Revealing a critical role of the incomplete spindle assembly checkpoint in zebrafish development through an optochemical approach

Revealing a critical role of the incomplete spindle assembly checkpoint in zebrafish development through an optochemical approach | 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 Revealing a critical role of the incomplete spindle assembly checkpoint in zebrafish development through an optochemical approach Ryota Uehara, Akira Matsura, Miyu Hosono, Kazuya Mastuo, Nobuyuki Tamaoki, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7071246/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Early animal embryos must balance the efficiency with the accuracy of mitotic control. However, the extent of mitotic errors that can be safely endured at different stages of development is unclear. In this study, using a recently developed photoswitchable CENP-E inhibitor, we introduced transient mitotic errors at various developmental windows and systematically addressed their organismal effects. Upon CENP-E inhibition in the pre-gastrula period, embryos suffered gradual aggravation of developmental defects as the duration of the inhibition extended. Conversely, embryos tolerated several hours of consecutive CENP-E inhibition in the gastrula period, frequently achieving full development. Live imaging revealed that chromosome misalignment caused by CENP-E inhibition resulted in a modest mitotic delay in the gastrula, but not in the early pre-gastrula period, suggesting the functionalization of the spindle assembly checkpoint (SAC) at this stage. This mitotic delay helped alleviate, though not perfectly resolve, polar chromosome misalignment before anaphase onset. Importantly, pharmacological suppression of SAC rendered gastrula embryos inviable upon CENP-E inhibition. Therefore, despite its leaky nature, the embryonic SAC contributed to partial mitotic error correction, which proved essential to manage consecutive mitotic perturbations. Our results demonstrate the power of optochemical approaches in understanding the robust control of dynamic processes in development. Biological sciences/Developmental biology/Cell proliferation Biological sciences/Cell biology/Cell division/Checkpoints Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Full Text Additional Declarations There is NO Competing Interest. Supplementary Files nrreportingsummary250705250707am.pdf ng_reporting_summery MovieS11.mp4 Movie S1. Fluorescence imaging of a DMSO-treated 3.5-hpf embryo Confocal slice sections of a DMSO-treated 3.5-hpf embryo. DNA (gray) and F-actin (magenta) were stained with DAPI and Acti-stain 555 phalloidin dye, respectively. Depth from the top surface of the embryo is indicated. Bar, 100 μm. MovieS21.mp4 Movie S2. Fluorescence imaging of a PCEI-HU Vis -treated 3.5-hpf embryo Confocal slice sections of a PCEI-HU Vis -treated 3.5-hpf embryo. DNA (gray) and F-actin (magenta) were stained with DAPI and Acti-stain 555 phalloidin dye, respectively. Depth from the top surface of the embryo is indicated. Bar, 100 μm. Fig7.jpg Figure S1. The time course of the viable embryonic proportion after PCEI-HU treatment Viability of embryos treated with PCEI-HU Vis during the time windows indicated at the top of the graphs. Means ± s.e. of at least three independent experiments, except for the 3-12 hpf condition tested in two independent experiments. Identical data for the 60-hpf time point are shown in Fig. 2B and 4B. At least 20 embryos were analyzed for each condition. Fig8.jpg Figure S2. SAC-dependent mitotic delay upon CENP-E inhibition in the gastrula period Histograms of mitotic duration in early pre-gastrula or gastrula embryonic cells in each drug treatment condition. At least 128 cells from at least three embryos in three independent experiments were analyzed for each condition. Fig9.jpg Figure S3. Enlarged views of cleaved caspase-3 and phospho-histone H3 immunostaining A merged image of DAPI-stained DNA, immunostained pH3 and cleaved caspase-3 in PCEI-HU Vis (5-12 hpf) and reversine-co-treated embryos at 24 hpf. The identical embryo is also shown in Fig. 6D. An example of the whole-head view of the staining is shown at the top. The box represents the region of the embryo enlarged in the bottom panels. Fig10.jpg Figure S4. Apoptotic and mitotic cell distribution at 72 hpf after CENP-E inhibition during