Introduction

Studies from the past few decades have shown the apparent role of ROS in influencing various biological processes^1-3. ROS are usually produced in specific cellular compartments close to their target molecules to regulate important signaling pathways.⁴. Mitochondria, a major source of oxidant species^5,6, localize dynamically towards nucleus, effecting oxidation induced reshaping of gene expression profiles⁷. Localized ROS production are contributed by NADH oxidases distributed across different cellular regions⁸. Intracellular hydrogen peroxide gradients maintained by thioredoxin system allow intercompartmental exchanges among endoplasmic reticulum (ER), mitochondria and peroxisomes^9-11.

The process of redox relays is conserved and plays a fundamental role in self-renewal and differentiation of stem cell populations¹². Stem cells whether embryonic or adult maintain a low redox profile, characterized by subdued mitochondrial respiration^13-17. Levels of ROS are tightly regulated and elevated amounts promote early differentiation and atypical stem cell behaviour¹⁸. However, leading evidences suggest that many transcription factors require oxidative environment for the maintenance of pluripotent states. For instance, physiological ROS levels play a crucial role in genome maintenance of embryonic stem cells¹³. Multipotent hematopoietic progenitors, intestinal stem cells ¹⁹ and neural stem cells require relatively high baseline redox for their maintenance^14,16,20. Suppression of Nox system or mitochondrial ROS compromises the maintenance of these self-renewing populations and promote their differentiation or death¹⁸. These evidences point towards the essentiality of a well-tuned redox state for balancing the pluripotent and differentiated states. However, how stem cells regulate their redox potential by possessing a restrained oxidant system is not very clear.

We addressed this fundamental question in two-stem cell population-based niche architecture in Drosophila testis. The testicular stem cell niche is composed of a central cluster of somatic cells called the hub which contacts eight to eleven GSCs arranged in a round array^21,22. A pair of cyst stem cells (CySCs) enclose each GSC and make their independent connections with the hub and GSCs via adherens junctions^23-28. The hub cells secrete self-renewal factors essential for both GSCs and CySCs maintenance involving signalling cascades like Jak-Stat signalling^29,30, BMP (Bone Morphogenetic Signalling)³¹, Hedgehog signalling³². The hub and CySCs produce BMPs which repress GSC differentiation by suppressing transcription of bag-of-marbles (Bam)^31,33. Concerted asymmetric division of both GSC and CySC is essential for proper cyst formation³⁴. A developing cyst contains a dividing and differentiating gonialblast encircled by cyst cells that provide nourishment to developing spermatids^35,36. However, the maintenance of GSCs in the spatially controlled microenvironment is still not very clear.

We found the existence of a balanced differential redox state between GSC and CySC essential for niche homeostasis. CySCs by virtue of their higher redox threshold and more clustered mitochondria generate an intercellular redox state that affects the physiological ROS levels in GSCs. Alterations in CySC redox state resulted in self-renewal and differentiation propensities of the GSCs. Intercellular redox-imbalance induced niche disequilibrium through precocious differentiation of GSCs and deregulated CySC proliferation, due to activation of pro-proliferative transductions and loss of cell-cell contact signalling. Our results indicate a sophisticated interplay in these two stem cell populations where a higher redox state in CySCs not only supports their self-renewing processes but also non-autonomously promotes the maintenance of GSCs.

Results

CySCs maintain higher differential ROS in comparison to adjacent GSCs

The apical region of the Drosophila testis incorporates a cluster of differentiated cells, the hub surrounded by GSCs in a rosette arrangement with each GSC being enclosed by two CySCs (Fig. 1A). Immunostaining ATP5A subunit of mitochondrial ATPase and marking cell boundary by discs-large (Dlg) indicated different mitochondria morphology (Fig. 1G) and distribution among these two stem cell populations. In wild type adult testis, we observed fragmented mitochondria in Vasa⁺ GSCs as compared to a fused organization in CySCs (Fig. 1B#, C, D, and S1B-B’’). The number of mitochondria per cell was higher in CySC (Fig 1F) and had a more elongated shape (reduced circularity) than the GSCs (Fig 1G). The ATP5A labelling overlapped with TFAM-GFP, a mitochondrial transcription factor³⁷, further confirming the patterning observed between these two stem cell populations (Fig. S1A). Mitochondria localization and dispersion pattern in GSCs and CySCs, corresponded with the intensity variance of the ROS reporter line gstD1-GFP³⁸ at the niche (Fig. 1L and S1C’’’). CySCs exhibited a higher baseline level of ROS compared to GSCs (Fig.1R).

