Steroid hormone-induced wingless ligands tune female intestinal size in Drosophila

Steroid hormone-induced wingless ligands tune female intestinal size in Drosophila | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Steroid hormone-induced wingless ligands tune female intestinal size in Drosophila Lisa Zipper, Bernat Corominas Murtra, Tobias Reiff This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3500287/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 06 Jan, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Female reproduction comes at great expense to energy metabolism compensated by extensive organ adaptations including intestinal size. Upon mating, Drosophila ovaries release the steroid hormone Ecdysone that stimulates a 30% net increase of absorptive epithelium by intestinal stem cell (ISC) divisions. Here, we uncover the transcription factor crooked legs (crol) as intraepithelial coordinator of Ecdysone-induced ISC mitosis by establishing Rapport, the first spatiotemporally-controlled dual expression and tracing system for the analysis of paracrine effects on ISC behaviour. Rapport tracing revealed that Ecdysone-induced Crol controls mitogenic Wnt/wg-ligand release from epithelial enterocytes towards ISC, which is counterbalanced by Crol-repression of string/CDC25 and Cyclin-B directly in ISC. Rapport-based ISC tumours confirm paracrine stimulation through the Ecdysone-Crol-Wg axis on mitotic behaviour. Finally, mathematical modelling corroborates increasing enterocyte numbers and Wnt/wg-degradation to set a stable post-mating intestinal size. Together, our findings provide insights into complex endocrine growth control mechanisms during mating-induced adaptations and gastrointestinal cancer. Biological sciences/Stem cells/Intestinal stem cells Health sciences/Endocrinology/Endocrine system and metabolic diseases/Endocrine cancer Health sciences/Diseases/Cancer/Gastrointestinal cancer/Colorectal cancer Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Generation of offspring is an energetically costly process that triggers multiple physiological adaptations of organs such as liver, pancreas and gastrointestinal tract in various species 1 , 2 . Survival and fitness of progeny relies on tight control of alimentary tract adaptations to metabolic demands in small rodents, in which daily food uptake during lactation can equal the mother´s body weight 1 , 3 . It is key to understand regulatory mechanisms for hyperplasia and -trophy of the intestine, as it underlies both, physiological tissue functionality and potential malfunctioning in common diseases such as diabetes, obesity and cancer. Physiological adaptations to mating and pregnancy offer a unique opportunity to explore the nature of the underlying interorgan communication. In Drosophila melanogaster females, gut size is increased to match energy consumption when egg production is initiated 4 – 8 . Endocrine interorgan communication orchestrates this organ size re-set yielding an enlarged intestine with about a third more absorptive enterocytes (EC) 7 , 8 . This expansion is orchestrated by systemic release of juvenile hormone (JH) from the neuroendocrine corpora allata and the steroid hormone 20-Hydroxy-Ecdysone (20HE) from the ovaries. Both hormones converge on intestinal progenitors increasing intestinal stem cell (ISC) proliferation and enteroblast (EB) differentiation towards EC fate 4 , 7 – 10 . 20HE-dependent increases in ISC proliferation depend on presence of the Ecdysone-receptor (EcR) and early response genes Broad, Eip75B and Hr3 4,8 , but mating-dependent molecular control mechanisms of how active 20HE-signalling affects the cell cycle in ISC, remained unknown. Here, we report and characterize a novel molecular player crooked legs ( crol)/ZNF267 that relays endocrine 20HE into local intraepithelial ISC division control. We detected 20HE-dependent crol activation in ISC and epithelial EC. Interestingly, functional experiments revealed antagonizing mitogenic effects of crol manipulation in the ISC population on one hand and the EC population on the other hand. This observation prompted us to design and establish ‘Rapport’, a bipartite spatiotemporally controlled dual binary tracing and expression system, which enabled us to manipulate EC while tracing non-autonomous effects on labelled stem cell progeny. We designed Rapport to offer highest flexibility and compatibility with existing genetic tools and combined it with a driver for epithelial enterocytes. Using Rapport, we discover that Crol relays systemic 20HE signalling in ECs into locally acting paracrine Wnt/Wg ligands. This finding is of high interest as EC are the largest cell population in the midgut and Wnt/wg signalling pathway is central in homeostasis and malignancies of the fly and mammalian gut 11 12 . Manipulation of the 20HE-Crol-Wg axis in microenvironmental EC non-autonomously controls ISC proliferation during mating-dependent intestinal growth and in neoplastic tumours. Quite the contrary, crol expression in ISC acts antiproliferative through the CDC25-orthologue string and the mitotic cyclin CyclinB , suggestive for a Crol-dependent mitotic balance. Mathematical modelling supports our hypothesis of opposing autonomous and non-autonomous mitogenic effects of Crol on ISC and that EC numbers are indeed stabilizing mating-adapted organ size depending on 20HE levels. Interestingly, our discovered dynamic pattern is highly robust and can be derived from the fundamental properties of diffusion and degradation of Wnt/wg ligands. These opposing cell type-dependent consequences of a single hormonal stimulus on stem cell proliferation inside the same epithelium underpin complex hormonal action on epithelial growth during pregnancy-induced hyperplasia and pleiotropic effects observed in cancer of the intestine. Results Crooked legs responds to mating-dependent 20HE steroid hormone release The female fly intestine undergoes a variety of post-mating adaptations including a net increase of the absorptive epithelium 5 – 7 . Mating-dependent enteroplasticity is orchestrated by two hormones, JH and 20HE, which act directly on ISC mitosis 4 , 7 , 8 . Aiming to elaborate Ecdysone-responsive genes exerting ISC division control, we followed leads from developmental studies 13 – 15 and sequencing approaches that suggested expression of crooked legs in the adult Drosophila midgut 16 , 17 . Transgenic flies in which Crol is GFP-tagged (Crol::GFP) confirmed crol -expression in the adult female midgut. We detected GFP-signal in ISC positive for the Notch-ligand Delta (N and Dl, Fig. S1 A-A’’), in EB identified by N-activity (N-reporter GBE + Su(H)-dsRed, Fig. S1 B-B’’), in EE positive for Pros (Prospero, Fig. S1 C-C’’) and EC, positive for the septate junction marker Dlg1 (Discs-large-1, Fig. S1 C-C’’). Mating induces ovary-to-gut 20HE release 8 and also significantly increased Crol::GFP levels in adult mated females (MF) compared to virgins (VF) (Fig. 1A-A’,E). Next, we confirmed crol responsiveness to pharmacological EcR activation by feeding the EcR agonist RH5849 (Fig. 1D-D’) 8 , which significantly increased Crol::GFP fluorescence intensity compared to controls (Fig. 1B-B’,E). Similar increases in Crol::GFP fluorescence were observed upon genetic ovariectomy using the dominant ovoD1 stock (Fig. 1C-C’,E) abolishing the ovaries as a sink for 20HE 8 , 18 . Interestingly, crol responded in both, ISC/EB as well as epithelial EC populations (Fig. 1A-C’,E). To investigate the role for crol in intestinal tissue homeostasis, we separately addressed its role in ISC/EB progenitors and epithelial EC (Fig. 1D’). Crol and its functional human orthologue ZNF267 control proliferation of intestinal stem cells First, we manipulated crol autonomously in ISC and EB using the ‘ReDDM’ (Repressible Dual Differential Marker, Fig. S2 A) tracing method to observe overall impact on tissue renewal with spatiotemporal control of tracing onset and gene manipulation 19 . Briefly, ReDDM differentially marks cells having active or inactive Gal4 expression with fluorophores of different stability. 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 , ‘>’ abbreviates Gal4/UAS regulation and ‘>>’ lexA/AoP regulation in the following) with short half-life and > H2B::RFP with long half-life. Upon epithelial replenishment, newly differentiated EC and EE stemming from ISC divisions retain an RFP + -nuclear stain due to fluorophore stability 19 . 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 temporal control of esg ReDDM -tracing and additional UAS-driven transgenes in progenitors (Fig. S2 A). After seven days of tracing adult female intestines using esg ReDDM , we found that overexpression of crol has an antiproliferative effect decreasing the number of progenitor cells (Fig. 2B) about 12-fold (Fig. 2E) compared to controls (Fig. 2A). Reciprocally, RNAi-knockdown (Fig. 2C) and guideRNA (gRNA) mediated excision of crol (Fig. S2 F) using esg ReDDMCas 9 tracing significantly increased the number of progenitors (Fig. 2D, S2 G), new epithelial cells (Fig. S2 H) and stimulated ISC division (Fig. S2 I) compared to controls (Fig. S2 B). Similar results were obtained using independent overexpression and loss-of-function transgenics of crol (Fig. S2 C-I). Underlining functionality of both, the reporter and RNAi stock, crol-RNAi driven by esg > in ISC and EB reduces Crol::GFP fluorescence (Fig. S2 J-L’’). We next sought to identify human orthologues of crooked legs and by mining databases for zinc finger transcription factors with a high degree of sequence homology to Crol, we isolated the human Krüppel-like zinc finger transcription factor ZNF267. To explore the ability of human ZNF267 to substitute Crol function in intestinal progenitors, we depleted endogenous crol by RNAi and expressed human ZNF267 at the same time (Fig. 2D) and observed rescue of crol-RNAi- induced progenitor accumulations by ZNF267 (Fig. 2D-E). In line with these observations, ZNF267 regulates cell proliferation and differentiation in liver tissue 20 , 21 . Like Crol (Fig. S1 ), ZNF267 shows wide-ranged expression across human intestinal cell types (GTEx, Proteinatlas) and is induced through oestrogen signalling 22 , which prompted us to investigate how steroid hormones control ISC proliferation downstream of Crol and ZNF267. Crol controls ISC proliferation through String and Cyclin B During Drosophila development, it was shown that the tyrosine protein phosphatase string ( stg , CDC25-orthologue) and the mitotic B-type Cyclin CycB are targets of EcR activity 23 , 24 . Supporting the idea that EcR-signalling controls ISC proliferation through Stg and CycB, EcR agonists not only increase ISC mitosis 4 , 8 , but also stg and CycB transcript levels (Fig. 2F). In line with previous observations 25 , 26 , up- and downregulation of stg levels in esg ReDDM traced guts (Fig. 2G,2I) reciprocally controlled progenitor (Fig. 2K) and epithelial cell production (Fig. 2L). Confirming a function of Stg downstream of EcR-signalling, ISC proliferation and subsequent increase in progenitor number upon > crol-RNAi is abolished when > stg-RNAi is co-expressed (Fig. 2I-L,Fig. S3 A) and vice versa , co-expression of > crol and > stg (Fig. 2G-H) sufficed to rescue progenitor number (Fig. 2K) and new EC production (Fig. 2L). Comparable results were obtained when we investigated the epistasis of Crol and CycB (Fig. S3 B-G), suggesting an endocrine control of ISC cell cycle exit by Crol (Fig. 2M) as observed in development 24 . An anti-proliferative role of Crol in ISC is further supported by high Crol::GFP levels in EB (Fig. S3 H) that become postmitotic during lineage progression 27 . As genetically shown for Crol and CycB (Fig. S3 ) 24 , the EB lineage-specifying transcription factor klumpfuss (klu) binds CycB regulatory regions 27 . Pointing to a role in ISC, EB-lineage specific crol manipulation using klu ReDDM (Fig. S3 I) 28 reveals an increase in number of new EC upon forced expression of crol (Fig. S3 J-L,N), but no significant changes on EB numbers (Fig. S3 J-M), even though mitotic pH3-positive EB are occasionally observed upon crol-RNAi (Fig. S3 O) and CycB expression (Fig. S3 P). Together, our data highlights crooked legs as effector of Ecdysone-signalling that autonomously promotes ISC cell cycle exit through stg and CycB (Fig. 2M). The 20HE-Crol-Wg axis in Enterocytes controls non-autonomous Wnt/wg activity in intestinal stem cells Intriguingly, ZNF267 20,21 as well as crol 24 connect steroid hormone and Wnt/wg-signalling, which prompted us to investigate whether Crol controls Wnt/wg-expression downstream of systemic 20HE-signalling during physiological mating-induced intestinal adaptations. Using transgenic flies in which Wg, the primary