Unraveling the role of Djstat5 in regulating planarian regeneration through modulating cell proliferation and apoptosis

Unraveling the role of Djstat5 in regulating planarian regeneration through modulating cell proliferation and apoptosis | 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 Research Article Unraveling the role of Djstat5 in regulating planarian regeneration through modulating cell proliferation and apoptosis Du Wang, Hanxue Zheng, Jiayi Chen, Mengdi Cheng, Linfeng Li, Yujie Nie, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6401380/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Signal transducer and activator of transcription (STAT) proteins are critical for converting extracellular stimuli into transcriptional programs that govern cellular homeostasis. Although extensively studied in mammals, the functions of STAT genes in invertebrates remain poorly understood. In this study, we investigated the role of Djstat5, a STAT5 homolog in the planarian Dugesia japonica, revealing its dynamic activation during wound response and regeneration initiation. The RNA interference (RNAi) knockdown of Djstat5 severely disrupted tissue regeneration, manifesting as blastemal hypoplasia coupled with attenuated mitotic and apoptotic dynamics. The transcriptomic analysis revealed that Djstat5 knockdown led to decreased expression of Djcbp (a chromatin regulator) and increased expression of Djnlrc3 (an inflammatory modulator). Furthermore, the results of double RNAi experiments demonstrated a functional antagonism between these downstream effectors, with Djcbp knockdown exacerbating while Djnlrc3 silencing ameliorating Djstat5-deficient phenotypes. In conclusion, our study highlights the critical role of Djstat5 in planarian regeneration and provides new insights into the underlying molecular mechanisms. STAT planarian regeneration stem cells Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Full Text Supplementary Files SupplementaryInformation.pdf Cite Share Download PDF Status: Posted 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-6401380","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":450802829,"identity":"87f860f2-9e3c-47c7-ac7f-d2a25672dcb6","order_by":0,"name":"Du Wang","email":"","orcid":"","institution":"Northwest University","correspondingAuthor":false,"prefix":"","firstName":"Du","middleName":"","lastName":"Wang","suffix":""},{"id":450802830,"identity":"ba92e6da-8f4a-42f8-9fd2-02a74212402c","order_by":1,"name":"Hanxue Zheng","email":"","orcid":"","institution":"Northwest University","correspondingAuthor":false,"prefix":"","firstName":"Hanxue","middleName":"","lastName":"Zheng","suffix":""},{"id":450802831,"identity":"010fd0cc-06b6-4924-a01a-504932364dee","order_by":2,"name":"Jiayi Chen","email":"","orcid":"","institution":"Northwest University","correspondingAuthor":false,"prefix":"","firstName":"Jiayi","middleName":"","lastName":"Chen","suffix":""},{"id":450802832,"identity":"aec31eaf-2c0f-40dc-8843-6510290d8d97","order_by":3,"name":"Mengdi Cheng","email":"","orcid":"","institution":"Northwest University","correspondingAuthor":false,"prefix":"","firstName":"Mengdi","middleName":"","lastName":"Cheng","suffix":""},{"id":450802833,"identity":"f09d0b43-2440-45f4-8e5f-038a6bf55c2b","order_by":4,"name":"Linfeng Li","email":"","orcid":"","institution":"Northwest University","correspondingAuthor":false,"prefix":"","firstName":"Linfeng","middleName":"","lastName":"Li","suffix":""},{"id":450802834,"identity":"bb810e3f-e5e8-4996-9a98-af77f6ae108f","order_by":5,"name":"Yujie Nie","email":"","orcid":"","institution":"Northwest University","correspondingAuthor":false,"prefix":"","firstName":"Yujie","middleName":"","lastName":"Nie","suffix":""},{"id":450802835,"identity":"528e24f3-73fb-4f27-93fd-c10f539fe5cd","order_by":6,"name":"Yanmei Qiang","email":"","orcid":"","institution":"Northwest University","correspondingAuthor":false,"prefix":"","firstName":"Yanmei","middleName":"","lastName":"Qiang","suffix":""},{"id":450802836,"identity":"7736190b-a833-4cd2-b9d1-ac23b03b3d58","order_by":7,"name":"Wenjuan Xue","email":"","orcid":"","institution":"Northwest