The Drosophila Su(var)3-7 gene is required for oogenesis and female fertility, genetically interacts with piwi and aubergine, but impacts only weakly transposon silencing

Abstract

Heterochromatin is made of repetitive sequences, mainly transposable elements (TEs), the regulation of which is critical for genome stability. We have analyzed the role of the heterochromatin-associated Su(var)3-7 protein in Drosophila ovaries. We present evidences that Su(var)3-7 is required for correct oogenesis and female fertility. It accumulates in heterochromatic domains of ovarian germline and somatic cells nuclei, where it co-localizes with HP1. Homozygous mutant females display ovaries with frequent degenerating egg-chambers. Absence of Su(var)3-7 in embryos leads to defects in meiosis and first mitotic divisions due to chromatin fragmentation or chromosome loss, showing that Su(var)3-7 is required for genome integrity. Females homozygous for Su(var)3-7 mutations strongly impair repression of P-transposable element induced gonadal dysgenesis but have minor effects on other TEs. Su(var)3-7 mutations reduce piRNA cluster transcription and slightly impact ovarian piRNA production. However, this modest piRNA reduction does not correlate with transposon de-silencing, suggesting that the moderate effect of Su(var)3-7 on some TE repression is not linked to piRNA production. Strikingly, Su(var)3-7 genetically interacts with the piwi and aubergine genes, key components of the piRNA pathway, by strongly impacting female fertility without impairing transposon silencing. These results lead us to propose that the interaction between Su(var)3-7 and piwi or aubergine controls important developmental processes independently of transposon silencing.

Figure 1. Su(var)3–7 is expressed in somatic and germline cells of wild type ovaries and colocalizes with HP1.

(A–C) Confocal images of wild type ovary stained with anti-Su(var)3–7 (green) and DAPI (blue) for DNA visualisation. Same procedure on Su(var)3–7^R2a8 homozygous ovaries shows complete absence of Su(var)3–7 staining (not shown). (D–F) Focus on somatic follicular cells and (G–I) germline cells co-stained with anti Su(var)3–7 (green) and anti-HP1 (red) antibodies. Arrows indicate the germinal vesicle with the karyosome. (J–M) Polytene chromosomes from otu¹¹ pseudonurse cells labelled with anti-Su(var)3–7 (green) and DAPI (blue). otu mutation causes polytenization of nurse cells chromosomes allowing mapping of chromosome-associated proteins . Su(var)3–7 binds centromeric heterochromatin (bracket) and several euchromatic sites scattered along the chromosome arms (arrowheads).

Figure 2. Su(var)3–7 is required for oogenesis, embryogenesis and female fertility.

(A) Fertility test of control (w¹¹¹⁸) and Su(var)3–7^R2a8 and Su(var)3–7⁹ homozygous mutant females. Bars represent the rate of laid eggs per female and the viability at pupal and adult stages. Error bars indicate the standard deviation, n = 40. (B) DAPI staining of a 3 days old Su(var)3–7^R2a8 mutant ovary. Arrows indicate degenerated egg chambers. (C) Confocal pictures of stage 5 egg chambers labelled with anti-H3K14ac antibody (green), DNA was visualized by DAPI (blue) staining. The round-shaped oocyte nucleus (karyosome) observed in the control (w¹¹¹⁸, left panel) is altered in Su(var)3–7 mutant ovary (right panel). (D) Phase-contrast images of mature eggs produced by control (w¹¹¹⁸) and Su(var)3–7^R2a8 mutant females. (E) Su(var)3–7 loss-of-function causes meiosis defects and embryonic development arrest. (a–e) Confocal images of (a) control (w¹¹¹⁸) and (b–e) Su(var)3–7^R2a8 mutant embryos stained with an anti-H3S10P (green) used as mitotic marker and anti-core histone proteins (red) antibodies. Embryos were examined 60′ to 120′ AED to ensure that control embryos have reach and exceeded mitotic cycle 6. (a) Mitotic cycle 7/8 control embryo. The nuclei are uniformly distributed within the embryo and the three female polar bodies are assembled into a single rosette. (b) Mutant embryo arrested at mitotic cycle 1. The rosette is misassembled and fragmented. (c) Mutant embryo arrested at mitotic cycle 3. The nuclei remain localized in the anterior part of the embryo and some nuclei contain a single set of chromosomes (1 n) suggesting cases of haploid mitotic cycles. (d) Mutant embryo arrested at mitotic cycle 4. The nuclei divided asynchronously. (e) Mitotic cycle 6 mutant embryo. Arrowheads point damaged mitotic nuclei. R, rosette.

Figure 3. Su(var)3–7 has a weak impact on transposon silencing, but regulates piRNA clusters transcription.

(A) Quantitative RT-PCR analysis on 22 retrotransposons in Su(var)3–7^R2a8 homozygous mutant ovaries (grey bars) and female carcasses (black bars). Histograms represent the fold changes in RNA levels relative to Su(var)3–7^R2a8/TM6 siblings; error bars indicate the standard deviation of triplicate samples (n = 3). Differences in the fold changes were tested by a Welch t-test (* : p<0,05; ** : p<0,01). (B) Quantitative RT-PCR analysis of cluster 2 and flamenco from control (w¹¹¹⁸) and Su(var)3–7^R2a8 heterozygote and homozygote mutant ovaries. Shown are the fold changes in RNA levels relative to the control (n = 3; * : p<0,05). The position of the primer sets used for qRT-PCR are indicated by bars named 1, 2 and 3 along the map above. Coordinates of the clusters along the X chromosome are indicated in Mb. Boxes indicate protein coding genes (blue) and transposon fragments in sense (black) and antisense (red) orientation. (C) Quantitative RT-PCR analysis of cluster1 from control (w¹¹¹⁸) and Su(var)3–7^R2a8 heterozygote and homozygote mutants. Shown are the fold changes in RNA levels relative to the control (n = 3; * : p<0,05). A map of the cluster1/42AB locus with position of the qPCR primer sets 4 and 5 is shown above. (D) Histograms show the log2 fold ratios of normalized ovarian piRNAs mapping antisense to transposons (left) and uniquely mapping piRNAs (sense plus antisense) over piRNA clusters (right) between homozygous and heterozygous Su(var)3–7 mutants. Up to 1 mismatch was allowed between reads and transposon sequences.

Figure 4. Su(var)3–7 does not interact with piwi and aubergine for transposon silencing in ovaries.

Quantitative RT-PCR analysis on the indicated transposons in (A) piwi²/+; Su(var)3–7^R2a8 and (B) aub^QC42/+; Su(var)3–7^R2a8 ovaries. Bars represent the fold changes in RNA levels relative to Su(var)3–7^R2a8 siblings (n = 3; ** : p<0,01).