The intestinal stem cell/enteroblast-GAL4 driver, escargot-GAL4, also manipulates gene expression in the juvenile hormone-synthesizing organ of Drosophila melanogaster

The intestinal stem cell/enteroblast-GAL4 driver, escargot-GAL4, also manipulates gene expression in the juvenile hormone-synthesizing organ of Drosophila melanogaster | 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 The intestinal stem cell/enteroblast-GAL4 driver, escargot-GAL4, also manipulates gene expression in the juvenile hormone-synthesizing organ of Drosophila melanogaster Yoshitomo Kurogi, Yosuke Mizuno, Takumi Kamiyama, Ryusuke Niwa This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3856222/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Apr, 2024 Read the published version in Scientific Reports → Version 1 posted 7 You are reading this latest preprint version Abstract Intestinal stem cells (ISCs) of the fruit fly, Drosophila melanogaster , offer an excellent genetic model to explore homeostatic roles of ISCs in animal physiology. Among available genetic tools, the escargot ( esg ) -GAL4 driver, expressing the yeast transcription factor gene, GAL4 , under control of the esg gene promoter, has contributed significantly to ISC studies. This driver facilitates activation of a gene of interest in proximity to a GAL4-binding element, UAS, in ISCs and progenitor enteroblasts (EBs). While esg-GAL4 has been considered an ISC/EB-specific driver, its actual specificity remains unexplored. In this study, we reveal esg-GAL4 expression in the corpus allatum (CA), responsible for juvenile hormone (JH) production. When driving the oncogenic gene Ras V 12 , esg-GAL4 induces overgrowth in ISCs/EBs as reported, but also increases CA cell number and size. Consistent with this observation, animals alter expression of JH-response genes. Our data show that esg-GAL4 -driven gene manipulation can systemically influence JH-mediated animal physiology, arguing for cautious use of esg-GAL4 as a “specific” ISC/EB driver to examine ISC/EB-mediated animal physiology. Biological sciences/Genetics Biological sciences/Genetics/Gene expression Biological sciences/Zoology/Animal physiology Biological sciences/Biological techniques/Genetic techniques Figures Figure 1 Figure 2 Figure 3 Introduction Precise overexpression of genes in specific cell types and time windows is crucial to discover essential functions of those genes in multicellular organisms. Among model organisms, the fruit fly, Drosophila melanogaster , is the one for which such gene expression manipulation techniques are best developed. In particular, the GAL4-UAS system is a powerful binary gene expression system in D. melanogaster for targeted genetic manipulation in a spatio-temporal specific manner to reveal gene functions 1 . This system utilizes the yeast transcription factor GAL4, controlled by a tissue-specific enhancer/promoter sequence, in combination with a GAL4-biding element called Upstream Activating Sequence (UAS), inserted upstream of the gene of interest, either endogenously or exogenously. The impact of the GAL4-UAS system on D. melanogaster genetics research is immeasurable. However, despite its utility, a potential drawback of the GAL4-UAS system is the possibility of incomplete cell type- or tissue-specific expression patterns, complicating interpretation of results. D. melanogaster escargot (esg)-GAL4 , formally known as P{GawB}NP5130 (RRID:BDSC_93857) 2 , has widely been used as the fundamental GAL4 driver to manipulate genes “specifically” in intestinal stem cells (ISCs) and enteroblasts (EBs) (Fig. 1 a). In D. melanogaster , ISCs regulate gut homeostasis by maintaining themselves and also by giving rise to other essential gut epithelial cells, including EBs, enteroendocrine cells, and enterocytes. Dysfunction of ISCs results in severe malfunctions of age-associated tissue integrity in the gut 3 . By virtue of convenient tools to analyze functions and roles of genes, D. melanogaster ISCs have served as a useful model system to study the homeostatic role of ISCs in gut physiology. Notably, the esg-GAL4 driver has contributed to overexpressing genes or other constructs to study fundamental roles of ISCs and EBs. For example, researchers have heavily used esg-GAL4 to generate ISC tumors by overexpressing oncogenic genes such as the gain-of-function transgenes, Ras and yokie(yki) . esg-GAL4 -driven ISC tumor animals have advanced our understanding of tumor-dependent impairment of systemic physiology, such as cachexia and the bloating phenotype. The crucial assumption for interpreting results of these studies as a phenotype originating from ISCs and EBs is that esg-GAL4 manipulates gene expression only in these cells in adults. However, a recent study reported that esg-GAL4 is expressed at least in brain neurons 4 . Therefore, it is apparent that characteristics of esg-GAL4 have not yet been sufficiently investigated. In this study, we report that esg-GAL4 is also expressed in the insect endocrine organ, the corpus allatum (CA), which is essential for synthesizing insect juvenile hormones (JHs). Our data show that esg-GAL4 -driven gene manipulation can systemically influence JH-mediated animal physiology, arguing for cautious use of esg-GAL4 as a “specific” ISC/EB driver to examine ISC/EB-mediated animal physiology. Result Esg-GAL4 is expressed in the endocrine corpus allatum We conducted experiments using the esg-GAL4 driver combined with tubulin promoter-driven temperature-sensitive GAL80 ( tubP-GAL80 ts ). Hereafter, esg-GAL4; tubP-GAL80 ts is designated “ esg ts -GAL4” or “ esg ts > ”. This strain has widely been used for adult stage-specific gene manipulation in ISCs and EBs 5 – 8 . In all experimental conditions in this study, we reared esg ts > flies at a permissive temperature (21°C) during development, such that esg-GAL4 activity is suppressed by GAL80 right before eclosion. Then, after eclosion, we subjected these flies to a restrictive temperature (29°C) to activate esg-GAL4 only in the adult stage. We realized by chance that esg ts -GAL4 was active in the tissue located between the brain and proventriculus (Fig. 1 b). This tissue was co-immunostained with an antibody against Juvenile hormone acid O -methyltransferase (JHAMT), the essential enzyme that synthesizes JHs in the CA 9 , 10 . This result strongly indicates that the esg ts -GAL4 -positive tissue is the CA. We also confirmed that esg ts -GAL4 was expressed in the CA of both male and female adult flies (Fig. 1 c). Moreover, esg ts -GAL4 was expressed in the ring gland, particularly in the CA of wandering 3rd -instar larva, as well as of adults (Fig. 1 d). These results suggest that esg ts -GAL4 labels the CA in both male and female larvae and adults. To confirm whether the esg gene itself is expressed in the CA, we used the esg-knock-in-GFP ( esg-GFP ) line 11 . As with esg ts > GFP expression, esg-GFP was expressed not only in a certain cells in the midgut, which seem to be ISCs/EBs (Fig. 1 e) 11 , but also in the CA of both male and female adults (Fig. 1 f). Furthermore, esg-GFP was expressed in the CA of 3rd instar wandering larvae, while considerable expression of esg-GFP was also detected in other ring gland cells (Fig. 1 g). These results suggest that esg is endogenously expressed in the CA. RNAi of JH-biosynthetic enzyme by esg-GAL4 also impairs oogenesis Next, we explored the possibility that esg ts -GAL4- driven transgenic RNAi suppresses gene expression in the CA. To examine this point, we conducted an RNAi experiment with esg ts -GAL4 to target jhamt , which is expressed explicitly in the CA 10 . Immunostaining signals of anti-JHAMT antibody were drastically decreased by jhamt RNAi, compared to controls (Fig. 2 a). In many insects, including D. melanogaster , JH promotes ovarian development by accumulating