the gastrula period (A) Immunostaining of cleaved caspase-3 and pH3 in the whole-mount embryos at 72 hpf in each condition. The box represents the region of the embryo enlarged in the bottom panels. DNA was stained with DAPI. (B, C) The density (cell number per volume) of cleaved caspase-3- or pH3-positive cells in the left midbrain in embryos in D. Ten confocal slice sections from the top of the midbrain were used for counting the number of positive cells. Means ± s.e. of at least 8 embryos from three independent experiments. There is no statistically significant difference from control (n.s.: not significant, Welch t-test). Cite Share Download PDF Status: Under Review 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. 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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-7071246","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":490791681,"identity":"89510001-36da-46bf-970b-ee19a8649857","order_by":0,"name":"Ryota Uehara","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABB0lEQVRIiWNgGAWjYHACNhAhx8DA2MDMwJDAwHCAsQEkwkxIizHpWhIbIKpAWgi4ynxG8rEHP/fYpc9vP9zAXFCRlth3/HADw48aBnZzHFpkbqSlG/Y8S87dcCaxgXnGmZzEmUAGY88xBmbLBuxaJCRyzCR4DjDnbpAA+oW3rSJxwwGgI3kbGJgNcLhQQiL/m+SfA/Xp8jNgWs4/bGD8i1dLDps0z4HDCQw3wFpyEjfcALoQry08z8ykZQ4cNwT55TDPmTTjmTceNhyWOSaB2y/syc8k3xyolpdvP/7wMU9Fsmzf+fSHD9/U2CTjCjEUcACJIZFsQIwWFGBHupZRMApGwSgYpgAAlA5dGJYAyM4AAAAASUVORK5CYII=","orcid":"","institution":"Faculty of Advanced Life Science, Hokkaido University, Japan","correspondingAuthor":true,"prefix":"","firstName":"Ryota","middleName":"","lastName":"Uehara","suffix":""},{"id":490791682,"identity":"63148493-0c2c-49bd-8775-d929e4550f5b","order_by":1,"name":"Akira Matsura","email":"","orcid":"","institution":"Hokkaido University","correspondingAuthor":false,"prefix":"","firstName":"Akira","middleName":"","lastName":"Matsura","suffix":""},{"id":490791683,"identity":"19a7bd7f-9da5-4a3c-8004-05e4c68d32ff","order_by":2,"name":"Miyu Hosono","email":"","orcid":"","institution":"Hokkaido University","correspondingAuthor":false,"prefix":"","firstName":"Miyu","middleName":"","lastName":"Hosono","suffix":""},{"id":490791684,"identity":"b7b49565-eb45-462c-9e0e-61a245f176fd","order_by":3,"name":"Kazuya Mastuo","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Kazuya","middleName":"","lastName":"Mastuo","suffix":""},{"id":490791685,"identity":"72ee7cfb-85ea-477b-9298-913edad54af6","order_by":4,"name":"Nobuyuki Tamaoki","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Nobuyuki","middleName":"","lastName":"Tamaoki","suffix":""},{"id":490791686,"identity":"e8fcf4c6-8d37-47eb-97a2-0204167c877a","order_by":5,"name":"Tomoya Kotani","email":"","orcid":"https://orcid.org/0000-0003-1930-6635","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Tomoya","middleName":"","lastName":"Kotani","suffix":""}],"badges":[],"createdAt":"2025-07-08 06:30:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7071246/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7071246/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87905053,"identity":"92e70626-b924-40eb-9d97-b1991a176585","added_by":"auto","created_at":"2025-07-30 08:42:49","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":541663,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eApplication of PCEI-HU to early zebrafish embryos\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003ePhotoisomerization between the non-inhibitory \u003cem\u003ecis \u003c/em\u003eand inhibitory \u003cem\u003etrans \u003c/em\u003estate of PCEI-HU. The photoswitchable azobenzene-derived moiety is color-labeled. \u003cstrong\u003e(B, D) \u003c/strong\u003eFluorescence microscopy of cells on the outermost embryonic layer during mitosis (B) or interphase (D) in 3-hpf embryos treated with or without PCEI-HU\u003csup\u003e\"is\u003c/sup\u003e. DNA and F-actin were stained with DAPI and Acti-stain 555 phalloidin dye, respectively. The arrow indicates polar misaligned chromosomes in B, or micronuclei in D. \u003cstrong\u003e(C, E) \u003c/strong\u003eFrequency of polar chromosome misalignment in B (C) or micronuclei in D (E). At least 40 cells from 8 embryos of two independent experiments were analyzed. Asterisks indicate statistically significant difference among conditions (n.s.: not significant, ***\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001, the Fisher exact test with the Benjamini-Hochberg multiple testing correction). \u003cstrong\u003e(F) \u003c/strong\u003eThe time course of the viable embryonic proportion in the cultures treated with DMSO (vehicle control), non-irradiated, or pre-irradiated PCEI-HU. Means ± s.e. of three independent experiments. At least 26 embryos were analyzed for each condition. Asterisks indicate statistically significant difference from control at 60 hpf (***\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001, the Steel test).