Autophagy controls differentiation of Drosophila blood cells by regulating Notch levels in response to nutrient availability

Autophagy controls differentiation of Drosophila blood cells by regulating Notch levels in response to nutrient availability | 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 Autophagy controls differentiation of Drosophila blood cells by regulating Notch levels in response to nutrient availability Pablo Wappner, Maximiliano Katz, Felipe Rodríguez, Fermín Evangelisti, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4707838/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Jul, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Drosophila larval hematopoiesis takes place at the lymph gland, where blood cell progenitors differentiate into two possible cell types: plasmatocytes, analogous to mammalian macrophages, or crystal cells that share features with mammalian megakaryocytes; a third cell type, the lamellocytes, can develop only upon specific immune challenges. In this work, we investigate the role of autophagy in Drosophila hematopoiesis. We found that autophagy inhibition in blood cell progenitors results in augmented crystal cell differentiation due to accumulation of high levels of Notch protein. Notch activation during hematopoiesis depends on the endocytic pathway, which cross-talks with autophagy: While endocytosis and endosomal maturation are essential for Notch activation, autophagosomes are required for Notch lysosomal degradation. TOR signaling inhibits autophagosome biogenesis, which in turn prevents the formation of Notch-containing amphisomes, being the latter necessary for Notch lysosomal destruction. Reduction of Notch lysosomal degradation shifts the balance towards Notch activation at late endosomal membranes, thereby enhancing differentiation of crystal cells. Our work defines a novel mechanism of regulation of immune cell differentiation in response to the nutritional status of the organism: High nutrient availability induces TOR activation, thereby inhibiting autophagy, hindering lysosomal degradation of Notch, and promoting crystal cell differentiation. Biological sciences/Developmental biology/Differentiation Biological sciences/Cell biology/Autophagy/Macroautophagy Biological sciences/Developmental biology/Haematopoiesis Biological sciences/Cell biology/Cell signalling/Extracellular signalling molecules Biological sciences/Cell biology/Membrane trafficking/Endocytosis Blood cell progenitors Drosophila lymph gland Autophagy Notch signaling TOR pathway. Full Text Additional Declarations There is NO Competing Interest. Supplementary Files SupplementalInformationKatzetal.pdf Cite Share Download PDF Status: Published Journal Publication published 01 Jul, 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. 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-4707838","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":329901056,"identity":"4e6840cb-2b88-4363-a3c0-c1a15eaacdb0","order_by":0,"name":"Pablo Wappner","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7ElEQVRIiWNgGAWjYDADfnYGxgMMBqRokWxmYCBRi8FhkBZigLlE8rPHhTts8o0PMx848KHgHgO/9PELDD/34NZiOSPN3HjmmTTLbYfZEg7OMChmkOzLKWDseYbHPbcTzKR52w4bmB3mMQCiBAaDMzwJDDx43GhwO/0bUMt/A+Nm/g9wLYx/8GrJAdlywMCAmYcBqoX9ADM+Wyznvyk3ntmWbCBxmM0A6JcEHskeoF4ZPFrMeY5ve1zYZmfA39788MGHPwly/DzsDx++wecwBgY2ZmQBHiAywKMBixYgYH+AT8coGAWjYBSMPAAAaMJN7+dlSmAAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-1517-0742","institution":"Instituto Leloir","correspondingAuthor":true,"prefix":"","firstName":"Pablo","middleName":"","lastName":"Wappner","suffix":""},{"id":329901057,"identity":"1a29e85f-8721-404b-adbe-5f781170137a","order_by":1,"name":"Maximiliano Katz","email":"","orcid":"","institution":"Instituto de Fisiología y Biofísica Bernardo Houssay","correspondingAuthor":false,"prefix":"","firstName":"Maximiliano","middleName":"","lastName":"Katz","suffix":""},{"id":329901058,"identity":"10ccf271-4acf-4eed-864e-a092d36e83b1","order_by":2,"name":"Felipe Rodríguez","email":"","orcid":"","institution":"Fundación Instituto Leloir","correspondingAuthor":false,"prefix":"","firstName":"Felipe","middleName":"","lastName":"Rodríguez","suffix":""},{"id":329901059,"identity":"1439f676-7663-4274-9ee0-62c592aa9980","order_by":3,"name":"Fermín Evangelisti","email":"","orcid":"","institution":"Fundación Instituto Leloir","correspondingAuthor":false,"prefix":"","firstName":"Fermín","middleName":"","lastName":"Evangelisti","suffix":""},{"id":329901060,"identity":"0738a740-e15e-4392-9532-8f1507c18749","order_by":4,"name":"Sebastián Perez-Pandolfo","email":"","orcid":"","institution":"Fundación Instituto Leloir","correspondingAuthor":false,"prefix":"","firstName":"Sebastián","middleName":"","lastName":"Perez-Pandolfo","suffix":""},{"id":329901061,"identity":"4cdbeb65-6f6b-45db-a86d-4f63a44b89cc","order_by":5,"name":"Natalia Sommario","email":"","orcid":"https://orcid.org/0009-0006-6725-2829","institution":"Fundación Instituto Leloir","correspondingAuthor":false,"prefix":"","firstName":"Natalia","middleName":"","lastName":"Sommario","suffix":""},{"id":329901062,"identity":"4acb8862-68bf-43b2-9746-a9e730569fd1","order_by":6,"name":"Agustina