{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,10,15]],"date-time":"2026-10-15T02:24:37Z","timestamp":1792031077866,"version":"4.4.0"},"posted":{"date-parts":[[2026,5,5]]},"group-title":"Developmental Biology","reference-count":46,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,5,5]],"date-time":"2026-05-05T00:00:00Z","timestamp":1777939200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"}],"funder":[{"award":["R01 EY016134"],"award-info":[{"award-number":["R01 EY016134"]}],"id":[{"id":"https:\/\/ror.org\/03wkg3b53","id-type":"ROR","asserted-by":"publisher"}]},{"award":["R01 EY036440"],"award-info":[{"award-number":["R01 EY036440"]}],"id":[{"id":"https:\/\/ror.org\/03wkg3b53","id-type":"ROR","asserted-by":"publisher"}]},{"award":["NEI-EY015387"],"award-info":[{"award-number":["NEI-EY015387"]}],"id":[{"id":"https:\/\/ror.org\/03wkg3b53","id-type":"ROR","asserted-by":"publisher"}]},{"award":["R03 DE032047-01"],"award-info":[{"award-number":["R03 DE032047-01"]}],"id":[{"id":"https:\/\/ror.org\/004a2wv92","id-type":"ROR","asserted-by":"publisher"}]},{"award":["2143217"],"award-info":[{"award-number":["2143217"]}],"id":[{"id":"https:\/\/ror.org\/021nxhr62","id-type":"ROR","asserted-by":"publisher"}]},{"id":[{"id":"https:\/\/ror.org\/02pjt4q95","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,5,5]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>The formation of functional corneal endothelial cells during development requires tight coordination between tissue-scale growth and cell-scale organization, yet how these processes are integrated in three dimensions remains poorly understood. Here, we combine high-resolution confocal imaging with quantitative analysis to reconstruct the morphogenesis of the chick corneal endothelium across embryonic development. Using a pipeline integrating 3D nuclear segmentation, Voronoi-based topological mapping, and spatial statistics, we link macroscopic globe expansion to single-cell geometry and lattice organization. We identify a multiphasic relationship between tissue growth and cell density, driven by temporal decoupling of organ expansion and proliferation. During early development, rapid globe expansion induces cellular stretching and spatial heterogeneity, followed by a phase of density accumulation and geometric refinement. Despite these dynamic conditions, the endothelial sheet maintains a robust monolayer architecture with minimal z-axis stratification. Quantitative topological analysis reveals that hexagonal packing is preserved from early stages and progressively refined through reduction of area variability and spatial clustering. Nearest-neighbor and Clark-Evans analyses demonstrate a transition from localized clustering to a more uniform spatial distribution, consistent with increasing packing regularity. Transient out-of-plane deviations coincide with key mechanical transitions, suggesting a role for 3D remodeling in accommodating mechanical stress. Concomitantly, junctional and cytoskeletal organization undergo progressive maturation. N-cadherin is established early at cell-cell interfaces, while Zonula Occludens-1 (ZO-1) transitions from diffuse localization to apically enriched tight junctions aligned with cortical actin. In parallel, microtubule organization becomes increasingly polarized to the apical domain, coinciding with the emergence of primary cilia. Together, these changes reflect coordinated establishment of epithelial polarity, barrier function, and mechanical stability. Overall, our study provides a multiscale, imaging-driven framework for understanding how epithelial tissues achieve and maintain geometric order under mechanical strain, establishing the corneal endothelium as an exemplar for linking developmental mechanics, 3D architecture, and epithelial topology.