The Drosophila proventriculus lacks stem cells but compensates for age-related cell loss via endoreplication-mediated cell growth

Domain: neuroscience
Published: 2026-01-27
Editorial outcome: minor_revision

Abstract
The Drosophila proventriculus is a bulb-shaped structure at the juncture of the foregut and the midgut, which plays important roles in ingestion, peritrophic membrane synthesis, and the immune response to oral pathogens. A previous study identified a population of cells in the proventriculus which incorporate bromodeoxyuridine (BrdU), a marker of DNA synthesis, and proposed that these cycling cells are multipotent stem cells that replace dying cells elsewhere in the tissue. Here, we re-investigate these cycling cells and find that they do not undergo mitosis, do not generate clonal lineages, and do not proliferate in response to tissue damage, and are therefore not stem cells. Instead, we find that these cells continually endocycle throughout the fly’s life, increasing their ploidy and size, while at the same time cells in this tissue are lost into the gut lumen as the fly ages. Functionally, these cells play a critical role in the synthesis of peritrophic membrane components, and we show that when their endocycling is experimentally increased or decreased, there is a concomitant change in ploidy, tissue size, and peritrophic membrane synthesis. Further, we show that inhibition of endocycling makes flies more susceptible to orally infectious bacteria. Altogether, we show that continual endocycling of these cells is critical for maintaining tissue size and function in the face of cell loss due to aging or tissue damage.

Introduction
The Drosophila gut is a highly regionalized organ with distinct functional domains along its length1–3. Within each domain, in addition to primary roles such as nutrient absorption and immunity, there are cellular mechanisms to ensure that the gut is repaired and renewed following tissue damage or cell loss. In the midgut, there are multipotent intestinal stem cells (ISCs) that continually divide to replace the differentiated cell types of the gut, both during homeostasis4,5 and in response to tissue damage6,7. Analogous ISCs are present in the mammalian small intestine8,9, which is functionally comparable to the Drosophila midgut10.

In contrast to the midgut, in the hindgut, there is no evidence of multipotent stem cells11,12. Instead, hindgut cells respond to tissue damage by undergoing extensive endocycling, a specialized cell cycle in which DNA is replicated without undergoing mitosis, thus increasing the ploidy and nuclear size of hindgut cells11. In Drosophila, this endocycling response to tissue damage or wounding is conserved in the adult epidermis11,13,14, and is important to maintain tissue integrity in the absence of stem cell activity.

At the anterior of the gut, where the foregut meets the midgut, there is a specialized bulb-shaped structure called the proventriculus (also known as the cardia, based on its shape). A previous study15 identified a ring of cells in the proventriculus that incorporates the nucleotide analog BrdU, a marker of S phase, and proposed that these cycling cells are stem cells that replace dying cells elsewhere in the proventriculus.

Here, we re-investigate the function of these previously identified cycling cells in the proventriculus. We find no evidence that these cells undergo mitosis, generate clonal lineages, or proliferate in response to tissue damage in other regions of the proventriculus. Instead, we find that these cells continually endocycle throughout the fly’s life, at a rate that varies with systemic nutritional signals. Further, we find that cells from this region of the proventriculus are progressively lost into the gut lumen as the fly ages, and that this cell loss drives nearby cells to endocycle and thereby increase in ploidy and size. Lastly, we show that blocking endocycling in these cells reduces peritrophic membrane synthesis, whereas inappropriately enhancing endocycling leads to increased peritrophic membrane synthesis. We conclude that the proventriculus does not contain stem cells, but that it instead compensates for age-related cell loss by endocycling-mediated cell growth to maintain tissue function.

Results
A ring of cycling cells in Zone 4
The proventriculus consists of a doubly-folded tubular epithelium and associated muscles and neuronal structures (Fig. 1A)16–18. It forms during embryogenesis by the invagination of an anterior, ectodermal portion of the embryonic gut (the future esophagus) into an adjacent segment of endodermal gut, creating a bulb structure with an internal lumen19–21. In addition to serving as a muscular valve surrounding the esophagus, the proventriculus plays a critical role in the synthesis of the peritrophic membrane, a chitin- and mucin-rich structure that lines the midgut16–18. Studies in other Dipterans have shown that the proventriculus is capable of producing peritrophic membrane at a rate exceeding 6 mm/h at 25°C, representing an enormous capacity for synthesis and secretion22,23. In addition to peritrophic membrane synthesis, the proventriculus plays important roles in the immune system24,25, and as a site for regulation of the microbiota26.

Morphological studies have identified six distinct “zones” arranged linearly along the proventriculus epithelium, based upon cellular morphology and on their proposed role in the synthesis of the peritrophic membrane16. These anatomical zones have been shown via single cell RNAseq (scRNAseq) to correspond to transcriptionally distinct cell types25.

Zone 4 is a ring-shaped structure that encircles the esophagus at the anterior of the proventriculus (Fig. 1A). A previous study identified a ring of cells located at the posterior limit of Zone 4 which consistently incorporate BrdU, indicating that these cycles progress through S phase in adult flies15. This study proposed that these cycling cells are multipotent stem cells that proliferate and replace dying cells elsewhere in the proventriculus15. An alternative hypothesis, not explored by Singh et al.15, is that these cells are endocycling and thus undergoing repeated S phases without mitosis.

Functionally, Zone 4 cells are highly secretory and play an important role in the synthesis of chitin and other peritrophic membrane components16,25,27. scRNAseq studies indicate it is enriched for genes involved in chitin synthesis and chitin modifications, including chitin synthase 2 (Chs2)25 which can be visualized with Chs2-Gal4 > UAS:2XEGFP (Fig. 1B, left). Studies in larval flies confirm that mutations in Chs2 lead to defects in peritrophic membrane synthesis27, and visualizing the peritrophic membrane with fluorescently labeled Helix pomatia (HPA) lectin28,29 confirms previous observations that peritrophic membrane components is highly enriched in the lumen directly abutting Zone 4 cells (Fig. 1B, middle.) Zone 4 cells also express high levels of the WNT pathway ligand Wingless (Fig. 1C, right), as previously reported15.

To re-investigate the function of these cycling cells, we fed flies with EdU for 2 days and confirmed the presence of a ring of EdU+ cells at the posterior edge of Zone 4 (Fig. 1C, left). EdU+ cells are occasionally observed elsewhere in the proventriculus (see e.g., Figs. 1F and 2A), but here we focus exclusively on the EdU+ population in Zone 4. Previous studies suggested that, in addition to Wg expression, JAK-STAT signaling is also enriched in this area15, and when we co-labeled proventriculi for EdU and 10XSTAT:GFP, a JAK-STAT pathway reporter, we found that the EdU+ population is directly anterior to STAT+ cells, suggesting that EdU+ cells are enriched at the posterior edge of Zone 4, with 10XSTAT:GFP expression representing the anterior limit of Zone 3 (Fig. 1C, right).

To characterize the dynamics of EdU incorporation over time, we fed flies with EdU for 1, 3, or 5 days. We performed this experiment with two standard lab genotypes, w1118 and OreR, and observed that the number of labeled cells increased linearly with time (Fig. 1D, E; simple linear regression, p < 0.0001 that slope is non-zero for both genotypes) at a rate of between 3.3 (w1118) and 4.1 (OreR) EdU+ cells per day, suggesting that cells in this region are continually entering S phase at a relatively constant rate. The number of EdU+ cells detected for these two genotypes were not significantly from one another at each time point (Fig. 1E).

To test whether individual cells cycle continuously over time or whether they cease cycling after a discrete number of S phases, we performed a pulse-chase experiment by feeding EdU for 3 days, followed by a 20 day chase in the absence of EdU (Supplementary Fig. 1). After 20 days, we observed significantly fewer in EdU+ cells in Zone 4, suggesting that the EdU signal was diluted by continued cycling (Supplementary Fig. 1). This dilution over time is similar to what is observed for the continually cycling ISCs, in which EdU labeling is diluted through mitosis, and in enterocytes (ECs), which endocycle and thereby dilute the proportion of Edu-labeled DNA in each nucleus over time (Supplementary Fig. 1). In contrast, these results differ from what is observed in the hindgut, where BrdU signal is retained for 20 days due to a lack of continual cycling under homeostasis11.

It has previously been shown that systemic nutritional signals tightly correlate with endocycle progression in a variety of cellular contexts in the Drosophila larva, whereas mitotic activity is not clearly linked to nutritional status30,31. We tested whether the EdU+ cells in Zone 4 are responsive to nutrition by varying the amount of dry active yeast, a primary source of protein and other macronutrients for Drosophila, added to a standard minimal lab food. We found that the number of EdU+ cells steadily increased in response to additional yeast added to the fly’s food (Fig. 1F, G; one-phase association model, rate constant K = 0.5689 [95% CI = 0.1639–1.463]), suggesting that the cycling of these cells is influenced by systemic nutritional cues.

We further tested this sensitivity to systemic nutrition using a genetic tumor model in the midgut (esg-Gal4, tubGal80ts > UAS:yki3SA; note that esg-Gal4 is not expressed in the proventriculus), which promotes systemic muscle wasting and cachexia via the upregulation of the systemic insulin antagonist Impl2, the cytokine upd3, and other cytokines32–34. In cachectic flies, we observed a significant decrease in the number of EdU+ cells in the proventriuclus (Supplementary Fig. 2A, B), suggesting that cycling in these cells responds to systemic insulin signaling. We then tested whether over-expressing the insulin antagonists Impl2, upd3, or both together, would lead to a similar reduction in EdU+ cells in the proventriculus. Indeed, when we expressed these factors from ISCs using esg-Gal4, we observed a significant decrease in EdU+ cells of the proventriculus (Supplementary Fig. 2C). Previous studies have shown that, in addition to ISCs, esg-Gal4 is also expressed in the corpus allatum, located near the proventriculus. We thus used an independent Gal4, dMef2-Gal4, to over-express secreted factors from the muscles, and observed a significant decrease in EdU+ cells in the proventriculus (Supplementary Fig. 2D).

Altogether, these observations confirm that the adult proventriculus contains a ring of cells at the posterior of Zone 4 which consistently cycle through S phase, and that they do so in response to systemic nutritional signals.

Single nucleus atlas and Gal4 toolkit for the proventriculus
In order to functionally characterize these EdU+ cells in vivo, we generated a toolkit of Gal4 lines to label and manipulate various cell types of the proventriculus, building on a recent single cell atlas generated for this tissue25. First, we screened the K-Gut database35 for Gal4 lines that specifically label Zone 4. We identified four lines that labeled Zone 4, inclusive of EdU+ cells, (GMR41F06-GAL4, GMR26D04-GAL4, GMR56D02-GAL4, and GMR71F06-Gal4) and selected a particularly specific and consistent line, 71F06-Gal4, for further study (Fig. 2A).

Next, to identify molecular markers for other proventriculus cell types and to serve as a foundation for further molecular study, we created a single cell transcriptional atlas of the proventriculus using single nucleus RNAseq (snRNAseq). We sequenced a total of 22,332 nuclei from approximately 600 dissected proventriculus tissues, which ultimately resolved into 14 transcriptionally-defined clusters (Fig. 2B–D). This atlas is available via an online data portal (https://www.flyrnai.org/scRNA/proventriculus/).

We mapped the identity of these snRNAseq clusters to in vivo anatomy using cluster-specific marker genes. One cluster was enriched for molecular markers of muscles (e.g., Mhc, up) and two clusters were enriched for pan-neuronal markers (e.g., nSyb, elav, brp), one of which was enriched for Akh- and Impl2-expressing cells and therefore likely represents the corpora cardiaca36–38. To map the remaining 11 clusters to the in vivo proventriculus, we examined the expression of top marker genes for each cluster using either Gal4 lines or split-intein Gal4 lines crossed to a ubiquitous reporter (Fig. 2C–E)39.

We were able to identify the remaining clusters as anatomically distinct epithelial cell types. One cluster corresponded to the esophagus (marked by Elovl7-Gal4), another to Zone 1 (marked by CG11550-Gal4 and Cht7-Gal4). At the clustering resolution we selected, Zone 2 and 3 cells were both found in a single cluster (enriched for Ppn-Gal4 and Cyp49a1-Gal4). We observed a distinct cluster enriched for Lgr1 and Delta that corresponds to a population of cells specifically at the anterior border of Zone 3, immediately posterior to Zone 4. We recovered distinct clusters corresponding to Zone 4 (Wnt4-Gal4) and Zone 5 (Ca-Ma2d-Gal4), as well as a cluster that corresponds to a discrete band of cells at the widest diameter of the proventriculus, at the border of Zone 5 and 6 (marked by CG10702-Gal4, Notum-Gal4, Mco4-Gal4N-int). A cluster enriched for SecCl-Gal4 corresponded to Zone 6, and a cluster enriched mex1-Gal4 and P5CS-Gal4 corresponded to the anterior midgut. Two additional minor clusters (labeled “unknown” and “anterior midgut 1” in Fig. 2B) were enriched for markers corresponding to multiple cell types and were excluded from further analysis.