Borrat","email":"","orcid":"","institution":"Fundación Instituto Leloir","correspondingAuthor":false,"prefix":"","firstName":"Agustina","middleName":"","lastName":"Borrat","suffix":""},{"id":329901063,"identity":"87aa1912-062f-4c55-b165-717ca681e138","order_by":7,"name":"Mariana Melani","email":"","orcid":"","institution":"Fundación Instituto Leloir","correspondingAuthor":false,"prefix":"","firstName":"Mariana","middleName":"","lastName":"Melani","suffix":""}],"badges":[],"createdAt":"2024-07-08 20:45:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4707838/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4707838/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-025-58389-y","type":"published","date":"2025-07-01T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":85828712,"identity":"fb4f84d4-a8b1-4346-9403-34ea729b7d2d","added_by":"auto","created_at":"2025-07-02 07:31:23","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2597856,"visible":true,"origin":"","legend":"","description":"","filename":"Katzetal.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4707838/v1_covered_c1c94398-075e-44a2-a3b0-79665f6dedea.pdf"},{"id":60866870,"identity":"59fc4215-e044-42e8-b8ba-d8748063ebc0","added_by":"auto","created_at":"2024-07-23 03:37:00","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1147723,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"SupplementalInformationKatzetal.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4707838/v1/5fa498264529083da6c0cce6.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Autophagy controls differentiation of \u003ci\u003eDrosophila\u003c/i\u003e blood cells by regulating Notch levels in response to nutrient availability","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"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":"Blood cell progenitors, Drosophila lymph gland, Autophagy, Notch signaling, TOR pathway.","lastPublishedDoi":"10.21203/rs.3.rs-4707838/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4707838/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ci\u003eDrosophila\u003c/i\u003e larval hematopoiesis takes place at the lymph gland, where blood cell progenitors differentiate into two possible cell types: plasmatocytes, analogous to mammalian macrophages, or crystal cells that share features with mammalian megakaryocytes; a third cell type, the lamellocytes, can develop only upon specific immune challenges. In this work, we investigate the role of autophagy in \u003ci\u003eDrosophila\u003c/i\u003e hematopoiesis. We found that autophagy inhibition in blood cell progenitors results in augmented crystal cell differentiation due to accumulation of high levels of Notch protein. Notch activation during hematopoiesis depends on the endocytic pathway, which cross-talks with autophagy: While endocytosis and endosomal maturation are essential for Notch activation, autophagosomes are required for Notch lysosomal degradation. TOR signaling inhibits autophagosome biogenesis, which in turn prevents the formation of Notch-containing amphisomes, being the latter necessary for Notch lysosomal destruction. Reduction of Notch lysosomal degradation shifts the balance towards Notch activation at late endosomal membranes, thereby enhancing differentiation of crystal cells. Our work defines a novel mechanism of regulation of immune cell differentiation in response to the nutritional status of the organism: High nutrient availability induces TOR activation, thereby inhibiting autophagy, hindering lysosomal degradation of Notch, and promoting crystal cell differentiation.","manuscriptTitle":"Autophagy controls differentiation of Drosophila blood cells by regulating Notch levels in response to nutrient availability","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-23 03:36:55","doi":"10.21203/rs.3.rs-4707838/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":"ec8e0531-0304-4fcf-9a65-8083cc36b2ad","owner":[],"postedDate":"July 23rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":34918515,"name":"Biological sciences/Developmental biology/Differentiation"},{"id":34918516,"name":"Biological sciences/Cell biology/Autophagy/Macroautophagy"},{"id":34918517,"name":"Biological sciences/Developmental biology/Haematopoiesis"},{"id":34918518,"name":"Biological sciences/Cell biology/Cell signalling/Extracellular signalling molecules"},{"id":34918519,"name":"Biological sciences/Cell biology/Membrane trafficking/Endocytosis"}],"tags":[],"updatedAt":"2025-07-02T07:31:16+00:00","versionOfRecord":{"articleIdentity":"rs-4707838","link":"https://doi.org/10.1038/s41467-025-58389-y","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-07-01 04:00:00","publishedOnDateReadable":"July 1st, 2025"},"versionCreatedAt":"2024-07-23 03:36:55","video":"","vorDoi":"10.1038/s41467-025-58389-y","vorDoiUrl":"https://doi.org/10.1038/s41467-025-58389-y","workflowStages":[]},"version":"v1","identity":"rs-4707838","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4707838","identity":"rs-4707838","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}