\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7071246/v1/944c47818a88a170520782fc.jpg"},{"id":87905055,"identity":"7813ac48-c58e-4548-b3f7-49fe892dd470","added_by":"auto","created_at":"2025-07-30 08:42:49","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":66865,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGradual aggravation of embryonic defects by extension of CENP-E inhibition time window in pre-gastrula embryos\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eA schematic image of time schedules for PCEI-HU treatment and photoswitching. Lights were shortly irradiated at the indicated times to convert the inhibitor between inhibitor and non-inhibitory states. Double arrows indicate the time windows for transient CENP-E inhibition (treated with PCEI-HU\u003csup\u003e\"is\u003c/sup\u003e). \u003cstrong\u003e(B) \u003c/strong\u003eFrequency of viable embryos at 60 hpf after the treatment with PCEI-HU according to the light irradiation schedule in A. Means ± s.e. of three independent experiments. At least 24 embryos were analyzed for each condition. Asterisks indicate statistically significant difference from control for each light irradiation schedule (n.s.: not significant, *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep \u003c/em\u003e\u0026lt;\u003c/p\u003e\n\u003cp\u003e0.01, Welch t-test). \u003cstrong\u003e(C) \u003c/strong\u003eTransparent microscopy of embryos at 60 hpf after the treatment with PCEI-HU according to the schedule in A. The dorsal views of the whole body (left) or the lateral view of the head region (right). \u003cstrong\u003e(D, E) \u003c/strong\u003eMeasurement of body axis length (D) and eye surface area (E) in control and PCEI-HU-treated embryos in C. Box plots and beeswarm plots of at least 12 embryos (24 eyes) from at least two independent experiments, except for the 2-4 hpf condition. For the 2-4 hpf condition, at least 4 embryos (8 eyes) from only one experiment were analyzed, as all embryos in the other two experiments died by 60 hpf after PCEI-HU treatment in this condition. Asterisks indicate statistically significant difference from control for each light irradiation schedule (n.s.: not significant, **\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001, Welch t-test). Time windows for PCEI-HU\u003csup\u003e\"is\u003c/sup\u003e treatment are indicated in the figure or graph captions.\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7071246/v1/4cd2cfd7d8504a814a5df28c.jpg"},{"id":87905447,"identity":"c331bd30-7b5d-49bc-b17e-5fe494e45b45","added_by":"auto","created_at":"2025-07-30 08:50:49","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":508828,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEmbryos survive through a few rounds of mitotic errors in the pre-gastrula period (A) \u003c/strong\u003eA schematic image of time schedules for PCEI-HU treatment, photoswitching, and live imaging. The time window for PCEI-HU\u003csup\u003e\"is\u003c/sup\u003e treatment (also corresponding to the live imaging time window) is indicated by the double arrow. \u003cstrong\u003e(B) \u003c/strong\u003eLive images of embryos at the 11\u003csup\u003eth\u003c/sup\u003e division expressing histone H2B-mCherry during PCEI-HU\u003csup\u003e\"is\u003c/sup\u003e treatment in A. Projected images of five Z-slices. Arrows indicate polar chromosome misalignment. \u003cstrong\u003e(C) \u003c/strong\u003eMitotic phenotype in each division cycle during the early pre-gastrula period in live imaging in B and the subsequent fate of the embryo at 30 dpf. Each row represents a single embryo. Mitotic phenotype is categorized as indicated in B. Data were obtained from at least two independent experiments for each condition.