yolk components such as yolk protein and vitellogenin 12 , 13 . In D. melanogaster , a previous study reported that loss of jhamt activity results in smaller ovaries and reduced egg numbers 14 . Therefore, we observed ovary morphology and counted the number of mature eggs in adult females expressing esg ts > jhamt RNAi. We found that esg ts > jhamt RNAi flies had smaller ovaries than controls (Fig. 2 b). Consistent with this observation, the number of mature eggs was significantly decreased in RNAi flies (Fig. 2 c). These results suggest that esg ts -GAL4 -driven RNAi suppresses gene expression in the CA and influences JH-mediated biological events such as oogenesis. Oncogenic Ras V 12 expression by esg-GAL4 causes CA hypertrophy and abnormal expression of JH-responsive genes In some recent studies, esg ts -GAL4 and UAS-Ras V 12 have been utilized to induce ISC/EB tumors to investigate cell turnover in the midgut and tumor-mediated systemic physiology 6 , 15 , 16 . Since esg-GAL4 is also expressed in the CA, esg ts > Ras V12 might affect both ISC/EB and CA cells. Notably, esg ts > Ras V12 resulted not only in abnormal expansion of the esg ts -GAL4 -driven GFP -positive area in the midgut (Fig. 3 a) 15 , but also increased CA size and cell number (Fig. 3 b-d). Considering morphological abnormalities in the CA, it seemed possible that esg ts > Ras V12 expression enhances JH biosynthesis in the CA. Therefore, we next performed quantitative PCR on three JH-responsive genes, Krüppel-homolog 1 ( Kr-h1 ), Jonah 25Bii ( jon25Bii ), Odorant-binding protein 99b ( Obp99b ), to estimate the amount of JH in Ras V 12 overexpressors and controls 17 . Previous studies have shown that expression levels of Kr-h1 and jon25Bii correlate positively with the amount of JH in the body, while Obp99b correlates negatively 17 , 18 . Our qPCR results showed that expression levels of Kr-h1 and jon25Bii were increased by Ras V 12 , while Obp99b was decreased (Fig. 3 e-g). These results strongly suggest that esg ts > Ras V12 leads to abnormalities in CA cells and increased JH biosynthesis. Discussion In this study, we found that esg-GAL4 , which is widely used to label midgut ISC/EB 2 , was also expressed in the CA. Genetic manipulation with esg ts -GAL4 , such as overexpression of Ras V 12 , caused CA hypertrophy and influenced JH-responsive gene expression (Fig. 3 b-g), suggesting that esg ts > Ras V12 increases JH biosynthesis. Our data suggest that esg itself is expressed in the CA in both males and females of both larvae and adults. Enrichment of esg expression in the larval ring gland was suggested by a previous microarray analysis comparing gene expression between the ring glands and whole body 19 . esg encodes a Snail-type transcription factor that contributes to cell cycle regulation, cell differentiation, and cell-cell adhesion in many cell types in D. melanogaster 20 – 23 . However, functions of Esg that regulate differentiation and morphogenesis of the CA have not been studied. Thus, additional studies are needed to clarify how Esg is involved in CA cell regulation, especially whether it regulates JH biosynthesis. In D. melanogaster , one of the reported functions of JHs is that these hormones directly act on ISCs and EBs through the nuclear JH receptors, Methoprene-tolerant (Met) and Germ cell expressed (Gce), to regulate gut remodeling in mated or aged females 5 , 6 . Interestingly, previous studies reported that esg ts > jhamt RNAi reduces numbers of ISCs and EBs. This phenotype is also observed in esg ts > Met or Gce RNAi animals. Since these studies use esg ts -GAL4 as the ISC/EB-“specific” GAL4 driver, these papers propose that JHs are biosynthesized in ISCs and EBs outside the CA, and cell-autonomously regulate maintenance of ISCs and EBs during aging 6 . However, our data strongly indicate that esg-GAL4 is also expressed in the CA. More importantly, esg ts > jhamt RNAi causes a decrease in JHAMT protein in the CA (Fig. 2 a), which implies decreased JH biosynthesis in the CA, hence the systemic decrease in JH titer. We emphasize that although these previous papers did not examine jhamt expression in ISCs and EBs, they carefully evaluated functions of JHAMT in ISCs and EBs with additional experiments in which they utilize other GAL4 drivers to knock down jhamt in ISCs and EBs via Delta-GAL4 and Su(H)-GAL4 , respectively 6 . Therefore, we argue that although JH biosynthesis most likely occurs in ISCs and EBs, we cannot rule out the possibility that JHs are also supplied from the CA for maintenance of ISCs and EBs. In the last decade, esg-GAL4 and esg-LexA , the other esg promoter-driven binary transcription factors, have widely been used to generate ISC tumors by overexpressing oncogenic genes such as the gain-of-fucntion transgenes, Ras , Raf , and yki 15 , 24 , 25 . In particular, very recently, a number of studies have utilized esg promoter -driven oncogenic gene models to study how ISC tumors impact gut homeostasis, as well as systemic physiology. However, based on our results, when interpreting these esg promoter-driven phenotypes, we should consider not only effects of ISC/EB tumors, but also effects of JH biosynthesis abnormalities caused by CA hypertrophy. For example, some intestinal cells receive JH from the CA through Met and Gce, influencing gut remodeling 5 , 26 . In addition, esg-LexA -driven Ras V 12 results in severe wasting phenotypes in ovaries 27 . Beside Ras V 12 , recent studies have shown that esg ts > yki 3SA leads to severe cachexia and a bloating phenotype, mediated by abnormal hormone secretion from several organs, such as Malpighian tubules and midgut 7 , 24 , 28 . Considering the systemic nature of these ISC/EB tumor-associated phenotypes, it may be necessary to consider the function of JHs, which have major impacts on insect physiology. Generally speaking, it will be important to examine phenotypes using more than just one GAL4 driver. Methods Drosophila strains and maintenance D . melanogaster flies were raised on a standard yeast-corn meal-glucose fly medium (0.275 g agar, 5.0 g glucose, 4.5 g cornmeal, 2.0 g yeast extract, 150 µL propionic acid, and 175 µL 10% butyl p-hydroxybenzoate (in 70% ethanol) in 50 mL water) at 25 ºC under a 12:12 h light/dark cycle. Throughout this study, we used esg ts -GAL4 flies (a gift from Fumiaki Obata, RIKEN Center for Biosystems Dynamics Research) that carried both esg-GAL4 (RRID:BDSC_93857) 2 and tub-GAL80 ts . The following transgenic strains were also used: esg-GFP (BDSC #78333), UAS - jhamt - IR KK (VDRC #103958), and UAS-RasV12 (BDSC #4847). For adult-specific GAL4 activation, flies carrying esg ts -GAL4 were reared at 21°C from embryos to newly eclosed adults. 