Fig 1.
(A) Schematic diagram of the cell cycle mode in the PG during larval development. (B) Adult sperm cells (left panel), larval PG (middle panel) and CA cells (right panel) at 84 hAH of wild-type strain Oregon R. DNA was stained by Hoechst. The mean C value is shown below each panel (see Fig 1C). Scale bars: 10 μm. (C) Scatter plots with a mean value of relative signal intensity of Hoechst in Oregon R sperm, PG, and CA cells. The mean C value in sperm cells was normalized to 1, and accordingly, the mean C values in PG and CA cells at 84 hAH were set to 58 and 8, respectively. Sample sizes (the number of sperm, PG, and CA cells) are shown above each column. (D) The PG (upper panels) and PG cells in higher magnification (lower panels) of Oregon R at indicated time points. PG cells, mitotic pH3-positive cells, and DNA were detected by anti-Sro antibody (green), anti-pH3 antibody (magenta), and Hoechst (blue), respectively. The arrows and arrowheads indicate pH3-positive PG cells and the nuclei of CA cells, respectively. Scale bars: 50 μm (upper panels) and 10 μm (lower panels). (E) Fzr-GFP expression in the PG at indicated time points. Fzr-GFP (green and white in the upper and lower panels, respectively) was detected using anti-GFP antibody at indicated stages. PG cells are labeled by mCherry.nls driven by PG-selective phm-Gal4 (magenta in the upper panels). The PGs are indicated by dotted lines. The asterisks indicate CC cells expressing DsRed driven by eyeless promoter: 3xP3-dsRed marker in Fzr-GFP transgene. Scale bars: 10 μm. (F–H) Scatter and box plots showing the cell number (F), the C value (G), and the percentage of pH3-positive cells (H) in the PG of Oregon R at indicated time points. Different lowercase letters indicate statistically significant differences (P < 0.05; Steel–Dwass test; see S1 Table). ns, not significant (P > 0.05). Box plots indicate the median (bold line), the 25th and 75th percentiles (box edges), and the range (whiskers). Dot plots show all data points individually. The mean C value at 84 hAH was normalized to 58 (see Fig 1C), and all data points were normalized accordingly. Sample sizes (the number of PGs) are shown above each column. (I) Scatter and box plots showing the relative expression level of Fzr-GFP in the PG at indicated stages. Different lowercase letters indicate statistically significant differences (P < 0.05; Steel–Dwass test; see S1 Table). Box and dot plots as in Fig 1C–1E. Sample sizes (the number of PGs) are shown above each column.
Fig 2.
Fzr is required for MES in the PG.
(A) The PG of the controls (phm>dicer2 mCherry.nls) (upper panels) and fzr RNAi (phm>dicer2 mCherry.nls fzr-RNAi) (lower panels) at indicated time points. pH3 and DNA were detected by anti-pH3 antibody (green) and Hoechst (blue), respectively, and the nuclei of PG cells were labelled by mCherry.nls (magenta). The PGs are indicated by dotted lines. The asterisks indicate nonspecific anti-pH3 antibody signal around CC cells. Scale bars: 50 μm. (B and C) Scatter and box plots showing the cell number (B) and the C value (C) in the PG of the controls (red) and fzr RNAi animals (blue) at indicated stages. P-values for all pairwise comparisons are shown in S1 Table (Steel–Dwass test). Box and dot plots as in Fig 1C–1E. The mean C value of control at 96 hAH was set to 54 (see S1B Fig). Sample sizes (the number of PGs) are shown above each column. (D) PG cells in higher magnification of the controls (upper panels) and fzr RNAi animals (lower panels) at indicated stages. The arrows indicate pH3-positive PG cells. Scale bars: 10 μm. (E) Scatter and box plots showing the percentage of pH3-positive PG cells of the controls (red) and fzr RNAi animals (blue) at indicated stages. P-values for all pairwise comparisons are shown in S1 Table (Steel–Dwass test). Sample sizes are the same as in B and C. (F) Schematic diagram of FLP/FRT system. Heat-shock-induced FLP recombinase recognizes FRT sites to induce trans-chromosome recombination between chromosome arms carrying fzrG0418 and mCherry.nls, which leads to wild-type and homozygous mutant progenies. (G) Diagram of temperature-shift experiment. Newly-hatched larvae are heat-shocked at 37°C for 30 min, and then recovered at 18°C until 120 hAH. (H) fzrG0418/fzrG0418 clone PG cells. fzrG0418/fzrG0418 homozygous mutant cells (indicated by dotted lines) do not express mRFP.nls (magenta and white in the left and middle panels, respectively). The PG and nuclear DNA were labelled by anti-Dib antibody (green in the left panels) and Hoechst (blue and white in the left and right panels, respectively), respectively. Scale bars: 10 μm.