\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7071246/v1/e42b8b954eece92a5abe8988.jpg"},{"id":87905449,"identity":"ff29f5fd-4c24-4531-93b9-6369df77e74e","added_by":"auto","created_at":"2025-07-30 08:50:50","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":562765,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTolerance of embryos to consecutive CENP-E inhibition during the gastrula period (A) \u003c/strong\u003eA schematic image of time schedules for PCEI-HU treatment and photoswitching. \u003cstrong\u003e(B) \u003c/strong\u003eFrequency of viable embryos at 60 hpf after PCEI-HU treatment according to the schedule in A. Means ± s.e. of three independent experiments, except for the 3-12 hpf condition tested with two independent experiments. At least 20 embryos were analyzed for each condition, except for the 3-12\u003c/p\u003e\n\u003cp\u003ehpf condition, which was tested with at least 17 embryos. Asterisks indicate statistically significant difference from control for each light irradiation schedule (n.s.: not significant, **\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01, Welch t-test). \u003cstrong\u003e(C) \u003c/strong\u003eTransparent microscopy of embryos at 60 hpf after the treatment with PCEI-HU according to the schedule in A. The dorsal views of the whole body (left) or the lateral view of the head region (right). \u003cstrong\u003e(D, E) \u003c/strong\u003eMeasurement of body axis length (D) and eye surface area (E) in control and PCEI-HU-treated embryos in C. Box plots and beeswarm plots of at least 9 embryos (18 eyes) from at least two independent experiments, except for the 2.5-12 hpf condition. For the 2.5-12 hpf condition, at least 2 embryos (4 eyes) from only one experiment were analyzed, as all embryos in the other two experiments died by 60 hpf after PCEI-HU treatment in this condition. Asterisks indicate statistically significant difference from control for each light irradiation schedule (n.s.: not significant, *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, ***\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001, Welch t-test). Note that, due to the drastic embryonic death by 2.5-12 hpf PCEI-HU\u003csup\u003e\"is\u003c/sup\u003e treatment, the sample size of body length is insufficient for statistical analysis for this condition. \u003cstrong\u003e(F) \u003c/strong\u003eImages of embryos at 7.5 dpf after PCEI-HU\u003csup\u003e\"is\u003c/sup\u003e (5-12 hpf) treatment. Arrowheads indicate edema formation. \u003cstrong\u003e(G) \u003c/strong\u003eFrequency of embryos with or without edema formation at 7.5 dpf. Means ± s.e. of three independent experiments. At least 29 embryos were analyzed for each condition. There is no statistically significant difference between the conditions (n.s.: not significant, Welch t-test). \u003cstrong\u003e(H) \u003c/strong\u003eExamples of zebrafish individuals (1 year old) fully developed after PCEI-HU\u003csup\u003e\"is\u003c/sup\u003e (5-12 hpf) treatment.\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7071246/v1/316c109a383590d8a2b9612d.jpg"},{"id":87905066,"identity":"9e5521cd-c172-4a73-aa75-a89bf1477e1c","added_by":"auto","created_at":"2025-07-30 08:42:50","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":570284,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFunctionalization of incomplete SAC in the gastrula period\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eLive images of mitotic cells in early pre-gastrula or gastrula embryos expressing H2B-mCherry. Embryos were treated with DMSO (vehicle control) or PCEI-HU\u003csup\u003e\"is\u003c/sup\u003e in combination with or without reversine. Images were taken at 2-min intervals. Arrows indicate polar chromosome misalignment. The timing of NEBD is set as 0 min. Data for early pre-gastrula embryos were obtained from the identical experiments to those in Fig. 3. \u003cstrong\u003e(B) \u003c/strong\u003eFrequency of polar chromosome misalignment in A. Pooled data from at least 81 cells in at least 2 embryos from at least two independent experiments.