We queried our atlas for markers of stem cells. In a previous scRNAseq atlas of the Drosophila midgut40, midgut ISC clusters were enriched for the stem cell marker esg, as well as cell-cycle related genes PCNA and stg. We did not observe notable expression of any of these genes, nor any cluster-specific enrichment for any Cyclin genes, in our proventriculus atlas, whether in our Zone 4 cluster or elsewhere.

Our snRNAseq atlas is largely concordant with a recently published scRNAseq atlas of the proventriculus25, with minor deviations in the proportions of cells present in each cluster (Supplementary Fig. 3). This high degree of concordance suggests that the cell types we identify are robust to experimental approach, as these two atlases were created using entirely different single cell protols; Zhu (2024) sequenced single cells from freshly dissected and chemically dissociated suspensions, while we sequenced isolated nuclei from flash frozen tissues. We also note that our atlas was generated from two biological samples: proventriculi from control flies and from cachectic flies, and thus includes cells across a range of nutritional status (see “Methods”). This snRNAseq atlas thus serves as a resource for studies of the proventriculus, and allows us to identify cell type-specific Gal4 driver lines for functional experiments.

No evidence for mitosis in the proventriculus
Stem cells are defined as multipotent precursor cells that undergo repeated asymmetric mitosis, giving rise to one daughter cell that retains a stem identity and another daughter cell capable of differentiation. Thus, mitosis is a critical feature of any stem cell system. A previous study of the proventriculus suggested that rare mitoses do occur in this tissue, albeit at a rate that is seemingly out of proportion with the steady numbers of cells passing through S phase15.

To re-examine whether these cells undergo mitosis, we first stained guts from two different control genotypes (20 OreR wild type guts, 36 71F06-Gal4 > UAS:2XEGFP guts, total n=56 guts) for phosphorylated Histone 3 (pH3+), a marker of mitosis. We fed these flies colchicine overnight prior to staining, which enriches for pH3+ cells by blocking exit from mitosis11. We observed zero pH3+ cells in the epithelia of Zone 4 in these flies, or anywhere else in the proventriculus.

The cell cycle can be experimentally driven forward using a variety of genetic manipulations, and in cells that are capable of mitosis these manipulations will dramatically increase M phase. For example, in ISCs of the posterior midgut, over-expression of UAS-yki3SA41–43 or UAS-Ras1A44–46, or Notch-RNAi4,5 all lead to dramatic increases in mitosis, leading to massive tumor growth. We hypothesized that, if Zone 4 cells are competent to undergo mitosis, over expression of such cell cycle-promoting factors should lead to an increase in mitotic cells.

To test whether these factors can drive mitotic proliferation in the proventriculus, we used 71F06-Gal4ts to drive UAS-yki3SA, UAS-Ras1A, or Notch-RNAi in Zone 4 cells, and then stained for pH3 as a marker of mitosis following enrichment via colchicine (here and below, 71F06-Gal4TS refers to flies containing both the 71F06-Gal4 driver and tubGal80ts, to restrict Gal4 function to adult flies).

All of these genetic manipulations led to significant increases in the number of EdU+ cells and the size of Zone 4 measured as the cross-sectional area at the tissue’s widest point (Figs. 3, S4A–D and S4F–H). Under these staining conditions, we could detect over 600 mitotic ISCs in the midgut that stained positively for pH3 (Fig. 3B). In contrast, we observed zero pH3+ cells in the proventriculus under any of these conditions (Fig. 3B). Interestingly, however, we did observe a significant increase in the ploidy of Zone 4 nuclei following over-expression of both UAS-yki3SA and UAS-Ras1A, indicating that these manipulations promote endocycling (Supplementary Fig. 4E and see below.) In contrast, while Notch-RNAi did increase the number of EdU+ cells, it did not increase ploidy, suggesting Notch signaling in the proventriculus may function to restrict inappropriate entry into S phase and/or endocycle progression, but not completion of the endocycle.

These results demonstrate that EdU+ cells located in Zone 4 of the proventriculus do not undergo mitosis either under homeostatic conditions or when experimentally driven through cell cycle progression via ectopic oncogene expression. Furthermore, we note that despite morphological deformities caused by over-expressing activated Yki or Ras1, we never saw GFP+ cells located outside of Zone 4, which provides further evidence that cycling cells in Zone 4 do not migrate to replace damaged or dead cells in other regions of the tissue as suggested by Singh et al.15.

No evidence for clonal growth of Zone 4 cells
Despite the absence of pH3 staining, it is formally possible that rare mitoses occur. Another hallmark of adult stem cells is the ability to generate multipotent clones in the adult organism. For example, in the adult posterior midgut, MARCM-based lineage tracing of ISCs leads to the presence of GFP-labeled clones throughout the midgut that contain all of the epithelial cell types that can be generated by ISC differentiation4,5. We therefore used MARCM-based lineage tracing to test whether there is clonal growth in the adult proventriculus.

As a positive control for our labeling protocol, we heat-shocked MARCM larvae during the L3 larval stage, when this tissue is still undergoing developmental proliferation. We observed large GFP+ clones throughout the proventriculus, both in Zone 4 and elsewhere, in every sample examined, demonstrating that MARCM labeling functions as expected in this tissue (Fig. 4A). As a negative control, we examined GFP+ patterns in both midguts and proventriculi from non-heat-shocked flies, and observed small numbers of scattered GFP+ cells throughout the proventriculus, as well as in the midgut. We scored proventriculi as having either no GFP+ clones, between 1–5 small groups of GFP+ cells (typically 1–2 cells each), or having large GFP+ clones that include >10 cells (Fig. 4D).

To test for clonal growth during the adult stage, we heat-shocked MARCM flies several days after eclosion, then reared them for varying lengths of time before scoring for GFP+ clones. We challenged an additional cohort of flies with a 2-day starvation followed by re-feeding, which increases ISC proliferation in the midgut47,48.

As expected, in the posterior midgut we observed GFP+ clones in the midguts of heat-shocked flies after 14 days, and we also found that these clones tended to be larger in flies that experienced a 2 day starvation (Fig. 4B). In contrast, in the proventriculus of these flies, we observed scattered singlet and doublet GFP+ cells throughout the tissue at roughly similar rates between negative controls and heat-shocked flies (Fig. 4C, D). This was true at 14 days after heat-shock, 19 days after heat-shock, and 30 days at 18°C after heat-shock (Fig. 4C, D). We did observe a single example of a large clone in one cohort of heat-shocked animals (Fig. 4D), which was likely a rare example of a MARCM clone being formed via leaky FLP expression during earlier development.

In the midgut, ISC divisions increase dramatically upon tissue damage6,7. To test whether there may be damage-responsive proliferation in the proventriculus, we repeated these MARCM experiments in flies subjected to a variety of gut damage conditions: bleomycin treatment, infection with pathogenic ECC15 bacteria, and 2000 RADs of X-rays6,7. We also performed MARCM experiments with 10% yeast supplementation because we had observed that EdU incorporation rates increased with yeast supplementation. We did not observe discernable GFP+ clones emanating from Zone 4 or any other area of the proventriculus in any of these conditions (Supplementary Fig. 5). We note the caveat that, unlike enterocytes of the midgut, Zone 4 cells do not directly contact the contents gut lumen and therefore likely do not experience the same degree of damage that ECs experience in contact with bleomycin or ECC15. Altogether, we find no evidence for clonal growth of the EdU+ cells in Zone 4 of the proventriculus.

No proliferation in response to distant tissue damage
Singh et al. proposed that the EdU+ cells in the proventriculus are proliferating for the purpose of migrating to replace dying cells elsewhere in the tissue15. This hypothesis implies that cells originating in Zone 4 proliferate in response to cell death in nearby tissues, and then migrate to such nearby tissues as the esophagus, anterior midgut, and crop.

To directly test how the EdU+ cells of Zone 4 respond to cell death in nearby tissues, we used the Gal4-UAS system to induce cell death in various regions of the proventriculus by overexpressing the pro-apoptotic factor UAS-rpr. We induced cell death in three different cell types of the proventriculus: in Zone 5 (Ca-Ma2d-Gal4), in Zone 1 (CG11550-Gal4), and in the esophagus (Evolv7-Gal4) (Fig. 4). In these experiments, we silenced UAS-rpr throughout development using tubGal80ts and raised flies at 18°C until several days post-eclosion. We activated tissue damage by shifting adult flies to 29°C for 48 h, and placed flies on EdU-containing food for the duration of the tissue damage, and throughout a recovery phase of 2 days or 5 days at 18°C following damage. If these EdU+ cells in Zone 4 proliferate to compensate for dying cells, we would expect to see an increase in EdU+ cells in response to damage in neighboring cells, and likely EdU+ cells migrating towards the area of damage.

In all three experiments, we observed pyknotic nuclei, indicative of apoptosis, in the intended cell type in UAS-rpr flies, and not in control flies (Fig. 5A–C), indicating that this system induces apoptosis as expected. However, in all three experiments we observed not an increase in EdU+ cells, but rather a significant decrease (Fig. 5.) These results are inconsistent with the hypothesis that these cells proliferate in response to tissue damage elsewhere in the proventriculus. The decrease in EdU+ cells support our previous observation that these cells cycle in response to cues indicative of organismal health.

Interestingly, when we drove apoptosis in the esophagus with Elovl7-Gal4, while we observed a decrease in EdU+ cells in Zone 4, we did observe indications of massive endoreplication in the esophagus itself: unusually large, EdU+ nuclei (Fig. 5C, asterisks). This is similar to what has been reported in the hindgut11. In contrast, tissue damage to Zone 1 or Zone 5 did not cause an obvious increase in endoreplication in the damaged tissue (Fig. 5A, B).

Genetic lineage tracing of EdU+ cells in Zone 4
As a final test for lineage tracing independent of MARCM, we wished to genetically label the descendants of Zone 4 EdU+ cells using a Gal4 line specific to these cycling cells. However, we did not observe any discernible transcriptional signatures of the Zone 4 EdU+ cells, and were initially unable to identify a Gal4 driver to specifically label these cells within Zone 4.

We thus used the split-intein Gal4 system to generate a genetic driver that specifically label the EdU+ population of cells in Zone 439. We generated a split-intein Gal4C-int line using the 71F06 enhancer fragment (Supplementary Fig. 6A; referred to here as 71F06Gal4-C-int), and then used our snRNAseq atlas to identify Delta as a candidate gene which is primarily enriched in Zones 1–3 and in Zone 3–4 border cells, with some expression as well in Zone 4 (Supplementary Fig. 6A). When we examined the intersectional labeling of 71F06Gal4-C-int ∩ DeltaGal4-N-int > UAS:2xEGFP, we observed specific expression in a band of cells at the posterior edge of Zone 4 (Supplementary Fig. 6A). We confirmed that these cells contain the large majority of EdU+ cells (Supplementary Fig. 6A).

To lineage trace these cells, we used the G-TRACE system combined with tubGal80ts, which labels current expression in RFP and lineage expression in GFP49. After development at 18°C, we placed 71F06Gal4-C-int ∩ DeltaGal4-N-int, tubGal80ts > G-TRACE at 29°C to initiate lineage tracing and examined the proventriculus after 1 day, 6 days, and 10 days (Fig. 6C, D). If these cells are proliferating and generating clones, we would expect to see progressively more GFP+/RFP− cells, as descendants of these cells would grow into the rest of the tissue. However, while we did observe small numbers of GFP+/RFP− cells immediately adjacent to the GFP+/RFP+ domain, the number of these cells did not increase or move over time, but were constant over a 10 day period (Fig. 6C, D). These results are consistent with leaky and/or minor dynamic changes in the expression pattern of our split-intein Gal4 line, rather than with proliferation of cells in this domain.

Taken together, we find no evidence that the EdU+ cells located in Zone 4 are stem cells. We do not observe mitosis, nor the generation of MARCM clones, nor a proliferative response to nearby tissue damage, nor any evidence of genetic cell lineages growing over time. We thus propose that these cells must be endocycling rather than undergoing mitosis.