Wnt-ligand in Drosophila , is GFP-tagged (Wg::GFP), we detected GFP signal in EC anterior to the mid-/hindgut boundary (MHB, Fig. 3A) 29 , a gut region known to be patterned by Wnt/Wg-signalling 30 . Secreted Wg is thought to act in a paracrine manner on ISC, which we analysed using the established frizzled3 ( fz3) sensor flies for Wnt-activity (Fig. 3K) 31 , 32 . Examining fz3-RFP intensity in posterior midguts, we confirmed an active Wnt-signalling crescendo towards the MHB 32 . More importantly, we detected fz3-RFP signal in intestinal progenitors along the midgut (Fig. 3E) and occasional Wg::GFP positive EC close to ISC with fz3-activity (Fig. S4 A) under homeostatic conditions. Similar to 20HE-induced Crol::GFP (Fig. 1A-C’’,E), the EcR agonist RH5849 and ovoD1 increased Wg::GFP fluorescence in EC (Fig. 3A-D,I-J) and Wnt/Wg-activity in ISC/EB (Fig. 3E-J), suggestive for active Wnt/Wg-signalling from EC to ISC (Fig. 3K). Mating also increased Wg::GFP and fz3-RFP levels (Fig. S4 B-F), which further supports a role of Wnt/wg-signalling during physiological midgut adaptations. Previous work in challenged guts showed autocrine Wnt/Wg-signalling between ISC and EB 33 . In contrast, depletion of wg using > wg-RNAi driven in esg ReDDM flies under homeostatic conditions 28 did not significantly alter intestinal turnover (Fig. S5 A-E) and direct assessment of Wnt/wg signalling activity using fz3-RFP sensor flies (Fig. S5 F-I). Together these findings prompted us to search for another source for Wg-ligands under homeostatic conditions. Given Crol::GFP (Fig. S1 C-C’’) and Wg::GFP (Fig. S4 A) signal in EC, we examined whether wg expression is controlled by 20HE and Crol in EC. Therefore, we combined the established EC-driver ( mex>) 34 with fz3-RFP to enable Wnt-activity assessment in ISC/EB from non-autonomous sources (Fig. 4A). In line with the idea of a 20HE-Crol-Wg axis, we found that increasing 20HE signalling pathway activity by EC-specific expression of EcI ( Ecdysone importer ) 8 , 35 , crol as well as wg significantly increased fz3 activity in adjacent ISC/EB (Fig. 4B, Fig. S5 J-L’,N-N’). Reciprocally, depletion of EcI , crol and wg in EC, non-autonomously reduced fz3 activity measured in ISC/EB (Fig. 4B, Fig. S5 O-P’,R-R’). Consequently, Wg-depletion downstream of forced crol expression reduced paracrine fz3-activity measured in ISC/EB (Fig. 4C,Fig. S5 M-M’), whereas ZNF267 expression in endogenous crol-depleted EC (Fig. 4D, Fig. S5 Q-Q’) stimulated wg expression, further supporting Crol and its human orthologue ZNF267 acting on the wg expression control 14 , 24 . Combined, these findings suggest wg as a central transcriptional target of Crol in EC that non-autonomously stimulates fz3-activity in ISC. Intrigued by these observations, we investigated whether Wnt/Wg activation in epithelial EC through the 20HE-Crol-Wg axis translates into stem cell driven intestinal homeostasis and size adaptation 4 , 7 , 8 . ‘Rapport’ tracing reveals non-autonomous control of intestinal homeostasis through 20HE-Crol-Wg For the investigation of paracrine effects on stem cell behaviour, we developed ‘Rapport’ (‘Repressible activity paracrine reporter’), a dual binary expression system that combines spatiotemporally controlled transgene expression with ReDDM tracing of stem cell progeny. To preserve advantage of the existing established Gal4/UAS drivers and toolbox, we created an entirely new and Gal4-independent lexA/Aop-based ‘ esg lexReDDM ’ ( esg > > CD8::GFP, >>H2B::RFP, tub-Gal80 ts ) tracing system. Importantly, when combined with mex-Gal4 ( mex> ) for EC specific expression 34 , the lexA-operator driven in esg lexReDDM as well as Gal4 driven by mex are repressed by temperature-sensitive Gal80 ts , which allows simultaneous temporally controlled onset of UAS-transgenes as well as esg lexReDDM tracing and Aop-transgenes (Fig. 5A) 36 . We confirmed Rapport tracing functionality by tracing outcrossed controls over three weeks (Fig. S6 A-C) and observed an expected linear increase in intestinal renewal (Fig. S6 E), while ISC/EB numbers remained constant (Fig. S6 D) 19 . Previous reports described autocrine EGFR-stimulation resulting in ISC proliferation 25 , which we confirmed by crossing Rapport to flies expressing the EGF ligand Spitz (> spi , TGF alpha homologue, Fig. S6 F) 37 . Next, we assessed whether 20HE and Crol controlled Wnt/wg activity (Fig. 4) stimulates ISC division resulting in EC production and organ size adaptation (Fig. 5A). Strikingly, forced expression of EcI, crol and wg (Fig. 5C-F) non-autonomously increased progenitor cell number (Fig. 5J) and ISC progeny (Fig. 5K) compared to controls (Fig. 5B) when midguts were traced with Rapport. Reciprocally, depletion of EcI, crol and wg reduced progenitor (Fig. 5G-J) and progeny cell numbers (Fig. 5K). These data reveal direct relay of 20HE activity in enterocytes into stem cell production mediated by Crol and paracrine Wnt/Wg-signal. The 20HE-Crol-Wg mitotic balance is conserved in intestinal tumours Wnt-signalling is a well-known driver of tumorigenesis with a key role in cancers of the intestine. ZNF267 is upregulated in colorectal cancer (CRC) and regulates cell proliferation and differentiation in epithelial cancer entities 20 , 21 . We found that ZNF267 expression levels positively and negatively correlate with members of the Wnt-signalling pathway (Fig. S7 A). CRC originates from ISC 38 , which prompted us to investigate crol and ZNF267 in two established intestinal tumour models. Investigating the autonomous role of Crol/ZNF267 in N loss-of-function (LOF, Fig. S7 B) tumours 25 , 39 – 41 showed that forced expression of crol and ZNF267 within ISC reduced tumour number (Fig. 6A-E) further underlining an anti-proliferative function in ISC. Growing evidence proves that microenvironmental Wnt/wg-ligands are an important contributor to the multifaceted process of colorectal tumorigenesis 42 – 46 . We thus extended Rapport with ISC-specific N-LOF that renders ISC incapable of EC lineage production and instead accumulate ISC- and EEP-like tumoral cells 25 , 39 – 41 (Fig. S7 C). When we investigated Crol/ZNF267 in EC, we found that > crol boosts ISC tumour cell mass by 4-fold leading to confluent tumours along the midgut (Fig. 6F-J) comparable to tumour-induction by microenvironmentally-derived mitogenic EGF ligands 37 , 47 . Even though Notch-tumours recapitulate important steps of CRC tumorigenesis, N is not frequently mutated in CRC. Therefore, we also investigated an autonomous function for Crol/ZNF267 in a CRISPR-Cas9 based model of sporadic CRC 48 targeting the most frequently mutated genes with a multiplex guideRNA array (Fig. 6K) 48 – 50 . CRC in ISC has severe and pleiotropic cellular phenotypes and results in early fly demise 48 . Underlining the antiproliferative autonomous role of crol and ZNF267, their forced expression significantly improved fly survival (Fig. S6 L) as well as midgut length as a readout for epithelial hypotrophy and deterioration (Fig. 6M) 8 . An established measure for epithelial integrity in intestinal tumour models is multilayering of epithelial cells 50 – 52 , which is significantly reduced in tumoral midguts expressing > crol and > ZNF267 (Fig. S7 D). These functional experiments in colorectal tumour paradigms additionally support the idea of a mitotic balance controlling intestinal growth. Overall, steroidal input on Crol in ISC (Fig. 2) and EC (Fig. 5) provides evidence for an endocrine intestinal size control implicated in mating hyperplasia. Indeed, mitogenic crol -depletion in ISC with esg ReDDM increases the size of the mating-responsive R5 posterior midgut region (Fig. 7A-A’) 7 , whereas forced crol expression in EC using Rapport elicits R5 growth (Fig. 7B-B’). Finally, we tested our hypothesis of hormonally controlled intestinal size in a mathematical model. Mathematical modelling of endocrine relay by Crol in the control of ISC proliferation underlines complex hormonal actions on intestinal size adaptation Our model tests whether it is mathematically plausible that these opposing trends between mitotic and antiproliferative stimuli based on the molecular mechanisms found in this study induce stable population sizes that change consistently with 20HE levels. Assuming constant 20HE levels in VF and a higher constant 20HE level in MF 8 , we tested the hypothesis whether an 20HE increment is capable to yield a stable, larger organ by temporarily boosting ISC mitosis (Fig. 7D), which then declines through the increment of EC numbers and their anti-proliferative effect on ISC mitosis. Constant hormonal input produces equal amounts of mitogenic Wnt/Wg independent of EC numbers. As a logical consequence of the increase in EC numbers 7 , 8 and constant ISC numbers (Fig. S7 E), the average EC to ISC distance increases (Fig. 7C-C’). In consequence, the amount of Wnt/Wg produced in the direct neighbourhood of an ISC decays with the increase of EC 53 . Considering a section of the intestine as a 1D ring, the exponential functional shape of the decay of morphogen gradient described in detail for Wg 54 (see supplementary material) holds. Therefore, the average amount of mitogenic Wnt/wg-ligand reaching equidistantly scattered ISC 39 , 41 will decline sharply when intestinal size increases (Fig. 7C-C’). Thus, only the close EC neighbourhood of the ISC effectively contributes to the Wnt/Wg levels playing an active role in ISC proliferation. Consequently, in the equation of evolution for the number of EC, we have a mitotic term that is proportional to the concentration of 20HE —and, consequently, inverse to the number of EC — and an anti-proliferative term that can be assumed to be constant or, in a more general setting, declining slower than the mitotic term as a function of the concentration. Overall, the equation of evolution for the number of EC reads: $$\frac{\varDelta E}{\varDelta t}=\beta C\frac{\gamma }{E}I-\alpha I$$ where \(E\) is the number of EC, \(C\) is a constant reproducing the effect of the neighbourhood, \(\gamma\) (t) the net amount of hormone, \(I\) the number of ISC cells and \(\alpha\) the anti-proliferative rate (Fig. 7E). Qualitatively, the key result is that the above equation has a stable, fixed point for the amount of EC that grows and declines depending on whether the net amount of 20HE hormone grows or declines (Fig. 7F). We provide detailed information about the construction and mathematical properties of the model in the supplementary information 55 , 56 . In a second modelling approach, we hypothetically explored how constant EC numbers as generated in N-LOF tumours intestines would affect ISC division dynamics (Fig. S7 B,F). With constant EC numbers and hormone level (Fig. 7D), ISC counts in our model increase in a square-root-like manner (Fig. S7 H) as the ISC population provides a growing sink for Wg. In vivo , block of EC generation and organ size is recapitulated in N-LOF tumours using Rapport (Fig. 6F), where upregulation of Crol/ZNF267 dramatically increases ISC numbers (Fig. 6J). Our discovered interdependencies between cell population sizes, Wnt/wg-degradation and their endocrine mitotic balance shed light on the complex endocrine involvement when tumour growth mechanisms are investigated. Together, our model is capable of capturing the emergence of a stable organ size from the antagonism of mitogenic and anti-proliferative hormonal input on ISC thresholding organ size as suggested by our functional data. Discussion Here we identify the transcription factor crooked legs as coordinator of endocrine input into intestinal organ size. The discovered molecular mechanisms underline the complexity of heterologous cellular interactions: a hormonal stimulus bifurcates on stem cells and microenvironment, where it is relayed differently into an antiproliferative and a mitogenic stimulus. This interdependent opposing crosstalk of forces balances stem cell divisions and ultimately stabilizes organ size, which is sustained by both, empirical observations and mathematical modelling. Our novel Rapport system contributes to disentangle the underlying endocrine and local signalling organ size control mechanisms, by allowing precise genetic intervention in cell types surrounding the ISC. The independent tracing of the whole stem cell population and easy fluorophore identification of different fate choices ensures robust progeny counts in fluctuating demand situations compared to previous systems 25 , 37 , 47 . Endocrine actions on ISC are complex and involve hormonal dosage and mating status 4 , 7 , 8 , sex differences 57 and feeding 58 . Hypertrophy upon pregnancy is described in the mammalian gut 1 , 3 and is well-studied in reproductive organs such as the mammary epithelium where steroid hormones induce dramatic remodelling and cancer susceptibility 59 – 61 . In gastrointestinal tumours such as CRC, epidemiological evidence about the role of steroid hormones remains controversial and ranges from favourable to detrimental 62 – 68 . Functional studies of both mammalian oestrogen receptors (ER) in rodents underline the complexity of oestrogen signalling in gut tumorgenicity 69 , 70 and reveal further complexities as pharmacological (E2/P4) and endogenous oestradiol levels differentially affect patient outcome 62 , 65 , 71 . Our functional and modelling data provides an initial logic to disentangle complex observations and involves the heterogeneity of tumour cell composition and its capacity to contribute to mitotic signals. A targeted therapeutic intervention of steroid hormone signalling is supported by: i) an overall protective tendency of ER signalling in CRC 68 . ii) Like Crol, ZNF267 is stimulated by ER 22 and is involved in Wnt signalling 20 , 21 suggesting conservation of the 20HE-Crol-Wg axis. iii) Wnt/wg signalling hyperactivation is central to CRC malignancy and Wnt-ligands remain indispensable for CRC growth albeit absence of APC 72 . iv) Effectors of steroid signalling like PPARγ/Eip75B play protective roles in fly pathophysiology 4 , 8 and human disease 73 , 74 . Our findings of antagonizing autonomous and paracrine effects of Crol and ZNF267 on tumour growth (Fig. 6G-K, Fig. S7 ) emphasize that targeted genetic investigation is of key importance to understand how mutational heterogeneity and cell type composition differentially affect the proliferative response to hormonal input. Precise intervention and tracing methods such as Rapport open the door for untangling heterogenous findings of epidemiological and functional studies. 