Ecdysone steroid hormone remote controls intestinal stem cell fate decisions via the PPARγ-homolog Eip75B in Drosophila

Developmental studies revealed fundamental principles on how organ size and function is achieved, but less is known about organ adaptation to new physiological demands. In fruit flies, juvenile hormone (JH) induces intestinal stem cell (ISC) driven absorptive epithelial expansion balancing energy uptake with increased energy demands of pregnancy. Here, we show 20-Hydroxy-Ecdysone (20HE)-signaling controlling organ homeostasis with physiological and pathological implications. Upon mating, 20HE titer in ovaries and hemolymph are increased and act on nearby midgut progenitors inducing Ecdysone-induced-protein-75B (Eip75B). Strikingly, the PPARγ-homologue Eip75B drives ISC daughter cells towards absorptive enterocyte lineage ensuring epithelial growth. To our knowledge, this is the first time a systemic hormone is shown to direct local stem cell fate decisions. Given the protective, but mechanistically unclear role of steroid hormones in female colorectal cancer patients, our findings suggest a tumor-suppressive role for steroidal signaling by promoting postmitotic fate when local signaling is deteriorated.


Introduction
Reproduction is an energetically costly process triggering multiple physiological adaptations of organs such as liver, pancreas and gastrointestinal tract upon pregnancy in various species (Hammond, 1997;Roa and Tena-Sempere, 2014). As a part of the hormonal response to mating and increased metabolic energy consumption, the female Drosophila melanogaster midgut is remodeled in size and physiology by stimulating intestinal stem cell (ISC) driven epithelial expansion to achieve an even energy balance (Cognigni et al., 2011;Klepsatel et al., 2013;Reiff et al., 2015).
Since the discovery of adult intestinal stem cells (Micchelli and Perrimon, 2006;Ohlstein and Spradling, 2006), local signaling pathways such as Notch (N), Jak/Stat, EGFR, Wnt/wingless, Insulinreceptor, Hippo/Warts and Dpp-signaling were shown to contribute to intestinal homeostasis under physiological and challenged conditions like bacterial infections (Miguel-Aliaga et al., 2018)(and references therein). The midgut epithelium is maintained by ISC giving rise to only two types of differentiated cells: enteroendocrine cells (EE) and absorptive enterocytes (EC) ( Figure 1A). Pluripotent ISC are able to self-renew or divide asymmetrically into either committed EC precursor cells called enteroblasts (EB) or enteroendocrine precursor (EEP) cells. EEP, upon timely activation of scute in ISC, divide once more prior to terminal differentiation yielding a pair of EE (Chen et al., 2018;Micchelli and Perrimon, 2006;Ohlstein and Spradling, 2006;Ohlstein and Spradling, 2007). Nine out of ten ISC mitosis give rise to EB specified by N-activation in EB daughters (Micchelli and Perrimon, 2006;Ohlstein and Spradling, 2006;Ohlstein and Spradling, 2007). Post-mitotic EB retain a certain degree of plasticity by: (1) delaying their terminal differentiation through mesenchymal-toepithelial transition (MET) (Antonello et al., 2015a), (2) changing their fate to EE upon loss of the  (3) undergoing apoptosis as an additional homeostatic mechanism (Korzelius et al., 2019;Reiff et al., 2019).
Apart from aforementioned local signaling pathways, systemic hormones are released into the hemolymph and act on distant organs (Figueroa-Clarevega and Bilder, 2015;Kwon et al., 2015;Reiff et al., 2015). Upon mating, JH released by the neuroendocrine corpora allata is able to control ISC proliferation through heterodimers of Met (methoprene-tolerant) and gce (germ cells expressed) nuclear hormone receptors (Reiff et al., 2015). JH coordinates Drosophila larval development in concert with the steroid hormone 20-hydroxy-ecdysone (20HE) and both hormones stimulate egg production in adult females (Bownes et al., 1984;Gilbert et al., 2002;Kozlova and Thummel, 2000;Truman and Riddiford, 2002). In mated adult female flies, we confirmed an increase of 20HE titers in ovary and detected a similar increase of hemolymph 20HE titers compared to virgin females (Ameku and Niwa, 2016;Gilbert and Warren, 2005;Harshman et al., 1999). The anatomical proximity of ovaries and the posterior midgut (PMG) prompted us to investigate a role for the Ecdysonereceptor (EcR) signaling cascade in organ plasticity during reproduction. Downstream of EcR activation, we detected upregulation of Ecdysone-induced protein 75B (Eip75B) protein isoforms ensuring absorptive EC production upon mating. Using the established Notch tumor paradigm, we found that 20HE through Eip75B/PPARg remote controls EB differentiation and suppresses N-loss of function driven hyperplasia. The mechanism identified in this study not only plays a role in the physiology of mating, but also contributes to our understanding of the protective effects of steroid hormone signaling in the pathophysiology of human colorectal cancer.