<\/jats:p>\n                <jats:sec>\n                  <jats:title>Summary Statement<\/jats:title>\n                  <jats:p>Using 3D imaging and quantitative analysis, this work reveals how corneal endothelial cells stay organized and form a regular pattern during growth, despite ongoing changes in tissue size and shape.<\/jats:p>\n                <\/jats:sec>","DOI":"10.64898\/2026.05.01.722310","type":"posted-content","created":{"date-parts":[[2026,5,6]],"date-time":"2026-05-06T06:00:16Z","timestamp":1778047216000},"source":"Crossref","is-referenced-by-count":1,"title":["Quantitative imaging of corneal endothelial development reveals dynamic but resilient monolayer"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9126-1585","authenticated-orcid":false,"given":"R","family":"Ramarapu","sequence":"first","affiliation":[{"name":"Department of Anatomy, Physiology, and Cell Biology, University of California, Davis, Davis, CA, USA"},{"name":"Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California, 1 Shields Ave., Davis, CA, 95616, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0003-4977-2570","authenticated-orcid":false,"given":"WR","family":"Stoehr","sequence":"additional","affiliation":[{"name":"Department of Anatomy, Physiology, and Cell Biology, University of California, Davis, Davis, CA, USA"},{"name":"Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California, 1 Shields Ave., Davis, CA, 95616, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"M","family":"Miesen","sequence":"additional","affiliation":[{"name":"Department of Anatomy, Physiology, and Cell Biology, University of California, Davis, Davis, CA, USA"},{"name":"Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California, 1 Shields Ave., Davis, CA, 95616, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"N","family":"Amro","sequence":"additional","affiliation":[{"name":"Department of Anatomy, Physiology, and Cell Biology, University of California, Davis, Davis, CA, USA"},{"name":"Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California, 1 Shields Ave., Davis, CA, 95616, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5617-9677","authenticated-orcid":false,"given":"SM","family":"Thomasy","sequence":"additional","affiliation":[{"name":"Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California, 1 Shields Ave., Davis, CA, 95616, USA"},{"name":"Department of Ophthalmology and Vision Science, School of Medicine, University of California Davis, Davis, CA, 95616, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9549-1089","authenticated-orcid":false,"given":"CD","family":"Rogers","sequence":"additional","affiliation":[{"name":"Department of Anatomy, Physiology, and Cell Biology, University of California, Davis, Davis, CA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026050911201596000_2026.05.01.722310v1.1","doi-asserted-by":"publisher","DOI":"10.1016\/j.biocel.2004.01.006"},{"key":"2026050911201596000_2026.05.01.722310v1.2","doi-asserted-by":"publisher","DOI":"10.1016\/j.ydbio.2020.06.012"},{"key":"2026050911201596000_2026.05.01.722310v1.3","doi-asserted-by":"crossref","unstructured":"Baddeley A R. E. ,, Turner R. (2015). Spatial Point Patterns: Methodology and Applications with R. Chapman and Hall\/CRC Press, London.","DOI":"10.1201\/b19708"},{"key":"2026050911201596000_2026.05.01.722310v1.4","doi-asserted-by":"publisher","DOI":"10.1016\/j.cub.2005.07.062"},{"key":"2026050911201596000_2026.05.01.722310v1.5","first-page":"1","article-title":"Fitting Linear Mixed-Effects Models Using lme4","volume":"67","year":"2015","journal-title":"Journal of Statistical Software"},{"key":"2026050911201596000_2026.05.01.722310v1.6","doi-asserted-by":"crossref","first-page":"912","DOI":"10.1038\/sj.eye.6700559","article-title":"Biology of the corneal endothelium in health and disease","volume":"17","year":"2003","journal-title":"Eye (Lond)"},{"key":"2026050911201596000_2026.05.01.722310v1.7","doi-asserted-by":"publisher","DOI":"10.1016\/j.yexcr.2017.03.061"},{"key":"2026050911201596000_2026.05.01.722310v1.8","doi-asserted-by":"publisher","DOI":"10.1016\/0014-4835(88)90021-8"},{"key":"2026050911201596000_2026.05.01.722310v1.9","doi-asserted-by":"publisher","DOI":"10.1167\/iovs.05-0806"},{"key":"2026050911201596000_2026.05.01.722310v1.10","doi-asserted-by":"crossref","first-page":"250","DOI":"10.1001\/archopht.1957.00930050260015","article-title":"The role of intraocular pressure in the development of the chick eye. II. Control of corneal size","volume":"57","year":"1957","journal-title":"AMA Arch Ophthalmol"},{"key":"2026050911201596000_2026.05.01.722310v1.11","doi-asserted-by":"publisher","DOI":"10.1111\/j.1469-7580.2005.00485.x"},{"key":"2026050911201596000_2026.05.01.722310v1.12","doi-asserted-by":"publisher","DOI":"10.1387\/ijdb.041937sc"},{"key":"2026050911201596000_2026.05.01.722310v1.13","doi-asserted-by":"publisher","DOI":"10.1002\/bies.20009"},{"key":"2026050911201596000_2026.05.01.722310v1.14","first-page":"92","article-title":"Meninges: from protective membrane to stem cell niche","volume":"1","year":"2012","journal-title":"Am J Stem Cells"},{"key":"2026050911201596000_2026.05.01.722310v1.15","doi-asserted-by":"crossref","first-page":"100180","DOI":"10.1016\/j.medntd.2022.100180","article-title":"Effect of mechanical stretching and substrate stiffness on the morphology, cytoskeleton and nuclear shape of corneal endothelial cells","volume":"16","year":"2022","journal-title":"Medicine in Novel Technology and Devices"},{"key":"2026050911201596000_2026.05.01.722310v1.16","doi-asserted-by":"publisher","DOI":"10.7554\/elife.10036.016."},{"key":"2026050911201596000_2026.05.01.722310v1.17","doi-asserted-by":"publisher","DOI":"10.1016\/j.cub.2007.11.049"},{"key":"2026050911201596000_2026.05.01.722310v1.18","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1917838117"},{"key":"2026050911201596000_2026.05.01.722310v1.19","doi-asserted-by":"crossref","first-page":"7402","DOI":"10.1038\/s41598-020-64311-x","article-title":"Phenotypic and functional characterization of corneal endothelial cells during in vitro expansion","volume":"10","year":"2020","journal-title":"Sci Rep"},{"key":"2026050911201596000_2026.05.01.722310v1.20","doi-asserted-by":"publisher","DOI":"10.1167\/iovs.05-0691"},{"key":"2026050911201596000_2026.05.01.722310v1.21","doi-asserted-by":"crossref","unstructured":"Ghosh, B. , Nishida, K. , Chandrala, L. , Mahmud, S. , Thapa, S. , Swaby, C. , Chen, S. , Khosla, A. A. , Katz, J. and Sidhaye, V. K. (2022). Epithelial plasticity in COPD results in cellular unjamming due to an increase in polymerized actin. J Cell Sci 135.","DOI":"10.1242\/jcs.258513"},{"key":"2026050911201596000_2026.05.01.722310v1.22","doi-asserted-by":"crossref","first-page":"331","DOI":"10.1016\/0014-4835(79)90051-4","article-title":"Contact inhibition and the regulation of endocytosis in the corneal endothelium: correlation with a restricted surface receptor lateral mobility and the appearance of a fibronectin meshwork","volume":"29","year":"1979","journal-title":"Exp Eye Res"},{"key":"2026050911201596000_2026.05.01.722310v1.23","first-page":"31","article-title":"Corneal endothelial cells possess an elaborate multipolar shape to maximize the basolateral to apical membrane area","volume":"22","year":"2016","journal-title":"Mol Vis"},{"key":"2026050911201596000_2026.05.01.722310v1.24","first-page":"1","article-title":"Fine structure of the developing avian cornea","volume":"1","year":"1969","journal-title":"Monogr Dev Biol"},{"key":"2026050911201596000_2026.05.01.722310v1.25","doi-asserted-by":"publisher","DOI":"10.1016\/S0074-7696(08)62339-6"},{"key":"2026050911201596000_2026.05.01.722310v1.26","doi-asserted-by":"crossref","first-page":"539","DOI":"10.1002\/jemt.24293","article-title":"Morphogenetic events influencing corneal maturation, development, and