Cells in Zone 4 increase in ploidy as flies age
The consistency with which EdU is incorporated into cells in Zone 4 suggests that these cells are continually endocycling throughout the fly’s life, which should lead to increased ploidy as flies age. To test this directly, we performed a time-course experiment using confocal microscopy to quantify the ploidy of Zone 4 nuclei over the course of fly aging. In this experiment, 71F06-Gal4 > UAS:GFP stinger flies (hereafter, UAS:GFPNUC), which express nuclear-localized GFP in Zone 4, were split into vials either containing dried active yeast powder ad libitum or in standard food lacking additional yeast powder, and were examined at six time points between 3 days and 35 days after eclosion. At each time-point, we collected 5-6 confocal z-stacks and quantified the ploidy and the volume of these nuclei (Supplementary Fig. 7).

We observed a consistent increase in ploidy and nuclear volume over the course of the fly’s life, supporting the hypothesis that these cells are continually endocycling and increasing in ploidy (Figs. 6A–C and S7A, B; relationship between age and ploidy was significantly different than zero; simple linear regression; n = 960/1064 cells [no yeast/yeast], p < 0.0001 for both no yeast and yeast).

Consistent with the observation that supplemental yeast increases EdU incorporation, we found that the 7 and 14 day time points, ploidy was significantly higher in flies which received dried active yeast, but that by 21 days the average ploidy was not significantly different between these treatments (Fig. 6A–C). Over this time-course, the mean ploidy of Zone 4 cells increased from ~4C at 3 days (2.1–2.2× diploid, with or without yeast) to ~10C at 35 days (4.8–4.9× diploid), while the maximum ploidy amongst Zone 4 cells increased from ~9C (4.3–4.6× diploid) to ~22–26C (11.1–13.0× diploid) between these time points. We found no evidence that Zone 4 cells become multinucleate, based on co-staining these samples with the f-actin marker, even at the latest time point (35d) when the tissue begins to lose a clearly monolayered structure (Supplementary Fig. 6C).

Zone 4 cells are progressively lost into the gut lumen
Surprisingly, we observed that while the volume of Zone 4 nuclei progressively increased, the total number of GFP+ nuclei steadily decreased (Fig. 6D). Over the first 14 days, cell numbers decreased at equivalent rates for flies fed with or without yeast, but beginning at 21 days the number fell faster for flies without added yeast (Fig. 6D). Remarkably, we could observe “snapshots” of this cell loss in fixed samples, visible as GFP+ nuclei being shed into the gut lumen at the time of dissection (Figs. 6E and S7). We quantified the number of GFP+ nuclei present in the lumen outside the Zone 4 epithelium in these fixed tissues and found that the number of GFP+ cells first started to increase at 14 days after eclosion, and subsequently increased until reaching a maximum at 28 days (Fig. 6F). We did not observe any obvious morphological differences in the cells being lost, and the mechanism by which these cells exit the epithelium is not known, but we observed holes forming in the F-actin-rich barrier separating Zone 4 cells from the gut lumen, with GFP+ nuclei passing through these holes into the lumen (Supplementary Fig. 8). We note that the patterns of cell loss neatly with our total cell counts, with the greatest decreases in cell numbers corresponding to those time-points when more nuclei were being lost.

Together, these data suggest that Zone 4 nuclei are endocycling and thus increasing in ploidy and nuclear volume while cells from this region are being lost into the lumen. A simple explanation for these observations is that ploidy is increasing to maintain metabolic output and tissue integrity as cells are progressively lost during aging. Alternatively, the opposite could be true: as endocycling increases tissue size, this increased tissue size drives the loss of cells to maintain tissue size.

To distinguish between these possibilities, we returned to the genetic cell ablation strategy described above, this time focusing on Zone 4 itself. We raised 71F06-Gal4ts > rpr and control flies (71F06-Gal4ts > +; Fig. 7A) at 18°C until 2–3 days post-eclosion, then shifted them to 29°C for either 2 or 5 days to induce cell death in Zone 4. During this damage period, flies were fed with EdU-containing food to monitor cell cycling in response to tissue damage. We observed small, pycnotic nuclei throughout Zone 4, indicative of cells in Zone 4 (Fig. 7B, arrowheads).

After 2 days of rpr expression in Zone 4 the number of EdU+ cells decreased significantly compared to control flies, but after 5 days this number had rebounded to control levels (Fig. 7C). Notably, the ploidy of these EdU+ cells following 5 days of tissue damage was significantly increased (Fig. 7D). Thus, when cell loss is ectopically induced in Zone 4 via tissue injury, the remaining cells in the tissue endocycle to increase their ploidy. These results are consistent with the hypothesis that Zone 4 cells detect the loss of nearby cells and increase their ploidy via the endocycle in response.

These results indicate that Zone 4 cells can endocycle in response to exogenous damage, yet our previous observations suggest that cells in this tissue also endocycle continuously throughout the fly’s life even in the absence of exogenous damage (Figs. 1 and 7). Specifically, we observe continuous endocycling during the first two weeks of the fly’s life when there is only a modest amount of cell loss (Fig. 7D). To reconcile these observations, we hypothesized that there are two phases of endocycling in this tissue: first, in young flies, endocycling occurs in the absence of cell loss, and is spatially restricted to the posterior edge of the tissue. Subsequently, when naturally occurring cell loss from Zone 4 begins to accelerate in ~2–3 week old flies, a second phase of endocycling begins in response to cell loss, and is spatially distributed throughout Zone 4, in areas nearby the sites of cell loss.

A prediction of this hypothesis is that the spatial distribution of EdU+ cells should be markedly different in the proventriculi of young vs old flies: restricted to the posterior in young flies, and distributed throughout the tissue in older flies. To test this, we collected OreR flies at five time points between 4 and 31 days old, and measured the spatial distribution of EdU+ cells relative to the posterior edge of Zone 4.

Remarkably, in younger flies (4 and 11 days), EdU+ cells were almost entirely restricted to 1–2 cell diameters from posterior of the tissue (Fig. 7E, F). However, in all three of the older time-points (18, 25, 31 days), in addition to those EdU+ cells located at the posterior of the tissue, there were also many EdU+ cells scattered throughout Zone 4, and these distributions were significantly different from the younger time-points (Fig. 7E, F). These results suggest that, in wildtype files under unperturbed conditions, there are two distinct phases of endocycling in this tissue: a steady, continuous endocycling in younger flies at the posterior of the tissue, and subsequently throughout the tissue in response to increased cell loss as the fly ages.

Excess endocycling increases peritrophic membrane synthesis
Increasing genomic copy number is a widespread mechanism to enhance synthesis of proteins and other biological components in non-proliferative tissues31,50,51. Given the role that Zone 4 cells play in the synthesis and secretion of chitin and other peritrophic membrane components25,27, we hypothesized that endocycling in this tissue is linked to peritrophic membrane synthesis. We therefore used genetic manipulations to both increase and decrease endocycling and then visualized the peritrophic membrane and tested for functional defects.

To increase endocycling, we overexpressed the cell cycle regulators UAS:CycE or UAS:Myc in Zone 4 cells using 71F06-Gal4ts. CycE is a key regulator of both endocycling and the mitotic cell cycle, and drives entry into S phase31,52, and Myc is a transcription factor whose overexpression is sufficient to drive endocycling and increased nuclear volume in multiple contexts31,51,53,54. In Zone 4 cells, both manipulations led to significantly enlarged nuclei after 7–9 days at 29°C (Fig. 8A, B), as well as significantly increased ploidy (Fig. 8F), indicative of increased endocycling. As predicted, these manipulations also led to significant increase in the tissue size of Zone 4 (Fig. 8G).

We tested whether increased ploidy led to altered peritrophic membrane synthesis by staining with a panel of molecular markers. Confocal projections of HPA lectin-stained tissues revealed large areas of glycosylated material emanating from Zone 4, forming thickened cords that were not observed in controls (Fig. 8B). To quantify this phenotype, we measured the cross-sectional area of HPA lectin-stained material adjacent to Zone 4 and found a significant increase in UAS:CycE and UAS:Myc flies (Fig. 8C, H). We then stained these tissues for calcofluor, which labels chitin, a major component of the layer of peritrophic membrane synthesized by Zone 4. We observed a significant increase in calcofluor-positive signal adjacent to Zone 4 in UAS:CycE and UAS:Myc flies, indicative of increased chitin synthesis (Fig. 8D, I). Lastly, we stained these tissues with wheat germ agglutinin (WGA), an additional marker for glycosylated targets, and which has been used as a peritrophic membrane marker55, and observed a significant increase in WGA staining adjacent to Zone 4 as well (Fig. 8E, J).

Together, these results suggest that increasing endocycling in Zone 4 leads to increased synthesis of peritrophic membrane in the proventriculus.

These experiments with UAS-CycE and UAS-Myc also allowed us to perform an independent test of whether endocycling was functionally causal for the loss of cells from Zone 4 during aging. If cell loss was driven by the process of tissue growth via endocycling, then experimentally increasing endocycling via UAS:Myc or UAS:CycE should lead to enhanced cell loss. We counted the number of GFP+ cells in these genotypes 1 day after shifting to 29°C, and after 12 days (Supplementary Fig. 9). The number of cells in each genotype decreased after 12 days by between 21 and 31%, but the percent decrease between genotypes were not significantly different (Supplementary Fig. 9), despite the fact that ploidy, nuclear size, and tissue size is significantly increased in these genotypes compared to controls (Fig. 8). This suggests that the loss of Zone 4 cells during aging is not driven by endocycling itself, and that a distinct mechanism must govern the loss of cells.

Blocking endocycling reduces peritrophic membrane synthesis
To inhibit endocycling, we used RNAi to knockdown several candidate genes implicated in the regulation of the endocycle in Drosophila: fizzy-related (fzr), E2F1, myc, CycE, and the insulin receptor (InR)12,56–64. Of these candidates, InR-RNAi was by far the most potent reagent to block endocycling and reduce ploidy in Zone 4 (Supplementary Fig. 10 and Fig. 9). fzr-RNAi did not reduce ploidy (Supplementary Fig. 10A), E2F1-RNAi caused a statistically significant yet relatively modest reduction in ploidy (Supplementary Fig. 10B), and myc-RNAi led to dramatic disruptions to Zone 4 structure beyond simply blocking endocycling (Supplementary Fig. 10C). While cycE-RNAi did reduce ploidy and tissue size (Supplementary Fig. 10D, F, G), this effect was muted compared to InR-RNAi and caused large aggregations of peritrophic membrane material that complicated further analysis (Supplementary Fig. 10D, E). Thus, we focused our analysis of inhibited endocycling to InR-RNAi, and we note the caveat that Insulin signaling has additional effects of cellular metabolism in addition to simply regulating the cell cycle.

Using two independent RNAi constructs, we observed an near total loss of EdU staining in 71F06-Gal4ts > InR-RNAi animals (Fig. 9A, B) and a concordant failure to increase ploidy during aging (Fig. 9C). In control flies (71F06-Gal4ts > w1118 or mCherry-RNAi), Zone 4 nuclei steadily increase in ploidy between 6 days and 21 days at 29°C, where in 71F06-Gal4ts > InR-RNAi flies, ploidy did not increase (Fig. 9C). The overall size of Zone 4, visualized with cytoplasmic UAS:2XEGFP, was dramatically reduced by InR-RNAi (Fig. 9D, H). These experiments also demonstrate that the link between systemic nutrition and endocycling in Zone 4 (e.g., Fig. 1F, G) is at least partially regulated cell autonomously via insulin signaling directly interpreted by these cells.

We visualized the peritrophic membrane in 71F06-Gal4ts > InR-RNAi flies using the same three molecular markers as above. Consistent with the reduced nuclear volume and tissue size, the quantity of peritrophic membrane emanating from Zone 4 in InR-RNAi animals was significantly reduced, as revealed by staining for HPA lectin, calcofluor, and WGA (Fig. 9E–G, I–K). These alterations in peritrophic membrane morphology are consistent with the hypothesis that increased genomic copies allow for increased synthesis of peritrophic membrane components.

The peritrophic membrane is continuously synthesized in the proventriculus, then migrates posteriorly to line the midgut, where it serves as a protective barrier for the absorptive surface of enterocytes65,66. To assess the ultimate functional consequences of reduced endocycling in Zone 4 cells, we performed a number of assays to characterize the structure and function of the peritrophic membrane in the midguts of 71F06-Gal4ts > InR-RNAi flies. When viewed using transmission electron microscopy (TEM), the peritrophic membrane can be seen to contain four distinct layers16. Zone 4 is thought to synthesize the contents the second layer, L2, which is chitin-rich and appears as a thick, electron-dense layer abutting the lumen16 (Supplementary Fig. 11A). We analyzed the peritrophic membrane of InR-RNAi flies using TEM (14 days at 29°C), and did not observe any gross morphological defects or alterations to the four-layered structure (Supplementary Fig. 11A), indicating that L2-containing peritrophic membrane is still produced in flies with reduced endocycling in Zone 4.

