**3. Discussion**

It is well established that CPT-induced toxicity is dependent upon top1, which is capable of introducing a transient single-strand break in DNA, through which another strand can pass, thereby reducing DNA supercoiling [37,38]. Due to the specific function of top1, the single-strand cleavage/rejoining activity of top1 suggests that it may serve as a swivel for unwinding and rewinding of DNA helices associated with many critical cellular processes, including DNA replication, transcription, repair, and recombination induction of cell cycle arrest [39,40]. Therefore, CPT and CPT-derived chemicals were reported to cause severe toxicity in the hematopoietic system, lymphatic tissue, gastrointestinal tract, and reproductive organs, and have been developed for cancer therapy [41–43]. Our data demonstrated that CPT is deleterious to *D. melanogaster* germline development, and it leads to GSC loss and concomitantly blocks CB and two-cell cyst differentiation by ectopically inducing Bam expression and regulating CycA. Other than growing evidence on intrinsic factors-dependent mechanisms regulating the GSC lineage in *D. melanogaster* [44], we reported the toxicological effects of CPT on GSCs and two-cells for the first time, highlighting the potential of its pharmacophores to be developed in stem cell-based therapies.

It is well documented that top1 is the only cellular target of CPT [2]. Top1 activity is robust in malignant cells and correlates with disease progression in colorectal and ovarian cancers [45], making CPT a potent agent for anticancer chemotherapy. We found that the inhibition of the expression of *top1* led to a large number of germ cell loss without CPT treatment, and the phenotype is similar to that of CPT treatment. At the same time, *top1RNAi* flies with CPT treatment could exacerbate GSC loss and two-cell accumulation phenotypes induced by CPT treatment alone, exhibiting more severe abnormal germarium and triggering almost the entire GSC and CB loss. Therefore, we suggested CPT affected GSC maintenance and two-cell accumulation through inhibiting the expression of *top1*. Based on the current study, it is noteworthy that we found oral ingestion of CPT led to GSC loss in *D. melanogaster* probably via interfering with the cell cycle, which serves as a critical aspect in the decision between GSC maintenance and differentiation. Our results suggest that during germline differentiation, CPT-mediated changes in CycA expression have a role in reprogramming self-renewal, leading to precocious GSC differentiation, and eventually contributing to GSC loss. A previous study indicated that overexpression of a stable form of CycA led to severe *Drosophila* GSCs loss [46], and stabilized CycA could prevent exiting from the cell cycle and entering into G1 at an appropriate developmental stage [47]. Thus, CycA plays an important role in cell cycle in *Drosophila* GSCs, and we also found CycB overexpression has no detectable phenotype in GSCs. In addition, we found CycA regulated GSC differentiation in a Bam-dependent manner, which is evident by the results that ectopic expression of Bam in GSCs would increase the stability of CycA, and down-regulation of CycA antagonized the function of ectopic Bam in GSCs. These results suggested that changes in CycA level are sufficient to explain the loss of GSCs when expressing Bam ectopically. Consistent with the previous study, they found that ectopic expression of the stable form of CycA in germ cells caused GSC loss, which is similar to the phenotype resulting from ectopic expression of Bam in GSCs [36,48]. Ji et al. convincingly showed that ectopic expression of Bam in GSC could be enhanced by coexpression of CycA, and suppressed by CycA reduction [24]. Furthermore, they found CycA can be coimmunoprecipitated with Bam from S2 cells and ovarian extracts [24,46,49]. The relationship between Bam and CycA might be interpreted as the way that Bam functions as a ubiquitin-associated protein to deubiquitinate and stabilize CycA, thereby balancing GSC self-renewal [24]. The results in this study indicated that the Bam–CycA regulatory axis plays an important role in GSC differentiation and cell-cycle alterations in response to the cytotoxicity of CPT, but the detailed regulatory mechanism remains to be further elucidated.

In the female germline, Bam is a key intrinsic regulator of differentiation [50] because Bam RNA appeared shortly after the differentiation of a stem cell that produces new CBs [51]. In the absence of Bam activity, GSC daughters failed to differentiate, and ectopic expression of Bam in GSCs was sufficient to induce GSC loss and led to the accumulation of undifferentiated germ cell tumors [52,53]. In our study, CPT treatment also resulted in accumulation of two-cell cysts, and such differentiation defects were further enhanced by the heterozygous mutation of *bam*. By contrast, heat-shock-induced Bam expression can sufficiently promote the differentiation of two-cell cysts, while reducing CycA can rescue two-cell accumulation caused by CPT treatment, indicating the Bam–CycA axis might also contribute to the two-cell accumulation phenotype. Previous study revealed the *bam* gene was required for the differentiation of CBs from the stem cells, perhaps by altering the cell cycle and stabilizing differentiation factors, such as CycA [51]. Moreover, Bam-dependent deubiquitinase complex can disrupt GSC maintenance by targeting CycA, and DNA damage could disrupt Bam-dependent differentiation pathways and cause the accumulation of CB-like cells in a Lok-dependent manner [54]. Based on those facts, we hypothesized that CPT treatment could regulate the expression of Bam, and the alterations in Bam expression are sufficient to trigger the transcription program of the cysts following differentiation, leading to cell arrest at G1/S and two-cell accumulation probably via affecting CycA.

#### **4. Materials and Methods**

#### *4.1. Drosophila Stocks*

*D. melanogaster* was reared by standard *Drosophila* medium. All fly stocks were maintained at 25 ◦C and a related humidity of 60% with 1:1 (light:dark) photoperiod. For each treatment group, newly emerged adults (<24 h) were placed into a vial containing standard media and were applied 100 mg/L CPT for 9 days. DMSO treated flies were considered as the control. The genotypes of the mutant lines used in this study were: *w1118* (used as wild-type control), *dpp2.0-lacZ* [55], *Bam-GFP* [50], *pz1444*, *nos-gal4 vp16*, *hs-bam* (a gift from Yu Cai [Temasek Life Sciences Laboratory, National University of Singapore, Singapore), *uas-top1*RNAi (Bloomington, BL#55314), *bam<sup>86</sup>* (Bloomington, BL#5427), FUCCI (*UASp-GFP-E2F11–230*; *UASp-mRFP1-cycB1–266*, Bloomington, BL#55101), *hs-cycA* (Bloomington, BL#91660), *hs-cycB* (Bloomington, BL#91664), *UAS-cycE*RNAi (Bloomington, BL#29314). All crosses were maintained at room temperature. For heat-shock stock, crosses were maintained at 25 ◦C. The flies were heat-shocked for 1 h at 37 ◦C following CPT treatment for 9 days, followed by 24 h recovery at room temperature. Additionally, the dissection and immunostaining were performed.
