Abstract
Epithelial cell division maintains tissue architecture through coordinated nuclear migration, cell shape changes, and spindle orientation. In columnar epithelia, interkinetic nuclear migration (INM) involves apical nuclear translocation in G2 phase and basal return post-mitosis, yet its regulation remains incompletely understood in vertebrates, in part due to limited in vivo live-imaging models. In this methodological study, we adapted the zebrafish embryonic otic vesicle as an in vivo model to investigate cell division dynamics in simple columnar epithelium using high-resolution live imaging and genetic tools. We demonstrated that apical INM initiates in mid-to-late G2 and is driven by dynein, not myosin II. Mitotic rounding is achieved via actomyosin-mediated basolateral constriction while maintaining basal attachment. Inhibiting myosin II impairs rounding and planar division, causing apical retention of daughter cells, suggesting planar division ensures proper integration. We additionally analyzed the mouse epididymal epithelium, a simple columnar epithelial tissue, to allow cross-species comparison of nuclear migration dynamics. Together, these optimized in vivo vertebrate models uncover conserved and tissue-specific mechanisms underlying epithelial organization and function, and importantly, provide tools for deeper mechanistic dissection in the future.
Data availability
Data generated in this study are included in the Supplementary file.
References
Rodriguez-Boulan, E. & Macara, I. G. Organization and execution of the epithelial polarity programme. Nat. Rev. Mol. Cell. Biol. 15, 225–242 (2014).
Morin, X. & Bellaiche, Y. Mitotic spindle orientation in asymmetric and symmetric cell divisions during animal development. Dev. Cell. 21, 102–119 (2011).
Godard, B. G. & Heisenberg, C. P. Cell division and tissue mechanics. Curr. Opin. Cell. Biol. 60, 114–120 (2019).
Tsue, T. T., Watling, D. L., Weisleder, P., Coltrera, M. D. & Rubel, E. W. Identification of hair cell progenitors and intermitotic migration of their nuclei in the normal and regenerating avian inner ear. J. Neurosci. 14, 140–152 (1994).
Lee, C., Goolsby, C. L. & Sensibar, J. A. Cell cycle kinetics in rat prostatic epithelia: nuclear migration during G2 phase. J. Urol. 152, 2294–2299 (1994).
Fleming, E. S. et al. Planar spindle orientation and asymmetric cytokinesis in the mouse small intestine. J. Histochem. Cytochem. 55, 1173–1180 (2007).
Robaire, B. & Hinton, B. T. The Epididymis: From Molecules to Clinical Practice (Kluwer Academic and Plenum, 2002).
Fujita, S. Mitotic pattern and histogenesis of the central nervous system. Nature 185, 702–703 (1960).
Bagley, D. C. et al. Bronchoconstriction damages airway epithelia by crowding-induced excess cell extrusion. Science 384, 66–73 (2024).
Taverna, E. & Huttner, W. B. Neural Progenitor Nuclei IN Motion. Neuron 67, 906–914 (2010).
Baye, L. M. & Link, B. A. Nuclear migration during retinal development. Brain Res. 1192, 29–36 (2008).
Rujano, M. A., Sanchez-Pulido, L., Pennetier, C., le Dez, G. & Basto, R. The microcephaly protein Asp regulates neuroepithelium morphogenesis by controlling the spatial distribution of myosin II. Nat. Cell Biol. 15, 1294–1306 (2013).
Meyer, E. J., Ikmi, A. & Gibson, M. C. Interkinetic nuclear migration is a broadly conserved feature of cell division in pseudostratified epithelia. Curr. biology: CB. 21, 485–491 (2011).
Sauer, F. C. Mitosis in the neural tube. J. Comp. Neurol. 62, 377–397 (1935).
Fujita, S. Kinetics of cellular proliferation. Exp. Cell. Res. 28, 52–60 (1962).
Leung, L., Klopper, A. V., Grill, S. W., Harris, W. A. & Norden, C. Apical migration of nuclei during G2 is a prerequisite for all nuclear motion in zebrafish neuroepithelia. Development 138, 5003–5013 (2011).
Ahringer, J. Control of cell polarity and mitotic spindle positioning in animal cells. Curr. Opin. Cell. Biol. 15, 73–81 (2003).
Nakajima, Y. I. et al. Junctional tumor suppressors interact with 14-3-3 proteins to control planar spindle alignment. J. Cell. Biol. 218, 1824–1838 (2019).
