Loss of core PRC1 induces tumor-like clusters in adult ISC

To study PRC1 loss of function in the gut, we first used the chromosomal deficiency XL26 encompassing the Psc gene and its redundant paralog Su(z)2 [14,33]. We generated fluorescently labelled Psc/Su(z)2 homozygous mutant clones for 14 days using the FLP-FRT-based MARCM (Mosaic Analysis with a Repressible Cell Marker) system [34]. With this technique, Psc/Su(z)2 clones are derived from a single mitotic escargot (esg)-positive progenitor and labelled with GFP. The differentiated cells, ECs and EEs, were distinguished as Prospero-negative large cells (only polyploid cells with large nuclei) and Prospero-positive small-nucleated cells, respectively. In control, neutral clones, we observed both EC and EE lineages derived from the mitotic stem cells, as expected (Fig 1A-1C). In contrast, the Psc/Su(z)2 loss-of-function clones consist only of Prospero-negative cells with small nuclei, indicating that they acquire neither absorptive (large nuclei) nor secretory (Pros-positive) identity (Fig 1B, 1C). These results show that Psc/Su(z)2-deficient cells (esg-positive) fail to differentiate properly in either lineage.

Download:

• PNG
  larger image
• TIFF
  original image

Fig 1. Loss of Psc/Su(z)2 induces tumor-like clusters in adult ISCs.

A. Lineage of Drosophila intestinal epithelial cells and their specific markers. B. Control and Psc/Su(z)2^XL26 mutant MARCM clones expressing GFP (yellow). The differentiated EE were marked with the anti-Prospero antibody (red). Clones and clusters are delimited by yellow lines. C. Plot on the left shows the percentage (%) of small nuclei in control and mutant clones. Chi²-test p-value < 2.2e-16. n = 20, one dot represents one measure. Plot on the right shows the % of the number of clones containing differentiated cells (large nucleus EC and/or Prospero-positive EE). Chi²-test p-value < 2.2e-16. Septuplicate for a total of n = 203 (control) and 151 (mutant) clones. One dot represents the % in one replicate. D. esg-positive cells expressing GFP (yellow) alone (control; upper panels) or together with Psc/Su(z)2^RNAi (bottom panels). Z-projections of the region traced by the brown lines appear below the merged images. E. Quantification of guts with tumor phenotype in %. Anova-Tukey multiple comparisons. One dot represents one replicate with n = 8–12 guts each. F. Control and Psc/Su(z)2^XL26 mutant MARCM clones expressing GFP (yellow) and stained with the anti-Delta antibody (red). Close-up images of selected clones are also shown (red squares). G. Quantification of Delta-positive cells per clone. Chi²-test p-value < 2.2e-16; n = 37 and 45. H. esg-positive cells expressing GFP (yellow) alone (control; upper panels) or with the double dominant negative (DDN) forms of both Psc and Su(z)2 proteins (lower panels). Mitotic cells are stained with the anti-PH3 antibody (purple). I. Quantification of PH3 counts per gut from 3d to 14d after induction of expression. Kruskall-Wallis and Wicolxon test p-values at 3, 7, 10 and 14 days are 0.00028; 5.7e-07; 5.2e-10; 2.2e-08, respectively, for comparison between control and cluster conditions. Different letters indicate that conditions belong to statistically different groups; n = 67, 52, 27, 29, 28, 31, 30, 25. J. Expression of Psc/Su(z)2^RNAi in ISCs (Dl-GAL4^ts,UAS-GFP), EBs (Su(H)-GAL4^ts,UAS-GFP) and EEps (Piezo-GAL4^ts,UAS-GFP). K. Quantification of guts with tumor phenotype in %. Anova-Tukey multiple comparisons. One dot represents one replicate of n = 8–12 guts each. Dl-esg = n.s (1.0); Su(H)-Piezo = n.s. (0.0643278); Su(H)-Dl and Su(H)-esg = p < 0.00000001; Piezo-Dl and Piezo-esg = p < 0.00000001. DNA (turquoise): stained with DAPI. Scale bar, 50 μm.

