PMC

Phenotypic description of mutants of Class II. (A−F) Mutant germline clones labeled by the lack of GFP (green). (A, B) Arrested pecan germline clones (arrows), in which the oocyte is not correctly polarized: Orb (red) (A) and centrosomes (red) (B) remain at the anterior of the oocyte. (C) macadamia germline clones stained for γ-tubulin (red). Centrosomes (arrow) remain at the anterior of the oocyte. macadamia germline clones rapidly degenerate. (D) Flies with somatic clones mutant for macadamia, often present short bristles (inset). (E) The development of germline clones mutant for noix is delayed (arrows). Orb protein (red) can be found at the anterior of the oocyte, migrating (arrow) to the posterior and in some cases at the posterior of the oocyte. (F) The development of germline clones mutant for nut is delayed (arrow). Orb (red) migration to the posterior is mainly delayed (arrow) in mutant germline clones.

Phenotypic description of the mutants of Class VI. (A−B) Mutant follicular clones labeled by the lack of GFP (green). (A, A′) Ovariole stained for the Orb protein (red). The oocyte (arrow) is mislocalized to one side of the egg chamber, when all follicle cells are homozygous mutant for pistachio. The two white bars represent the anterior-posterior axis as determined by the stalk cells. (B) pistachio homozygous mutant follicle cells (white dotted lines). (B′) DE-cadherin channel is shown on its own. DE-cadherin is mislocalized in the cytoplasm and basal side of follicle cells mutant for pistachio (arrowhead), instead of being restricted to the apical cortex.

Crossing scheme for isolating mutants affecting early oogenesis on 2L. An asterisk indicates the EMS-treated chromosome. Males carrying an FRT40A chromosome were mutagenized with EMS and crossed in mass to females carrying the FLP () and a lethal w+ mutation balanced over CyO. Each mutation is recovered over a balancer chromosome. In the progeny, males were crossed individually to five females to generate clones with the FLP/FRT system on the left arm of the chromosome 2. Larvae were heat-shocked at 37° for 2 hr on 3 consecutive days. In the progeny, ovaries of five females were dissected for each cross. Interesting mutations were recovered through the brothers, which were backcrossed to the same lethal w+ mutation balanced over CyO.

Phenotypic description of the mutants of Classes VII, VIII. (A−D) Mutant germline clones labeled by the lack of GFP (green). (A) cacahuète mutant germline clone stained for actin (red) and DAPI (blue). Germline clones contain 15 nurse cells and one tiny oocyte (arrow). Mutant egg chambers have thus the shape of lozenge. (B−D) coconut mutant germline clones stop their development around stages 4-5 (arrows). (B, C) Orb protein (red) is correctly restricted to one cell, but forms a round sphere and never makes a tight crescent along the posterior cortex (arrows). (D) In some cases, Orb (red) and the oocyte are mislocalized in the egg chamber (arrow).

Phenotypic description of mutants of Classes IV and V. (A−D) Follicular and germline clones labeled by the lack of GFP (green). (A, B) All follicle cells are mutant for gevuinanut. Germline cells are wild type (green) and stained for Orb (red), which labels the oocyte. Mutant follicle cells overproliferate (A) and trigger encapsulation defects. In some cases, follicular tumor invades in between germline cells (B). (C, D) hazelnut mutant germline clones are made of only eight cells. (C) Synaptonemal complex (red) is restricted to one cell (arrow), suggesting that the oocyte is correctly determined. (D) The oocyte is connected to the nurse cells by only three ring canals (arrows) as shown by actin staining (red).

Overview of the screen. (A−C) Ovarioles observed by Nomarski microscopy. (A) Wild-type ovariole. O, oocyte (B) Mosaic ovariole that contains some arrested egg chambers (black arrow). The mutant egg chamber is smaller than the younger one that precedes it. (C) Egg chamber in which the oocyte is absent and replaced by a nurse cell. In this case, the egg chamber takes the characteristic shape of a lozenge (black arrows). (D−E) Mutant germline clones labeled by the lack of GFP (green). Their development is stopped at stage 2. (D) Orb protein (red) remains at the anterior of the oocyte (arrow). (E) Centrosomes (red), stained with an anti-γ-tubulin antibody, remain at the anterior of the oocyte (arrow).

Phenotypic description of the mutants of Class III and mutants of the insulin pathway. (A−F) Mutant germline clones labeled by the lack of GFP (green). (A, B) Class III mutant germline clones stop their development very early between stages 2 and 3. (A) Orb protein (red) is correctly localized at the posterior of the oocyte (arrow). (B) Centrosomes (red) are also at the posterior of the oocyte (arrow). (C) Simplified scheme of the Drosophila insulin pathway. (D) trans-heterozygous flies obtained during complementation tests are reduced in size. m1 and m2 are any two different alleles of these 19 mutants. (E) p60 mutant germline clones. (F) dp110 mutant germline clones (E, F) Orb protein (red) is correctly localized at the posterior of the oocyte (arrows) as in wild type in both p60 and dp110 mutant germline clones.