Kosodo, Y. Interkinetic nuclear migration: beyond a hallmark of neurogenesis. Cell. Mol. Life Sci. 69, 2727–2738 (2012).
Tsai, J. W., Lian, W. N., Kemal, S., Kriegstein, A. R. & Vallee, R. B. Kinesin 3 and cytoplasmic dynein mediate interkinetic nuclear migration in neural stem cells. Nat. Neurosci. 13, 1463–1471 (2010).
Xie, Z. et al. Cep120 and TACCs control interkinetic nuclear migration and the neural progenitor pool. Neuron 56, 79–93 (2007).
Ge, X., Frank, C. L., Calderon de Anda, F. & Tsai, L. H. Hook3 interacts with PCM1 to regulate pericentriolar material assembly and the timing of neurogenesis. Neuron 65, 191–203 (2010).
Gambello, M. J. et al. Multiple dose-dependent effects of Lis1 on cerebral cortical development. J. Neurosci. 23, 1719–1729 (2003).
Zhang, X. et al. SUN1/2 and Syne/Nesprin-1/2 complexes connect centrosome to the nucleus during neurogenesis and neuronal migration in mice. Neuron 64, 173–187 (2009).
Schenk, J., Wilsch-Brauninger, M., Calegari, F. & Huttner, W. B. Myosin II is required for interkinetic nuclear migration of neural progenitors. Proc. Natl. Acad. Sci. U S A. 106, 16487–16492 (2009).
Norden, C., Young, S., Link, B. A. & Harris, W. A. Actomyosin is the main driver of interkinetic nuclear migration in the retina. Cell 138, 1195–1208 (2009).
Norden, C. Pseudostratified epithelia - cell biology, diversity and roles in organ formation at a glance. J. Cell Sci. 130, 1859–1863 (2017).
Carroll, T. D. et al. Interkinetic nuclear migration and basal tethering facilitates post-mitotic daughter separation in intestinal organoids. J. Cell. Sci. 130, 3862–3877 (2017).
Saleh, J. et al. Length limitation of astral microtubules orients cell divisions in murine intestinal crypts. Dev. Cell. 58, 1519–1533e1516 (2023).
Spear, P. C. & Erickson, C. A. Apical movement during interkinetic nuclear migration is a two-step process. Dev. Biol. 370, 33–41 (2012).
Megason, S. G. In toto imaging of embryogenesis with confocal time-lapse microscopy. Methods Mol. Biol. 546, 317–332 (2009).
Mosaliganti, K. R. et al. Size control of the inner ear via hydraulic feedback. Elife 8 (2019).
Hoijman, E., Rubbini, D., Colombelli, J. & Alsina, B. Mitotic cell rounding and epithelial thinning regulate lumen growth and shape. Nat. Commun. 6, 7355 (2015).
Hendzel, M. J. et al. Mitosis-specific phosphorylation of histone H3 initiates primarily within pericentromeric heterochromatin during G2 and spreads in an ordered fashion coincident with mitotic chromosome condensation. Chromosoma 106, 348–360 (1997).
Hirashima, T. & Adachi, T. Procedures for the quantification of whole-tissue immunofluorescence images obtained at single-cell resolution during murine tubular organ development. PLoS One. 10, e0135343 (2015).
Navarro, C. et al. Junctional recruitment of mammalian Scribble relies on E-cadherin engagement. Oncogene 24, 4330–4339 (2005).
Lickert, H., Bauer, A., Kemler, R. & Stappert, J. Casein kinase II phosphorylation of E-cadherin increases E-cadherin/beta-catenin interaction and strengthens cell-cell adhesion. J. Biol. Chem. 275, 5090–5095 (2000).
Bertocchi, C., Vaman Rao, M. & Zaidel-Bar, R. Regulation of adherens junction dynamics by phosphorylation switches. J. Signal Transduct. 125295 (2012).
Burkel, B. M., von Dassow, G. & Bement, W. M. Versatile fluorescent probes for actin filaments based on the actin-binding domain of utrophin. Cell. Motil. Cytoskeleton. 64, 822–832 (2007).
Kosodo, Y. et al. Cytokinesis of neuroepithelial cells can divide their basal process before anaphase. EMBO J. 27, 3151–3163 (2008).
Straight, A. F. et al. Dissecting temporal and spatial control of cytokinesis with a myosin II Inhibitor. Science 299, 1743–1747 (2003).