https://doi.org/10.1371/journal.pgen.1012226.g001

To confirm this phenotype, we induced loss of function of both Psc and Su(z)2 either by RNAi (two different lines of Psc/Su(z)2^RNAi) [35] or by co-expression of dominant negative forms of both proteins (Psc/Su(z)2^DDN,“double dominant negative”) [14] in progenitor cells using the esg-GAL4^ts,UAS-GFP driver. In all cases, we observed epithelial disorganization, visualized by clusters of small nuclei that were Prospero negative and located mainly in the posterior midgut (Figs 1D, 1E and S1A, top three rows). Interestingly, large clusters no longer showed the GFP reporter, suggesting a progressive loss of esg-GAL4 driver activity. To better follow the cells in which we induced Psc/Su(z)2 loss of function, we then used an alternative FLP-FRT system: the esgF/O line. This line allows cells to permanently express the transgenes (e.g., GFP and RNAi constructs) under the ubiquitous actin promoter once recombination is induced by the esg promoter. When using this tool with either Psc/Su(z)2^RNAi or Psc/Su(z)2^DDN, we observed large, GFP-positive, Prospero-negative clones with small nuclei that originated from progenitor cells (S1A Fig, bottom two rows). In agreement with the previous observations, this establishes that Psc/Su(z)2 loss of function in intestinal progenitors generates clusters of small undifferentiated cells.

The disappearance of GFP reporter gene expression with the esg-GAL4 driver observed above (Figs 1D, S1A, top three rows) suggests that the cell clusters lose expression of the esg gene and thus stem cell identity. We tested this hypothesis by analyzing the presence of the ISC specific marker Delta (Dl) in Psc/Su(z)2 loss-of-function (MARCM or RNAi) cell clusters. As expected, control MARCM clones contain ISCs identified by anti-Dl staining. However, the mutant MARCM clones Psc/Su(z)2^XL26 are not composed of any ISCs, as indicated by the absence of Dl expression within the clones (Fig1F, 1G). In accordance with this result, we observed loss of Dl-positive cells with Psc/Su(z)2^RNAi or Psc/Su(z)2^DDN (S1B Fig, top three rows). We also confirmed this result using the Dl.LacZ reporter gene (S1B Fig, fourth row). Occasionally, we observed rare MARCM clusters of two or three cells expressing Dl, likely reflecting emerging clones that have not yet lost its expression (Fig 1G). These results support the notion that Dl expression is rapidly lost during cluster formation. We then assessed the expression of Su(H), which is specifically expressed in enteroblasts [36]. The results showed no expression of the Su(H)-lacZ reporter in Psc/Su(z)2^XL26 MARCM clones, indicating that cell clusters are not composed of enteroblasts (S1B Fig, fifth row). We also quantified the mitotic activity of the mutant clusters compared to control progenitors using PH3 immunostaining. Control progenitors have a basal level of mitotic activity, allowing a continuous and tightly controlled epithelial renewal throughout life (Fig 1H, 1I). We detected strong mitotic activity of the Psc/Su(z)2^DDN progenitor cells compared to the control cells as early as three days after induction. Therefore, Psc/Su(z)2 loss of function generates clusters that are mitotically active and rapidly lose stem cell identity, suggesting that they may have tumor-like characteristics.

As esg-positive progenitors include ISCs, EBs and EEps, we asked which cell population was at the origin of the Psc/Su(z)2 mutant clusters. We used specific drivers allowing the expression of Psc/Su(z)2 RNAi in each cell type: Dl-GAL4^ts or esg-GAL4^ts,Su(H)-GAL80 in ISCs, Su(H)-GAL4^ts in EBs and Piezo-GAL4^ts in EEps. Both ISC drivers showed clusters with small nuclei and Prospero-negative cells, as previously obtained with esg-GAL4^ts (Figs 1J, 1K; 1F and S1B, bottom panels). Again, we observed the disappearance of GFP, supporting a loss of driver activity. In contrast, Psc/Su(z)2 loss of function in EBs does not produce clusters (Fig 1J, 1K), whereas in EEps, some guts exhibit the cluster-like phenotype (Fig 1J, 1K). This weakly penetrant phenotype observed with the EEp driver is likely due to Piezo expression in a subpopulation of Dl-positive cells [37]. Taken together, our results demonstrate that Psc and Su(z)2 act in a cell-autonomous manner in ISCs to prevent the formation of tumor-like clusters.