University","correspondingAuthor":false,"prefix":"","firstName":"Wenjuan","middleName":"","lastName":"Xue","suffix":""},{"id":450802837,"identity":"08fb6a0d-9992-4104-b08d-18f69bc6b368","order_by":8,"name":"Ruohan Lin","email":"","orcid":"","institution":"Northwest University","correspondingAuthor":false,"prefix":"","firstName":"Ruohan","middleName":"","lastName":"Lin","suffix":""},{"id":450802838,"identity":"449ecf56-18b4-4cd5-be9f-a811fa445673","order_by":9,"name":"Xin Leng","email":"","orcid":"","institution":"Northwest University","correspondingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Leng","suffix":""},{"id":450802839,"identity":"552690d8-b38e-47f3-b041-0dbad3b04f62","order_by":10,"name":"Fulin Chen","email":"","orcid":"","institution":"Northwest University","correspondingAuthor":false,"prefix":"","firstName":"Fulin","middleName":"","lastName":"Chen","suffix":""},{"id":450802840,"identity":"e383b0ac-ab85-45ab-a75f-f2defd79161e","order_by":11,"name":"Yuan Yu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAsklEQVRIiWNgGAWjYPACGwjFQ4KWNNK1HCZBi8GNHDOJnzvO5xncSGB88LaNQd6cGC2SvWduFwO1MBvObWMw3NlAjC28bbcTt91IYJPmbWNIMDhAjC1/286BtLD/JloL0PADYFuYidIieeZZsbVsW3Li/jMPmyXnnJMw3EBIC9/x5I0337bZJc5sTz744U2ZjTxBWxQOcBhAmYwNQEKCgHogkG9gf0BY1SgYBaNgFIxsAACbaEL0pgDrSAAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-7123-669X","institution":"Northwest University","correspondingAuthor":true,"prefix":"","firstName":"Yuan","middleName":"","lastName":"Yu","suffix":""}],"badges":[],"createdAt":"2025-04-08 09:06:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6401380/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6401380/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82056533,"identity":"9b4bbbb6-51b8-40e2-b597-e731a965d379","added_by":"auto","created_at":"2025-05-06 10:37:55","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2377940,"visible":true,"origin":"","legend":"\u003cp\u003eSpatiotemporal expression of \u003cem\u003eDjstat5 \u003c/em\u003ein planarians\u003cstrong\u003e. A \u003c/strong\u003eWISH analysis of \u003cem\u003eDjstat5 \u003c/em\u003eexpression in intact planarians. Scale bars = 1 mm. \u003cstrong\u003eB \u003c/strong\u003eViolin plots depicting the distribution of \u003cem\u003eSmed-stat5 \u003c/em\u003eexpression levels across individual cells (represented as dots) within 12 neoblast clusters and 10 clusters of sub-lethally irradiated X1 and X2 cells. \u003cstrong\u003eC \u003c/strong\u003eRepresentative WISH of \u003cem\u003eDjstat5 \u003c/em\u003ein intact animals, with confocal images of fluorescent \u003cem\u003ein situ \u003c/em\u003ehybridization for \u003cem\u003eDjstat5 \u003c/em\u003e(red) and H3P antibody (green). White arrows indicate double-positive cells. Confocal images are partial projections of the dashed box regions. Scale bar = 10 pm. \u003cstrong\u003eD \u003c/strong\u003eSpatiotemporal expression pattern of \u003cem\u003eDjstat5 \u003c/em\u003eduring planarian regeneration assessed by WISH. Scale bars = 1 mm.\u003c/p\u003e","description":"","filename":"Fig1.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6401380/v1/b96b3a88a98de9b0a3889c2c.jpg"},{"id":82055923,"identity":"55950c96-e44d-4db2-9d12-8fb07799354a","added_by":"auto","created_at":"2025-05-06 10:29:55","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2241836,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of \u003cem\u003eDjstat5 knockdown \u003c/em\u003eon the regeneration of planarians. \u003cstrong\u003eA \u003c/strong\u003ePlanarians were subjected to RNAi feeding every other day for a total of 5 feedings, followed by amputation and assessment of knockdown efficiency after a 3-day interval. \u003cstrong\u003eB \u003c/strong\u003eWestern blot analysis of nuclear proteins extracted from tail fragments at 2 dpa to confirm the levels of p-Stat5. Histone H3 was used as an interanal control. \u003cstrong\u003eC \u003c/strong\u003eDorsal views of tail fragments undergoing regeneration in both control and \u003cem\u003eDjstat5 \u003c/em\u003eRNAi animals, captured at various time points: 0 hpa, 6 hpa, 1, 3, 5, and 7 dpa. The newly