EcR controls intestinal stem cell proliferation and progenitor differentiation
The female fly intestine undergoes various physiological post-mating adaptations including a size increase of the absorptive epithelium (Cognigni et al., 2011;Klepsatel et al., 2013;Reiff et al., 2015). Intrigued by post-mating increases of 20HE titers (Ameku and Niwa, 2016;Harshman et al., 1999), we explored a role for EcR-signaling in mating adaptations of the adult Drosophila melanogaster intestine.
EcR encodes for three different splice variants: EcR.A, EcR.B1 and EcR.B2 (Cherbas et al., 2003;Talbot et al., 1993), which we detected by PCR in intestinal tissue with highest expression for EcR. B2 (Figure 1-figure supplement 1A). Using EcR antibodies detecting all splice variants, we found EcR in ISC (positive for the Notch ligand Delta + ), EB (Notch responsive element, NRE-GFP + ) and EC (Discs-large-1, Dlg-1 + , Figure 1-figure supplement 1B-D''' and Figure 1A for an overview). To investigate a role for the EcR in intestinal tissue homeostasis, we first manipulated EcR function in ISC and EB using the 'ReDDM' (Repressible Dual Differential Marker, Figure 1B) tracing method to ISC and EB, esg ReDDM double marks ISC and EB driving the expression of UAS-CD8::GFP (membrane CD8::GFP, green), UAS-H2B::RFP (nuclear H2B:: RFP, red) and further UAS-driven transgenes (UAS abbreviated as >hereafter in Figure panels). Newly differentiated EC and EE with inactive esg-Gal4 are RFP + -only owing to protein stability of H2B::RFP. Flies are grown at permissive 18˚C in which transgene expression is repressed by ubiquitous tubulin-driven Gal80 ts . By shifting adult females to the restrictive temperature of 29˚C, Gal80 ts is destabilized, in turn enabling ReDDM-tracing marking progeny (EE and EC with H2B::RFP nuclear stain) and in parallel manipulation by allowing transactivation of UAS-sequences through esg-driven Gal4expression (Antonello et al., 2015a). Posterior midguts (PMG) after seven days of esg ReDDM tracing of control (crossed with w 1118 ) adult MF (D) show mating dependent addition of new EC compared to control VF (C) (Reiff et al., 2015). (E) Knockdown of EcR using UAS-driven RNAi abolishes mating induced new EC generation in MF. (F+G) Overexpression of >EcR.B2 in VF (F) and >EcRFlyORF FlyORF840 (G) does not induce proliferation or differentiation of progenitors (ISC+EB). (H-I) Quantification of progenitor numbers (H) and traced progeny encompassing EC and EE (I) in R5 PMG (n = 24,17,17,17, 8). Error bars are Standard Error of the Mean (SEM) and asterisks denote significances from one-way ANOVA with Bonferroni's Multiple Comparison Test (*p<0.05, **p<0.01; ***p<0,001; ****p<0.0001). (J) Cartoon depicting experimental manipulations on EcR signaling pathway investigated with esg ReDDM . Scale bars = 100 mm. The online version of this article includes the following source data and figure supplement(s) for figure 1: Source data 1. Data from Figure 1.  observe its overall impact on tissue renewal (Antonello et al., 2015a). Briefly, ReDDM differentially marks cells having active or inactive Gal4 expression with fluorophores of different stability over a defined period of time. Combined with the enhancer trap esg-Gal4, active in progenitors (ISC and EB), esg ReDDM double marks ISC and EB driving the expression of UAS-CD8::GFP (>CD8::GFP) with short half-life and >H2B::RFP with long half-life. Upon epithelial replenishment, CD8::GFP signal is lost and new terminally differentiated EC (Dlg-1 + ) and EE (Prospero, Pros + ) stemming from ISC divisions retain a RFP + -nuclear stain due to fluorophore stability ( Figure 1A,B; Antonello et al., 2015a). Crosses are grown at 18˚C in which transgene expression is repressed by ubiquitous tubulin-driven temperature sensitive Gal80 ts . By shifting adult females to 29˚C, Gal80 ts is destabilized, in turn enabling spatiotemporal control of esg ReDDM -tracing and additional UAS-driven transgenes in progenitors ( Figure 1B).
After seven days of esg ReDDM -tracing, mated females (MF, Figure 1D) showed increases of progenitor numbers ( Figure 1H) and newly generated progeny (EE+EC, Figure 1I) in the R5 region of the PMG over virgin females (VF, Figure 1C) confirming previous observations (Reiff et al., 2015). Reducing EcR-levels with two different >EcR RNAi stocks in MF resulted in a reduction of ISC/EB numbers ( Figure 1E,H, Figure 1-figure supplement 1L) and newly generated progeny ( Figure 1E, I, Figure 1-figure supplement 1L) to levels comparable to VF controls ( Figure 1C,H,I). We confirmed knockdown efficiency of >EcR RNAi in esg ReDDM by measuring fluorescence intensity of EcR in progenitor cells in situ and found EcR-protein levels significantly decreased in both RNAi lines (Figure 1-figure supplement 1E-H).
Independent of EcR-abundance, we found that expressing dominant-negative EcR.B2 isoforms or EcR-heterozygosity using EcR M554fs , a well described loss-of-function (LOF) allele, phenocopy >EcR RNAi (Figure 1-figure supplement 1I-K). Generally, EcR LOF leads to a similar phenotype as JHreceptor knockdown in MF (Reiff et al., 2015). To investigate whether EcR-levels affect progenitor behavior, we overexpressed wildtype EcR.B2 and pan-EcR using esg ReDDM in VF. We found neither induction of progenitor numbers nor an increase in new EC ( Figure 1F-I), suggesting that EcRdependent proliferation and differentiation of progenitors might be limited by 20HE availability ( Figure 1J).

