When an animal is developing as an embryo, different cells start to specialize into the specific cell types needed to form the tissues and organs of the body. How an individual cell commits to become a certain type of cell is mostly determined by which of the genes in its DNA are active. In animal cells, DNA is wrapped around proteins called histones, and one way that cells can maintain their distinct pattern of gene activity is via chemical tags on the histones. These tags can switch nearby genes on or off, and are added or removed by other proteins called epigenetic regulators. The epigenetic tags are also stably inherited when the cell divides, meaning that a cell’s identity can be maintained over many cell generations.

If epigenetic regulators fail to work properly or get disrupted, the pattern of gene activity in a cell becomes altered. As a consequence, that cell can lose its identity and will often turn into a cancer cell. In fact, mutations in epigenetic regulators are found in several human cancers. It is not yet understood how these changes in gene expression lead cells to become cancerous.

Torres et al. have now analyzed an epigenetic regulator called Polyhomeotic in developing larvae of the fruit fly, Drosophila melanogaster. The results show that when Polyhomeotic is not produced the fly larvae develop tumors. Moreover, the mutant cells without Polyhomeotic had different gene expression profiles compared to normal cells. This in turn caused the mutant cells, which had previously committed to a certain fate, to become more like the unspecialized cells found in early embryos.

Torres et al. next showed that, among the genes that were incorrectly regulated when Polyhomeotic’s activity was compromised, one gene called knirps was switched on by mistake, which led the mutant cells to become tumor cells. When the activity of knirps was reduced instead, almost no tumors grew. Additionally, Torres et al. found that the protein encoded by knirps activates a signaling pathway that keeps tumor cells unspecialized by blocking their normal progress to a more mature and specialized state – a process known as differentiation. Experimentally raising the levels of a different molecule that ultimately promotes differentiation caused the tumor cells to grow less.

These findings suggest that tumors caused when epigenetic regulation goes awry may be reversed by targeting key genes such as knirps. Further work is now needed to test whether these findings will also extend to humans. Forcing cancer cells from a highly dividing, non-specialized state into a dead-end, mature state may lead to new ways to treat cancer.

During development, epigenetic regulators are responsible for controlling and restraining cellular plasticity. This tight regulation allows cells to differentiate faithfully and heritably towards a specific fate (Roy and Hebrok, 2015; Wainwright and Scaffidi, 2017). An appropriate balance between proliferation and differentiation is fundamental and multiple regulatory layers of transcription factors and epigenetic regulators are employed to accomplish the underlying transcriptional control (Gonda and Ramsay, 2015; Piunti and Shilatifard, 2016).

Many epigenetic regulators are evolutionary conserved and among the most commonly mutated genes in human cancer (Piunti and Shilatifard, 2016). Disruption of epigenetic constraints leads to global reorganization of the epigenome and changes in transcriptional profiles, which might provide a cellular state permissive for tumorigenesis (Wainwright and Scaffidi, 2017). Indeed, disturbed transcriptional profiles and putative oncogenic transcriptional regulators have recently gained significance as better alternatives for therapeutic targets in comparison to signaling pathways. Transcription factors (TFs) are less prone to be bypassed by alternative mutational events, and their perturbation can affect several cancer hallmarks. In addition, due to the complexity of transcriptional networks, it is unlikely that one TF functioning as an oncogenic driver can be entirely replaced by another (Gonda and Ramsay, 2015). For these reasons it is fundamental to identify the core transcriptional networks defining cancer cell types, and target those regulators crucial for survival (Bonifer and Cockerill, 2015).

Epigenetic regulators involved in preserving cellular identity are composed of two classes of chromatin proteins, the Polycomb (PcG) and the Trithorax group (TrxG), whose complementary functions maintain the repressed and active gene expression state, respectively (Geisler and Paro, 2015). PcG proteins are organized into two basic complexes, Polycomb repressive complex 1 and 2 (PRC1 and PRC2) (Piunti and Shilatifard, 2016). One example of classical PcG targets are homeotic genes encoding Homeobox TFs, first identified in Drosophila and responsible for correct spatial body development in flies (Shah and Sukumar, 2010; Abate-Shen, 2002). The altered expression of Hox genes in human tumors suggests important roles for both oncogenesis and tumor suppression (Shah and Sukumar, 2010), which further hints towards an role for PcG proteins in oncogenesis.

