Nuclear receptor Ftz-f1 promotes follicle maturation and ovulation partly via bHLH/PAS transcription factor Sim
- Department of Physiology & Neurobiology, University of Connecticut, United States
- Institute for Systems Genomics, University of Connecticut, United States
Figures
Figure 1 with 3 supplements
Ftz-f1 is induced in stage-10B follicle cells through ecdysteroid signaling.
(A–F) The expression of Ftz-f1 protein in late oogenesis. Ftz-f1 protein is detected by anti–Ftz-f1 antibody shown in green. Hnt expression (shown in red) is used to mark stage-10A (A) and stage-14 (F) follicles. The insets are higher magnification of Ftz-f1 expression (white) in outlined areas. All images from A-F are acquired using the same microscopic settings. (G–H) Ftz-f1 protein expression (red in G-H) in stage-10B egg chambers with flip-out Gal4 clones (marked by green GFP in G-H) overexpressing EcRDN (act >EcRDN in G) or ttkRNAi (act >ttkRNAi in H). Insets show higher magnification of Ftz-f1 expression in squared area. The clone boundary is outlined by red dashed line. (I–J) Ttk69 expression (red in I) and EdU staining (red in J) in stage-10B egg chambers with ftz-f1ex7 mutant follicle cell clones (marked by green GFP). Insets show the higher magnification of Ttk69 expression (I) and EdU staining (J) in squared areas with the clone boundary marked by red dashed line. Nuclei are marked by DAPI in blue in all figures.
Figure 1—figure supplement 1
Expression pattern of ftz-f1::GFP.FLAG and ftz-f1-lacZ in late oogenesis.
(A–E) Ftz-f1::GFP.FLAG expression (detected by anti-GFP antibody shown in green) in stage-10A (A), stage-10B (B), stage-11 (C), stage-12 (D), and stage-13 (E) egg chambers from ftz-f1::GFP.FLAG females. Hnt expression (shown in red) is used to mark stage-10A egg chambers (A). (F–J) β-Gal expression (red in F-J) in stage-10A (F), stage-10B (G), stage-11 (H), stage-12 (I), and stage-13 (J) egg chambers from ftz-f1fs(3)2877 females. Ftz-f1 protein detected by anti-Ftz-f1 antibody is shown in green. Nuclei are marked by DAPI in blue.
Figure 1—figure supplement 2
Ftz-f1 expression is not sensitive to the ecdysone level.
(A) EcR ligand sensor (hsGal4DBD-EcRLBD, UAS-nlacZ) can be activated prematurely in stage-8 egg chambers (marked by β-Gal expression (red in A and white in A’)) with exogenous 20E treatment. (B) Exogenous 20E is not sufficient to induce premature expression of Ftz-f1 protein (green in B and white in B’) in wild-type egg chambers before stage-10B. Hnt expression in red is used to mark stages 7-10A. (C–F) Ftz-f1 protein expression (green in C-F, and white in C’-F’) in stage-10A (C,E) and stage-10B (D,F) egg chambers with flip-out Gal4 clones (marked by red RFP in C-F) overexpressing Cyp18a1 (act >cyp18a1 in C-D) or Cyp18a1RNAi (act >cyp18a1RNAi in E-F). The clone boundary is outlined by red dashed line. Nuclei are marked by DAPI in blue.
Figure 1—figure supplement 3
Ttk69 and Sim are efficiently knocked down by overexpression of ttkRNAi and simRNAi, respectively.
(A) Ttk69 protein expression (red) in stage-10B egg chambers with flip-out Gal4 clones (marked by green GFP) overexpressing ttkRNAi. (B–D) Sim protein (red in B-D) in stage-10B (B), stage-12 (C), and stage-14 (D) egg chambers with flip-out Gal4 clones (marked by green GFP) overexpressing simRNAi. Insets show higher magnification of Ttk69 (A) and Sim (B–D) expression in squared area. The clone boundary is outlined by red dashed line. Nuclei are marked by DAPI in blue in all figures.
Figure 2 with 1 supplement
Ftz-f1 is required for ovulation and follicle rupture.