3451 words References Hammond, K.A. Adaptation of the maternal intestine during lactation. J Mammary Gland Biol Neoplasia 2 , 243-52 (1997). Roa, J. & Tena-Sempere, M. Connecting metabolism and reproduction: roles of central energy sensors and key molecular mediators. Mol Cell Endocrinol 397 , 4-14 (2014). Speakman, J.R. The physiological costs of reproduction in small mammals. Philosophical Transactions of the Royal Society B: Biological Sciences 363 , 375-398 (2008). Ahmed, S.M.H. et al. Fitness trade-offs incurred by ovary-to-gut steroid signalling in Drosophila. Nature 584 , 415-419 (2020). Cognigni, P., Bailey, A.P. & Miguel-Aliaga, I. Enteric neurons and systemic signals couple nutritional and reproductive status with intestinal homeostasis. Cell Metab 13 , 92-104 (2011). Klepsatel, P. et al. Reproductive and post-reproductive life history of wild-caught Drosophila melanogaster under laboratory conditions. J Evol Biol 26 , 1508-20 (2013). Reiff, T. et al. Endocrine remodelling of the adult intestine sustains reproduction in Drosophila. Elife 4 , e06930 (2015). Zipper, L., Jassmann, D., Burgmer, S., Gorlich, B. & Reiff, T. Ecdysone steroid hormone remote controls intestinal stem cell fate decisions via the PPARgamma-homolog Eip75B in Drosophila. Elife 9 , e55795 (2020). Ameku, T. & Niwa, R. Mating-Induced Increase in Germline Stem Cells via the Neuroendocrine System in Female Drosophila. PLoS Genet 12 , e1006123 (2016). Harshman, L.G., Loeb, A.M. & Johnson, B.A. Ecdysteroid titers in mated and unmated Drosophila melanogaster females. J Insect Physiol 45 , 571-577 (1999). Lin, G., Xu, N. & Xi, R. Paracrine Wingless signalling controls self-renewal of Drosophila intestinal stem cells. Nature 455 , 1119-23 (2008). Radtke, F. & Clevers, H. Self-renewal and cancer of the gut: two sides of a coin. Science 307 , 1904-9 (2005). D'Avino, P.P. & Thummel, C.S. crooked legs encodes a family of zinc finger proteins required for leg morphogenesis and ecdysone-regulated gene expression during Drosophila metamorphosis. Development 125 , 1733-45 (1998). Mitchell, N., Cranna, N., Richardson, H. & Quinn, L. The Ecdysone-inducible zinc-finger transcription factor Crol regulates Wg transcription and cell cycle progression in Drosophila. Development 135 , 2707-16 (2008). Mitchell, K.J. et al. Genetic analysis of Netrin genes in Drosophila: Netrins guide CNS commissural axons and peripheral motor axons. Neuron 17 , 203-15 (1996). Dutta, D. et al. Regional Cell-Specific Transcriptome Mapping Reveals Regulatory Complexity in the Adult Drosophila Midgut. Cell Rep 12 , 346-58 (2015). Hung, R.J., Li, J.S.S., Liu, Y. & Perrimon, N. Defining cell types and lineage in the Drosophila midgut using single cell transcriptomics. Curr Opin Insect Sci 47 , 12-17 (2021). Busson, D., Gans, M., Komitopoulou, K. & Masson, M. Genetic Analysis of Three Dominant Female-Sterile Mutations Located on the X Chromosome of DROSOPHILA MELANOGASTER. Genetics 105 , 309-25 (1983). Antonello, Z.A., Reiff, T., Ballesta-Illan, E. & Dominguez, M. Robust intestinal homeostasis relies on cellular plasticity in enteroblasts mediated by miR-8-Escargot switch. EMBO J 34 , 2025-41 (2015). Schnabl, B., Valletta, D., Kirovski, G. & Hellerbrand, C. Zinc finger protein 267 is up-regulated in hepatocellular carcinoma and promotes tumor cell proliferation and migration. Exp Mol Pathol 91 , 695-701 (2011). Yang, H. et al. Knockdown of zinc finger protein 267 suppresses diffuse large B-cell lymphoma progression, metastasis, and cancer stem cell properties. Bioengineered 13 , 1686-1701 (2022). Stossi, F. et al. Transcriptional profiling of estrogen-regulated gene expression via estrogen receptor (ER) alpha or ERbeta in human osteosarcoma cells: distinct and common target genes for these receptors. Endocrinology 145 , 3473-86 (2004). Li, T.R. & White, K.P. Tissue-specific gene expression and ecdysone-regulated genomic networks in Drosophila. Dev Cell 5 , 59-72 (2003). Mitchell, N.C. et al. The Ecdysone receptor constrains wingless expression to pattern cell cycle across the Drosophila wing margin in a Cyclin B-dependent manner. BMC Dev Biol 13 , 28 (2013). Patel, P.H., Dutta, D. & Edgar, B.A. Niche appropriation by Drosophila intestinal stem cell tumours. Nat Cell Biol 17 , 1182-92 (2015). Zhang, P. et al. An SH3PX1-Dependent Endocytosis-Autophagy Network Restrains Intestinal Stem Cell Proliferation by Counteracting EGFR-ERK Signaling. Dev Cell 49 , 574-589 e5 (2019). Korzelius, J. et al. The WT1-like transcription factor Klumpfuss maintains lineage commitment of enterocyte progenitors in the Drosophila intestine. Nat Commun 10 , 4123 (2019). Reiff, T. et al. Notch and EGFR regulate apoptosis in progenitor cells to ensure gut homeostasis in Drosophila. EMBO J 38 , e101346 (2019). Port, F., Chen, H.M., Lee, T. & Bullock, S.L. Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila. Proc Natl Acad Sci U S A 111 , E2967-76 (2014). Takashima, S., Mkrtchyan, M., Younossi-Hartenstein, A., Merriam, J.R. & Hartenstein, V. The behaviour of Drosophila adult hindgut stem cells is controlled by Wnt and Hh signalling. Nature 454 , 651-5 (2008). Olson, E.R. et al. Yan, an ETS-domain transcription factor, negatively modulates the Wingless pathway in the Drosophila eye. EMBO reports 12 , 1047-1054 (2011). Tian, A. et al. Intestinal stem cell overproliferation resulting from inactivation of the APC tumor suppressor requires the transcription cofactors Earthbound and Erect wing. PLoS Genet 13 , e1006870 (2017). Cordero, J.B., Stefanatos, R.K., Scopelliti, A., Vidal, M. & Sansom, O.J. Inducible progenitor-derived Wingless regulates adult midgut regeneration in Drosophila. EMBO J 31 , 3901-17 (2012). Phillips, M.D. & Thomas, C.M. Brush border spectrin is required for early endosome recycling in Drosophila. J Cell Sci 119 , 1361-70 (2006). Okamoto, N. et al. A Membrane Transporter Is Required for Steroid Hormone Uptake in Drosophila. Dev Cell 47 , 294-305 e7 (2018). Yagi, R., Mayer, F. & Basler, K. Refined LexA transactivators and their use in combination with the Drosophila Gal4 system. Proc Natl Acad Sci U S A 107 , 16166-71 (2010). Liang, J., Balachandra, S., Ngo, S. & O'Brien, L.E. Feedback regulation of steady-state epithelial turnover and organ size. Nature 548 , 588-591 (2017). Barker, N. et al. Crypt stem cells as the cells-of-origin of intestinal cancer. Nature 457 , 608-11 (2009). Micchelli, C.A. & Perrimon, N. Evidence that stem cells reside in the adult Drosophila midgut epithelium. Nature 439 , 475-9 (2006). Ohlstein, B. & Spradling, A. Multipotent Drosophila intestinal stem cells specify daughter cell fates by differential notch signaling. Science 315 , 988-92 (2007). Ohlstein, B. & Spradling, A. The adult Drosophila posterior midgut is maintained by pluripotent stem cells. Nature 439 , 470-4 (2006). Vermeulen, L. et al. Wnt activity defines colon cancer stem cells and is regulated by the microenvironment. Nat Cell Biol 12 , 468-76 (2010). Hanahan, D. & Weinberg, Robert A. Hallmarks of Cancer: The Next Generation. Cell 144 , 646-674 (2011). Patel, S., Alam, A., Pant, R. & Chattopadhyay, S. Wnt Signaling and Its Significance Within the Tumor Microenvironment: Novel Therapeutic Insights. Front Immunol 10 , 2872 (2019). McAllister, S.S. & Weinberg, R.A. The tumour-induced systemic environment as a critical regulator of cancer progression and metastasis. Nat Cell Biol 16 , 717-27 (2014). Hanahan, D. Hallmarks of Cancer: New Dimensions. Cancer Discovery 12 , 31-46 (2022). Ngo, S., Liang, J., Su, Y.H. & O'Brien, L.E. Disruption of EGF Feedback by Intestinal Tumors and Neighboring Cells in Drosophila. Curr Biol 30 , 1537-1546 e3 (2020). Zipper, L., Batchu, S., Kaya, N.H., Antonello, Z.A. & Reiff, T. The MicroRNA miR-277 Controls Physiology and Pathology of the Adult Drosophila Midgut by Regulating the Expression of Fatty Acid beta-Oxidation-Related Genes in Intestinal Stem Cells. Metabolites 12 (2022). Hanahan, D. & Weinberg, R.A. The hallmarks of cancer. Cell 100 , 57-70 (2000). Bangi, E., Murgia, C., Teague, A.G., Sansom, O.J. & Cagan, R.L. Functional exploration of colorectal cancer genomes using Drosophila. Nat Commun 7 , 13615 (2016). Martorell, O. et al. Conserved mechanisms of tumorigenesis in the Drosophila adult midgut. PLoS One 9 , e88413 (2014). Zhai, Z. et al. Accumulation of differentiating intestinal stem cell progenies drives tumorigenesis. Nature Communications 6 , 10219 (2015). Dubois, L., Lecourtois, M., Alexandre, C., Hirst, E. & Vincent, J.P. Regulated endocytic routing modulates wingless signaling in Drosophila embryos. Cell 105 , 613-24 (2001). Dubois, L., Lecourtois, M., Alexandre, C., Hirst, E. & Vincent, J.-P. Regulated Endocytic Routing Modulates Wingless Signaling in Drosophila Embryos. Cell 105 , 613-624 (2001). Wartlick, O., Kicheva, A. & González-Gaitán, M. Morphogen gradient formation. Cold Spring Harb Perspect Biol 1 , a001255 (2009). Berg, H.C. Random Walks in Biology , (Princeton University Press, 1993). Hudry, B., Khadayate, S. & Miguel-Aliaga, I. The sexual identity of adult intestinal stem cells controls organ size and plasticity. Nature 530 , 344-8 (2016). O'Brien, L.E., Soliman, S.S., Li, X. & Bilder, D. Altered modes of stem cell division drive adaptive intestinal growth. Cell 147 , 603-14 (2011). Joshi, P.A., Di Grappa, M.A. & Khokha, R. Active allies: hormones, stem cells and the niche in adult mammopoiesis. Trends in Endocrinology & Metabolism 23 , 299-309 (2012). Russo, J. & Russo, I.H. The role of estrogen in the initiation of breast cancer. J Steroid Biochem Mol Biol 102 , 89-96 (2006). Yager, J.D. & Davidson, N.E. Estrogen carcinogenesis in breast cancer. N Engl J Med 354 , 270-82 (2006). Barzi, A., Lenz, A.M., Labonte, M.J. & Lenz, H.J. Molecular pathways: Estrogen pathway in colorectal cancer. Clin Cancer Res 19 , 5842-8 (2013). Chlebowski, R.T. et al. Estrogen plus progestin and colorectal cancer in postmenopausal women. N Engl J Med 350 , 991-1004 (2004). Foster, P.A. Oestrogen and colorectal cancer: mechanisms and controversies. Int J Colorectal Dis 28 , 737-49 (2013). Manson, J.E. et al. Menopausal Hormone Therapy and Long-term All-Cause and Cause-Specific Mortality: The Women's Health Initiative Randomized Trials. JAMA 318 , 927-938 (2017). Nie, X., Xie, R. & Tuo, B. Effects of Estrogen on the Gastrointestinal Tract. Dig Dis Sci 63 , 583-596 (2018). Simon, M.S. et al. Estrogen plus progestin and colorectal cancer incidence and mortality. J Clin Oncol 30 , 3983-90 (2012). Nakhostin, L., Stadler, A. & Stute, P. Impact of menopausal hormone therapy on colorectal cancer risk-A systematic review. Clin Endocrinol (Oxf) 95 , 390-397 (2021). Campbell-Thompson, M., Lynch, I.J. & Bhardwaj, B. Expression of estrogen receptor (ER) subtypes and ERbeta isoforms in colon cancer. Cancer Res 61 , 632-40 (2001). Hoff, M.B., Chang, W.W. & Mak, K.M. Effect of estrogen on cell proliferation in colonic mucosa of the mouse. Virchows Arch B Cell Pathol Incl Mol Pathol 35 , 263-73 (1981). Charlton, B.M. et al. Oral contraceptive use and colorectal cancer in the Nurses' Health Study I and II. Cancer Epidemiol Biomarkers Prev 24 , 1214-21 (2015). Voloshanenko, O. et al. Wnt secretion is required to maintain high levels of Wnt activity in colon cancer cells. Nat Commun 4 , 2610 (2013). Lecarpentier, Y., Claes, V., Vallée, A. & Hébert, J.L. Interactions between PPAR Gamma and the Canonical Wnt/Beta-Catenin Pathway in Type 2 Diabetes and Colon Cancer. PPAR Res 2017 , 5879090 (2017). Sabatino, L. et al. Emerging role of the beta-catenin-PPARgamma axis in the pathogenesis of colorectal cancer. World J Gastroenterol 20 , 7137-51 (2014). Additional Declarations There is NO Competing Interest. Supplementary Files SupplementalFigures.docx NatureCellBiologyZipperetalMaterialandMethods.pdf NatureCellBiologyZipperetalmathmodel.pdf NatureCellBiologyZipperetalmathmodel.pdf Cite Share Download PDF Status: Published Journal Publication published 06 Jan, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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20:24:40","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":561954,"visible":true,"origin":"","legend":"","description":"","filename":"NatureCellBiologyZipperetalmathmodel.