0–12 hours after eclosion, flies were moved to 29°C. To visualize esg ts > GFP , wandering 3rd -inster larvae were used. Larvae were reared at 25°C until the middle 3rd-larval instar and transferred to 29°C for 24 h before dissection. Heterozygous controls were obtained by crossing w 1118 with strains of GAL4 drivers. Immunohistochemistry Tissues were dissected in PBS and fixed in 4% paraformaldehyde in PBS for 30–60 min at 25–27°C. Fixed samples were rinsed thrice in PBS, washed for 15 min with PBS containing 0.3% Triton X-100 (PBT), and treated with a blocking solution (2% bovine serum albumin in PBT; Sigma #A9647) for 1 h at 25–27°C or overnight at 4°C. Samples were incubated with a primary antibody in blocking solution overnight at 4°C. Primary antibodies used were as follows: chicken anti-GFP antibody (Abcam #ab13970, 1:2,000), guinea pig anti-JHAMT antibody (1:2,000) 29 , rabbit anti-JHAMT antibody (1:1,000) 10 , guinea pig anti-Sro antibody (1:400) 30 . Samples were rinsed thrice with PBS and then washed for 15 min with PBT, followed by incubation with fluorophore (Alexa Fluor 488, 555, and 633)-conjugated secondary antibodies (Thermo Fisher Scientific #A32931, #A21435, #A32732, and #A21105; 1:200), in blocking solution for 2 h at RT or overnight at 4°C. Nuclear stains used in this study were 4',6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich #D9542; final concentration 1 µg/ml). For DAPI staining, after incubation with secondary antibodies, samples were washed and then incubated with DAPI for 1 h. After another round of washing, all samples were mounted on glass slides using FluorSave reagent (Merck Millipore, #345789). Quantification of immunostaining signals was conducted using ImageJ software version 1.53q (Schneider et al., 2012). Reverse transcription-quantitative PCR (RT-qPCR) Total RNA was extracted from whole bodies of 4-day-old adult virgin female flies. RNA was reverse-transcribed using ReverTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO #FSQ-301). Synthesized cDNA samples were used as templates for quantitative PCR using THUNDERBIRD SYBR qPCR Mix (TOYOBO #QPS-201) on a Thermal Cycler Dice Real Time System (Takara Bio #TP870). The amount of target RNA was normalized to the endogenous control ribosomal protein 49 gene ( rp49 ) and the relative change was calculated. Expression levels of each gene were compared using the ΔΔCt method. The following primers were used for this analysis: rp49 F (5'-CGGATCGATATGCTAAGCTGT-3'), rp49 R (5'-GCGCTTGTTCGATCCGTA-3'), kr-h1 F (5'-TCACACATCAAGAAGCCAACT-3'), kr-h1 R (5'-GCTGGTTGGCGGAATAGTAA-3'), obp99b F (5'-AGCACGGATTCGATGTCCACAAGA-3'), obp99b R (5'-TTGGAGTTCATGAAGCACATGCCG-3'), jon25Bii F (5'-CAGGCTCAGTACACCCACAC-3'), jon25Bii R (5'-TGGTGTTGTAGTCCGAGTGC-3'), Statistical analysis All experiments were performed independently at least twice. Sample sizes were chosen based on the number of independent experiments required for statistical significance and technical feasibility. Experiments were not randomized, and investigators were not blinded. All statistical analyses were performed using “R”, software version 4.0.3. Details of statistical analyses are described in figure legends. Declarations Acknowledgements We thank Yuto Yoshinari for critical input during the initial stage of this work. We also thank Fumiaki Obata, Bloomington Stock Center, Vienna Drosophila Resource Center for fly strains, and Yuichiro Nakajima for helpful discussions. This work was supported by the Japan Society of the Promotion of Science KAKENHI (21J20365 to YK and 23KJ0252 to YM) and by the Japan Science and Technology Agency grant SPRING JPMJSP2124. YK, YM, and TK received fellowships from the JSPS. Author contributions (names must be given as initials) T.K. and R.N. designed the research. Y.K., Y.M., and T.K. conceived of the experiment. Y.K. and Y.M. acquired the data. All authors analyzed the data. Y.K., Y.M., and R.N. wrote the manuscript and prepared figures. All authors reviewed the manuscript. Y.K., Y.M., and T.K. equally contributed to this work. Data availability statement (mandatory) All numerical data are available in Supplementary Tables S1 and S2. All other data are available upon request to R.N. Additional Information (including a Competing Interests Statement) The authors declare no competing or financial interests. References Brand, A. H. & Perrimon, N. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118 , 401–415 (1993). Goto, S. & Hayashi, S. Proximal to distal cell communication in the Drosophila leg provides a basis for an intercalary mechanism of limb patterning. Development 126 , 3407–3413 (1999). Jasper, H. Intestinal Stem Cell Aging: Origins and Interventions. Annu. Rev. Physiol. 82 , 203–226 (2020). Weaver, L. 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Supplementary Files KurogiMizunoKamiyamaSciRepSupplementarytables.xlsx Cite Share Download PDF Status: Published Journal Publication published 26 Apr, 2024 Read the published version in Scientific Reports → Version 1 posted Reviewers agreed at journal 18 Jan, 2024 Reviewers agreed at journal 18 Jan, 2024 Reviewers invited by journal 18 Jan, 2024 Editor assigned by journal 18 Jan, 2024 Editor invited by journal 17 Jan, 2024 Submission checks completed at journal 17 Jan, 2024 First submitted to journal 12 Jan, 2024 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3856222","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":267939539,"identity":"8f4370da-f4a5-4a96-adcd-326c75a30b95","order_by":0,"name":"Yoshitomo Kurogi","email":"","orcid":"","institution":"University of Tsukuba","correspondingAuthor":false,"prefix":"","firstName":"Yoshitomo","middleName":"","lastName":"Kurogi","suffix":""},{"id":267939540,"identity":"fae17572-8028-4ffb-a592-c70ebe744e41","order_by":1,"name":"Yosuke Mizuno","email":"","orcid":"","institution":"University of Tsukuba","correspondingAuthor":false,"prefix":"","firstName":"Yosuke","middleName":"","lastName":"Mizuno","suffix":""},{"id":267939541,"identity":"b6eeae95-f928-4d11-badd-5f4212cf0199","order_by":2,"name":"Takumi Kamiyama","email":"","orcid":"","institution":"University of Tsukuba","correspondingAuthor":false,"prefix":"","firstName":"Takumi","middleName":"","lastName":"Kamiyama","suffix":""},{"id":267939542,"identity":"bb3b05bf-c184-43b3-8e95-3fe4dcd3e13b","order_by":3,"name":"Ryusuke Niwa","email":"data:image/png;base64,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","orcid":"","institution":"University of Tsukuba","correspondingAuthor":true,"prefix":"","firstName":"Ryusuke","middleName":"","lastName":"Niwa","suffix":""}],"badges":[],"createdAt":"2024-01-12 08:20:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3856222/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3856222/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-60269-2","type":"published","date":"2024-04-26T19:06:50+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":49854301,"identity":"406fb34c-a6ad-4e79-ac8b-873f9688454c","added_by":"auto","created_at":"2024-01-19 07:00:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2513110,"visible":true,"origin":"","legend":"\u003cp\u003eTranscription factor \u003cem\u003eesg\u003c/em\u003e was expressed in the CA. (\u003cstrong\u003ea\u003c/strong\u003e) (Left) \u003cem\u003eGFP\u003c/em\u003e (green) driven by \u003cem\u003eesg-GAL4\u003c/em\u003e with \u003cem\u003etubP-GAL80\u003c/em\u003e\u003csup\u003e\u003cem\u003ets\u003c/em\u003e\u003c/sup\u003e (\u003cem\u003eesg\u003c/em\u003e\u003csup\u003e\u003cem\u003ets\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u0026gt;\u003c/em\u003e) was expressed in a subpopulation of adult midgut cells. (Right) magnified view of the area enclosed by the white square in the left figure. Blue is the DAPI signal. (\u003cstrong\u003eb\u003c/strong\u003e) \u003cem\u003eesg\u003c/em\u003e\u003csup\u003e\u003cem\u003ets\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u0026gt; GFP\u003c/em\u003e (green) was expressed not only in the part of midgut cells but also in the CA (arrowhead). The CA was labeled with anti-JHAMT antibody (magenta). Two inset images correspond to a region marked with a dashed line surrounding the brain, CA, and proventriculus. Both \u003cem\u003eesg\u003c/em\u003e\u003csup\u003e\u003cem\u003ets\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u0026gt; GFP \u003c/em\u003eand anti-JHAMT immunoreactive signals were observed in the CA. (\u003cstrong\u003ec\u003c/strong\u003e) GFP (green) driven by \u003cem\u003eesg-GAL4\u003c/em\u003e labeled the CA in both adult males (upper) and females (lower). The CA was labeled with anti-JHAMT antibody (magenta). (\u003cstrong\u003ed\u003c/strong\u003e) GFP (green) driven by \u003cem\u003eesg-GAL4\u003c/em\u003e labeled the CA and a part of the PG in wandering L3 larva. The CA was labeled with anti-JHAMT antibody (magenta) and the PG was labeled with anti-Shroud antibody (blue). (\u003cstrong\u003ee\u003c/strong\u003e) (Left) \u003cem\u003eesg-knock in-GFP\u003c/em\u003e (green) was expressed in a subpopulation of adult midgut cells. (Right) A magnified view of the area encircled with a white line in the left figure. Blue is the DAPI signal. (\u003cstrong\u003ef\u003c/strong\u003e) \u003cem\u003eesg-knock in-GFP\u003c/em\u003e (green) was expressed in the CA in both males (upper) and females (lower). The CA was labeled with anti-JHAMT antibody (magenta). (\u003cstrong\u003eg\u003c/strong\u003e) \u003cem\u003eesg-knock in-GFP\u003c/em\u003e (green) was expressed in the CA in wandering L3 larve. The CA was labeled with anti-JHAMT antibody (magenta) and the PG was labeled with anti-Sro antibody (blue).