The tumor suppressive role of PcG proteins, in particular PRC1 members in Drosophila imaginal discs, has been extensively investigated (Classen et al., 2009; Martinez et al., 2009). However, the effects on the transcriptional landscape after PRC1 deregulation in tumorigenesis has only recently started to be assessed (Bunker et al., 2015; Loubière et al., 2016). Here, we show that loss of Polyhomeotic (ph), a member of the PRC1 complex, in eye-antennal imaginal discs of larvae leads to a reprogramming of cellular identity towards an embryonic state and a concomitant loss of differentiation markers. Among the reactivated genes is knirps, an orphan nuclear hormone receptor. Depletion of knirps revealed its vital role in tumor maintenance, while misexpression showed its capacity to drive tumorigenesis in otherwise wild-type tissues. Tumors initiated by ph disruption or misexpression of knirps share features such as ectopic activation of JAK/STAT signaling and a differentiation block. We conclude that the embryonic TF knirps is an oncogene in eye-antennal imaginal discs and is crucial for the tumorigenic capacity of the epigenetic tumor under study. Additionally, we found that overexpressing a pro-neural TF leads to reduction of proliferation and suppression of the tumor phenotype.

ph⁵⁰⁵ mitotic mutant clones were generated using the Flp/FRT system, allowing for specific tumor growth within eye-antennal imaginal discs (from here on referred to as ph⁵⁰⁵-tumor). Mutant clones were fluorescently labeled with GFP, by using the mosaic analysis with a repressible cell marker (MARCM) system (Wu and Luo, 2006).

Reduced expression of Ph in the mutant clones compared to tissues carrying FRT19A (neutral) mitotic clones was observed (Figure 1—figure supplement 1A–B). Only a small number of larvae carrying ph⁵⁰⁵-tumors reached adulthood (Figure 1—figure supplement 1C). The majority of tumors arising in these epithelial tissues display disrupted cell polarity (Martinez et al., 2009), which was confirmed using the cell adhesion marker Armadillo (Figure 1—figure supplement 1D–E). Additionally, we observed ectopic expression of Matrix metalloprotease 1 (MMP1) (Figure 1—figure supplement 1F–G), which is required for matrix degradation and the invasive potential of tumor cells (Christofi and Apidianakis, 2013; Uhlirova and Bohmann, 2006).

The tumorigenic capacity of PcG mutant tissues has been previously reported (Classen et al., 2009; Martinez et al., 2009). Here we show for the first time that neoplastic growth of ph⁵⁰⁵ mutant clones in eye-antennal imaginal discs can be rescued by wild-type ph (via UAS-ph) co-expression. The resulting tissues with restored Ph levels harbored smaller clones compared to the tissues containing ph⁵⁰⁵ clones (Figure 1—figure supplement 1H–I). To better quantify changes of tumor volume in these tissues we developed an image analysis pipeline (see Materials and methods). This enabled us to concomitantly measure the volume of mutant clones (GFP-labeled) as well as the volume of the tissue. We found that in tissues with ph⁵⁰⁵ clones 46% of the disc was composed of tumor cells, while in the rescue experiment this ratio was reduced to only 7% (Figure 1—figure supplement 1K–N). Consistent with these observations, a reduced number of larvae carrying ph⁵⁰⁵-tumors reached adulthood (eclosion rate <20%). In contrast the eclosion rate of ph⁵⁰⁵, UAS-ph pupae (97%) was similar to control flies (99%) (Figure 1—figure supplement 1J).