Figure 1.

To affirm the role of CySC in influencing GSC redox state, we asked if disrupting the dismutases in CySCs would influence the redox state of GSCs. We depleted the expression of superoxide dismutase 1 (Sod1) in the hub, CySCs and early differentiating cyst cells (CCs) using Tj-Gal4³⁹ driver (Tj>Sod1i) that resulted in a net increase in gstD1-GFP intensity (Fig. 1Q, D’’ and S1E). The elevated redox state in CySCs led to a corresponding increase in ROS levels in adjacent GSCs, as evidenced by the increased gstD1-GFP intensity (Fig.1Q and S) and an overall rise in superoxide levels, confirmed through DHE fluorescence analysis (Fig.S1F). This suggests a potential redox crosstalk between these stem cell populations, where the redox state of CySCs influences the oxidative status of neighbouring GSCs.

Balanced CySC redox profile is crucial for its proliferation and GSC maintenance

Alongside the differential redox profile, we observed that elevated ROS levels in Tj>Sod1i had a striking effect on the increase of DAPI⁺ nuclei at the testis tip (Fig. 2A’’). This overcrowding was attributed to both an increase in the number of Tj⁺ CySCs and early differentiating CCs, as well as their positional shift (Fig. 2A’, B’, and E). To further validate these findings, we used an alternative driver line, C-587-Gal4, which specifically drives expression in CySCs and CCs. This also resulted in an increase in the total number of Tj⁺ cells (Fig. S1H’, and M) although to a lesser extent than Tj-Gal4, suggesting that signals from the hub may also partially influence CySC proliferation. Given the stronger phenotypic effect observed with Tj-Gal4, we proceeded with this driver line for subsequent experiments.

Figure 2.

The enhancement in the number of CySCs was also reflected by ∼2-fold change in Zfh1⁺ cells (Fig. 2F, S2R-S). However, Vasa⁺ cells flanking the hub (GSCs) showed a substantial reduction in number (Fig. 2C’’-D’’ and G). This decline was further validated by western blot analysis, which revealed lower overall detectable levels of Vasa protein, indicating a potential impact of the altered redox state on GSC maintenance and stability. (Fig. S2N). C-587-Gal4 also showed a reduction in the number of neighbouring GSCs (Fig.S1N). The observations were further verified using Sod1i localized in different chromosome to avoid any chromosome or balancer-based biasness (Fig. S1I-L and O-P). However, the phenotypic effect of higher ROS was limited to its origin in CySCs only because ablation of GSC redox status did not cause any marked change in niche composition. Nos-Gal4 driven knock-down of Sod1 in GSCs did not result in significant change in CySC number (Fig. S2A’, B’ and E) but effected a considerable reduction on Vasa⁺ cells (Fig. S2C’, D’ and F), aligning with previous observations¹⁷. Although Sod1 is the predominant enzyme which also localizes in the intermembrane space of mitochondria, we observed a similar result upon depleting the matrix localized Sod2 in both GSCs and CySCs (Fig. S2G-M), indicating the involvement of mitochondrial ROS for the observed cellular phenotypes.

