The follicle epithelium of the Drosophila ovary has been a widely used and informative model for understanding epithelial tissue biology within the native, in vivo, environment (Sahai-Hernandez et al., 2012). First described over 60 years ago as a single layered epithelium that encapsulates developing germ cell cysts (Demerec, 1950; King et al., 1956), studies of this tissue have revealed insights into many aspects of epithelial biology, including diverse mechanisms that regulate the specification of cell fate in an epithelial stem cell lineage (Assa-Kunik et al., 2007; Chang et al., 2013; González-Reyes and St Johnston, 1998; Johnston et al., 2016; Pocha and Montell, 2014; Song and Xie, 2003), the establishment and maintenance of cell polarity (Bilder et al., 2000; Castanieto et al., 2014; Kronen et al., 2014; Mirouse et al., 2007; St Johnston and Ahringer, 2010), and the discovery of a novel mechanism for establishing planar polarity (Cetera et al., 2014; Chen et al., 2016).

A distinct advantage of the Drosophila ovary as an experimental model is that it has a highly consistent and well-described organization that facilitates the study of tissue biology with precise spatial and temporal resolution. Each Drosophila ovary is composed of long chains of developing follicles, called ovarioles (Miller, 1950), and oogenesis begins at the anterior tip of each ovariole in a structure called the germarium (Koch and King, 1966). The germarium has a stereotypical organization with four morphologically distinct regions, numbered from anterior to posterior as Regions 1, 2a, 2b, and 3 (Figure 1—figure supplement 1A). Germline stem cells (GSCs) reside at the anterior end of the germarium (Carpenter, 1975; Koch and King, 1966), in Region 1, and divide during adulthood to self-renew and produce daughter cells called cystoblasts. Cystoblasts undergo four rounds of mitosis with incomplete cytokinesis, as they move through Region 1 into Region 2a, which is defined by the presence of two 16 cell cysts that span the width of the germarium. Throughout Regions 1 and 2a, the germ cell cysts are covered by a population of somatic cells, referred to as inner germarial sheath (IGS) cells or escort cells. These cells provide a differentiation niche for the germ cells during these early stages of oogenesis (Kirilly et al., 2011), and may also help to propel the germ cells toward the posterior (Morris and Spradling, 2011). At the Region 2a/2b border, the cysts shed their IGS cell layer and move one at a time into Region 2b, where they become encapsulated by the follicle cell layer and take on a characteristic lens shape. Next, the cysts become more spherical in Region 3 (which is also referred to as Stage 1) and then bud off the germarium as a Stage 2 follicle. After budding, follicles rapidly grow and develop into a fully mature Stage 14 follicle that is ready for ovulation. This process, which takes approximately 8–9 days total under normal laboratory conditions (King, 1970), proceeds continuously during the first half of adult life, producing an organized tissue in which cells across the entire continuum of oogenesis are present simultaneously and arranged in order from the anterior to the posterior.

This thorough characterization of the ovarian structure and germ cell biology has facilitated the use of the ovary to study somatic cell biology as well. In a landmark study (Margolis and Spradling, 1995), the follicle epithelium was found to be maintained by a population of follicle stem cells (FSCs) in the germarium. This study was among the first to identify adult stem cells in vivo using site-specific DNA recombination to label clones of cells with a genetically heritable marker. In Drosophila studies, this is typically achieved by expressing flippase (Flp) to induce mitotic recombination between FRT sites on homologous chromosomes, whereas studies of mammalian tissues typically use cre-recombinase to induce intrachromosomal recombination at lox sites. This form of lineage tracing has become a gold standard for identifying adult stem cells (Fox et al., 2008; Simons and Clevers, 2011), and the methods used in the identification of FSCs helped to establish principles that have been applied to the study of many other Drosophila and mammalian adult stem cell lineages. Specifically, it is important to use a reliable clonal marking system in which the clonal marker is unambiguous, the method of clone induction does not significantly perturb tissue function or development, and the rate of background or ‘leaky’ clone induction is low. In addition, it is important to induce clones only sparsely, so that the majority of stem cells in the organ are not labeled with the clonal marker, to maximize the chance that each patch of labeled cells is one clone that originated from a single stem cell. With these quality control measures in place, the analysis of clone size and frequency at multiple time points can be used to infer the number of stem cells in the tissue as well as properties of the lineage such as the rate and number of transit amplifying divisions, the location of the stem cells, and the rate of stem cell turn over.

Using these principles, Margolis and Spradling induced sparse clones during adulthood and observed that single FSC clones contribute to an average of 50% of the follicle cell population on Stage 10 follicles, leading to the conclusion that each ovariole contains two actively dividing FSCs. In addition, they noticed that follicle cell clones remain coherent as a single patch of contiguous cells, even as the follicle grows and develops, and proposed that the FSCs reside at the Region 2a/2b border because persistent clones of contiguous cells at every time point always included one or more cells in this position. In contrast, a recent study concluded that there are 15–16 actively dividing FSCs per germarium that are organized into three rings, with one ring at the Region 2a/2b border, and two additional rings of cells in the adjacent positions immediately to the anterior, within Region 2a (Reilein et al., 2017). In addition, they proposed that, though all 16 FSCs are mitotically active, each FSC does not necessarily contribute to every follicle, so individual FSC clones can be discontinuous. The majority of the evidence in this study came from the use of a novel clonal marking system in which three clonal markers and two pairs of FRT sites are combined onto a single chromosome (Figure 1—figure supplement 1B). One homolog of Chromosome II has a constitutive GFP distal to FRT40A and a constitutive RFP distal to FRT42B, and the other homolog also has an FRT40A and an FRT42B with a constitutive Lac-Z that is distal to the FRT 40A. In this system, all cells initially express all three clonal markers, and heat shock-induced expression of Flp promotes recombination at one or both FRT sites, which has the potential to produce cells with 6 combinations of clonal markers (LacZ⁺, GFP^–, RFP⁺; LacZ⁺, GFP^–, RFP^–; LacZ^–, GFP⁺, RFP⁺; LacZ^–, GFP⁺, RFP^–; LacZ⁺, GFP⁺, RFP^–; LacZ⁺, GFP⁺, RFP⁺) (Figure 1—figure supplement 1B).

Here, we evaluate the conclusions of the Reilein et al. study by testing the clonal system that was reported in this study and repeating key experiments from the Margolis and Spradling study using quantitative microscopy and two standard single color marking systems: a negatively marked clonal system and a MARCM system. We find that the multicolor system used in Reilein, et al. is highly unreliable, producing an unpredictable pattern of clones across multiple time points and a high rate of ‘leaky’ clone induction in the absence of heat shock. In addition, we identify several logical flaws in the Reilein et al. study and performed new experiments to test the assumptions that underlie these flaws. Overall, our findings contradict the findings of Reilein et al. and instead support the original finding that the follicle epithelium is typically produced by two active FSCs residing at the FasIII border (Margolis and Spradling, 1995; Nystul and Spradling, 2010; Nystul and Spradling, 2007; Spradling et al., 1997). However, our quantitative approach revealed that the contribution from these two lineages is more variable than previously thought, leaving open the possibility that the number of active FSCs is not fixed but instead may fluctuate within a narrow range of one to four.