Psc and Su(z)2 have previously been reported to act redundantly in the Drosophila ovary and testis to restrain stem cell proliferation [14,15]. We used both RNAi and the MARCM system to induce knockdown or knockout of only one of the two genes. Single Psc or Su(z)2 RNAi does not form tumor-like clusters, and the single MARCM clones harbor Dl-positive ISCs and generate differentiated cells (large-nucleated ECs and Pros-expressing EEs) (S1C-S1E Fig). These results reveal that Psc and Su(z)2 act redundantly in ISCs, reminiscent of what has been reported in other Drosophila organs like the ovary and testis [14,15].

We then tested by RNAi whether the selective knockdown of the other core PRC1 subunits Sce, Ph, and Pc, as well as of the substoichiometric subunit Scm, could phenocopy the loss of Psc/Su(z)2. We observed that loss of function of the catalytic subunit Sce or the subunit Ph (depletion of both Ph-d and Ph-p) is sufficient to induce the tumor-like phenotype and the disappearance of GFP expression. However, loss of the Pc or substoichiometric Scm subunits does not produce the Psc/Su(z)2 loss-of-function phenotype (S2A,S2B Fig). This is a surprising observation since Pc and Scm, like Psc/Su(z)2, Ph and Sce, are part of the core PRC1. Nevertheless, this result is in accordance with previously published data in imaginal discs showing that the loss of Pc and Scm subunits have no or less severe phenotypes compared to the loss of other core PRC1 subunits [33,38]. To fully explore the complexity of PRC1 subunit association in this context, we investigated whether a non-canonical PRC1 complex could be involved in ISCs to prevent cluster formation. Therefore, we tested RNAi-induced loss of function of the known non-canonical PRC1 subunits RYBP and Kdm2 and did not observe the formation of cell clusters in any of these conditions (S2A, S2B Fig). Overall, our results point to an essential role for the core PRC1 complex, comprising Sce, Ph and Psc/Su(z)2 but independently of Pc, in maintaining ISC identity and ensuring the full differentiation capacity of the stem cell lineage in the adult Drosophila intestine. The loss of core PRC1 function in adult ISCs results in the formation of clusters with tumor-like characteristics, which include the loss of cell identity, loss of differentiation and unchecked proliferation.

Psc/Su(z)2 loss of function triggers intestinal neoplastic transformation and impairs intestinal response to stress

The tumor-like characteristics of the PRC1 mutant clusters prompted us to investigate whether they were potentially malignant. Neoplastic malignant tumors are expected to be immortal and to acquire metastatic behavior after serial transplantations (allografts) in adult abdomens [39]. We conducted allograft experiments with GFP-positive clusters coming from esgF/O > Psc/Su(z)2^RNAi flies, described above (S1A Fig), allowing co-expression throughout the process of Psc/Su(z)2^RNAi for cluster formation and of GFP for tracking. We performed two series of allografts, involving 18 transplants from 6 independent Psc/Su(z)2^RNAi adult midguts into the abdomens of healthy adult female hosts. Before killing their hosts, two independent transplants (tumor 1 and tumor 2) were extracted, and portions were re-transplanted into new healthy flies until the fifth generation of transplantation (G1 to G5). The Psc/Su(z)2^RNAi transplants maintained their ability for sustained growth in host flies over the five generations (about 1,5 month) (Fig 2A). Furthermore, in contrast to control esgF/O cells, esgF/O > Psc/Su(z)2^RNAi cells exhibited a propensity to invade the whole host body as early as G1, disseminating from the initial injection site and infiltrating the entire host organism (Fig 2A). We observed a significant decline in host fly survival that gradually worsened through transplantations, demonstrating that Psc/Su(z)2-depleted transplants become more aggressive upon serial transplantation (Fig 2B-2D). These results imply that the PRC1 mutant clusters are neoplastic tumors that exhibit continuous and autonomous growth and are hereafter referred to as tumor-initiating intestinal cells (TIICs).

Download:

• PNG
  larger image
• TIFF
  original image

Fig 2. Psc/Su(z)2 loss of function triggers intestinal neoplastic transformation and impairs intestinal response to stress.