We then performed two independent assays for intestinal barrier integrity on 71F06-Gal4ts > InR-RNAi flies. First, we subjected 71F06-Gal4ts > InR-RNAi flies to the “smurf assay”, in which flies are fed a blue dye which leaks from the gut into the rest of the body in flies with a compromised gut barrier67,68. In flies which had been aged for 24 days at 29°C, we did not observe any gut leakage either in controls or 71F06-Gal4ts > InR-RNAi flies (Supplementary Fig. 11B), indicating that these guts retained a baseline of functional integrity. Similarly, when we fed flies with fluorescently-labeled beads (diameter 0.056 µm) to test for leakage55,66, we did not detect a statistically significant increase in the proportion of 71F06-Gal4ts > InR-RNAi flies with leaky peritrophic membranes compared to controls (Supplementary Fig. 11C). These two assays suggest that flies with inhibited endocycling in the proventriculus are still capable of producing sufficient peritrophic membrane to function under standard laboratory conditions.

The peritrophic membrane is a critical barrier to pathogens66,69,70, so we hypothesized that reduced endocycling in Zone 4 cells may ultimately lead to functional defects in pathogen resistance. Under standard conditions at 29°C, the lifespan 71F06-Gal4 > InR-RNAi flies was indistinguishable from control flies (Fig. 9L), consistent with the above observations that peritrophic membrane is not grossly malformed or dysfunctional. However, when these flies were challenged with the pathogenic bacteria Pseudomonas aeruginosa (PA14), InR-RNAi flies were significantly more susceptible to infection than controls (Fig. 9M), indicating that the peritrophic membrane has compromised function. These results suggest that while continual endocycling of Zone 4 cells is not strictly required for peritrophic membrane synthesis, it instead provides robustness in the face of environmental challenges such as pathogenic bacteria.

Methods
Experimentalanimals
Genotypes and sources for Drosophila melanogaster lines used in this study are provided in Supplementary Table 1. Flies were maintained at 18°C, 25°C, and 29°C, as indicated in the text, on standard laboratory cornmeal food, except for EdU treatments, yeast supplementation experiments, or drug treatments, which were maintained on Formula 4–24 Instant Blue Food (“Instant Blue Food”, Carolina Biological Supply) as described below. All experiments were performed on female flies. No ethical approval was required for studies on Drosophila melanogaster.

Except where noted in the text, all experiments were initiated on flies within 1 week of eclosion, and all flies were age-matched within an experiment. For temperature shift experiments with tubGal80ts, flies were reared throughout development at 18°C and were shifted to 29°C 1–4 days after eclosing.

EdU feeding and EdU+ cell counting
EdU (Thermo Fisher) was diluted to 0.2 mM in water, then used to hydrate an equal volume of Carolina Instant Blue Food, supplemented with yeast powder as indicated in the text. Adult flies were transferred to EdU-containing food and flipped to fresh EdU-containing food every 2 days for any experiment lasting longer than 2 days. EdU signal was detected using Click-iT™ EdU Cell Proliferation Kit (Thermo Fisher). Except where noted in the text, 5% yeast was used in EdU experiments (100 mg dried yeast per 2 mL of food), and flies were < 2 weeks old. EdU+ cells were manually counted on a compound epifluorescent microscope, specifically focusing on those EdU+ cells located in Zone 4. Unless otherwise noted in the text, flies were fed EdU for 2 days prior to dissection.

Yeast variation experiments
To test the effect of supplemental dietary yeast on EdU incorporation, dried active yeast was added as a percentage by weight of Instant Blue Food.

Colchicine treatment
To enrich mitotic nuclei, flies were fed with 0.2 mg/mL colchicine (EMD Millipore) for 12 h prior to dissection. Colchicine was diluted in water and used to dilute Instant Blue Food supplemented with 10% yeast.

Gut damage with X-rays, bleomycin, and ECC15 infection
For X-ray damage, adult flies were irradiated with 0, 1000, or 2000 RADs in a TORREX 120D X-ray Inspection System (ScanRay Corporation). For gut damage with bleomycin, bleomycin (Sigma) was diluted to 25 ng/µL in water and used to rehydrate 2 mL of Instant Blue Food. Flies were fed bleomycin-containing food for 2 days at 29°C to increase ingestion, and were flipped to fresh food each day, with water as a control. For ECC15 treatment, 100 mL of LB medium was inoculated with 20 µL of a ECC15 glycerol stock and grown at 30°C for 20–24 h. ECC15 cultures were then centrifuged for 30 min at 1810 x g and resuspended in 1.4 mL of a 5% sucrose. Flies were starved for 2 h at 29°C, and then flipped to a vial containing only a kimwipe soaked in 700 µL of the ECC15 solution, where they fed for 24 h at 29°C to induce gut damage. After gut damage, flies were maintained on standard food at 25°C for the indicated amount of time.

Immunohistochemistry and confocal imaging
Tissues were dissected in 1× phospho-buffered saline (PBS), fixed for 25–30 min in 4% paraformaldehyde in PBS, permeabilized in PBS with 0.3% Triton-X (PBST), and stained following standard antibody staining procedures using 5% normal goat serum as a blocking agent. Filamentous actin was stained using Alexa 555-coupled phalloidin (Molecular Probes), peritrophic membranes were stained with Alexa647-coupled HPA lectin (1:100, Thermo Fisher), calcofluor white stain (1:100, Sigma-Aldrich), Alexa647-coupled wheat germ agglutinin (1:400, Fisher Scientific), and nuclei were stained using DAPI. For HPA Lectin, we found that an incubation of at least two nights at 4°C, and up to five nights, was necessary for reliable staining, whereas calcofluor and WGA were stained for two nights, and that these peritrophic membrane stainings were improved by isolating the anterior midgut below the proventriculus from the rest of the midgut. Stained tissues were mounted in Vectashield (Vector Labs), except for Alexa647-labeled tissues, which were mounted in Prolong Gold (Thermo Fisher Scientific). Rabbit anti-GFP AlexaFluor488 conjugate (Molecular Probes, 1:300) was used to visualize GFP expression, Wg expression was detected using mouse anti-Wg (Developmental Studies Hybridoma Bank 4D4, 1:100) and phospho-Histone 3 was detected using rabbit anti-PH3 (Cell Signaling Technologies 9701S, 1:500). Primary antibodies were detected with Alexa Fluor 488- or 555-coupled secondary antibodies (1:400). Confocal images were captured on a Zeiss LSM780, LSM980, or an Olympus IX83 confocal microscope, through the Microscopy Resources of the North Quad (MicRoN) facility at Harvard Medical School.

Single-nucleus RNAseq (snRNAseq) atlas
To capture proventriculus transcriptional states in both well-fed flies and flies with reduced systemic insulin signaling, single nuclei suspensions were generated from proventriculus samples from both control flies (esg-Gal4, tubGal80ts, UAS:GFP/+) and cachexic flies (esg-Gal4, tubGal80ts, UAS:GFP; UAS-yki3SA), after 8 or 9 days at 29°C. We verified via confocal analysis that esg-Gal4 is not expressed in the proventriculus itself, which was also confirmed via snRNA sequencing. Proventriculus samples were manually dissected from adult female flies in cold Schneider’s medium and immediately flash frozen on dry ice in Eppendorf tubes until a total of approximately 600 samples were collected (~300 of each genotype). Proventriculi were manually severed from the crop and esophagus, and from the midgut just posterior to the proventriculus, anterior to where esg-Gal4 is detected in midgut ISCs.

Single nuclei were isolated following the protocol described in ref. 76, derived originally from refs. 77, 78, and stained with DAPI immediately before FACS sorting, performed at the Immunology Flow Cytometry Core at Harvard Medical School. Sorted nuclei were resuspended in 1× PBS supplemented with 0.5% BSA with RNAse inhibitor (Promega) at a concentration of ~2000 cells/µl.

snRNAseq was performed following the 10X Genomics Chromium protocol (Chromium Next GEM 551 Single Cell 3’_v3.1_Rev_D), and sequenced using a Illumina NovaSeq 6000 at the Harvard Medical School Biopolymers Facility. 10,525 nuclei from esgts flies and 11,801 nuclei from esgts > yki flies were sequenced (total = 22,332 nuclei) and reads were mapped to BDGP6.32.

Bioinformatic analysis was performed as follows, and the analysis pipeline is available here: https://github.com/MujeebQadiri/SingleNucleiProventriculusAnalysis/tree/main. Raw data were processed with Cell Ranger software (10x Genomics, v7.0.0) to generate a single-cell matrix for each sample. The count matrices were imported into Seurat (v4.3.0) and cells with less than 1300 or greater than 60,000 UMIs were filtered out, and low-abundance genes with less than 10 counts were removed from further analysis. The two biological samples were bioinformatically pooled for PCA and clustering analysis to generate a single atlas including cells from both conditions. After a PCA reduction, the first 40 components were used for UMAP and clustering analysis, at a 0.2 resolution. We removed from further analysis one cluster that appeared to be an artifact, as it contained markers for multiple different unrelated cell-types as well as stress and heat shock proteins. Differentially expressed genes were calculated using the Wilcox Rank Sum Test across each cluster and were then further selected using a Log2FC > X cut-off and adjusted p value < 0.05. All downstream analysis was performed in R (v4.3).

To map snRNAseq clusters to proventriculus anatomy, we examined the expression pattern of Gal4 lines for top-ranking marker genes using confocal microscopy.

MARCM analysis
Flies of the genotype hsFLP, FRT19A, tubGal80; tub-Gal4, UAS-GFP were crossed to FRT19A flies (BL1709), and offspring were heat-shocked in a 37°C water bath either during the L3 stage for during the adult stage. Two 1 h heat-shocks were administered, separated by 2–3 h at 25°C. MARCM crosses were maintained at either 25°C or 18°C as indicated in the text.

Tissue damage protocol using UAS-rpr
We induced ectopic apoptosis by crossing Gal4 drivers to flies containing UAS-rpr; tubGal80ts or, for non-damaged controls, to w1118 flies. These crosses were maintained at 18°C until several days after eclosion. Adult flies were then transferred to EdU+ food and placed at 29°C for 48 h to induce apoptosis. For recovery experiments, flies then returned to recover at 18°C for either 2 days or 5 days, on EdU+ food throughout. For continuous damage experiments, flies were maintained at 29°C for either 2 or 5 days. Thus, all EdU+ incorporation events that occurred during the damage or during the recovery period could be visualized and quantified.

Creation of split-intein Gal4 line
To generate GMR71F06::Gal4-C-int, the 3547 bp enhancer fragment from GMR71F06 FlyLight line79 was amplified using the following primers: caccTGCCCCGAGCAGCAAAAGAAGTCCG and TCTCCGAAATCTGCGCCAATTCCAG, and cloned into the pENTR-dTOPO vector (Thermo Fisher Scientific). This fragment was cloned into the pBP-Gal4C-int backbone39 (Addgene 199202) via LR gateway cloning, and was inserted into the attP40 landing site using standard phiC31 transgenesis.

Time series of nuclear morphology and EdU incorporation
To quantify ploidy and nuclear morphology over the course of the fly’s life, age matched flies of the genotype GMR7106-Gal4/UAS-Stinger; MKRS/+ were collected on eclosion day, and maintained at 25°C in standard fly vials on standard lab food (approximately 10–15 adult flies per vial), either with or without dried active yeast sprinkled on the food’s surface. In a pilot experiment, we observed that UAS-Stinger is tightly nuclear-localized in this tissue, whereas other common nuclear-localized fluorophores transgenes (UAS:GFPNLS [BL4776], UAS-unc84-GFP [lab stock] and UAS:RedStinger [BL8545]) were not. Flies were flipped onto fresh food every 2 or 3 days. On each day of sampling (3, 7, 14, 21, 28, and 35 days), approximately 6–8 cardia were dissected and stained overnight with Alexa 488-coupled anti-GFP, DAPI, and phalloidin as described above. At each time-point, confocal z-stacks were collected through the entirety of Zone 4 (n = 5–6 proventriculus per time point) and analyzed as described below. We also collected samples for eclosion day and for 1 day later, but found that the Gal4 expression pattern extended beyond Zone 4 at these time points, so they were excluded from further analysis.