Overall, these results show that the tumorigenic phenotype of ph⁵⁰⁵ tissues is primarily due to the loss of ph since it can be rescued by the expression of the wild-type protein. We had shown previously that tumors with impaired ph do not accumulate genetic instabilities, even when cultivated for prolonged time in adult hosts (Sievers et al., 2014). Hence, the observed neoplastic behavior can be solely restricted to the loss of the silencer ph and the ensuing deregulation of the transcriptional state described below.

ph⁵⁰⁵-tumor cells exhibit global gene expression deregulation

To better understand the molecular consequences of polyhomeotic loss-of-function in vivo, we analyzed the transcriptome of ph⁵⁰⁵-tumor cells by RNA sequencing (RNA-seq). We used fluorescence activated cell sorting (FACS) of dissociated eye-antennal imaginal discs to separate GFP-labeled tumor cells from surrounding unlabeled and non-mutant cells (Martinez et al., 2009; Dutta et al., 2013; Harzer et al., 2013). This proved to be an essential step for an accurate diversification of the tumor transcriptome. Transcriptome analysis showed substantial deregulation of gene expression in mutant cells compared to neighboring non-mutant cells. We identified 1337 differentially expressed genes (Benjamini adjusted p-value, padj. <0.01), with 275 genes being upregulated in ph⁵⁰⁵-tumor cells (Figure 1A, Figure 1—figure supplement 2A, Figure 1—source data 1). Furthermore, gene set enrichment analysis revealed that neurogenesis-related genes were mainly downregulated in ph⁵⁰⁵-tumor cells, while genes regulating transcription were upregulated (Figure 1B). Since PcG target genes encode crucial developmental regulators, such as TFs (Simon and Kingston, 2009), our expression data corroborates its impaired function. Moreover, we observed deregulation of genes involved in tissue development (e.g., GO-terms for genital disc development, imaginal disc development) and enrichment for TFs among the upregulated genes (GO-term sequence-specific DNA binding transcription factor activity) (Figure 1B, Figure 1—source data 2). The observed global modulation of transcription output is in agreement with PcG proteins constituting a global regulatory system (Simon and Kingston, 2009).

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Figure 1—source data 1

List of differentially expressed genes.

https://doi.org/10.7554/eLife.32697.008

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Figure 1—source data 2

Gene Ontology (GO) analysis.

https://doi.org/10.7554/eLife.32697.009

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Figure 1—source data 3

Information relative to samples used for clustering (83 samples).

https://doi.org/10.7554/eLife.32697.010

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Figure 1—source data 4

List of genes used for hierarchical clustering analysis.

https://doi.org/10.7554/eLife.32697.011

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Figure 1—source data 5

List of Genotypes.

https://doi.org/10.7554/eLife.32697.012

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TFs as key regulators of tumorigenesis – candidate-hit validation in vivo

In order to pinpoint key regulators of tumorigenesis in ph⁵⁰⁵-tumors, we performed an in vivo screen for a subset of selected candidates. Among all the TFs upregulated in ph⁵⁰⁵-tumor cells, we chose, based on literature search, 24 to assess their importance in promoting tumorigenic potential in these cells. Our approach to test the ability of candidate genes playing a key role in ph⁵⁰⁵-tumorigenesis was to combine generation of ph⁵⁰⁵-tumor clones with RNAi-mediated knock-down (KD) of a target of interest within these clones. We compared effects of RNAi to the baseline ph⁵⁰⁵ neoplastic phenotype and observed that some RNAi lines targeting TFs (in ph⁵⁰⁵ clones) resulted in a strong increase in viability (close to 90–100%, for example cad, drm, kni, bgcn), while others did not change or only slightly changed pupal viability (Figure 2A). We further characterized 6 RNAi lines: crocodile (croc), lateral muscles scarcer (lms), caudal (cad), drumstick (drm), knirps (kni) and benign gonial cell neoplasm (bgcn) (Figure 2B–H), which showed significant differences in eclosion rate in comparison to flies carrying ph⁵⁰⁵ clones (Figure 2B and Figure 2—figure supplement 1A). The eclosion rate for three of these perturbations (drm-, kni- and bgcn-KD) reached similar levels as control flies (carrying FRT19A neutral clones) and rescue experiment (ph⁵⁰⁵, UAS-ph). By quantifying tumor volumes relative to tissue size of the six above-mentioned perturbations, we observed that only cad-, drm-, kni- and bgcn-KD showed a significant difference compared to the baseline of 46% tumor volume in the ph⁵⁰⁵ condition (14, 13, 5 and 14% tumor volume, respectively). In addition, the two perturbations (croc- and lms-KD) that did not show a significant effect on tumor volume were also those with less remarkable differences in eclosion rate (Figure 2B and Figure 2—figure supplement 1B–D). These results suggest that higher eclosion rate is a good approximation for decreased tumor volume. Furthermore, the tissue volume of the eye-antennal imaginal disc in the drm-, kni- and bgcn-KD conditions was closer to the control tissue volume than the ph⁵⁰⁵ condition (Figure 2—figure supplement 1C).