A. Visualization of the GFP signal (green) after serial transplantation (allograft) of esgF/O > GFP intestinal cells into the abdomens of adult female hosts. Left panels: images showing two control flies in which the GFP signal of esgF/O midguts (i.e., GFP-positive midguts of siblings coming from the cross allowing the generation of intestinal TIICs) is only visible close to the injection site after the first generation of transplantation. Right panels: Images showing the proliferative and invasive properties of two GFP-labeled esgF/O > Psc/Su(z)2^RNAi intestinal tumors. In the first two generations of transplantation (G1 and G2), the GFP-positive cells already have the ability to disperse in the abdomen. By G5, Psc/Su(z)2^RNAi tumors have acquired the ability to invade different sites, even outside the abdomen region (arrows). The abdomen are limited by white dotted lines. B. Survival rate as a percentage of flies after the first generation of transplantation (G1). Two independent transplantation series (series n°1 in pink and series n°2 in green, respectively) were performed and compared to the control flies. C. Survival rate (%) after the fifth generation of control flies (G5 control in black; injected with PBS) and Psc/Su(z)2^RNAi tumor-transplanted flies (in pink, green and purple). D. Survival (%) of host flies 7 days after control allograft (black) and after allograft of Psc/Su(z)2^RNAi knockdown tumors (red) during 5 rounds of transplantation. In all allograft experiments, significant differences were assessed using the Log-rank test. E. Survival curves (%) upon DSS challenge of control flies (esg^ts) or flies expressing Psc/Su(z)2^RNAi (esg^ts > Psc/Su(z)2^RNAi). Statistics with Pairwise CoxPH comparisons.

https://doi.org/10.1371/journal.pgen.1012226.g002

We next investigated whether TIICs compromise the overall health of the flies in response to extrinsic challenges. To this end, we exposed the flies to dextran sulfate sodium (DSS), which induces gut damages similar to human ulcerative colitis [40]. The survival curves of unchallenged esg-GFP^ts/+ (control) or esg-GFP^ts > Psc/Su(z)2^RNAi (tumor-bearing) flies did not appear to be significantly different, indicating that TIIC tumors had no direct impact on Drosophila lifespan (Fig 2E). However, esg-GFP^ts > Psc/Su(z)2^RNAi flies treated with DSS exhibited a significant decrease in survival compared to control flies treated with DSS. These results demonstrate that Psc/Su(z)2 loss of function impairs intestinal response to colitogenic stress in flies, leading to a shorter lifespan upon challenge. Altogether, these results highlight that core PRC1 is required to maintain ISC identity, thereby ensuring full capacity for gut regeneration. Moreover, preserving normal levels of PRC1 activity is crucial for safeguarding the adult intestine against malignant neoplastic tumors.

Recent studies showed that transient depletion of PRC1 subunits in the larval eye imaginal disc is sufficient to induce a neoplastic transformation, indicating that epigenetic modifications can initiate tumorigenesis independently of genetic mutations [35]. To investigate whether epigenetically induced tumors can also be generated in adult tissues, we used the thermosensitive RNAi system enabling the reversible knockdown of Psc/Su(z)2 in adult intestinal progenitors, as performed in the eye imaginal disc. We observed that a transient loss of Psc/Su(z)2 for 24 or 48 hours led to the formation of TIIC tumors, with the 48-hour condition achieving an efficacy close to that of continuous 14-day depletion (S2C, S2D Fig). Reminiscent of the observations made in the larval eye imaginal disc, these results further demonstrate that a transient loss of the epigenetic modulator PcG genes is also sufficient to induce neoplasia in an adult tissue and that epigenetic alteration can lead to adult cell reprogramming.

TIIC transcriptomic signatures

To uncover key players leading to the formation of PRC1-dependent intestinal tumors, we compared the transcriptome signatures of control progenitors to the FACS-sorted Psc/Su(z)2^RNAi TIICs. The RNA sequencing data showed that 2488 genes are defined as significantly differentially expressed, with 1268 downregulated genes and 1220 upregulated genes (Fig 3A; S1 Table). The efficacy of the knockdown of Psc and Su(z)2 in TIIC tumors is validated by the reduction of their expression (Fig 3B). As expected, several canonical Homeotic (Hox) PcG targets are derepressed, such as abd-A, Abd-B, Antp, pb, Dfd and Scr (Fig 3B). Our data confirmed the concomitant loss of esg transcription, which is consistent with our previous observation that esg expression disappeared in TIICs (Fig 3B). We also detected decreased expression of several validated ISC markers: mira, insc, sox21a and zfh2 [41–43]. These data, together with our previous findings (Fig 1), confirm that TIICs lose stem cell identity.