Bulinski, J. C., Gruber, D., Faire, K., Prasad, P. & Chang, W. GFP chimeras of E-MAP-115 (ensconsin) domains mimic behavior of the endogenous protein in vitro and in vivo. Cell. Struct. Funct. 24, 313–320 (1999).
Faire, K. et al. E-MAP-115 (ensconsin) associates dynamically with microtubules in vivo and is not a physiological modulator of microtubule dynamics. J. Cell. Sci. 112 (Pt 23), 4243–4255 (1999).
Wuhr, M., Obholzer, N. D., Megason, S. G., Detrich, H. W., Mitchison, T. J. & 3rd & Live imaging of the cytoskeleton in early cleavage-stage zebrafish embryos. Methods Cell. Biol. 101, 1–18 (2011).
Liang, Y. et al. Nudel modulates kinetochore association and function of cytoplasmic dynein in M phase. Mol. Biol. Cell. 18, 2656–2666 (2007).
Echeverri, C. J., Paschal, B. M., Vaughan, K. T. & Vallee, R. B. Molecular characterization of the 50-kD subunit of dynactin reveals function for the complex in chromosome alignment and spindle organization during mitosis. J. Cell. Biol. 132, 617–633 (1996).
Burkhardt, J. K., Echeverri, C. J., Nilsson, T. & Vallee, R. B. Overexpression of the dynamitin (p50) subunit of the dynactin complex disrupts dynein-dependent maintenance of membrane organelle distribution. J. Cell. Biol. 139, 469–484 (1997).
Asakawa, K. et al. Genetic dissection of neural circuits by Tol2 transposon-mediated Gal4 gene and enhancer trapping in zebrafish. Proc. Natl. Acad. Sci. USA 105, 1255–1260 (2008).
Gavet, O. & Pines, J. Activation of cyclin B1-Cdk1 synchronizes events in the nucleus and the cytoplasm at mitosis. J. Cell. Biol. 189, 247–259 (2010).
Santos, S. D., Wollman, R., Meyer, T. & Ferrell, J. E. Jr. Spatial positive feedback at the onset of mitosis. Cell 149, 1500–1513 (2012).
Kosodo, Y. et al. Regulation of interkinetic nuclear migration by cell cycle-coupled active and passive mechanisms in the developing brain. EMBO J. 30, 1690–1704 (2011).
Packard, A. et al. Luminal Mitosis Drives Epithelial Cell Dispersal within the Branching Ureteric Bud. Dev. Cell. 27, 319–330 (2013).
Qin, Y., Capaldo, C., Gumbiner, B. M. & Macara, I. G. The mammalian Scribble polarity protein regulates epithelial cell adhesion and migration through E-cadherin. J. Cell. Biol. 171, 1061–1071 (2005).
O’Connell, C. B. & Wang, Y. L. Mammalian spindle orientation and position respond to changes in cell shape in a dynein-dependent fashion. Mol. Biol. Cell. 11, 1765–1774 (2000).
Lu, M. S. & Johnston, C. A. Molecular pathways regulating mitotic spindle orientation in animal cells. Development 140, 1843–1856 (2013).
Siller, K. H. & Doe, C. Q. Spindle orientation during asymmetric cell division. Nat. Cell. Biol. 11, 365–374 (2009).
Campinho, P. et al. Tension-oriented cell divisions limit anisotropic tissue tension in epithelial spreading during zebrafish epiboly. Nat. Cell Biol. 15, 1405–1414 (2013).
Murciano, A., Zamora, J., Lopez-Sanchez, J. & Frade, J. M. Interkinetic nuclear movement may provide spatial clues to the regulation of neurogenesis. Mol. Cell. Neurosci. 21, 285–300 (2002).
Tsai, J. W., Chen, Y., Kriegstein, A. R. & Vallee, R. B. LIS1 RNA interference blocks neural stem cell division, morphogenesis, and motility at multiple stages. J. Cell. Biol. 170, 935–945 (2005).
Del Bene, F., Wehman, A. M., Link, B. A. & Baier, H. Regulation of neurogenesis by interkinetic nuclear migration through an apical-basal notch gradient. Cell 134, 1055–1065 (2008).
Ueno, M., Katayama, K., Yamauchi, H., Nakayama, H. & Doi, K. Cell cycle progression is required for nuclear migration of neural progenitor cells. Brain Res. 1088, 57–67 (2006).
Strzyz, P. J. et al. Interkinetic nuclear migration is centrosome independent and ensures apical cell division to maintain tissue integrity. Dev. Cell. 32, 203–219 (2015).