To confirm the active proliferation of cells and rule out the possibility of arrested growth, we utilized fly-Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) system⁴⁰ which comprises of two reporter constructs marking G1/S transition (green), S (red) and G2/early mitosis (yellow) (Fig. 2H). Testes from Tj>Sod1i exhibited an approximately two-fold increase in cell number across all stages of the cell cycle (Fig. S2O). However, the percentage of cells in each phase remained unchanged (Fig 2I), indicating that the observed increase in different phases is a consequence of more cells entering proliferation rather than alterations in the duration of different cell-cycle stages. The enhanced number of dividing cells was contributed mainly by progressive division of CySCs, showing >2-fold difference in the accumulation of Zfh1⁺ nuclei in S and G2/M phases (Fig. 2J, S2RV-VII and SV-VII). The number of Zfh1 labelled cells in G1/S transition was also substantially more (Fig. S2RIII and SIII), indicating dysregulated redox ensued an increased mitotic index of CySCs. The enhanced mitotic activity of these proliferating niche cells is further supported by increased phospho-histone H3 (PH3) incorporation, indicating a higher mitotic frequency compared to the control (Fig. S2T, U), along with elevated cyclin D expression (Fig. S2Q). Downregulation of Sod1 in GSCs through Nos-Gal4 demonstrated no significant change in G1/S, S and G2/M phase numbers, confirming our previous observation that altering ROS levels in GSCs does not have any non-autonomous effect on CySCs (Fig. S2P).

CySC induced ROS couples precocious differentiation of stem cell lineages

Since in Tj>Sod1i testes, the increased number of Tj⁺ cells, marking CySCs and early differentiating cells, exceeded the number of CySC-specific Zfh1⁺ cells (Fig. 2J and S2O), we checked for parallel enhancement of cellular differentiation using the corresponding marker, Eya (Eyes absent).

We found that Eya⁺ cells clustered towards the hub along with a significant increase in their number in Sod1 depleted CySCs (Fig. 3A, B and D). This increased number of Eya⁺ cells does not suggest that these differentiating cells are proliferative. Instead, we propose that the knockdown of Sod1 may alter the timing or regulation of cyst cell differentiation, leading to an accumulation of Eya⁺ cells near the niche. The supposed premature expression of Eya resulted in a population of differentiating cyst precursor cells co-expressing Eya-Zfh1 (Fig. 3A’’, B’’ and D). This population was found to be deviating from the normal partitioning of wild-type representative populations (Fig. 3C). In contrast, depletion of Sod1i in the germline lineage using Nos-Gal4 did not result in a similar phenotypic arrangement (Fig. S3A-D). These findings suggest that alterations in the redox state of GSCs can impact their numbers, even when the changes are induced non-autonomously through CySCs.

Figure 3.

Together with reduction in Vasa⁺ GSCs shown earlier (Fig. 2G), we also detected gonialblast expressing differentiation-promoting factor Bam to be much closer to the hub in Tj>Sod1i testes (Fig. 3E’’ and F’’). The mean distance between the hub and Bam⁺ cells was found to be shortened by ∼7 microns (Fig 3G). The relative advancement of spermatogonial differentiation towards the hub was also ascertained by tracking the transformation of GSC-specific spectrosome into branched fusomes connecting the multi-cell gonialblast (Fig. 1A). The branching fusomes were found more proximal to hub than controls (Fig. 3H, I’’ and J’’), along with parallel reduction in number of spectrosomes, implying a decline in early-stage germ cells (Fig. S3F). This loss could possibly be attributed to loss of intercellular contacts, particularly with hub cells (Fig. 2C# and D#). The observation corroborated with decreased expression pattern of E-cadherin in Tj>Sod1i flanking the hub and GSCs (Fig. 3K-M) and could be one of the reasons for GSCs dissociation and differentiation (Fig. 2D#), Disengagement of GSCs from the hub caused reduction in Socs36E and Ptp61F transcripts that act as drivers of E-cadherin expression (Fig. 3N and O). These results suggest that disruption of intercellular redox gradient affected the premature differentiation of stem cells in the niche.