Fig 3.
Fzr-mediated downregulation of mitotic cyclins is required for MES and ecdysteroidogenesis in the PG.
(A and B). CycA (A) and B expression (B) in the PG of the controls (phm>dicer2 mCD8::GFP) and fzr RNAi larvae (phm>dicer2 mCD8::GFP fzr-RNAi) at 96 hAH. PG cells were labelled by mCD8::GFP (green in the upper panels) and CycA and B were detected by specific antibodies against each cyclin (magenta and white in the upper and lower panels, respectively). The PGs are indicated by dotted lines. Scale bars = 50 μm. (C and D) Scatter and box plots showing the percentage of CycA (C) and CycB (D)-positive PG cells of the controls (red) and fzr RNAi animals (blue) at indicated stages. Different lowercase letters indicate statistically significant differences (P < 0.05; Steel–Dwass test; see S1 Table). Box and dot plots as in Fig 1C–1E. Sample sizes (the number of PGs) are shown above each column. (E) The PG (upper panels) and PG cells in higher magnification (lower panels) of fzr RNAi (phm>dicer2 mCherry.nls fzr-RNAi), fzr RNAi + cycA RNAi (phm>dicer2 mCherry.nls fzr-RNAi cycA-RNAi), and fzr RNAi + cycB RNAi (phm>dicer2 mCherry.nls fzr-RNAi cycB-RNAi) at 96 hAH. pH3 and DNA were detected by anti-pH3 antibody (green) and Hoechst (blue), respectively, and the nuclei of PG cells were labelled by mCherry.nls (magenta). The PGs are indicated by dotted lines. The arrows indicate pH3-labelled mitotic PG cells. Scale bars: 50 μm (upper panels) and 10 μm (lower panels). (F–H) Scatter and box plots showing the cell number (F), the C value (G), and the percentage of pH3-positive cells (H) in the PG of fzr RNAi, fzr RNAi + cycA RNAi, and fzr RNAi + cycB RNAi animals at 96 hAH. Different lowercase letters indicate statistically significant differences (P < 0.05; Steel–Dwass test; see S1 Table). The mean C value of fzr RNAi at 96 hAH was set to 4 according to its mean C value at the same time point (Fig 2C). Sample sizes (the number of PGs) are shown above each column.(I) Percentages of pupariated fzr RNAi (phm>dicer2 fzr-RNAi), fzr RNAi + cycA RNAi (phm>dicer2 fzr-RNAi cycA-RNAi), and fzr RNAi + cycB RNAi animals (phm>dicer2 fzr-RNAi cycB-RNAi) are shown. The asterisks indicate statistically significant differences between fzr RNAi and fzr RNAi + cycA RNAi or fzr RNAi + cycB RNAi (P < 0.05; Fisher’s test). Sample sizes (the number of animals) are shown above each column. (J) fzr RNAi animals arrested at the L3 stage and pupariated fzr RNAi + cycA RNAi and fzr RNAi + cycB RNAi animals at 120 hAH. (K) Whole-body ecdysteroid levels in fzr RNAi, fzr RNAi + cycA RNAi, and fzr RNAi + cycB RNAi animals at 96 hAH were measured using ELISA. Scatter and box plots show ecdysteroid level of five independent data sets (10 larvae were pooled in each datum). Different lowercase letters indicate statistically significant differences (P < 0.05; Steel–Dwass test; see S1 Table). (L) The expression level of ecdysone biosynthetic genes at 96 hAH was measured using qPCR. Scatter plots with average values of triplicate data sets are shown with SE (ten to fifteen larvae were pooled in each datum). Different lowercase letters indicate statistically significant differences (P < 0.05; Tukey’s multiple comparison test; see S1 Table).
Fig 4.