(A–C) Representative images show Ftz-f1 protein expression (green in A-C) in stage-10B egg chambers of control (A), ftz-f1RNAi1 (B), and ftz-f1RNAi2 (C) females with Vm26Aa-Gal4. The insets are higher magnification of Ftz-f1 expression in squared areas. (D) Quantification of egg laying in control or ftz-f1RNAi females with Vm26Aa-Gal4 and Oamb-RFP. The number of females is noted above each bar. (E) Quantification of OA-induced (light grey bars) and Ionomycin-induced (dark grey bars) follicle rupture using mature follicles isolated from control or ftz-f1RNAi females with Vm26Aa-Gal4 and Oamb-RFP. Mature follicles were isolated according to Oamb-RFP expression. The number of mature follicles analyzed is noted above each bar. (F–G) Representative images show Mmp2::GFP expression (green in F-G) in stage-14 egg chambers from control (F) or ftz-f1RNAi 1 (G) females with Vm26Aa-Gal4. Insets show higher magnification of Mmp2::GFP expression in posterior follicle cells in squared areas. Oocytes are outlined in cyan. (H–I) Representative images show Oamb-RFP expression (red) in stage-14 egg chambers from control (H) and ftz-f1RNAi 1 (I) females with Vm26Aa-Gal4. (J–K) Quantification L-012 luminescent signal (indicating superoxide production) in stage-14 egg chambers from control (black), ftz-f1RNAi1(dark blue), and ftz-f1RNAi2 (light blue) females with VM26Aa-Gal4 and Oamb-RFP. Follicles are either stimulated with OA (J) or Ionomycin (K). Nuclei are marked by DAPI in blue. ***p<0.001 (Student’s t-test).
Figure 2—figure supplement 1
ftz-f1 knockdown causes defects in ovulation and egg morphology.
(A–C) Ftz-f1 protein (green in A-C) in stage-12 egg chambers from control (A), ftz-f1RNAi1 (B), and ftz-f1RNAi2 (C) females with Vm26Aa-Gal4, UAS-dcr2; Oamb-RFP. The insets are higher magnification of Ftz-f1 expression in squared areas. (D–E) Quantification of egg laying (D) and mature follicles in females post egg laying (E) in control or ftz-f1RNAi females with Vm26Aa-Gal4, UAS-dcr2; 47A04-LexA, lexAop2-6XGFP. The number of females is noted above each bar. (F) Quantification of OA- and Ionomycin-induced follicle rupture using mature follicles isolated from control or ftz-f1RNAi females. Mature follicles were isolated according to 47A04-lexA > 6 XGFP expression. The number of mature follicles analyzed is noted above each bar. (G–I) Representative images show follicles from control (G), ftz-f1RNA1i (H), and ftz-f1RNAi2 (I) females after 3 hr culture with OA. Follicles were isolated according to Oamb-RFP expression (red). Ruptured follicles are marked by arrowheads. (J–L) Representative images show follicles from control (J), ftz-f1RNA1i (K), and ftz-f1RNAi2 (L) females after 3 hr culture with OA. Follicles were isolated according to 47A04-lexA > 6 XGFP expression (green). Ruptured follicles are marked by arrowheads. 6XGFP forms puncta inside follicle cells. (M–O) Representative DIC images show dorsal appendage morphology in control (M), ftz-f1RNA1i (N), and ftz-f1RNAi2 (O) stage-14 egg chambers. Blue arrowheads indicate stunted dorsal appendage formation. ***p<0.001 (Student’s t-test).
Figure 3 with 2 supplements
Ftz-f1 promotes follicle cell differentiation into the final maturation stage.
(A–F) Representative images show the expression of Hnt (A), Cut (B), Ttk69 (C), EcR.A (D), EcR.B1 (E), and Br-C (F) in stage-14 egg chambers with ftz-f1ex7 mutant follicle cell clones (marked by green GFP). Insets show higher magnification of Hnt (A), Cut (B), Ttk69 (C), EcR.A (D), EcR.B1 (E), and Br-C (F) in squared areas with the clone boundary marked by red dashed line. (G) Edu staining (red in G) in stage-14 egg chambers with ftz-f1ex7 mutant follicle cell clones (marked by green GFP). The inset shows the higher magnification of Edu staining. (H–I) Hnt expression (red in H-I) in stage-10B (H) and stage-12 (I) egg chambers with ftz-f1ex7 mutant follicle cell clones (marked by green GFP). Insets show the higher magnification of Hnt expression (H–I). (J–K) Cut expression (red in J-K) in stage-10B (J) and stage-12 (K) egg chambers with ftz-f1ex7 mutant follicle cell clones (marked by green GFP). Insets show the higher magnification of Cut expression (J–K) in squared areas with the clone boundary marked by red dashed line. Nuclei are marked by DAPI in blue.