Disrupted niche architecture compromises GSC-CySC communication

GSCs and CySCs intercommunicate through several factors such as EGFR, PI3K/Tor, Notch⁴¹ to support CySC maintenance⁴². EGF ligands secreted by spermatogonia maintain differences in cell polarity and segregates self-renewing from differentiating populations⁴³. Tj>Sod1i cells showed lower levels of cell-polarity marker Dlg (Fig. 4A”, B” and E), that expanded more into the differentiated zone (Fig. S4A). Since, Dlg expression is dependent on EGFR⁴⁴, impairment in its reception was reflected in net reduction in pErk levels across sections in Sod1i testis (Fig. 4C’, D’ and F). This reduction in overall Erk levels may be attributed to the loss of cell contact and altered GSC-CySC balance in Sod1i testis, probably resulting in lower EGF response. The uncontrolled proliferation of cyst cells correlates with increased levels of self-renewal inducers, including the elevated expression of Hedgehog (Hh) pathway components ³² in Tj>Sod1i testis. The depletion of Sod1 in CySC resulted in elevated expression of Hh receptor Patched (Ptc) (Fig. 4I, S4V) which extended to regions farther from the hub, overlapping with expanded Tj⁺ cells (Fig. 4G and H). Since, Ptc itself is a transcriptional Hh target, its expression in CySCs suggests active Hh signalling⁴⁵, further supported by higher levels and a similar pattern of Hh transcriptional effector Cubitus interruptus (Ci) in Sod1i testis (Fig. 4M’’, N’’, J and S4W). Since, Ptc, Ci, and a GPCR-like signal transducer Smoothened (Smo) are all transcriptionally driven by Hh, and Hh itself is regulated by higher levels of Ci^46,47, we expectedly found higher levels of transcripts for these pathway genes (Fig. S4C-F). The overt proliferation of CySCs under high ROS conditions was suppressed by reducing gene dosage using Hh-RNAi (Fig. 4L and Fig. S4G-I), which was associated with rescue in the GSC population (Fig. 4K and S4J-L). The levels of Sod1 transcripts under single and Sod1, hh double depletion were almost similar (Fig. S4M). To determine whether the changes observed in the germ cell population were a sole consequence of Sod1 knockdown in CySC, we manipulated CySC numbers using UAS-Ci and assessed their impact on neighbouring GSCs. Ci overexpression driven by Tj-Gal4 led to a higher number of Tj⁺ cells (Fig. S4N, and R’’) and a complete loss of Vasa⁺ GSCs (Fig. S4O, and R’’’). However, the effect on CySC and GSC population was less severe when driven by C-587 Gal4 (Fig.S4N, O, Q’’and Q’’’).

Figure 4.

Elevating CySC antioxidant defence promotes GSC self-renewal

To further substantiate the role of ROS in coordinating the stem cell populations, we strengthened the cellular defences against oxidants by overexpressing Sod1 in CySCs. We checked the overall ROS level using DHE (Fig. S5I) and monitored the relative GSC/CySC populations. Together with reduction in superoxide levels, we observed an increase in the number of Vasa⁺ cells (Fig. 5A and S5A’’-B’’), and a slight reduction in Tj⁺ cells (Fig. 5B and S5E’’-F’’). Any morphological anomalies associated with cell-cell adhesion or abnormal cellular dispersion, was not observed (Fig. S5A-B and E-F). Enhancing the levels of Sod1 in GSCs promoted the growth of GSC-like cells (Fig. S5C’’, D’’ and J) but did not have any prominent effect on CySCs (Fig. S5G’’, H’’ and K). The increment in Vasa⁺ GSCs under CySC-induced low redox conditions was also represented by a parallel increase in spectrosome number (Fig. 5C-E). The uptick in GSC number due to scavenging of ROS in CySC, might be a result of delayed differentiation as indicated by displacement of Bam⁺ zone away from hub; thus, confirming non-cell-autonomous role of CySC ROS in maintaining GSC fate (Fig. 5F-H). The data suggest that reducing redox profile of CySCs differentially affected their own self-renewing propensities along with GSC maintenance and optimum redox state of both the stem-cell populations is controlled by CySCs.

Figure 5.

Figure 6.

Discussion

ROS is a crucial mediator of stem cell maintenance where these species act as second messengers to induce different post-translational protein modifications thereby, affecting cell fate^48,49. The effect of ROS is mainly considered to be autocrine in nature, where generation of these oxidative species is usually restricted to specific cellular compartments, ensuring they act close to their targets. Recent studies have reported non-autonomous generation of ROS by NOX enzymes or upon stimulation by growth factors, to play a critical role in regenerative growth^50-52. However, in either of the cases, the target cell serves as the source and the site of response. Among ROS, hydrogen peroxide (H₂O₂) stands out due to its stability and membrane permeability⁵³, allowing it to diffuse from its source⁵⁴. Our observations in Drosophila testicular stem cell niche suggested a previously unaccounted role of superoxide dismutases in maintaining niche homoeostasis. The redox perturbations affected cyst stem cell dynamics which indirectly influenced the germline. The higher ROS threshold of CySCs maintained the physiological redox state of GSCs. Reduced ROS in GSC was accompanied with its proliferation⁵⁵, phenocopied when Sod1 was overexpressed in its neighbours (Fig. 5). However, we feel that net alterations in GSC state were the combinatorial effect of oxidative stress and niche alterations brought about by CySC deregulation.