To test the LacZ, GFP, RFP (LGR) clonal marking system developed in Reilein, et al., we heat shocked the flies of the appropriate genotype for 1 hr at 37°C four times over 3 days, as described (Reilein et al., 2017), and assayed clone frequency at 5, 7, 9, 12, 14, 20, and 25 days post heat shock (dphs). Consistent with Reilein et al., we identified all six of the expected label combinations, and observed a progressive decrease in clonal diversity over the time course (Figure 1A–D and Figure 1—figure supplement 2). However, we observed less clonal diversity overall than reported in Reilein et al. For example, Reilein et al. reported that the majority of ovarioles had three uniquely labeled lineages at nine dphs, while the majority of ovarioles in our study had just two uniquely labeled lineages at this time point (Figure 1E). Notably, whereas the Reilein et al. study found a small number of ovarioles with five or six uniquely labeled lineages (8 and 4, respectively, out of 50 total at nine dphs), we never observed more than four uniquely marked clones within the same ovariole among the more than 300 ovarioles we imaged for this time course.

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Frequency of clones over a time course, LRG system, 4x heat shocks.

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Comparison of this study with Reilein et al. (2017).

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Frequency of clones over a time course, LGR system, 1x heat shock.

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Importantly, we also noticed that, in the large majority (88.7%, n = 62) of ovarioles with more than two distinctly labeled lineages, only 1 or 2 of the lineages spanned the entire ovariole, while the other lineage(s) covered only part of one follicle (Figure 1F) or consisted of a small patch of cells in the germarium. This suggests that just one or two FSC lineages predominated in these cases. According to the criteria used in the Reilein et al. study (Reilein et al., 2017), the small clones in these ovarioles would also be considered FSC clones. However, there are several problems with this interpretation. First, it is generally inappropriate to use multiple heat shocks with a clonal marking system that has more than one pair of FRT sites because the later heat shocks may produce subclones within clones that were induced by the earlier heat shock treatments. Thus, at least some of the cases in which small and large clones coexist in the same ovariole may have been due to the production of subclones induced by the later heat shocks. Consistent with this, clone induction with a single 1 hr heat shock substantially reduced the number of distinctly labeled clones per ovariole at 9 and 14 dphs (Figure 1G). However, this may also be due to an overall reduction in the frequency of clone induction, and we still observed small and large clones coexisting in the same ovariole with this heat shock regimen in some cases.

Second, these small clones may have arisen independent of heat shock. To test this possibility, we compared the rate of clone induction with the LGR clonal marking system to a commonly used GFP negative clonal marking system with FRT19A (Haelterman et al., 2014; Yamamoto et al., 2014) in the absence of heat shock at 7 days after eclosion (Figure 2A). As expected, we found that the rate of clone induction was very low in the GFP negative system (<2%, n = 188). However, with the LGR clonal marking system, we found that, on average, 25% of ovarioles had FSC clones (with or without transient clones), and an additional 22% had transient clones without an FSC clone (n = 112) (Figure 2A–B). This contrasts with a frequency of 2% clone induction in the absence of heat shock in the Reilein et al. study. It is unclear why we observed different distributions of clone frequencies and rates of clone induction in the absence of heat shock, though differences in experimental conditions may have been a contributing factor. Nonetheless, our inability to reproduce their results suggests that a low rate of background clone induction, which is important for any clonal marking system, is not a robust feature of the LGR system.

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No heat shock control, LGR system vs GFPneg system.

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Surprisingly, in flies with the LGR system, we identified clones that lacked expression of all three markers (Figure 2C–D) and others that expressed RFP but not LacZ or GFP (Figure 2E). This is unexpected because LacZ and GFP are both on the left arm of Chromosome II (2L), so all cells should express at least one of these two markers. We observed these types of clones in both heat-shocked and non-heat shocked cohorts. In all cases, other cells in the same ovariole clearly expressed all three markers, serving as an internal control for our ability to detect marker expression. In addition, we ruled out the possibility that the absence of LacZ and GFP signal was due to cell death because cells in the clone had normal nuclear morphology, and the clones often contained a dozen or more cells (Figure 2D), indicating that cells with these marker patterns were healthy enough to divide several times. We also ruled out the possibility that the absence of a LacZ and GFP signal was because the cells were in mitosis, when nuclear markers are more difficult to detect, because the DNA did not appear condensed. In addition, we sometimes observed this clonal pattern in post-mitotic follicle cells (Figure 2D) and multiple adjacent cells, which are unlikely to all be in mitosis at the same time, shared the same marker pattern. Taken together, these findings reveal significant flaws with the LGR clonal system that severely reduce its reliability and reproducibility. This may account for a large part of the discrepancies between the Reilein et al. study and prior studies, and strongly suggests that the LGR system is not an appropriate tool for determining the number of FSCs per ovariole.

An assumption that was integral to the calculations of FSC number in the Reilein et al. study was that the clone induction protocol induced recombination at both FRT sites in every mitotic follicle cell. Specifically, they explain that recombination at both FRT sites followed by segregation of sister chromatids at mitosis would produce nine possible genotypes with equal frequency, and that these nine genotypes would produce six detectable phenotypes (marker combinations) at a 1:1:1:2:2:2 frequency. They then stated “We therefore assumed in our statistical modeling that the different colors of FSC clone were present in those same proportions (B:G:BG:BR:GR:LGR = 1:1:1:2:2:2)’ (Reilein et al., 2017). However, we are not aware of any other example where recombination between FRT sites is reported to occur in every mitotic cell after heat shock. Cells must be in the S or G2 phase of the cell cycle for the recombination event to produce a detectable chromosomal rearrangement, and it is very unlikely that all cells will enter into this part of the cell cycle during the time that flippase is present after heat shock. Indeed, even in cases where the Flp is expressed constitutively, such as with eyelss-Flp, FRT recombination does not occur in all cells (Menut et al., 2007; Moberg et al., 2005). Using hs-Flp to generate negatively marked clones in the follicle epithelium, we typically find that only 25–50% of ovarioles have FSC clones (Castanieto et al., 2014; Johnston et al., 2016; Kim-Yip and Nystul, 2018; Kronen et al., 2014). To investigate whether FRT recombination occurs in all cells in the LGR system, we quantified the frequency of each marker combination at multiple time points after either four 1 hr heat shocks or one 1 hr heat shock. We found that the most common marker combination at every time point was the unrecombined phenotype, LacZ⁺, GFP⁺, RFP⁺, followed by LacZ⁺, GFP^–, RFP⁺, while the remaining four marker combinations were present at much lower frequencies (Figure 3A). Unexpectedly, we found that clones marked by the absence of LacZ were less frequent than clones marked by the absence of GFP, indicating that cells lacking these two markers are not equally fit. These findings challenge the underlying assumptions of the analysis in the Reilein et al. study, and suggest that, even if the LGR system were reliable, the interpretation of the data leading to their conclusions about the number of FSCs per germarium are also flawed.

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Frequency of each type of clone over a time course, LGR system, 4x heat shocks.

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Frequency of each type of clone over a time course, LGR system, 1x heat shock.

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Frequency of each type of clone pattern, GFPneg system.

