The dysplasia phenotypes observed upon EGFR or JAK-STAT manipulation are often portrayed as models for early stages of tumor development, and these can be distinguished from the phenotype observed in the midgut upon manipulation of Notch, another important developmental pathway. Notch is an evolutionarily conserved cell signaling pathway, essential for stem cell maintenance (Liu et al., 2010) and cell fate determination across different developing tissues and organs (Artavanis-Tsakonas et al., 1999; Siebel and Lendahl, 2017). In the Drosophila midgut, the level of Notch activity between ISCs that contain the Delta ligand, and the neighboring EB containing the Notch receptor has a determinant role in stem cell fate (Ohlstein and Spradling, 2007; Perdigoto et al., 2011). Mutations that inhibit differentiation in stem cell lineages have been reported in early steps of cancer development (Huang et al., 2015), and in the fly intestine, it has been reported that suppression of Notch signaling results in tumor initiation (Patel et al., 2015). More particularly, in the Drosophila midgut, Notch loss-of-function has been shown to lead to the formation of clusters of ISC-like cells that fail to differentiate and proliferate at a very high rate. While EGFR or Jak/Stat overactivation have been associated with a dysplastic phenotype in the fly midgut, Notch downregulation has been characterized as a neoplastic growth. Therefore, decided to address whether aneuploidy could also have an impact on this model. Firstly, and to test whether we could observe the reported Notch loss-of-function epithelial phenotypes (Patel et al., 2015), we expressed a UAS-notchRNAi construct in ISCs/EBs during the first 10 days of the adult life. After 10 days of expressing UAS-notchRNAi in ISCs/EBs, we observed a very strong epithelial phenotype, characterized by a striking accumulation of GFP positive cells that formed several clusters across the midgut, and a very high number of mitotic cells per intestine (Fig. 3A,D–F). This phenotype could easily be distinguished from the ones observed upon aneuploidy induction or EGFR or JAK-STAT manipulation (Fig. 1A), as clusters of a very high number of ISCs/EBs were found in the midgut epithelium (Fig. 3A). The quantification of ISCs/EBs in this phenotype proved to be impossible due to the large number of GFP positive cells, therefore we opted to quantify the severity of the tumorigenic phenotype by the number of intestines that presented a clear tumorigenic appearance (several clusters with large numbers of ISCs/EBs), the percentage of these areas per total area of the midgut, and the number of mitotic cells per midgut. After NotchRNAi expression, we found that 80% of the intestines analyzed presented the phenotype previously described in the literature, with ISCs-like clusters across the midgut (Fig. 3A,D). On average, 20% of the total area of the midgut was occupied by these clusters (Fig. 3E). Moreover, the majority of the Notch depleted intestines analyzed had a number of mitotic cells significantly higher when compared to controls and to the other dysplastic conditions (Figs 1H and 3A,F). We then proceeded to induce Notch downregulation while simultaneously inducing aneuploidy, as describe before for the JAK-STAT and EGFR experiments. Aneuploidy induction in ISCs/EBs had a strong and synergistic impact on the severity of the phenotype caused by Notch downregulation. All intestines had a tumorigenic phenotype (Fig. 3A–D), presented a clear increase in the area occupied by these tumorigenic clusters (Fig. 3A–C,E), and in the number of mitotic cells per intestine (Fig. 3A–C,F). According to a previous study, Notch-defective ISCs require stress-induced divisions for tumor initiation (Patel et al., 2015). Based on these results, we can speculate that aneuploidy might be acting as a source of stress and increase the malignant behavior of ISCs/EBs. Importantly, we could conclude that aneuploidy also exacerbated the development of a neoplastic phenotype. In our experiments, we observed that in EGFR, JAK-STAT tumor models it revealed to have an additive effect, while it had a clear synergistic effect in the case of Notch tumors. Since the biology of these tumors is well understood to be different, being the EGFR, JAK-STAT tumor phenotypes characterized as dysplastic, while Notch tumors are characterized as neoplastic, we speculate this might explain the difference between the additive and synergistic effects of aneuploidy. Future studies should focus on what types of aneuploid genotypes (specific unbalanced chromosomes) in particular are responsible for this effect and on how different pathways crosstalk to increase ISC proliferation. One possible mechanism, might involve the JNK pathway as both aneuploidy induction and EGFR activation have been shown to lead to overactivation of this stress pathway and this overactivation was shown to be necessary for ISC proliferation (Resende et al., 2018; Biteau and Jasper, 2011).

In this work, we characterized the impact of inducing aneuploidy in ISCs, under homeostatic conditions and under contexts of misregulation of developmental pathways associated with dysplastic and tumorigenic phenotypes in the gut. We show that aneuploidy induction in ISCs potentiates the development of intestinal dysplasia and tumorigenic phenotypes driven by misregulation of pathways such as JAK-STAT, EGFR and Notch. Aneuploidy is a source of genomic variability, which has been suggested to confer phenotypic advantages allowing a better adaptation of malignant cells to changing environments (Negrini et al., 2010). Consistently, aneuploidy correlates with resistance to antineoplastic treatments (Lee et al., 2011) and metastatic behavior (Bakhoum et al., 2018). However, there is a great complexity of the phenotypes conveyed by aneuploidy, since it is highly dependent on the type of cancer cells, tissue type and on the tumor microenvironment (Hoevenaar et al., 2020; Ben-David and Amon, 2020). Our work strongly suggests that Drosophila midgut stem cells might play a key role in the unveiling of this paradox. In the context of the experiments described here, and the impact of aneuploidy under dysplastic/tumorigenic contexts, we have not addressed a putative non-autonomous contribution of aneuploid stem cell progeny on promoting tumor progression. However, we have previously shown that under homeostatic conditions, the impact of aneuploidy in ISC behavior is driven by an autonomous upregulation of JNK (Resende et al., 2018), as this damage sensing pathway was found to be overactivated in ISCs upon aneuploidy induction and preventing this upregulation specifically in ISCs was sufficient to rescue the dysplastic phenotype. This, together with the fact that ISCs are the only dividing cells in the Drosophila intestinal epithelium, represents evidence towards an autonomous effect of aneuploidy to increase the severity of the tumor models here addressed, while future experiments could be planned to address a putative contribution of secreted signals from differentiated progeny to ISCs. When comparing and contrasting our findings in Drosophila with mammalian models, one factor to consider is the fact that the Drosophila genome is distributed in four chromosomes, a reduced number compared to 23 in humans, or 20 in mice. However, this difference does not seem to have a major impact on how aneuploidy impacts cell biology as Drosophila was used as a model for seminal discoveries on the impact of aneuploidy in cell physiology (Birchler, 2013; Milán et al., 2014), and also on the link between aneuploidy and tumor development. Further studies should focus on the characterization of the specific response of stem cells to chromosomal imbalances in different tissues and model organisms, contributing to a better understanding of how aneuploidy impacts human pathologies.