Figure 3—figure supplement 1
ftz-f1 knockdown disrupts follicle cell transition into stage 14.
(A–F) Representative images show Hnt protein (green in A-B), Cut protein (green in C-D), and Ttk69 protein (green in E-F) in stage-14 follicles from control (A,C,E) and ftz-f1RNAi1 (B,D,F) females with Vm26Aa-Gal4, UAS-dcr2; Oamb-RFP. Note the uniform and patchy expression of Oamb-RFP (red in A-F) in control and ftz-f1-knockdown follicles. The insets are higher magnification of Hnt (A–B), Cut (C–D) and Ttk69 (E–F) expression in squared areas. Nuclei are marked by DAPI in blue.
Figure 3—figure supplement 2
Analysis of Cut expression in ftz-f1 mutant clones.
(A) Cut expression (red in A and white in A’) is not detected in stage-10A egg chambers with ftz-f1ex7 mutant follicle cell clones (marked by green GFP). (B–C) Representative egg chambers used for quantifying Cut expression (red in B and C, white in B’ and C’) in D. ftz-f1 mutant clones were marked by green GFP. Nuclei are marked by DAPI in blue. (D) Quantification of Cut protein level in wild-type (black) and ftz-f1ex7 (green) mutant follicle cells. The average Corrected Total Cellular Fluorescence (CTCF) (McCloy et al., 2014) was quantified for nuclear Cut expression. Seven cells of each clone and four clones for each genotype were used. The error bar indicates the standard deviation.
Figure 4 with 1 supplement
RNA-seq and CUT&RUN analyses indicate sim as a direct target of Ftz-f1.
(A) Principle component analysis of RNA-seq data. (B) A volcano plot shows the differentially expressed genes between control and ftz-f1RNAi1 egg chambers. The significantly upregulated and downregulated genes were marked red and green, respectively. (C) The comparison of de novo-identified Ftz-f1-binding motifs and the canonical Ftz-f1 motif. The number of peaks used for motif search was listed at the upper-right corner of each motif. (D) A motif footprint plot for the Ftz-f1-binding motif in replicate 2. (E) An IGV plot shows the narrow peaks and motif sites in the gene region of sim. The motif sequences are shown in the magnified area. (F) The quantification of individual sim transcript expression in control and ftz-f1RNAi1 egg chambers. The transcript expression is mined from RNA-seq data.
Figure 4—figure supplement 1
Analysis of CUT&RUN narrow peaks.
(A) The chromosomal distribution of narrow peaks in the three biological replicates. (B) The average profile of narrow peaks binding to TSS region. The shaded regions show 95% confidence interval. (C) The distribution of narrow peaks relative to TSS.
Figure 5 with 2 supplements
Ftz-f1 promotes Sim expression in stage-10B follicle cells.
(A–F) The expression of Sim protein in late oogenesis. Sim protein is detected by anti-Sim antibody shown in green. Hnt expression (shown in red) is used to mark stage-10A (A) and stage-14 (F) follicles. The insets are higher magnification of Sim expression in squared areas. All images from A-F are acquired using the same microscopic settings. (G–H) Sim expression (red in G,H and white in G’,H’) in stage-10B (G) and stage-12 (H) egg chambers with ftzf1ex7 mutant clones (marked by green GFP and outlined by dashed lines). (I) Sim expression (red in I and white in I’) in stage-10B egg chambers with flip-out Gal4 clones (marked by green GFP and outlined by dashed line) overexpressing ttkRNAi. Nuclei are marked by DAPI in blue.
Figure 5—figure supplement 1
Sim is detected in stalk follicle cells.
