All flies were maintained on the standard Bloomington Formulation diet (Nutri-Fly® BF, Cat #: 66–112, Genesee Scientific Inc.) in a climate-controlled environment at 24 °C under a 12 h light-dark cycle and 65%–70% humidity. Crosses with GAL80 ^TS , which were maintained at 18 °C until eclosion, and the adult flies were transferred to 29 °C. Flies were reared at the permissive temperature (18 °C) and adult males were collected and shifted to the restrictive temperature of 29 °C to allow GAL4 activity. Rescue experiments without GAL80 were raised at room temperature until eclosion and shifted to 26 °C for the specified period to ensure maximum GAL4 activity. nos-GAL4::VP16 dependent mei-P26 overexpression phenotype is not suppressible with GAL80. Therefore, to ensure that the phenotype observed with germline-specific overexpression and somatic cell-specific overexpression is only optimally induced in adulthood, crosses with nos-GAL4 were also made at 18 °C until eclosion and then adult male F1s were transferred to 26 °C prior to dissection.

Fly strains used in this research were obtained from Bloomington Drosophila Stock Center (BDSC) and RNAi lines were from the Vienna Drosophila RNAi Center (VDRC). The following fly lines were used in this study: w ¹¹¹⁸ (BDSC# 3605), UAS-mei-P26 (BDSC# 25771, #25772), c587-GAL4 (BDSC# 67747), nos-GAL4::VP16 (BDSC# 64277), UAS-mei-P26-RNAi (VDRC# 101060).

The testes of aged (5–7 days after eclosion) unmated F1s with desired genotype were dissected in ice cold PBSTX (1X PBS with 0.2% Triton X-100, 0.1% Tween-20). The dissected testes were fixed in 4% paraformaldehyde (PFA) for 30 min, washed thrice with PBSTX, and blocked in PBSTX +5% NGS (5% normal goat serum) in PBSTX for an hour. The blocked samples were then nutated with primary antibodies at 4 °C overnight. The testes samples were then washed thrice in PBSTX, followed by blocking for 1 hour in PBSTX +5% NGS before incubating with secondary antibodies at room temperature for 2.5 h. The primary antibodies used in this study were guinea pig anti-Traffic jam (1:400; a kind gift from Dorothea Godt, University of Toronto) (Li et al., 2003), rabbit anti-Mei-P26 (1:400; a kind gift from Paul Lasko, McGill University) (Liu et al., 2009), mouse anti-1B1 (HTS; 1:50; Developmental Studies Hybridoma Bank [DSHB]), mouse anti-α-Spectrin (α-Spec; 1:100; DSHB) mouse anti-dMyc (1:1; DSHB), mouse anti-Eya (1:10; DSHB), mouse anti-LamC (1:50; DSHB), rat anti-Vasa (1:10; Santa Cruz), rabbit anti-Phospho-Histone H3 (Ser10; 1:500, Invitrogen), rabbit anti-Cleaved Caspase-3 (1:400; Cell Signaling Technology) rabbit anti-pSmad (1:100; Cell Signaling Technology) and rabbit anti-Phospho-S6 Ribosomal Protein (Ser235/236; 1:800, Cell Signaling Technology). The secondary antibodies used in this study were goat anti-rabbit IgG Alexa Fluor 488, goat anti-mouse IgG Alexa Fluor 488, goat anti-mouse IgG Alexa Fluor 594, goat anti-guinea pig IgG Alexa Fluor 488, and goat anti-guinea pig IgG Alexa Fluor 594 (Invitrogen). All secondary antibodies were used at dilution of 1:500. Then, the testes samples were washed thrice in PBSTX and mounted on glass slide with 10 μL SlowFade mounting medium with DAPI (Biotium).

Bromodeoxyuridine (BrdU) is a S-phase marker which is used to identify proliferating cells. The adult aged unmated male F1 flies with desired genotype were starved for 2 h before feeding with 2.5 mM BrdU (SIGMA, Germany) in DMSO with 6% (v/v) red food coloring to monitor feeding for 24 h at 26 °C (Singh et al., 2016). Flies that have red abdominal pigmentation were selected and the testes were dissected immediately for BrdU labelling. The protocol described by Brawley and Matunis was followed with a slight modification (Brawley and Matunis, 2004). Briefly, the testes were fixed using 4% PFA immediately after dissection for 30 min and DNA was subsequently denatured using 2N HCl for 30 min (Boyle et al., 2007). This is followed by standard immunostaining protocol mentioned above using mouse anti-BrdU (1:100; DSHB) primary antibody, and goat anti-mouse AlexaFluor 594 (1:500; Invitrogen) secondary antibodies. The number of BrdU labelled cells were counted.