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From all the RNAi conditions tested, kni-KD in ph⁵⁰⁵ mutant clones showed the most striking decrease in tumor volume (9.2 fold decrease), similar to the rescue experiment (ph⁵⁰⁵, UAS-ph) (Figure 2B). Additionally, the phenotype of adult eyes of this genotype suggests a recovery of the differentiation program (Figure 2—figure supplement 2A–D). This is supported by immunostaining against ELAV showing that it is no longer disrupted when expression of kni is blocked in ph⁵⁰⁵ (Figure 2—figure supplement 2E). Altogether, this shows that the differentiation block observed in ph⁵⁰⁵-tumors is prevented upon reducing the level of knirps expression by RNAi KD. We confirmed that a second, independent RNAi line against kni also led to a significant decrease of tumor volume (14% of tumor volume vs. 46% in ph⁵⁰⁵) and an increase in eclosion rate (85%) (Figure 2—figure supplement 2F–J). We can thus minimize the chance that the effects observed using either kni-RNAi were due to off-target effects.

knirps-KD reduces ph⁵⁰⁵ tumorigenic capacity

We characterized the tumorigenic potential of ph⁵⁰⁵-tumors and ph⁵⁰⁵, kni-KD by conducting transplantation assays (Rossi and Gonzalez, 2015) of these tissues into the abdomen of adult host flies (Figure 3A–D). In the case of the ph⁵⁰⁵-tumors the percentage of tumor-bearing hosts increased from 40% to 60%, from the first week to subsequent weeks after transplantation indicating hyperproliferation of the transplanted tissues (Figure 3A). The tumorigenic potential of ph⁵⁰⁵-transplanted tissue was already detected on day seven after transplantation (Figure 3B). By contrast, when transplanting ph⁵⁰⁵, kni-KD clones we did not observe any tumors in the host flies within the first three weeks. Even after up to 5 weeks post-transplantation, we could only find a single fly with GFP⁺ tissue overgrowth (Figure 3C–D). Our data demonstrate that the TF Knirps plays a crucial role in tumorigenesis of ph⁵⁰⁵-tumors given that kni-KD in these tissues not only led to a reduction of tumor volume but also the remaining clones were not able to proliferate in the host fly abdomen.

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Evasion of apoptosis is one of the hallmarks of cancer (Hanahan and Weinberg, 2000). As we observed a significant reduction of tumor volume upon depleting kni in ph⁵⁰⁵ mutant cells, we hypothesized that kni-KD could trigger cell death of tumor cells. We blocked apoptosis within mutant clones (via expression of anti-apoptotic protein p35 [Hay et al., 1994]). Levels of apoptosis as assessed by immunostaining against Death caspase-1 (Dcp-1) confirmed a decrease in apoptosis in tissues where p35 was expressed in ph⁵⁰⁵, kni-KD clones (Figure 3E–F and Figure 3—figure supplement 1A–B). However, we observed that blocking apoptosis in ph⁵⁰⁵, kni-KD clones was not sufficient to revert the anti-oncogenic effects of kni-KD (Figure 3G–H and Figure 3—figure supplement 1C–F). Furthermore, the tumor volume of ph⁵⁰⁵, kni-KD, UAS-p35 was similar to ph⁵⁰⁵, kni-KD (Figure 3G and Figure 3—figure supplement 1D–F). We also tested the effect of this particular RNAi line in the context of neutral clones generated with the same driver as for ph⁵⁰⁵-tumors. These FRT19A, kni-KD flies showed neither difference in eclosion rate nor in the adult eye phenotype, compared to control flies (Figure 3E–F and Figure 3—figure supplement 1C). This suggests that the RNAi line targeting kni does not per se affect eye-antennal imaginal disc development.