PG-selective RNAi screen to identify novel MES regulator.
(A) Schematic of PG-selective RNAi screen design. In “step 1”, UAS-RNAi males against each 703 candidate gene are crossed with females carrying two copies of phm>mCherry.nls to obtain F1 animals in which target gene is knocked down selectively in the PG. Octβ3R and tor were included in candidate gene sets as positive control showing L3 arrest phenotype. In “step 2”, PGs of RNAi animals, in which developmental defect was confirmed, were observed by histochemistry to investigate their cell number and the DNA content. (B) Pie chart showing the distribution of the phenotypic categories in step 1. The number of RNAi lines showing each phenotype is shown in the legend of each pie. NOP, no obvious phenotype. (C) Schematic of developmental transition of the PG cell number and the DNA content. PG cells at L1 stage (indicated as “Early PG cells”) undergo mitotic cell cycle to increase their number, and subsequent MES initiates endocycling which leads to mature PG cells with higher DNA content (indicated as “Late PG cells”). When MES regulator was inhibited in the PG, the PG cell number was increased and an increase in the DNA content was blocked (indicated as “Defects in MES”). Defects in endocycle progression results in decrease in the DNA content, whereas inhibition of downstream effector of endocycle should display no significant decrease in the DNA content. (D) Dot-plots showing the mean values of the cell number and the DNA content in the PG of control and RNAi animals. Each plot shows the average values of triplicate data sets in each group. Green and black plots indicate control animals which were cultured in standard culture medium and nutrient-rich German food (GF), respectively. Numbers indicate time points for sampling of control animals cultured in GF. All other data were obtained at day 6 after crossing in standard culture medium, including control shown by green plot. Purple and magenta plots show RNAi animals in which the PG cell number was significantly increased compared to the controls (P < 0.05; Dunnett’s multiple comparisons test). Magenta indicates RNAi against cct subunit genes.
Fig 5.
cct subunit genes are required for ecdysone biosynthesis in the PG.
(A) The PG (upper panels) and PG cells in higher magnification (lower panels) of the controls (phm>mCherry.nls) and RNAi against each cct subunit genes (phm>mCherry.nls cct-RNAi) at 96 hAH. pH3 and DNA were detected by anti-pH3 antibody (green) and Hoechst (blue), respectively, and the nuclei of PG cells were labelled by mCherry.nls (magenta). The PGs are indicated by dotted lines. The arrows indicate pH3-positive PG cells. Scale bars: 50 μm (upper panels) and 10 μm (lower panels). (B–D) Scatter and box plots showing the cell number (B), the C value (C), and the percentage of pH3-positive cells (D) in the PG of control and cct1-8 RNAi animals at 96 hAH. The asterisks indicate statistically significant differences between the control and cct RNAi (P < 0.05; Steel–Dwass test). P-values for all pairwise comparisons are shown in S1 Table. ns, not significant (P > 0.05). Box and dot plots as in Fig 1C–1E. The mean C value of control at 96 hAH was set to 53 (see S1B Fig). Sample sizes (the number of PGs) are shown in right side of each column. (E) Percentages of pupariated and L1/L2- and L3-arrested animals in the controls (phm>+) and cct1–8 RNAi (phm>cct-RNAi) are shown. Sample sizes (the number of animals) are shown above each column. (F) Pupariated control and cct4 RNAi arrested at the L3 stage. (G) The expression level of ecdysone biosynthetic genes was measured using qPCR. Scatter plots with average values of triplicate data sets are shown with SE (ten to fifteen larvae were pooled in each datum). Different lowercase letters indicate statistically significant differences (P < 0.05; Tukey’s multiple comparison test; see S1 Table). (H) Percentages of pupariated cct1–8 RNAi animals, cultured on the medium with 20E (0.5 mg/g) or without 20E from 48 hAH are shown. The asterisks indicate statistically significant differences between 20E-fed and no 20E groups (P < 0.05; Fisher’s test). Sample sizes (the number of animals) are shown above each column. (I) cct4 RNAi larva fed on -20E medium and pupariated cct4 RNAi animal fed on +20E medium.
Fig 6.
TRiC is required for proper progression of MES and endocycle in the PG.