\u003c/p\u003e\n\u003cp\u003eAsterisks indicate statistically significant difference from control for each light irradiation schedule (n.s.: not significant, **\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001, Fisher exact test with or without the Benjamini-Hochberg multiple testing correction for gastrula or pre-gastrula embryos, respectively. \u003cstrong\u003e(C) \u003c/strong\u003eMitotic duration in A. Means ± s.e. of at least 3 embryos from three independent experiments. At least 128 cells were analyzed for each condition. Asterisks indicate statistically significant difference from control for each light irradiation schedule (n.s.: not significant, ***\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001, Wilcoxon test for pre-gastrula and Steel-Dwass test for gastrula embryos). Distributions of mitotic duration in individual cells are shown in Fig. S3. \u003cstrong\u003e(D) \u003c/strong\u003eConfocal microscopy of mitotic cells in gastrula embryos expressing H2B-mCherry. Embryos were treated with DMSO, PCEI-HU\u003csup\u003eVis\u003c/sup\u003e, or a combination of PCEI-HU\u003csup\u003eVis\u003c/sup\u003e and reversine. Images were taken at 2-min intervals. Arrows indicate polar chromosome misalignment. The arrowhead indicates lagging chromosomes. The timing of NEBD is set as 0 min. The time frames used for the quantification in E are color-labeled. \u003cstrong\u003e(E) \u003c/strong\u003eQuantification of number of misaligned chromosomes at early prometaphase (early PM, 4 min after NEBD; only for the PCEI-HU\u003csup\u003eVis\u003c/sup\u003e-treated condition) and late prometaphase (late PM, right before anaphase onset; for all conditions) in D. Because of the short time window of prometaphase in DMSO-, or PCEI-HU\u003csup\u003eVis\u003c/sup\u003e and reversine-co-treated cells, we could not distinguish early prometaphase from late one in many cases in these conditions. At least 46 cells in 3 embryos from 3 independent experiments were analyzed.\u003c/p\u003e","description":"","filename":"Fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7071246/v1/e3af595277207dab70d7d4f7.jpg"},{"id":87905065,"identity":"6e9fe641-9516-42e6-a1d7-250f8bbfed60","added_by":"auto","created_at":"2025-07-30 08:42:50","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":594394,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSAC inactivation renders gastrula embryos inviable upon consecutive CENP-E inhibition\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eThe time course of the viable embryonic proportion in the cultures treated with DMSO, reversine, PCEI-HU\u003csup\u003eVis\u003c/sup\u003e (5-12 hpf), or reversine and PCEI-HU\u003csup\u003eVis\u003c/sup\u003e (5-12 hpf). Means ± s.e. of three independent experiments. At least 28 embryos were analyzed for each condition. Asterisks indicate statistically significant difference from control at 60 hpf (***\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001, the Steel test). \u003cstrong\u003e(B, C) \u003c/strong\u003eMeasurement of body axis length (B) and eye surface area (C) in each condition. Box plots and beeswarm plots of at least 4 embryos (8 eyes) from two independent experiments. Asterisks indicate\u003c/p\u003e\n\u003cp\u003estatistically significant difference among conditions (n.s.: not significant, ***\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001, Tukey-Kramer test). \u003cstrong\u003e(D) \u003c/strong\u003eImmunostaining of cleaved caspase-3 and phospho-histone H3 (pH3) in whole-mount embryos at 24 hpf in each condition. DNA was stained with DAPI. An example of enlarged view of the staining is shown in Fig. S3. \u003cstrong\u003e(E, F) \u003c/strong\u003eThe density (cell number per volume) of cleaved caspase-3- or pH3-positive cells in the whole-head region in embryos in D. Five confocal slice sections from the top of the head were used for counting the number of positive cells. Means ± s.e. of at least 5 embryos from two independent experiments. Asterisks indicate statistically significant difference among conditions (n.s.: not significant, *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, ***\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001, Tukey-Kramer test).\u003c/p\u003e","description":"","filename":"Fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7071246/v1/5a2741b823525f5ee27b5146.jpg"},{"id":87906457,"identity":"11c93603-3568-4c78-9b91-e7b05a98bf83","added_by":"auto","created_at":"2025-07-30 08:58:53","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3543005,"visible":true,"origin":"","legend":"Article File","description":"","filename":"MSMatsurazebra2507084.