Wnt signaling-mediated redox regulation maintains the germ line stem cell differentiation niche
- Stowers Institute for Medical Research, United States
- University of Kansas School of Medicine, United States
- Tsinghua University, China
Figures
Figure 1 with 1 supplement
Canonical Wnt signaling in ISCs promotes germ cell differentiation.
(A) The Drosophila germarium dividing into three regions 1, 2a, 2b and 3. Abbreviations: TF-terminal filament; CPC-cap cell; ISC-inner germarial sheath cell; FC-follicle cell; GSC-germ line stem cell; CB-cystoblast; DC-developing cyst; SS-spectrosome; FS-fusome. In B–L, cap cells are highlighted by broken ovals, whereas CBs and cysts are indicated by arrowheads and arrows, respectively. (B) In the c587>>UAS-GFP germarium containing two GSCs (spectrosomes indicated by arrowheads) close to cap cells, one CB and a few differentiated cysts are surrounded by GFP-positive ISCs. (C–E) In armKD1 (C) dshKD1 (D) germaria, many spectrosome-containing CBs accumulate far away from cap cells. (E) Quantification results on the percentages of the germaria exhibiting the germ cell differentiation defect (≥4 CBs). (F–H) fz fz2 double knockdown (F), axn- (G), and sgg-overexpressing (H) germaria contain excess CBs. (I–L) In arm*-overexpressing control (I) and dshKD (K, L) germaria, GSC progeny differentiate into cysts containing a branched fusome (arrow). J: Quantification results.
Figure 1—figure supplement 1
Wnt receptors FZ and FZ2 function redundantly in ISCs to promote germ cell differentiation.
Broken ovals highlight cap cells and GSCs, while arrowheads denote spectrosomes in CBs (A–C, E). (A–D) fzKD (B) and fz2KD (C) germaria contain 0 and 1 CB, respectively, in comparison with the gfpKD germarium carrying one CB (A). (D) The quantification results on CB numbers in one-week-old (1w) and two-week-old (2w) control and single knockdown germaria. (E, F) fzKD fz2KD (E) germarium contains significantly more CBs. (F) The quantification results on CB numbers.
Figure 2
Wnt signaling is required in adult ISCs to promote germ cell differentiation.
Broken ovals highlight germ line stem cells (GSCs), whereas arrowheads indicate cystoblasts (CBs). Two experimental regimens 25–25 and 25–29 mean the females cultured under 25°C or 29°C for one additional week after reaching adulthood at 25°C, respectively. (A, A′) Control c587>>gal80ts germaria contain one or two CBs under the 25–25 (A) or 25–29 (A′) condition. (B) Quantification results on the percentages of the germaria carrying 4 or more CBs show that adult stage-specific arm or dsh knockdown causes an accumulation of more CBs in comparison with the control. (C–F) armRANi- (C, D) or dshRNAi- (E, F) carrying germaria contain one or two CBs under 25–25, indicating that RNAi-mediated arm or dsh knockdown in adult inner germarial sheath cells (ISCs) under 25°C is very limited based on the germ cell differentiation phenotype. (C′–F′) armRANi- (C′, D′) or dshRNAi- (E′, F′) carrying germaria contain many more CBs under 25–29, indicating that RNAi-mediated arm or dsh knockdown under 29°C in adult ISCs leads to the severe germ cell differentiation defects.
Figure 3 with 1 supplement
Wnt signaling maintains the differentiation niche by promoting cell proliferation and survival.
Cap cells are highlighted by broken ovals. (A) The control germarium contains PZ1444-labeled cap cells and ISCs (one by arrow). (B–D) The armKD (B) and dshKD (D) germaria maintain zero and one ISC (arrow), respectively. (C) Quantification results on ISC numbers (mean ± standard deviation; Student's t-test is used to calculate p values). (E, F) c587-directed axn- (E) and sgg- (F) overexpressing germaria contain no ISCs (E) and a few ISCs (F, arrows), respectively. (G) c587-directed arm* expression drastically increases the ISC number (one by arrow). (H–L) c587-mediated knockdown of arm (I) and dsh significantly decreases BrdU-positive PZ1444-labeled ISCs (arrows), whereas c587-directed arm* expression (K) significantly increases BrdU-labeled ISCs in comparison with the control (H). The arrowhead in I indicates a BrdU-positive germ cell cyst. J and L show quantification results. (M–Q) c587-mediated knockdown of arm and dsh (N) significantly increases TUNEL-positive PZ1444-labeled ISCs (arrows), whereas c587-directed arm* expression (P) significantly decreases TUNEL-positive ISCs in comparison with the control (M). (O, Q) Quantification results.