(A) The PG of the controls (phm>mCherry.nls), cct4 RNAi (phm>mCherry.nls cct4-RNAi), and cct8 RNAi (phm>mCherry.nls cct8-RNAi) at indicated time points. pH3 and DNA were detected by anti-pH3 antibody (green) and Hoechst (blue), respectively, and the nuclei of PG cells were labelled by mCherry.nls (magenta). The PGs are indicated by dotted lines. Scale bars: 50 μm. (B and C) Scatter and box plots showing the cell number (B) and the C value (C) in the PG of the controls (red), cct4 RNAi (green), and cct8 RNAi animals (blue) at indicated stages. P-values for all pairwise comparisons are shown in S1 Table (Steel–Dwass test). Box and dot plots as in Fig 1C–1E. The mean C value of control at 96 hAH was set to 53 (see S1B Fig), and all data points were normalized accordingly. Sample sizes (the number of PGs) are shown above each column. (D) PG cells in higher magnification of control, cct4 RNAi, and cct8 RNAi animals at indicated stages. The arrows indicate pH3-positive mitotic PG cells. Scale bars: 10 μm. (E) Scatter and box plots showing the percentage of pH3-positive PG cells of the controls (red), cct4 RNAi (green), and cct8 RNAi animals (blue) at indicated stages. P-values for all pairwise comparisons are shown in S1 Table (Steel–Dwass test). Sample sizes are the same as in B and C. (F) cct4KG092808/cct4KG092808 FLP-out clone induced by heat shock. Newly-hatched larvae are heat-shocked at 37°C for 30 min, and then recovered at 18°C until 120 hAH. FRT40A site was used in this clonal analysis. cct4KG092808 homozygous mutant cell (indicated by dotted line) is mRFP.nls-negative (magenta in left panel and middle panel). The PG and nuclear DNA were labelled by anti-Dib antibody (green in left panel) and Hoechst (blue in left panel and right panel), respectively. Scale bars: 10 μm.
Fig 7.
TRiC regulates expression of Fzr and mitotic cyclin in the PG.
(A and B) Fzr-GFP expression (green and white in the upper and lower panels, respectively) was observed in the controls (phm>mCherry.nls, fzr-GFP), cct4 RNAi (phm>mCherry.nls cct4-RNAi, fzr-GFP), and cct8 RNAi (phm>mCherry.nls cct8-RNAi, fzr-GFP) using anti-GFP antibody at 24 (A) and 48 hAH (B). The nuclei of PG cells are labeled by mCherry.nls (magenta in the upper panels). The PGs are indicated by dotted lines. The asterisks indicate CC cells expressing 3xP3-dsRed marker in Fzr-GFP transgene. Scale bars: 10 μm. (C) Scatter and box plots showing the nuclear/cytoplasmic ratio of Fzr-GFP expression of the controls (red), cct4 RNAi (green), and cct8 RNAi animals (blue) at indicated stages. Different lowercase letters indicate statistically significant differences (P < 0.05; Steel–Dwass test; see S1 Table). Sample sizes (the number of PGs) are shown above each column. (D and E) CycA (D) and B expression (E) in the PG of the controls (phm>mCD8::GFP), cct4 RNAi (phm>mCD8::GFP cct4-RNAi), and cct8 RNAi animals (phm>mCD8::GFP cct8-RNAi) at 24 hAH. PG cells were labelled by mCD8::GFP (green in the upper panels), and CycA and B were detected by specific antibodies to each cyclin (magenta and white in the upper and lower panels, respectively). The PGs are indicated by dotted lines. The arrows indicate PG cells expressing CycA. Scale bars = 10 μm. (F and G) Scatter and box plots showing the percentage of CycA-positive (F) and CycB-positive PG cells (G) of the controls (red), cct4 RNAi (green), and cct8 RNAi animals (blue) at indicated stages. Different lowercase letters indicate statistically significant differences (P < 0.05; Steel–Dwass test; see S1 Table). ns, not significant. Sample sizes (the number of PGs) are shown above each column.
Fig 8.
Current working model for TRiC-mediated MES regulation in the PG PG cells undergo MES in Fzr-dependent manner.
Molecular chaperonin TRiC downregulates CycA at least in part by regulating Fzr nuclear translocation to induce MES and subsequent endocycling. Proper progression of MES and endocycle leads to the terminal differentiation of the PG, activation of ecdysone biosynthesis.