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 – Cystic Stem Cell). (B-E) Differential distribution of mitochondria labelled with ATP5A (green) in Vasa+ GSCs and Tj+ CySCs of wild-type fly testis; B# shows digitally zoomed image of the dotted area. (F) Relative mitochondria density profile of GSCs and CySCs denoted through cooccurrence of ATP5A with Tj or Vasa. A.U. – arbitrary units. (G-P) Redox profiling of testicular stem cell niche using gstD1-GFP as intrinsic ROS reporter in control (G-K) and Tj-Gal4 driven Sod1RNAi (Sod1i) testis (L-P). (Q) Gaussian distribution curve for the variations in gstD1-GFP fluorescence at CySC-GSC intersection region (inset) in Tj-Gal4 control and Sod1i testis. (R) Quantification of mean gstD1-GFP fluorescence intensity (MFI) upon Sod1 knockdown represented as fold change over control. Data denotes mean ± s.e.m, n = 25 (S). Colocalization intensity between Vasa and gstD1-GFP expressed through Manders’ co-localization coefficient (MCC) among control and Sod1i cells (G). 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 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. (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 total cell population (I) or Zfh1+ cells (J) present in G1, S and G2/M phase at the niche zone between control and Tj>Sod1i. Data points denotes 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 cyst cells (Eya+) (A-B) and Zfh1, Eya co-expressing population were imaged (A’’-B’’), represented schematically (C) and quantified with respect to control and Sod1RNAi lines (D). Data points represent mean ± s.e.m, n = 10. (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. (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. 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) 3D surface intensity histogram for E-cadherin expression near the hub. (N-O) Fold change in expression of Stat dependent transcripts Socs36E (N) and Ptp61F (O) among control and Sod1i niche. Data is shown as mean ± s.e.m, n = 3. 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. (E) Segregated pErk expression patterns in CySCs near the hub and differentiating cyst cells (CC), mean ± s.e.m, n = 200. (F) Estimation of the differentiation zone through quantification of high pErk expressing regions. (G-I) Activation of PI3K/Tor in somatic lineage was adjudged through reporter p4E-BP in Tj>Sod1i (G’-H’) and levels of the active form was quantified (I), MFI – mean fluorescence intensity shown as mean ± s.e.m, n = 40. (J) Induction of Cyclin D in Sod1i represented as fold change over controls by mean ± s.e.m, n = 3. (K-L) The number of Vasa+ (K) and Tj+ (L) cells across control, Sod1i, Sod1i/PI3KDN (dominant negative allele) and Sod1i/Hhi rescue samples depicted as mean ± s.e.m, n = 10. (M’-N’) Representative image showing distribution of Patched (Ptc) in Tj+ cells in control and Tj>Sod1i. (O-P) Quantification of Ptc (O) and Hh effector Ci (P) expression through fluorescence intensity as mean ± s.e.m, n (Ptc) = 90, n (Ci) = 200. 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. (C-E) The spectrosomes labelled with α-spectrin (α-spec) were imaged (C’-D’) and their number was quantified (E). (F-H) The initiation of gonialblast differentiation was determined by measuring the distance of Bam+ zone from hub (red), n = 10 (F), (G’’-H’’) shows distribution of Bam+ reporter expressing cells. Scale bar – 10 µm. See also Figure S5.

Proposed role of intercellular redox gradients in GSC maintenance and differentiation

Mitochondrial abundance in GSC-CySC boundary maintains redox differential between two stem cell population. Disequilibrated intercellular redox gradient enhances precocious GSC differentiation and enhances CySC proliferation, thereby disrupting the homeostatic balance between the two stem populations.