Figure 1.
Follicle cells maintain monolayer during Drosophila oogenesis. (A–C) Surface view of a wild-type ovariole containing different stages of egg chambers. The ovariole is subjected to immunofluorescence analysis with antibodies against CTPS (green) and Hts (red, labelling cell membranes). DNA is labelled by Hoechst 33342 (blue). (A) Cytoophidia are distributed almost at different stages of each follicle cell. (B) The boundaies of follicle epithelia are displayed by a single projection of Hts staining. (C) CTPS staining shows the distribution of cytoophidia on the surface of egg chambers. (D–F) Side view of the same ovariole in (A–C). (D) Monolayer follicle cells envelop germline cells. (E) A single projection of Hts staining shows the monolayer structure of follicle epithelia. (F) CTPS staining shows the distribution of cytoophidia in follicle cells and germline cells. Scale bars, 50 μm.
Figure 1.
Follicle cells maintain monolayer during Drosophila oogenesis. (A–C) Surface view of a wild-type ovariole containing different stages of egg chambers. The ovariole is subjected to immunofluorescence analysis with antibodies against CTPS (green) and Hts (red, labelling cell membranes). DNA is labelled by Hoechst 33342 (blue). (A) Cytoophidia are distributed almost at different stages of each follicle cell. (B) The boundaies of follicle epithelia are displayed by a single projection of Hts staining. (C) CTPS staining shows the distribution of cytoophidia on the surface of egg chambers. (D–F) Side view of the same ovariole in (A–C). (D) Monolayer follicle cells envelop germline cells. (E) A single projection of Hts staining shows the monolayer structure of follicle epithelia. (F) CTPS staining shows the distribution of cytoophidia in follicle cells and germline cells. Scale bars, 50 μm.
Figure 2.
CTPS-mCh ovaries have same morphology as wild-type ovaries. (A–C) Surface view of a stage 8 CTPS-mCh egg chamber. The CTPS signal (green) shown is obtained using mCherry-tagged CTPS. Hts (red) staining marks cell membranes and Hoechst (blue) for DNA. (A) mCherry moiety doesn’t affect CTPS assembly. (B) Merged panel of Hts and CTPS to display the cytoophidia location. (C) Single panel of the nucleus. (D) Lateral view of a stage 8 CTPS-mCh egg chamber. (E) Merged panel of Hts and CTPS to show the monolayer structure of follicle epithelia as well as cytoophidia distribution. (F) Single panel of nucleus to stress the single-layer follicle epithelia.
Figure 2.
CTPS-mCh ovaries have same morphology as wild-type ovaries. (A–C) Surface view of a stage 8 CTPS-mCh egg chamber. The CTPS signal (green) shown is obtained using mCherry-tagged CTPS. Hts (red) staining marks cell membranes and Hoechst (blue) for DNA. (A) mCherry moiety doesn’t affect CTPS assembly. (B) Merged panel of Hts and CTPS to display the cytoophidia location. (C) Single panel of the nucleus. (D) Lateral view of a stage 8 CTPS-mCh egg chamber. (E) Merged panel of Hts and CTPS to show the monolayer structure of follicle epithelia as well as cytoophidia distribution. (F) Single panel of nucleus to stress the single-layer follicle epithelia.
Figure 3.
Follicle cell ingression in CTPSH355A mutant in early and middle stages of oogenesis. (A) Cross section of a CTPSH355A stage 6 egg chamber. The egg chamber is labeled with CTPS (green), Armadillo (red) for apical complex in follicle cells and Hoechst 33342 (blue) for DNA. The white rectangle emphasizes the ingression of a follicle cell. (B–E) Close-up images of the ingressive follicle cell, indicated by the yellow arrow. No cytoophidium is formed due to the H355A mutation in CTPS. (F) Cross section of a CTPSH355A egg chamber at stage 9. The white rectangle emphasizes the ingression of a follicle cell. (G–J) Close-up images of the ingressive follicle cell, indicated by the yellow arrow. Scale bars, 20 μm.
Figure 3.
