Fig 1.
c-Fos reduction partially suppressed defects in piwi mutant ovaries.
(A) DAPI-stained image of a wild-type ovary, a diagramed ovariole, and a diagramed germarium. Labeled cell types are, respectively, TF, terminal filament; CC, cap cell; GSC, germline stem cell; CB, cystoblast; EC, escort cell; SSC, somatic stem cell; FC, follicle cell; NC, nurse cell; oocyte. (B) Images of DAPI-stained ovaries from wild-type and mutant animals. (C) Quantification of Drosophila females with large (partially suppressed ovariole defects as shown in 1Biii) ovaries in piwi[1/2] and c-Fos/+;piwi[1/2]. (D) Vasa (green) and Hts (magenta) IF and DAPI (blue) staining of a wild-type germarium, with white dashed circles around GSCs. (E) Quantification of GSCs per germarium. (F) Quantification of Drosophila females with large (partially suppressed ovariole defects as shown in 1Biii) ovaries in animals with C587:Gal4 (escort cell-specific) driving Piwi and c-Fos shRNAs. Error bars represent standard deviation, and the chi-square test was used for statistical comparison.
Fig 2.
Piwi represses c-Fos in ovarian somatic and germ cells.
(A) Average mRNA levels of c-Fos and Piwi across 26 distinct adult and larval tissue types, from 4 replicate data with Affymetrix Dros Genome 2 chips [34]. The Grubb’s test shows that in the ovary c-Fos and Piwi are strongly negatively correlated in the ovary (P < 10−7). (B) RT-qPCR quantitation of RPL40 and c-Fos (standardized by rp49) mRNAs in ovarian cells or larval cells from the wild-type and piwi[1/2] mutant. c-Fos 1 and c-Fos 2 are 2 different sets of RT-qPCR primers used to quantitate the c-Fos cDNA. c-Fos (magenta) and Piwi (blue) IF of (C) a wild-type or (E) piwi[1/2] ovariole. Labeled cell types are, respectively, TF, terminal filament; CC, cap cell; GSC, germline stem cell; CB, cystoblast; EC, escort cell; SSC, somatic stem cell; FC, follicle cell; NC, nurse cell; oocyte. (D) Piwi, c-Fos, fibrillarin, and Gapdh WB analysis of cytoplasmic and nuclear fractionation of wild-type ovarian cells. (F) Quantitation of c-Fos and Piwi IF signals (intensity/μm2) in wild-type and piwi[1/2] mutant ovarian cells. (G) Left: representative c-Fos and α-tubulin WB of wild-type and piwi ovarian extract. Right: quantitation of data from triplicate c-Fos WB using α-tubulin for normalization. Error bars represent standard deviation, and the Student’s t test was used for statistical comparison.
Fig 3.
The 3′ UTR from the c-Fos mRNA is a primary piRNA precursor.
(A) Diagram of the c-Fos locus. Published piRNA sequences are represented by black dashes. (B) Piwi and Aub WB of Piwi and IgG IP from wild-type ovarian cells. (C) TaqMan RT-qPCR quantitation of putative piRNAs 1–3 (unique to the c-Fos 3′ UTR) and 2S rRNA. The 5% input serves as normalization. (D) TaqMan RT-qPCR analysis of putative piRNAs 1–3 in ovarian cells from wild-type and 3 piwi mutant allele combinations. 2S rRNA served as normalization. Values represent means of 3 RT reactions, with error bars representing standard deviation and the Student’s t test used for statistical comparison.
Fig 4.
The 3′ UTR of c-Fos mRNA induces gene repression in the Drosophila ovarian cells.
(A) Confocal images of GFP (green) IF of ovaries from Tj:Gal4; GFP-UTR (K10 control or c-Fos). (B) Quantitation of GFP IF signals in germarium or first germ cyst of Tj:Gal4; GFP-UTR. Quantitation of triplicate GFP WB and representative WB from (C) Tj:Gal4; GFP-UTR, (D) Actin:Gal4; GFP-UTR larval cells, or (E) Tj:Gal4; GFP-c-Fos UTR with or without Piwi shRNA.GFP-K10UTR was the control for (C) and (D). GFP-K10UTRs II-III and GFP-c-Fos UTRs 1–3 were generated by random integration. GFP-ss-Ftz-intron and GFP-ss-c-FosUTR were generated by PhiC31-mediated, site-specific integration. Error bars represent standard deviation, and the Student’s t test was used for statistical comparison.
Fig 5.
The 3′ UTR of c-Fos recruits Piwi.