Download:

• PNG
  larger image
• TIFF
  original image

Fig 3. Psc/Su(z)2 TIIC transcriptomic signature reveals activation of the Toll and Imd pathways.

A. Volcano plot of differentially expressed genes between Psc/Su(z)2^RNAi tumors and control progenitors. Each star represents a gene whose expression is significantly modified with a log2 fold-change of less than -1 (purple) and more than +1 (orange). A set of candidate genes is indicated (see main text for details). B. Heatmap showing normalized expression of key genes in control progenitors and Psc/Su(z)2^RNAi tumors (see main text for details). C. Representative Gene Ontology terms enriched in downregulated genes. The full chart is available in S3 Fig. D. Control and Psc/Su(z)2^XL26 mutant MARCM clones expressing GFP (yellow). The adherent junctions were marked with the anti-Armadillo antibody (red). Clones and clusters are delimited by yellow lines. Scale bar, 50 μm. E. Quantification of the intensity of the Armadillo signal in control (orange) and Psc/Su(z)2^XL26 mutant (purple) clones. n = 10 guts across 2 independent experiments for a total of 30 measurements for each condition. F. Representative Gene Ontology terms enriched in upregulated genes. The full chart is available in S3 Fig. G. Heatmap showing normalized expression of genes of the Imd and Toll signaling pathways in control progenitors and Psc/Su(z)2^RNAi tumors.

https://doi.org/10.1371/journal.pgen.1012226.g003

Gene ontology analysis revealed that downregulated genes are enriched for functions such as “epithelial cell differentiation”, “cell adhesion” and “maintenance of epithelial integrity” (Figs 3C, S3 and S2 Table), including genes encoding for Armadillo/β-Catenin and cadherins (Fig 3B). Armadillo/β-Catenin, a component of the adherent junctions in the intestinal epithelium that is enriched in ISC [44], shows reduced expression in Psc/Su(z)2^XL26 MARCM clones, as evidenced by immunostaining, which validates the loss of epithelial identity in TIIC tumors (Fig 3D, 3E). Conversely, the upregulated genes are enriched for ontologies associated with proliferation control like polo, stg and Cdk1 (Figs 3F, S3 and S3 Table), which is in accordance with proliferative features and the loss of differentiation that are characteristic of a tumoral phenotype. Data also showed a strong upregulation of genes involved in the immune response against bacteria and fungi (Fig 3F; S3 Table), which was not observed in control midguts dissected under the same conditions. This suggests that the activated immune response is specific to PRC1 downregulation. In particular, several members and target genes of the Toll and Imd signaling pathways showed an increase in transcription in TIICs, including the PGRP-SA, spz, Toll, Drs and Def for the Toll pathway and DptB for the Imd pathway (Figs 3G, S3). A recent study used the CUT&Tag methodology to determine the direct targets of the Pc-containing complexes in the whole Drosophila gut [45]. Re-analysis of their data indicates that several genes of the Toll/Imd pathways are indeed direct targets of Pc, suggesting that the upregulation of the Toll/Imd pathways in TIIC tumors may be a direct effect of the PRC1 loss (S3E, S3F Fig). Interestingly, these pathways were not found to be upregulated upon depletion of PRC1 subunits in the larval eye imaginal disc [35], indicating that this response is specific to the gut.

Activation of the Toll and Imd pathways supports an antitumoral compensatory mechanism independently of cell death

To assess the potential involvement of immune activation through the Toll and Imd pathways in TIICs, we investigated whether RNAi-induced knockdown of the two downstream NF-κB transcription factors dorsal (Toll pathway) or Relish (Imd pathway) affects the development of TIIC tumors. We observed that the concomitant loss of Psc/Su(z)2 and Relish, or more notably Psc/Su(z)2 and dorsal, induced massive tumors (Fig 4A, 4B). In contrast, the loss of Relish or dorsal alone does not result in tumor formation (Fig 4A, last two rows). The number of mitotic cells per gut increased significantly in both conditions compared to normal TIIC tumors (Fig 4C). Drosophila Toll and Imd signaling, and their downstream effectors NF-κB dorsal and Relish, have been shown to exert tumor suppressive roles through pro-apoptotic cell death [46–51]. We therefore tested whether a compensatory mechanism takes place in TIICs that reduces tumor expansion by promoting cell death. We expressed p35 or reaper^RNAi in TIICs but observed neither a significant change in the severity of the tumor phenotype compared to Psc/Su(z)2-depleted tumors nor in mitotic activity (Fig 4A-4C). In addition, we used the TUNEL assay to detect dying cells, and the results showed no differences in the proportion of cell death between control and TIIC conditions (Fig 4D, 4E). Our findings thus indicate that Dorsal of the Toll pathway and Relish of the Imd pathway are both cell-autonomously activated in TIIC tumors to restrict their expansion independently of cell death. However, these compensatory mechanisms are not sufficient to prevent neoplastic tumor formation and progression after transplantation.

