 4
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  		Fig. 4. The postmigratory germ cell marker GCNA1 is precociously expressed in premigratory germ cells in Dnmt1-deficient embryos.

Embryos were immunostained for SSEA1 with TG-1 (red) and for GCNA1 (black). (A) TG-1 and GCNA1 immunohistochemistry of a 12.5 dpc embryo, demonstrating that both markers specifically recognize germ cells. The inset shows that GCNA1 reactivity at this time is normally restricted to the TG-1-positive population. Arrow and arrowhead indicate GCNA1-expressing and non-expressing germ cells, respectively. (B) 8.5 dpc Dnmt1c/c yolk sac with a TG-1 and GCNA1-expressing cell. The inset shows a higher magnification view of the doubly stained cell. (C) Numerous TG-1- and GCNA1-positive cells in a 9.5 dpc Dnmt1c/c embryo.

 

Figure 5
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  		Fig. 5. The postmigratory germ cell marker GCNA1 is ectopically expressed in somatic cells of Dnmt1-deficient embryos. All embryos were immunostained for GCNA1 expression. (A) 9.5 dpc Dnmt1+/n embryo. (B) Enlarged view of the same embryo. (C) 9.5 dpc Dnmt1n/n embryo (n=4/4). (D) Enlarged view of embryo in C, demonstrating scattered GCNA1 expression throughout the embryo. (E) 9.5 dpc Dnmt1+/c embryo. (F) 9.5 dpc Dnmt1c/c embryo (n=5/5). (G) Enlarged view of embryo in F. (H) 8.5 dpc Dnmt+/c embryo. Micrographs in A,C,E,F are at the same magnification. Ectopic GCNA1 expression was not detected in any of 11 control littermate embryos stained in parallel (not shown).

 

Figure 6
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  		Fig. 6. Postmigratory germ cell-specific genes are prematurely expressed in Dnmt1-deficient embryos. RT-PCR gene expression analysis for Dazl, Mvh, Mageb4 and Scp3, and was performed on 9.5 dpc wild-type, heterozygous and mutant Dnmt1n and Dnmt1c embryos. Hprt amplification was used as a loading control.

 

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© The Company of Biologists Ltd 2006