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7071246/v1_covered_1e11aca6-fb73-4d71-9c5b-e08376413b52.pdf"},{"id":87905057,"identity":"4903795e-49e9-42a4-a5ee-7e80ae0ee044","added_by":"auto","created_at":"2025-07-30 08:42:49","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1666331,"visible":true,"origin":"","legend":"ng_reporting_summery","description":"","filename":"nrreportingsummary250705250707am.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7071246/v1/208d83598f6c152fc4392d1b.pdf"},{"id":87905450,"identity":"c5fcf669-873c-4f5b-8b03-5e57d228f2d0","added_by":"auto","created_at":"2025-07-30 08:50:50","extension":"mp4","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":12102988,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMovie S1. Fluorescence imaging of a DMSO-treated 3.5-hpf embryo\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConfocal slice sections of a DMSO-treated 3.5-hpf embryo. DNA (gray) and F-actin (magenta) were stained with DAPI and Acti-stain 555 phalloidin dye, respectively. Depth from the top surface of the embryo is indicated. Bar, 100 μm.\u003c/p\u003e","description":"","filename":"MovieS11.mp4","url":"https://assets-eu.researchsquare.com/files/rs-7071246/v1/bfed662a5e1573df931ca9d0.mp4"},{"id":87905068,"identity":"e471d762-bf89-4b5f-a4fc-63bf496afcfa","added_by":"auto","created_at":"2025-07-30 08:42:50","extension":"mp4","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":10857688,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMovie S2. Fluorescence imaging of a PCEI-HU\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eVis\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e-treated 3.5-hpf embryo\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConfocal slice sections of a PCEI-HU\u003csup\u003eVis\u003c/sup\u003e-treated 3.5-hpf embryo. DNA (gray) and F-actin (magenta) were stained with DAPI and Acti-stain 555 phalloidin dye, respectively. Depth from the top surface of the embryo is indicated. Bar, 100 μm.\u003c/p\u003e","description":"","filename":"MovieS21.mp4","url":"https://assets-eu.researchsquare.com/files/rs-7071246/v1/856e433e3632f02390e70f65.mp4"},{"id":87905064,"identity":"59d40766-46b5-4864-9af3-d966b13af8d4","added_by":"auto","created_at":"2025-07-30 08:42:50","extension":"jpg","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":471567,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S1. The time course of the viable embryonic proportion after PCEI-HU treatment \u003c/strong\u003eViability of embryos treated with PCEI-HU\u003csup\u003eVis\u003c/sup\u003e during the time windows indicated at the top of the graphs. Means ± s.e. of at least three independent experiments, except for the 3-12 hpf condition tested in two independent experiments. Identical data for the 60-hpf time point are shown in Fig. 2B and 4B. At least 20 embryos were analyzed for each condition.\u003c/p\u003e","description":"","filename":"Fig7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7071246/v1/f96c233000a28003a2ae5f18.jpg"},{"id":87905451,"identity":"493292c6-91a5-41b5-b73c-08ee45e8da54","added_by":"auto","created_at":"2025-07-30 08:50:50","extension":"jpg","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":313322,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S2. SAC-dependent mitotic delay upon CENP-E inhibition in the gastrula period \u003c/strong\u003eHistograms of mitotic duration in early pre-gastrula or gastrula embryonic cells in each drug treatment condition. At least 128 cells from at least three embryos in three independent experiments were analyzed for each condition.\u003c/p\u003e","description":"","filename":"Fig8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7071246/v1/a8f5e051cba43867419878c9.jpg"},{"id":87905062,"identity":"4b394bdc-7755-4dc5-bed1-e3d8031777fe","added_by":"auto","created_at":"2025-07-30 08:42:50","extension":"jpg","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":258612,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S3. Enlarged views of cleaved caspase-3 and phospho-histone H3 immunostaining \u003c/strong\u003eA merged image of DAPI-stained DNA, immunostained pH3 and cleaved caspase-3 in PCEI-HU\u003csup\u003eVis\u003c/sup\u003e (5-12 hpf) and reversine-co-treated embryos at 24 hpf. The identical embryo is also shown in Fig. 6D. An example of the whole-head view of the staining is shown at the top. The box represents the region of the embryo enlarged in the bottom panels.