Follicle cell ingression in CTPSH355A mutant in early and middle stages of oogenesis. (A) Cross section of a CTPSH355A stage 6 egg chamber. The egg chamber is labeled with CTPS (green), Armadillo (red) for apical complex in follicle cells and Hoechst 33342 (blue) for DNA. The white rectangle emphasizes the ingression of a follicle cell. (B–E) Close-up images of the ingressive follicle cell, indicated by the yellow arrow. No cytoophidium is formed due to the H355A mutation in CTPS. (F) Cross section of a CTPSH355A egg chamber at stage 9. The white rectangle emphasizes the ingression of a follicle cell. (G–J) Close-up images of the ingressive follicle cell, indicated by the yellow arrow. Scale bars, 20 μm.
Figure 4.
Three dimensional view of follicle cell ingression. (A) Cross section of a three-dimensional CTPSH355A egg chamber. A three-dimensional view of DNA stacked in layers. The interval between each layer is 0.5 μm, and a total of 12 layers are superimposed. The color from red to blue indicates the depth of DNA. (B) Side view of an ingressive nucleus. (C) On the xz plane, with the yellow dotted line marking the ingressive cell. (D) Quantification of the ingression frequency, 20 stage 8 egg chambers were counted per genotype.
Figure 4.
Three dimensional view of follicle cell ingression. (A) Cross section of a three-dimensional CTPSH355A egg chamber. A three-dimensional view of DNA stacked in layers. The interval between each layer is 0.5 μm, and a total of 12 layers are superimposed. The color from red to blue indicates the depth of DNA. (B) Side view of an ingressive nucleus. (C) On the xz plane, with the yellow dotted line marking the ingressive cell. (D) Quantification of the ingression frequency, 20 stage 8 egg chambers were counted per genotype.
Figure 5.
Ingression of follicle cells labeled with different membrane proteins. (A) Lateral view of a stage 8 egg chamber. Hts (red) labels the hulitaishao protein mainly presents at the lateral of follicle cell membranes. mCherry-tagged CTPS is shown green, and Hoechst (blue) for DNA. (B) A stage 8 egg chamber of CTPSH355A Drosophila. mCherry-tagged CTPSH355A is diffused. (C) The Hts pattern of an ingressive follicle cell. (D) Yellow arrow pointed out the ingressive cell nuclear. (E) Lateral view of a stage 10A egg chamber. Dlg (red) labels the discs large protein, which presents in the lateral and basal side of follicle cell. CTPS-mCherry cytoophidia can be observed, and Hoechst (blue) for DNA. (F) Lateral view of the CTPSH355A egg chamber at stage 10A. (G) The ingressive cell nuclear is stressed by the yellow arrow. (H) The Dlg pattern of the follicle cell ingression.
Figure 5.
Ingression of follicle cells labeled with different membrane proteins. (A) Lateral view of a stage 8 egg chamber. Hts (red) labels the hulitaishao protein mainly presents at the lateral of follicle cell membranes. mCherry-tagged CTPS is shown green, and Hoechst (blue) for DNA. (B) A stage 8 egg chamber of CTPSH355A Drosophila. mCherry-tagged CTPSH355A is diffused. (C) The Hts pattern of an ingressive follicle cell. (D) Yellow arrow pointed out the ingressive cell nuclear. (E) Lateral view of a stage 10A egg chamber. Dlg (red) labels the discs large protein, which presents in the lateral and basal side of follicle cell. CTPS-mCherry cytoophidia can be observed, and Hoechst (blue) for DNA. (F) Lateral view of the CTPSH355A egg chamber at stage 10A. (G) The ingressive cell nuclear is stressed by the yellow arrow. (H) The Dlg pattern of the follicle cell ingression.
Figure 6.
DCAD2 distribution is disturbed in CTPSH355A follicle epithelia. (A) Cross section of Drosophila egg chamber expressing CTPS labelled by mCherry. The part of the follicle cell adjacent to the nurse cell is called apical, and the DE-Cadherin labeled by DCAD2 (red) is located at the apical of follicle cells. (B) DCAD2 together with CTPS as control to show the normal distribution of DCAD2. (C) Lateral view of a CTPSH355A–mCh egg chamber with abnormal follicle cell. (D) Yellow arrow pointed to the follicle cell ingression. (E) Ingressive folllicle cell pointed to by yellow arrows show abnormal distribution of DCAD2.
