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X-chromosome silencing in the germline of C. elegans

¹ Biology Department, Emory University, Atlanta, GA 30322, USA
² Lawrence Berkeley National Laboratory, One Cyclotron Road MS-84-171 and Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA
³ Department of Genetics, Washington University School of Medicine, St. Louis, MO 63110, USA
⁴ Departments of Developmental Biology and Genetics, Stanford University School of Medicine, Stanford, CA 94305
⁵ Department of Genetics, Yale University School of Medicine, New Haven, CN 06520, USA

View larger version (109K): [in a new window]   Fig. 1. Transgene expression, histone modification and morphology in germ cells. (A,B) Differential interference contrast (top panels) and GFP fluorescence (bottom panels) microscopy of hermaphrodite ovaries from transgenic line PD7271, which carries an extrachromosomal, multi-copy array of a let-858:gfp reporter transgene, pBK48.1 (A), and line KW1336, which carries an extrachromosomal ‘complex’ array with pBK48.1 (B). Brackets indicate germ cell nuclei in one hermaphrodite ovary arm. The arrowheads in the lower panels illustrate GFP fluorescence in intestinal cell nuclei. The lower panel in A represents a longer exposure than that shown in B to demonstrate a complete lack of detectable nuclear GFP expression in germ cells in this transgenic line (brackets; the few fluorescent nuclei within the brackets are from somatic components of the gonad). (C,D) Paraformaldehyde-fixed oocytes from PD7271 (C) and KW1336 (D) hermaphrodites were stained with -H3 dimethyl-K4 antibody (green) and counterstained with DAPI (red), arrowheads indicate transgene arrays. (E,F) Paraformaldehyde-fixed pachytene stage nuclei from transgenic PD7271 hermaphrodites (E) and N2 males (F) were stained with DAPI and examined by deconvolution fluorescence microscopy. Au, autosomes; X, X chromosome; Tgn, transgene, Scale bars: 5 µm in C-F.

View larger version (98K): [in a new window]   Fig. 2. Histone H3 lysine 4 methylation in male germ cells. Gonads from transgenic PD7271 males were fixed in paraformaldehyde and stained with the -H3 dimethyl K4 antibody (green) and counterstained with DAPI (red). A1-A3 illustrate each signal separately and after merging; (B-F) the merged signals only. Arrows in all panels point to the X chromosome in each nucleus. (A) Pachytene stage; (B-D) progressive stages of primary spermatocytes; (E) condensing secondary spermatocytes; (F) spermatids. Arrowheads in B,C indicate transgenes. Transgenes were identified in all experiments by z-axis optical scanning through nuclei to rule out confusion with chromosome ends. Scale bars: 5 µm.

View larger version (108K): [in a new window]   Fig. 3. Histone H3 lysine 9 methylation in male germ cells. Methanol/acetone fixed gonads from N2 (A-C) and PD7271 transgenic males (D) were stained with -H3 methyl-K9 alone (green) and counter stained with DAPI (red; A,B), or stained with both -H3 methyl-K9 (red) and -phosphoacetyl H3 (green; C,D). The curved arrow in A shows the direction of meiotic progression. Arrows in B-D indicate a chromosome enriched in H3 modified by methyl-K9 (green in B, red in C), and under-modified by the phosphoacetyl epitope (green in C). Arrowheads in D indicate inactive transgenes. Scale bars: 5 µm.

View larger version (25K): [in a new window]   Fig. 4. Reduced expression of X-linked oocyte-specific genes. Relative abundance of autosomal- and X-linked oocyte-enriched mRNAs (A) and autosomal- and X-linked somatic mRNAs (B) were determined for each chromosome as described in Materials and Methods, and plotted with standard errors. The number of genes used in the calculation for each chromosome is listed in parentheses.