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"KurogiMizunoKamiyamaSciRepFigures131.png","url":"https://assets-eu.researchsquare.com/files/rs-3856222/v1/723fe720343b4544968b42e2.png"},{"id":49854298,"identity":"91efb5bb-91ed-4c5b-9d2f-c2a1a950641e","added_by":"auto","created_at":"2024-01-19 07:00:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":618241,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003ejhamt\u003c/em\u003e-RNAi driven by \u003cem\u003eesg-GAL4\u003c/em\u003e inhibited \u003cem\u003ejhamt\u003c/em\u003e in the CA and reduced the number of eggs in the ovary. (\u003cstrong\u003ea\u003c/strong\u003e) Immunostaining signal of anti-JHAMT (magenta) in the CA was drastically decreased by jhamt-RNAi, driven by \u003cem\u003eesg-GAL4\u003c/em\u003e. Upper panels are controls (\u003cem\u003eesg\u003c/em\u003e\u003csup\u003e\u003cem\u003ets\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u0026gt; GFP\u003c/em\u003e) and lower panels are \u003cem\u003ejhamt\u003c/em\u003e-RNAi (\u003cem\u003eesg\u003c/em\u003e\u003csup\u003e\u003cem\u003ets\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u0026gt; GFP, jhamt-IR\u003c/em\u003e\u003csup\u003e\u003cem\u003eKK\u003c/em\u003e\u003c/sup\u003e) flies. The CA is encircled with a dashed line. (\u003cstrong\u003eb\u003c/strong\u003e) Ovaries of control (upper: \u003cem\u003eesg\u003c/em\u003e\u003csup\u003e\u003cem\u003ets\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u0026gt; GFP\u003c/em\u003e) and \u003cem\u003ejhamt\u003c/em\u003e-RNAi (lower: \u003cem\u003eesg\u003c/em\u003e\u003csup\u003e\u003cem\u003ets\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u0026gt; GFP, jhamt-IR\u003c/em\u003e\u003csup\u003e\u003cem\u003eKK\u003c/em\u003e\u003c/sup\u003e) virgin females. (\u003cstrong\u003ec\u003c/strong\u003e) Numbers of mature eggs in virgin females were significantly decreased by adult-specific \u003cem\u003ejhamt-\u003c/em\u003eRNAi driven by\u003cem\u003e esg-GAL4\u003c/em\u003e. The Wilcoxon rank sum test was used for these data. **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"KurogiMizunoKamiyamaSciRepFigures132.png","url":"https://assets-eu.researchsquare.com/files/rs-3856222/v1/6a796796e79880e87fdec553.png"},{"id":49855267,"identity":"c501e01e-17e1-4db1-826d-ee47283f0025","added_by":"auto","created_at":"2024-01-19 07:08:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1228569,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eRas\u003c/em\u003e\u003csup\u003e\u003cem\u003eV12\u003c/em\u003e\u003c/sup\u003e driven by \u003cem\u003eesg-GAL4\u003c/em\u003e caused hypertrophy of the CA. (\u003cstrong\u003ea\u003c/strong\u003e) Ectopic expression of \u003cem\u003eRasV\u003c/em\u003e\u003csup\u003e\u003cem\u003e12\u003c/em\u003e\u003c/sup\u003e induced overproliferation of \u003cem\u003eesg-GAL4\u003c/em\u003e positive midgut cells (GFP: green). (\u003cstrong\u003eb\u003c/strong\u003e) The CA of control (\u003cem\u003eesg\u003c/em\u003e\u003csup\u003e\u003cem\u003ets\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u0026gt; GFP\u003c/em\u003e) adult virgin females (upper) and RasV\u003csup\u003e12 \u003c/sup\u003eoverexpression (\u003cem\u003eesg\u003c/em\u003e\u003csup\u003e\u003cem\u003ets\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u0026gt; GFP, RasV\u003c/em\u003e\u003csup\u003e\u003cem\u003e12\u003c/em\u003e\u003c/sup\u003e) adult virgin females (lower).\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ec\u003c/strong\u003e) The CA was enlarged by adult-specific \u003cem\u003eRasV\u003c/em\u003e\u003csup\u003e\u003cem\u003e12\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003eoverexpression. (d) Numbers of CA cells were increased by adult-specific \u003cem\u003eRasV\u003c/em\u003e\u003csup\u003e\u003cem\u003e12\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003eoverexpression. (\u003cstrong\u003ee-g\u003c/strong\u003e) Expression levels of JH-responsive genes (e: \u003cem\u003ekr-h1\u003c/em\u003e, f: \u003cem\u003eJon25Bii\u003c/em\u003e, g: \u003cem\u003eObp99b\u003c/em\u003e) were significantly changed by adult-specific \u003cem\u003eRasV\u003c/em\u003e\u003csup\u003e\u003cem\u003e12\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003eoverexpression. The Wilcoxon rank sum test was used for (\u003cstrong\u003ec,d\u003c/strong\u003e). Student’s\u003cem\u003e t\u003c/em\u003e-test was used for (\u003cstrong\u003ee-g\u003c/strong\u003e). *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"KurogiMizunoKamiyamaSciRepFigures133.png","url":"https://assets-eu.researchsquare.com/files/rs-3856222/v1/37cf010e0472a3e09a7714fc.png"},{"id":55546217,"identity":"e6dc6887-3df2-46e2-97ae-0813d871f47f","added_by":"auto","created_at":"2024-04-29 19:07:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2211205,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3856222/v1/6b7930ad-1b13-4030-96a5-bb5beff42091.pdf"},{"id":49854299,"identity":"ddd68a4c-79a2-4a0d-960e-c530a91a276e","added_by":"auto","created_at":"2024-01-19 07:00:02","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":14244,"visible":true,"origin":"","legend":"","description":"","filename":"KurogiMizunoKamiyamaSciRepSupplementarytables.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3856222/v1/03a922e2f8c60f86983d3bde.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"The intestinal stem cell/enteroblast-GAL4 driver, escargot-GAL4, also manipulates gene expression in the juvenile hormone-synthesizing organ of Drosophila melanogaster","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePrecise overexpression of genes in specific cell types and time windows is crucial to discover essential functions of those genes in multicellular organisms. Among model organisms, the fruit fly, \u003cem\u003eDrosophila melanogaster\u003c/em\u003e, is the one for which such gene expression manipulation techniques are best developed. In particular, the GAL4-UAS system is a powerful binary gene expression system in \u003cem\u003eD. melanogaster\u003c/em\u003e for targeted genetic manipulation in a spatio-temporal specific manner to reveal gene functions \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. This system utilizes the yeast transcription factor GAL4, controlled by a tissue-specific enhancer/promoter sequence, in combination with a GAL4-biding element called Upstream Activating Sequence (UAS), inserted upstream of the gene of interest, either endogenously or exogenously. The impact of the GAL4-UAS system on \u003cem\u003eD. melanogaster\u003c/em\u003e genetics research is immeasurable. However, despite its utility, a potential drawback of the GAL4-UAS system is the possibility of incomplete cell type- or tissue-specific expression patterns, complicating interpretation of results.