Ecdysone titers are increased upon mating and actively transported into progenitors to adapt intestinal physiology
In close anatomical proximity to the PMG, the ovaries are an established ecdysteroidogenic tissue. Determining 20HE titers 48 hr after mating using enzyme immunoassays, we confirmed previous reports of mating dependent increases of ovarian 20HE titers (Figure 2A; Ameku and Niwa, 2016;Harshman et al., 1999) and observed a similar increase in the hemolymph ( Figure 2A). As a study investigating the ecdysoneless mutants suggested that the ovary is the only source for 20HE in adult females (Garen et al., 1977), we sought to diminish 20HE titers in adult females. Therefore, we genetically ablated the ovaries using the dominant sterile ovo D1 allele in which egg production is blocked prior to vitellogenesis (Busson et al., 1983;Oliver et al., 1987;Reiff et al., 2015; Figure 2D). 20HE titers in the hemolymph of ovo D1 females are reduced around 40-50% compared to wild-type females ( Figure 2B,A). Interestingly, 20HE titers in hemolymph and remnants of the ovaries are still significantly increased upon mating of ovo D1 females ( Figure 2C). Consequently, 20HE titers in sterile esg ReDDM /ovo D1 MF increase the number of progenitors ( Figure 2E) and progeny ( Figure 2F). This suggests that remaining 20HE levels are sufficient to elicit mating related midgut adaptations.
20HE is a polar steroid, which disperses through hemolymph and binds to EcR to activate target gene transcription ( Figure 2D; Bownes et al., 1984;Gilbert et al., 2002). Using an established reporter for the activation of 20HE signaling, we found mating induced increases of EcR activity in PMG and ovaries using qPCR ( Figure 2J). As orally administered 20HE is metabolized and cleared of rapidly, we used the potent non-steroidal EcR agonist RH5849. RH5849 is used as pest control due to its stability, specificity and a 30-60 times higher efficacy compared to 20HE (Robinson et al., 1987;Wing et al., 1988). Testing concentrations from 1 to 100 mg/ml in timed egg-layings, we observed expected larval molting defects for concentrations from 50 mg/ml upwards (Figure 2-figure supplement 1A). Feeding control esg ReDDM VF 50 mg/ml with RH5849, we traced intestinal progenitors with pharmacologically activated EcR-signaling for seven days. RH5849 strongly induces ISC mitosis, reflected by a tenfold increase in newly generated progeny over mating induction  Figure 2G-I,Q). Interestingly, RH5849 mediated EcR-activation leads to no accumulation of progenitors ( Figure 2P) as observed in oncogenic manipulations or disruptions of intestinal homeostasis, suggesting a role for EcR in both, proliferation and differentiation (Antonello et al., 2015a;Chen et al., 2016;Patel et al., 2015;Reiff et al., 2019). Given the role of 20HE induced programmed cell death (PCD) in larval metamorphosis and the recently discovered role of EB PCD in adult midgut homeostasis (Jiang et al., 1997;Jiang et al., 2000;Reiff et al., 2019), we addressed PCD by activated caspase-3 staining, but found no increase of PCD by RH5849 (data not shown). Cellular 20HE uptake was recently shown to depend on Ecdysone Importer (EcI) belonging to the evolutionary conserved SLCO superfamily of solute carrier transporters. EcI LOF causes phenotypes indistinguishable from 20HE and EcR deficiencies in vivo (Okamoto et al., 2018). To investigate a role of EcI, we confirmed membrane localization of >EcI tagged with HA by immunostaining in progenitors using esg ReDDM (Figure 2-figure supplement 1B). After seven days, forced expression of >EcI using esg ReDDM in VF lead to no increase in progenitor numbers and new EC, underlining that VF 20HE levels are low ( Figure 2K,P,Q). Further supporting this hypothesis,>EcI in MF lead to an increase of newly generated EC exceeding typical mating induction of MF controls by 4.8 fold ( Figure 2L,P,Q). Blocking 20HE uptake by EcI-RNAi in MF abolished ecdysone induced tissue expansion to VF control levels ( Figure 2N,P,Q). In addition, we tested a function of the EcI gene in mating induction, but found no change in EcI expression upon mating ( Figure 2O). Both, pharmacological and genetic experiments, suggest that 20HE titer and import control EcR-activity upon mating ( Figure 2D).

Ecdysone-induced protein 75b protein isoforms Eip75B-A and Eip75B-C control enteroblast differentiation
During Drosophila development, 20HE pulses lead to direct binding of EcR to regulatory regions of early ecdysone response genes Ecdysone-induced protein 74A (Eip74EF) and Ecdysone-induced protein 75B (Eip75B, Figure 3A; Bernardo et al., 2014;Karim and Thummel, 1991;Segraves and Hogness, 1990). First, we performed conventional PCR to analyze JH-and Ecdysone target gene expression. Signal for Kr-h1-A (Kr-h1 in the following), but not Kr-h1-B, and for all isoforms of Eip74EF and Eip75B was found ( Figure 3B). Using quantitative real time PCR (qPCR) analysis, we confirmed an increase of JH-pathway activity upon mating using Kr-h1 as control ( Figure 3C; Reiff et al., 2015). To our surprise, we found Eip74EF expression unchanged, in contrast to its prominent role in the control of germline stem cell (GSC) proliferation in oogenesis (Ables and Drummond-Barbosa, 2010). Instead, we found induction of Eip75B-A and -C isoforms ( Figure 3C). Eip75B encodes for three protein isoforms (Eip75B-A, -B and -C) that differ in their N-terminal domain structure (Segraves and Hogness, 1990) and Eip75B-B lacks one of the two zinc-finger DNA binding domains rendering it incapable of direct DNA binding (White et al., 1997).
Intrigued by mating increases of Eip75B-A /-C levels, we manipulated Eip75B function using esg ReDDM . Reducing Eip75B levels with RNAi, we found a significant increase in ISC and EB numbers ( Figure 3D,E,L), but not in EC generation compared to VF controls ( Figure 3M). In MF, Eip75B knockdown reduced newly generated EC compared to MF controls, pointing to a role of Eip75B in EB differentiation downstream of EcR-activation ( Figure 3E,M). Interestingly, feeding flies with RH5849 lacking Eip75B in their intestinal progenitors ( Figure 3F) did not result in newly generated progeny ( Figure 3M compare to Figure 2Q), suggesting a central role for Eip75B in the RH5849 response. Additionally, we investigated homozygous Eip75B-A mutants using MARCM clonal analysis with a known LOF allele (Rabinovich et al., 2016). MARCM clones of Eip75B-A (Eip75B A81 ) are significantly larger compared to controls (Figure 3-figure supplement 1A-D; Lee and Luo, 1999). Eip75B A81 deficient clones contain few differentiated EC (GFP + /Dlg-1 + , Figure 3-figure supplement 1C) suggesting disturbed EB to EC differentiation as observed for esg ReDDM >Eip75 B-RNAi ( Figure 3E). Together, these LOF experiments hint to a role for mating induced Eip75B-A /-C isoforms in EC differentiation of EB downstream of EcR.
Forced expression of Eip75B variants with esg ReDDM resulted in three prominent phenotypes: (1) Eip75B-A and Eip75B-C strongly drive differentiation into EC (Dlg-1 + , Figure 3G (Hudry et al., 2016) and in line with this, Eip75B manipulations result in a shorter midgut as EC make up around 90% of the whole midgut epithelium (Figure 3-figure supplement 1G). Eip75B affects intestinal homeostasis by either slowing down terminal EB to EC differentiation (Eip75B-B and Eip75B-RNAi) or depleting the ISC and EB pool (Eip75B-A /-C, Figure 3-figure supplement 1G) preventing sufficient EC production along the gut. When EC production is blocked using >N RNAi, the midgut shrinks around one third over a period of seven days (Chen et al., 2018;Guo and Ohlstein, 2015;Micchelli and Perrimon, 2006;Ohlstein and Spradling, 2006;Ohlstein and Spradling, 2007;Patel et al., 2015). Thus, all Eip75B manipulations ultimately lead to an insufficient number of new absorptive EC resulting in a shorter midgut.
Eip75B is a long term predicted PPARg-homologue (peroxisome proliferator-activated receptor gamma) in Drosophila. A close functional homology got recently confirmed in pharmacogenetic approaches demonstrating that Eip75B-mutant flies are irresponsive to PPARg activating drugs Values are normalized to VF levels (horizontal line = 1) and statistically analyzed using student´s t-test (n = 6; *p<0.05, Figure 3 continued on next page (Joardar et al., 2015;King-Jones and Thummel, 2005). This homology is of particular interest, as human PPARg plays a role in i) pregnancy related adaptations of lipid metabolism (Waite et al., 2000) and ii) as target in colorectal cancer (CRC) (Sarraf et al., 1999). Thus, we tested pharmacological activation of Eip75B/PPARg with the established agonist Pioglitazone, a drug used in Diabetes mellitus treatment (Gillies and Dunn, 2000;Jafari et al., 2007). Flies fed with food containing Pioglitazone show a strong increase in the number of progenitor cells ( Figure 4B,E) and newly generated EC ( Figure 4F) compared to controls ( Figure 4A). Strikingly, knockdown of Eip75B led to irresponsiveness to Pioglitazone ( Figure 4C,D,F) suggesting that Pioglitazone activates Eip75B in intestinal progenitors. Eip75B-C and EcI-RNAi (K) after seven days of tracing with esg ReDDM . Inset in (F) depicts epithelial integration of newly generated Dlg-1 + /RFP + -EC. (I-J) Quantification of progenitor numbers (I) and traced progeny encompassing EC and EE (J) in R5 PMG (n = 12,13,10,12,11,11,14,8,10,10,10,5,11). Error bars are Standard Error of the Mean (SEM) and asterisks denote significances from one-way ANOVA with Bonferroni's Multiple Comparison Test (*p<0.05, **p<0.01; ***p<0,001; ****p<0.0001, identical p-values are marked by # when compared to MF). Scale bars = 100 mm. The online version of this article includes the following source data and figure supplement(s) for figure 3: Source data 1. Data from Figure 3.    Taken together, these findings strongly suggested a role for Eip75B in EB differentiation. EB are specified by N-signaling and their lineage is maintained by the transcription factor klumpfuss (klu). klu + -EB retain some plasticity to change fate to EE upon reduction of klu-levels (Korzelius et al., 2019;Reiff et al., 2019). We used klu ReDDM to explore the function of EcR and Eip75B in EB lineage identity ( Having identified Eip75B-A /-C as mating induced differentiation effector of 20HE signaling, we hypothesized and investigated a synergism of 20HE and JH hormonal signaling pathways controlling epithelial expansion upon mating.