Ectopic expression of knirps is sufficient to drive tumorigenesis

Ectopic expression of cell fate-specifying TFs was recently shown to lead to the formation of epithelial cysts (Bielmeier et al., 2016). Cyst formation in wing and eye imaginal discs represents a response to cell fate mis-specification, compromising tissue integrity and potentially promoting precancerous lesions. We thus assessed the effect of ectopic kni expression in eye-antennal imaginal discs by generating mitotic clones using again the eyFlp system. We observed that FRT19A clones expressing kni (Figure 4A) displayed a more pronounced round shape in comparison to the notchy-shape of FRT19A neutral clones (Figure 1—figure supplement 1A). Additionally, ectopic kni expression compromised the viability of the flies, evidenced by an eclosion rate of only 35% (Figure 4B), and the defective development of the adult eye structures (Figure 4C). Furthermore, we confirmed that ectopic expression of kni leads to the formation of cysts (Figure 4D) and thus interferes with epithelial polarity (Figure 4D and Figure 5—figure supplement 1A).

Figure 4

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To test if ectopic expression of kni alone is sufficient to drive tumorigenesis we conducted transplantations of eye-antennal imaginal disc tissues ectopically expressing kni. We observed that knirps is sufficient to generate tumors in the host flies, visible 3 weeks after transplantation (ranging from 15–50%, from week 3 to 5 after transplantation respectively) (Figure 4E–F). Our data suggest that ectopic expression of knirps interferes with the normal course of development and that knirps is a new oncogene, possibly acting in a context/tissue-dependent manner.

Knirps activates JAK/STAT pathway and blocks cellular differentiation

Since knirps-KD alone was sufficient to reduce the tumorigenic potential of ph⁵⁰⁵-tumors, we hypothesized that some features of clones ectopically expressing knirps in a wild-type context could resemble ph⁵⁰⁵-tumor clones. We therefore evaluated the activation of signaling pathways in this context. We observed that of the JNK, JAK/STAT and Notch signaling pathways (all are activated in ph⁵⁰⁵-tumors [Classen et al., 2009; Martinez et al., 2009; Beira et al., 2018]), only JAK/STAT was ectopically activated, particularly in knirps cyst-like clones (Figure 5A–C). This observation suggests that ectopic expression of knirps alone is sufficient to activate the JAK/STAT pathway in mitotic clones. Hence, ectopic activation of the other signaling pathways in ph⁵⁰⁵-tumors is likely attributable to other factors regulated by Ph and independent of knirps.

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In light of the compromised eye development seen in kni-ectopic flies, and suggestions that the JAK/STAT pathway needs to be switched off to allow differentiation (Amoyel and Bach, 2012), we investigated the expression of a number of neurogenesis-related markers in kni-ectopic eye-antennal imaginal discs. Similarly to what we observed with ph⁵⁰⁵ clones, ELAV expression was disrupted in kni-expressing cyst-like structures, as shown in Figure 5D, as well as Eya (Figure 5E), without ectopic activation of Hth (Figure 5F). These observations are thus in agreement with the hypothesis that knirps alone is sufficient to initiate tumorigenesis.

Our data argue in favor of a role for JAK/STAT in contributing to the differentiation block in ph⁵⁰⁵ and kni-ectopic tumors. We decided to block this pathway in ph⁵⁰⁵-tumors (ph⁵⁰⁵, dome^∆CYT) and examine cellular differentiation in these eye-antennal imaginal discs. Upon blocking JAK/STAT in ph⁵⁰⁵-tumors, we observed that ELAV expression is re-established almost to a normal situation, even in the presence of clones (Figure 5—figure supplement 1B in comparison to Figure 1—figure supplement 3B). Moreover, the viability of these flies is increased, close to normal levels (Figure 5—figure supplement 1C, eclosion rate 85%) and some adult flies presented eye structures similar to wt individuals Figure 5—figure supplement 1D).

Overexpression of a pro-neural TF in ph⁵⁰⁵-clones suppresses the tumorigenic phenotype

Blockage of normal differentiation appears to be a common feature between ph⁵⁰⁵ and kni-ectopic tumors in eye tissues, suggesting that kni expression in the ph⁵⁰⁵-tumors contributes to the differentiation defects observed (Figure 1—figure supplement 3 and Figure 2—figure supplement 2C). Hence, we expected that apart from the knock-down of an embryonic TF with tumorigenic capacity, forcing differentiation of tumor cells could restrain the tumorigenic phenotype.