formed photoreceptors are highlighted with yellow arrows. The scale bar = 1 mm. \u003cstrong\u003eD \u003c/strong\u003eQuantitative analysis of blastema growth is presented in Fig. 2C. Blastema length was measured and normalized to the overall body length of the trunk. \u003cstrong\u003eE \u003c/strong\u003eImmunostaining of the regenerated central nervous system (CNS) at 7 dpa using an anti-SYNORF1 antibody. Scale bar = 100 pm. \u003cstrong\u003eF \u003c/strong\u003eWISH analysis of intestine (\u003cem\u003eDjporcn\u003c/em\u003e), pharynx (\u003cem\u003eDjmhc\u003c/em\u003e) and epidermis (\u003cem\u003eDjifb\u003c/em\u003e) regeneration in control and \u003cem\u003eDjstat5 \u003c/em\u003eRNAi animals at 7 dpa. Scale bar = 1 mm. \u003cstrong\u003eG \u003c/strong\u003eRepresentative images of mitotic activity (green dots, H3P positive cells) at 6 and 48 hpa. Scale bar = 100 pm. \u003cstrong\u003eH \u003c/strong\u003eTUNEL staining of apoptotic nuclei (red dots) in tail fragments at 6 and 48 hpa. Scale bar = 100 pm.\u003c/p\u003e","description":"","filename":"Fig2.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6401380/v1/38d6db05fb020cd7a3f4e5f9.jpg"},{"id":82055925,"identity":"9e1ad794-6b44-4b46-88e8-224b2d3f8d4d","added_by":"auto","created_at":"2025-05-06 10:29:55","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1450879,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification and analysis of \u003cem\u003eDjstat5 \u003c/em\u003edownstream genes. \u003cstrong\u003eA \u003c/strong\u003eSchematic diagram of RNA-seq sampling strategy. \u003cstrong\u003eB \u003c/strong\u003eVolcano plot showing the significance (-log10 (p-value)) versus fold change (log2 (Fold Change)) for gene expression between the control and \u003cem\u003eDjstat5 \u003c/em\u003eRNAi samples. Blue dots indicate significantly upregulated genes, red dots indicate significantly downregulated genes, and gray dots represent non­significant genes. Dashed lines mark the significance and fold change thresholds. \u003cstrong\u003eC \u003c/strong\u003eBar chart of KEGG pathway enrichment analysis for differentially expressed genes. The x-axis represents the -log10 (p-value) of enrichment significance, and the y-axis displays the KEGG pathways. Each bar represents a specific pathway, with its lenth indicating the level of enrichment significance. \u003cstrong\u003eD \u003c/strong\u003eGO analysis of differentially expressed genes. The x-axis represents Rich factor, and the y-axis shows the GO term. Dot size indicates the number of associated genes, and color represents significance, with redder colors indicating higher significance. \u003cstrong\u003eE \u003c/strong\u003eqRT-PCR validation of selected differentially expressed genes identified through transcriptomic sequencing.\u003c/p\u003e","description":"","filename":"Fig3.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6401380/v1/8c3e8b595d4c49a71dba262f.jpg"},{"id":82056534,"identity":"327b031c-d557-4fbd-a487-e61bf6b6ded6","added_by":"auto","created_at":"2025-05-06 10:37:55","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3389665,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eDjstat5 \u003c/em\u003eregulates planarian regeneration through \u003cem\u003eDjcbp \u003c/em\u003eand \u003cem\u003eDjnlrc3. \u003c/em\u003e\u003cstrong\u003eA \u003c/strong\u003eDorsal views of tail fragments undergoing regeneration in control, \u003cem\u003eDjstat5 \u003c/em\u003e(RNAi), \u003cem\u003eDjstat5\u003c/em\u003e+\u003cem\u003eDjcbp \u003c/em\u003e(RNAi) and \u003cem\u003eDjstat5\u003c/em\u003e+\u003cem\u003eDjnlrc3 \u003c/em\u003e(RNAi) animals at 7 dpa. Yellow arrows indicate newly formed photoreceptors. Scale bar = 1 mm. \u003cstrong\u003eB \u003c/strong\u003eImmunostaining of the regenerated CNS at 7 dpa using an anti-SYNORF1 antibody. Scale bar = 100 μm. \u003cstrong\u003eC \u003c/strong\u003eWISH analysis of intestine (\u003cem\u003eDjporcn\u003c/em\u003e), pharynx (\u003cem\u003eDjmhc\u003c/em\u003e) and epidermis (\u003cem\u003eDjifb\u003c/em\u003e) regeneration in control and RNAi animals at 7 dpa. Scale bar = 1 mm. \u003cstrong\u003eD \u003c/strong\u003eRepresentative images of mitotic activity (green dots, H3P positive cells) at 6 and 48 hpa. Scale bar = 100 μm. \u003cstrong\u003eE \u003c/strong\u003eTUNEL staining of apoptotic nuclei (red dots) in tail fragments at 6 and 48 hpa. Scale bar = 100 μm.