GSCs are anchored to the hub cells via adherens junctions, allowing them to receive crucial maintenance signals²⁸. These include Bone Morphogenetic Proteins (BMPs) such as Decapentaplegic (Dpp) and Glass bottom boat (Gbb), which activate the BMP pathway³¹ required for GSC self-renewal, and Unpaired (Upd), which triggers the JAK-STAT pathway to promote GSC adhesion to the hub⁵⁶. The suppression of these pathways activates Bam, acting as a switch from transit-amplifying cells to spermatocyte differentiation^57,58. E-cadherin levels ensure only undifferentiated stem cells persist within the niche, while differentiating GSCs lose the competition for niche occupancy²⁶. Elevated ROS in CySC possibly affected STAT phosphorylation through S-glutathionylation^59,60, reducing the levels of STAT-dependent transcripts such as Socs36E⁶¹, Ptp61F and E-cadherin. This depletion compromises GSC adhesion, resulting in detachment from neighboring cells, similar to E-cadherin loss conditions. ^56,62-64. This resulted in compromised GSC-CySC paracrine receptions such as EGFR which play a crucial role in the maintenance of cell-polarity that segregates self-renewing CySC populations from the ones receiving differentiation cues ^43,65,66, leading to the accumulation of cells co-expressing both stemness and differentiation markers. In contrast to GSCs, ROS imbalance in CySCs induced accelerated proliferation as well as differentiation due to deregulation of EGFR and Hedgehog pathways. Although, redox modulation of EGFR pathway components has been previously demonstrated⁶⁷, in this study we found Hedgehog to be probably susceptible to redox regulation. The redox dependent induction of Hedgehog probably, contributed to enhancement in CySC numbers which partly contributed to depletion of GSCs.

However, we do not rule out the possibility of hub cells playing an equivalent role in GSC maintenance, given their parallel effect in suppressing GSC differentiation³⁰. Tj-Gal4 depleting Sod1 in both the hub and CySCs, presented a stronger phenotype that CySC specific driver C587-Gal4, indicating potential hub contributions. We had also tried using E-132 Gal4 particularly expressed in the hub to express Sod1i lines in hub cells but were challenged by substantial adverse effect on fly viability. While ROS accumulation typically enhances EGFR signaling promoting premature GSC differentiation⁶⁸, the loss of EGFR in somatic cells increases the number of GSCs⁶⁵ and reduced EGFR activity leads to fewer somatic cells ⁶⁹. However, we feel that the response of germline to EGFR modulation is more context-dependent as Sod1 depletion caused parallel reduction of pErk level and GSC number. We tried an EGFR gain-of-function rescue of CySC depletion but the driven progenies were lethal in the absence of Sod1. We also avoided usage of Gal80 dependent clonal populations to maintain a homogenous genetic background and prevent false readouts due to diffusion of ROS signals in unaltered neighbourhood.

The proposed concept of intercellular ROS communication can be of importance in deciphering the biochemical adaptability and plasticity of different niches influencing stem cell fate, ensuring niche size and architecture to prevent stem cell loss and aging. This has been observed in neural stem cells where inflammation-induced quiescence recovers the regenerating capacity of aging brain⁷⁰. In addition to metabolic variations, dependence of the germline on somatic neighbours for its redox state might be one the reasons behind its presumed immortal nature and resistance to aging⁷¹. The same can also be applied to cancer stem cells, which maintain niche occupancy and resist oxidative stress-induced apoptosis, probably by receiving redox cues from the environment. However, further work is required to elucidate the myriads of driver mechanisms intersecting in the realm of redox regulation that extend beyond the present system into broader translational areas.