\u003c/p\u003e \u003cp\u003e \u003cem\u003eD. melanogaster escargot (esg)-GAL4\u003c/em\u003e, formally known as P{GawB}NP5130 (RRID:BDSC_93857)\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, has widely been used as the fundamental GAL4 driver to manipulate genes \u0026ldquo;specifically\u0026rdquo; in intestinal stem cells (ISCs) and enteroblasts (EBs) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). In \u003cem\u003eD. melanogaster\u003c/em\u003e, ISCs regulate gut homeostasis by maintaining themselves and also by giving rise to other essential gut epithelial cells, including EBs, enteroendocrine cells, and enterocytes. Dysfunction of ISCs results in severe malfunctions of age-associated tissue integrity in the gut\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. By virtue of convenient tools to analyze functions and roles of genes, \u003cem\u003eD. melanogaster\u003c/em\u003e ISCs have served as a useful model system to study the homeostatic role of ISCs in gut physiology. Notably, the \u003cem\u003eesg-GAL4\u003c/em\u003e driver has contributed to overexpressing genes or other constructs to study fundamental roles of ISCs and EBs. For example, researchers have heavily used \u003cem\u003eesg-GAL4\u003c/em\u003e to generate ISC tumors by overexpressing oncogenic genes such as the gain-of-function transgenes, \u003cem\u003eRas\u003c/em\u003e and \u003cem\u003eyokie(yki)\u003c/em\u003e. \u003cem\u003eesg-GAL4\u003c/em\u003e-driven ISC tumor animals have advanced our understanding of tumor-dependent impairment of systemic physiology, such as cachexia and the bloating phenotype. The crucial assumption for interpreting results of these studies as a phenotype originating from ISCs and EBs is that \u003cem\u003eesg-GAL4\u003c/em\u003e manipulates gene expression only in these cells in adults. However, a recent study reported that \u003cem\u003eesg-GAL4\u003c/em\u003e is expressed at least in brain neurons\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Therefore, it is apparent that characteristics of \u003cem\u003eesg-GAL4\u003c/em\u003e have not yet been sufficiently investigated.\u003c/p\u003e \u003cp\u003eIn this study, we report that \u003cem\u003eesg-GAL4\u003c/em\u003e is also expressed in the insect endocrine organ, the \u003cem\u003ecorpus allatum\u003c/em\u003e (CA), which is essential for synthesizing insect juvenile hormones (JHs). Our data show that \u003cem\u003eesg-GAL4\u003c/em\u003e-driven gene manipulation can systemically influence JH-mediated animal physiology, arguing for cautious use of \u003cem\u003eesg-GAL4\u003c/em\u003e as a \u0026ldquo;specific\u0026rdquo; ISC/EB driver to examine ISC/EB-mediated animal physiology.\u003c/p\u003e"},{"header":"Result","content":"\u003cp\u003e \u003cb\u003eEsg-GAL4\u003c/b\u003e \u003cb\u003eis expressed in the endocrine\u003c/b\u003e \u003cb\u003ecorpus allatum\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe conducted experiments using the \u003cem\u003eesg-GAL4\u003c/em\u003e driver combined with \u003cem\u003etubulin\u003c/em\u003e promoter-driven temperature-sensitive \u003cem\u003eGAL80\u003c/em\u003e (\u003cem\u003etubP-GAL80\u003c/em\u003e\u003csup\u003e\u003cem\u003ets\u003c/em\u003e\u003c/sup\u003e). Hereafter, \u003cem\u003eesg-GAL4; tubP-GAL80\u003c/em\u003e\u003csup\u003e\u003cem\u003ets\u003c/em\u003e\u003c/sup\u003e is designated \u0026ldquo;\u003cem\u003eesg\u003c/em\u003e\u003csup\u003e\u003cem\u003ets\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-GAL4\u0026rdquo;\u003c/em\u003e or \u0026ldquo;\u003cem\u003eesg\u003c/em\u003e\u003csup\u003e\u003cem\u003ets\u003c/em\u003e\u003c/sup\u003e \u003cem\u003e\u0026gt;\u003c/em\u003e\u0026rdquo;. This strain has widely been used for adult stage-specific gene manipulation in ISCs and EBs\u003csup\u003e\u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. In all experimental conditions in this study, we reared \u003cem\u003eesg\u003c/em\u003e\u003csup\u003e\u003cem\u003ets\u003c/em\u003e\u003c/sup\u003e \u003cem\u003e\u0026gt;\u003c/em\u003e flies at a permissive temperature (21\u0026deg;C) during development, such that \u003cem\u003eesg-GAL4\u003c/em\u003e activity is suppressed by GAL80 right before eclosion. Then, after eclosion, we subjected these flies to a restrictive temperature (29\u0026deg;C) to activate \u003cem\u003eesg-GAL4\u003c/em\u003e only in the adult stage.\u003c/p\u003e \u003cp\u003eWe realized by chance that \u003cem\u003eesg\u003c/em\u003e\u003csup\u003e\u003cem\u003ets\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-GAL4\u003c/em\u003e was active in the tissue located between the brain and proventriculus (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). This tissue was co-immunostained with an antibody against Juvenile hormone acid \u003cem\u003eO\u003c/em\u003e-methyltransferase (JHAMT), the essential enzyme that synthesizes JHs in the CA\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. This result strongly indicates that the \u003cem\u003eesg\u003c/em\u003e\u003csup\u003e\u003cem\u003ets\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-GAL4\u003c/em\u003e-positive tissue is the CA. We also confirmed that \u003cem\u003eesg\u003c/em\u003e\u003csup\u003e\u003cem\u003ets\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-GAL4\u003c/em\u003e was expressed in the CA of both male and female adult flies (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Moreover, \u003cem\u003eesg\u003c/em\u003e\u003csup\u003e\u003cem\u003ets\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-GAL4\u003c/em\u003e was expressed in the ring gland, particularly in the CA of wandering 3rd -instar larva, as well as of adults (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). These results suggest that \u003cem\u003eesg\u003c/em\u003e\u003csup\u003e\u003cem\u003ets\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-GAL4\u003c/em\u003e labels the CA in both male and female larvae and adults.\u003c/p\u003e \u003cp\u003eTo confirm whether the \u003cem\u003eesg\u003c/em\u003e gene itself is expressed in the CA, we used the \u003cem\u003eesg-knock-in-GFP\u003c/em\u003e (\u003cem\u003eesg-GFP\u003c/em\u003e) line\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. As with \u003cem\u003eesg\u003c/em\u003e\u003csup\u003e\u003cem\u003ets\u003c/em\u003e\u003c/sup\u003e \u003cem\u003e\u0026gt; GFP\u003c/em\u003e expression, \u003cem\u003eesg-GFP\u003c/em\u003e was expressed not only in a certain cells in the midgut, which seem to be ISCs/EBs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee) \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, but also in the CA of both male and female adults (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). Furthermore, \u003cem\u003eesg-GFP\u003c/em\u003e was expressed in the CA of 3rd instar wandering larvae, while considerable expression of \u003cem\u003eesg-GFP\u003c/em\u003e was also detected in other ring gland cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg). These results suggest that \u003cem\u003eesg\u003c/em\u003e is endogenously expressed in the CA.