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First published online November 3, 2003
doi: 10.1242/10.1242/dev.00817

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Cct1, a phosphatidylcholine biosynthesis enzyme, is required for Drosophila oogenesis and ovarian morphogenesis

Howard Hughes Medical Institute, Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA

View larger version (55K): [in a new window]   Fig. 1. Map of Cct1 mutations and alignment of predicted CCT protein sequences. (A) Deletions were generated in Cct1 and Cct2 by mobilization of the P-element BN81 (indicated by green arrowhead), which is inserted at nucleotide 57 in the 5'UTR of Cct1 (AY051993). Excisions are shown as green bars. The genomic rescue construct is shown as a blue bar. (B) Predicted protein sequences of Drosophila melanogaster Cct1 (NP_728628), Drosophila melanogaster Cct2 (NP_647622), Saccharomyces cerevisiae CCT (NP_011718) and Rattus norvegicus CCT (NP_511177) were aligned using ClustalW. Identical residues are indicated in bold and dark gray; similar residues are indicated in light gray.

View larger version (59K): [in a new window]   Fig. 2. Expression pattern of Cct1 in wild-type ovaries. (A) X-gal staining showing BN81 enhancer trap expression in the anterior follicle cells from stage 7 to stage 10. At stage 10, expression is seen in the border cells, nurse cell associated follicle cells and centripetally migrating follicle cells. (B-D) Cct1 expression detected by whole-mount in situ hybridization. (B) Cct1 is first expressed in the follicle cells in region 2B of the germarium. (C) During mid-oogenesis, Cct1 expression is detected in the anterior follicle cells. Expression begins during stage 7 in the follicle cells at the anterior-most end of the egg chamber. At stage 10B, Cct1 expression is seen in the nurse cell associated and centripetally migrating follicle cells. (D) From stages 12-13, Cct1 is expressed in the dorsoanterior follicle cells and the posterior follicle cells. Cct1 expression can also be detected in the germline cells at various levels throughout oogenesis.

View larger version (42K): [in a new window]   Fig. 3. Defect in germline stem cell maintenance in Cct1 mutants. (A) Wild-type ovariole labeled with an antibody to Vasa protein, which marks the germline cells (green) and phalloidin to visualize the actin cytoskeleton (red). Ovaries from Cct199 mutants contain ovarioles with only one or two egg chambers. (B,C) Germaria stained with antibodies to Vasa (green) and -Spectrin (red), which marks the spectrosomes and fusomes. (B) Wild-type germaria contain two or three germline cells, marked by the presence of spectrosomes, and seven or eight dividing germline cysts with fusomes. Arrowhead indicates a spectrosome. (C) Cct199 mutant germaria sometimes contain few or no germline cells with spectrosomes. Scale bars: 20 µm.

View larger version (64K): [in a new window]   Fig. 4. Packaging defects and mispositioned oocytes in Cct1 mutant ovaries. (A-E) Egg chambers stained with Orb antibody (red) to label the oocyte, phalloidin (green) to visualize the actin cytoskeleton, and Hoechst (blue) to label the DNA. (A,D,F) Wild-type egg chambers. (C,G) Cct1BN81 mutant egg chambers. (B,E) Cct199 mutant egg chambers. (A,D) Wild-type egg chambers consist of 15 nurse cells and an oocyte positioned at the posterior. (B) Cct1 mutant egg chambers have packaging defects, with multiple oocytes in one egg chamber. (C and E) Egg chambers from Cct1 mutant ovaries contain randomly positioned oocytes. Packaging defect and mispositioned oocyte phenotypes were also seen with other alleles. (F,G) Egg chambers labeled with antibody to Fas3 (green) to mark the polar follicle cells and Encore antibody (red) to label the oocyte. (F) Wild-type ovaries have a pair of polar follicle cells at each end of the egg chamber. (G) Polar follicle cells are present at both ends of mutant egg chambers with mispositioned oocytes. (H) Follicle cell clone mutant for Cct1179 marked by absence of Myc protein expression (green). Asterisk indicates position of oocyte at posterior of egg chamber. (I,J) Egg chambers stained with antibody to Orb protein (red) to mark the oocyte, Hoechst (blue) to label the DNA, and GFP (green) to mark wild-type cells. (I) Egg chambers with complete follicle cell clones mutant for Cct1 (marked by absence of GFP expression in the follicle cell epithelium) showing mispositioned oocyte phenotype. (J) Egg chamber in which both the germline and follicle cell epithelium is mutant for Cct1 with a mispositioned oocyte phenotype.