Download:

• PNG
  larger image
• TIFF
  original image

Fig 4. Activation of the Toll and Imd pathways acts as a compensatory mechanism that is independent of cell death.

A. esg-positive cells expressing GFP (yellow) and Psc/Su(z)2^RNAi along with dorsal^RNAi, Relish^RNAi, p35 or reaper^RNAi. Tumors are outlined with yellow lines. In contrast, the esg^ts>dorsal^RNAi and esg^ts>Relish^RNAi controls showed no tumors (n = 10 for each). B. Quantification of the tumor burden following 5 grades of severity (0 = no tumor to 4 = large tumors) as described in S4 Fig. P-value 1.6586.10^-5 and 8.098.10^-5, respectively, compared to the Psc/Su(z)2^RNAi control condition. Statistics were obtained using a contingency table and Chi-sq. tests. C. Quantification of the mitotic cells (PH3-positive) per midgut in the conditions indicated in A. Anova-Tukey multiple comparisons. P-value <0.0000000 and 0.0000002, respectively, compared to Psc/Su(z)2^RNAi control condition. D. esg-positive cells expressing GFP only (yellow) or with Psc/Su(z)2^RNAi stained by the TUNEL method (red). Close-up images are also shown (red squares). Tumors are outlined with yellow lines. E. Quantification of the TUNEL-positive cells per normalized area in the 2 conditions. Student T-test shows no significant differences. DNA (turquoise): stained with DAPI. Scale bar, 50 μm.

https://doi.org/10.1371/journal.pgen.1012226.g004

Activation of JAK/STAT signaling sustains TIIC tumor growth

Finally, we went a step further to find the mechanisms that might support tumor formation. Analysis of our RNA sequencing data yielded a marked increase in the expression levels of chinmo and zfh1 (Fig 5A; S1 Table). These two genes have been shown to be direct PcG targets and to promote tumor growth [33,35,52–58]. Of particular interest, the mammalian homologue of zfh1, ZEB1, is an oncogene that has been reported in various human cancers and has the capacity to induce epithelial-to-mesenchymal transition [59]. Interestingly, chinmo and zfh1 have been identified as direct transcriptional targets of the JAK/STAT pathway [55], prompting us to evaluate the role of this pathway in TIIC tumors. Consistent with this, our data showed decreased expression of ken, a known negative regulator of the JAK/STAT pathway (Fig 5A). We first monitored the activity of the pathway using the 10XSTAT92E>GFP reporter transgene. Tumors obtained either by expression of the DDN forms of Psc/Su(z)2 proteins or by Psc/Su(z)2 RNAi showed consistent activation of JAK/STAT signaling (Fig 5B). In the eye imaginal disc, loss of PRC1 drives tumor growth partly in a JAK/STAT-dependent manner through the production of the Upd ligands [33,35]. We re-analyzed the Pc-profiling CUT&Tag performed in intestinal cells [45], focusing on the JAK/STAT genes found regulated in TIIC tumors. The results indicated that the genes coding for the ligands Upd2 and Upd3 (but not Upd1) can be potentially directly regulated by PRC1 (S3G Fig). In our RNA sequencing data, however, we did not detect transcriptional upregulation of these ligands, suggesting that JAK/STAT activation in the TIIC tumors does not occur through derepression of the upd genes. To directly address whether expression of JAK/STAT pathway ligands is indeed unchanged, we analyzed upd3-lacZ expression in guts bearing tumors (esg^ts,GFP,upd3-lacZ > Psc-Su(z)2^DDN). The results indicate no change in expression of upd3 in TIICs or adjacent cells compared to control guts, suggesting that JAK/STAT activation is independent of the Upd ligands (Fig 5C). Finally, an RNAi loss-of-function experiment targeting the JAK/STAT transcription factor Stat92E in Psc/Su(z)2 TIICs caused a significant decrease in the number and size of the tumors, even though we still observed the loss of the esg > GFP marker (Fig 5D, 5E). This indicates that the tumor burden of Psc/Su(z)2 clusters is reduced upon Stat92E knockdown. Collectively, these observations indicate that JAK/STAT signaling contributes to the growth of Psc/Su(z)2-induced TIIC tumors.