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRNAi of JH-biosynthetic enzyme by\u003c/b\u003e \u003cb\u003eesg-GAL4\u003c/b\u003e \u003cb\u003ealso impairs oogenesis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eNext, we explored the possibility that \u003cem\u003eesg\u003c/em\u003e\u003csup\u003e\u003cem\u003ets\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-GAL4-\u003c/em\u003edriven transgenic RNAi suppresses gene expression in the CA. To examine this point, we conducted an RNAi experiment with \u003cem\u003eesg\u003c/em\u003e\u003csup\u003e\u003cem\u003ets\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-GAL4\u003c/em\u003e to target \u003cem\u003ejhamt\u003c/em\u003e, which is expressed explicitly in the CA\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Immunostaining signals of anti-JHAMT antibody were drastically decreased by \u003cem\u003ejhamt\u003c/em\u003e RNAi, compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn many insects, including \u003cem\u003eD. melanogaster\u003c/em\u003e, JH promotes ovarian development by accumulating yolk components such as yolk protein and vitellogenin\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. In \u003cem\u003eD. melanogaster\u003c/em\u003e, a previous study reported that loss of \u003cem\u003ejhamt\u003c/em\u003e activity results in smaller ovaries and reduced egg numbers\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Therefore, we observed ovary morphology and counted the number of mature eggs in adult females expressing \u003cem\u003eesg\u003c/em\u003e\u003csup\u003e\u003cem\u003ets\u003c/em\u003e\u003c/sup\u003e \u003cem\u003e\u0026gt; jhamt\u003c/em\u003e RNAi. We found that \u003cem\u003eesg\u003c/em\u003e\u003csup\u003e\u003cem\u003ets\u003c/em\u003e\u003c/sup\u003e \u003cem\u003e\u0026gt; jhamt\u003c/em\u003e RNAi flies had smaller ovaries than controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Consistent with this observation, the number of mature eggs was significantly decreased in RNAi flies (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). These results suggest that \u003cem\u003eesg\u003c/em\u003e\u003csup\u003e\u003cem\u003ets\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-GAL4\u003c/em\u003e-driven RNAi suppresses gene expression in the CA and influences JH-mediated biological events such as oogenesis.\u003c/p\u003e \u003cp\u003e \u003cb\u003eOncogenic\u003c/b\u003e \u003cb\u003eRas\u003c/b\u003e\u003csup\u003e\u003cb\u003eV\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eexpression by\u003c/b\u003e \u003cb\u003eesg-GAL4\u003c/b\u003e \u003cb\u003ecauses CA hypertrophy and abnormal expression of JH-responsive genes\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn some recent studies, \u003cem\u003eesg\u003c/em\u003e\u003csup\u003e\u003cem\u003ets\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-GAL4\u003c/em\u003e and \u003cem\u003eUAS-Ras\u003c/em\u003e\u003csup\u003e\u003cem\u003eV\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e have been utilized to induce ISC/EB tumors to investigate cell turnover in the midgut and tumor-mediated systemic physiology\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Since \u003cem\u003eesg-GAL4\u003c/em\u003e is also expressed in the CA, \u003cem\u003eesg\u003c/em\u003e\u003csup\u003e\u003cem\u003ets\u003c/em\u003e\u003c/sup\u003e \u003cem\u003e\u0026gt; Ras\u003c/em\u003e\u003csup\u003e\u003cem\u003eV12\u003c/em\u003e\u003c/sup\u003e might affect both ISC/EB and CA cells. Notably, \u003cem\u003eesg\u003c/em\u003e\u003csup\u003e\u003cem\u003ets\u003c/em\u003e\u003c/sup\u003e \u003cem\u003e\u0026gt; Ras\u003c/em\u003e\u003csup\u003e\u003cem\u003eV12\u003c/em\u003e\u003c/sup\u003e resulted not only in abnormal expansion of the \u003cem\u003eesg\u003c/em\u003e\u003csup\u003e\u003cem\u003ets\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-GAL4\u003c/em\u003e-driven \u003cem\u003eGFP\u003c/em\u003e-positive area in the midgut (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea) \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, but also increased CA size and cell number (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb-d).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eConsidering morphological abnormalities in the CA, it seemed possible that \u003cem\u003eesg\u003c/em\u003e\u003csup\u003e\u003cem\u003ets\u003c/em\u003e\u003c/sup\u003e \u003cem\u003e\u0026gt; Ras\u003c/em\u003e\u003csup\u003e\u003cem\u003eV12\u003c/em\u003e\u003c/sup\u003e expression enhances JH biosynthesis in the CA. Therefore, we next performed quantitative PCR on three JH-responsive genes, \u003cem\u003eKr\u0026uuml;ppel-homolog 1\u003c/em\u003e (\u003cem\u003eKr-h1\u003c/em\u003e), \u003cem\u003eJonah 25Bii\u003c/em\u003e (\u003cem\u003ejon25Bii\u003c/em\u003e), \u003cem\u003eOdorant-binding protein 99b\u003c/em\u003e (\u003cem\u003eObp99b\u003c/em\u003e), to estimate the amount of JH in \u003cem\u003eRas\u003c/em\u003e\u003csup\u003e\u003cem\u003eV\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e overexpressors and controls\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Previous studies have shown that expression levels of \u003cem\u003eKr-h1\u003c/em\u003e and \u003cem\u003ejon25Bii\u003c/em\u003e correlate positively with the amount of JH in the body, while \u003cem\u003eObp99b\u003c/em\u003e correlates negatively\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Our qPCR results showed that expression levels of \u003cem\u003eKr-h1\u003c/em\u003e and \u003cem\u003ejon25Bii\u003c/em\u003e were increased by \u003cem\u003eRas\u003c/em\u003e\u003csup\u003e\u003cem\u003eV\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e, while \u003cem\u003eObp99b\u003c/em\u003e was decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee-g). These results strongly suggest that \u003cem\u003eesg\u003c/em\u003e\u003csup\u003e\u003cem\u003ets\u003c/em\u003e\u003c/sup\u003e \u003cem\u003e\u0026gt; Ras\u003c/em\u003e\u003csup\u003e\u003cem\u003eV12\u003c/em\u003e\u003c/sup\u003e leads to abnormalities in CA cells and increased JH biosynthesis.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we found that \u003cem\u003eesg-GAL4\u003c/em\u003e, which is widely used to label midgut ISC/EB\u003csup\u003e2\u003c/sup\u003e, was also expressed in the CA. Genetic manipulation with \u003cem\u003eesg\u003c/em\u003e\u003csup\u003e\u003cem\u003ets\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-GAL4\u003c/em\u003e, such as overexpression of \u003cem\u003eRas\u003c/em\u003e\u003csup\u003e\u003cem\u003eV\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e, caused CA hypertrophy and influenced JH-responsive gene expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb-g), suggesting that \u003cem\u003eesg\u003c/em\u003e\u003csup\u003e\u003cem\u003ets\u003c/em\u003e\u003c/sup\u003e \u003cem\u003e\u0026gt; Ras\u003c/em\u003e\u003csup\u003e\u003cem\u003eV12\u003c/em\u003e\u003c/sup\u003e increases JH biosynthesis.\u003c/p\u003e \u003cp\u003eOur data suggest that \u003cem\u003eesg\u003c/em\u003e itself is expressed in the CA in both males and females of both larvae and adults. Enrichment of \u003cem\u003eesg\u003c/em\u003e expression in the larval ring gland was suggested by a previous microarray analysis comparing gene expression between the ring glands and whole body\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eesg\u003c/em\u003e encodes a Snail-type transcription factor that contributes to cell cycle regulation, cell differentiation, and cell-cell adhesion in many cell types in \u003cem\u003eD. melanogaster\u003c/em\u003e\u003csup\u003e\u003cspan additionalcitationids=\"CR21 CR22\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. However, functions of Esg that regulate differentiation and morphogenesis of the CA have not been studied. Thus, additional studies are needed to clarify how Esg is involved in CA cell regulation, especially whether it regulates JH biosynthesis.