View larger version (27K): [in a new window]   Fig. 5. Mutations in Cct1 do not affect levels of Arm protein. (A-D) Germaria were stained with Encore antibody (green) to mark the oocyte and Armadillo antibody (red). (A,C) Merge. (B,D) Arm staining only. (A,B) In wild-type germaria, Arm protein becomes transiently enriched in the oocyte and in the anterior and posterior follicle cells. (C,D) In ovaries from Cct1BN81 mutant females with mispositioned oocytes, accumulation of Arm protein is still seen in the oocyte and anterior and posterior follicle cells. Similar results were obtained using an antibody to DE-cadherin.

View larger version (62K): [in a new window]   Fig. 6. Branched ovariole and shortened ovariolar sheath in Cct1 and Drosophila Wnt4 mutant ovaries. Ovaries were stained with Orb antibody (red) to label the oocyte, phalloidin (green) to visualize the actin cytoskeleton and Hoechst (blue) to label the DNA. (A) Cct199 mutant ovary with branched ovarioles. (B) Branched ovarioles in Drosophila Wnt4C1 mutant ovary. (C) Wild-type ovariole showing egg chambers stretched out within ovariolar sheath. (D) Cct199 mutant ovariole with egg chambers curled up within the sheath.

View larger version (74K): [in a new window]   Fig. 7. Mutations in Cct1 affect ovarian morphogenesis. (A-C) Figures adapted from King (King, 1970) depicting stages of ovarian morphogenesis at 2 hours, 24 hours and 36 hours APF. Apical cells are labeled in green, terminal filaments in purple, germline cells and follicle cell precursors in yellow and basal cells in red. (D-G) Pupal ovaries labeled with an antibody to Fas3 (red), which marks the basal cells and phalloidin (green) to visualize the actin cytoskeleton. (D) Wild-type ovary 20-22.5 hours APF with completely separated ovarioles and partially formed basal stalks. (E) Cct199 mutant ovary 20-22.5 hours APF. Apical cells have not migrated fully and basal stalks have not started to form. (F) Wild-type ovary 39-40 hours APF. Basal stalks have formed and become long and thin. (G) Cct199 mutant ovary 39-40 hours APF. Basal stalks have started to form but are incompletely separated. (H) Pupal ovary 20-21 hours APF with positively marked apical cell clones mutant for Cct1179. Arrowheads indicate mutant cells, which are marked by expression of GFP (green). Ovaries are also stained with Fas3 antibody (red) to mark the basal stalks and phalloidin (blue) to visualize the actin cytoskeleton.

View larger version (64K): [in a new window]   Fig. 8. Shortened operculum phenotype and regulation of expression by dpp signaling. (A,B) Chorion phenotypes. (A) Wild-type egg. (B) Egg laid by Cct1124/Cct1299 mutant. Females hypomorphic for mutations in Cct1 lay eggs with shortened opercula. (C,D) In situ hybridizations showing Cct1 expression. (C) Cct1 expression in anterior follicle cells in a wild-type egg chamber. (D) Cct1 expression in a dpp misexpression background. When dpp is ectopically expressed in all of the follicle cells, Cct1 is also expressed in all the follicle cells. (Uneven staining caused by patchy expression of Gal4 driver.) (E) Expression of the enhancer trap BN81 (green) in a follicle cell clone mutant for Mad12, which is marked by the loss of Myc expression (red). (F) Expression of phosphorylated-SMAD (green) in a Cct199 mutant follicle cell clone, indicated by lack of Myc expression (red).

View larger version (37K): [in a new window]   Fig. 9. Regulation of Cct1 expression by Egfr signaling and fused appendage and collapsed eggs produced by Cct1 mutant females. (A-C) In situ hybridization showing Cct1 expression during stages 12 and 13. Gap between Cct1-expressing follicle cells is indicated with brackets. (A) Wild-type egg chamber showing expression in dorsoanterior follicle cells. (B) Cct1 expression in a fs(1)K10 mutant background. (C) Cct1 expression in a background in which there are four copies of grk. (D) Wild-type egg. (E) Fused appendage phenotype in egg laid by a Cct1124 female. (F) Collapsed egg phenotype in egg laid by a Cct116919 female.