Drosophila AATF/Che-1 is not involved in meiotic DSB repair dependent on Mei-41 and plays a moderate antiapoptotic role in polar cells. (A) Mosaic ovariole dissected from hsFLP; AATF/Che-1^21.3 FRT40A/nlsGFP-FRT40A flies were fixed and stained for γ-H2AV to detect DSB foci (red) and DAPI (blue). Mutant chambers are distinguished by the absence of nls-GFP (green). Magnifications correspond to: a clonal (1) and a WT (2) chamber in region 2 of the germarium; (3) a clonal chamber in region 3 of the germarium. (B) Ovarioles from the same flies as in A also mutated for mei-41^D3 were dissected, fixed, and stained for Orb (red) and for DAPI (blue). Mutant chambers are distinguished by the absence of NLS-GFP (green). Magnification corresponds to the indicated mutated chamber (white arrow) where Orb remains to the anterior of the oocyte. (C) Ovarioles from flies overexpressing CG11188-GFP (green) in polar cells under the control of unpaired (upd-Gal4;UASp::CG11188-GFP) were dissected, fixed and stained for Fas-III to detect polar cells (red) and DAPI (blue). (D) The percentage of egg chambers with more than two polar cells was calculated in ovarioles from flies overexpressing CG11188 (UAS::AATF) or the caspase inhibitor p35 (UAS::p35).

Drosophila early oogenesis. Each ovariole is made of a chain of progressively more mature egg chambers toward the posterior (P). An egg chamber comprises 16 germline cells surrounded by a monolayer of follicle cells. The egg chambers are produced at the anterior (A) of the ovariole in the germarium, which is divided into four morphological regions along the anterior-posterior axis. The germline stem cells reside at the anterior tip of the germarium (left) and divide to produce cystoblasts, which divide four more times in region 1 to produce 16 cell germline cysts that are connected by ring canals. The stem cells and cystoblasts contain a spectrosome (red circles), which develops into a branched structure called the fusome, which orients each division of the cyst. In late region 2a, the synaptonemal complex (red lines), which is a marker of meiosis, is restricted to the two cells with four ring canals (pro-oocytes, yellow). By region 2b, the oocyte has been selected and is the only cell to remain in meiosis. In region 2a, cytoplasmic proteins, mRNAs and mitochondria (green), and the centrosomes (blue circles) progressively accumulate at the anterior of the oocyte. The follicle cells (gray) also start to migrate and surround the germline cells. As the cyst moves down to region 3, the oocyte adheres strongly to the posterior follicle cells and repolarizes along its anterior-posterior axis, with the microtubule minus-ends and specific cytoplasmic components now localized at the posterior cortex (adapted from ).

CG11188 corresponds to Drosophila AATF/Che-1, a nucleolar protein involved in oogenesis. (A) Schematic representation of CG11188 (red arrow) genomic region and genes located nearby (gray arrows) in 2L chromosome. The deficiency leading to lethality when crossed with mutated alleles of CG11188 is shown by a red line. (B) Schematic representation of CG11188 gene. Black arrow shows the sense of transcription. White boxes correspond to 3′UTR and 5′UTR. Gray box corresponds to the coding sequence. CG11188^29.3 allele codes for a truncated protein (Q235stop) and available transposon insertions are shown. (C) Drosophila AATF/Che-1 conserved protein domains are depicted: acidic domains are in light pink; leucine zipper is in red; nuclear localization signals (NLS) are in gray; putative serine (S) phosphorylated by ataxia telengiectasia (ATM) or by checkpoint kinase 2 (Chk2) are indicated by a yellow or an orange star, respectively. (D) Mosaic ovarioles dissected from hsFLP; AATF/Che-1^21.3 FRT40A flies were fixed and stained for Orb (red) and DAPI (blue). Mutant egg chambers can be easily detected by the absence of NLS-GFP. Magnification shows Orb mislocalized to the anterior of the oocyte in a mutant arrested chamber indicated by a white arrow. (E−F) Same flies as in D overexpressing CG11188-GFP were dissected. Ovarioles were fixed and stained for Orb (red) and for DAPI (blue). (E) WT egg chambers express nls-GFP and CG11188-GFP. (F) Mutant chambers only express CG11188-GFP. Magnifications correspond to: 1, GFP signal in a nurse cell. 2, GFP and Orb localization in the oocyte. CG11188-GFP rescues Orb localization and egg chamber growth. (G) Same flies as in E−F were stained for the nucleolar protein Wicked. Magnifications correspond to nurse cells in which nucleolar GFP and wicked signals can be discriminated from nuclear DAPI signal. CG11188-GFP and Wicked perfectly colocalized in the germline.