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Therefore, to reexamine the original claims of the Margolis and Spradling study, we switched to the use of the FRT19A GFP negative clonal marking system that we found had a very low rate of background clone induction in the absence of heat shock (Figure 2A). Importantly, this system uses only one pair of FRT sites so there is no risk of generating subclones with multiple heat shocks. In addition, the generation of GFP negative clones with FRT19A is one of the most commonly used methods to label clones in studies of the FSC lineage (Cook et al., 2017; Dai et al., 2017; Nystul and Spradling, 2010; O'Reilly et al., 2008). With this system, all cells initially express GFP, and FRT recombination produces clones that are clearly marked by the lack of GFP expression. One of the original observations in the Margolis and Spradling study is that an FSC clone forms a coherent, contiguous patch of cells that extends from the Region 2a/2b border out to the posterior edge of the clone boundary, contributing to each new follicle that is formed after clone induction. In contrast, Reilein et al. posited that each FSC does not normally contribute to every follicle, so discontinuous clone patterns with labeled cells at the Region 2a/2b border followed by intermittent, non-contiguous patches of labeled cells downstream should be expected. However, with the GFP negative clonal marking system, we found that the clone patterns in 94% of ovarioles with clones did not fit this expectation. Specifically, 61.5% of the ovarioles with clones had large FSC clones that extended from the Region 2a/2b border through the germarium and over one or more follicles (Figure 3B–C), 20.6% had transient clones that covered only a half of a follicle or less (Figure 3B,D), and 3.6% had large clones that spanned multiple follicles but did not extend back to the Region 2a/2b border (Figure 3B,E), which is the predicted pattern for a recent FSC replacement event (Song and Xie, 2003). The remaining 14.3% of ovarioles had one of two unexpected clone patterns: 8.3% had small clones within the germarium that typically covered only a small portion of one cyst (Figure 3B,F), and 6.0% had a discontinuous pattern in which a clone near the Region 2a/2b border coexisted with a larger, non-contiguous clone of cells further downstream in the ovariole (Figure 3B,G). These two clone categories occurred frequently enough that they seem unlikely to be due solely to background recombination events, though this remains a formal possibility. Alternatively, they may be evidence that FSC lineage dynamics are not as predictable as previously thought, and that rare events cause some cells to become quiescent or delay differentiation for a period of time. Therefore, although up to 6% of the ovarioles in this population may have had discontinuous clones, our results confirm that the large majority of FSC clones typically form coherent, contiguous units and contribute to every follicle within the boundaries of the clone.

Next, we sought to estimate stem cell number by measuring clone sizes at multiple time points. In the Margolis and Spradling study, this was achieved by measuring the size of clones on Stage 10 follicles, which provides a snapshot of the events that contributed to a single follicle in each ovariole. In that case, the authors reported that an average of 50% of the follicle cell population was labeled by each FSC clone. To assay FSC behavior during the production of multiple successive follicles, we used quantitative image analysis to count the number of GFP positive and GFP negative cells in mosaic ovarioles at two time points after a standard heat shock regimen of four 1 hr heat shock treatments over 2 days (Figure 4A–B). This heat shock regimen produced sparse clonal labeling, with only 33.9% and 35.0% ovarioles containing FSC clones at 7 and 14 dphs, respectively (Figure 4C). In mosaic ovarioles with GFP negative clones that extended from the Region 2a/2b border into at least one budded follicle, we observed an average clone size of 50.1 ± 14.1% at 7dphs and 51.3 ± 18.0% at 14dphs. We took two additional approaches to further test this result. First, we quantified FSC clone sizes at 7 days after a single 30 min heat shock. In these flies, the frequency of FSC clones was much lower (11.9%), yet the size of the FSC clones in mosaic ovarioles were not significantly different (48.2 ± 14.8%) from the average sizes of clones in mosaic ovarioles at 7 or 14 days after four 1 hr heat shock treatments (Figure 4C–D). Second, we repeated this test with another clonal marking system, MARCM (Lee and Luo, 2001), that uses a different mechanism to label clones. With this system, cells are initially GFP negative and mitotic recombination at FRT sites generates GFP positive clones. Again, we found that the average size of MARCM clones in mosaic ovarioles was not significantly different (44.7 ± 10.4%) from the average sizes of clones we observed with the GFP negative system (Figure 4C–D, and Figure 4—figure supplement 1). These measurements are remarkably similar to those reported in the Margolis and Spradling study, though we observed more variability in FSC clone size than was reported previously. Thus, our data provide strong support for the conclusion that the follicle epithelium is typically maintained by just two active FSCs. However, considering values that fall within two standard deviations of the means, and the fact that we excluded non-mosaic ovarioles from our analysis, our data are consistent with the possibility that FSC number fluctuates within a range of approximately one to four FSCs per ovariole.

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Frequency of clones in each experimental condition, GFPneg system and MARCM system.

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Clone sizes in each experimental condition, GFPneg system and MARCM system.

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Lastly, to determine where the FSCs are located, we stained ovarioles with GFP negative clones at 7dphs for GFP and FasIII. The Reilein et al. study reported that the anterior-most cells labeled with the LGR clonal marker system were located within one of three adjacent positions leading up to the FasIII border, and the authors interpreted this as evidence that FSCs are located within three rings, two FasIII negative rings in Region 2a, and one ring at the boundary of FasIII expression. With the GFP negative clonal marking system, we found that the anterior-most GFP negative cell was FasIII positive and located at the boundary of FasIII expression in the large majority of ovarioles with FSC clones (85.7%, n = 63). In these germaria, all GFP negative somatic cells formed a single, contiguous clone and there were no GFP negative somatic cells anterior to the boundary of FasIII expression (Figure 5A and D), indicating that the FSC must be FasIII positive. This observation is consistent with other studies that have demonstrated that the anterior border of an FSC clone coincides with the boundary of FasIII expression (Chang et al., 2013; Kirilly et al., 2005; Nystul and Spradling, 2010; Nystul and Spradling, 2007; Spradling et al., 1997). In 7.9% of the ovarioles, the anterior-most GFP negative cell was contiguous with the rest of the clone but was located on the anterior side of the FasIII border. Thus, FasIII staining was detectable on the posterior surface of the cell (which is likely to be signal from the adjacent cell) but not the anterior surface (Figure 5B and D). IGS cells in Region 2a are also mitotic during adulthood, and labeling systems that use mitotic recombination occasionally produce clones in Region 2a (Kirilly et al., 2011). Therefore, the anterior-most GFP negative cell in these germaria could be either an FSC with little or no FasIII expression or an IGS cell that became labeled through an independent recombination event from the one that produced the FSC clone. In the remaining 6.3% of ovarioles, the anterior-most GFP negative cell was FasIII negative and was not adjacent to either the FasIII border or the large clone of contiguous GFP negative follicle cells (Figure 5C–D). Notably, we found that cells in this position could be GFP negative even in germaria that did not have an FSC clone (Figure 5E), strongly suggesting that these cells are IGS cells, not FSCs. These observations confirm the conclusion that FSCs are usually, if not always, FasIII positive, and reside at the FasIII expression boundary.

