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First published online December 12, 2006
doi: 10.1242/10.1242/dev.02698

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Histone methylation is required for oogenesis in Drosophila

Emily Clough, Woongjoon Moon^*, Shengxian Wang, Kathleen Smith and Tulle Hazelrigg

Department of Biological Sciences, Columbia University, New York, NY 10027, USA.

View larger version (18K): [in this window] [in a new window]   Fig. 1. The Drosophila eggless (egg) gene encodes a SET domain protein similar to human/mouse SETDB1/ESET. (A) Northern blot of ovary poly(A+) RNA hybridized with a probe made from the 5' end of the egg gene. A 3' probe labels the same 3.9 kb band. (B) Western blot of ovary extracts, labeled with an antibody to Egg. Two major protein bands, 170 and 140 kDa, are detected in wild-type ovaries. These proteins are strongly reduced in egg235 ovaries, and replaced with a smaller 59 kDa protein. (C) Proposed structure of the egg transcript. This structure is based on the assembly of the complete sequences of three overlapping ovary cDNAs and two embryonic cDNAs. (D)The predicted protein structures of Egg and SETDB1, showing the identified protein domains within each protein. (E) The egg235 allele contains two nucleotide substitutions near the 5' end of intron 4 (shown in large bold letters). Failure to splice out this intron introduces a premature stop codon (shown in uppercase letters). (F) The predicted protein product of the egg1473 allele. This allele contains an 856 bp in-frame deletion that removes the entire SET domain. WT, wild type.

View larger version (60K): [in this window] [in a new window]   Fig. 2. Expression of Egg protein during oogenesis. Ovaries labeled with an antibody to Egg. The left panels show Egg alone, and the right panels show Egg (green) and DNA (red). (A) Germarium and stage 2 egg chamber. The regions of the germarium are labeled. In germ stem cells (white arrow), cystoblasts and dividing cystocytes, the protein is both cytoplasmic and nuclear. In 16-cell germline cysts, Egg is concentrated in the germ cell nuclei (white arrowhead). Egg is not detected in somatic cells at the tip of the germarium (magenta arrow), but is present at low levels in prefollicular cells, and follicle cells of stage 1 egg chambers (green arrow). (B) Stage 8 egg chamber. In mid- and late stages of oogenesis, Egg is present throughout the nurse cell nuclei, and localizes to distinct foci associated with the DNA in the oocyte nucleus (white arrow). Egg expression increases in the follicle cells, where the protein is nuclear (green arrow). G, germarium; nc, nurse cell nuclei; s2, stage 2 egg chamber.

View larger version (46K): [in this window] [in a new window]   Fig. 3. Oogenesis arrest in egg mutants. (A) Ovaries labeled for the germ-cell-specific protein Vasa (green) and a DNA dye (red). Regions of the wild-type germarium are indicated in the top panel. egg1473 ovaries consist of germaria that fail to bud off normal egg chambers. These germaria often exhibit no recognizable regions and are filled with germ cells and germline cysts that do not form apparent egg chambers (middle panels). In some cases an incompletely budded single egg chamber lies at the posterior end of the mutant germarium (white arrow; bottom panel). (B) Ovaries labeled with an antibody to Fas III (Fas3, green) and a DNA dye (red). Incomplete encapsulation of germline cysts occurs in egg1473 germaria, and the prefollicular (arrowhead) and follicle cells (arrow) appear reduced in number. WT, wild type.

View larger version (68K): [in this window] [in a new window]   Fig. 4. Patterns of Histone H3 Lysine 9 (H3K9) methylation during oogenesis. Wild-type ovaries labeled with antibodies to dimethyl-H3K9 (H3K9me2) or trimethyl-H3K9 (H3K9me3), and an antibody to Vasa, which labels germ cells, or a DNA dye. Left panels, methylated H3K9 alone; right panels, merged images of H3K9me2 or H3K9me3 (green) and Vasa (red) or DNA (red). (A) H3K9me2 is present in both the germ cells and somatic cells in the germarium and newly budded egg chambers (upper panels). As oogenesis proceeds (stage 4 and 6 egg chambers in bottom panels), the H3K9me2 signal increases in the somatic cells relative to the germ cells. A nurse cell nucleus, germ stem cell nucleus (arrowhead) and follicle cell nuclei (arrows) are indicated. (B) In the germarium (top panels) H3K9me3 is present in both the germ cells and somatic cells, with higher levels apparent in the germ cells. A germ stem cell (arrowhead) and somatic cells (arrow) are indicated. In later stages (stage 4 and 6 egg chambers in bottom panels), H3K9me3 persists in the germ cells but is reduced in somatic cells. The signal is relatively weak in nurse cell nuclei compared with the oocyte nucleus (*). G, germarium; NC, nurse cell nuclei; S2/S4/S6, stage 2/4/6 egg chambers.

View larger version (76K): [in this window] [in a new window]   Fig. 5. egg is required for trimethylation but not dimethylation of H3K9 in germ and somatic cells during oogenesis. Left panels, methylated H3K9 alone; right panels, merged images of H3K9me3 or H3K9me2 (green) and Vasa (red). (A) H3K9me3 is absent or severely reduced in both the germ and somatic cell nuclei of egg1473 germaria. (B) High levels of H3K9me2 are present in both germ and somatic cell nuclei of egg1473 germaria.

View larger version (29K): [in this window] [in a new window]   Fig. 6. Proliferation of somatic cells is reduced in egg1473 germaria. Ovaries were labeled with antibodies to phosphorylated H3S10 (phospho-H3), to detect mitotic cells, and Vasa. (A,B) Left panels, phospho-H3 alone; right panels, merged images of phospho-H3 (green) and Vasa (red). (A) Wild-type germarium and budding egg chamber. The arrowhead indicates a group of germ cells in mitosis and the arrow indicates a mitotic somatic cell. (B) egg1473 germarium. In this example, a single germ cell is undergoing mitosis. (C,D) Histograms showing the number of wild-type and egg1473 germaria with the indicated number of mitoses in germ or somatic cells. (C) Germ cell mitosis. See text for an explanation of the germ cell categories. (D) Somatic cell mitosis. WT, wild type.

View larger version (72K): [in this window] [in a new window]   Fig. 7. Abundant apoptotic cell death occurs in egg1473 germaria. Ovaries were labeled with antibodies to cleaved Caspase-3, a marker of apoptotic cell death, and Vasa. Left panels, cleaved Caspase-3 alone; right panels, merged images of cleaved Caspase-3 (green) and Vasa (red). (A) Wild-type germarium. No apoptotic cells are present in this example, and were only rarely seen in our sample. (B) egg1473 germarium. Several apoptotic somatic cells are present in this example (arrows). See text for a full description. WT, wild type.

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