Cutoff and aubergine mutations result in retrotransposon upregulation and checkpoint activation in Drosophila

Abstract

Gametogenesis is a highly regulated process in all organisms. In Drosophila, a meiotic checkpoint which monitors double-stranded DNA breaks and involves Drosophila ATR and Chk2 coordinates the meiotic cell cycle with signaling events that establish the axis of the egg and embryo. Checkpoint activity regulates translation of the transforming growth-factor-alpha-like Gurken signaling molecule which induces dorsal cell fates in the follicle cells [1-3]. We found that mutations in the Drosophila gene cutoff (cuff) affect germline cyst development and result in ventralized eggs as a result of reduced Grk protein expression. Surprisingly, cuff mutations lead to a marked increase in the transcript levels of two retrotransposable elements, Het-A and Tart. We found that small interfering RNAs against the roo element are still produced in cuff mutant ovaries. These results indicate that Cuff is involved in the rasiRNA pathway and most likely acts downstream of siRNA biogenesis. The eggshell and egg-laying defects of cuff mutants are suppressed by a mutation in chk2. We also found that mutations in aubergine (aub), another gene implicated in the rasiRNA pathway, are significantly suppressed by the chk2 mutation. Our results indicate that mutants in rasiRNA pathways lead to elevated transposition incidents in the germline, and that this elevation activates a checkpoint that causes a loss of germ cells and a reduction of Gurken protein in the remaining egg chambers.

Fig. 1. Grk expression in cuff mutant egg chambers

grk RNA in situ hybridization (A, C) and Grk antibody staining (B, D). (A, B) Cuff heterozygous females were used as wild type control: In Stage 9 wild type egg chambers, grk transcripts form a tight cap around the oocyte nucleus at the dorsal cortex (A). Grk protein is translated from the localized mRNA, and is also restricted to the dorsal anterior region of the oocyte (B). At this stage of oogenesis, the chromatin of the oocyte nucleus forms a compact, round structure termed the karyosome (B inset). (C, D) cuff ^WM25/KG05951. In Stage 9 cuff mutant egg chambers, grk transcript localization appears mostly normal (C), however, in 10–40% of the egg chambers, Grk protein is undetectable (D). Some 10–20% of the oocyte nuclei at this stage also assume a defective morphology. Often, the DNA seems to localize to the periphery of the nucleus (D inset). In B and D, the oocyte nucleus is marked by dotted lines.

Fig. 2. Germline cyst development in cuff mutants

(A, B) Using an antibody against α-Spectrin (red), we always observed highly branched fusomes (arrow) in wild type germaria in the anterior regions (A). In contrast, in cuff mutant germaria, many of the cysts fail to divide normally and form branched fusomes, instead often retaining a round spectrosome-like fusome (arrowheads) (B). (C, D) Using an antibody against C(3)G (red), we monitored meiotic progression in wild type and cuff mutant backgrounds. In wild type germaria, we consistently observed multiple cysts initiating meiosis (C); while in cuff mutant background, although germ cells are present (as marked by Vas staining in blue), the cysts lack C(3)G staining, indicating an early arrest (D). (E, F) In wild type germaria, developing germline cysts migrate to the posterior of the germaria (E), and subsequently bud off, forming an egg chamber. In cuff mutants, this process is disrupted. In corresponding regions marked by the bracket (region 2b and 3 as marked by FasIII staining in red), there are few if any normal looking cysts in cuff mutant germaria (F). (G, H) The defects in cyst development of cuff mutants is partially suppressed by a mutation in chk2. In chk2 cuff double mutants, we observed many highly branched fusomes (arrows in G), and females also lay many more eggs than the corresponding cuff mutant. All flies were growing under optimal nutritional conditions. Cuff heterozygous females were used as wild type control, while cuff ^WM25/KG0595 and chk2 cuff ^WM25females were used for phenotypic analysis.

Fig. 3. Characterization of cuff

A. Sequencing of cuff mutants. cuff^WM25, cuff ^RN48, and cuff^WL52 were induced by EMS [6]. cuff^KG05951 was derived by the BDGP Gene Disruption Project. B–C. In order to analyze Cuff localization, we expressed HA-tagged Cuff protein under the control of nanos-Gal4 VP16. When expressed in this manner, Cuff shows a prominent peri-nuclear pattern, co-localizing with Vas protein in the germarium (B) and early stage egg chambers (C). D. In cuff mutants, Vas protein electorophoretic migration is slower than in wild type. A very similar phenotype was observed in spnB mutants (Ghabrial and Schupbach, 1999). The Vas migration defect is suppressed by a chk2 mutation, but not by a mutation in the Drosophila ATR homolog mei41. E. Mutations in cuff lead to a strong up-regulation of Het-A and Tart retrotransposable elements in the Drosophila germline. In cuff mutants, Het-A displays a 800-fold increase in transcript levels, while Tart is up-regulated approximately 20 fold. A deregulation of these elements can also be detected in the germline of spnE and aub mutants (see also [31]). In spnE ovaries both Het-A and Tart are up-regulated approximately 10 fold, while in aub only Het-A levels are significantly increased. F. rasiRNAs production is not affected in cuff mutant ovaries. Similar levels of the roo rasiRNA can be detected in ovaries heterozygous (lane 1) and homozygous (lane 2) mutant for cuff. In contrast, roo rasiRNAs are expressed in ovaries heterozygous mutant for aub (lane 3), but are absent from the homozygous mutant.