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3500287/v1/f7fed845d0c98f66dd58e436.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Steroid hormone-induced wingless ligands tune female intestinal size in Drosophila","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGeneration of offspring is an energetically costly process that triggers multiple physiological adaptations of organs such as liver, pancreas and gastrointestinal tract in various species\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Survival and fitness of progeny relies on tight control of alimentary tract adaptations to metabolic demands in small rodents, in which daily food uptake during lactation can equal the mother\u0026acute;s body weight\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. It is key to understand regulatory mechanisms for hyperplasia and -trophy of the intestine, as it underlies both, physiological tissue functionality and potential malfunctioning in common diseases such as diabetes, obesity and cancer. Physiological adaptations to mating and pregnancy offer a unique opportunity to explore the nature of the underlying interorgan communication.\u003c/p\u003e \u003cp\u003eIn \u003cem\u003eDrosophila melanogaster\u003c/em\u003e females, gut size is increased to match energy consumption when egg production is initiated\u003csup\u003e\u003cspan additionalcitationids=\"CR5 CR6 CR7\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Endocrine interorgan communication orchestrates this organ size re-set yielding an enlarged intestine with about a third more absorptive enterocytes (EC)\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. This expansion is orchestrated by systemic release of juvenile hormone (JH) from the neuroendocrine corpora allata and the steroid hormone 20-Hydroxy-Ecdysone (20HE) from the ovaries. Both hormones converge on intestinal progenitors increasing intestinal stem cell (ISC) proliferation and enteroblast (EB) differentiation towards EC fate\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. 20HE-dependent increases in ISC proliferation depend on presence of the Ecdysone-receptor (EcR) and early response genes \u003cem\u003eBroad, Eip75B\u003c/em\u003e and \u003cem\u003eHr3\u003c/em\u003e\u003csup\u003e4,8\u003c/sup\u003e, but mating-dependent molecular control mechanisms of how active 20HE-signalling affects the cell cycle in ISC, remained unknown.\u003c/p\u003e \u003cp\u003eHere, we report and characterize a novel molecular player \u003cem\u003ecrooked legs\u003c/em\u003e (\u003cem\u003ecrol)/ZNF267\u003c/em\u003e that relays endocrine 20HE into local intraepithelial ISC division control. We detected 20HE-dependent \u003cem\u003ecrol\u003c/em\u003e activation in ISC and epithelial EC. Interestingly, functional experiments revealed antagonizing mitogenic effects of \u003cem\u003ecrol\u003c/em\u003e manipulation in the ISC population on one hand and the EC population on the other hand. This observation prompted us to design and establish \u0026lsquo;Rapport\u0026rsquo;, a bipartite spatiotemporally controlled dual binary tracing and expression system, which enabled us to manipulate EC while tracing non-autonomous effects on labelled stem cell progeny. We designed Rapport to offer highest flexibility and compatibility with existing genetic tools and combined it with a driver for epithelial enterocytes. Using Rapport, we discover that Crol relays systemic 20HE signalling in ECs into locally acting paracrine Wnt/Wg ligands. This finding is of high interest as EC are the largest cell population in the midgut and Wnt/wg signalling pathway is central in homeostasis and malignancies of the fly and mammalian gut\u003csup\u003e11 12\u003c/sup\u003e. Manipulation of the 20HE-Crol-Wg axis in microenvironmental EC non-autonomously controls ISC proliferation during mating-dependent intestinal growth and in neoplastic tumours. Quite the contrary, \u003cem\u003ecrol\u003c/em\u003e expression in ISC acts antiproliferative through the CDC25-orthologue \u003cem\u003estring\u003c/em\u003e and the mitotic cyclin \u003cem\u003eCyclinB\u003c/em\u003e, suggestive for a Crol-dependent mitotic balance.\u003c/p\u003e \u003cp\u003eMathematical modelling supports our hypothesis of opposing autonomous and non-autonomous mitogenic effects of Crol on ISC and that EC numbers are indeed stabilizing mating-adapted organ size depending on 20HE levels. Interestingly, our discovered dynamic pattern is highly robust and can be derived from the fundamental properties of diffusion and degradation of Wnt/wg ligands. These opposing cell type-dependent consequences of a single hormonal stimulus on stem cell proliferation inside the same epithelium underpin complex hormonal action on epithelial growth during pregnancy-induced hyperplasia and pleiotropic effects observed in cancer of the intestine.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCrooked legs responds to mating-dependent 20HE steroid hormone release\u003c/h2\u003e \u003cp\u003e The female fly intestine undergoes a variety of post-mating adaptations including a net increase of the absorptive epithelium\u003csup\u003e\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Mating-dependent enteroplasticity is orchestrated by two hormones, JH and 20HE, which act directly on ISC mitosis\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Aiming to elaborate Ecdysone-responsive genes exerting ISC division control, we followed leads from developmental studies\u003csup\u003e\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e and sequencing approaches that suggested expression of \u003cem\u003ecrooked legs\u003c/em\u003e in the adult \u003cem\u003eDrosophila\u003c/em\u003e midgut\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTransgenic flies in which Crol is GFP-tagged (Crol::GFP) confirmed \u003cem\u003ecrol\u003c/em\u003e-expression in the adult female midgut. We detected GFP-signal in ISC positive for the Notch-ligand Delta (N and Dl, Fig.\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA-A\u0026rsquo;\u0026rsquo;), in EB identified by N-activity (N-reporter GBE\u0026thinsp;+\u0026thinsp;Su(H)-dsRed, Fig.\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB-B\u0026rsquo;\u0026rsquo;), in EE positive for Pros (Prospero, Fig.\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC-C\u0026rsquo;\u0026rsquo;) and EC, positive for the septate junction marker Dlg1 (Discs-large-1, Fig.\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC-C\u0026rsquo;\u0026rsquo;). Mating induces ovary-to-gut 20HE release\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e and also significantly increased Crol::GFP levels in adult mated females (MF) compared to virgins (VF) (Fig.\u0026nbsp;1A-A\u0026rsquo;,E). Next, we confirmed \u003cem\u003ecrol\u003c/em\u003e responsiveness to pharmacological EcR activation by feeding the EcR agonist RH5849 (Fig.\u0026nbsp;1D-D\u0026rsquo;)\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, which significantly increased Crol::GFP fluorescence intensity compared to controls (Fig.\u0026nbsp;1B-B\u0026rsquo;,E). Similar increases in Crol::GFP fluorescence were observed upon genetic ovariectomy using the dominant \u003cem\u003eovoD1\u003c/em\u003e stock (Fig.\u0026nbsp;1C-C\u0026rsquo;,E) abolishing the ovaries as a sink for 20HE\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Interestingly, \u003cem\u003ecrol\u003c/em\u003e responded in both, ISC/EB as well as epithelial EC populations (Fig.\u0026nbsp;1A-C\u0026rsquo;,E). To investigate the role for \u003cem\u003ecrol\u003c/em\u003e in intestinal tissue homeostasis, we separately addressed its role in ISC/EB progenitors and epithelial EC (Fig.\u0026nbsp;1D\u0026rsquo;).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCrol and its functional human orthologue ZNF267 control proliferation of intestinal stem cells\u003c/h2\u003e \u003cp\u003eFirst, we manipulated \u003cem\u003ecrol\u003c/em\u003e autonomously in ISC and EB using the \u003cem\u003e\u0026lsquo;ReDDM\u0026rsquo;\u003c/em\u003e (Repressible Dual Differential Marker, Fig.\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA) tracing method to observe overall impact on tissue renewal with spatiotemporal control of tracing onset and gene manipulation\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Briefly, \u003cem\u003eReDDM\u003c/em\u003e differentially marks cells having active or inactive \u003cem\u003eGal4\u003c/em\u003e expression with fluorophores of different stability. Combined with the enhancer trap \u003cem\u003eesg-Gal4\u003c/em\u003e, active in progenitors (ISC and EB), \u003cem\u003eesg\u003c/em\u003e\u003csup\u003e\u003cem\u003eReDDM\u003c/em\u003e\u003c/sup\u003e double marks ISC and EB driving the expression of \u003cem\u003eUAS-CD8::GFP\u003c/em\u003e (\u003cem\u003e\u0026gt;\u0026thinsp;CD8::GFP\u003c/em\u003e, \u0026lsquo;\u0026gt;\u0026rsquo; abbreviates Gal4/UAS regulation and \u0026lsquo;\u0026gt;\u0026gt;\u0026rsquo; lexA/AoP regulation in the following) with short half-life and \u003cem\u003e\u0026gt;\u0026thinsp;H2B::RFP\u003c/em\u003e with long half-life. Upon epithelial replenishment, newly differentiated EC and EE stemming from ISC divisions retain an RFP\u003csup\u003e+\u003c/sup\u003e-nuclear stain due to fluorophore stability\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Crosses are grown at 18\u0026deg;C in which transgene expression is repressed by ubiquitous tubulin-driven temperature sensitive Gal80\u003csup\u003ets\u003c/sup\u003e. By shifting adult females to 29\u0026deg;C, Gal80\u003csup\u003ets\u003c/sup\u003e is destabilized, in turn enabling temporal control of \u003cem\u003eesg\u003c/em\u003e\u003csup\u003e\u003cem\u003eReDDM\u003c/em\u003e\u003c/sup\u003e-tracing and additional UAS-driven transgenes in progenitors (Fig.\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eAfter seven days of tracing adult female intestines using \u003cem\u003eesg\u003c/em\u003e\u003csup\u003e\u003cem\u003eReDDM\u003c/em\u003e\u003c/sup\u003e, we found that overexpression of \u003cem\u003ecrol\u003c/em\u003e has an antiproliferative effect decreasing the number of progenitor cells (Fig.\u0026nbsp;2B) about 12-fold (Fig.\u0026nbsp;2E) compared to controls (Fig.\u0026nbsp;2A). Reciprocally, RNAi-knockdown (Fig.\u0026nbsp;2C) and guideRNA (gRNA) mediated excision of \u003cem\u003ecrol\u003c/em\u003e (Fig.\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eF) using \u003cem\u003eesg\u003c/em\u003e\u003csup\u003e\u003cem\u003eReDDMCas\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e tracing significantly increased the number of progenitors (Fig.\u0026nbsp;2D, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eG), new epithelial cells (Fig.\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eH) and stimulated ISC division (Fig.\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eI) compared to controls (Fig.\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eB). Similar results were obtained using independent overexpression and loss-of-function transgenics of \u003cem\u003ecrol\u003c/em\u003e (Fig.\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eC-I). Underlining functionality of both, the reporter and RNAi stock, \u003cem\u003ecrol-RNAi\u003c/em\u003e driven by \u003cem\u003eesg\u0026thinsp;\u0026gt;\u003c/em\u003e\u0026thinsp;in ISC and EB reduces Crol::GFP fluorescence (Fig.\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eJ-L\u0026rsquo;\u0026rsquo;).