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An understanding about the number and position of FSCs in each germarium is an important foundation for the diverse range of studies that use the follicle epithelium as a model. Indeed, many studies have relied on the conclusions of the Margolis and Spradling study, and both the early descriptions of the ovariole and decades of subsequent work have reinforced these conclusions. For example, the prediction that there are just two active FSCs per germarium aligns well with the published rates of oogenesis and follicle cell number. Specifically, a careful measurement of cell number revealed that there are approximately 900 follicle cells per egg chamber at Stage 6, when the follicle cells exit mitosis (Kolahi et al., 2009), and measurements of the rate of oogenesis (King, 1970; Lin and Spradling, 1993; Margolis and Spradling, 1995) suggest that the transit time from the Region 2a/2b border to Stage 6 is approximately 100 hr. Two progenitor cells dividing continuously with a 9.6 hour cell cycle (King and Vanoucek, 1960; Margolis and Spradling, 1995) would take approximately 85 hr to produce 900 cells (log₂(900/2) x 9.6 = 85). Since the part of the lineage that differentiates into polar and stalk cells ceases division well before Stage 6, the actual time to produce 900 main body follicle cells would be somewhat longer. In contrast, 16 progenitor cells would take only 56 hr (log₂(900/16) x 9.6 = 56) at this average rate of division. In addition, multiple studies have found that transient clones are capable of covering up to half of a follicle (Nystul and Spradling, 2010; Skora and Spradling, 2010; Song and Xie, 2003), consistent with the idea that the follicle cell population of each follicle is typically founded by two progenitors. Moreover, images of very large persistent clones from many different studies using a wide variety of single and multicolor labeling strategies and the analysis of the clonal data are consistent with a small number of actively dividing FSCs per germarium (Dai et al., 2017; Kronen et al., 2014; Nystul and Spradling, 2007; O'Reilly et al., 2008; Song and Xie, 2002). With respect to the position of the FSCs, the supposition that some cells in Region 2a are FSCs is inconsistent with many other studies that have demonstrated that somatic cells in this region have a markedly different shape, function, and gene expression pattern than cells in Region 2b (Decotto and Spradling, 2005; Huang et al., 2014; Kirilly et al., 2011; Rust et al., 2019; Song and Xie, 2002; Spradling et al., 1997; Wang et al., 2015; Wang and Page-McCaw, 2018; Upadhyay et al., 2018). Thus, the claim that there are 15–16 FSCs per germarium at both the Region 2a/2b border and within Region 2a is incompatible with a wide range of studies that provide independent orthogonal support for the conclusions of the Margolis and Spradling study.

Our study identifies several reasons for the discrepancies between the current paradigm and the Reilein et al study. First, their estimates of FSC number relied primarily on the use of the LGR clonal marking system, which we found to be unreliable because it had a high rate of background clone induction in our hands and produced clones with marker combinations such as LacZ^–, GFP^–, RFP⁺ and LacZ^–, GFP^–, RFP^– that are not predicted by the genotype. This raises the possibility that at least some of the clones generated by this system arise through a non-canonical event, such as recombination between an FRT40A and an FRT42D. This type of recombination would produce genome rearrangements that are not likely to be compatible with clone growth and, indeed, some clones of this type contained less than five cells. However, others, such as the clone shown in Figure 2D, were larger and the cells looked healthy. Thus, an alternative hypothesis is that these clones are not the product of an FRT recombination event but instead are due to epigenetic silencing of one or more marker transgenes. Consistent with this interpretation, high levels of fluorophore expression can reduce cellular fitness in some cases, so it may be that constitutive expression of three separate markers creates a selective pressure to silence one or more markers. Moreover, epigenetic silencing of transgenes has been described in follicle cells previously (Skora and Spradling, 2010). Although this study focused on transgenes with a UAS promoter rather than a constitutive promoter like those in the LGR system, it provides clear evidence that follicle cells actively silence transgene promoters and that the changes are heritable, producing a clone of cells with similar levels of transgene expression. Regardless of the cause, a high rate of clone induction in the absence of heat shock and the presence of clones with unexpected marker combinations are problematic for the use of clone size and frequency measurements to infer stem cell number. The Reilein et al. study also used a MARCM system as an independent test of their conclusions for this part of the study, but the number of clones analyzed was significantly lower and images of the MARCM clones were not provided so it is difficult to fully assess these data.

Second, we found that the assumptions and analysis of FSC clone number was flawed. The authors arrived at an estimate of 15–16 FSCs per germarium by quantifying the number of distinct lineages at 5, 7, 14, 21, and 30 dphs and then extrapolating back to 0 dphs using neutral drift modeling, but do not explain why the average number of uniquely labeled lineages would decline from more than four at five dphs to two at 15 dphs, and then remain at approximately two for the subsequent 15 days. Neutral competition models predict that mosaic tissues will drift toward monoclonality, not biclonality. Related to this, we found no evidence for the claim that individual FSC clones can be non-contiguous and contribute only sporadically to follicles that form after clone induction. On the contrary, we found that the large majority of FSC clones labeled with the GFP negative marking system in our study closely resembled the large contiguous clones described originally (Margolis and Spradling, 1995) and reported in many subsequent studies. Indeed, if FSC clones were typically discontinuous, it would be very difficult to distinguish between persistent FSC clones and transient clones because, even at late time points, it would appear as if ‘transient’ clones had not moved out of the tissue. Thus, much of the published work on persistent FSC clones would not have been possible (Buszczak et al., 2009; Kirilly et al., 2005; O'Reilly et al., 2008; Song and Xie, 2003; Song and Xie, 2002; Su et al., 2018). Therefore, rather than interpret the decrease in clonal diversity at early time points in the Reilein et al. study as evidence for neutral drift among a large pool of FSCs, we favor the alternative hypothesis that the clonal diversity at early time points is due to the presence of transient clones that had not yet moved out of the tissue. Because the authors allowed for the possibility that FSC clones are not contiguous, these transient clones may have been misinterpreted as evidence for persistently labeled FSC lineages.

A parallel approach used in the Reilein et al. study to determine the number of FSCs per germarium was to induce LGR clones sparsely and measure the fraction of a follicle labeled by a rare FSC clone. This is a valid approach if one assumes that all stem cells in the tissue have an equal chance of becoming labeled. However, in Reilein et al., the authors argue that there are significantly different rates of proliferation among the 15–16 FSCs in each germarium. Since mitosis is required to label clones with the LGR system, more rapidly proliferating FSCs would be more likely to be labeled, though it would be difficult to predict how much the differences in proliferation rate would affect the chances of becoming labeled. The rapidly dividing FSCs would also presumably contribute a disproportionately high number of cells to the tissue, so the overrepresentation of clones from these FSCs in the dataset would skew the estimate of stem cell number by an unknown amount. Therefore, within the framework of the Reilein et al study, this would not be an appropriate approach to determine stem cell number.

Third, we challenged the assumption in the Reilein et al. study that FRT recombination at both sites occurs in 100% of mitotic follicle cells. This assumption does not accord with either our experience with the Flp/FRT system or the frequencies of marker combinations that we observed with the LGR system. Moreover, if both FRT sites always undergo recombination in every FSC, the same should occur in every mitotic follicle cell. Each ovariole has thousands of mitotic follicle cells, so at early time points such as 5 dphs, when cells produced in the germarium would not have moved out of the ovariole, every ovariole would have all six clonal marker phenotypes. Yet this clearly was not the case in either our study or in the Reilein et al. study. By assuming that the recombined phenotypes (marker combinations) should be more common than they actually are, the neutral drift analysis in Reilein et al. may have overestimated the amount of clonal diversity that was present prior to the first point of analysis (5 dphs), thus contributing to their overestimate of the number of FSCs.