(A) Sim protein (red in A and white in A’) is detected in stalk follicle cells that connects two adjacent egg chambers in the ovariole. White arrowheads point to stalk follicle cells. Nuclei are marked by DAPI in blue.
Figure 5—figure supplement 2
Overexpression of ftz-f1 induces premature Sim expression at stage 10A and disrupts follicle cell transition into stage 10B.
Flip-out Gal4 clones with ftz-f1 overexpression are marked by green GFP and outlined in red. (A–B) Ftz-f1 (red in A,B and white in A’,B’) was overexpressed in flip-out Gal4 clones in stage-8 egg chambers (A) and stage-10B egg chambers (B). (C–D) Sim (red in C,D and white in C’,D’) is prematurely expressed in flip-out Gal4 clones at stage 10A (C) and continues to be expressed at stage 10B (D). (E–F) Cut (red in E,F and white in E’,F’) is neither detected in flip-out Gal4 clones at stage 10A (E) nor at stage 10B (F). (G–H) Hnt (red in G,H and white in G’,H’) is properly expressed in stage-10A clones (G), and is continuously expressed in clones cells at stage 12 (H). Nuclei are marked by DAPI in blue.
Figure 6 with 2 supplements
Sim promotes follicle cell differentiation into the final maturation stage.
(A–F) The expression of Hnt (A), Cut (B), Ttk69 (C), EcR.A (D), EcR.B1 (E), and Br-C (F) in stage-14 egg chambers with flip-out Gal4 clones overexpressing simRNAi (marked by green GFP). Insets show higher magnification of Hnt (A), Cut (B), Ttk69 (C), EcR.A (D), EcR.B1 (E), and Br-C (F) expression in squared areas with the clone boundary marked by red dashed line. (G–H) Hnt expression (red in G-H) in stage-10B (G) and stage-12 (H) egg chambers with flip-out Gal4 clones overexpressing simRNAi. Insets show higher magnification of Hnt expression in squared areas with the clone boundary marked by red dashed line. (I–J) Cut expression (red in I-J) in stage-10B (I) and stage-12 (J) egg chambers with flip-out Gal4 clones overexpressing simRNAi. Insets show higher magnification of Cut expression. (K) Ftz-f1 expression (red in K) in stage-10B egg chambers with flip-out Gal4 clones overexpressing simRNAi. Insets show higher magnification of Ftz-f1 expression in squared areas with the clone boundary marked by red dashed line. Nuclei are marked by DAPI in blue.
Figure 6—figure supplement 1
Overexpression of sim disrupts earlier follicle cell differentiation.
Flip-out Gal4 clones with sim overexpression and ftz-f1 knockdown are marked by green GFP and outlined. (A–B) Hnt (red in A-B, and white in B’) is detected in clone cells in stage-9 (A) and stage-10A (B) egg chambers. Clone cells exhibit smaller nuclei and abnormal cell organization. (C) Hnt (red in C and white in C’) is continuously expressed in clone cells at stage 13. (D–E) Cut (red in D,E and white in D’,E’) is properly downregulated in flip-out Gal4 clones at stage 8 (D) and is not upregulated in clone cells at stage 12 (E). Nuclei are marked by DAPI in blue.
Figure 6—figure supplement 2
Rescue ftz-f1 defect with Vm26Aa-Gal4 driving sim overexpression.
(A–H) Representative images show Cut expression (green in A-D and white in A’-D’) and Hnt expression (green in E-H and white in E’-H’) in stage-10B/11 egg chambers from control (A and E), sim (B and F), sim;ftz-f1RNAi2 (C and G), and ftz-f1RNAi2 (D and H) females with Vm26Aa-Gal4, UAS-dcr2; Oamb-RFP. (I–P) Representative images show Cut (green in I-L and white in I’-L’) and Hnt (green in M-P and white in M’-P’) expression in stage-14 follicles marked by Oamb-RFP expression (shown in red). Nuclei are marked by DAPI in blue.
Figure 7 with 1 supplement
The role of Ftz-f1 in follicle cell maturation can be replaced by mSF-1.