Materials and methods

Fly strains

Fly stocks were maintained and crosses were set at 25°C on normal corn meal and yeast medium unless otherwise indicated. All fly stocks (BL-24493) UAS SOD1 RNAi, (BL-32909) UAS SOD1 RNAi, (BL-29389) UAS SOD1 RNAi, (BL-32983) UAS SOD2 RNAi, (BL-24754) UAS SOD1, (BL-32489) UAS hhRNAi, (BL-25751) UAS Dcr-nos Gal4, (BL-55122) UAS-FUCCI, were obtained from Bloomington Drosophila Stock Centre. Nos-Gal4, Tj-Gal4, Tj-Gal4-BamGFP, gstD1-GFP, TFAM-GFP, UAS-Ci were kind gift from U. Nongthomba, P. Majumder, K. Ray, B. C. Mandal, Hong Xu, LS Shahidhara labs respectively. Parents were maintained at 25°C, and crosses were set at the same temperature. To ensure optimal Gal4 activity, progenies were shifted to 29°C until eclosion, with aging also conducted at 29°C. Males aged 3–5 days were used for all experiments. The control lines for all experiments are the corresponding Gal4-line crossed with wild type OregonR⁺.

Dissection and immunostaining

Anesthetized flies were dissected in 1X Phosphate Buffer Saline (PBS). All incubations were carried out at room temperature (25°C) unless otherwise mentioned. Testes were fixed in 4% paraformaldehyde for 30 minutes, followed by three washes with 0.3% PBTX (PBS + TritonX 100). Post-blocking in 0.5% Bovine Serum Albumin, testis was incubated overnight at 4°C for primary antisera, followed by washing in 0.3% PBTX 3 times 15 minutes each before incubating with secondary antibody. The tissues were counterstained with DAPI (4,6-diamidino-2-phenylindole) for 20 minutes, followed by three washes in 0.3% PBTX for 15 minutes each. For Patched antibody staining, a modified protocol utilizing PIPES-EGTA buffer was used as described⁷². For Eya staining, the tissues were incubated in primary antibody for 2 days. The dpErk was labelled by dissecting fly testes in Schneider’s media, followed by fixation in 4% PFA for 30 min, 3x wash in 0.1% PBTX, blocked and incubated in primary antibody overnight; each step supplemented with phosphatase inhibitor cocktail 2 (1:100, Sigma, cat#P5726). Samples were mounted in DABCO anti-fade medium prior imaging.

The following primary antibodies were used in the experiments - anti-FasIII (7G10) (1:120, DSHB (Developmental Studies Hybridoma Bank)), anti-Eya (1:50, DSHB), anti-DEcad (DCAD2) (1:50, DSHB), anti-α-Spectrin (3A9) (1:20, DSHB), anti-Ptc (1:50,DSHB), anti-Ci (1:50, DSHB), anti -Dlg (1:50), anti-βtubulin (1:300, DSHB), anti-pERK (1:100, Cell Signaling (4370)), ATP5A (1:700, Abcam (ab14748)), anti-Tj (1:5000), anti-Vasa (1:4000), anti-Zfh1(1:2000). The primary labelling was detected using appropriate Alexa-Flour tagged secondary antibody (ThermoFisher Scientific).

To assess superoxide levels, testes were dissected in Schneider’s medium (SM) and immediately incubated in 30 µM Dihydroxyethidium (DHE) at 25 °C in the dark. Testes were washed thrice with SM for 7 minutes each before quantifying the emitted fluorescence using SpectraMax iD5 (Molecular Devices).

Quantitative Reverse transcription–PCR

For semi-quantitative RT-PCR and qRT-PCR analyses, total RNA was isolated from testes of 3-5 days old male flies using Qiagen RNA extraction kit. RNA pellets were resuspended in nuclease-free water and quantity of RNA was spectrophotometrically estimated. First-strand cDNA was synthesized from 1-2 µg of total RNA as described earlier. The prepared cDNAs were subjected to real time PCR using forward and reverse primer pairs as listed below, using 5 µl qPCR Master Mix (SYBR Green, ThermoFisher Scientific), 2 pmol/µl of each primer per reaction for 10 µl final volume in ABI 7500 Real time PCR machine. The fold change in expression was calculated through 2^-ΔΔCt method. The primers used are listed in Supplementary Table S1.