Fourth, we used a standard, reliable clonal marking system and quantitative analysis to measure FSC clone size at two time points after clone induction. Our measurements were consistent with the original finding that each FSC typically produces approximately half of the follicle cells in each ovariole, though we found a wider range of clone sizes than expected. This finding, combined with the unexpected clone patterns that we observed (Figure 3C and G–H) suggest that FSC lineages are not rigid but instead exhibit some flexible, stochastic behaviors that allow for a variable number of FSCs or cause individual FSC lineages to contribute to the tissue unequally. An interesting possibility to consider in future studies is that unequal contributions between two stem cell lineages in the same germarium may precede an FSC loss and replacement event. Nonetheless, despite this variation in clone size and pattern, our findings reinforce the idea that two FSCs produce the large majority of the follicle epithelium in most cases.

Lastly, our analysis established that FSCs reside at the FasIII expression boundary. Somatic cells in Region 2a have a shape, function and gene expression pattern that is distinct from the follicle epithelium, so it would be surprising if the population of FSCs spanned this border, with some FSCs that have IGS cell characteristics and others that have follicle cell characteristics. Live imaging in the Reilein et al. study demonstrated that labeled cells near the Region 2a/2b border can move toward the anterior, but these cells could not be simultaneously labeled with cell type specific markers, so their identity is unclear. Further analysis will be required to determine whether there are any conditions in which cells convert from the IGS cell identity into a follicle cell identity or vice versa, but our data suggest that, at least under normal laboratory conditions, persistent FSC clones are typically contained entirely within the FasIII expression domain. Taken together, our study clarifies and reaffirms the current paradigm for the fundamental features of the Drosophila FSC lineage, and provides important context for a wide variety of studies that use the follicle epithelium as a model system.

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 1-5.

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Acceptance summary:

This work revisited earlier studies regarding the behavior and characteristics of FSCs (follicle stem cells) in the Drosophila ovary, and identified a few potential flaws in the previous study, which concluded that there the number of FSCs was higher than previously thought. The current study presents data that are more in line with earlier studies that concluded that there are ~2 FSCs. The reviewers felt that the authors thoroughly examined the FSC clone behavior and provided potential explanations as to why previous and present studies differ in conclusion. The reviewers recognize that it may be sometimes difficult to reconcile differences between multiple studies, but the authors at least provided sufficient information such that this study itself can be thoroughly revisited/reproduced by anybody who wishes so. In summary, the reviewers agreed that this is an important study to characterize the clonal behavior of FSCs.

Decision letter after peer review:

Thank you for submitting your article "The follicle epithelium in the Drosophila​ ovary is maintained by a small number of stem cells" for consideration by eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by and Michael Eisen as the Senior Editor. The reviewers have opted to remain anonymous.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

This manuscript by Fadiga and Nystul re-assessed the work by Reilein et al. from Kalderon laboratory regarding the characteristics and the behavior of follicle stem cells (FSCs) in the Drosophila ovary. There have been discrepancies in the field, mainly regarding the number and location of FSCs: former work suggested that there are ~2 FSCs/ovariole which locates at the boundary of region 2a/b, and Reilein et al. suggested that there are ~16 FSCs and some of them locate more anteriorly. By pointing out a few flaws in the previous study by Reilein et al., the present manuscript confirms the older work and conclude that there are only ~2 FSCs.

Overall, the reviewers found that the present work is generally convincing, but raised inter-related issues regarding whether or not this paper should be published in eLife. If the present study clearly points out the logical flaws and explains why the Reilein et al. study was incorrect, then the reviewers agreed that this study is worth publishing in eLife.

However, currently, this manuscript simply presents discrepancies between the present study and Reilein et al. paper, and if it is simply reporting discrepancies, it should be dealt as 'matters arising' 'communication' sort of publication at Nature Cell Biology, where Reilein et al. work was originally published.

To warrant the publication in eLife as a stand-alone work, the reviewers felt that the present work needs more experiments, so that it would go beyond simply reporting discrepancies and provide clearly higher level of rigor compared to Reilein work. To this end, the reviewers raised several important points to be addressed as following.

Major comments:

– The manuscript does not provide a clear view of the source of discrepancies between the present study and the Reilein et al. study, and it was very unclear whether the authors are claiming that Reilein et al.'s logic was flawed (e.g. insufficient control etc) or that Reilein et al.'s data and their data do not match. If the former, it can be strongly concluded that the present study is correct. But if the data do not match between independent experiments between two labs do not match, then, the readers need to be presented with additional evidence which study to be believed. For example, Figure 1E show that a simple discrepancy between two labs' data, without providing the reason why Reilein et al. was wrong. "We did not reproduce it" is not a strong enough argument to deny preceding work. In particular, it is well known that slight changes in experimental conditions influence the outcome of heatshock experiments (e.g. the temperature at which flies were raised prior to heatshock). Therefore, we cannot/should not scientifically judge who is right at this point. Similarly, although the present study concludes that multi-color lineage tracing system is unreliable using their own number of clone induction without heatshock (25%), Reilein et al. reports that such background clone induction is only 2%. Therefore, it is not that Reilein et al. was careless in doing their own experiments, and Reilein et al.'s interpretation itself was reasonable based on their own data. At least the authors have to precisely describe the nature of issues with the Reilein paper (flawed data interpretation vs. discrepancy with the author's own data). This confusion (unclear about flawed interpretation vs. discrepancy) is seem throughout the manuscript, and it has to be corrected such that the readers can understand exactly why the authors are stating Reilein et al., is not correct.

– If simply reporting discrepancies between authors' experiments vs. Reilein et al's experiments, it would be more suited for matters arising type publication at the journal of Reilein et al. (Nature Cell Biology). To make this paper beyond that level (i.e. worth of a stand-alone publication), the reviewers would like to see independent method to verify the authors' conclusion. In parallel, the reviewers noted that the heatshock treatments that span the duration of two days (for multicolor system), which were used by Reilein et al., as well as the present study, will produce many clones with considerably different birthtime. This will likely complicate the interpretation. Therefore, experiments that uses single heatshock is highly recommended. In this regard, this paper does not describe their heatshock scheme (how long, how many times) for any other heatshock experiments, thus it was hard for the reviewers to evaluate whether other experiments must give more reliable numbers or not.

– The reviewers discussed what would be the ideal 'independent' experiments that can provide independent testing of authors' vs. Reilein's conclusion. MARCM technique may be a choice (it still relies on mitotic recombination, but at least address the possibility of 'difficult to see' negative clonal marking). Additionally, FRT-stop-FRT type of constructs (which is already available: such has act-FRT-stop-FRT-lacZ or gal4) does not rely on mitotic recombination, thus it may be considered better as an independent method. Similarly, the use of Flybow system might work as well.

[Editors' note: further revisions were requested prior to acceptance, as described below.]

Thank you for submitting your article "The follicle epithelium in the ​ Drosophila​ ovary is maintained by a small number of stem cells" for consideration by eLife. Your article has been reviewed by two peer reviewers, including Yukiko M Yamashita as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Michael Eisen as the Senior Editor.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

The reviewers agreed that this is an important work trying to reconcile some discrepancies in the field regarding the number of follicle stem cells in Drosophila ovary. The authors did thorough analysis and identified a few possible flaws in the previous studies. Although this study might not completely settle the entire discrepancy, it at least provides a critical starting point for a better understanding.

As an essential revision, we would like you to present the following results more thoroughly, as these are critical information in assessing this work as well as comparing this work to others.