The interplay between JH and 20HE in intestinal progenitors
Our data suggest that mating induced JH and 20HE signaling affect ISC proliferation and EB differentiation through their effectors Kr-h1 and Eip75B-A /-C ( Figure 5K; Reiff et al., 2015). In VF lacking mating induction of both hormones (Figure 2A-C; Ameku and Niwa, 2016;Harshman et al., 1999;Reiff et al., 2015), forced >Kr h1 expression doubles progenitor numbers reproducing previous results (Figure 5B,I; Reiff et al., 2015). Initially, we aimed to perform a full genetic epistasis analysis for Eip75B and Kr-h1, but this analysis was hampered as we failed to recombine Kr-h1-RNAi with Eip75B isoform expressing stocks.
However, we were able to analyze midguts of MF with forced Kr-h1-expression and simultaneous Eip75B-RNAi ( Figure 5C,D), which increases progenitor numbers ( Figure 5I) with only few newly generated EC ( Figure 5J). This finding supports the requirement of Eip75B in EB differentiation ( Figure 3E) and Kr-h1 in ISC proliferation ( Figure 5A). Simultaneous double knockdown of >Kr-h1-RNAi/Eip75B-RNAi ( Figure 5H) in MF did not show mating induction. Progenitor numbers and differentiated progeny are reduced ( Figure 5I,J) phenocopying single >Kr h1-RNAi (Reiff et al., 2015). Surprisingly, we observed a strong increase of progenitor numbers forcing Kr-h1 and Eip75B-B expression ( Figure 5F,I,J). These results indicate an additive role of forced Eip75B-B and Kr-h1expression on ISC proliferation, whereas the latter transduces the mating related proliferation response ( Figure 3C). The stimulus inducing Eip75B-B expression yet needs to be identified ( Figure 5K).
Most interestingly, Eip75B-A and -C, despite of raised Kr-h1-levels, drive progenitors into EC differentiation largely phenocopying sole Eip75B-A and -C expression (compare Figures 3G,J and 5E, G,I,J). Our data on Eip75B-A and -C corroborate a potent role for Ecdysone-induced EB differentiation. ISC fate decisions in the adult midgut rely on N-signaling (Micchelli and Perrimon, 2006;Ohlstein and Spradling, 2006;Ohlstein and Spradling, 2007). During Drosophila follicle cell development N opposes EcR-function (Sun et al., 2008), which prompted us to investigate Eip75B-A /-C as effectors of EcR-signaling in the context of N-dependent EB differentiation.
Using N-LOF tumors as an experimental paradigm lacking EC production, we sought to investigate the differentiation inducing properties of Eip75B-A /-C. Reduction of N by RNAi or a dominant-negative N-receptor using esg ReDDM lead to tumors of different sizes, which we quantified and classified according to their number ( Figure 6B). We predominantly found tumors of 5-10 cells  Figure 6B, Figure 6-figure supplement 1A), but also few tumors containing more than 100 ISC/ EE-like cells with indistinguishable single or multiple ISC origin (class IV, Figure 6B, Figure 6-figure  supplement 1A).
Co-expression of Eip75B-A /-C with N-LOF strongly reduced total tumor number ( Figure 6C,D, F) and smaller class I, II and III tumors ( Figure 6-figure supplement 1A), whereas Eip75B-RNAi results in confluent class IV tumors all along the PMG ( Figure 6E). Although tumor number is strongly reduced (Figure 6F), occasional class IV tumors (<1/PMG) are able to escape suppressive Eip75B-A /-C showing no reduction in tumor size ( Figure 6-figure supplement 1A). Additional tumorigenic mechanisms such as Upd-or EGF-ligand upregulation may counteract the tumor suppressive function of 20HE signaling in class IV tumors (Patel et al., 2015).
Since Eip75B-A and Eip75B-C re-enable EC differentiation despite the lack of N ( Figure 6C,D; Dlg-1 + /RFP + ), we investigated whether the EcR-pathway and pharmacological Eip75B-activation are able to trigger EC fate in a N-LOF context. Activation of Eip75B using Pioglitazone led to more newly generated EC ( Figure 6H,I) compared to controls ( Figure 6G). In addition, we observed a slight, but significant reduction in the number of N-tumors upon feeding Pioglitazone ( Figure 6J).
Given the protective role of steroid hormone signaling in colorectal cancer (CRC), we sought to investigate N tumor numbers upon EcR-signaling activation (Chen et al., 1998;Hendifar et al., 2009;Lin et al., 2012). We activated EcR-signaling with RH5849 ( Figure 7B) or by raising intracellular levels of 20HE expressing > EcI in esg ReDDM ( Figure 7D). Either manipulation resulted in numerous confluent tumors reflecting the previously observed role of 20HE in proliferation ( Figure 7F). In line with this,>EcI RNAi-and >EcR RNAi significantly reduced tumor burden ( Figure 7C,E,F).
More importantly, ecdysone-pathway activation led to significantly higher numbers of newly generated EC (arrowheads in Figure 7B,D,G), suggesting an upregulation of Eip75B-A and Eip75B-C in Notch tumors upon EcR-activation through RH5849 and > EcI. To exclude remaining N activity, we generated MARCM clones mutant for N (N 55e11 ) (Guo and Ohlstein, 2015). In agreement with our previous observations, activating 20HE signaling in N clones led to a tenfold increase in EC numbers over controls (Figure 7-figure supplement 1A-C) and forced expression of > E75 A/-C to instant EC formation (Figure 7-figure supplement 1D-E). Interestingly, in the presence of N, its activity is increased upon RH5849 feeding (Figure 7-figure supplement 1F), which suggests an interplay between Notch and EcR-signaling that might converge on Eip75B (this study) and klu (Korzelius et al., 2019;Reiff et al., 2019).