transparency: Light and electron microscopic study","volume":"86","year":"2023","journal-title":"Microsc Res Tech"},{"key":"2026050911201596000_2026.05.01.722310v1.27","doi-asserted-by":"publisher","DOI":"10.1016\/j.exer.2009.03.019"},{"key":"2026050911201596000_2026.05.01.722310v1.28","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-023-42825-y"},{"key":"2026050911201596000_2026.05.01.722310v1.29","doi-asserted-by":"publisher","DOI":"10.1016\/j.bpj.2016.11.004"},{"key":"2026050911201596000_2026.05.01.722310v1.30","doi-asserted-by":"crossref","first-page":"2647","DOI":"10.1038\/s41598-017-02755-4","article-title":"Morphologic and biometric evaluation of chick embryo eyes in ovo using 7 Tesla MRI","volume":"7","year":"2017","journal-title":"Sci Rep"},{"key":"2026050911201596000_2026.05.01.722310v1.31","doi-asserted-by":"publisher","DOI":"10.1016\/bs.pmbts.2015.04.003"},{"key":"2026050911201596000_2026.05.01.722310v1.32","doi-asserted-by":"publisher","DOI":"10.1113\/jphysiol.1957.sp005758"},{"key":"2026050911201596000_2026.05.01.722310v1.33","doi-asserted-by":"publisher","DOI":"10.1046\/j.0960-7722.2001.00216.x"},{"key":"2026050911201596000_2026.05.01.722310v1.34","doi-asserted-by":"crossref","unstructured":"Pachitariu, M. , Rariden, M. and Stringer, C. (2025). Cellpose-SAM: superhuman generalization for cellular segmentation. bioRxiv, 2025.04.28.651001.","DOI":"10.1101\/2025.04.28.651001"},{"key":"2026050911201596000_2026.05.01.722310v1.35","doi-asserted-by":"publisher","DOI":"10.1076\/ceyr.18.1.10.5392"},{"key":"2026050911201596000_2026.05.01.722310v1.36","doi-asserted-by":"publisher","DOI":"10.1083\/jcb.201408044"},{"key":"2026050911201596000_2026.05.01.722310v1.37","doi-asserted-by":"crossref","unstructured":"Ramarapu, R. , Stoehr, W. R. , Miesen, M. , Border, S. , Thomasy, S. and Rogers, C. D. (2026). A molecular and spatial resource defining tubulin isotype organization during corneal development. bioRxiv, 2026.02.19.706651.","DOI":"10.64898\/2026.02.19.706651"},{"key":"2026050911201596000_2026.05.01.722310v1.38","doi-asserted-by":"publisher","DOI":"10.1167\/iovs.14-16166"},{"key":"2026050911201596000_2026.05.01.722310v1.39","doi-asserted-by":"publisher","DOI":"10.1038\/nrn1221"},{"key":"2026050911201596000_2026.05.01.722310v1.40","doi-asserted-by":"publisher","DOI":"10.1117\/1.1606461"},{"key":"2026050911201596000_2026.05.01.722310v1.41","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.2089"},{"key":"2026050911201596000_2026.05.01.722310v1.42","doi-asserted-by":"publisher","DOI":"10.1016\/j.exer.2009.08.004"},{"key":"2026050911201596000_2026.05.01.722310v1.43","first-page":"11","article-title":"Scanning electron microscopy (SEM) of cranial neural crest migration in chick embryos","volume":"15","year":"1979","journal-title":"Birth Defects Orig Artic Ser"},{"key":"2026050911201596000_2026.05.01.722310v1.44","doi-asserted-by":"publisher","DOI":"10.1186\/s12859-021-04344-9"},{"key":"2026050911201596000_2026.05.01.722310v1.45","doi-asserted-by":"publisher","DOI":"10.3109\/02713688509017661"},{"key":"2026050911201596000_2026.05.01.722310v1.46","doi-asserted-by":"crossref","first-page":"475","DOI":"10.3389\/fbioe.2020.00475","article-title":"Investigate the Odontogenic Differentiation and Dentin-Pulp Tissue Regeneration Potential of Neural Crest Cells","volume":"8","year":"2020","journal-title":"Front Bioeng Biotechnol"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.64898\/2026.05.01.722310","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,5,9]],"date-time":"2026-05-09T18:20:25Z","timestamp":1778350825000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.64898\/2026.05.01.722310"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,5,5]]},"references-count":46,"URL":"https:\/\/doi.org\/10.64898\/2026.05.01.722310","relation":{},"subject":[],"published":{"date-parts":[[2026,5,5]]},"subtype":"preprint"}}