\u003c/p\u003e \u003cp\u003eWe next sought to identify human orthologues of \u003cem\u003ecrooked legs\u003c/em\u003e and by mining databases for zinc finger transcription factors with a high degree of sequence homology to Crol, we isolated the human Kr\u0026uuml;ppel-like zinc finger transcription factor ZNF267. To explore the ability of human ZNF267 to substitute Crol function in intestinal progenitors, we depleted endogenous crol by RNAi and expressed human ZNF267 at the same time (Fig.\u0026nbsp;2D) and observed rescue of \u003cem\u003ecrol-RNAi-\u003c/em\u003einduced progenitor accumulations by ZNF267 (Fig.\u0026nbsp;2D-E). In line with these observations, ZNF267 regulates cell proliferation and differentiation in liver tissue\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Like Crol (Fig.\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), ZNF267 shows wide-ranged expression across human intestinal cell types (GTEx, Proteinatlas) and is induced through oestrogen signalling\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, which prompted us to investigate how steroid hormones control ISC proliferation downstream of Crol and ZNF267.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCrol controls ISC proliferation through String and Cyclin B\u003c/h3\u003e\n\u003cp\u003eDuring \u003cem\u003eDrosophila\u003c/em\u003e development, it was shown that the tyrosine protein phosphatase \u003cem\u003estring\u003c/em\u003e (\u003cem\u003estg\u003c/em\u003e, CDC25-orthologue) and the mitotic B-type Cyclin \u003cem\u003eCycB\u003c/em\u003e are targets of EcR activity\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Supporting the idea that EcR-signalling controls ISC proliferation through Stg and CycB, EcR agonists not only increase ISC mitosis\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, but also \u003cem\u003estg\u003c/em\u003e and \u003cem\u003eCycB\u003c/em\u003e transcript levels (Fig.\u0026nbsp;2F). In line with previous observations\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, up- and downregulation of \u003cem\u003estg\u003c/em\u003e levels in \u003cem\u003eesg\u003c/em\u003e\u003csup\u003e\u003cem\u003eReDDM\u003c/em\u003e\u003c/sup\u003e traced guts (Fig.\u0026nbsp;2G,2I) reciprocally controlled progenitor (Fig.\u0026nbsp;2K) and epithelial cell production (Fig.\u0026nbsp;2L). Confirming a function of Stg downstream of EcR-signalling, ISC proliferation and subsequent increase in progenitor number upon \u003cem\u003e\u0026gt;\u0026thinsp;crol-RNAi\u003c/em\u003e is abolished when \u003cem\u003e\u0026gt;\u0026thinsp;stg-RNAi\u003c/em\u003e is co-expressed (Fig.\u0026nbsp;2I-L,Fig.\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eA) and \u003cem\u003evice versa\u003c/em\u003e, co-expression of \u003cem\u003e\u0026gt;\u0026thinsp;crol\u003c/em\u003e and \u003cem\u003e\u0026gt;\u0026thinsp;stg\u003c/em\u003e (Fig.\u0026nbsp;2G-H) sufficed to rescue progenitor number (Fig.\u0026nbsp;2K) and new EC production (Fig.\u0026nbsp;2L). Comparable results were obtained when we investigated the epistasis of Crol and CycB (Fig.\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eB-G), suggesting an endocrine control of ISC cell cycle exit by Crol (Fig.\u0026nbsp;2M) as observed in development\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAn anti-proliferative role of Crol in ISC is further supported by high Crol::GFP levels in EB (Fig.\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eH) that become postmitotic during lineage progression\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. As genetically shown for Crol and CycB (Fig.\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e)\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, the EB lineage-specifying transcription factor \u003cem\u003eklumpfuss (klu)\u003c/em\u003e binds CycB regulatory regions\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Pointing to a role in ISC, EB-lineage specific \u003cem\u003ecrol\u003c/em\u003e manipulation using \u003cem\u003eklu\u003c/em\u003e\u003csup\u003e\u003cem\u003eReDDM\u003c/em\u003e\u003c/sup\u003e (Fig.\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eI)\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e reveals an increase in number of new EC upon forced expression of crol (Fig.\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eJ-L,N), but no significant changes on EB numbers (Fig.\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eJ-M), even though mitotic pH3-positive EB are occasionally observed upon \u003cem\u003ecrol-RNAi\u003c/em\u003e (Fig.\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eO) and \u003cem\u003eCycB\u003c/em\u003e expression (Fig.\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eP). Together, our data highlights \u003cem\u003ecrooked legs\u003c/em\u003e as effector of Ecdysone-signalling that autonomously promotes ISC cell cycle exit through \u003cem\u003estg\u003c/em\u003e and \u003cem\u003eCycB\u003c/em\u003e (Fig.\u0026nbsp;2M).\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eThe 20HE-Crol-Wg axis in Enterocytes controls non-autonomous Wnt/wg activity in intestinal stem cells\u003c/h2\u003e \u003cp\u003eIntriguingly, \u003cem\u003eZNF267\u003c/em\u003e\u003csup\u003e20,21\u003c/sup\u003e as well as \u003cem\u003ecrol\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e connect steroid hormone and Wnt/wg-signalling, which prompted us to investigate whether Crol controls Wnt/wg-expression downstream of systemic 20HE-signalling during physiological mating-induced intestinal adaptations. Using transgenic flies in which Wg, the primary Wnt-ligand in \u003cem\u003eDrosophila\u003c/em\u003e, is GFP-tagged (Wg::GFP), we detected GFP signal in EC anterior to the mid-/hindgut boundary (MHB, Fig.\u0026nbsp;3A)\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, a gut region known to be patterned by Wnt/Wg-signalling\u003csup\u003e30\u003c/sup\u003e. Secreted Wg is thought to act in a paracrine manner on ISC, which we analysed using the established \u003cem\u003efrizzled3\u003c/em\u003e (\u003cem\u003efz3)\u003c/em\u003e sensor flies for Wnt-activity (Fig.\u0026nbsp;3K)\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Examining \u003cem\u003efz3-RFP\u003c/em\u003e intensity in posterior midguts, we confirmed an active Wnt-signalling crescendo towards the MHB\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. More importantly, we detected fz3-RFP signal in intestinal progenitors along the midgut (Fig.\u0026nbsp;3E) and occasional Wg::GFP positive EC close to ISC with fz3-activity (Fig.\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eA) under homeostatic conditions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSimilar to 20HE-induced Crol::GFP (Fig.\u0026nbsp;1A-C\u0026rsquo;\u0026rsquo;,E), the EcR agonist RH5849 and \u003cem\u003eovoD1\u003c/em\u003e increased Wg::GFP fluorescence in EC (Fig.\u0026nbsp;3A-D,I-J) and Wnt/Wg-activity in ISC/EB (Fig.\u0026nbsp;3E-J), suggestive for active Wnt/Wg-signalling from EC to ISC (Fig.\u0026nbsp;3K). Mating also increased Wg::GFP and fz3-RFP levels (Fig.\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eB-F), which further supports a role of Wnt/wg-signalling during physiological midgut adaptations. Previous work in challenged guts showed autocrine Wnt/Wg-signalling between ISC and EB\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. In contrast, depletion of wg using\u0026thinsp;\u003cem\u003e\u0026gt;\u0026thinsp;wg-RNAi\u003c/em\u003e driven in \u003cem\u003eesg\u003c/em\u003e\u003csup\u003e\u003cem\u003eReDDM\u003c/em\u003e\u003c/sup\u003e flies under homeostatic conditions\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e did not significantly alter intestinal turnover (Fig.\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003eS5\u003c/span\u003eA-E) and direct assessment of Wnt/wg signalling activity using \u003cem\u003efz3-RFP\u003c/em\u003e sensor flies (Fig.\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003eS5\u003c/span\u003eF-I). Together these findings prompted us to search for another source for Wg-ligands under homeostatic conditions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGiven Crol::GFP (Fig.\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC-C\u0026rsquo;\u0026rsquo;) and Wg::GFP (Fig.\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eA) signal in EC, we examined whether \u003cem\u003ewg\u003c/em\u003e expression is controlled by 20HE and Crol in EC. Therefore, we combined the established EC-driver (\u003cem\u003emex\u0026gt;)\u003c/em\u003e\u003csup\u003e34\u003c/sup\u003e with \u003cem\u003efz3-RFP\u003c/em\u003e to enable Wnt-activity assessment in ISC/EB from non-autonomous sources (Fig.\u0026nbsp;4A). In line with the idea of a 20HE-Crol-Wg axis, we found that increasing 20HE signalling pathway activity by EC-specific expression of \u003cem\u003eEcI\u003c/em\u003e (\u003cem\u003eEcdysone importer\u003c/em\u003e)\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003ecrol\u003c/em\u003e as well as \u003cem\u003ewg\u003c/em\u003e significantly increased \u003cem\u003efz3\u003c/em\u003e activity in adjacent ISC/EB (Fig.\u0026nbsp;4B, Fig.\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003eS5\u003c/span\u003eJ-L\u0026rsquo;,N-N\u0026rsquo;). Reciprocally, depletion of \u003cem\u003eEcI\u003c/em\u003e, \u003cem\u003ecrol\u003c/em\u003e and \u003cem\u003ewg\u003c/em\u003e in EC, non-autonomously reduced \u003cem\u003efz3\u003c/em\u003e activity measured in ISC/EB (Fig.\u0026nbsp;4B, Fig.\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003eS5\u003c/span\u003eO-P\u0026rsquo;,R-R\u0026rsquo;). Consequently, Wg-depletion downstream of forced \u003cem\u003ecrol\u003c/em\u003e expression reduced paracrine fz3-activity measured in ISC/EB (Fig.\u0026nbsp;4C,Fig.\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003eS5\u003c/span\u003eM-M\u0026rsquo;), whereas \u003cem\u003eZNF267\u003c/em\u003e expression in endogenous crol-depleted EC (Fig.\u0026nbsp;4D, Fig.\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003eS5\u003c/span\u003eQ-Q\u0026rsquo;) stimulated \u003cem\u003ewg\u003c/em\u003e expression, further supporting Crol and its human orthologue ZNF267 acting on the \u003cem\u003ewg\u003c/em\u003e expression control \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCombined, these findings suggest \u003cem\u003ewg\u003c/em\u003e as a central transcriptional target of Crol in EC that non-autonomously stimulates fz3-activity in ISC. Intrigued by these observations, we investigated whether Wnt/Wg activation in epithelial EC through the 20HE-Crol-Wg axis translates into stem cell driven intestinal homeostasis and size adaptation\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003e\u0026lsquo;Rapport\u0026rsquo; tracing reveals non-autonomous control of intestinal homeostasis through 20HE-Crol-Wg\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFor the investigation of paracrine effects on stem cell behaviour, we developed \u0026lsquo;Rapport\u0026rsquo; (\u0026lsquo;Repressible activity paracrine reporter\u0026rsquo;), a dual binary expression system that combines spatiotemporally controlled transgene expression with ReDDM tracing of stem cell progeny. To preserve advantage of the existing established Gal4/UAS drivers and toolbox, we created an entirely new and Gal4-independent lexA/Aop-based \u0026lsquo;\u003cem\u003eesg\u003c/em\u003e\u003csup\u003e\u003cem\u003elexReDDM\u003c/em\u003e\u003c/sup\u003e\u0026rsquo; (\u003cem\u003eesg\u0026thinsp;\u0026gt;\u0026thinsp;\u0026gt;\u0026thinsp;CD8::GFP, \u0026gt;\u0026gt;H2B::RFP, tub-Gal80\u003c/em\u003e\u003csup\u003e\u003cem\u003ets\u003c/em\u003e\u003c/sup\u003e) tracing system. Importantly, when combined with \u003cem\u003emex-Gal4\u003c/em\u003e (\u003cem\u003emex\u0026gt;\u003c/em\u003e) for EC specific expression\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, the lexA-operator driven in \u003cem\u003eesg\u003c/em\u003e\u003csup\u003e\u003cem\u003elexReDDM\u003c/em\u003e\u003c/sup\u003e as well as Gal4 driven by \u003cem\u003emex\u003c/em\u003e are repressed by temperature-sensitive Gal80\u003csup\u003ets\u003c/sup\u003e, which allows simultaneous temporally controlled onset of UAS-transgenes as well as \u003cem\u003eesg\u003c/em\u003e\u003csup\u003e\u003cem\u003elexReDDM\u003c/em\u003e\u003c/sup\u003e tracing and Aop-transgenes (Fig.\u0026nbsp;5A)\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWe confirmed Rapport tracing functionality by tracing outcrossed controls over three weeks (Fig.\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003eS6\u003c/span\u003eA-C) and observed an expected linear increase in intestinal renewal (Fig.\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003eS6\u003c/span\u003eE), while ISC/EB numbers remained constant (Fig.