Figure 6.
DCAD2 distribution is disturbed in CTPSH355A follicle epithelia. (A) Cross section of Drosophila egg chamber expressing CTPS labelled by mCherry. The part of the follicle cell adjacent to the nurse cell is called apical, and the DE-Cadherin labeled by DCAD2 (red) is located at the apical of follicle cells. (B) DCAD2 together with CTPS as control to show the normal distribution of DCAD2. (C) Lateral view of a CTPSH355A–mCh egg chamber with abnormal follicle cell. (D) Yellow arrow pointed to the follicle cell ingression. (E) Ingressive folllicle cell pointed to by yellow arrows show abnormal distribution of DCAD2.
Figure 7.
Morphological comparision of follicles between wild-type and CTPSH355A. (A–F) Surface view of stage 8 egg chambers. Membrane was labeled by Armadillo (red), and CTPS is shown in green. (A,C) w1118 egg chamber. Scale bars, 50 μm. (B,D) CTPSH355A egg chamber. Scale bars, 20 μm. (E) Zoom-in view of (C). (F) Zoom-in view of (D). Redlines in E and F outline a central follicle cell with neighboring follicle cells in numbers. Note that the number of neighboring cells reflects the number of sides of the central polygonal follicle cells. (G) Quantitative analysis of the morphological difference between the wild-type control and CTPSH355A follicle cells (6 egg chambers were quantified per genotype, biological repeats = 3).
Figure 7.
Morphological comparision of follicles between wild-type and CTPSH355A. (A–F) Surface view of stage 8 egg chambers. Membrane was labeled by Armadillo (red), and CTPS is shown in green. (A,C) w1118 egg chamber. Scale bars, 50 μm. (B,D) CTPSH355A egg chamber. Scale bars, 20 μm. (E) Zoom-in view of (C). (F) Zoom-in view of (D). Redlines in E and F outline a central follicle cell with neighboring follicle cells in numbers. Note that the number of neighboring cells reflects the number of sides of the central polygonal follicle cells. (G) Quantitative analysis of the morphological difference between the wild-type control and CTPSH355A follicle cells (6 egg chambers were quantified per genotype, biological repeats = 3).
Figure 8.
Quantitative morphological analysis of follicle cells. (A) Surface view of a stage 8 wild-type egg chamber. CTPS-mCherry (green), Hts (red) and DNA (blue, labeled with Hoechst 33342). (B) Morphology of cell membrane. The yellow rectangle highlights an area of the follicle epithelium. (C) Enlarged part of the framed region in B. Larger follicular cells are circled in red, and smaller follicular cells are circled in blue. (D) CTPS-mCherry (green) and Hts (red) of the same egg chamber shown in A. (E) The area of each cell was measured after dividing each cell along the cell membrane. (F) corresponds to (D). (G) Surface view of a CTPSH355A stage 8 egg chamber. CTPSH355A-mcherry knock-in (green), Hts (red, labels cell membrane), and DNA (blue, labeled with Hoechst 33342). (H) Morphology of the cell membrane. A yellow rectangle highlights an area of the follicle epithelium. (I) Enlarged part of the framed region in (H). Larger follicle cells are circled in red, and smaller follicle cells are circled in blue. (J) CTPSH355A-mCherry (green) and Hts (red) of the same egg chamber shown in (E). Surface view of the cell membrane with CTPS. (K) The area of each cell was measured after dividing each cell along the cell membrane. (L) corresponds to (K). Scale bars, 20 μm. (M) The ratio of the average area of the three largest cells to the average area of the three smallest cells in an egg chamber. N = 6, ***, p < 0.0001. Mann-Whitney U test. (5 stage 7–8 egg chambers/genotypes, 3 biological replicates) (N) Average follicle cell surface. N = 3.
Figure 8.