\u003c/p\u003e \u003cp\u003eIn \u003cem\u003eD. melanogaster\u003c/em\u003e, one of the reported functions of JHs is that these hormones directly act on ISCs and EBs through the nuclear JH receptors, Methoprene-tolerant (Met) and Germ cell expressed (Gce), to regulate gut remodeling in mated or aged females\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Interestingly, previous studies reported that \u003cem\u003eesg\u003c/em\u003e\u003csup\u003e\u003cem\u003ets\u003c/em\u003e\u003c/sup\u003e \u003cem\u003e\u0026gt; jhamt\u003c/em\u003e RNAi reduces numbers of ISCs and EBs. This phenotype is also observed in \u003cem\u003eesg\u003c/em\u003e\u003csup\u003e\u003cem\u003ets\u003c/em\u003e\u003c/sup\u003e \u003cem\u003e\u0026gt; Met\u003c/em\u003e or \u003cem\u003eGce\u003c/em\u003e RNAi animals. Since these studies use \u003cem\u003eesg\u003c/em\u003e\u003csup\u003e\u003cem\u003ets\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-GAL4\u003c/em\u003e as the ISC/EB-\u0026ldquo;specific\u0026rdquo; GAL4 driver, these papers propose that JHs are biosynthesized in ISCs and EBs outside the CA, and cell-autonomously regulate maintenance of ISCs and EBs during aging\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. However, our data strongly indicate that \u003cem\u003eesg-GAL4\u003c/em\u003e is also expressed in the CA. More importantly, \u003cem\u003eesg\u003c/em\u003e\u003csup\u003e\u003cem\u003ets\u003c/em\u003e\u003c/sup\u003e \u003cem\u003e\u0026gt; jhamt\u003c/em\u003e RNAi causes a decrease in JHAMT protein in the CA (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), which implies decreased JH biosynthesis in the CA, hence the systemic decrease in JH titer. We emphasize that although these previous papers did not examine \u003cem\u003ejhamt\u003c/em\u003e expression in ISCs and EBs, they carefully evaluated functions of JHAMT in ISCs and EBs with additional experiments in which they utilize other GAL4 drivers to knock down \u003cem\u003ejhamt\u003c/em\u003e in ISCs and EBs via \u003cem\u003eDelta-GAL4\u003c/em\u003e and \u003cem\u003eSu(H)-GAL4\u003c/em\u003e, respectively\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Therefore, we argue that although JH biosynthesis most likely occurs in ISCs and EBs, we cannot rule out the possibility that JHs are also supplied from the CA for maintenance of ISCs and EBs.\u003c/p\u003e \u003cp\u003eIn the last decade, \u003cem\u003eesg-GAL4\u003c/em\u003e and \u003cem\u003eesg-LexA\u003c/em\u003e, the other \u003cem\u003eesg\u003c/em\u003e promoter-driven binary transcription factors, have widely been used to generate ISC tumors by overexpressing oncogenic genes such as the gain-of-fucntion transgenes, \u003cem\u003eRas\u003c/em\u003e, \u003cem\u003eRaf\u003c/em\u003e, and \u003cem\u003eyki\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. In particular, very recently, a number of studies have utilized \u003cem\u003eesg promoter\u003c/em\u003e-driven oncogenic gene models to study how ISC tumors impact gut homeostasis, as well as systemic physiology. However, based on our results, when interpreting these \u003cem\u003eesg\u003c/em\u003e promoter-driven phenotypes, we should consider not only effects of ISC/EB tumors, but also effects of JH biosynthesis abnormalities caused by CA hypertrophy. For example, some intestinal cells receive JH from the CA through Met and Gce, influencing gut remodeling\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. In addition, \u003cem\u003eesg-LexA\u003c/em\u003e-driven \u003cem\u003eRas\u003c/em\u003e\u003csup\u003e\u003cem\u003eV\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e results in severe wasting phenotypes in ovaries\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Beside \u003cem\u003eRas\u003c/em\u003e\u003csup\u003e\u003cem\u003eV\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e, recent studies have shown that \u003cem\u003eesg\u003c/em\u003e\u003csup\u003e\u003cem\u003ets\u003c/em\u003e\u003c/sup\u003e \u003cem\u003e\u0026gt; yki\u003c/em\u003e\u003csup\u003e\u003cem\u003e3SA\u003c/em\u003e\u003c/sup\u003e leads to severe cachexia and a bloating phenotype, mediated by abnormal hormone secretion from several organs, such as Malpighian tubules and midgut\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Considering the systemic nature of these ISC/EB tumor-associated phenotypes, it may be necessary to consider the function of JHs, which have major impacts on insect physiology. Generally speaking, it will be important to examine phenotypes using more than just one GAL4 driver.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e \u003cb\u003eDrosophila\u003c/b\u003e \u003cb\u003estrains and maintenance\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eD\u003c/em\u003e. \u003cem\u003emelanogaster\u003c/em\u003e flies were raised on a standard yeast-corn meal-glucose fly medium (0.275 g agar, 5.0 g glucose, 4.5 g cornmeal, 2.0 g yeast extract, 150 \u0026micro;L propionic acid, and 175 \u0026micro;L 10% butyl p-hydroxybenzoate (in 70% ethanol) in 50 mL water) at 25 \u0026ordm;C under a 12:12 h light/dark cycle.\u003c/p\u003e \u003cp\u003eThroughout this study, we used \u003cem\u003eesg\u003c/em\u003e\u003csup\u003e\u003cem\u003ets\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-GAL4\u003c/em\u003e flies (a gift from Fumiaki Obata, RIKEN Center for Biosystems Dynamics Research) that carried both \u003cem\u003eesg-GAL4\u003c/em\u003e (RRID:BDSC_93857)\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e and \u003cem\u003etub-GAL80\u003c/em\u003e\u003csup\u003e\u003cem\u003ets\u003c/em\u003e\u003c/sup\u003e. The following transgenic strains were also used: \u003cem\u003eesg-GFP\u003c/em\u003e (BDSC #78333), \u003cem\u003eUAS\u003c/em\u003e-\u003cem\u003ejhamt\u003c/em\u003e-\u003cem\u003eIR\u003c/em\u003e\u003csup\u003eKK\u003c/sup\u003e (VDRC #103958), and \u003cem\u003eUAS-RasV12\u003c/em\u003e (BDSC #4847). For adult-specific \u003cem\u003eGAL4\u003c/em\u003e activation, flies carrying \u003cem\u003eesg\u003c/em\u003e\u003csup\u003e\u003cem\u003ets\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-GAL4\u003c/em\u003e were reared at 21\u0026deg;C from embryos to newly eclosed adults. 0\u0026ndash;12 hours after eclosion, flies were moved to 29\u0026deg;C. To visualize \u003cem\u003eesg\u003c/em\u003e\u003csup\u003e\u003cem\u003ets\u003c/em\u003e\u003c/sup\u003e \u003cem\u003e\u0026gt; GFP\u003c/em\u003e, wandering 3rd -inster larvae were used. Larvae were reared at 25\u0026deg;C until the middle 3rd-larval instar and transferred to 29\u0026deg;C for 24 h before dissection. Heterozygous controls were obtained by crossing \u003cem\u003ew\u003c/em\u003e\u003csup\u003e\u003cem\u003e1118\u003c/em\u003e\u003c/sup\u003e with strains of \u003cem\u003eGAL4\u003c/em\u003e drivers.\u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry\u003c/h2\u003e \u003cp\u003eTissues were dissected in PBS and fixed in 4% paraformaldehyde in PBS for 30\u0026ndash;60 min at 25\u0026ndash;27\u0026deg;C. Fixed samples were rinsed thrice in PBS, washed for 15 min with PBS containing 0.3% Triton X-100 (PBT), and treated with a blocking solution (2% bovine serum albumin in PBT; Sigma #A9647) for 1 h at 25\u0026ndash;27\u0026deg;C or overnight at 4\u0026deg;C. Samples were incubated with a primary antibody in blocking solution overnight at 4\u0026deg;C. Primary antibodies used were as follows: chicken anti-GFP antibody (Abcam #ab13970, 1:2,000), guinea pig anti-JHAMT antibody (1:2,000)\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, rabbit anti-JHAMT antibody (1:1,000)\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, guinea pig anti-Sro antibody (1:400)\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Samples were rinsed thrice with PBS and then washed for 15 min with PBT, followed by incubation with fluorophore (Alexa Fluor 488, 555, and 633)-conjugated secondary antibodies (Thermo Fisher Scientific #A32931, #A21435, #A32732, and #A21105; 1:200), in blocking solution for 2 h at RT or overnight at 4\u0026deg;C. Nuclear stains used in this study were 4',6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich #D9542; final concentration 1 \u0026micro;g/ml). For DAPI staining, after incubation with secondary antibodies, samples were washed and then incubated with DAPI for 1 h. After another round of washing, all samples were mounted on glass slides using FluorSave reagent (Merck Millipore, #345789). Quantification of immunostaining signals was conducted using ImageJ software version 1.53q (Schneider et al., 2012).