\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003eS6\u003c/span\u003eD)\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Previous reports described autocrine EGFR-stimulation resulting in ISC proliferation\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, which we confirmed by crossing Rapport to flies expressing the EGF ligand \u003cem\u003eSpitz\u003c/em\u003e (\u0026gt;\u0026thinsp;\u003cem\u003espi\u003c/em\u003e, TGF alpha homologue, Fig.\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003eS6\u003c/span\u003eF)\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eNext, we assessed whether 20HE and Crol controlled Wnt/wg activity (Fig.\u0026nbsp;4) stimulates ISC division resulting in EC production and organ size adaptation (Fig.\u0026nbsp;5A). Strikingly, forced expression of \u003cem\u003eEcI, crol\u003c/em\u003e and \u003cem\u003ewg\u003c/em\u003e (Fig.\u0026nbsp;5C-F) non-autonomously increased progenitor cell number (Fig.\u0026nbsp;5J) and ISC progeny (Fig.\u0026nbsp;5K) compared to controls (Fig.\u0026nbsp;5B) when midguts were traced with Rapport. Reciprocally, depletion of \u003cem\u003eEcI, crol\u003c/em\u003e and \u003cem\u003ewg\u003c/em\u003e reduced progenitor (Fig.\u0026nbsp;5G-J) and progeny cell numbers (Fig.\u0026nbsp;5K). These data reveal direct relay of 20HE activity in enterocytes into stem cell production mediated by Crol and paracrine Wnt/Wg-signal.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eThe 20HE-Crol-Wg mitotic balance is conserved in intestinal tumours\u003c/h2\u003e \u003cp\u003eWnt-signalling is a well-known driver of tumorigenesis with a key role in cancers of the intestine. ZNF267 is upregulated in colorectal cancer (CRC) and regulates cell proliferation and differentiation in epithelial cancer entities\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. We found that ZNF267 expression levels positively and negatively correlate with members of the Wnt-signalling pathway (Fig.\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003eS7\u003c/span\u003eA). CRC originates from ISC\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, which prompted us to investigate \u003cem\u003ecrol\u003c/em\u003e and ZNF267 in two established intestinal tumour models.\u003c/p\u003e \u003cp\u003eInvestigating the autonomous role of Crol/ZNF267 in N loss-of-function (LOF, Fig.\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003eS7\u003c/span\u003eB) tumours\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan additionalcitationids=\"CR40\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e showed that forced expression of \u003cem\u003ecrol\u003c/em\u003e and ZNF267 within ISC reduced tumour number (Fig.\u0026nbsp;6A-E) further underlining an anti-proliferative function in ISC. Growing evidence proves that microenvironmental Wnt/wg-ligands are an important contributor to the multifaceted process of colorectal tumorigenesis\u003csup\u003e\u003cspan additionalcitationids=\"CR43 CR44 CR45\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. We thus extended Rapport with ISC-specific N-LOF that renders ISC incapable of EC lineage production and instead accumulate ISC- and EEP-like tumoral cells\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan additionalcitationids=\"CR40\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e (Fig.\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003eS7\u003c/span\u003eC). When we investigated Crol/ZNF267 in EC, we found that \u0026gt;\u0026thinsp;\u003cem\u003ecrol\u003c/em\u003e boosts ISC tumour cell mass by 4-fold leading to confluent tumours along the midgut (Fig.\u0026nbsp;6F-J) comparable to tumour-induction by microenvironmentally-derived mitogenic EGF ligands\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eEven though Notch-tumours recapitulate important steps of CRC tumorigenesis, N is not frequently mutated in CRC. Therefore, we also investigated an autonomous function for Crol/ZNF267 in a CRISPR-Cas9 based model of sporadic CRC\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e targeting the most frequently mutated genes with a multiplex guideRNA array (Fig.\u0026nbsp;6K)\u003csup\u003e\u003cspan additionalcitationids=\"CR49\" citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. CRC in ISC has severe and pleiotropic cellular phenotypes and results in early fly demise\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Underlining the antiproliferative autonomous role of \u003cem\u003ecrol\u003c/em\u003e and ZNF267, their forced expression significantly improved fly survival (Fig.\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003eS6\u003c/span\u003eL) as well as midgut length as a readout for epithelial hypotrophy and deterioration (Fig.\u0026nbsp;6M)\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. An established measure for epithelial integrity in intestinal tumour models is multilayering of epithelial cells\u003csup\u003e\u003cspan additionalcitationids=\"CR51\" citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e, which is significantly reduced in tumoral midguts expressing\u0026thinsp;\u003cem\u003e\u0026gt;\u0026thinsp;crol\u003c/em\u003e and \u003cem\u003e\u0026gt;\u0026thinsp;ZNF267\u003c/em\u003e (Fig.\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003eS7\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eThese functional experiments in colorectal tumour paradigms additionally support the idea of a mitotic balance controlling intestinal growth. Overall, steroidal input on Crol in ISC (Fig.\u0026nbsp;2) and EC (Fig.\u0026nbsp;5) provides evidence for an endocrine intestinal size control implicated in mating hyperplasia. Indeed, mitogenic \u003cem\u003ecrol\u003c/em\u003e-depletion in ISC with \u003cem\u003eesg\u003c/em\u003e\u003csup\u003e\u003cem\u003eReDDM\u003c/em\u003e\u003c/sup\u003e increases the size of the mating-responsive R5 posterior midgut region (Fig.\u0026nbsp;7A-A\u0026rsquo;)\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, whereas forced \u003cem\u003ecrol\u003c/em\u003e expression in EC using Rapport elicits R5 growth (Fig.\u0026nbsp;7B-B\u0026rsquo;). Finally, we tested our hypothesis of hormonally controlled intestinal size in a mathematical model.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMathematical modelling of endocrine relay by Crol in the control of ISC proliferation underlines complex hormonal actions on intestinal size adaptation\u003c/b\u003e \u003c/p\u003e \u003cp\u003eOur model tests whether it is mathematically plausible that these opposing trends between mitotic and antiproliferative stimuli based on the molecular mechanisms found in this study induce stable population sizes that change consistently with 20HE levels. Assuming constant 20HE levels in VF and a higher constant 20HE level in MF\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, we tested the hypothesis whether an 20HE increment is capable to yield a stable, larger organ by temporarily boosting ISC mitosis (Fig.\u0026nbsp;7D), which then declines through the increment of EC numbers and their anti-proliferative effect on ISC mitosis.\u003c/p\u003e \u003cp\u003eConstant hormonal input produces equal amounts of mitogenic Wnt/Wg independent of EC numbers. As a logical consequence of the increase in EC numbers\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e and constant ISC numbers (Fig.\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003eS7\u003c/span\u003eE), the average EC to ISC distance increases (Fig.\u0026nbsp;7C-C\u0026rsquo;). In consequence, the amount of Wnt/Wg produced in the direct neighbourhood of an ISC decays with the increase of EC\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. Considering a section of the intestine as a 1D ring, the exponential functional shape of the decay of morphogen gradient described in detail for Wg\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e(see supplementary material) holds. Therefore, the average amount of mitogenic Wnt/wg-ligand reaching equidistantly scattered ISC\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e will decline sharply when intestinal size increases (Fig.\u0026nbsp;7C-C\u0026rsquo;). Thus, only the close EC neighbourhood of the ISC effectively contributes to the Wnt/Wg levels playing an active role in ISC proliferation. Consequently, in the equation of evolution for the number of EC, we have a mitotic term that is proportional to the concentration of 20HE \u0026mdash;and, consequently, inverse to the number of EC \u0026mdash; and an anti-proliferative term that can be assumed to be constant or, in a more general setting, declining slower than the mitotic term as a function of the concentration. Overall, the equation of evolution for the number of EC reads:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\frac{\\varDelta E}{\\varDelta t}=\\beta C\\frac{\\gamma }{E}I-\\alpha I$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(E\\)\u003c/span\u003e\u003c/span\u003e is the number of EC, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(C\\)\u003c/span\u003e\u003c/span\u003e is a constant reproducing the effect of the neighbourhood, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\gamma\\)\u003c/span\u003e\u003c/span\u003e(t) the net amount of hormone, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(I\\)\u003c/span\u003e\u003c/span\u003e the number of ISC cells and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\alpha\\)\u003c/span\u003e\u003c/span\u003e the anti-proliferative rate (Fig.\u0026nbsp;7E). Qualitatively, the key result is that the above equation has a stable, fixed point for the amount of EC that grows and declines depending on whether the net amount of 20HE hormone grows or declines\u003c/p\u003e \u003cp\u003e(Fig.\u0026nbsp;7F). We provide detailed information about the construction and mathematical properties of the model in the supplementary information\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e,\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn a second modelling approach, we hypothetically explored how constant EC numbers as generated in N-LOF tumours intestines would affect ISC division dynamics (Fig.\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003eS7\u003c/span\u003eB,F). With constant EC numbers and hormone level (Fig.\u0026nbsp;7D), ISC counts in our model increase in a square-root-like manner (Fig.\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003eS7\u003c/span\u003eH) as the ISC population provides a growing sink for Wg. \u003cem\u003eIn vivo\u003c/em\u003e, block of EC generation and organ size is recapitulated in N-LOF tumours using Rapport (Fig.\u0026nbsp;6F), where upregulation of Crol/ZNF267 dramatically increases ISC numbers (Fig.\u0026nbsp;6J). Our discovered interdependencies between cell population sizes, Wnt/wg-degradation and their endocrine mitotic balance shed light on the complex endocrine involvement when tumour growth mechanisms are investigated. Together, our model is capable of capturing the emergence of a stable organ size from the antagonism of mitogenic and anti-proliferative hormonal input on ISC thresholding organ size as suggested by our functional data.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eHere we identify the transcription factor \u003cem\u003ecrooked legs\u003c/em\u003e as coordinator of endocrine input into intestinal organ size. The discovered molecular mechanisms underline the complexity of heterologous cellular interactions: a hormonal stimulus bifurcates on stem cells and microenvironment, where it is relayed differently into an antiproliferative and a mitogenic stimulus. This interdependent opposing crosstalk of forces balances stem cell divisions and ultimately stabilizes organ size, which is sustained by both, empirical observations and mathematical modelling.