Imaging and image analysis

Confocal imaging was carried out using Zeiss LSM 900 confocal microscope, using appropriate dichroics and filters. Images were further analyzed and processed for brightness and contrast adjustments using ImageJ (Fiji). Mean intensity measurements were carried out using standard Fiji plug-ins,The relative distance from the two reference points in the images was estimated through Inter-edge Distance ImageJ Macro v2.0 from Github. All images were assembled using Adobe Photoshop version 21.2.1.

Quantification of GSCs, CySCs and CCs

For GSC quantification, only cells in direct contact with the hub were considered. While single slices are shown for representation, counting was performed on individual slices of z-stacks using the semi-automated Cell Counter plugin in Fiji for all specified genotypes.

For CySC quantification, all Tj-positive and Zfh1-positive cells present in each testis were counted and represented. For cyst cells quantification, only those near the hub were considered, which does not reflect the total number of Eya-positive cells. All quantifications were performed using the Cell Counter plugin in Fiji.

Mitochondrial and GstD-GFP quantification

The mitochondrial analyzer first generates 2D mitochondrial regions of interest (ROIs), followed by the measurement of their total area and circularity. Initially, this is done using a thresholded image of a single slice. For GstD-GFP quantification, we have manually demarcated the cell, taking Dlg as a reference, and quantified the GFP intensity.

Immunoblot Analysis

Protein was extracted from the dissected testes using RIPA buffer as described previously and quantified using Bradford reagent (Biorad). Equivalent concentration of lysate was denatured using 1X Laemelli Buffer with 1 M DTT at 95°C, separated in 10% SDS-PAGE and transferred onto PVDF membranes. Membranes were blocked with commercial blocking solution in TBST base before sequential primary antibody incubation with anti-Vasa and anti-β-Tubulin (DSHB, E7). Secondary detection was performed using HRP-tag anti-mouse antibody (GE Amersham).

Statistical analyses

The sample sizes carrying adequate statistical power are mentioned in the figure legends. Statistical significance for each experiment was calculated using two-tailed Student’s t-test, unless otherwise mentioned, using GraphPad Prism 8 software. The P values were calculated through pairwise comparison of the data with wild type or driver alone and driven RNAi lines. Significance values for sample sizes mentioned in figure legends were represented as *P < 0.01, **P <0.001, or ***P < 0.0001.

Data availability

The data that support the findings of this study are available within the main text and its Supplementary Information file. The lead contact will share all raw data associated with this paper upon request. This study did not utilize or generate any unique datasets or codes.

Acknowledgements

We thank P. Majumder, U. Nongthomba, K. Ray, G. Ratnaparkhi, B. C. Mandal, Hong Xu lab and S. C. Lakhotia for kindly sharing some of the transgenic fly lines. Christian Bökel for sharing experimental protocols. Anti-Tj antibody was a generous gift from Prof. D. Godt, University of Toronto. Anti-Vasa and Anti-Zfh1 were kindly gifted by Prof. R. Lehmann, New York University. We also thank Dr. Bama Charan Mandal for generously sharing his Confocal microscope imaging system and Sudeshna Majumder for preliminary data. We acknowledge BDSC for fly stocks and DSHB for antibodies. This work is supported by Innovative Young Biotechnologist Award by Department of Biotechnology (BT/12/IYBA/2019/01), Early Career Research Award by Science and Engineering Research Board (ECR/2018/000009), BHU-Institute of Eminence grant (R/Dev/IoE/Incentive/2021-22/32452). The authors also acknowledge financial support from DST-INSPIRE fellowship (to O.M.) and UGC-CAS doctoral fellowship (to T.C.).

Additional information

Author contribution

D.S. and O.M conceived the study and designed the experiments. O.M, A.C and T.C performed the experiments. D.S and O.M analyzed the data. D.S, O.M. and A.C. wrote the manuscript.

Additional files

Fig S1

Fig S2

Fig S3

Fig S4

Fig S5

Supplemental Table S1