Knopf, F. et al. Bone regenerates via dedifferentiation of osteoblasts in the zebrafish fin. Dev. Cell. 20, 713–724 (2011).
Dzafic, E., Strzyz, P. J., Wilsch-Brauninger, M. & Norden, C. Centriole Amplification in Zebrafish Affects Proliferation and Survival but Not Differentiation of Neural Progenitor Cells. Cell. Rep. 13, 168–182 (2015).
Compagnon, J. et al. The notochord breaks bilateral symmetry by controlling cell shapes in the zebrafish laterality organ. Dev. Cell. 31, 774–783 (2014).
Behrndt, M. et al. Forces driving epithelial spreading in zebrafish gastrulation. Science 338, 257–260 (2012).
Bouldin, C. M., Snelson, C. D. & Farr, G. H. 3rd & Kimelman, D. Restricted expression of cdc25a in the tailbud is essential for formation of the zebrafish posterior body. Genes Dev. 28, 384–395 (2014).
Sakaue-Sawano, A. et al. Visualizing spatiotemporal dynamics of multicellular cell-cycle progression. Cell 132, 487–498 (2008).
Bouldin, C. M. & Kimelman, D. Dual fucci: a new transgenic line for studying the cell cycle from embryos to adults. Zebrafish 11, 182–183 (2014).
Iida, A., Wang, Z., Hondo, E. & Sehara-Fujisawa, A. Generation and evaluation of a transgenic zebrafish for tissue-specific expression of a dominant-negative Rho-associated protein kinase-2. Biochem. Biophys. Res. Commun. 525, 8–13 (2020).
Westerfield, M. The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio)., Edn. 4th (University of Oregon, 2000).
Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B. & Schilling, T. F. Stages of embryonic development of the zebrafish. Dev. Dyn. 203, 253–310 (1995).
Aronheim, A. et al. Membrane targeting of the nucleotide exchange factor Sos is sufficient for activating the Ras signaling pathway. Cell 78, 949–961 (1994).
Hancock, J. F., Cadwallader, K., Paterson, H. & Marshall, C. J. A CAAX or a CAAL motif and a second signal are sufficient for plasma membrane targeting of ras proteins. EMBO J. 10, 4033–4039 (1991).
Li, Y., Yu, W., Liang, Y. & Zhu, X. Kinetochore dynein generates a poleward pulling force to facilitate congression and full chromosome alignment. Cell. Res. 17, 701–712 (2007).
Kawakami, K. et al. A transposon-mediated gene trap approach identifies developmentally regulated genes in zebrafish. Dev. Cell. 7, 133–144 (2004).
Shepard, J. L., Stern, H. M., Pfaff, K. L. & Amatruda, J. F. Analysis of the cell cycle in zebrafish embryos. Methods Cell. Biol. 76, 109–125 (2004).
Wang, F., Zhang, Q., Cao, J., Huang, Q. & Zhu, X. The microtubule plus end-binding protein EB1 is involved in Sertoli cell plasticity in testicular seminiferous tubules. Exp. Cell. Res. 314, 213–226 (2008).
Wloga, D. et al. TTLL3 Is a tubulin glycine ligase that regulates the assembly of cilia. Dev. Cell. 16, 867–876 (2009).
Acknowledgements
We thank A. Afolalu, C. Shapiro, S. Hosten, and C. Quaies for fish care; Dr. Koichi Kawakami (Division of Molecular and Developmental Biology, National Institute of Genetics, Japan) for providing the pT2KhspGFF and pCS-TP constructs; Yanan Xu, Zhili Wu, and Yiping Li for technical support; and Xueliang Zhu for guidance and comments on the manuscript.
Funding
This work was supported by The Louis and Rachel Rudin Foundation fellowship to Y.X., a New York State Stem Cell Science program (NYSTEM) predoctoral training grant position to B.P., and National Institutes of Health (NIH) grants R01HL155607 and R01HL166518 to J.C.
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Conceptualization: J.C.Methodology: Y.X. and J.C.Investigation: Y.X., B.P., A.G.C.Y., M.M., and M.Q.Resources: G.S.P. and J.C.Writing and editing: Y.X. and J.C.Funding Acquisition: J.C.
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Xia, Y., Perder, B., Yao, A.G.C. et al. Zebrafish otic vesicle and mouse epididymis as model systems for studying columnar epithelial cell division. Sci Rep (2026). https://doi.org/10.1038/s41598-026-42729-z
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DOI: https://doi.org/10.1038/s41598-026-42729-z