Apoptotic cells were detected using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay with the OneStep TUNEL Apoptosis Kit [Red, 594] (Novus Biologicals; Cat. No. NBP3-11959). Male unmated F1s with desired genotype were dissected in PBSTX followed by immediate fixing for 30 min in 4% PFA, washed thrice, and labelled according to the manufacturer’s instructions with slight modification. Briefly, after washing, fixed testes samples were incubated in the TdT buffer for 30 min at 37 °C. Then the samples were nutated in dark at 37 °C for at least 2 h with a cocktail of TdT buffer, staining solution and terminal deoxynucleotidyl transferase (TdT). The samples were washed thrice (15 min each) in dark. Then the samples were directly mounted on glass slide with SlowFade mounting medium with DAPI (Biotium) as described above or standard immunostaining protocol was followed for counterstaining.

Adult unmated males of 5–7 days in age were dissected quickly in filter sterilized ice cold PBSTX. A maximum of thirty males were processed per round. As much of the dissecting medium was removed prior to the addition of 100 μL of TRIzol (Invitrogen). Tubes were immediately placed in the −80 °C refrigerator for flash freezing. Three batches of testes (60 pairs each batch) were consolidated for a singular RNA extract. Total RNA from tissue samples was extracted with an RNAqueous Micro Kit (Thermo Fisher–Life Technologies) according to the manufacturer’s instructions. First-strand cDNA was synthesized from total RNA using the SuperScript™ VILO™ cDNA Synthesis Kit (Thermo Fisher Scientific; Cat. No. 11754250). Real-time PCR reaction on cDNA was performed using the TB Green Advantage qPCR Premix Kit (Takara Bio; Cat. No. 639676) on qTOWER³ iris 384 PCR System (Analytik Jena) according to the manufacturer’s instructions.

The following primers were used: mei-P26-specific Fw-5′-TCCGGGGATTCCCAATCTGAA and Rev-5′-GGAGCTAGAGCTGCTAGAACT, RpL32-specific Fw-5′-GACGCTTCAAGGGACAGTATCTG and Rev-5′- AAA​CGC​GGT​TCT​GCA​TGA​G which was used as a reference gene.

Images were acquired using an Olympus FV3000 laser-scanning confocal microscope controlled by CellSens software (Olympus). Z-stack images were obtained using an Olympus fluorescence microscope (Olympus). Acquired images were converted to grayscale, and the number of cells and fluorescence intensity were quantified using ImageJ software. All image adjustments, i.e., brightness and contrast were applied uniformly across experimental groups.

Samples were collected from at least five independent males grown under equivalent environmental conditions. Statistical significance between control and experimental groups was determined using the non-parametric Mann–Whitney U test. For comparisons between more than two datasets, a non-parametric Kruskal–Wallis one-way analysis of variance test followed by a Dunn’s multiple correction was used. Significance levels are indicated as follows: * = p < 0.05; ** = p < 0.005; *** = p < 0.0005; **** = p < 0.0001. All statistical analyses were performed using GraphPad Prism 10 software.

To explore how increased expression of mei-P26 impacts male germline cells in Drosophila, we overexpressed mei-P26 in the germline using nos-GAL4. We found that nos > mei-P26 expressing flies exhibited shorter, truncated testes (n = 65) compared to nos > w1118 control males (n = 70), with 100% penetrance, as observed across three independent biological replicates (Figures 1A–C). Although nos > w1118 control flies exhibit a low baseline incidence of truncated testes (∼10%), this phenotype is substantially more penetrant and severe upon mei-P26 overexpression. A similar developmental defect was observed in females, where mei-P26 overexpression caused rudimentary ovarian formation in the unmated females (Supplementary Figure S1). These findings suggested an arrest or reduction in germ cell proliferation during gonadal development. To determine if testis truncation can be rescued by knocking down mei-P26 in the germline alongside overexpression, we used a genetic fly construct containing mei-P26 RNAi combined with UAS-mei-P26. Knocking down mei-P26 alone was sufficient to suppress approximately 50% (n=60) of the testis truncation caused by mei-P26 overexpression in the germline (Supplementary Figures S2A, B).