1) Multicolor labeling with a single heat shock

2) MARCM experiment

The authors may already have the results, but the results are not thoroughly described/discussed in the current manuscript.

Individual comments are provided as a guideline, but the above two points are the most critical ones.

Reviewer #1:

This is a revised manuscript from Nystul lab that tried to clarify the number of follicle stem cells (FSCs) in the Drosophila ovary. It has been postulated that there are ~2 FSCs/ovariole for a very long time, until a recent study proposed there are ~16 FSCs/ovariole. This manuscript re-evaluated the conclusion of the recent study to conclude that there is an average of ~2 FSCs (with variation range possibly 1-4 FSCs).

In the original manuscript, all the reviewers felt that it was unclear whether this manuscript should be published as a stand-alone paper, if it is only reporting a discrepancy from the previous paper. Reviewers commented that this paper will require clear explanation(s) as to why the previous paper might have been flawed to be acceptable in eLife as a stand-alone paper.

In this revised version, the authors generally made convincing case. However, I remain troubled by the use of multiple heatshock regimen by this paper as well as earlier Reilein paper. In my opinion, for multi-color system, multiple heatshock MUST NOT be applied, NEVER EVER. This is because later heatshock event will provide an opportunity to create 'subclones' within the original clone. For example, for the LGR (LacZ, Red, Green) scheme, the first heatshock may create LacZ, Green clone (losing Red). Then after this LacZ-Green clone proliferated a few times, second heatshock can hit to convert a subset of cells to become LacZ only (losing Green). If this happens, originally marked LacZ Green clone will be further subdivided to LacZ Green and LacZ only clone, as if there are two clones (instead of original one clone). For this reason, I have insisted for the longest time that multi color labelling should not be combined with multiple heatshocks (for single color, multiple heatshok is OK, because there is no chance to generate subclone within a clone'). I understand that the authors have already invested quite a bit of effort and time in this, but I need to insist NOT to use multiple heatshocks. The authors claims that this is 'to make the experimental condition consistent', but if such experimental condition is the very cause of logical flaw, it should not be repeated. Or at least, the results with single heatshock must be more thoroughly presented.

The presence of clones that lack both LacZ and GFP is indeed indicates a big flaw in the experimental scheme (I suspect that it may represent an event of recombination between FRT40 and FRT42).

The results of MRCM clone analysis must be more thoroughly showed, as this is provided as 'key experimental results' from an orthogonal testing.

Overall, I feel this is worth publishing, but the above comments should be addressed even if this goes to the second round of revision, because I think it is fair to provide more thorough information on this paper such that Reilein paper's authors can repeat and refute as necessary/appropriate, if they choose so. Although I think that it is generally not advisable or productive to allow multiple schools of scientists to keep refuting each other, sometimes it is inevitable, until the entire field settles. And at the least everyone should do their best to ensure everything (from reasoning to experimental condition) is clear to readers.

Reviewer #2:

I have seen the resubmission of the manuscript by Fadiga and Nystul readersand believe they have adequately addressed the reviewer's concerns. As I mentioned previously, I highly recommend publication of this paper, not only because it helps to clarify our understanding of FSC biology, but it also serves as an excellent primer for researchers from different backgrounds to learn how to properly carry out lineage studies.

1) In Figure 1D and 1E from what part of the ovary are the authors gathering data? The germarium? Region 2? Region 3? Stage 1? Stage 2?

This manuscript by Fadiga and Nystul re-assessed the work by Reilein et al. from Kalderon laboratory regarding the characteristics and the behavior of follicle stem cells (FSCs) in the Drosophila ovary. There have been discrepancies in the field, mainly regarding the number and location of FSCs: former work suggested that there are ~2 FSCs/ovariole which locates at the boundary of region 2a/b, and Reilein et al. suggested that there are ~16 FSCs and some of them locate more anteriorly. By pointing out a few flaws in the previous study by Reilein et al., the present manuscript confirms the older work and conclude that there are only ~2 FSCs.

Overall, the reviewers found that the present work is generally convincing, but raised inter-related issues regarding whether or not this paper should be published in eLife. If the present study clearly points out the logical flaws and explains why the Reilein et al. study was incorrect, then the reviewers agreed that this study is worth publishing in eLife.

We thank the reviewers for their input and are pleased to hear that the reviewers agree that our work is generally convincing. Our study clearly identifies several logical flaws that may explain why the Reilein et al. study was incorrect. We have revised the text in several places to make this point clearer. The following sentences, some of which were also present in the original manuscript, are examples of places where we clearly indicate that our study identifies logical flaws in the Reilein et al. study:

1) In addition, we identify several logical flaws in the Reilein et al. study and performed new experiments to test the assumptions that underlie these flaws. Introduction paragraph five.

2) Taken together, these findings reveal significant flaws with the LGR clonal system that severely reduce its reliability and reproducibility. This may account for a large part of the discrepancies between the Reilein et al. study and prior studies, and strongly suggests that the LGR system is not an appropriate tool for determining the number of FSCs per ovariole. Results paragraph three.

3) They then stated “We therefore assumed in our statistical modeling that the different colors of FSC clone were present in those same proportions (B:G:BG:BR:GR:LGR = 1:1:1:2:2:2)”.

4) However, we are not aware of any other example where recombination between FRT sites is reported to occur in every mitotic cell after heat shock.

These findings challenge the underlying assumptions of the analysis in the Reilein et al. study, and suggest that, even if the LGR system were reliable, the interpretation of the data leading to the conclusion that there are 16 FSCs per germarium are also flawed. Results paragraph four.

5) One of the original observations in the Margolis and Spradling study is that an FSC clone forms a coherent, contiguous patch of cells that extends from the Region 2a/2b border out to the posterior edge of the clone boundary, contributing to each new follicle that is formed after clone induction. In contrast, Reilein et al. posited that each FSC does not normally contribute to every follicle, so discontinuous clone patterns with labeled cells at the Region 2a/2b border followed by intermittent, non-contiguous patches of labeled cells downstream should be expected. Using the GFP negative clonal marking system, we found that 85.7% of the ovarioles had typical clone patterns that had been described previously. Results paragraph five.

6) Thus, the claim that there are 16 FSCs per germarium at both the Region 2a/2b border and within Region 2a is incompatible with a wide range of studies that provide independent orthogonal support for the conclusions of the Margolis and Spradling study. Discussion paragraph one.

7) Our study identifies several reasons for the discrepancies between the current paradigm and the Reilein et al. study. First, their estimates of FSC number relied primarily on the use of the LGR clonal marking system, which we found has a high rate of background clone induction…Second, we found that the assumptions and analysis of FSC clone number was flawed…Third, we challenged the assumption in the Reilein et al. study that FRT recombination at both sites occurs in 100% of mitotic follicle cells. Discussions paragraphs one-three.

However, currently, this manuscript simply presents discrepancies between the present study and Reilein et al. paper, and if it is simply reporting discrepancies, it should be dealt as 'matters arising' 'communication' sort of publication at Nature Cell Biology, where Reilein et al. work was originally published.

Although we do report discrepancies between our study and the Reilein et al. study, we also used new approaches and experiments that went beyond simply repeating the experiments of Reilein et al.

These include the following:

1) The use of an independent and well-established clonal marking system, the GFP negative system on FRT19A, to confirm FSC clone size and dynamics.