Download:

• PNG
  larger image
• TIFF
  original image

Fig 5. Activation of JAK/STAT signaling supports tumor growth.

A. Heatmap showing normalized expression of the negative regulator ken and the target genes chinmo and zfh1 of the JAK/STAT pathway in control progenitors and Psc/Su(z)2^RNAi tumors. B. 10XSTAT92E>GFP reporter gene expression (yellow) in esg^tsNP7097 > Psc/Su(z)2^DDN (top panels) or Psc/Su(z)2^RNAi tumors (bottom panels). C. upd3-LacZ reporter gene expression (red) in control guts (esg^ts,UAS-GFP>+; top panels; n = 14) and TIIC-containing guts (esg^ts,UAS-GFP > Psc/Su(z)2^DDN; bottom panels; n = 23). D. esg-positive cells expressing GFP (yellow) and Psc/Su(z)2^RNAi only or with STAT92E^RNAi. E. Tumor burden in Psc/Su(z)2^RNAi (control) and Psc/Su(z)2^RNAi,STAT^RNAi. Chi-sq. test p-value = 0.0006242. Tumor burden was evaluated as described in Figs 4 and S4. Tumors are outlined with yellow lines. DNA (turquoise): stained with DAPI. Scale bar, 50 μm.

https://doi.org/10.1371/journal.pgen.1012226.g005

Ethics statement

We have received authorization from the French ministry of higher education, research and innovation “for the contained use of genetically modified organisms for research, development or educational purposes”, which allows us to handle genetically modified Drosophila (DUO number 12508).

Reagents and biological resources

Experimental methods

Survival experiment

DSS was prepared by dissolving DSS in 1X-PBS/5% sucrose solution, filter sterilized, and stored at 4°C for up to two weeks. For DSS survival, 20 virgin females per vial were reared at 29°C and transferred three times per week to fresh vials containing 5% DSS/5% sucrose or 5% sucrose alone. Dead flies were counted every 1–3 days.

Gut dissection and staining

Dissections and immunostainings were performed as described [92] using mouse anti-Prospero at 1:200, rabbit anti-PH3 at 1:500, mouse anti-Armadillo at 1:25 and chicken Anti-β-Galactosidase at 1:1000 for the primary antibodies. For Delta staining, we used the previously described protocol [41] using mouse anti-Delta at 1:2500. Samples were mounted with DAPI-containing Fluoroshield medium. Cell death was detected using the TMR red In Situ Cell Death Detection Kit (Roche) following the kit’s standard staining protocol. Briefly, guts were washed in PBS after secondary antibody, then stained with 100uL of TUNEL solution for 1hr at 37°C and washed twice with PBS before mounting.

Allograft assay

Allografts were performed as previously described [39]. Briefly, UAS-RNAi Psc/Su(z)2 #44 female flies were crossed with esgF/O males at 18°C. After 3 weeks, 0–3 day old female flies were collected along with a few males, and the intestines were allowed to mature for 3 or 4 days at 18°C. The flies were then transferred to 29°C for 14 days for tumor formation. Tumors were positively labeled with GFP. Only female midguts were dissected, cut into pieces and injected into the abdomens of RFP+ adult female hosts (genotype RRID:BDSC_23650). Flies were monitored every two days, and tumors were dissected and re-injected when the host abdomen was fully GFP. For the first generation of transplantation (G1), the midguts of esgF/O > GFP-positive CyO siblings were injected as controls. For subsequent transplantations (G2 to G5), controls were injected with PBS.

