Mitochondrial Distribution and Redox Profile of GSCs and CySCs in Adult Drosophila Testes

(A) Schematic representation of adult Drosophila testicular niche showing arrangement of different stem cell populations (GSC – Germinal Stem Cell, CySC – Cyst Stem Cell). (B-E) Differential distribution of mitochondria labelled with ATP5A (monochrome/green) in Vasa+ GSCs of wild-type fly testis with cellular boundaries marked by Dlg; B# shows digitally zoomed image of the dotted area. (F) Quantification of mitochondrial size per cell. Bars represent mean ± s.e.m., n = 10 fields of view; ***P (unpaired t-test)<0.0001. (G) Representation of mitochondrial shape,. that is circular or elongated (less circular) in two stem cell populations. Bars represent mean ± s.e.m., n = 10 fields of view; ***P (unpaired t-test)<0.0001. (H-Q) Redox profiling of testicular stem cell niche using gstD1-GFP as intrinsic ROS reporter in control (H-L) and Tj-Gal4 driven Sod1RNAi (Sod1i) testis (M-Q). (R) Quantification of gstD1-GFP mean fluorescence intensity (MFI) represented as fold change between GSCs and CySCs in control. Data denotes mean ± s.e.m,, n = 25, ***P(unpaired t-test)<0.0001. (S) Quantification of cell-specific ROS content. Data denotes mean ± s.e.m, n = 25, ***P(unpaired t-test)<0.0001. Controls are the indicated driver line crossed with Oregon R+. Scale bar - 10 µm. See also Figure S1.

Redox disequilibrium in Cyst stem cell lineage deregulates early CySCs and decreases the GSC number

(A-B) Distribution of Tj+ CySC/early cyst cell around the hub (FasIII) in control and Tj driven Sod1RNAi testis. (C-D) Represents the rosette arrangement of Vasa+ GSCs flanking the hub. The digitally magnified region around the hub (dotted line) as seen in control (C#) and Sod1RNAi (D#). (E-G) Mean number of Tj+ cells, Zfh1+ early CySCs and GSCs in control and Sod1RNAi lines shown as mean ± s.e.m, n = 10, ***P(unpaired t-test)<0.0001, **P(unpaired t-test)<0.0001. (H) Construct design of the FUCCI reporter line for tracking the cell cycle stages in Drosophila tissues, containing degrons of Cyclin B and E2F1 proteins fused with RFP or GFP. G1, S and G2/M is represented by GFP+, RFP+ and dual labelled cells respectively. (I-J) Comparative changes in the cell cycle phases (I) or Zfh1+ cells (J) present in G1, S and G2/M phase at the niche zone between control and Tj>Sod1i. Data denote mean ± s.e.m, n = 10, ***P(unpaired t-test)<0.0001, **P(unpaired t-test)<0.001. (K) Mean number of pH3+ cells, a mitotic marker. Data points denote mean ± s.e.m, n = 10. Scale bar - 10 µm. See also Figure S2.

ROS imbalance in CySCs promotes differentiation of both GSCs and CySCs

(A-D) Number of early CySCs (Zfh1+) (A’-B’), late differentiating CCs (Eya+) (A - B) and Zfh1, Eya co-expressing population (A’’-B’’) were imaged, represented schematically (C) and quantified with respect to control and Sod1RNAi lines (D). Data points represent mean ± s.e.m, n = 10, ***P(unpaired t-test)<0.0001. (E-F) The effect of Sod1 depletion in CySCs on the differentiation status of GSCs as observed using Bam-GFP reporter line. (G) The relative distance of the differentiation initiation zone (Bam+) from the hub (red) in control and Sod1i testis shown as mean ± s.e.m, n = 10, ***P(unpaired t-test)<0.0001. (H-J) The shape and size of the spectrosomes marked with α-spectrin (green) were imaged (I’’-J’’) and quantified for their distance from the hub (marked with asterisk) (H), n = 10, ***P(unpaired t-test)<0.0001. Branched fusome marks differentiating populations (I’-J’). (K-L) Comparative staining of CySC-GSC contacts through adherens junction using E-cadherin (monochrome/green). Dotted area near the hub has been expanded as inset to show loss of E-cadherin network. (M) E-cadherin intensity histogram plot generated from ImageJ representing the mean intensity of expression in the region flanking the hub and GSCs. (N-O) Fold change in expression of Stat dependent transcripts Socs36E (N) and Ptp61F (O) among control and Sod1i niche, obtained through qPCR. Data is shown as mean ± s.e.m, n = 3, ***P(unpaired t-test)<0.0001. ns – not significant. Scale bar - 10 µm. See also Figure S3.

Altered CySC redox state affects niche maintenance signals.

(A-B) The expression of cell polarity marker Dlg (red) in control and Sod1i stem-cell niche (A’’-B’’), (C-D) Representative image showing pErk distribution parallelly with Tj+ cells and its expression pattern through Fire LUT (C’’-D’’). Scale bar - 10 µm. Mean florescence intensity (MFI) corresponding to Dlg level (E) was quantified, n = 75, ***P(unpaired t-test)<0.0001. (F) MFI of total pErk expression was quantified, n =200, ***P(unpaired t-test)<0.0001. (G’-H’) Representative image showing distribution of Patched (Ptc) in Tj+ cells in control and Tj>Sod1i. (I-J) Quantification of Ptc (I) and Hh effector Ci (J) expression through fluorescence intensity as mean ± s.e.m, n (Ptc) = 90, n (Ci) = 200, ***P(unpaired t-test)<0.0001 (K-L) The number of Vasa+ (K) and Tj+ (L) cells across control, Sod1i, and Sod1i/Hhi rescue samples depicted as mean ± s.e.m, n = 10, ***P(unpaired t-test)<0.0001. See also Figure S4.

Low CySC ROS sustains GSC maintenance

(A-B) Bar graphs illustrating variations in Vasa+ GSC (A) and Tj+ CySCs (B) numbers upon overexpressing Sod1 (Tj>SodOE), represented as mean ± s.e.m, n = 10, ***P(unpaired t-test)<0.0001, *P(unpaired t-test)<0.01. (C-E) The spectrosomes labelled with α-spectrin (α-spec) were imaged (C’-D’) and their number was quantified (E), n = 10, **P(unpaired t-test)<0.001. (F-H) The initiation of gonialblast differentiation was determined by measuring the distance of Bam+ zone from hub (red), n = 10, *P(unpaired t-test)<0.01 (F), (G’’-H’’) shows distribution of Bam+ reporter expressing cells. Scale bar - 10 µm. See also Figure S5.

Proposed role of intercellular redox balance in germline maintenance and differentiation

GSC and CySC are associated with different mitochondria abundance which correspond to the presence of a redox differential between two stem cell populations. Disequilibrated redox potential among the two populations due to depletion of Superoxide dismutase, enhances CySC proliferation and precocious GSC differentiation, thereby disrupting the homeostatic balance between the two stem populations.