For Oregon R aging experiments with EdU, batches of flies were collected on the day that they eclosed and then aged at 25°C on standard food with yeast powder until the indicated ages. Flies were transferred to EdU+ food 2 days prior to EdU+ food. Anterior-posterior position was calculated from confocal stacks by measuring the shortest distance between the center of each EdU+ and the vertical center of the posterior-most row of cells in Zone 4, hence, EdU+ cells located in the posterior-most row were measured as zero.

Image quantification
Ploidy was analyzed from DAPI-stained confocal z-stacks, following a protocol modified from ref. 80 and described below, using FIJI software81. A subset of the confocal z-stack containing only a single layer of nuclei from the surface of Zone 4 was generated using the “sum intensity” projection method, and region-of-interests were manually drawn for each visible Zone 4 nucleus using 71F06-Gal4 > GFP signal to ensure that all nuclei measured were in Zone 4. Summed DAPI intensity was calculated using the “integrated density” function. To normalize these values, we used diploid nuclei from Zone 3 as an internal control within each sample. We determined Zone 3 cells to be diploid by direct comparison to diploid ISCs captured under identical imaging parameters. Thus, a diploid standard was calculated for each sample from an averaged value of approximately 15–25 diploid cells within each confocal stack.

Additional nuclear quantification, including cell volume and cell counts were performed with arivis Vision4D software (Zeiss, v4.1.2). GFP+ nuclei were segmented using the Cellpose-based Segmenter, and segmentation was then manually corrected for each sample by splitting or removing nuclei that were erroneously fused and removing artifacts that did not correspond to nuclei. Nuclear volume was extracted for all objects > 10 µm3. For cell count, those nuclei missed by Cellpose-based segmentation were manually counted using the “add marker” function while viewing the tissue in 3D. To quantify cell loss during aging, cells that had been shed into the lumen were manually counted in FIJI81.

As a measure of tissue size for Zone 4, we measured the area of Zone 4 at the widest point of the proventriculus. Each sample value represents the averaged area of two independent measurements of cross-sections of Zone 4, on either side of the esophagus. For area measurements of peritrophic membrane features stained with HPA lectin, WGA, or calcofluor, we made a maximum intensity projection of 3–4 confocal z-slices at the equator of the proventriculus, and measured the area of stained material, averaging two measurements taken for each sample on either side of the esophagus.

Peritrophic membrane electron microscopy and leakiness assays
“Smurf” assays were performed as previously described67, as follows. Flies transferred to standard lab food containing 2.5% FD&C Blue #1 dye (Spectrum) by weight, and were examined for gut leakiness after 24 and 48 h. Any flies where blue food was restricted to the proboscis, crop, and gut was scored as wild type.

Fluorescent bead leakiness analysis were performed similar to previous reports55,66. Flies were starved in empty vials for 2 h at 29°C, then transferred to vials containing half of a standard kimwipe saturated with a 1:50 dilution of Fluoresbrite YG microbeads (diameter 0.056 µm; Polysciences) in 5% sucrose. Flies were dissected between 30–60 min after feeding, and whole guts were fixed and stained with DAPI and phalloidin and/or HPA lectin as described above. Guts were scored under 40X magnification as either intact or containing leaky regions where fluorescent beads were visible outside of the peritrophic membrane.

Electron microscopy was performed in the Harvard Electron Micrscopy Facility. Tissues were prepared as described69, and longitudinally sectioned anterior midguts were imaged using a Tecnai G2 BioTwin microscope.

Lifespan and Pseudomonas survival experiments
Lifespan experiments were conducted at 29°C, with 15 mated females per vial on standard fly food supplemented with dried yeast powder, and flipped every 1–2 days. Flies of the genotype w; tubGal80ts, UAS:GFP; 71F06-Gal4 were crossed to control or UAS:RNAi lines, and the offspring were reared at 18°C until eclosion. Adult were collected and mated over a 4-day period at 18°C, then shifted to 29°C 3 days later to begin life-span measurements. The number of dead flies was recorded every day.

Infection with Pseudomonas aeruginosa (PA14) followed a protocol similar to ref. 69. Five milliliters of LB medium was seeded from a glycerol stock of PA14 and grown overnight in a shaking 37°C incubator. The following morning, 1 mL of this culture was diluted in 50 mL of fresh LB and grown for an additional 6 h, shaking at 37°C. This culture was then diluted 1:3 in 5% sucrose solution. Fly vials containing half of a standard kimwipe, compressed into the bottom of the vial, were loaded with 1.5 mL of this bacterial suspension. In order to promote feeding on the bacteria, flies were starved for 2 h in empty vials immediately prior to bacterial treatment.

These experiments were performed at 29°C on flies that had been maintained at 29°C for 15 days prior to PA14 infection. After a 24 h infection, flies were flipped to fresh vials containing Kimwipes saturated with 5% sucrose solution, and flipped to fresh 5% sucrose solution daily after that. The number of dead flies was recorded approximately every 24 h. Four vials of 8–10 flies each were used per genotype, and the entire experiment was performed in biological duplicate, with similar results in both experiments.