Figure 3—figure supplement 1
Hyperactive Wnt signaling increases the ISC population.
Cap cells are highlighted by ovals, while some of the ISCs are denoted by arrowheads. (A) In the control germarium, PZ1444 labels cap cells and ISCs. (B–D) The axnKD (B) and sggKD (C, D) germaria accumulate more ISCs.
Figure 4
Wnt signaling in ISCs prevents BMP signaling in the differentiated germ cell zone and maintains long ISC cellular processes.
Ovals indicate GSCs, whereas arrows in A-F′ denote CBs or CB-like single germ cells. (A′–F′) Images only show green fluorescence of (A–F). (A, A′) In the control germarium, GSCs are positive for pMad, but one CB is negative. (B–C′) In the armKD (B, B′) and dshKD (C, C′) germaria, GSCs are pMad-positive as in the control. Although most of the accumulated CBs are negative for pMad, some CBs are pMad-positive (arrows). (D, D′) The control germarium shows that GSCs are positive for Dad-lacZ expression, but one CB is negative. (E–F′) In the armKD (E, E′) and dshKD (F, F′) germaria, GSCs are Dad-lacZ-positive as in the control. Some of the accumulated CBs are negative for Dad-lacZ, but the other ones exhibit low Dad-lacZ expression (arrows). (G) RNA sequencing (RNA-seq) results on the purified ISCs show that mRNA levels for the known BMP pathway components, including dpp, gbb (also encoding a BMP ligand) and dally, remain largely unchanged in axn-overexpressing ISCs or dskKD ISCs in comparison with the control (FKPM = reads per kilobase per million mapped reads). (H, H′) Our CB and GSC quantification results indicate that the heterozygous dpp mutations have partial suppression on the germ cell differentiation defects caused by dshKD, and the suppression is not due to the changes in GSC numbers (50 germaria examined for each genotype). (I–K′) armKD (J, J′) and dshKD (K, K′) ISCs lack their CD8GFP-positive cellular processes extending into the accumulated CBs (arrowheads) in contrast with the control ISCs extending the cellular processes wrapping up underneath germ cells (arrowheads, I′). (I′–K′) A higher magnification of highlighted areas in (I–K).
Figure 5
Piwi mRNA and protein expression remain normally expressed in the Wnt signaling-defective ISCs.
PZ1444 is used to highlight ISCs. (A) The control germarium shows that ISCs (arrow) express more Piwi proteins than cap cells (oval) and underneath germ cells. (B, C) The remaining armKD (B) and dshKD (C) ISCs (arrows) still retain high Piwi protein expression. (D) Quantification results show that Piwi protein levels remain largely unchanged in the remaining armKD1, armKD2, and dshKD2 ISCs in comparison with the wild-type control ISCs, but it appears to increase its expression in dshKD1 ISCs. (E) RNA-seq results on the purified ISCs show that piwi mRNA expression levels are not changed in axn-overexpressing ISCs, but increase in dskKD ISCs, in comparison with the wild-type control ISCs.
Figure 6 with 2 supplements
Wnt signaling maintains the reduced redox state in ISCs, promoting germ cell differentiation.