2) The use of MARCM as a second, independent system, which we now include in response to reviewers’ comments. Our results provide further confirmation of our original conclusions about the number of FSCs per ovariole.

3) The use of 3D image segmentation methods to measure clone size as a percentage of total cell number in the tissue.

4) Quantification of images co-stained with FasIII and a clonal marker to precisely determine the position of the clone boundary relative to the FasIII expression boundary

To warrant the publication in eLife as a stand-alone work, the reviewers felt that the present work needs more experiments, so that it would go beyond simply reporting discrepancies and provide clearly higher level of rigor compared to Reilein work. To this end, the reviewers raised several important points to be addressed as following.

Major comments:

– The manuscript does not provide a clear view of the source of discrepancies between the present study and the Reilein et al. study, and it was very unclear whether the authors are claiming that Reilein et al.'s logic was flawed (e.g. insufficient control etc) or that Reilein et al.'s data and their data do not match. If the former, it can be strongly concluded that the present study is correct. But if the data do not match between independent experiments between two labs do not match, then, the readers need to be presented with additional evidence which study to be believed.

Although we cannot know precisely how or why the authors of the Reilein et al. study obtained each of the results they report, we have identified several instances in which the logic in the Reilein et al. paper is flawed and have pointed this out in our manuscript. These include:

1) The assumption that mitotic recombination produces clonal labeling in all mitotic cells. A significant flaw in the logic of the Reilein et al. study that was integral to their analysis was the assumption that their clone induction protocol induced recombination at both FRT sites in every mitotic follicle cell. Specifically, as we described in the manuscript, they explain that recombination at both FRT sites followed by segregation of sister chromatids at mitosis would produce 9 possible genotypes with equal frequency, and that these 9 genotypes would produce 6 detectable phenotypes (marker combinations) at a 1:1:1:2:2:2 frequency. They then stated “We therefore assumed in our statistical modeling that the different colors of FSC clone were present in those same proportions (B:G:BG:BR:GR:LGR = 1:1:1:2:2:2)”. There is no evidence that FRT recombination in a clonal marking system that relies on mitosis is 100% efficient in any fly tissue. Indeed, because cells must be in S phase or G2 for the recombination event to produce a detectable chromosomal rearrangement, it is extremely unlikely that all cells will be in the correct phase of the cell cycle at the time of the heat shock treatments to be susceptible to clonal labeling in this way.

2) The assumption that a quantification of the number of differentially labeled FSC clones per ovariole alone is sufficient to infer the number of FSCs per ovariole. This logic is stated in the Reilein et al. paper as follows: “The number of FSC colours observed (Figure 2J) and the inferred number of surviving FSC lineages (Figure 2C–K; see Supplementary Note) declined rapidly over time and, by extrapolation, indicates the initial presence of about 16 FSCs”. However, we found that, in 88.7% of ovarioles with more than two distinct lineages (n = 62), only 1 or 2 of the lineages spanned the entire ovariole while the other lineage(s) were very small, covering only part of one follicle or a small patch of cells in the germarium (Figure 1F). In Reilein et al., the authors did not comment on whether the clones they analyzed in this experiment were different sizes, so we do not know if this was also true in their hands, but our observations raise the possibility that they may have been. If clone sizes do differ in this way, it suggests that either the smaller clones are not FSC clones or that different FSCs within the same ovariole contribute substantially different numbers of cells to the tissue. Either of these possibilities, if true, would significantly undermine the logic used in Reilein et al. Specifically, if they interpreted non-FSC clones as FSC clones, this would lead to an overestimate of the number of FSCs in the tissue. Alternately, if some FSCs divide more often than others, then FSCs clones would be unequal sizes, which would contradict the assumption made elsewhere in the paper that clone size is proportional to stem cell number. Therefore, the failure to consider clone size in these experiments is a logical flaw of the Reilein et al. study that undermines their conclusions.

3) The assumption that FSC clones are commonly discontinuous. There is no evidence from the literature that this is the case and, in fact, several studies, including Margolis and Spradling, 1995, Nystul and Spradling, 2010, and Skora and Spradling, 2010 clearly demonstrate the contrary, that clones in the FSC lineage remain contiguous. In addition, as we described in the manuscript, if FSC clones were typically discontinuous, it would be very difficult to distinguish between persistent FSC clones and transient clones because, even at late time points, it would appear as if small “transient” clones had not washed out of the tissue. This would have made much of the published work on persistent FSC clones not possible. Lastly, we provide new evidence in this study that only 6% of ovarioles had clones that could be interpreted as discontinuous while 85.7% of the ovarioles had typical clone patterns that had been described previously. Interpreting patches of clonally marked cells that do not extend back to the Region 2a/2b border as FSC clones would lead to an inaccurate count of the number of FSC clones per ovariole. Therefore, the assumption that FSC clones are commonly discontinuous is a logical flaw of the Reilein et al. study that undermines their conclusions.

We have revised the text throughout the manuscript to make these points clearer.

For example, Figure 1E show that a simple discrepancy between two labs' data, without providing the reason why Reilein et al. was wrong. "We did not reproduce it" is not a strong enough argument to deny preceding work. In particular, it is well known that slight changes in experimental conditions influence the outcome of heatshock experiments (e.g. the temperature at which flies were raised prior to heatshock). Therefore, we cannot/should not scientifically judge who is right at this point. Similarly, although the present study concludes that multi-color lineage tracing system is unreliable using their own number of clone induction without heatshock (25%), Reilein et al. reports that such background clone induction is only 2%. Therefore, it is not that Reilein et al. was careless in doing their own experiments, and Reilein et al.'s interpretation itself was reasonable based on their own data.

We apologize for the confusion. By providing the comparison in Figure 1E, we did not intend to imply that our results with the LGR system should be considered to be more accurate than the results reported in Reilein et al. Instead, we provided this comparison to indicate that the LGR system, which they created and based most of their conclusions on, is apparently not reliable since we are unable to reproduce their results. In fact, we spent several months varying experimental conditions, such as culture temperature, heat shock conditions, and feeding, in an attempt to reproduce the results of the Reilein et al. study. However, in our hands, regardless of the experimental conditions we used, we consistently found that the numbers of clones labeled with different marker combinations were lower than those reported in Reilein et al. and the frequency of clones in the absence of heat shock was higher than the frequency reported in Reilein et al. In addition, when working with the LGR system, we were very surprised to discover that some cells could lack both LacZ and GFP expression (Figure 2C-E). Both of these markers are on the left arm of Chromosome 2 so, even after recombination, every cell should have at least one of these two markers. The emergence of LacZ-, GFP- clones is not likely to be due to variations in experimental conditions and instead suggests a fundamental flaw in the LGR system, thus further undermining its reliability.

Based on these observations, we concluded that the results with the LGR system from both our study and the Reilein et al. study could not be relied upon and thus a different clonal marking system that is more reliable and reproducible was necessary. We chose to work with the GFP negative marking system on FRT19A because it has been used by many different labs to study the ovary and we found the rate of background recombination to be very low (as is the case in the ovary for most GFP negative marking systems). We have now revised the text Results paragraph two and three to clarify our interpretation of our results with the LGR system. In response to the reviewers’ comments, we now indicate in this revised text that differences in experimental conditions may be one explanation for the differences in results between the two studies Results paragraph two. We hope this resolves the confusion about this issue.