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eReverse transcription-quantitative PCR (RT-qPCR)\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from whole bodies of 4-day-old adult virgin female flies. RNA was reverse-transcribed using ReverTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO #FSQ-301). Synthesized cDNA samples were used as templates for quantitative PCR using THUNDERBIRD SYBR qPCR Mix (TOYOBO #QPS-201) on a Thermal Cycler Dice Real Time System (Takara Bio #TP870). The amount of target RNA was normalized to the endogenous control \u003cem\u003eribosomal protein 49\u003c/em\u003e gene (\u003cem\u003erp49\u003c/em\u003e) and the relative change was calculated. Expression levels of each gene were compared using the ΔΔCt method. The following primers were used for this analysis: \u003cem\u003erp49\u003c/em\u003e F (5'-CGGATCGATATGCTAAGCTGT-3'), \u003cem\u003erp49\u003c/em\u003e R (5'-GCGCTTGTTCGATCCGTA-3'), \u003cem\u003ekr-h1\u003c/em\u003e F (5'-TCACACATCAAGAAGCCAACT-3'), \u003cem\u003ekr-h1\u003c/em\u003e R (5'-GCTGGTTGGCGGAATAGTAA-3'), \u003cem\u003eobp99b\u003c/em\u003e F (5'-AGCACGGATTCGATGTCCACAAGA-3'), \u003cem\u003eobp99b\u003c/em\u003e R (5'-TTGGAGTTCATGAAGCACATGCCG-3'),\u003c/p\u003e \u003cp\u003e \u003cem\u003ejon25Bii\u003c/em\u003e F (5'-CAGGCTCAGTACACCCACAC-3'), \u003cem\u003ejon25Bii\u003c/em\u003e R (5'-TGGTGTTGTAGTCCGAGTGC-3'),\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll experiments were performed independently at least twice. Sample sizes were chosen based on the number of independent experiments required for statistical significance and technical feasibility. Experiments were not randomized, and investigators were not blinded. All statistical analyses were performed using \u0026ldquo;R\u0026rdquo;, software version 4.0.3. Details of statistical analyses are described in figure legends.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Yuto Yoshinari for critical input during the initial stage of this work. We also thank Fumiaki Obata, Bloomington Stock Center, Vienna \u003cem\u003eDrosophila\u003c/em\u003e Resource Center for fly strains, and Yuichiro Nakajima for helpful discussions. This work was supported by the Japan Society of the Promotion of Science KAKENHI (21J20365 to YK and 23KJ0252 to YM) and by the Japan Science and Technology Agency grant SPRING JPMJSP2124. YK, YM, and TK received fellowships from the JSPS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions (names must be given as initials)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eT.K. and R.N. designed the research. Y.K., Y.M., and T.K. conceived of the experiment. Y.K. and Y.M. acquired the data. All authors analyzed the data. Y.K., Y.M., and R.N. wrote the manuscript and prepared figures. All authors reviewed the manuscript. Y.K., Y.M., and T.K.\u0026nbsp;equally contributed to this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement (mandatory)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll numerical data are available in Supplementary Tables S1 and S2. All other data are available upon request to R.N.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional Information (including a Competing Interests Statement)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing or financial interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBrand, A. H. \u0026amp; Perrimon, N. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. \u003cem\u003eDevelopment\u003c/em\u003e \u003cstrong\u003e118\u003c/strong\u003e, 401\u0026ndash;415 (1993).\u003c/li\u003e\n\u003cli\u003eGoto, S. \u0026amp; Hayashi, S. Proximal to distal cell communication in the Drosophila leg provides a basis for an intercalary mechanism of limb patterning. \u003cem\u003eDevelopment\u003c/em\u003e \u003cstrong\u003e126\u003c/strong\u003e, 3407\u0026ndash;3413 (1999).\u003c/li\u003e\n\u003cli\u003eJasper, H. 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Growth Differ.\u003c/em\u003e \u003cstrong\u003e63\u003c/strong\u003e, 249\u0026ndash;261 (2021).\u003c/li\u003e\n\u003cli\u003eShimada-Niwa, Y. \u0026amp; Niwa, R. Serotonergic neurons respond to nutrients and regulate the timing of steroid hormone biosynthesis in Drosophila. \u003cem\u003eNat. Commun.\u003c/em\u003e\u003cstrong\u003e5\u003c/strong\u003e, (2014).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3856222/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3856222/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIntestinal stem cells (ISCs) of the fruit fly, \u003cem\u003eDrosophila melanogaster\u003c/em\u003e, offer an excellent genetic model to explore homeostatic roles of ISCs in animal physiology. Among available genetic tools, the \u003cem\u003eescargot\u003c/em\u003e (\u003cem\u003eesg\u003c/em\u003e)\u003cem\u003e-GAL4\u003c/em\u003e driver, expressing the yeast transcription factor gene, \u003cem\u003eGAL4\u003c/em\u003e, under control of the \u003cem\u003eesg\u003c/em\u003e gene promoter, has contributed significantly to ISC studies. This driver facilitates activation of a gene of interest in proximity to a GAL4-binding element, UAS, in ISCs and progenitor enteroblasts (EBs). While \u003cem\u003eesg-GAL4\u003c/em\u003e has been considered an ISC/EB-specific driver, its actual specificity remains unexplored. In this study, we reveal \u003cem\u003eesg-GAL4\u003c/em\u003e expression in the \u003cem\u003ecorpus allatum\u003c/em\u003e (CA), responsible for juvenile hormone (JH) production. When driving the oncogenic gene \u003cem\u003eRas\u003c/em\u003e\u003csup\u003e\u003cem\u003eV\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003eesg-GAL4\u003c/em\u003e induces overgrowth in ISCs/EBs as reported, but also increases CA cell number and size. Consistent with this observation, animals alter expression of JH-response genes. Our data show that \u003cem\u003eesg-GAL4\u003c/em\u003e-driven gene manipulation can systemically influence JH-mediated animal physiology, arguing for cautious use of \u003cem\u003eesg-GAL4\u003c/em\u003e as a \u0026ldquo;specific\u0026rdquo; ISC/EB driver to examine ISC/EB-mediated animal physiology.\u003c/p\u003e","manuscriptTitle":"The intestinal stem cell/enteroblast-GAL4 driver, escargot-GAL4, also manipulates gene expression in the juvenile hormone-synthesizing organ of Drosophila melanogaster","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-19 06:59:58","doi":"10.21203/rs.3.rs-3856222/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"d1516f48-25d6-421a-b0b7-1c6d4dc44bfa","date":"2024-01-18T13:43:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"6ada5877-fd59-4ab0-bd3c-5bd982241012","date":"2024-01-18T13:31:01+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-01-18T13:22:51+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-01-18T13:08:43+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-01-17T20:29:45+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-01-17T20:28:58+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-01-12T08:18:14+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a4ef7d2c-dec8-48d4-b96d-4eb55018124d","owner":[],"postedDate":"January 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":28234329,"name":"Biological sciences/Genetics"},{"id":28234330,"name":"Biological sciences/Genetics/Gene expression"},{"id":28234331,"name":"Biological sciences/Zoology/Animal physiology"},{"id":28234332,"name":"Biological sciences/Biological techniques/Genetic techniques"}],"tags":[],"updatedAt":"2024-04-29T19:06:50+00:00","versionOfRecord":{"articleIdentity":"rs-3856222","link":"https://doi.org/10.1038/s41598-024-60269-2","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-04-26 19:06:50","publishedOnDateReadable":"April 26th, 2024"},"versionCreatedAt":"2024-01-19 06:59:58","video":"","vorDoi":"10.1038/s41598-024-60269-2","vorDoiUrl":"https://doi.org/10.1038/s41598-024-60269-2","workflowStages":[]},"version":"v1","identity":"rs-3856222","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3856222","identity":"rs-3856222","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}