View larger version (117K): [in a new window]   Fig. 5. Decreased activating histone modifications on one hermaphrodite chromosome pair. (A-E) PD7271 (A1-A3) or N2 (B-G) hermaphrodite ovaries were fixed with paraformaldehyde and stained with antibodies recognizing histone H3 methyl-K4 (A), H3 acetyl-K9, –14 (B), H3 phospho-S10 (C), histone H4 acetyl-K8 (D) and H4 acetyl-K16 (E). In A-E, antibody staining is false-colored green and DAPI counterstain is false-colored red. Arrowheads in A1-A3 point to transgenes identified as in Fig. 2. Arrows in A3-E point to the under-labeled chromosome; arrowhead in C points out a concentration of the H3 phospho-S10 modification that is otherwise absent from this chromosome. (F,G) N2 hermaphrodite germ cells were simultaneously stained with -H3 dimethyl-K4 (F; green) and mAb H5 (G; green), and counterstained with DAPI (F,G; red). Arrowheads indicate chromosomes under-stained by both antibodies. (H,I) -H3 dimethyl-K4 staining of pachytene nuclei in strain carrying autosomal duplication sDp1 (H) and the activated transgene (KW1336; I). The free duplication and the transgene are circled in H,I, respectively.

View larger version (31K): [in a new window]   Fig. 6. The under-modified chromosome is the X chromosome. (A) Hermaphrodite animals homozygous for an X:autosome reciprocal translocation [mnT10(X:V)] were stained with -H3 dimethyl-K4 (green) and counterstained with DAPI (red). Arrows point to chromosomes in a nucleus that are each half-labeled by the antibody. Scale bar: 5 µm. (B) Hermaphrodite germ cells were probed simultaneously by antibody -H4 acetyl-K12; (‘AcHis4’) and by fluorescent in situ hybridization (FISH) using probes to both ends of the X chromosome. Scale bar: 5 µm in A.

View larger version (77K): [in a new window]   Fig. 7. Histone H3 lysine 9 methylation in hermaphrodites. N2 (Bristol) (A,C) and transgenic strain PD7271 (B) hermaphrodite ovaries were fixed in methanol/acetone and stained with anti-H3 dimethyl-K9 antibody (green) and counter-stained with DAPI (red). The progression of the nuclei through meiotic prophase I is indicated by labels and arrows in A. Arrowheads in B indicate the major staining foci seen in early pachytene nuclei in all strains; the arrows show additional nuclear foci unique to the PD7271 strain, which correspond to the transgene arrays. (C) An enlargement of the pachytene-to-diplotene transition region from A. Scale bars: 30 µm in A; 5 µm in B,C.

View larger version (45K): [in a new window]   Fig. 8. Post-pachytene histone modification in hermaphrodites. Hermaphrodite ovaries were fixed in paraformaldehyde, stained with -H3 dimethyl-K4 (green) and counter-stained with DAPI (red). (A,B) Transgenic strain PD7271 with inactive transgene. (A) Nuclei progressing into diplotene, with the direction of progression indicated by the long arrow; arrowhead points to a transgene. (B) An oocyte in diakinesis with six attached homolog pairs and transgene indicated (arrow). (C) Oocyte in diakinesis from sDp1 strain, with duplication indicated (arrow). Scale bars: 5 µm.

View larger version (66K): [in a new window]   Fig. 9. In situ analysis of X-linked oocyte gene expression. (A-C) Antisense probes were generated for several X-linked genes that exhibited an oocyte-enriched profile by microarray analysis (Reinke et al., 2000). The probes were then hybridized to wild-type ovaries to determine where the corresponding mRNAs begin to accumulate. In each panel, the mitotic (distal) region is towards the left and maturing oocytes in the most proximal region of each ovary are towards the right. The arrowheads indicate the boundaries of the pachytene region of each ovary. DAPI and lacZ are shown separately in A; these signals have been merged in B,C. (D,E) Distribution of transcription-competent RNA POL II. A methanol/acetone fixed hermaphrodite gonad was stained with DAPI (D) and also with monoclonal antibody H14 (E).

View larger version (106K): [in a new window]   Fig. 10. Evolutionary conservation of meiotic silencing. Gonads from divergent species of nematodes were fixed in paraformaldehyde and stained with -H3 dimethyl-K4. Hermaphrodite species tested include Oscheius sp. (A), Pristionchus pacificus (B) and C. briggsae and Oschieus myriophila (not shown). Gonochoristic species include C. remanei (C), Caenorhabditis sp. (D) and Mesorhabditis longespicula (not shown). Arrows in each panel indicate the ‘under-modified’ chromosome.