\u003c/p\u003e \u003cp\u003eOur novel Rapport system contributes to disentangle the underlying endocrine and local signalling organ size control mechanisms, by allowing precise genetic intervention in cell types surrounding the ISC. The independent tracing of the whole stem cell population and easy fluorophore identification of different fate choices ensures robust progeny counts in fluctuating demand situations compared to previous systems\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Endocrine actions on ISC are complex and involve hormonal dosage and mating status\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, sex differences\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e and feeding\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. Hypertrophy upon pregnancy is described in the mammalian gut\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e and is well-studied in reproductive organs such as the mammary epithelium where steroid hormones induce dramatic remodelling and cancer susceptibility\u003csup\u003e\u003cspan additionalcitationids=\"CR60\" citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn gastrointestinal tumours such as CRC, epidemiological evidence about the role of steroid hormones remains controversial and ranges from favourable to detrimental\u003csup\u003e\u003cspan additionalcitationids=\"CR63 CR64 CR65 CR66 CR67\" citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e. Functional studies of both mammalian oestrogen receptors (ER) in rodents underline the complexity of oestrogen signalling in gut tumorgenicity\u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e,\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e and reveal further complexities as pharmacological (E2/P4) and endogenous oestradiol levels differentially affect patient outcome\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e,\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e,\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e. Our functional and modelling data provides an initial logic to disentangle complex observations and involves the heterogeneity of tumour cell composition and its capacity to contribute to mitotic signals.\u003c/p\u003e \u003cp\u003eA targeted therapeutic intervention of steroid hormone signalling is supported by: i) an overall protective tendency of ER signalling in CRC\u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e. ii) Like Crol, ZNF267 is stimulated by ER\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e and is involved in Wnt signalling\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e suggesting conservation of the 20HE-Crol-Wg axis. iii) Wnt/wg signalling hyperactivation is central to CRC malignancy and Wnt-ligands remain indispensable for CRC growth albeit absence of APC\u003csup\u003e\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e. iv) Effectors of steroid signalling like PPARγ/Eip75B play protective roles in fly pathophysiology\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e and human disease\u003csup\u003e\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e,\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOur findings of antagonizing autonomous and paracrine effects of Crol and ZNF267 on tumour growth (Fig.\u0026nbsp;6G-K, Fig.\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003eS7\u003c/span\u003e) emphasize that targeted genetic investigation is of key importance to understand how mutational heterogeneity and cell type composition differentially affect the proliferative response to hormonal input. Precise intervention and tracing methods such as Rapport open the door for untangling heterogenous findings of epidemiological and functional studies.\u003c/p\u003e \u003cp\u003e3451 words\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eHammond, K.A. Adaptation of the maternal intestine during lactation. \u003cem\u003eJ Mammary Gland Biol Neoplasia\u003c/em\u003e \u003cstrong\u003e2\u003c/strong\u003e, 243-52 (1997).\u003c/li\u003e\n \u003cli\u003eRoa, J. \u0026amp; Tena-Sempere, M. Connecting metabolism and reproduction: roles of central energy sensors and key molecular mediators. \u003cem\u003eMol Cell Endocrinol\u003c/em\u003e \u003cstrong\u003e397\u003c/strong\u003e, 4-14 (2014).\u003c/li\u003e\n \u003cli\u003eSpeakman, J.R. The physiological costs of reproduction in small mammals. \u003cem\u003ePhilosophical Transactions of the Royal Society B: Biological Sciences\u003c/em\u003e \u003cstrong\u003e363\u003c/strong\u003e, 375-398 (2008).\u003c/li\u003e\n \u003cli\u003eAhmed, S.M.H.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Fitness trade-offs incurred by ovary-to-gut steroid signalling in Drosophila. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e584\u003c/strong\u003e, 415-419 (2020).\u003c/li\u003e\n \u003cli\u003eCognigni, P., Bailey, A.P. \u0026amp; Miguel-Aliaga, I. Enteric neurons and systemic signals couple nutritional and reproductive status with intestinal homeostasis. \u003cem\u003eCell Metab\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 92-104 (2011).\u003c/li\u003e\n \u003cli\u003eKlepsatel, P.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Reproductive and post-reproductive life history of wild-caught Drosophila melanogaster under laboratory conditions. \u003cem\u003eJ Evol Biol\u003c/em\u003e \u003cstrong\u003e26\u003c/strong\u003e, 1508-20 (2013).\u003c/li\u003e\n \u003cli\u003eReiff, T.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Endocrine remodelling of the adult intestine sustains reproduction in Drosophila. \u003cem\u003eElife\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, e06930 (2015).\u003c/li\u003e\n \u003cli\u003eZipper, L., Jassmann, D., Burgmer, S., Gorlich, B. \u0026amp; Reiff, T. Ecdysone steroid hormone remote controls intestinal stem cell fate decisions via the PPARgamma-homolog Eip75B in Drosophila. \u003cem\u003eElife\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, e55795 (2020).\u003c/li\u003e\n \u003cli\u003eAmeku, T. \u0026amp; Niwa, R. Mating-Induced Increase in Germline Stem Cells via the Neuroendocrine System in Female Drosophila. \u003cem\u003ePLoS Genet\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, e1006123 (2016).\u003c/li\u003e\n \u003cli\u003eHarshman, L.G., Loeb, A.M. \u0026amp; Johnson, B.A. Ecdysteroid titers in mated and unmated Drosophila melanogaster females. \u003cem\u003eJ Insect Physiol\u003c/em\u003e \u003cstrong\u003e45\u003c/strong\u003e, 571-577 (1999).\u003c/li\u003e\n \u003cli\u003eLin, G., Xu, N. \u0026amp; Xi, R. Paracrine Wingless signalling controls self-renewal of Drosophila intestinal stem cells. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e455\u003c/strong\u003e, 1119-23 (2008).\u003c/li\u003e\n \u003cli\u003eRadtke, F. \u0026amp; Clevers, H. Self-renewal and cancer of the gut: two sides of a coin. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e307\u003c/strong\u003e, 1904-9 (2005).\u003c/li\u003e\n \u003cli\u003eD\u0026apos;Avino, P.P. \u0026amp; Thummel, C.S. crooked legs encodes a family of zinc finger proteins required for leg morphogenesis and ecdysone-regulated gene expression during Drosophila metamorphosis. \u003cem\u003eDevelopment\u003c/em\u003e \u003cstrong\u003e125\u003c/strong\u003e, 1733-45 (1998).\u003c/li\u003e\n \u003cli\u003eMitchell, N., Cranna, N., Richardson, H. \u0026amp; Quinn, L. The Ecdysone-inducible zinc-finger transcription factor Crol regulates Wg transcription and cell cycle progression in Drosophila. \u003cem\u003eDevelopment\u003c/em\u003e \u003cstrong\u003e135\u003c/strong\u003e, 2707-16 (2008).\u003c/li\u003e\n \u003cli\u003eMitchell, K.J.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Genetic analysis of Netrin genes in Drosophila: Netrins guide CNS commissural axons and peripheral motor axons. \u003cem\u003eNeuron\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, 203-15 (1996).\u003c/li\u003e\n \u003cli\u003eDutta, D.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Regional Cell-Specific Transcriptome Mapping Reveals Regulatory Complexity in the Adult Drosophila Midgut. \u003cem\u003eCell Rep\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 346-58 (2015).\u003c/li\u003e\n \u003cli\u003eHung, R.J., Li, J.S.S., Liu, Y. \u0026amp; Perrimon, N. Defining cell types and lineage in the Drosophila midgut using single cell transcriptomics. \u003cem\u003eCurr Opin Insect Sci\u003c/em\u003e \u003cstrong\u003e47\u003c/strong\u003e, 12-17 (2021).\u003c/li\u003e\n \u003cli\u003eBusson, D., Gans, M., Komitopoulou, K. \u0026amp; Masson, M. Genetic Analysis of Three Dominant Female-Sterile Mutations Located on the X Chromosome of DROSOPHILA MELANOGASTER. \u003cem\u003eGenetics\u003c/em\u003e \u003cstrong\u003e105\u003c/strong\u003e, 309-25 (1983).\u003c/li\u003e\n \u003cli\u003eAntonello, Z.A., Reiff, T., Ballesta-Illan, E. \u0026amp; Dominguez, M. Robust intestinal homeostasis relies on cellular plasticity in enteroblasts mediated by miR-8-Escargot switch. \u003cem\u003eEMBO J\u003c/em\u003e \u003cstrong\u003e34\u003c/strong\u003e, 2025-41 (2015).\u003c/li\u003e\n \u003cli\u003eSchnabl, B., Valletta, D., Kirovski, G. \u0026amp; Hellerbrand, C. Zinc finger protein 267 is up-regulated in hepatocellular carcinoma and promotes tumor cell proliferation and migration. \u003cem\u003eExp Mol Pathol\u003c/em\u003e \u003cstrong\u003e91\u003c/strong\u003e, 695-701 (2011).\u003c/li\u003e\n \u003cli\u003eYang, H.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Knockdown of zinc finger protein 267 suppresses diffuse large B-cell lymphoma progression, metastasis, and cancer stem cell properties. \u003cem\u003eBioengineered\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 1686-1701 (2022).\u003c/li\u003e\n \u003cli\u003eStossi, F.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Transcriptional profiling of estrogen-regulated gene expression via estrogen receptor (ER) alpha or ERbeta in human osteosarcoma cells: distinct and common target genes for these receptors. \u003cem\u003eEndocrinology\u003c/em\u003e \u003cstrong\u003e145\u003c/strong\u003e, 3473-86 (2004).\u003c/li\u003e\n \u003cli\u003eLi, T.R. \u0026amp; White, K.P. Tissue-specific gene expression and ecdysone-regulated genomic networks in Drosophila. \u003cem\u003eDev Cell\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 59-72 (2003).\u003c/li\u003e\n \u003cli\u003eMitchell, N.C.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e The Ecdysone receptor constrains wingless expression to pattern cell cycle across the Drosophila wing margin in a Cyclin B-dependent manner. \u003cem\u003eBMC Dev Biol\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 28 (2013).\u003c/li\u003e\n \u003cli\u003ePatel, P.H., Dutta, D. \u0026amp; Edgar, B.A. Niche appropriation by Drosophila intestinal stem cell tumours. \u003cem\u003eNat Cell Biol\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, 1182-92 (2015).\u003c/li\u003e\n \u003cli\u003eZhang, P.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e An SH3PX1-Dependent Endocytosis-Autophagy Network Restrains Intestinal Stem Cell Proliferation by Counteracting EGFR-ERK Signaling. \u003cem\u003eDev Cell\u003c/em\u003e \u003cstrong\u003e49\u003c/strong\u003e, 574-589 e5 (2019).\u003c/li\u003e\n \u003cli\u003eKorzelius, J.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e The WT1-like transcription factor Klumpfuss maintains lineage commitment of enterocyte progenitors in the Drosophila intestine. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 4123 (2019).\u003c/li\u003e\n \u003cli\u003eReiff, T.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Notch and EGFR regulate apoptosis in progenitor cells to ensure gut homeostasis in Drosophila. \u003cem\u003eEMBO J\u003c/em\u003e \u003cstrong\u003e38\u003c/strong\u003e, e101346 (2019).\u003c/li\u003e\n \u003cli\u003ePort, F., Chen, H.M., Lee, T. \u0026amp; Bullock, S.L. Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e \u003cstrong\u003e111\u003c/strong\u003e, E2967-76 (2014).\u003c/li\u003e\n \u003cli\u003eTakashima, S., Mkrtchyan, M., Younossi-Hartenstein, A., Merriam, J.R. \u0026amp; Hartenstein, V. The behaviour of Drosophila adult hindgut stem cells is controlled by Wnt and Hh signalling. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e454\u003c/strong\u003e, 651-5 (2008).\u003c/li\u003e\n \u003cli\u003eOlson, E.R.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Yan, an ETS-domain transcription factor, negatively modulates the Wingless pathway in the Drosophila eye. \u003cem\u003eEMBO reports\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 1047-1054 (2011).\u003c/li\u003e\n \u003cli\u003eTian, A.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Intestinal stem cell overproliferation resulting from inactivation of the APC tumor suppressor requires the transcription cofactors Earthbound and Erect wing. \u003cem\u003ePLoS Genet\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, e1006870 (2017).\u003c/li\u003e\n \u003cli\u003eCordero, J.B., Stefanatos, R.K., Scopelliti, A., Vidal, M. \u0026amp; Sansom, O.J. Inducible progenitor-derived Wingless regulates adult midgut regeneration in Drosophila. \u003cem\u003eEMBO J\u003c/em\u003e \u003cstrong\u003e31\u003c/strong\u003e, 3901-17 (2012).\u003c/li\u003e\n \u003cli\u003ePhillips, M.D. \u0026amp; Thomas, C.M. Brush border spectrin is required for early endosome recycling in Drosophila. \u003cem\u003eJ Cell Sci\u003c/em\u003e \u003cstrong\u003e119\u003c/strong\u003e, 1361-70 (2006).\u003c/li\u003e\n \u003cli\u003eOkamoto, N.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e A Membrane Transporter Is Required for Steroid Hormone Uptake in Drosophila. \u003cem\u003eDev Cell\u003c/em\u003e \u003cstrong\u003e47\u003c/strong\u003e, 294-305 e7 (2018).