Cell sorting and RNA sequencing

Female flies with the desired genotype (esg-GAL4^ts,UAS-GFP/+ to express GFP in control progenitors; esg^F/O/Psc-Su(z)2^RNAi#44 to induce constitutive GFP expression in TIICs) were collected within 4 days after hatching and allowed to age for 3–7 days at 18°C before temperature shift to 29°C to induce transgene expression for 14 days. 100–120 guts per condition were dissected and placed in a cold vial containing 0.4mL of 1X-PBS. The PBS was discarded and the guts were placed in 10µL of 10mg/mL elastase and 90µL of Tris-HCl pH = 8.8. Vials were incubated at 27°C, 60 rpm for 30 minutes with mixing every 10 minutes. Vials were centrifuged at 300g for 20 minutes at 4°C and the pellet was washed in 1X-PBS before reiterating the centrifugation step. Excess PBS was discarded and the pellet was resuspended in 500µL PBS and filtered with PluriStrainer Mini 40 µm filters before GFP-positive cells were sorted by cytometry. Cells were then snap frozen at -80°C and sent to Azenta Life Sciences for mRNA extraction, library preparation (ultra-low input) and sequencing (Illumina HiSeq 2x150bp).

RNA-sequencing analysis

Raw data were aligned to the D. melanogaster Dm6 (dmel_r6.32) genome assembly. Differential expression analysis of the data was performed using the DESeq2 R package v.1.40.2 (design= ~ condition). Genes were considered differentially expressed if Padj  <  0.05 and |log2fold2 fold change |  > 1. Principal component analysis (PCA) was performed for quality assessment (S3A Fig). The Volcano plot was generated using the EnhancedVolcano R library v.1.18.0. Gene Ontology analysis was performed on differentially expressed genes using the enrichGO function from the clusterProfiler R package v. 4.8.3, and plots were produced using the ggplot2 R library v. 3.5.1.

Pc-profiling CUT&Tag

We re-analyzed the Pc-profiling CUT&Tag dataset recently published [45] as follows. FASTQ data were downloaded from SRA run selector (GSE291173). Reads were aligned to the D. melanogaster reference genome dm6 using Bowtie 2 (v 2.4.2). Then, samtools (v1.9) was used to filter out low-quality reads (command ‘samtools view -b -q 30’) and sambamba (v 1.0.0) was used to sort (command ‘sambamba sort’), deduplicate and index BAM files (‘sambamba markdup –remove-duplicates’) with default parameters. For visualization, reads per kilobase per million mapped reads (RPKM)-normalized igwig binary files were generated using the bamCoverage function from deepTools2 (version 3.5.5). Peak calling was performed with each replicate as a separate input file using MACS2 with the following parameters: -g dm -f BAMPE -q 0.005. Only peaks from the merged replicates, detected in the four replicates and using merged replicates, were retained for further analysis. Gene Ontology analysis was performed on differentially expressed genes using the enrichGO function from the clusterProfiler R package v. 4.8.3, and plots were produced using the ggplot2 R library v. 3.5.1.

Imaging, quantification and statistics

Measurements

Fluorescence quantification of Armadillo staining was performed using FIJI/ImageJ. A total of 30 regions of interest (ROIs) per condition were analyzed, derived from n = 10 guts across 2 independent experiments (1–9 ROIs per gut). Two conditions were compared: (i) MARCM mutant clones and (ii) MARCM wild-type clones, which were selected manually based on clone morphology and GFP expression. For each ROI, mean fluorescence intensity was measured using FIJI/ImageJ. Mitotic indexes were determined by manually counting PH3-positive cells in the whole gut. The presence of differentiated cells in clones was evaluated by counting the number of clones containing at least one Prospero-positive cell (from the enteroendocrine lineage) and/or one large nucleus specific for polyploid enterocytes (from the absorptive lineage). In the same way, the presence of stem cells in clones was evaluated by counting the number of clones containing at least one Delta-positive cell. TUNEL-positive cells were counted in FIJI and normalized to area. Tumor burden was determined in a blinded analysis on FIJI following the chart in S4 Fig.

Statistics

For all experiments, flies were first selected by genotype and then randomly chosen for experimental analysis. All experiments were independently repeated at least 2–7 times. All statistical tests were performed with R software. Box-plots are defined as follows: center line, median; box limits, upper and lower quartiles; whiskers, 1.5x interquartile range; points, outliers. The statistical tests (all two-sided) used to determine the significance, the total number of guts analyzed (“n”), and the exact p-value are indicated in the legend of each figure.