Taken together, our data establish 20HE as a systemic signal controlling reproductive adaptations in synergism with JH ( Figure 7H). Focusing on EcR-signaling in ISC and EB, we found the mating induced Eip75B-A /-C isoforms acting as differentiation factors in EB. Eip75B-A /-C induces EC fate even in Notch-deficient midgut progenitors, an experimental paradigm used to recapitulate early steps of tumorigenesis. Notch dependent fate decisions in midgut precursor cells are conserved between flies and humans. Thus, our findings suggest a new mechanism how steroid hormone signaling might suppress tumor growth by promoting post-mitotic cell fate in intestinal neoplasms marked by a lack of fate specification.  (n = 12,5,9,8,7,13,8,8,13). Error bars are Standard Error of the Mean (SEM) and asterisks denote significances from one-way ANOVA with Bonferroni's Multiple Comparison Test (*p<0.05, **p<0.01; ***p<0,001; ****p<0.0001, identical p-values are marked by # when compared to MF). Scale bars = 50 mm (K) Cartoon depicting transcriptional effectors of JH-and Ecdysone signaling pathways. The JH receptor is formed by a heterodimer of Methoprene tolerant (Met) and germ cells expressed (gce). Ligand bound receptor activates the transcription of krüppel homolog 1 (Kr-h1) mediating mating effects in the adult intestine (Reiff et al., 2015) Forced expression of Eip75B-B affects proliferation and differentiation through an yet unknown stimulus. The online version of this article includes the following source data for figure 5: Source data 1. Data from Figure 5. Growing offspring involves significant metabolic adaptations to raised energy demands in mothers. Drosophila melanogaster uses a classical r-selected reproductive strategy with high number of offspring and minimal parental care (Pianka, 1970). Upon mating, egg production is increased tenfold supported by metabolic and behavioral adaptations such as food intake and transit, nutrient preference and a net increase of absorptive tissue in the PMG depending on JH release (Carvalho et al., 2006;Cognigni et al., 2011;Reiff et al., 2015;Ribeiro and Dickson, 2010).
Here, we describe a role for the steroid-hormone ecdysone controlling intestinal tissue remodeling upon mating through EcR-signaling. The unliganded heterodimer of EcR and ultraspiracle (USP) binds its DNA consensus sequences serving as a repressor. Upon ligand binding, the EcR/USP heterodimer turns into an activator inducing the expression of early response genes Eip74EF, Eip75B and broad (br) (Schwedes et al., 2011;Uyehara and McKay, 2019). Initially, we investigated knockdown of USP, br and Eip74EF in intestinal progenitors and observed effects on progenitor survival independent of mating status ( Figure 3C, Zipper and Reiff, unpublished data).
Our data support the idea that 20HE and JH synergistically concert intestinal adaptations balancing nutrient uptake to the increased energy demands after mating (Figures 1-5). We confirmed that mating induces ovarian ecdysteroid biosynthesis stimulating egg production by ovarian GSC (Figure 2A In accordance with an inter-organ signaling role for 20HE, we also detected increased 20HE titers in the hemolymph (Figure 2A). Surprisingly, we found this mating dependent increase of 20HE titers still present upon partial genetic ablation of the ovaries using ovo D1 ( Figure 2B; Reiff et al., 2015). This can be explained by either (i) other source(s) for 20HE in adult females like the brain (Chen et al., 2014;Itoh et al., 2011) or (ii) 20HE release from remaining germarial cells in stage 1-4 eggs of ovo D1 VF and MF. Indeed, germarial cells have been shown to express the ecdysteroidogenic Halloween genes (Ameku and Niwa, 2016;Ameku et al., 2017). In addition, ovo D1 MF lack ovarian 20HE uptake during vitellogenesis ( Figure 2D) of later egg stages, which potentially contributes to 20HE titer in the hemolymph of ovo D1 females and intestinal epithelium expansion ( Figure 2D,E,J,K; Enya et al., 2014).
The epithelial expansion upon mating of ovo D1 females has been observed before (Reiff et al., 2015) and might be comparably high due to the different genetic background of ovo D1 flies compared to w 1118 ( Figure 2Q). While this work was in review, genetic ablation of ovarian ecdysteroidogenic enzymes was performed in female flies and resulted in a reduction of mitotic ISC. However, a direct assessment of 20HE titers was not performed and a mating dependent context was not analyzed (Ahmed et al., 2020). Taken together, current data cannot clearly dissect whether the ovary is the exclusive source of 20HE (Garen et al., 1977). In future experiments, 20HE titers have to be directly addressed to investigate whether other sources like the brain might be involved in 20HE release upon mating and other physiological stimuli (Chen et al., 2014;Itoh et al., 2011).
In the adjacent posterior midgut, we describe the impact of 20HE on Eip75B-A /-C expression ( Figure 7H) and show that 20HE import triggers EcR-activation: i) in VF with low ovarian 20HE levels, overexpression of wild-type EcR and EcI are unable to elicit neither ISC proliferation nor differentiation effects (Figures 1-2). ii) pharmacological activation through RH5849 and Pioglitazone (Figures 2 Figure 6 continued Eip75B/PPARy agonist Pioglitazone to esg ReDDM > N DN flies leads to the generation of newly generated EC ((H,I), Dlg-1 + /RFP + ) after seven days of tracing compared to DMSO controls (G,I). (I-J) Quantification of ISC progeny encompassing ISC-like and EE total N-tumor number in R5 PMG (n = 5,5). Error bars are Standard Error of the Mean (SEM) and asterisks denote significances from one-way ANOVA with Bonferroni's Multiple Comparison Test (*p<0.05, **p<0.01; *** p$$BOX_TXT_END$$. <0,001; ****p<0.0001). Scale bars = 25 mm. The online version of this article includes the following source data and figure supplement(s) for figure 6: Source data 1. Data from Figure 6.   and 4) as well as (iii) forced EcI expression induce proliferation and EB differentiation in MF mediated by Eip75B-A /-C (Figures 2, 3, 6 and 7). Together with the previous findings on JH upon mating (Reiff et al., 2015), our current data suggest a concerted role for JH on proliferation and 20HE on differentiation of midgut progenitors.