\u003c/li\u003e\n \u003cli\u003eYagi, R., Mayer, F. \u0026amp; Basler, K. Refined LexA transactivators and their use in combination with the Drosophila Gal4 system. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e \u003cstrong\u003e107\u003c/strong\u003e, 16166-71 (2010).\u003c/li\u003e\n \u003cli\u003eLiang, J., Balachandra, S., Ngo, S. \u0026amp; O\u0026apos;Brien, L.E. Feedback regulation of steady-state epithelial turnover and organ size. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e548\u003c/strong\u003e, 588-591 (2017).\u003c/li\u003e\n \u003cli\u003eBarker, N.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Crypt stem cells as the cells-of-origin of intestinal cancer. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e457\u003c/strong\u003e, 608-11 (2009).\u003c/li\u003e\n \u003cli\u003eMicchelli, C.A. \u0026amp; Perrimon, N. Evidence that stem cells reside in the adult Drosophila midgut epithelium. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e439\u003c/strong\u003e, 475-9 (2006).\u003c/li\u003e\n \u003cli\u003eOhlstein, B. \u0026amp; Spradling, A. Multipotent Drosophila intestinal stem cells specify daughter cell fates by differential notch signaling. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e315\u003c/strong\u003e, 988-92 (2007).\u003c/li\u003e\n \u003cli\u003eOhlstein, B. \u0026amp; Spradling, A. The adult Drosophila posterior midgut is maintained by pluripotent stem cells. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e439\u003c/strong\u003e, 470-4 (2006).\u003c/li\u003e\n \u003cli\u003eVermeulen, L.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Wnt activity defines colon cancer stem cells and is regulated by the microenvironment. \u003cem\u003eNat Cell Biol\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 468-76 (2010).\u003c/li\u003e\n \u003cli\u003eHanahan, D. \u0026amp; Weinberg, Robert A. Hallmarks of Cancer: The Next Generation. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e144\u003c/strong\u003e, 646-674 (2011).\u003c/li\u003e\n \u003cli\u003ePatel, S., Alam, A., Pant, R. \u0026amp; Chattopadhyay, S. Wnt Signaling and Its Significance Within the Tumor Microenvironment: Novel Therapeutic Insights. \u003cem\u003eFront Immunol\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 2872 (2019).\u003c/li\u003e\n \u003cli\u003eMcAllister, S.S. \u0026amp; Weinberg, R.A. The tumour-induced systemic environment as a critical regulator of cancer progression and metastasis. \u003cem\u003eNat Cell Biol\u003c/em\u003e \u003cstrong\u003e16\u003c/strong\u003e, 717-27 (2014).\u003c/li\u003e\n \u003cli\u003eHanahan, D. Hallmarks of Cancer: New Dimensions. \u003cem\u003eCancer Discovery\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 31-46 (2022).\u003c/li\u003e\n \u003cli\u003eNgo, S., Liang, J., Su, Y.H. \u0026amp; O\u0026apos;Brien, L.E. Disruption of EGF Feedback by Intestinal Tumors and Neighboring Cells in Drosophila. \u003cem\u003eCurr Biol\u003c/em\u003e \u003cstrong\u003e30\u003c/strong\u003e, 1537-1546 e3 (2020).\u003c/li\u003e\n \u003cli\u003eZipper, L., Batchu, S., Kaya, N.H., Antonello, Z.A. \u0026amp; Reiff, T. The MicroRNA miR-277 Controls Physiology and Pathology of the Adult Drosophila Midgut by Regulating the Expression of Fatty Acid beta-Oxidation-Related Genes in Intestinal Stem Cells. \u003cem\u003eMetabolites\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e(2022).\u003c/li\u003e\n \u003cli\u003eHanahan, D. \u0026amp; Weinberg, R.A. The hallmarks of cancer. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e100\u003c/strong\u003e, 57-70 (2000).\u003c/li\u003e\n \u003cli\u003eBangi, E., Murgia, C., Teague, A.G., Sansom, O.J. \u0026amp; Cagan, R.L. Functional exploration of colorectal cancer genomes using Drosophila. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, 13615 (2016).\u003c/li\u003e\n \u003cli\u003eMartorell, O.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Conserved mechanisms of tumorigenesis in the Drosophila adult midgut. \u003cem\u003ePLoS One\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, e88413 (2014).\u003c/li\u003e\n \u003cli\u003eZhai, Z.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Accumulation of differentiating intestinal stem cell progenies drives tumorigenesis. \u003cem\u003eNature Communications\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, 10219 (2015).\u003c/li\u003e\n \u003cli\u003eDubois, L., Lecourtois, M., Alexandre, C., Hirst, E. \u0026amp; Vincent, J.P. Regulated endocytic routing modulates wingless signaling in Drosophila embryos. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e105\u003c/strong\u003e, 613-24 (2001).\u003c/li\u003e\n \u003cli\u003eDubois, L., Lecourtois, M., Alexandre, C., Hirst, E. \u0026amp; Vincent, J.-P. Regulated Endocytic Routing Modulates Wingless Signaling in Drosophila Embryos. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e105\u003c/strong\u003e, 613-624 (2001).\u003c/li\u003e\n \u003cli\u003eWartlick, O., Kicheva, A. \u0026amp; Gonz\u0026aacute;lez-Gait\u0026aacute;n, M. Morphogen gradient formation. \u003cem\u003eCold Spring Harb Perspect Biol\u003c/em\u003e \u003cstrong\u003e1\u003c/strong\u003e, a001255 (2009).\u003c/li\u003e\n \u003cli\u003eBerg, H.C. \u003cem\u003eRandom Walks in Biology\u003c/em\u003e, (Princeton University Press, 1993).\u003c/li\u003e\n \u003cli\u003eHudry, B., Khadayate, S. \u0026amp; Miguel-Aliaga, I. The sexual identity of adult intestinal stem cells controls organ size and plasticity. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e530\u003c/strong\u003e, 344-8 (2016).\u003c/li\u003e\n \u003cli\u003eO\u0026apos;Brien, L.E., Soliman, S.S., Li, X. \u0026amp; Bilder, D. Altered modes of stem cell division drive adaptive intestinal growth. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e147\u003c/strong\u003e, 603-14 (2011).\u003c/li\u003e\n \u003cli\u003eJoshi, P.A., Di Grappa, M.A. \u0026amp; Khokha, R. Active allies: hormones, stem cells and the niche in adult mammopoiesis. \u003cem\u003eTrends in Endocrinology \u0026amp; Metabolism\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e, 299-309 (2012).\u003c/li\u003e\n \u003cli\u003eRusso, J. \u0026amp; Russo, I.H. The role of estrogen in the initiation of breast cancer. \u003cem\u003eJ Steroid Biochem Mol Biol\u003c/em\u003e \u003cstrong\u003e102\u003c/strong\u003e, 89-96 (2006).\u003c/li\u003e\n \u003cli\u003eYager, J.D. \u0026amp; Davidson, N.E. Estrogen carcinogenesis in breast cancer. \u003cem\u003eN Engl J Med\u003c/em\u003e \u003cstrong\u003e354\u003c/strong\u003e, 270-82 (2006).\u003c/li\u003e\n \u003cli\u003eBarzi, A., Lenz, A.M., Labonte, M.J. \u0026amp; Lenz, H.J. Molecular pathways: Estrogen pathway in colorectal cancer. \u003cem\u003eClin Cancer Res\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 5842-8 (2013).\u003c/li\u003e\n \u003cli\u003eChlebowski, R.T.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Estrogen plus progestin and colorectal cancer in postmenopausal women. \u003cem\u003eN Engl J Med\u003c/em\u003e \u003cstrong\u003e350\u003c/strong\u003e, 991-1004 (2004).\u003c/li\u003e\n \u003cli\u003eFoster, P.A. Oestrogen and colorectal cancer: mechanisms and controversies. \u003cem\u003eInt J Colorectal Dis\u003c/em\u003e \u003cstrong\u003e28\u003c/strong\u003e, 737-49 (2013).\u003c/li\u003e\n \u003cli\u003eManson, J.E.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Menopausal Hormone Therapy and Long-term All-Cause and Cause-Specific Mortality: The Women\u0026apos;s Health Initiative Randomized Trials. \u003cem\u003eJAMA\u003c/em\u003e \u003cstrong\u003e318\u003c/strong\u003e, 927-938 (2017).\u003c/li\u003e\n \u003cli\u003eNie, X., Xie, R. \u0026amp; Tuo, B. Effects of Estrogen on the Gastrointestinal Tract. \u003cem\u003eDig Dis Sci\u003c/em\u003e \u003cstrong\u003e63\u003c/strong\u003e, 583-596 (2018).\u003c/li\u003e\n \u003cli\u003eSimon, M.S.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Estrogen plus progestin and colorectal cancer incidence and mortality. \u003cem\u003eJ Clin Oncol\u003c/em\u003e \u003cstrong\u003e30\u003c/strong\u003e, 3983-90 (2012).\u003c/li\u003e\n \u003cli\u003eNakhostin, L., Stadler, A. \u0026amp; Stute, P. Impact of menopausal hormone therapy on colorectal cancer risk-A systematic review. \u003cem\u003eClin Endocrinol (Oxf)\u003c/em\u003e \u003cstrong\u003e95\u003c/strong\u003e, 390-397 (2021).\u003c/li\u003e\n \u003cli\u003eCampbell-Thompson, M., Lynch, I.J. \u0026amp; Bhardwaj, B. Expression of estrogen receptor (ER) subtypes and ERbeta isoforms in colon cancer. \u003cem\u003eCancer Res\u003c/em\u003e \u003cstrong\u003e61\u003c/strong\u003e, 632-40 (2001).\u003c/li\u003e\n \u003cli\u003eHoff, M.B., Chang, W.W. \u0026amp; Mak, K.M. Effect of estrogen on cell proliferation in colonic mucosa of the mouse. \u003cem\u003eVirchows Arch B Cell Pathol Incl Mol Pathol\u003c/em\u003e \u003cstrong\u003e35\u003c/strong\u003e, 263-73 (1981).\u003c/li\u003e\n \u003cli\u003eCharlton, B.M.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Oral contraceptive use and colorectal cancer in the Nurses\u0026apos; Health Study I and II. \u003cem\u003eCancer Epidemiol Biomarkers Prev\u003c/em\u003e \u003cstrong\u003e24\u003c/strong\u003e, 1214-21 (2015).\u003c/li\u003e\n \u003cli\u003eVoloshanenko, O.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Wnt secretion is required to maintain high levels of Wnt activity in colon cancer cells. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, 2610 (2013).\u003c/li\u003e\n \u003cli\u003eLecarpentier, Y., Claes, V., Vall\u0026eacute;e, A. \u0026amp; H\u0026eacute;bert, J.L. Interactions between PPAR Gamma and the Canonical Wnt/Beta-Catenin Pathway in Type 2 Diabetes and Colon Cancer. \u003cem\u003ePPAR Res\u003c/em\u003e \u003cstrong\u003e2017\u003c/strong\u003e, 5879090 (2017).\u003c/li\u003e\n \u003cli\u003eSabatino, L.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Emerging role of the beta-catenin-PPARgamma axis in the pathogenesis of colorectal cancer. \u003cem\u003eWorld J Gastroenterol\u003c/em\u003e \u003cstrong\u003e20\u003c/strong\u003e, 7137-51 (2014).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3500287/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3500287/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Female reproduction comes at great expense to energy metabolism compensated by extensive organ adaptations including intestinal size. Upon mating, Drosophila ovaries release the steroid hormone Ecdysone that stimulates a 30% net increase of absorptive epithelium by intestinal stem cell (ISC) divisions. Here, we uncover the transcription factor crooked legs (crol) as intraepithelial coordinator of Ecdysone-induced ISC mitosis by establishing Rapport, the first spatiotemporally-controlled dual expression and tracing system for the analysis of paracrine effects on ISC behaviour. Rapport tracing revealed that Ecdysone-induced Crol controls mitogenic Wnt/wg-ligand release from epithelial enterocytes towards ISC, which is counterbalanced by Crol-repression of string/CDC25 and Cyclin-B directly in ISC. Rapport-based ISC tumours confirm paracrine stimulation through the Ecdysone-Crol-Wg axis on mitotic behaviour. Finally, mathematical modelling corroborates increasing enterocyte numbers and Wnt/wg-degradation to set a stable post-mating intestinal size. Together, our findings provide insights into complex endocrine growth control mechanisms during mating-induced adaptations and gastrointestinal cancer.","manuscriptTitle":"Steroid hormone-induced wingless ligands tune female intestinal size in Drosophila","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-11-28 19:22:45","doi":"10.21203/rs.3.rs-3500287/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"76491123-5b84-42ed-af38-4a3de9b0ce10","owner":[],"postedDate":"November 28th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":26203127,"name":"Biological sciences/Stem cells/Intestinal stem cells"},{"id":26203128,"name":"Health sciences/Endocrinology/Endocrine system and metabolic diseases/Endocrine cancer"},{"id":26203129,"name":"Health sciences/Diseases/Cancer/Gastrointestinal cancer/Colorectal cancer"}],"tags":[],"updatedAt":"2025-01-07T08:06:43+00:00","versionOfRecord":{"articleIdentity":"rs-3500287","link":"https://doi.org/10.1038/s41467-024-55664-2","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-01-06 05:00:00","publishedOnDateReadable":"January 6th, 2025"},"versionCreatedAt":"2023-11-28 19:22:45","video":"","vorDoi":"10.1038/s41467-024-55664-2","vorDoiUrl":"https://doi.org/10.1038/s41467-024-55664-2","workflowStages":[]},"version":"v1","identity":"rs-3500287","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3500287","identity":"rs-3500287","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}