Graphing and statistical analysis
All graphs were created and statistical tests performed using Prism (GraphPad, v10).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript, Ewen-Campen et al. re-investigated the proventriculus zone 4 cells, which were previously proposed to contain multipotent stem cells by Singh, Zeng, Zheng, and Hou. In contrast to the previous publication, the authors, through genetic and lineage analysis, found that the proventriculus zone 4 cells do not contain stem cells because they have no mitotic activity, do not generate clonal lineages, and do not respond to tissue damage. However, to put this in perspective, Sing et al.'s 2008 claim of PV4 stem cells was similar to a claim by Takashima et al. in 2008 for the hindgut. However, in 2009, Fox and Spradling 2009, disproved the claim and showed it regulated from defects in the lineage system. The field realized that Sing et al.'s data was similar but even lower quality that Takashima et al, and consequently this claim has not be believed for at least 15 years. There the paper needs more than just to correct the record on this old claim.\nThe authors also reported that the proventriculus zone 4 cells undergo continuous endoreplication in a nutrient-dependent manner. They concluded PV4 cells are lost as the fly ages, and claim this results in functional defects in the peritrophic membrane (PM) and an increased susceptibility to oral bacterial infections. There is a substantial literature on the early aging of the Drosophila gut, at least under lab conditions. These are potentially interesting findings, but they need to better supported experimentally in ways that add insight. Currently the novelty of the findings is shallow.\nThe descriptive data regarding proventriculus zone 4 cells number under homeostasis as well as in various nutrient and stress conditions, needs to be better supported with more data. What is the rate of polyploidization of PV4 and how does it vary with nutrition? Do these changes affect the number of PV4 cells relative to other cells contibuting to PM? Do you have information on what it responds? Insulin levels, or particular nutrients. Does the PV4 response change PM structure over time?\nThe experiments that utilize the tumor cachexia model contain a systematic flaw due to the use of esg-GAL4 to manipulate the proventriculus with the assumption that these effects act only through changes in nutrition via action on ISCs. It was demonstrated (ref 25) that esg-GAL4 is highly active in the corpus allatum (CA), the major source of adult juvenile hormone. JH is a major regulator of reproductive diapause during which its level is seen to decline sharply and possibly in circadian fluctuations in gut activity. JH might well effect PV cells since many express JH binding proteins as well.\nBelow are more detailed point-by-point comments.\n1. Figure 1F, zone 4 cells in the 5 days EdU image seem disorganized or “lose a clearly monolayered epithelial structure” as defined later in the text (Line 397). Is this representative? Given that the authors later reported an age-related phenotype, please clarify how the fly age is controlled for each experiment within the study.\n2. Please elaborate on the “observed a marked decrease in EdU+ cells in Zone 4, suggesting that the EdU signal was diluted by continued cycling” (Line 127)? If the proventriculus zone 4 cells do not have mitotic activity as the authors suggested later, would an accumulation of the EdU signal from the EdU exposure period (3 days in the case of Figure S1B) be expected?\n3. The authors observed (Line 133) that “after 1d of EdU feeding, Edu+ nuclei tended to be small in volume and located at the posterior limit of Zone 4 whereas after 5d of feeding labeled nuclei were larger and could be seen a few cell diameters anteriorly.” Please comment on whether this is indicative of the zone 4 cell migration.\n4. The authors concluded that the proventriculus zone 4 cells are sensitive to systemic nutrient status (Line 147). Under physiological conditions, nutrient status fluctuates over time, especially for animals like wild flies. How would the proventriculus zone 4 cells respond under physiological conditions, e.g. when switching between rich and poor nutrient status? Also, how are these signals transmitted to the proventriculus zone 4 cells? Do you find expression of any receptors or regulatory molecules responsible for nutrient status (e.g. insulin receptor) in these cells?\n5. Line 147, do you mean Figure 1H instead of Figure 1E?\n6. The manuscript can be written more concisely with a clearer focus on re-examining proventriculus zone 4 cells. The authors reported an interesting nutrient-dependent endoreplication phenomenon in the proventriculus zone 4 cells (Figure 1G&H) at the beginning, but quickly shifted gears and used a significant length of writing reporting major cell types, transcriptomes, and associated genetic lines for the proventriculus (Line 167) before coming back on examining the cell biology of proventriculus zone 4 cells. Much of this information is not directly relevant to the focus of the study, and some of the languages used in the manuscript have actually been learned by several previous studies in great depth. It would be more succinct to highlight findings that have not been reported or thoroughly examined in the past, such as zone 3-4 junctional cells, and reference what was known in the appropriate literature.\n7. Do you find any PM defects in esg-Gal4, tubGal80ts > UAS:yki3SA, and esg-Gal4 and dMef2-Gal4 driving ImpL2 and/or upd3 overexpression flies, where proventriculus zone 4 cells have slower endoreplication (Line 150-160)?\n8. Please provide quantifications for Figure 3A.\n9. In Figure 7D, zone 4 cells decrease by ~21 cells by 14d; however, Figure 7F only reports ~1 cell loss into the lumen by this time. Please clarify these quantifications.\n10. Line 394-395, do you mean Figure S7B instead of Figure S6B? Line 398, do you mean Figure S7C instead of Figure S6C?\n11. Do you find that the loss of proventriculus zone 4 cells correlates with any other cell biological characteristics of the cells?\n12. Line 411, do you mean Figure S8 instead of Figure S7?\n13. The authors reported alterations in the PM structure upon perturbations to endocycling in the proventriculus zone 4 cells (Line 430-450 and Figure 8). PM is a dynamic structure and varies by animal and tissue location. More precise and quantitative approaches would be necessary to conclude the PM phenotype. For example, do you find changes in key PM-producing genes upon your treatments (e.g. using qRT-PCR or RNAseq)? Also, the PM is composed of multiple layers. Could you examine the detailed PM structure using electron microscopy? Given that the PM is largely produced in the proventriculus, but its main function occurs in the midgut, it would be informative to provide more characterizations of the PM defects in both the proventriculus and midgut in a layer-specific manner.\n14. Figure 8F, do the “Increased PM” conditions in Figure 8A show a better fly survival post-pathogen infection?\n15. Line 910, I am not sure what “single-cell epithelium” means.\n16. The authors conducted scRNAseq on well-fed flies and flies with reduced systemic insulin signal (Line 575). Did the authors compare the data from the two conditions? Where was the analysis for the cachexic flies? What datasets were used for the atlas analysis?\n17. In general, the figure legends need to be reorganized. Information regarding scale bar size and experimental replication needs to be precisely summarized. Currently, this information is flowing around and sometimes missing. Some of the experiments that led to the main conclusions in the paper need more replication.\n18. Please provide more detailed descriptions of statistical analysis in the materials and methods. In many places when t-tests were used, it was unclear whether the data passed the normality test.\n19. Please add languages to the discussion on how this study differs from the previous study by Singh, Zeng, Zheng, and Hou that led to the opposite conclusions.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_3": "Summary:\nIn this study, the authors reexamine an anterior region of the fruit fly intestine, known as the proventriculus, which was reported to be maintained by multipotent stem cells. They definitively show, using cell cycle markers and lineage tracing techniques, that stem cells do not exist in this tissue, correcting the initial report. Using a variety of genetic and genomic techniques the cell-types within proventriculus are clearly defined providing a new toolkit of Gal4 drivers for future studies. In addition, the authors discover that cells that cycle (S-phase+) in zone 4 are endocycling, instead of mitotically dividing. Endoreplication appears to be age and nutrition dependent, which may be functionally necessary for the synthesis of the peritrophic member. Secretion of the peritrophic membrane is essential to protect most insect intestines from damage upon ingestion of food as well as pathogenic microorganisms. This is further demonstrated by enhanced sensitivity to P. aeruginosa in flies with the insulin receptor knocked down in zone 4 cells of the proventriculus.\nOverall, this study provides a comprehensive analysis of the proventriculus cellular organization and gene expression program. However, many of the conclusions, particularly in regard to polyploidy are not supported by the experiments performed (i.e. nuclear volume, instead of DNA content measurements). Therefore, the following major and minor comments are strongly recommended to be addressed:\nMajor Comments:\n1. Nuclear volume does not equal nuclear ploidy and cannot be used as a proxy for concluding that a change has occurred. If the authors want to conclude that ploidy is changing with nutrition and age then it needs to be measured. Ploidy can be measured using confocal imaging of DAPI or Hoescht stained nuclei using a diploid cell type in the guts as an internal control (i.e. intestinal stem cells or enteroendocrine cells). Therefore, existing images acquired in this study could be used and reanalyzed to draw these conclusions.\n2. The ploidy measurements would be helpful to further understand the organization of EdU+ nuclei as it was noted that at 1d and 5d post EdU feeding the nuclei were localized in different regions of zone 4. Spatial ploidy, instead of nuclear volume, analysis is necessary to conclude that ploidy changes with age and nutrition in this region of the tissue.\n3. The authors meticulously and rigorously identify as well as generate cell-type specific Gal4 drivers in this study yet they are under-utilized. An example of this is in regard to the genetic ablation experiment in Figure 5. Cell death may need to be induced in zone 4 itself to activate the endocycle. That is the case for other fly tissues that induce polyploidy in response to injury/damage. Gal4 drivers specific to zones 2/3, 3/4, and/or 4 should be used to ablate cells and test if zone 4 cells are truly refractory to enhanced endoreplication following injury. In addition, the DEMISE system could be used to couple apoptosis with genetic manipulations (RNAi or overexpression) of other target genes (Cohen et al. 2018).\n4. This study concludes that the proventriculus response to damage is unique from other models of wound- or damage-induced polyploidy yet the assays used are not analogous to prior studies (local vs distal damage- comment #3) and the zone 4 cells appear to be polyploid (Figure S6). The authors may not see a response to X-rays, chemicals, or pathogens as polyploid cells are known to be more resilient to stress. Do the treatments (Figure S5) lead to local damage in zone 4? The high ploidy of zone 4 cells (4C to 40C) may inhibit further polyploidization, a distinction from the epithelium and hindgut pyloric cells which are diploid (2C) prior to injury.\n5. One of the most impactful parts of this study is the potential physiological role for endoreplication in the synthesis of the peritrophic membrane. However, the study lacks a direct demonstration of this conclusion. To do so, endoreplication in zone 4 cells should be directly inhibited using cycE RNAi or fzr RNAi, instead of knockdown of the insulin receptor. Additionally, it is unclear when in development the peritrophic membrane is produced or if it has to be continually maintained? If it has to be continuously synthesized during adult life than this may explain why the zone 4 cells continue to endocycle with age.\n6. The authors could use the dextran permeability assay (Kuraishi et al. PNAS 2011) to determine if the peritrophic membrane is compromised when endoreplication is upregulated or inhibited. It is unclear if the changes in lectin staining are indicative of a functional change.\nMinor Comments:\n1. The authors should consider moving Figure 6 (GTRACE) to the supplement as it is redundant with Figure 4 (MARCM).\n2. Line 333-335: Liver hepatocytes are derived from the endoderm and well-documented to endoreplicate in response to tissue damage, so there is insufficient evidence to make a claim of an ectoderm vs endoderm difference in endoreplication.\n3. Miss cited figures (i.e. Fig S6C – line 397-8 does not exist). Figure S7 legend is missing a description for part (C).\n4. Several graphs (i.e. Figure 1H and 6F) are missing significance and the statistical test if used it is not reported.\n5. Check image stains are labeled in all figures (i.e. Figure 2E is missing this info)\n6. It would be helpful to provide the guts (n) and PH3+ nuclei (#) for intestines in Figure 3B as well.", "reviewer_4": "In this thorough and carefully conducted study, the authors investigate the mechanism by which tissue homeostasis is maintained in the Drosophila adult proventriculus. A previous study reported on the presence of BrdU+ cells in the proventriculus and proposed that it therefore contains mitotically active stem cells. Here the authors reexamine this conclusion and provide convincing evidence that these cells are endocycling rather than undergoing mitosis and therefore are not stem cells. They reach this conclusion through a combination of approaches, including stains for mitotic markers and clonal analysis in a wide range of experimental conditions designed to enrich for mitotic cells if they exist in this tissue. However, despite this extensive search, they do not find evidence of cells in mitosis. Suitable positive controls are included to confirm that their methods are working as expected. They also generate a single cell atlas of the proventriculus and use the results, along with a screen and a recently developed split-Gal4 tool to specifically target the region of the tissue where cells in S-phase can be found. Interestingly, they find that cells in this region are polyploid and that the ploidy increases with age, suggesting that they continue to endocycle during adulthood. The authors propose that this may be a mechanism to compensate for age-dependent cell loss, which they provide evidence for. Overall, I think this study will be a valuable contribution to the field and I am supportive of publication. I have only a few minor comments, which I recommend the authors consider before publication:\n1. I am confused by the results described in the paragraph from lines 132-139 (Figure 1D is referenced but I think the authors are referring to Figure 1F): if the cells are continuously endocycling, why would the size of EdU+ nuclei increase over 5 days? Wouldn't there be a distribution of nuclear sizes at any given time point when EdU is administered such that both large and small nuclei could be labeled even at day 1?\n2. On line 147, it seems that Figure 1H should be referenced (not Figure 1E)\n3. On Line 252-253, the authors state that they never saw cells travel from Zone 4 to any other portion of the proventriculus but, without live imaging or clonal analysis, how is it possible to track cell movement?\n4. The data in Fig 4D show that there are, in fact, a small number of large clones in the proventriculus at 14 days after heat shock (dark grey bar at the top of the third column from the left) but this is not discussed in the text. How did these larger clones arise if the cells cannot undergo mitosis?\n5. The code used to analyze the scRNAseq data as well as the final Seurat objects should be made available."}, "author_response": "We thank the reviewers for this feedback, and respectfully disagree that there is clarity or agreement in the literature about whether or not the proventriculus contains stem cells. While the reviewers are correct that Fox & Spradling (2009) disproved the existence of stem cells in the hindgut, it does not follow that one can draw any conclusions about the proventriculus, a different tissue. And in fact, as we show in this manuscript, the cycling dynamics of these two gut regions differ: the hindgut only becomes polyploid in response to tissue injury, whereas in the proventriculus there is continual endocycling even in the absence of any exogenous damage.\nSecond, while it may be true that some researchers in the field viewed Singh et al. (2011) as unpersuasive, there have been zero publications re-addressing this issue to date. Clarifying this issue of whether or not stem cells are present in this tissue is a primary motivation for our manuscript, and has not been addressed in the literature to date.\nThe authors also reported that the proventriculus zone 4 cells undergo continuous endoreplication in a nutrient-dependent manner. They concluded PV4 cells are lost as the fly ages, and claim this results in functional defects in the peritrophic membrane (PM) and an increased susceptibility to oral bacterial infections. There is a substantial literature on the early aging of the Drosophila gut, at least under lab conditions. These are potentially interesting findings, but they need to better supported experimentally in ways that add insight. Currently the novelty of the findings is shallow.