The interplay between Eip75B and Kr-h1 controls intestinal size adaptation
Downstream of the EcR, we found differential regulation of Eip75B isoforms upon mating. Eip75B activates egg production in ovarian GSC (Ables and Drummond-Barbosa, 2010;Kö nig et al., 2011;Morris and Spradling, 2012;Uyehara and McKay, 2019) and its knockdown reduces germarial size and number. In accordance with differentiation defects found in EB in our work, the number of cystoblasts, the immediate daughters of GSC, is reduced as well (Ables and Drummond-Barbosa, 2010;Morris and Spradling, 2012).
In our study, we dissected specific roles for Eip75B isoforms. Eip75B-B expression is at the lower detection limit ( Figure 3B) and unchanged upon mating ( Figure 3C). Thus, our finding that forced Eip75B-B expression raised ISC mitosis in VF (Figure 3-figure supplement 1F) might be due to ectopic expression. Eip75B-B mutants are viable and fertile and Eip75B-B is interacting with DNA only upon forming heterodimers with Hormone receptor 3 (Hr3) temporarily repressing gene expression (Bialecki et al., 2002;Sullivan and Thummel, 2003;White et al., 1997). Further studies of Eip75B-B, especially its expression pattern and transcriptional regulation, are necessary to elucidate in which physiological context, else than mating, Eip75B-B controls ISC proliferation.
Mating induction of Eip75B-A /-C, but not Eip75B-B, underlines their differential transcriptional regulation (Segraves and Hogness, 1990). This idea is supported by the finding that larval epidermis from Manduca sexta exposed to heightened JH-levels is sensitized to 20HE, which results in a 10-fold increase of Eip75B-A, but not of Eip75B-B expression (Dubrovskaya et al., 2004;Zhou et al., 1998). An in silico analysis of 4 kb regulatory regions upstream of Kr-h1 using JASPAR revealed several EcR/USP and Met consensus sequences (Khan et al., 2018), suggesting that both hormonal inputs may also converge on Kr-h1 driving ISC proliferation ( Figure 5B; Reiff et al., 2015). We propose a synergistic interplay between JH-and 20HE-signaling upon mating, in which the JH pathway effector Kr-h1 increases proliferation in ISC and the EcR effectors Eip75B-A /-C drive EB into differentiation. The combined action of Kr-h1 and Eip75B-A /-C ensures mating induced organ size growth (Figures 5K and 7H). The experimental investigation of Eip75B and Kr-h1 regulatory regions incorporating endocrine signals from at least two different organs and leading to differential activity and function in ISC and EB will be a fascinating topic for future studies.  (n = 13,5,7,4,6,9). Error bars are Standard Error of the Mean (SEM) and asterisks denote significances from one-way ANOVA with Bonferroni's Multiple Comparison Test (*p<0.05, **p<0.01; ***p<0,001; ****p<0.0001). (G) Quantification of newly generated EC (Dlg-1 + /RFP + ) in R5 PMG (n = 13,5,7,4,6,9). Error bars are Standard Error of the Mean (SEM) and asterisks denote significances from one-way ANOVA with Bonferroni's Multiple Comparison Test (*p<0.05, **p<0.01; ***p<0,001; ****p<0.0001). (H) Model of 20HE and JH hormonal pathways influencing physiological and pathological turnover in the intestine. Upon mating, JH from the neuroendocrine CA (corpora allata, [Reiff et al., 2015]) as well as ovarian 20HE (a) synergize on progenitor cells in the posterior midgut of adult female flies. The source of 20HE could not be definitely determined in this current study and 20HE might as well stem from the brain (b). JH induces ISC proliferation through Kr-h1, whereas 20HE signaling transduced by Eip75B-A /-C/ PPARg, ensures that newly produced EB differentiate into EC. New EC lead to a net increase in absorptive epithelium and thus ensures physiological adaptation of intestinal size to the new metabolic energy requirements of pregnancy. In the adult intestine, early steps of tumor-pathology are recapitulated when EC fate is inhibited by the lack of Notch signaling in progenitors (Patel et al., 2015). Notch mutant ISC divisions only produces ISClike progenitors and EE. We show in this study that 20HE signaling, through Eip75B-A /-C/PPARg, is capable to alleviate Notch tumor growth by driving intestinal progenitors into post-mitotic absorptive EC fate. The online version of this article includes the following source data and figure supplement(s) for figure 7: Source data 1. Data from Figure 7.  Eip75B/PPARg and progenitor fate commitment in physiology and pathology A key discovery of our study is that systemic 20HE directs local intestinal progenitor differentiation. Upon EcR activation, we found Eip75B-A /-C promoting EB to EC differentiation. When tissue demand arises, EB are thought to physically separate from ISC and become independent of N. Detached EB acquire motility and are able to postpone their terminal epithelial integration after initial specification (Antonello et al., 2015a;Antonello et al., 2015b;Martin et al., 2018;Siudeja et al., 2015). Epithelial integration of progenitors can be initiated experimentally by nubbin (nub) mediated downregulation of esg and the nub-RB isoform is sufficient to initiate EC formation downstream of Notch signaling (Korzelius et al., 2014;Tang et al., 2018).
Our data suggest, that the ecdysone pathway acting through Eip75B-A /-C provides a similar, remotely inducible signal for terminal EB differentiation. This idea is supported by several lines of evidence: i) EB-specific knockdown of Eip75B stalls their differentiation using klu ReDDM (Figure 3figure supplement 2E), suggesting Eip75B functions after initial N-input. ii) klu + -EB do not change fate upon Eip75B knockdown, suggesting a more mature EB differentiation status that already lost plasticity (Figure 3-figure supplement 2A-D; Korzelius et al., 2019;Reiff et al., 2019). iii) Forced Eip75B-A /-C expression in EB leads to their immediate differentiation independent of N-input ( Figure 6C,D, Figure 7-figure supplement 1D,E). In addition, Eip75B was found to be highly expressed in EB in a recent study from the Perrimon lab (Hung et al., 2018), raising the question about its transcriptional regulation.
A plethora of studies established Eip75B as an early response gene downstream of the Ecdysone pathway (Ables and Drummond-Barbosa, 2010;Kö nig et al., 2011;Morris and Spradling, 2012;Thummel, 1996;Uyehara and McKay, 2019). Another pathway stimulating Eip75B expression in EB might be the N pathway. N activation leads to immediate EC differentiation of progenitors and thus phenocopies Eip75B-A /-C expression (Hudry et al., 2016;Reiff et al., 2019). Indeed, 20HE-and Notch-signaling have been shown to converge on target genes in various tissues and functions (Mitchell et al., 2013;Sun et al., 2008;Xu et al., 2018) and both pathways acetylate H3K56 modifying multiple regulatory genomic regions including Eip75B (Skalska et al., 2015). We observed that 20HE-signaling leads to the differentiation of progenitors to EC in a N LOF context (Figures 5 and  6, Figure 7-figure supplement 1). This is probably the result from strong genetic and pharmacological stimuli (Figures 6 and 7, Figure 7-figure supplement 1) acting through Eip75B. 20HE signaling might facilitate the expression of differentiation factors like Eip75B as well as klu in the presence of N signaling under normal physiological conditions (Figure 7-figure supplement 1F). Future in-depth analysis of Eip75B regulatory regions and the generation of Eip75B reporter flies will help to understand the interplay of 20HE and N signaling acting on Eip75B.
Confirming a close relationship between fly Eip75B and human PPARg, we found midgut progenitors responding to the known PPARg agonist Pioglitazone. In patients, PPARg plays a role in i) pregnancy related adaptations of lipid metabolism (Waite et al., 2000) and ii) as target in colorectal cancer (CRC) (Sarraf et al., 1999). Decreased PPARg levels are associated with development of insulin resistance and failure of lipolysis in obese pregnant women (Rodriguez-Cuenca et al., 2012;Vivas et al., 2016). Mating upregulates PPARg/Eip75B (Figure 3) and we also describe increased lipid uptake upon EcR-activation (Figure 2-figure supplement 1) in absorptive EC. Srebp, a gene with known function in fatty acid metabolism, is also activated (Horton et al., 2003;Reiff et al., 2015;Seegmiller et al., 2002). Studying this interplay between SREBP, PPARg and steroid hormone signaling will shed light on to which extent adaptation of intestinal size and lipid metabolism contribute to pathophysiological conditions like diabetes.
In a CRC mouse model, biallelic loss of PPARg leads to a 4-fold increase tumor incidence and reduced survival in female mice over males (Apc Min/+ /PPARg -/-) (McAlpine et al., 2006), whereas males develop around three times more colon tumors with wild type PPARg (Cooper et al., 2005).
In line with this, ovariectomized Apc Min/+ mice develop significantly more tumors and have decreased PPARg expression, highlighting a possible link between PPARg and estrogen signaling. Meta-analyses of hormone replacement studies have shown that estrogen confers a lower risk to develop CRC and better survival rates for female CRC patients (Chen et al., 1998;Hendifar et al., 2009;Lin et al., 2012).
It is tempting to speculate that this mechanism of steroid hormones signaling through EcR/ER (estrogen receptor) to activate Eip75B/PPARg is conserved from flies to humans. Indeed, human estradiol and 20HE activate the EcR with similar affinity and elicit Eip75B transcription by binding to EcRE (Ecdysone-Responsive Elements). EcRE can be converted to functional ERE (estrogen responsive elements) by a simple change of nucleotide spacing suggesting high conservation (Martinez et al., 1991;Schwedes et al., 2011).