To assess cell proliferation and differentiation status in these testes, S-phase cells were labeled with BrdU (bromodeoxyuridine), and mitotic cells were identified using phospho–Ser10 histone H3 (PH3). Germline-specific overexpression of mei-P26 showed an increase in BrdU-positive cells (Figure 1E’) compared to control testes (Figure 1D’), but the PH3-positive mitotic cells were markedly reduced and almost absent (Figure 1E) compared to control testes with PH3-positive mitotic cells (Figure 1D), indicating defective spermatogenesis where cells are arrested in the S-phase of the cell cycle. The Drosophila testis is tightly regulated by both cell-autonomous and non-cell-autonomous signaling to maintain controlled division and differentiation, especially at the transit-amplifying (TA) region where mitotic endoreplication occurs to produce enough spermatogonia that will further differentiate into spermatocytes through meiotic division. The endoreplication stage is a critical point where cells can accumulate damage during the four rounds of division and determine whether to enter meiosis, as only high-quality germ cells are committed to becoming sperm (Figure 1F) (Fabian and Brill, 2012).

Previous studies have demonstrated that Mei-P26 plays a pivotal role in balancing cell proliferation and differentiation within the Drosophila germline. It acts downstream of the bam pathway to limit the proliferative potential of germline stem cells (GSCs) and their progeny, while promoting the onset of differentiation through post-transcriptional regulation of target mRNAs (Insco et al., 2012; Neumüller et al., 2008). Consistent with its role as a tumor suppressor in the germline, loss of mei-P26 results in excessive proliferation of undifferentiated germ cells, whereas its overexpression can disrupt germline homeostasis and differentiation programs (Li et al., 2012; Page et al., 2000).

In our study, we observed that proliferating cells in the testes include both germline- and somatic-derived populations. This was evidenced by strong expression of the germline marker Vasa (Figure 2B) and the somatic cell marker Traffic Jam (Tj; Figure 2D’), in contrast to control testes, which showed Vasa expression restricted to germline cells (Figure 2A) and Tj expression restricted to somatic cells (Figure 2C’). The number of somatic cells undergoing TA divisions was quantified by counting Tj-positive cells and was significantly increased in germline-specific mei-P26 overexpressing testes compared to controls (Figure 2E), consistent with the elevated proliferation detected by BrdU S-phase labeling (Figure 1E’). No fusome structures were observed in these testes (Figure 2D) when stained with an adducin homolog, Hu-li tai shao (Hts), indicating there are no clustered germline cysts as seen in the control testis (Figure 2C). Nonetheless, the detection of Vasa-positive germline cells without fusomes suggests a failure in incomplete cytokinesis during TA division that forms spermatogonia cysts. Interestingly, the somatic cells showed excessive proliferation, resembling a tumor-like phenotype within the testis. The absence of differentiating spermatogonia may deprive somatic cells of essential germline-derived differentiation cues, leading to their arrest at the TA division stage, in agreement with prior studies demonstrating that germline-to-soma signaling is necessary for proper somatic differentiation during testis development (Kiger et al., 2001; Sarkar et al., 2007).

To confirm the defective differentiation of somatic cells, the truncated testes were stained with Tj to mark somatic cells at TA division stage of testis and Eyes absent (Eya) for differentiated somatic cells supporting meiotic spermatocytes. As expected, germline-specific mei-P26 overexpression resulted in the absence of Eya-positive cells, indicating a failure to generate spermatocytes and to enter meiotic division (Figures 3B–B"), in contrast to control testes, which exhibited a well-defined transition of somatic cells from Tj-positive to Eya-positive states (Figure 3A–A”), consistent with normal differentiation of spermatogonia into spermatocytes (sp). Mei-P26 expression is high in germline stem cells (GSCs) and decreases as bam expression increases until meiosis. During differentiation, bam is suppressed while mei-P26 levels increase in spermatocytes (Insco et al., 2012; Samuels et al., 2024).

Previous studies have shown that loss of BMP signaling in germline stem cells leads to upregulation of bam, and that bam and mei-P26 function in a closely linked regulatory pathway controlling exit from transit-amplifying (TA) divisions (Kawase et al., 2004; Salerno-Kochan et al., 2022). Based on these findings, we examined whether BMP signaling is altered in response to germline-specific mei-P26 overexpression by analyzing the distribution of phosphorylated Mad (pMad) in the apical region of the testis. In control testes, strong pMad staining was detected in germline stem cells surrounding the hub, consistent with active BMP signaling required for stem cell maintenance (Figure 3C’). In contrast, testes overexpressing mei-P26 in the germline exhibited a near-complete loss of detectable pMad signal in the apical region (Figure 3D’). This reduction in BMP signaling was accompanied by disorganization of somatic cells and the absence of fusome structures, indicating disruption of the germline niche and impaired maintenance of early germ cells. Together, these findings suggest that germline-specific mei-P26 overexpression suppresses BMP signaling, potentially through ectopic elevation of bam in early germ cells, thereby contributing to germline stem cell depletion and testis truncation.