(A–H) Hnt expression (red in A-C) and Cut expression (red in E-G) in stage-14 egg chambers with flip-out Gal4 clones (marked by green GFP) overexpressing ftz-f1RNAi2 (A,E), mSF-1;ftz-f1RNAi2(B,F), or mSF-1 (C,G). The insets show higher magnification of Hnt expression (A–C) and Cut expression (E–G) in squared areas with the clone boundary marked by red dashed line. Quantification of clone phenotype is show in D for Hnt expression and H for Cut expression. The number of clones analyzed is noted above each bar. (I–P) Hnt expression (red in I-L) and Cut expression (red in M-P) in stage-8 (I and M), stage-10A (J and N), stage-12 (K, and O) and stage-13 (L and P) egg chambers with flip-out Gal4 clones overexpressing mSF-1;ftz-f1RNAi2 (marked by green GFP). Insets show the higher magnification of Hnt expression (I–L) and Cut expression (M–P) in squared areas with the clone boundary marked by red dashed line. Nuclei are marked by DAPI in blue.
Figure 7—figure supplement 1
Ftz-f1 can rescue differentiation defects in ftz-f1–knockdown follicle cells.
Flip-out Gal4 clones with ftz-f1 and ftz-f1RNAi2 are marked by green GFP and outlined in red. (A–B) Ftz-f1 (red in A,B and white in A’,B’) is faintly detected in clone cells at stage 8 (A) and is moderately expressed at stage 10B (B). (C–D) Sim (red in C,D and white in C’,D’) is not ectopically expressed at stage 10A (C) and is properly upregulated at stage 10B (D) in the clone cells. (E–F) Cut (red in E,F and white in E’F’) is properly upregulated at stage 10B (E) and downregulated at stage 14 (F) in the clone cells. (G–H) Hnt (red in G,H and white in G’,H’) is properly downregulated at stage 13 (G) and properly upregulated at stage 14 (H). Nuclei are marked by DAPI in blue.
Figure 8
Sim expression can be rescued by mSF-1.
(A–D) Sim expression (red in A-C) in stage-14 egg chambers with flip-out Gal4 clones (marked by green GFP) overexpressing ftz-f1RNAi2 (A), mSF-1;ftz-f1RNAi2(B), or mSF-1 (C). Insets show higher magnification of Sim expression (A–C) in squared areas with the clone boundary marked by red dashed line. Quantification of Sim expression in these clones is shown in D. The number of clones analyzed is noted above each bar. (E–H) Sim expression (red in E-H) in stage-8 (E), stage-10A (F), stage-12 (G), and stage-13 (H) egg chambers with flip-out Gal4 clones (marked by green GFP in E-H) overexpressing mSF-1;ftz-f1RNAi2. Insets show higher magnification of Sim expression (E–H) in squared areas with the clone boundary marked by red dashed line. Nuclei are marked by DAPI in blue. (I) A schematic drawing shows the role of Ftz-f1 and Sim in follicle cell differentiation in late oogenesis. At stage-10B Ftz-f1 expression is required for induction of Sim. Expression of Cut, Ttk69, Br-C, and EcR.A/B1 are high in stage-10B follicle cells and downregulate by stage-13. Expression of Hnt, Oamb, and Mmp2 are absent in stages 10B-13, and are then robustly upregulated in stage-14 follicle cells. GA: gene amplification.
Additional files
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Supplementary file 1
Table of differentially expressed genes.
- https://cdn.elifesciences.org/articles/54568/elife-54568-supp1-v2.xlsx
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Supplementary file 2
Table of motif site analysis.
- https://cdn.elifesciences.org/articles/54568/elife-54568-supp2-v2.xlsx
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Supplementary file 3
Table of direct targets of Ftz-f1.
- https://cdn.elifesciences.org/articles/54568/elife-54568-supp3-v2.xlsx
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Supplementary file 4
Key Resources Table.
- https://cdn.elifesciences.org/articles/54568/elife-54568-supp4-v2.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/54568/elife-54568-transrepform-v2.docx
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Nuclear receptor Ftz-f1 promotes follicle maturation and ovulation partly via bHLH/PAS transcription factor Sim
eLife 9:e54568.
https://doi.org/10.7554/eLife.54568