(A) RNA-seq results of the purified ISCs show that GstD2, GstD4, GstD10, and GstE3 mRNA expression levels are significantly lower in axn-overexpressing or dskKD ISCs than the control ISCs. In B–B′″, D–I, L–N, and P–R, cap cells and GSCs are highlighted by broken ovals, some CBs are indicated by arrowheads and some PZ1444-positive ISCs are denoted by arrows. (B–B′″) The dshKD germarium (B′) exhibits a drastic increase of dihydroethidium (DHE) fluorescence in the anterior region, including ISCs, GSCs, and early GSC progeny, in comparison with the control germarium (B). GST2 (B″) or catalase (CAT) (B′″) overexpression in ISCs restores low DHE fluorescence in the dshKD germariun. (C–J) GST2 (D, G), CAT (E, H), or superoxide dismutase1 (SOD1) (F, I) overexpression in dshKD ISCs significantly decreases CBs (arrowheads; D–F) and significantly increase ISCs (arrows; G–I) in comparison with dshKD. (C, J) Quantification results on CB and ISC numbers, respectively (for each genotype, 50 or more germaria examined). (K) Quantitative RT-PCR results on the purified ISCs show that GstD2 knockdown by RNAi Line 1 (GstD2KD1), but not the line 2 (GstD2KD2), significantly decreases GstD2 mRNA levels in comparison to the control (c587). (L–O) GstD2KD1 (L) and CatKD germaria contain 4 CBs and 0 CB, respectively, but the GstD2KD1 CatKD germarium (N) contains 5 CBs (O: CB quantitative results). (P–S) GstD2KD1 CatKD germarium (R) contains fewer ISCs (arrows) than GstD2KD1 (P) and CatKD (Q) germaria (S: ISC quantitative results). Note: PZ1444 expression appears to be downregulated in GstD2KD1 CatKD ISCs.
Figure 6—figure supplement 1
Wnt signaling maintains the reduced redox state in ISCs.
(A) The control ovariole exhibit low DHE staining in the germarial region and gradually upregulates DHE staining in differentiated germ cells in egg chambers. B′–F′ only show DHE staining in B–F. In B–F′, broken ovals highlight cap cells, whereas arrowheads point to PZ1444-labeled ISCs. (B, B′) Control germarium shows low DHE staining in ISCs and cap cells (broken oval). (B–F′) armKD (C–D′) and dshKD (E–F′) ISCs as well as their underneath germ cells increase DHE staining. (G–I) c587-directed GST2 (H) or CAT (I) overexpression in ISCs restores low DHE fluorescence in the anterior region of the dshKD2 germariun (G). Bars in A and B (B–I in the same scale) represent 20 μm and 10 μm, respectively.
Figure 6—figure supplement 2
Reduced redox state is important for ISC maintenance.
In A–F, broken ovals highlight cap cells and GSCs, whereas arrowheads point to CBs. (A–G) c587-directed overexpressing GST2 (A), CAT (B), or SOD1 (C) in ISCs does not affect GSC maintenance and germ cell differentiation, but drastically reduces CBs in the dshKD2 germaria (D–F). (G) GSC quantification results. In H–M, broken ovals highlight cap cells and GSCs, whereas arrows point to PZ1444-labeled ISCs. (H–M) c587-directed overexpressing GST2 (H), CAT (I), or SOD1 (J) in ISCs does not affect ISC maintenance, but drastically rescues the ISC loss phenotype in the dshKD2 germaria (K–M).
Figure 7
Wnt2 and Wnt4 function redundantly in ISCs to promote germ cell differentiation.
(A) RNA-seq results show that Wnt2 and Wnt4 are highly expressed in the purified ISCs in comparison with Wnt5 and Wnt6. In B–C′ and E–H, cap cells and GSCs are highlighted by ovals, CBs are indicated by arrowheads. (B–D) Wnt2 (B–B″) or Wnt4 (C, C′) knockdown by independent RNAi lines causes a slight accumulation of CBs. (D) Quantification results on CB numbers (the numbers on the top of bars represent the p values in comparison with the wild-type control, whereas those on the lines indicate the p values between single and double knockdown). (E–H) Knocking down both Wnt2 and Wnt4 significantly increases CBs. (I) A schematic diagram showing that autocrine Wnt2 and Wnt4 signals control ISC maintenance and promote germ cell differentiation at least in part by maintaining the reduced redox state and preventing BMP signaling.
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Wnt signaling-mediated redox regulation maintains the germ line stem cell differentiation niche
eLife 4:e08174.
https://doi.org/10.7554/eLife.08174