Although truncated testes displayed extensive proliferation of Tj-positive somatic cyst cells (Figure 2B), these cells did not exhibit phospho-Histone H3 (PH3) staining (Figure 1B), indicating a lack of mitotic activity and suggesting a possible block in differentiation. To determine whether this absence of PH3 signal was associated with cell death, we performed TUNEL assays to detect apoptotic cells. Strikingly, truncated testes exhibited a high level of TUNEL-positive signal (Figures 4C,D) compared to control (Figures 4A,B), indicating widespread apoptosis as quantified by increased TUNEL fluorescence intensity (Figure 4E). These observations suggest that, in the absence of germline cells, somatic cyst cells fail to receive survival cues and undergo apoptosis, ultimately contributing to the formation of the truncated testis phenotype. Consistent with this, previous studies have shown that germline-derived signals, particularly those mediated through the EGFR and JAK/STAT pathways, are essential for cyst cell survival and differentiation, and disruption of these germline-to-soma interactions leads to apoptosis and testis degeneration (Amoyel, et al., 2016b; Kiger et al., 2001; Sarkar et al., 2007; Schulz et al., 2002).

Notably, apoptotic cells exhibited distinct spatial patterns between conditions. In control testes, apoptosis occurred predominantly within clustered spermatogonia (Figure 4B’), as previously reported (Lu and Yamashita, 2017). In contrast, germline-specific mei-P26 overexpressing testes displayed apoptotic events in isolated individual cells lacking organized clusters (Figure 4D’). This failure to form clustered spermatogonia is consistent with the absence of branching fusomes observed in mei-P26 overexpressing testes (Figure 2D), indicating an early defect in germ cell mitotic entry and cyst formation that likely underlies the dispersed pattern of apoptosis.

Together, these findings suggest that while somatic cyst cells and germline cyst cells initially proliferate, they ultimately undergo apoptosis due to the loss of germline-derived survival signals from spermatogonial cysts, culminating in the truncated and degenerative phenotype of the testis.

Mei-P26 overexpression in gonadal somatic cells results in a phenotype distinct from that observed with germline-specific mei-P26 overexpression, as it leads to impaired testis development in males. Although somatic cell specific mei-P26 overexpression induces lethality in males, the recovered unmated female progenies had enlarged ovaries (Supplementary Figure S3) (Steenwinkel et al., 2022), opposite to what has been observed with the germline-specific mei-P26 overexpression with rudimentary ovaries (Supplementary Figure S1). To circumvent this lethality in males, tubulin-Gal80 ^ts was used to suppress the expression of Gal4 until the eclosion of adult male flies in 18 °C and these flies were collected and transferred to 29 °C to express Gal4 to drive the expression of mei-P26 in testicular somatic cells. To evaluate whether this lethality is due to mei-P26 overexpression alone, mei-P26 was knocked down in the background of this somatic cell specific overexpression using mei-P26-RNAi line. The male-specific lethality was rescued, confirming that somatic cell-specific mei-P26 overexpression alone is sufficient to induce this male-specific lethality (Supplementary Figure S2C).

Surprisingly, overexpression of mei-P26 in somatic cells resulted in a strikingly different phenotype compared to germline overexpression. In c587>UAS-mei-P26 testes, a marked increase in the number of cells in S-phase was observed, as indicated by increased incorporation of BrdU throughout the apical region (Figure 5D) compared to the control testis (Figure 5B) with BrdU only incorporated in the apical tip of the testis (Figure 5B’). In addition, the number of cells that had entered mitosis was dramatically elevated, with abundant phospho-histone H3 (PH3)-positive cells (Figure 5C) compared to controls (Figure 5A).

This increase in both BrdU and PH3 positive cells reflects a robust elevation in proliferation, with more cells actively cycling through S and M phases (Figures 5E,F). Moreover, these proliferating cells also showed signs of rapid progression beyond S-phase, rather than cell-cycle arrest and germline depletion. Together, these findings reveal that mei-P26 overexpression in somatic cells drives a hyperproliferative, differentiation committed cells, representing a sharp contrasting fate to the proliferative arrest and germline loss observed when mei-P26 is overexpressed in germ cells.