\u003c/p\u003e","description":"","filename":"Fig4.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6401380/v1/963528e3012928d0f6116088.jpg"},{"id":82055927,"identity":"7040d893-54bb-4e50-bcf4-05bb21fb97da","added_by":"auto","created_at":"2025-05-06 10:29:55","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1176453,"visible":true,"origin":"","legend":"\u003cp\u003eModel of \u003cem\u003eDjstat5 \u003c/em\u003emechanisms in regeneration. Model of \u003cem\u003eDjstat5 \u003c/em\u003efunction in planarian regeneration. \u003cem\u003eDjstat5 \u003c/em\u003econtrols regenerative proliferation and apoptotic by upregulating \u003cem\u003eDjcbp \u003c/em\u003eand inversely regulating \u003cem\u003eDjnlrc3\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Fig5.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6401380/v1/9284ff3f2281effe8685c7b9.jpg"},{"id":83796943,"identity":"2da8df63-b7b4-4e28-ab5d-01eaf0465fd7","added_by":"auto","created_at":"2025-06-03 01:08:14","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11138952,"visible":true,"origin":"","legend":"","description":"","filename":"Manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6401380/v1_covered_5ef9ecc3-f866-4e11-bee9-c0c5244fb815.pdf"},{"id":82055931,"identity":"bab7d61d-d4a1-4e1c-b12f-7577b20a3dda","added_by":"auto","created_at":"2025-05-06 10:29:55","extension":"pdf","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":930891,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6401380/v1/c794b98ef26988c51b859107.pdf"}],"financialInterests":"","formattedTitle":"Unraveling the role of Djstat5 in regulating planarian regeneration through modulating cell proliferation and apoptosis","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"STAT, planarian, regeneration, stem cells","lastPublishedDoi":"10.21203/rs.3.rs-6401380/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6401380/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Signal transducer and activator of transcription (STAT) proteins are critical for converting extracellular stimuli into transcriptional programs that govern cellular homeostasis. Although extensively studied in mammals, the functions of STAT genes in invertebrates remain poorly understood. In this study, we investigated the role of Djstat5, a STAT5 homolog in the planarian Dugesia japonica, revealing its dynamic activation during wound response and regeneration initiation. The RNA interference (RNAi) knockdown of Djstat5 severely disrupted tissue regeneration, manifesting as blastemal hypoplasia coupled with attenuated mitotic and apoptotic dynamics. The transcriptomic analysis revealed that Djstat5 knockdown led to decreased expression of Djcbp (a chromatin regulator) and increased expression of Djnlrc3 (an inflammatory modulator). Furthermore, the results of double RNAi experiments demonstrated a functional antagonism between these downstream effectors, with Djcbp knockdown exacerbating while Djnlrc3 silencing ameliorating Djstat5-deficient phenotypes. In conclusion, our study highlights the critical role of Djstat5 in planarian regeneration and provides new insights into the underlying molecular mechanisms.","manuscriptTitle":"Unraveling the role of Djstat5 in regulating planarian regeneration through modulating cell proliferation and apoptosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-06 10:29:50","doi":"10.21203/rs.3.rs-6401380/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4810270b-8737-45ee-85f7-78de5b24408e","owner":[],"postedDate":"May 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-06-03T00:59:59+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-06 10:29:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6401380","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6401380","identity":"rs-6401380","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}