MARCM clones
Mosaic analysis with repressible cell marker (MARCM; Lee and Luo, 1999) clones were induced in midguts by Flippase under control of a heat-shock promoter. Expression of the Flippase was activated for 45 min in a 37˚C-water bath to induce positively marked clones. Guts were dissected 5 days after induction. Clones in experimental and control flies were induced in parallel.

Food composition and fly keeping
Fly food contained 1424 g corn meal, 900 g malt extract, 800 g sugar beet syrup, 336 g dried yeast, 190 g soy fluor, 100 g agarose, 90 ml propionic acid and 30 g NIPAGIN powder (antimycotic agent) in 20 l H 2 O. Food was cooked for about an hour to reduce bioburden, then filled into small plastic vials and cooled down to RT. Flies were kept at 25˚C except for crosses with temperature-sensitive GAL80 ts (GAL4 repressor) which were kept at 18˚C (permissive temperature) until shifted to 29˚C (restrictive temperature) to activate GAL4-mediated transgene expression. Crosses with esg ReDDM and klu ReDDM were carried out as described previously (Antonello et al., 2015a;Reiff et al., 2015;Reiff et al., 2019). Due to persisting problems with mucous formation on food surface in vials with VF, all experiments distinguishing between mated and virgin female flies were run on food with twice the amount of NIPAGIN. Mucous formation was avoided because of massive induction of tissue renewal by pathogenic stress.