To determine which cell populations were actively proliferating under somatic cell-specific overexpression of mei-P26, markers for germline and somatic lineages were tested. Notably, both germline and somatic cells exhibited strong proliferative activity accompanied by differentiation. Germline cells displayed increased numbers of branched fusomes (Figure 6B), a hallmark of differentiating spermatogonial cysts, indicating accelerated progression from stem cells toward differentiated states as observed in controls (Figure 6A). In parallel, there was a marked increase in Tj positive somatic cells, consistent with hyperproliferation of somatic lineages (Figures 6B,D). Together, these observations suggest that, in contrast to germline-specific mei-P26 overexpression which primarily results in germline depletion, somatic cell-specific overexpression drives hyperproliferation and differentiation of both germline and somatic cell populations. To test the efficiency of the Gal80 system, mei-P26 expression was quantified and an elevation of mei-P26 mRNA expression was observed in c587-Gal4; tubGal80>UAS-mei-P26 testes compared to control (Figure 6C).

Next, to determine whether the hyperproliferation and differentiation observed with somatic cell-specific mei-P26 overexpression are linked to TOR pathway activation, the distribution of pS6, a marker of mTORC1 activity involved in the TOR signaling pathway, was examined. A marked increase in pS6 signal intensity compared to controls was observed, indicating elevated TOR activity (Figures 7D’,E). Interestingly, this pS6 signal elevation was rescued with mei-P26 knockdown, suggesting suppression of Mei-P26 is sufficient to both rescue the lethality of the overexpression construct and repress the elevation of pS6 signal in the testis induced by mei-P26 overexpression (Figure 7E; Supplementary Figure S2C). Notably, the pS6 puncta appeared enriched closer to the apical tip of the mei-P26 overexpressed testis (Figure 7C’) compared to control (Figures 7A’,B’), indicating that TOR signaling is activated early upon mei-P26 overexpression. This elevated signaling coincided with accelerated cell-cycle entry, increased mitotic activity, and the appearance of smaller individual mitotic cells as can be seen in Figure 5C compared to control (Figure 5A), consistent with a hyper-proliferative state. These findings align with prior reports that TRIM-NHL proteins, including Mei-P26, can activate TOR signaling to promote differentiation and translational output (Gui et al., 2023).

On the other hand, pS6 puncta were almost absent (a maximum of 2-3 puncta) in the germline-specific mei-P26 overexpression (Figure 7G’) compared to the control testis with pS6 positive puncta (Figure 7F’), indicating repressed TOR signaling in the germline cells, similar to what has been observed previously in germline-specific TOR knockdown (Basar et al., 2019; Clémot et al., 2024).

Interestingly, even though the TOR signaling pathway activity was elevated in somatic cell-specific mei-P26 overexpressed testes, the dMyc signal was absent (Figures 7C,D). In contrast, the control testis showed a clear dMyc signal in the apical tip region (Figures 7A,G). This finding diverges from the usual positive correlation between TOR and Myc in proliferative tissues. The phenotype supports previous reports indicating that mei-P26 antagonizes dMyc by repressing its expression, thereby preventing excessive growth. (Herranz et al., 2010). The smaller cell size observed in somatic mei-P26 overexpression during TA endoreplication also supports the previously noted phenotype in dMyc mutants, which resulted in reduced cell size during endoreplication in ovarian follicle cells. (Tamori and Deng, 2013). Thus, Mei-P26 appears to promote TOR-driven proliferation that favours differentiation while suppressing Myc activity.

Additionally, ectopic overexpression of mei-P26 caused lethality in both males and females, while ectopic knockdown of mei-P26 did not induce lethality. Although germline-specific mei-P26 knockdown did not result in lethality, spermatocyte specific mei-P26 knockdown led to a reduction in spermatocytes (Supplementary Figure S4B) compared to controls (Supplementary Figure S4A), indicating a requirement for Mei-P26 during spermatocyte development. Despite this reduction, overall testis morphology remained largely intact. The relatively mild phenotype observed upon germline mei-P26 knockdown (Supplementary Figure S4D) may reflect functional redundancy with other RNA-binding proteins, including Held out wings (HOW), Benign gonial cell neoplasm (Bgcn), and Tumorous testis (Tut), which are known to regulate spermatogonial differentiation, especially expression of bam during transit-amplifying divisions (Chen et al., 2014; Harris et al., 2025). In contrast, somatic cell-specific mei-P26 knockdown did not produce detectable defects in testis morphology or germline organization under the conditions examined (Supplementary Figure S4F). Collectively, these findings support a model in which the phenotypes induced by mei-P26 overexpression reflect signaling-dependent gain-of-function effects, rather than loss of mei-P26 activity, which can be buffered by redundant RNA-binding proteins.