(A) RT-qPCR analysis of Piwi and IgG IP’d mRNAs. Diagram of the c-Fos mRNA indicates the regions amplified by primer sets a-d. (B) Western blot of Piwi and IgG IP from ovarian extract of Tj:GFP-ssFtz-UTR and RT-qPCR analysis of Piwi and IgG IP’d mRNAs. Diagram of the GFP mRNA indicates the regions amplified by primer sets 1 and 2. The 5% input served as normalization. (C) As in (B), using ovarian extract from Tj:GFP-c-Fos-UTR1 (generated by random integration). (D) as in (B), using Tj:GFP-ss-c-FosUTR (generated by site-specific integration). Error bars represent standard deviation, and the Student’s t test was used for statistical comparison.
Fig 6.
Gene repression mediated by the c-Fos 3′ UTR coincides with increased primary c-Fos-specific piRNAs.
(A) Model of primary piRNA generation as a mode of gene repression predicts that the GFP-c-Fos-UTR will generate additional c-Fos piRNAs. (B) TaqMan RT-qPCR quantitation of piRNAs 1–3 unique to the c-Fos 3′ UTR from GFP-K10 UTR and 4 lines of GFP-c-Fos UTR. The 2S rRNA served as normalization. Error bars represent standard deviation, and the Student’s t test was used for statistical comparison. (C) piRNA profiling by Illumina sequencing of 2 biological replicate samples from (i) Tj:Gal4, (ii) Tj:Gal4; GFP-K10UTR, or (iii) Tj:Gal4; GFP-c-Fos UTR. Error bars represent standard deviation, and one-sided Student’s t test was performed for statistical analysis.
Fig 7.
Ectopic expression of c-Fos in somatic stem cells and follicle cells results in animal infertility, ovarian tissue dysmorphogenesis, and somatic cell disorganization.
(A) Quantitation of eggs laid by animals with Tj:Gal4 driving control, c-Fos CDS with K10 UTR or c-Fos UTR. Median and twenty-fifth and seventy-fifth percentiles are indicated by circles and lines, respectively. n = sample size. (B) Vasa (green) IF and DAPI (blue) staining of ovaries, imaged by light sheet microscopy (C) Average number of egg chambers per ovariole. (D) Tj (magenta) and Vasa (green) IF and DAPI (blue) staining of germarium and egg chambers from wild-type or Tj:Gal4; c-Fos-K10 UTR. (i) Excessive and disorganized somatic cells in the germarium, (ii) germ-cell cyst accumulation in the germarium, and (iii) excessive and disorganized somatic cells that invade into the germ-cell compartment in the egg chambers are phenotypes observed in Tj:Gal4; c-Fos-K10 UTR. Dashed ovals indicate somatic cell disorganization or invasion. Quantitation of ovaries with (E) normal structure and organization, (F) excessive and disorganized somatic cells in the germarium, (G) >3 cysts in the germarium, and (H) egg chambers with excessive and disorganized somatic cells. In each graph, genotypes (color-coded as black, dark blue, and light blue) on panel E apply to F, G, and H, and numbers below the x-axis indicate the sample sizes. Error bars represent standard deviation. The Student’s t test was used for statistical analyses.
Fig 8.
The transcriptomes are similar between the piwi mutant and somatic c-Fos overexpressing ovarian cells.
Comparison of Gene expression profiles between (A) piwi mutant and wild type or (B) c-Fos overexpression and wild type by RNA-seq. c-Fos and Piwi are indicated in the comparative graphs. Upregulation and downregulation were determined by using a false discovery rate <0.01 and > 1.5-fold. Note that c-Fos is upregulated in both (A) and (B). (C) Venn diagrams indicate the overlap of upregulated and downregulated genes (compared to wild type) in c-Fos overexpression and piwi mutant ovarian cells. (D) Graphs indicate gene ontology categories and p-values of upregulated and downregulated genes in both c-Fos overexpression and piwi mutant. (E) Proposed model of c-Fos repression by Piwi in the somatic niche (cap cells and escort cells) to influence GSCs and in the somatic/follicle cells to regulate defined cellular processes. Labeled cell types are, respectively, TF, terminal filament; CC, cap cell; GSC, germline stem cell; CB, cystoblast; EC, escort cell; SSC, somatic stem cell; FC, follicle cell; NC, nurse cell; oocyte.
Fig 9.
Proposed mechanism by which Piwi mediates c-Fos repression.
Piwi protein and unknown associated factors are recruited by the c-Fos 3’UTR to bind the mRNA. The Piwi protein complex includes the 5’ and 3’ nucleases that generate the mature primary piRNAs. Processing of the c-Fos 3’UTR into mature piRNAs cause instability and degradation of the whole mRNA.