Quantitative morphological analysis of follicle cells. (A) Surface view of a stage 8 wild-type egg chamber. CTPS-mCherry (green), Hts (red) and DNA (blue, labeled with Hoechst 33342). (B) Morphology of cell membrane. The yellow rectangle highlights an area of the follicle epithelium. (C) Enlarged part of the framed region in B. Larger follicular cells are circled in red, and smaller follicular cells are circled in blue. (D) CTPS-mCherry (green) and Hts (red) of the same egg chamber shown in A. (E) The area of each cell was measured after dividing each cell along the cell membrane. (F) corresponds to (D). (G) Surface view of a CTPSH355A stage 8 egg chamber. CTPSH355A-mcherry knock-in (green), Hts (red, labels cell membrane), and DNA (blue, labeled with Hoechst 33342). (H) Morphology of the cell membrane. A yellow rectangle highlights an area of the follicle epithelium. (I) Enlarged part of the framed region in (H). Larger follicle cells are circled in red, and smaller follicle cells are circled in blue. (J) CTPSH355A-mCherry (green) and Hts (red) of the same egg chamber shown in (E). Surface view of the cell membrane with CTPS. (K) The area of each cell was measured after dividing each cell along the cell membrane. (L) corresponds to (K). Scale bars, 20 μm. (M) The ratio of the average area of the three largest cells to the average area of the three smallest cells in an egg chamber. N = 6, ***, p < 0.0001. Mann-Whitney U test. (5 stage 7–8 egg chambers/genotypes, 3 biological replicates) (N) Average follicle cell surface. N = 3.
Figure 9.
Follicle cell ingression occurs in egg chamber overexpressing CTPSH355A. (A,B) Side view of stage 8 egg chamber overexpressing CTPS. (C) Cross section of stage 8 egg chamber overexpressing CTPSH355A. The ingression is framed by a rectangle. (D–G) Zoom-in images of the ingression follicle cells in (C). Scale bars, 20. (H) Western blot is detected with antibodies against mCherry and tubulin on the ovarian lysates of CTPS-mCh and CTPSH355A-mCh mutants. Scale bars, 20 μm. (I) Quantitative analysis of the CTPS protein level of samples represented in (H), the mean and standard deviation. (J) Western blot of the ovarian lysates of Actin > UAS CTPS-OE and Actin > UAS CTPSH355A-OE mutants, detected with antibodies against mCherry and tubulin. (K) Quantitative analysis of the CTPS protein level of samples represented in (J), the mean and standard deviation. *, p < 0.05; Mann-Whitney U test.
Figure 9.
Follicle cell ingression occurs in egg chamber overexpressing CTPSH355A. (A,B) Side view of stage 8 egg chamber overexpressing CTPS. (C) Cross section of stage 8 egg chamber overexpressing CTPSH355A. The ingression is framed by a rectangle. (D–G) Zoom-in images of the ingression follicle cells in (C). Scale bars, 20. (H) Western blot is detected with antibodies against mCherry and tubulin on the ovarian lysates of CTPS-mCh and CTPSH355A-mCh mutants. Scale bars, 20 μm. (I) Quantitative analysis of the CTPS protein level of samples represented in (H), the mean and standard deviation. (J) Western blot of the ovarian lysates of Actin > UAS CTPS-OE and Actin > UAS CTPSH355A-OE mutants, detected with antibodies against mCherry and tubulin. (K) Quantitative analysis of the CTPS protein level of samples represented in (J), the mean and standard deviation. *, p < 0.05; Mann-Whitney U test.
Figure 10.
The integrity of follicle epithelium is compromised when overexpressing CTPSH355A. (A–C) Surface view of a stage 8 egg chamber with follicle cells overexpressing CTPS. Large cytoophidia are detectable in almost all follicle cells. (D–F) Surface view of a stage 8 egg chamber with follicle cells overexpressing CTPSH355A. Note that the heterogenous sizes of follicle cells and increased gaps between neighbouring follicle cells. CTPS-mCherry (green), Hts (red) and DNA (blue, labelled with Hoechst 33342). Scale bars, 20 μm.
Figure 10.