\u003c/p\u003e","description":"","filename":"Fig9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7071246/v1/afb4ab68c9ea646dfb272bd2.jpg"},{"id":87905069,"identity":"f77655ea-0b29-48d2-b4af-f7103d8de8a5","added_by":"auto","created_at":"2025-07-30 08:42:50","extension":"jpg","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":686152,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S4. Apoptotic and mitotic cell distribution at 72 hpf after CENP-E inhibition during the gastrula period\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eImmunostaining of cleaved caspase-3 and pH3 in the whole-mount embryos at 72 hpf in each condition. The box represents the region of the embryo enlarged in the bottom panels. DNA was stained with DAPI. \u003cstrong\u003e(B, C) \u003c/strong\u003eThe density (cell number per volume) of cleaved caspase-3- or pH3-positive cells in the left midbrain in embryos in D. Ten confocal slice sections from the top of the midbrain were used for counting the number of positive cells. Means ± s.e. of at least 8 embryos from three independent experiments. There is no statistically significant difference from control (n.s.: not significant, Welch t-test).\u003c/p\u003e","description":"","filename":"Fig10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7071246/v1/1f87c410f6ffbb2d06066c78.jpg"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Revealing a critical role of the incomplete spindle assembly checkpoint in zebrafish development through an optochemical approach","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"","lastPublishedDoi":"10.21203/rs.3.rs-7071246/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7071246/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Early animal embryos must balance the efficiency with the accuracy of mitotic control. However, the extent of mitotic errors that can be safely endured at different stages of development is unclear. In this study, using a recently developed photoswitchable CENP-E inhibitor, we introduced transient mitotic errors at various developmental windows and systematically addressed their organismal effects. Upon CENP-E inhibition in the pre-gastrula period, embryos suffered gradual aggravation of developmental defects as the duration of the inhibition extended. Conversely, embryos tolerated several hours of consecutive CENP-E inhibition in the gastrula period, frequently achieving full development. Live imaging revealed that chromosome misalignment caused by CENP-E inhibition resulted in a modest mitotic delay in the gastrula, but not in the early pre-gastrula period, suggesting the functionalization of the spindle assembly checkpoint (SAC) at this stage. This mitotic delay helped alleviate, though not perfectly resolve, polar chromosome misalignment before anaphase onset. Importantly, pharmacological suppression of SAC rendered gastrula embryos inviable upon CENP-E inhibition. Therefore, despite its leaky nature, the embryonic SAC contributed to partial mitotic error correction, which proved essential to manage consecutive mitotic perturbations. Our results demonstrate the power of optochemical approaches in understanding the robust control of dynamic processes in development.","manuscriptTitle":"Revealing a critical role of the incomplete spindle assembly checkpoint in zebrafish development through an optochemical approach","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-30 08:42:45","doi":"10.21203/rs.3.rs-7071246/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-biology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commsbio","sideBox":"Learn more about [Communications Biology](http://www.nature.com/commsbio/)","snPcode":"","submissionUrl":"","title":"Communications Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"5afdfb0f-c24d-4185-9ffb-f13b0ef201c2","owner":[],"postedDate":"July 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":52114591,"name":"Biological sciences/Developmental biology/Cell proliferation"},{"id":52114592,"name":"Biological sciences/Cell biology/Cell division/Checkpoints"}],"tags":[],"updatedAt":"2026-03-05T11:45:37+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-30 08:42:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7071246","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7071246","identity":"rs-7071246","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}