\nIn our revised manuscript we have deepened our analysis of the peritrophic membrane by performing multiple additional assays and quantification to describe the PM phenotype, now shown in revised Figure 8 and Figure 9, as well as Supplemental Figure S11. In our initial submission, our analysis was limited to HPA-Lectin staining; in our revised manuscript we have added stainings with calcofluor, which stains chitin, as well as wheat germ agglutinin, which stains a distinct set of glycosyllated targets. We have also now quantified the amount of stained PM material in the proventriculus, and showed consistent changes in all three labels following genetic manipulations to the endocycle, as would be predicted by a direct relationship between metabolic output of these cells and their ploidy state/cell size. As described below, we also examine several aspects of PM function in the midgut as well (using TEM, smurf assay, and a fluorescent bead retention assay.)\nThe descriptive data regarding proventriculus zone 4 cells number under homeostasis as well as in various nutrient and stress conditions, needs to be better supported with more data. What is the rate of polyploidization of PV4 and how does it vary with nutrition? Do these changes affect the number of PV4 cells relative to other cells contibuting to PM? Do you have information on what it responds? Insulin levels, or particular nutrients. Does the PV4 response change PM structure over time?\nWe have re-analyzed our time-course analysis of nuclear changes in Zone 4 in order to directly measure ploidy (rather than nuclear volume, as we had previously done.) This allowed us to directly show changes in ploidy over the first 35 days of the fly’s life, shown in a revised Figure 6 and described in the text. We show that both ploidy and cell number are influenced by yeast supplementation (Figure 6C,D), and we also now directly show that endocycling can be induced by cell loss from the tissue. Our RNAi experiments with InR (revised Figure 9) demonstrate that Zone 4 cells respond to nutritional signals in a cell autonomous fashion, which we also clarify in the text of the manuscript.\nThe experiments that utilize the tumor cachexia model contain a systematic flaw due to the use of esg-GAL4 to manipulate the proventriculus with the assumption that these effects act only through changes in nutrition via action on ISCs. It was demonstrated (ref 25) that esg-GAL4 is highly active in the corpus allatum (CA), the major source of adult juvenile hormone. JH is a major regulator of reproductive diapause during which its level is seen to decline sharply and possibly in circadian fluctuations in gut activity. JH might well effect PV cells since many express JH binding proteins as well.\nWe thank the authors for pointing out this caveat of our esg-Gal4 experiments, and have noted this in the text. We have now emphasized in the text that we also over-expressed Impl2 and Upd3 from muscles (dMef2-Gal4) also drives a significant decrease in cell cycling in the proventriculus, which provides independent support to the conclusion that systemic nutrition is influencing the cycling status of these cells.\nBelow are more detailed point-by-point comments.\n1. Figure 1F, zone 4 cells in the 5 days EdU image seem disorganized or “lose a clearly monolayered epithelial structure” as defined later in the text (Line 397). Is this representative? Given that the authors later reported an age-related phenotype, please clarify how the fly age is controlled for each experiment within the study.\nBased on helpful comments from all three reviewers, we have now removed the data shown in Figure 1F (comparing 1d EdU vs. 5d EdU feeding), as we agree no real conclusions can be drawn from this data.\nWe have also clarified in the Methods section that, except as noted in the text, all experiments show flies under 7 days post-eclosion.\n2. Please elaborate on the “observed a marked decrease in EdU+ cells in Zone 4, suggesting that the EdU signal was diluted by continued cycling” (Line 127)? If the proventriculus zone 4 cells do not have mitotic activity as the authors suggested later, would an accumulation of the EdU signal from the EdU exposure period (3 days in the case of Figure S1B) be expected?\nWe have clarified the text to explain that continual endocycling dilutes the EdU as the amount of EdU-labeled DNA decreases as a proportion of the total DNA in the nucleus, similar to what we and others have observed in midgut ECs, which endocycle.\n3. The authors observed (Line 133) that “after 1d of EdU feeding, Edu+ nuclei tended to be small in volume and located at the posterior limit of Zone 4 whereas after 5d of feeding labeled nuclei were larger and could be seen a few cell diameters anteriorly.” Please comment on whether this is indicative of the zone 4 cell migration.\nPlease see above, in our response to (1).\n4. The authors concluded that the proventriculus zone 4 cells are sensitive to systemic nutrient status (Line 147). Under physiological conditions, nutrient status fluctuates over time, especially for animals like wild flies. How would the proventriculus zone 4 cells respond under physiological conditions, e.g. when switching between rich and poor nutrient status? Also, how are these signals transmitted to the proventriculus zone 4 cells? Do you find expression of any receptors or regulatory molecules responsible for nutrient status (e.g. insulin receptor) in these cells?\nOur experiments with InR RNAi suggest that Zone 4 cells respond cell autonomously to insulin signaling, as knocking down the insulin receptor in these cells abolishes endocycling. We agree that in future studies it will be very interesting to characterize how major fluctuations in food availability are mechanistically linked to the peritrophic membrane machinery.\n5. Line 147, do you mean Figure 1H instead of Figure 1E?\nFixed.\n6. The manuscript can be written more concisely with a clearer focus on re-examining proventriculus zone 4 cells. The authors reported an interesting nutrient-dependent endoreplication phenomenon in the proventriculus zone 4 cells (Figure 1G&H) at the beginning, but quickly shifted gears and used a significant length of writing reporting major cell types, transcriptomes, and associated genetic lines for the proventriculus (Line 167) before coming back on examining the cell biology of proventriculus zone 4 cells. Much of this information is not directly relevant to the focus of the study, and some of the languages used in the manuscript have actually been learned by several previous studies in great depth. It would be more succinct to highlight findings that have not been reported or thoroughly examined in the past, such as zone 3-4 junctional cells, and reference what was known in the appropriate literature.\nWe agree with the Reviewer #1 that Zhu et al.’s 2024 PNAS paper established an immensely valuable single cell atlas of the proventriculus, which has provided many insights into the cell types and biology of this understudied tissue. We ultimately decided to include our own snRNAseq section for three reasons 1) because we wound up using different cell-type specific Gal4 lines than those described by Zhu et al. and we wanted to explain their origins; 2) because our atlas was generated independently yet was highly concordant, we feel that it serves to strengthen the conclusion that the major cell types of this tissue have indeed now been identified; 3) because we mention the lack of markers for stem cells, we wished to explain in some detail how we had generated our atlas.\nThat said, if the manuscript Editor agrees that this section is excessively detailed, we can easily shift parts of it to a supplement.\n7. Do you find any PM defects in esg-Gal4, tubGal80ts > UAS:yki3SA, and esg-Gal4 and dMef2-Gal4 driving ImpL2 and/or upd3 overexpression flies, where proventriculus zone 4 cells have slower endoreplication (Line 150-160)?\nWe attempted to analyze the PM in Yki gut tumor flies, but found that these tissues are extraordinarily malformed and disrupted by the presence of massive tumors, which makes this analysis exceedingly difficult to perform.\n8. Please provide quantifications for Figure 3A.\nWe have quantified these data, including # of EdU cells, ploidy, and tissue size, in a revised Supplemental Figure 4.\n9. In Figure 7D, zone 4 cells decrease by ~21 cells by 14d; however, Figure 7F only reports ~1 cell loss into the lumen by this time. Please clarify these quantifications.\nWe have clarified in the text that the images and counts of cell loss shown in 7E-7F (now Figure 6E-6F in the revised paper) are “snap-shots” of cells being actively shed at the time of dissection and staining, whereas the total cell counts are the true and complete number of cells remaining in Zone 4 and thus account for the full time period.\n10. Line 394-395, do you mean Figure S7B instead of Figure S6B? Line 398, do you mean Figure S7C instead of Figure S6C?\nThese and other Figure references have been corrected.\n11. Do you find that the loss of proventriculus zone 4 cells correlates with any other cell biological characteristics of the cells?\nWe have added a note in the text to state that we do not observe any obvious morphological changes in the cells being lost. We also note here for the reviewer that we are now researching the molecular factors involved in cell loss from the proventriculus for a future manuscript.\n12. Line 411, do you mean Figure S8 instead of Figure S7?\nFixed.\n13. The authors reported alterations in the PM structure upon perturbations to endocycling in the proventriculus zone 4 cells (Line 430-450 and Figure 8). PM is a dynamic structure and varies by animal and tissue location. More precise and quantitative approaches would be necessary to conclude the PM phenotype. For example, do you find changes in key PM-producing genes upon your treatments (e.g. using qRT-PCR or RNAseq)? Also, the PM is composed of multiple layers. Could you examine the detailed PM structure using electron microscopy? Given that the PM is largely produced in the proventriculus, but its main function occurs in the midgut, it would be informative to provide more characterizations of the PM defects in both the proventriculus and midgut in a layer-specific manner.\nWe thank the Reviewer for these helpful suggestions, and as the Reviewer correctly notes, the PM is a highly dynamic structure that varies between individuals and over time, and thus any description of a PM phenotype indeed requires quantification over multiple individuals.\nWe have now added a number of additional characterizations and quantifications of the peritrophic membrane phenotypes associated with increased and decreased endocycling.\nThese are included in revised Figure 8 and Figure 9, as well as new Figure S11.\nSpecifically, we now characterize the increase and decreased peritrophic membrane synthesis using three independent dyes: calcofluor (which binds chitin, a major component of one of the four PM layers), and the two lectins HPA-lectin and wheat germ agglutinin, which have distinct binding affinities for glycosylated molecules. We have quantified the amount of PM using each of these three stains by measuring the area of PM adjacent to Zone 4 at the widest point of the peritrophic membrane, representing a snapshot of the amount of PM material being secreted by these cells. These results all show significant support for the hypothesis that increasing or decreasing endocycling in Zone 4 leads to lower and higher levels of PM secretion, respectively.\nWe also performed three additional assays for PM structure and function in the midgut: we examined the PM via electron microscopy, we performed the “Smurf assay” for gut leakiness, and we performed a fluorescent bead retention assay (which was also suggested by other Reviewers.) These results are shown in Figure S11, and none of these assays revealed consistent or significant differences between control and InR-RNAi flies, which suggests that, under standard rearing conditions and in the absence of infection, the peritrophic membrane retains basic functionality and structure even when synthesis is reduced in Zone 4 by blocking endocycling. However, given the results with Pe infection, there must be some compromised activity of the PM in terms of infection resistance, suggesting that endocycling in this system confers robustness to the digestive system in the face of environmental challenges.\nWe also note that we made multiple attempts to perform qPCR for chs2 to address the Reviewer’s comment, but unfortunately found that technical challenges made these experiments excessively difficult for this manuscript. Specifically, we found that to obtain sufficient total RNA from these tissue would require ~>=100 dissected proventriculi for each sample, across three replicates and four genotypes, all of which would need to be aged to 14-21d at 29ºC (i.e. 1,200 dissections.) Ultimately, we believe that a directly demonstration of varying chitin levels (calcofluor staining), which is the functional product of Chs2, is more direct than the demonstration that transcription levels vary with ploidy, which is well established in the literature as a general principle.\n14. Figure 8F, do the “Increased PM” conditions in Figure 8A show a better fly survival post-pathogen infection?\nWe decided against performing a post-infection survival experiment in flies with increased endocyling (UAS-cycE, UAS-myc) because this manipulation is not physiologically realistic (whereas disrupting endocycling serves as a straightforward loss-of-function analysis), and therefore unlikely to meaningfully add to the conclusions of our study. We believe that the PM assays we perform directly support the hypothesis that endocycling levels correlate with synthetic and secretory activity of these cells. In contrast, it is not clear whether we would expect a non-physiologically high PM secretion to confer additional infection resistance.\n15. Line 910, I am not sure what “single-cell epithelium” means.\nThis typo has been corrected (now reads “simple epithelium”)\n16. The authors conducted scRNAseq on well-fed flies and flies with reduced systemic insulin signal (Line 575). Did the authors compare the data from the two conditions? Where was the analysis for the cachexic flies? What datasets were used for the atlas analysis?\nOur snRNAseq atlas contains all nuclei collected from both control and cachectic flies (see Methods), and in this manuscript we do not present bioinformatic analyses of differences between the two datasets as these were not relevant to the present study. Additional analyses of differences between these datasets will likely be the subject of a future manuscript.\n17. In general, the figure legends need to be reorganized. Information regarding scale bar size and experimental replication needs to be precisely summarized. Currently, this information is flowing around and sometimes missing. Some of the experiments that led to the main conclusions in the paper need more replication.\nWe have endeavored to fix this issue in all instances, and have added scale bar and replication information in those instances where it was lacking.\n18. Please provide more detailed descriptions of statistical analysis in the materials and methods. In many places when t-tests were used, it was unclear whether the data passed the normality test.\nWe have added additional information about all of the statistical tests performed, and have mentioned those cases where adjustments were used for non-normally distributed data.\n19. Please add languages to the discussion on how this study differs from the previous study by Singh, Zeng, Zheng, and Hou that led to the opposite conclusions.\nWe have added a section to the first paragraph of the Discussion addressing this.\nReviewer #2 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nWe thank Reviewers #1 and #2 for their helpful feedback.\nReviewer #3 (Remarks to the Author):\nSummary:\nIn this study, the authors reexamine an anterior region of the fruit fly intestine, known as the proventriculus, which was reported to be maintained by multipotent stem cells. They definitively show, using cell cycle markers and lineage tracing techniques, that stem cells do not exist in this tissue, correcting the initial report. Using a variety of genetic and genomic techniques the cell-types within proventriculus are clearly defined providing a new toolkit of Gal4 drivers for future studies. In addition, the authors discover that cells that cycle (S-phase+) in zone 4 are endocycling, instead of mitotically dividing. Endoreplication appears to be age and nutrition dependent, which may be functionally necessary for the synthesis of the peritrophic member. Secretion of the peritrophic membrane is essential to protect most insect intestines from damage upon ingestion of food as well as pathogenic microorganisms. This is further demonstrated by enhanced sensitivity to P. aeruginosa in flies with the insulin receptor knocked down in zone 4 cells of the proventriculus.\nOverall, this study provides a comprehensive analysis of the proventriculus cellular organization and gene expression program. However, many of the conclusions, particularly in regard to polyploidy are not supported by the experiments performed (i.e. nuclear volume, instead of DNA content measurements). Therefore, the following major and minor comments are strongly recommended to be addressed:\nMajor Comments:\n1. Nuclear volume does not equal nuclear ploidy and cannot be used as a proxy for concluding that a change has occurred. If the authors want to conclude that ploidy is changing with nutrition and age then it needs to be measured. Ploidy can be measured using confocal imaging of DAPI or Hoescht stained nuclei using a diploid cell type in the guts as an internal control (i.e. intestinal stem cells or enteroendocrine cells). Therefore, existing images acquired in this study could be used and reanalyzed to draw these conclusions.\nWe are very grateful to the Reviewer for this feedback, and have re-analyzed every relevant dataset using a direct measure of ploidy instead of nuclear volume. We validated that the nuclei directly posterior to Zone 4 (a region called Zone 3) are diploid and thus used internal ploidy controls within each confocal image that we captured. While these re-analyses did not change the overall conclusions of the paper, the use of a direct assay for ploidy is obviously preferable and more informative.\n2. The ploidy measurements would be helpful to further understand the organization of EdU+ nuclei as it was noted that at 1d and 5d post EdU feeding the nuclei were localized in different regions of zone 4. Spatial ploidy, instead of nuclear volume, analysis is necessary to conclude that ploidy changes with age and nutrition in this region of the tissue.