At least the authors have to precisely describe the nature of issues with the Reilein paper (flawed data interpretation vs. discrepancy with the author's own data). This confusion (unclear about flawed interpretation vs. discrepancy) is seem throughout the manuscript, and it has to be corrected such that the readers can understand exactly why the authors are stating Reilein et al., is not correct. If simply reporting discrepancies between authors' experiments vs. Reilein et al's experiments, it would be more suited for matters arising type publication at the journal of Reilein et al. (Nature Cell Biology). To make this paper beyond that level (i.e. worth of a stand-alone publication), the reviewers would like to see independent method to verify the authors' conclusion. The reviewers discussed what would be the ideal 'independent' experiments that can provide independent testing of authors' vs. Reilein's conclusion. MARCM technique may be a choice (it still relies on mitotic recombination, but at least address the possibility of 'difficult to see' negative clonal marking). Additionally, FRT-stop-FRT type of constructs (which is already available: such has act-FRT-stop-FRT-lacZ or gal4) does not rely on mitotic recombination, thus it may be considered better as an independent method. Similarly, the use of Flybow system might work as well.

In response to this comment, we have now included an independent method to verify our conclusions. We considered each of the options recommended by the reviewers and decided to use MARCM because it is commonly used and well-tested; uses a single fluorophore and thus avoids potential issues with multicolor systems as we described for the LGR system used in Reilein et al.; and because, unlike Flpout systems, requires mitosis and thus is less prone to background recombination and labeling of non-mitotic cells that are not relevant for our analysis. We now include these data in an updated version of Figure 4C-D.

In parallel, the reviewers noted that the heatshock treatments that span the duration of two days (for multicolor system), which were used by Reilein et al., as well as the present study, will produce many clones with considerably different birthtime. This will likely complicate the interpretation. Therefore, experiments that uses single heatshock is highly recommended. In this regard, this paper does not describe their heatshock scheme (how long, how many times) for any other heatshock experiments, thus it was hard for the reviewers to evaluate whether other experiments must give more reliable numbers or not.

We completely agree with the concern that multiple heat shock treatments may complicate the analysis. We used four heat shock treatments to induce clones with the LGR system in order to stay consistent with the approach taken in Reilein et al. However, when we used the GFP negative system, we used two clonal induction regimens: In one case we used a single 30 minute heat shock treatment, and in a second case, we used four 1 hour heat shock treatments (Figure 4C-D). We found no significant difference in clone size using these two different protocols, which strongly supports the conclusion that the follicle epithelium is typically maintained by just two FSCs. We thank the reviewers for noticing that we did not fully explain this in the Materials and methods section. In response to this comment, we have now added details for each heat shock regimen and clonal marking system into the Materials and methods section.

[Editors' note: further revisions were requested prior to acceptance, as described below.]

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

The reviewers agreed that this is an important work trying to reconcile some discrepancies in the field regarding the number of follicle stem cells in Drosophila ovary. The authors did thorough analysis and identified a few possible flaws in the previous studies. Although this study might not completely settle the entire discrepancy, it at least provides a critical starting point for a better understanding.

As an essential revision, we would like you to present the following results more thoroughly, as these are critical information in assessing this work as well as comparing this work to others.

1) Multicolor labeling with a single heat shock

2) MARCM experiment

The authors may already have the results, but the results are not thoroughly described/discussed in the current manuscript.

Individual comments are provided as a guideline, but the above two points are the most critical ones.

As requested, we now provide these data. The data for the use of multicolor labeling with a single heat shock are in Figures 1G and 3A. The new data from the MARCM experiment are in Figure 4—figure supplement 1.

Reviewer #1:

[…] In this revised version, the authors generally made convincing case. However, I remain troubled by the use of multiple heatshock regimen by this paper as well as earlier Reilein paper. In my opinion, for multi-color system, multiple heatshock MUST NOT be applied, NEVER EVER. This is because later heatshock event will provide an opportunity to create 'subclones' within the original clone. For example, for the LGR (LacZ, Red, Green) scheme, the first heatshock may create LacZ, Green clone (losing Red). Then after this LacZ-Green clone proliferated a few times, second heatshock can hit to convert a subset of cells to become LacZ only (losing Green). If this happens, originally marked LacZ Green clone will be further subdivided to LacZ Green and LacZ only clone, as if there are two clones (instead of original one clone). For this reason, I have insisted for the longest time that multi color labelling should not be combined with multiple heatshocks (for single color, multiple heatshok is OK, because there is no chance to generate subclone within a clone'). I understand that the authors have already invested quite a bit of effort and time in this, but I need to insist NOT to use multiple heatshocks. The authors claims that this is 'to make the experimental condition consistent', but if such experimental condition is the very cause of logical flaw, it should not be repeated. Or at least, the results with single heatshock must be more thoroughly presented.

We completely agree with the reviewer’s point that using multiple heat shocks with a clonal system that has more than one pair of FRT sites is problematic. In addition to adding new data for the use of multicolor labeling with a single heat shock in Figures 1G and 3A, we now also make this point in the text in the second paragraph of the Results section. Specifically, we state “…it is generally inappropriate to use multiple heat shocks with a clonal marking system that has more than one pair of FRT sites because the later heat shocks may produce subclones within clones that were induced by the earlier heat shock treatments. Thus, at least some of the cases in which small and large clones coexist in the same ovariole may have been due to the production of subclones induced by the later heat shocks.”

The presence of clones that lack both LacZ and GFP is indeed indicates a big flaw in the experimental scheme (I suspect that it may represent an event of recombination between FRT40 and FRT42).

We agree.

The results of MRCM clone analysis must be more thoroughly showed, as this is provided as 'key experimental results' from an orthogonal testing.

We now provide representative images of a 3D rendering of an ovariole with a MARCM clone, including the surfaces generated with Imaris and the associated scatter plot as Figure 4—figure supplement 1.

Reviewer #2:

I have seen the resubmission of the manuscript by Fadiga and Nystul readersand believe they have adequately addressed the reviewer's concerns. As I mentioned previously, I highly recommend publication of this paper, not only because it helps to clarify our understanding of FSC biology, but it also serves as an excellent primer for researchers from different backgrounds to learn how to properly carry out lineage studies.

1) In Figure 1D and 1E from what part of the ovary are the authors gathering data? The germarium? Region 2? Region 3? Stage 1? Stage 2?

These data are derived from an analysis of the germarium and the first 2-3 follicles downstream from the germarium. We have added this detail into the figure legend for Figure 1.

Author details

1. Jocelyne Fadiga

   1. Department of Anatomy, University of California, San Francisco, San Francisco, United States
   2. Department of OB/GYN-RS, Center for Reproductive Sciences, University of California, San Francisco, San Francisco, United States

   Contribution

   Data curation, Formal analysis, Investigation, Visualization

   Competing interests

   No competing interests declared

2. Todd G Nystul

   1. Department of Anatomy, University of California, San Francisco, San Francisco, United States
   2. Department of OB/GYN-RS, Center for Reproductive Sciences, University of California, San Francisco, San Francisco, United States

   Contribution

   Conceptualization, Data curation, Software, Formal analysis, Supervision, Funding acquisition, Investigation, Visualization, Methodology, Project administration

   For correspondence

   todd.nystul@ucsf.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-6250-2394

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