Hormone analogue treatments
A vial of fly food was reheated in the microwave until it became liquid, the hormone analogues were added, thoroughly mixed and filled into a new vial. For each ml of food 5 ml of RH5849 (340 mM final concentration; 20 mg/ml stock solution, diluted in MeOH; DRE-C16813000, DrEhrenstorfer) was added. As a control, an equivalent volume of carrier solution (MeOH) was added to the food. Hormone analogue treatments were performed for the period of the 7 days ReDDM (Antonello et al., 2015a) shift.

PPARg agonist treatments
A vial of fly food was reheated in the microwave until it became liquid, the PPARg agonist was added, thoroughly mixed and filled into a new vial. For each ml of food 2.5 ml Pioglitazone (14 mM final concentration, 2 mg/ml stock solution, diluted in DMSO; Sigma-Aldrich, St. Louis, USA) were added. The equivalent amount of DMSO served as control. Flies were starved in an empty vial for at least six hours to ensure synchronized feeding when set to Pioglitazone. Food was renewed after three days and fly midguts were dissected after five days.

Immunohistochemistry
Guts were dissected in PBS and transferred into 4% PFA immediately after dissection and staining was performed on an orbital shaker. After 45 min of fixation guts were washed once with PBS for 10 min. Antibodies were diluted in 0.5% PBT + 5% normal goat serum.  ). Oil-RedO staining intensity was analyzed using Fiji. RGB channels were split and the green channel was subtracted from the red channel to eliminate background signal. A fixed threshold was set, and guts were manually outlined as a ROI. The mean intensity of the resulting signal within the ROI was measured.

Quantification of proliferation and intensity measurements
Maximum intensity projections were calculated from Z-stack images of PMG by Fiji (ImageJ 1.51 n, Wayne Rasband, National Institutes of Health, USA). Total cell number and RFP-positive cell count of ReDDM (Antonello et al., 2015a) guts were analyzed semi-automatically by a self-written macro for Fiji whereas GFP-positive cells were counted manually (macro available from the authors).
For fluorescence or OilRedO intensity measurements, intestines were scanned with fixed laser/ exposure time settings and measured in Fiji. The region of interest was selected manually, and mean intensity of the area was determined. This way, relative EcR and ß-Galactosidase protein levels were measured in antibody-stainings, relative SREBP activity was analyzed in PMG cells expressing mCD8::GFP under the control of Srebp-GAL4, and amount of triglycerides was analyzed by OilRedO stainings.

Jaspar
The open-access webtool JASPAR (Khan et al., 2018) was utilized to predict transcription factor binding sites within the 5'-UTR of EcI. It was specifically scanned for binding motifs related to Ecdysone and Juvenile Hormone signaling.

RNA isolation and cDNA synthesis
The R5 regions or ovaries of at least three female flies were dissected and transferred into a droplet of RNAlater Solution (Invitrogen by Thermo Fisher Scientific) on ice. The tissue was homogenized in 100 ml peqGOLD TriFast (VWR Life Science) and total RNA was isolated as specified by the manufacturer. The following cDNA synthesis was performed with 250 ng of total RNA and the SuperScript IV Reverse Transcriptase (Invitrogen by Thermo Fisher Scientific) using a 1:1 mixture of oligo-dT primers and random hexamers directly upon RNA isolation. Prior to Real-time qPCR, cDNA samples were diluted 1:2 in dH 2 O.

Verification of gene expression in the adult midgut by PCR
To verify gene expression in the adult Drosophila midgut, PCRs were performed with primer pairs specific for ovo and the Svb isoform, or isoform-specific primer pairs for EcR spanning at least one exon-exon boundary. PCRs were performed with Q5 High-Fidelity DNA Polymerase (NEB) for at least 30 cycles. Reaction products were loaded on an agarose gel (1.5%) and separated by electrophoresis to verify lengths of PCR products.

Real-time qPCR
Expression levels of Ecdysone signaling pathway-associated genes were determined in VF and MF. Eclosed OregonR or EcRE-LacZ flies were aged for 4d before mating. After 72 hr of mating, RNA was isolated and cDNA synthetized before running qPCRs. After an enzyme activation step (20 s 95C ), 40 cycles of denaturation (2 s 95˚C), primer annealing (20 s 58˚C) and elongation (20 s 72˚C) were run. Primers were designed to anneal at 59˚C. Reaction was set up with KAPA SYBR FAST Universal (Roche) in a total volume of 10 ml. All qPCR results were normalized to the house-keeping gene rp49.

20-HE isolation and titer determination
20-HE titers were determined in VF and MF of the same age. Eclosed OregonR or heterozygous ovo D1 mutant flies were aged for 3d before mating. After 24 hr or 48 hr at least 20 adult female flies were pierced in the thorax with a needle and put into a 150 ml-PCR tube that was punctured in its very bottom. Hemolymph was harvested by centrifugation (5.000x g 5 min RT) and collected in a 1.5 ml-reaction tube. Total weight of flies was determined before and after centrifugation. Typical yields of hemolymph were around 0.6-1 mg. The isolated hemolymph was mixed with 500 ml MeOH, centrifuged (12.000x g 20 min 4˚C) and the supernatant was transferred into a new 1.5 ml-reaction tube.
MeOH was evaporated at 30˚C in a vacuum centrifuge (Eppendorf concentrator plus) and 20HE was resuspended in 100 ml EIA buffer and stored at À20˚C until usage. Ecdysone levels were determined using the 20-Hydroxyecdysone Enzyme Immunoassay kit according to manufacturer's instructions (#A05120.96 wells; Bertin Bioreagent). 20HE titer was normalized to hemolymph yield.

Statistical analysis
Figures of quantifications were assembled, and statistics were run in GraphPad Prism 6.01. For single comparisons, data sets were analyzed by two-sided unpaired t-test. For multiple comparisons, data sets were analyzed by one-way ANOVA and Tukey's post-hoc test. Significant differences are displayed as * for p 0.05, ** for p 0.01, *** for p 0.001 and **** for p 0.0001.