Atonal (ato), encoding a pro-neural TF, was previously shown to have an anti-oncogenic role in the fly retina, where it instructs tissue differentiation (Bossuyt et al., 2009). Notably, ato is also among our downregulated set of genes (padj. <0.01). We ectopically expressed ato in ph⁵⁰⁵ clones, which led to the rescue of the phenotype by a reduction of the tumor volume from 46% baseline to 3% and an increase in the eclosion rate from 12% to 84% (Figure 6A–C and Figure 6—figure supplement 1A–E). Hence, expression of ato in ph⁵⁰⁵ clones was sufficient to restore the normal pattern of differentiation of this tissue, as confirmed by the expression of ELAV (Figure 6D and Figure 6—figure supplement 1G). Indeed, also the eye phenotype of the hatched flies resembled the phenotype of wild-type flies (Figure 6—figure supplement 1F). We then asked whether these effects can be attributed to the capacity of atonal in preventing proliferation, as previously shown in a different tumor model (Bossuyt et al., 2009). To test this hypothesis, we assessed levels of phospho-histone H3 (pH3) as a measure of proliferation (Figure 6E–G and Figure 6—figure supplement 2). Quantitative analysis showed an overall increase in proliferation levels in ph⁵⁰⁵-tumor tissues in comparison to control tissues. This was largely due to an increase of proliferative cells outside of ph⁵⁰⁵ clones (Figure 6—figure supplement 2C). The analysis also showed a decrease in pH3+ cells inside clones co-expressing ph⁵⁰⁵ and atonal (in comparison to ph⁵⁰⁵ clones) (Figure 6G). Thus, atonal antagonizes ph tumor growth by counterbalancing proliferation, ultimately leading to a reduction of tumor burden and to a normal eye differentiation pattern.

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Author details

1. Joana Torres

   Department of Biosystems Science and Engineering, ETH Zürich, Basel, Switzerland

   Contribution

   Conceptualization, Investigation, Methodology, Writing—original draft

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-5651-4575

2. Remo Monti

   Department of Biosystems Science and Engineering, ETH Zürich, Basel, Switzerland

   Contribution

   Formal analysis, Writing—review and editing

   Competing interests

   No competing interests declared

3. Ariane L Moore

   1. Department of Biosystems Science and Engineering, ETH Zürich, Basel, Switzerland
   2. Swiss Institute of Bioinformatics, Basel, Switzerland

   Contribution

   Formal analysis

   Competing interests

   No competing interests declared

4. Makiko Seimiya

   Department of Biosystems Science and Engineering, ETH Zürich, Basel, Switzerland

   Contribution

   Investigation

   Competing interests

   No competing interests declared

5. Yanrui Jiang

   Department of Biosystems Science and Engineering, ETH Zürich, Basel, Switzerland

   Contribution

   Investigation

   Competing interests

   No competing interests declared

6. Niko Beerenwinkel

   1. Department of Biosystems Science and Engineering, ETH Zürich, Basel, Switzerland
   2. Swiss Institute of Bioinformatics, Basel, Switzerland

   Contribution

   Resources, Funding acquisition

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-0573-6119

7. Christian Beisel

   Department of Biosystems Science and Engineering, ETH Zürich, Basel, Switzerland

   Contribution

   Resources, Funding acquisition

   Competing interests

   No competing interests declared

8. Jorge V Beira

   Department of Biosystems Science and Engineering, ETH Zürich, Basel, Switzerland

   Contribution

   Investigation, Methodology, Supervision, Writing—review and editing

   Contributed equally with

   Renato Paro

   For correspondence

   jbeira@gmail.com

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-2884-4964

9. Renato Paro

   1. Department of Biosystems Science and Engineering, ETH Zürich, Basel, Switzerland
   2. Faculty of Science, University of Basel, Basel, Switzerland

   Contribution

   Conceptualization, Resources, Supervision, Funding acquisition, Methodology, Writing—review and editing

   Contributed equally with

   Jorge V Beira

   For correspondence

   renato.paro@bsse.ethz.ch

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-3308-2965

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