The integrity of follicle epithelium is compromised when overexpressing CTPSH355A. (A–C) Surface view of a stage 8 egg chamber with follicle cells overexpressing CTPS. Large cytoophidia are detectable in almost all follicle cells. (D–F) Surface view of a stage 8 egg chamber with follicle cells overexpressing CTPSH355A. Note that the heterogenous sizes of follicle cells and increased gaps between neighbouring follicle cells. CTPS-mCherry (green), Hts (red) and DNA (blue, labelled with Hoechst 33342). Scale bars, 20 μm.
Figure 11.
Follicle cell ingression occurs with follicle cell-specific overexpresson of CTPSH355A. (A,B) Side view of a stage 8 egg chamber overexpressing CTPS in follicle epithelium. (C) Cross section of stage 8 egg chamber after overexpression of CTPSH355A in follicle epithelium. The ingression is framed by a rectangle. (D–G) Zoom-in of the ingressive follicle cell in (C). (H–J) Surface view of a stage 8 egg chamber with follicle cell-specific overexpression of CTPS. Large cytoophidia are detectable in almost all follicle cells. Note that most cytoophidia are distributed on or near the cortex of follicle cells. (K–M) Surface view of a stage 8 egg chamber with follicle cell-specific overexpression of CTPSH355A. No cytoophidium is detectable. CTPS-mCherry (green), Hts (red) and DNA (blue, labelled with Hoechst 33342). Scale bars, 20 μm. (N) Quantitative analysis of the ratio of three largest cells versus three smallest cells (5 images/genotypes, 3 biological replicates). Mann-Whitney U test, **, p = 0.002.
Figure 11.
Follicle cell ingression occurs with follicle cell-specific overexpresson of CTPSH355A. (A,B) Side view of a stage 8 egg chamber overexpressing CTPS in follicle epithelium. (C) Cross section of stage 8 egg chamber after overexpression of CTPSH355A in follicle epithelium. The ingression is framed by a rectangle. (D–G) Zoom-in of the ingressive follicle cell in (C). (H–J) Surface view of a stage 8 egg chamber with follicle cell-specific overexpression of CTPS. Large cytoophidia are detectable in almost all follicle cells. Note that most cytoophidia are distributed on or near the cortex of follicle cells. (K–M) Surface view of a stage 8 egg chamber with follicle cell-specific overexpression of CTPSH355A. No cytoophidium is detectable. CTPS-mCherry (green), Hts (red) and DNA (blue, labelled with Hoechst 33342). Scale bars, 20 μm. (N) Quantitative analysis of the ratio of three largest cells versus three smallest cells (5 images/genotypes, 3 biological replicates). Mann-Whitney U test, **, p = 0.002.
Figure 12.
Compared with wild-type control, the space increases between muscle sheath and egg chambers in CTPSH355A. (A,B) A stage 8 egg chamber of the CTPSH355A mutant. Integrin is labeled with knock-in GFP (green). CTPSH355A is labeled with knock-in mCherry (red). Yellow arrows point to gap between egg chamber and the muscle sheath. (C,D) A stage 8 wild-type egg chamber. Integrin is labeled by knock-in GFP. CTPS is labeled by knock-in mCherry. Scale bars, 20 μm. (E) The ratio of GFP intensity of the integrin to DNA from (A–D). The value is normalized to the control (5 images/genotypes, 3 biological replicates). Mann-Whitney U test. ***, p = 0.0005.
Figure 12.
Compared with wild-type control, the space increases between muscle sheath and egg chambers in CTPSH355A. (A,B) A stage 8 egg chamber of the CTPSH355A mutant. Integrin is labeled with knock-in GFP (green). CTPSH355A is labeled with knock-in mCherry (red). Yellow arrows point to gap between egg chamber and the muscle sheath. (C,D) A stage 8 wild-type egg chamber. Integrin is labeled by knock-in GFP. CTPS is labeled by knock-in mCherry. Scale bars, 20 μm. (E) The ratio of GFP intensity of the integrin to DNA from (A–D). The value is normalized to the control (5 images/genotypes, 3 biological replicates). Mann-Whitney U test. ***, p = 0.0005.