\nAs mentioned above, all three Reviewers identified issues with drawing any conclusions from Figure 1F, and we have thus removed this from the revised manuscript. This removal does not change any of our conclusions.\n3. The authors meticulously and rigorously identify as well as generate cell-type specific Gal4 drivers in this study yet they are under-utilized. An example of this is in regard to the genetic ablation experiment in Figure 5. Cell death may need to be induced in zone 4 itself to activate the endocycle. That is the case for other fly tissues that induce polyploidy in response to injury/damage. Gal4 drivers specific to zones 2/3, 3/4, and/or 4 should be used to ablate cells and test if zone 4 cells are truly refractory to enhanced endoreplication following injury. In addition, the DEMISE system could be used to couple apoptosis with genetic manipulations (RNAi or overexpression) of other target genes (Cohen et al. 2018).\nWe are grateful for this suggestion, and have now performed genetic ablation experiments in Zone 4 itself, shown in a new Figure 7. These experiments led to an important new observation, which is that cell death within Zone 4 itself does result in increased endocycling. This result is an important improvement to our paper because it directly supports the hypothesis that endocycling is a compensatory mechanism to account for cell loss from the tissue.\nWe had initially focused on genetic ablation in other regions of the proventriculus specifically to address previous claims from (Singh et al 2011) who claimed that proventriculus stem cells divide in order to replace dying cells elsewhere in the tissue, a claim that our data refutes.\nHowever, the Reviewer’s suggestion to extend these to Zone 4 itself was very informative, as is the suggestion to use DEMISE in future experiments to understand the mechanism by which Zone 4 endocycles in response to damage.\nRelatedly, given the observation that endocycling both (1) occurs in the absence of exogenous damage but (2) can be induced by damage to the tissue, we used an aging experiment to reconcile these two facts. Specifically, we find that when cell loss ramps up in older flies, there is a distinct change in the spatial distribution of endocycling cells, which we propose is a response to cell loss in aging flies. This is described in the text and in Figure 7E-F.\n4. This study concludes that the proventriculus response to damage is unique from other models of wound- or damage-induced polyploidy yet the assays used are not analogous to prior studies (local vs distal damage- comment #3) and the zone 4 cells appear to be polyploid (Figure S6). The authors may not see a response to X-rays, chemicals, or pathogens as polyploid cells are known to be more resilient to stress. Do the treatments (Figure S5) lead to local damage in zone 4? The high ploidy of zone 4 cells (4C to 40C) may inhibit further polyploidization, a distinction from the epithelium and hindgut pyloric cells which are diploid (2C) prior to injury.\nSee response to #3 - we agree that direct damage to Zone 4 was an important addition to the manuscript.\nThe reason that we chose to include the X-ray, bleomycin, ECC15 experiments in S5 despite the caveats noted by the Reviewer is due to the inherent difficulty in proving a “negative” result - in this case, that there really are no MARCM clones generated from Zone 4. We believe that the more evidence we can provide that, under a wide range of experimental manipulations, the stronger our case is that previous observations were wrong and that our results are robust.\n5. One of the most impactful parts of this study is the potential physiological role for endoreplication in the synthesis of the peritrophic membrane. However, the study lacks a direct demonstration of this conclusion. To do so, endoreplication in zone 4 cells should be directly inhibited using cycE RNAi or fzr RNAi, instead of knockdown of the insulin receptor. Additionally, it is unclear when in development the peritrophic membrane is produced or if it has to be continually maintained? If it has to be continuously synthesized during adult life than this may explain why the zone 4 cells continue to endocycle with age.\nWe have now performed RNAi experiments using four additional candidates for blocking the endocycle: fzr, cycE, myc, and E2F, which we describe in a new supplemental figure, S10 (in fact, we had performed several of these experiments previously but due to the reasons described below had chosen not to include in our initial submission.)\nUnfortunately, these experiments were not informative for our study because none of these manipulations effectively blocked endocycling nearly as strongly as InR-RNAi. fzr-RNAi (using the same line used in e.g. PMID: 25142462) did not show any decrease in ploidy after 14 days of RNAi at 29ºC, indicating that this gene is not strictly required for endocycling in the proventriculus. E2F1 RNAi did cause a decrease in ploidy, but not as dramatically as InR-i and therefore not particularly informative. Myc plays many different roles in Drosophila, and we found that while myc RNAi did lead to dramatic loss of EdU+ cells, it also caused a severe morphological phenotype in which the tissue structure of Zone 4 itself was highly disrupted, with GFP+ cells nearly abolished. Thus, myc RNAi was not an effective means to cleanly block endocycling.\nFor cycE RNAi, we tested three independent RNAi lines (BL38902, BL33654, and BL29314). Two of these lines (BL38902 and BL33654) had no consistent effect on EdU+ cells or ploidy, and we do not include in the manuscript. The third, BL29314, produced a more complex phenotype than simply reducing endocycling, making these difficult to interpret in the context of our study. Specifically, while there was a reduction in endocycling and the overall tissue size of Zone 4, these effects were muted compared to InR RNAi, and we also observed large aggregates of peritrophic membrane components throughout Zone 4 (shown in Figure S10) which stained positive for all three markers of PM. While these results may be interesting in their own right, they do not allow us to cleanly assay the effect of blocking endocycling using cycE RNAi.\nIt is possible that there are technical explanations for all these results, such as perhaps that RNAi knockdowns of these factors are not sufficiently strong over 14-21d time period. However, the ultimate result is that at this point, the only genetic manipulation that we have found which consistently blocks endocycling in Zone 4 cells is InR RNAi. We have also added text in the manuscript to clarify that InR may play other roles in these cells in addition to solely blocking endocycling.\n6. The authors could use the dextran permeability assay (Kuraishi et al. PNAS 2011) to determine if the peritrophic membrane is compromised when endoreplication is upregulated or inhibited. It is unclear if the changes in lectin staining are indicative of a functional change.\nWe have now performed both the Smurf assay and a fluorescent bead assay similar to that performed by Kuraishi et al (2011), and both assays did not reveal a significant disruption to the gut barrier in flies with endocycling inhibited. Together with our finding that these flies are more susceptible to pathogenic infection, we conclude that endocycling (and the resultant increase in synthetic capacity) is not strictly required for the synthesis of a functional peritrophic membrane under standard conditions, but instead provides robustness in the face of environmental challenges that flies may encounter including infection.\nMinor Comments:\n1. The authors should consider moving Figure 6 (GTRACE) to the supplement as it is redundant with Figure 4 (MARCM).\nDone.\n2. Line 333-335: Liver hepatocytes are derived from the endoderm and well-documented to endoreplicate in response to tissue damage, so there is insufficient evidence to make a claim of an ectoderm vs endoderm difference in endoreplication.\nWe have removed this speculative statement from the manuscript.\n3. Miss cited figures (i.e. Fig S6C – line 397-8 does not exist). Figure S7 legend is missing a description for part (C).\nFixed.\n4. Several graphs (i.e. Figure 1H and 6F) are missing significance and the statistical test if used it is not reported.\nFixed.\n5. Check image stains are labeled in all figures (i.e. Figure 2E is missing this info)\nFixed.\n6. It would be helpful to provide the guts (n) and PH3+ nuclei (#) for intestines in Figure 3B as well.\nDone - under these conditions, >600 ph3+ ISCs are observed in midguts.\nReviewer #4 (Remarks to the Author):\nIn this thorough and carefully conducted study, the authors investigate the mechanism by which tissue homeostasis is maintained in the Drosophila adult proventriculus. A previous study reported on the presence of BrdU+ cells in the proventriculus and proposed that it therefore contains mitotically active stem cells. Here the authors reexamine this conclusion and provide convincing evidence that these cells are endocycling rather than undergoing mitosis and therefore are not stem cells. They reach this conclusion through a combination of approaches, including stains for mitotic markers and clonal analysis in a wide range of experimental conditions designed to enrich for mitotic cells if they exist in this tissue. However, despite this extensive search, they do not find evidence of cells in mitosis. Suitable positive controls are included to confirm that their methods are working as expected. They also generate a single cell atlas of the proventriculus and use the results, along with a screen and a recently developed split-Gal4 tool to specifically target the region of the tissue where cells in S-phase can be found. Interestingly, they find that cells in this region are polyploid and that the ploidy increases with age, suggesting that they continue to endocycle during adulthood. The authors propose that this may be a mechanism to compensate for age-dependent cell loss, which they provide evidence for. Overall, I think this study will be a valuable contribution to the field and I am supportive of publication. I have only a few minor comments, which I recommend the authors consider before publication:\nWe appreciate the Reviewer’s supportive feedback and helpful suggestions.\n11\n1. I am confused by the results described in the paragraph from lines 132-139 (Figure 1D is referenced but I think the authors are referring to Figure 1F): if the cells are continuously endocycling, why would the size of EdU+ nuclei increase over 5 days? Wouldn't there be a distribution of nuclear sizes at any given time point when EdU is administered such that both large and small nuclei could be labeled even at day 1?\nWe appreciate this helpful feedback, and as all Reviewers pointed out flaws with the analyses of these panels we have removed them from the revised manuscript.\n2. On line 147, it seems that Figure 1H should be referenced (not Figure 1E).\nFixed.\n3. On Line 252-253, the authors state that they never saw cells travel from Zone 4 to any other portion of the proventriculus but, without live imaging or clonal analysis, how is it possible to track cell movement?\nWe thank the Reviewer for raising this issue. We have clarified in the text that this conclusion is based on the fact that we never observed GFP+ cells located outside of Zone 4 despite increased cell cycling and tissue growth. Had these cells migrated outside of Zone 4 as claimed by Singh et al 2011, we would have expected to see GFP+ cells located elsewhere in the proventriculus.\n4. The data in Fig 4D show that there are, in fact, a small number of large clones in the proventriculus at 14 days after heat shock (dark grey bar at the top of the third column from the left) but this is not discussed in the text. How did these larger clones arise if the cells cannot undergo mitosis?\nWe have added text in the manuscript to clarify that we believe this single example of large clone was the result of a spontaneous clone formation earlier in development.\n5. The code used to analyze the scRNAseq data as well as the final Seurat objects should be made available.\nWe have now made this code available and provided a link to the scripts in the manuscript.\nSincerely,\nBen Ewen-Campen & Norbert Perrimon"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The changes made in the revised version have addressed the major concerns raised in the original review in a satisfactory manner. I now favor publication without further substantive changes.\nThe paper is framed as a re-investigation of a 2011 report describing proventriculus (PV) stem cells that were never confirmed and fell into obscurity. However, this paper goes beyond classical stem cells and provides convincing evidence of a different and very interesting alternative phenomenon related to \"wound-induced polyploidy,\" an alternative repair system based on induced endocycles rather than mitotic cycles. Midgut and hindgut enterocytes, and many other cells are known are known under some conditions to polyploidize in response to tissue damage, as has now been well studied previously by multiple groups including the authors. Here they show that PV4 cells undergo scattered endocycling in a ring-like pattern during much of adult life, in the absence of a known source of tissue damage.\nAn interesting potential explanation would be that active peritrophic membrane (PM) production is intrinsically damaging to the posterior rim of PV4 cells. The cells may be responding to cell loss with induced polyploidization of some remaining nearby PV4 cells to maintain the functional capacity of this region, which is thought to play a critical role by generating the major chitin-rich inner layer of the peritrophic membrane. This layer is involved in digestion, and restricting access of gut microbiota to enterocyte tissue. These findings recall the well known theme in studies of the gut that the normal tasks of some cells are intrinsically damaging, This is the generally accepted reason for the rapid turnover of midgut enterocytes and the presence of highly active stem cells to supply replacements. Damaging food and microorganisms are thought to be largely excluded from the upper PV, and this is a rationale for the absence of active stem cells. Something must be different in this region of PV4, and the capacity of the endocycle-based repair may eventually be inadequate and contribute to an age and metabolism-related loss of homeostasis and normal tissue structure. A similar scenario of polyploidization incompletely counteracting damage leading to slow progression of disease was described in the mouse model of Fuchs Endothelial Corneal Dystrophy analyzed in reference 14. Endocycling in the pattern described might serve as a marker for such early aging.\nThe authors in the revised version make a strong case for cell turnover and compensatory polyploidization (rather than cell generation) in PV4. They now show that the average ploidy as well as nuclear volume of zone 4 cells increases over the lifespan. They do not currently propose a reason for this behavior other than to call it a \"unique pattern of endoreplication.\" I think the paper would be further improved by a little more discussion of what factors might lead to such a unique pattern of cell cycle behavior.", "reviewer_3": "Summary:\nThis revised manuscript addresses the major and minor concerns of the original study. The ploidy analysis now proves that zone 4 cells in the proventriculus endocycle with age and appear to do so to compensate for cell loss. The authors find that cell loss occurs naturally with age as well as in response to genetically induced-apoptosis. This is consistent with other studies performed to date in fruit fly. This study also provides a comprehensive analysis of the proventriculus cellular organization, its gene expression program, as well as identification of a novel Gal4 driver to regulate gene expression in zone 4. The study debunks previous report of stem cells in the tissue and finds that tissue homeostasis appears to be maintained by cell growth, instead of cell division. Despite the many different genetic manipulations performed, the authors were not able to find a condition to directly inhibit endoreplication in zone 4 cells. Therefore, the physiological importance of endoreplication to the intestine’s peritrophic membrane synthesis as well as infection resistance remain inconclusive lessening the impact of this study.\nRemaining Comments:\n1. A major conclusion of this paper is that endoreplication with age or injury compensates for cell loss. The authors should be able to definitively show this by summing the nuclear ploidy in young vs old or uninjured vs repaired tissues. If ploidy is compensating for cell loss, then the total ploidy of the two conditions will remain unchanged despite a reduction in cell number.\n2. The authors still claim that their findings are unique (line 625-635) from other studies on injury or age-induced polyploidy. However, the uniqueness is subtle at best as Nandakumar et al. 2020 demonstrated that neuron ploidy increases with age via endocycle. As for nutrient dependance, endoreplication is well-characterized to be sensitive to nutritional inputs. Therefore, the changes observed are more likely tissue intrinsic and due to cell type specific differences in longevity/ turnover rate.", "reviewer_4": "With this revision, the authors have fully addressed my concerns."}, "author_response": "Author response: We are grateful for these positive comments and have now included additional discussion based on the suggestion of the reviewer’s interesting proposal that PM production may be intrinsically damaging.\nAuthor response:\nRe (1): We are grateful for these additional comments. While we would very much wish to calculate the total ploidy of Zone 4 during aging and damage, unfortunately our experimental setup does not allow this calculation. Specifically, due to the 3D shape of the tissue, we are unable to comprehensively measure the ploidy of Zone 4 because multiple nuclei overlap with each other in 2D and 3D, and with Zone 3 and the esophagus. Instead, we have relied on measuring the ploidy of as many nuclei as can be unambiguously scored in each sample.\nRe (2): We have modified our discussion in line with the reviewer’s context including Nandakumar."}]

Decision letter