Abstract
As an animal’s surface area expands during development, skin cell populations must quickly respond to maintain sufficient epithelial coverage. Despite much progress in understanding of skin cell behaviours in vivo1,2, it remains unclear how cells collectively act to satisfy coverage demands at an organismic level. Here we created a multicolour cell membrane tagging system, palmskin, to monitor the entire population of superficial epithelial cells (SECs) in developing zebrafish larvae. Using time-lapse imaging, we found that many SECs readily divide on the animal body surface; during a specific developmental window, a single SEC can produce a maximum of four progeny cells over its lifetime on the surface of the animal. Remarkably, EdU assays, DNA staining and hydroxyurea treatment showed that these terminally differentiated skin cells continue splitting despite an absence of DNA replication, causing up to 50% of SECs to exhibit reduced genome size. On the basis of a simple mathematical model and quantitative analyses of cell volumes and apical surface areas, we propose that ‘asynthetic fission’ is used as an efficient mechanism for expanding epithelial coverage during rapid growth. Furthermore, global or local manipulation of body surface growth affects the extent and mode of SEC division, presumably through tension-mediated activation of stretch-activated ion channels. We speculate that this frugal yet flexible mode of cell proliferation might also occur in contexts other than zebrafish skin expansion.
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Data availability
Microscopy data are available from the corresponding author on request, without any restrictions. Source data are provided with this paper.
Code availability
Custom code for image processing and analysis has been deposited in GitHub (https://github.com/ccsyan/labeling-cells-using-slic, https://github.com/peggyscshu/Analysis-on-membrane-labeled-cell and https://github.com/peggyscshu/Mitosis-phase-analysis).
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Acknowledgements
We thank J. Livet and Y. A. Pan for sharing the Palmbow construct, as well as C.-H. Wang and the Taiwan Zebrafish Core Facility (TZCF) for zebrafish care and services. TZCF is supported by the Ministry of Science and Technology, Taiwan (MOST 110-2740-B-400-001). We also thank S.-Y. Du and the Core Facility at ICOB for assistance with flow cytometry; L.-H. Yih, C.-H. Lee, Y.-H. Su, C.-F. Kao, J. Lu, H.-C. Kuo and members of the Chen laboratory for comments on the manuscript, and M. J. Calkins for English editing and comments. We acknowledge funding support from Academia Sinica to C.-H.C. (AS-CDA-109-L03) and C.-P.H. (AS-IA-106-M01) and grants from the Ministry of Science and Technology to C.-H.C. (MOST 106-2628-B-001-001-MY4) and C.-P.H. (MOST 105-2113-M-001-009-MY4 and 109-2113-M-001-022-MY4).
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K.Y.C. and C.-H.C. conceived the project, designed experiments, analysed data and prepared the manuscript. K.Y.C. performed experiments and imaging. K.Y.C., C.-C.S.Y., S.-C.H. and C.-P.H. performed quantitative analyses. C.-H.C. designed and H.-Y.R. generated palmskin lines. H.-Y.R. performed histology. T.-L.T. performed sgRNA experiments and RT–qPCR assays. C.-D.H. contributed the Tg(krt4:NLS-EGFP)cy34 line. All authors reviewed and edited the manuscript.
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Extended data figures and tables
Extended Data Fig. 1 Inducible control of Cre activity in the SECs population.
a, b, The transgenic constructs of SEC:iCre#1 (a) and SEC:iCre#2 (b). c, d, Cross-sections of 8 dpf palmskin larvae. Despite the Tet-on cassette being driven by the ubi or krt19 promoter, the Cre activity in both lines is restricted to the SEC layer (red). P63 Ab stains the BEC layer (green). DAPI stains all cells (white). White dashed boxes indicate magnified areas. White arrows point to mCherry-positive SECs. e, palmskin; SECiCre#2 images captured at 8 dpf from the posterior region. Three animals were examined for each treatment. f, g, Whole-animal view of a live, 8 dpf Tg(ubi:Switch; SEC:iCre#2) (f) and a palmskin; SEC:iCre#2 zebrafish larvae (g). Stitched images (e–g). Yellow dashed line marks body margins (e–g). Scale bars, 50 µm (c, d); 100 µm (e); 500 µm (f, g).
Extended Data Fig. 2 palmskin enables long-term tracking of SECs over their surface lifetime.
a, Magnified images of a body area from a live palmskin animal. Images were captured from the same individual at 8 and 17 dpf. SECs of the same color were labeled with a clone identification number. b, Analysis of total hue numbers at 8 and 10 dpf. c, palmskin images were captured at 8, 10, 14, and 21 dpf. Red dashed line marks trunk margins. d, Labeled SEC coverage ratio, as determined by SEC coverage area over Trunk surface area. Data are shown as mean ± s.d. Scale bars, 50 µm (a, c). n = number of larvae (b, d). Significance was examined by two-tailed Student’s t-test (b). P-values are shown above the horizontal lines indicating intergroup comparisons.
Extended Data Fig. 3 A single SEC readily divides twice to generate four progeny cells.
a, Schematic timeline of time-lapse imaging over a 12-hr period, starting at 8.5 dpf. b, Representative images of a dividing SEC. Arrowheads point to microridge pattern on the SEC. c, Representative images of a live palmskin animal. Images were captured from the same individual at 8 and 10 dpf. SECs of the same color were labeled with a clone identification number. d, Angles of the long axis of the cell and the cell division axis of the dividing SECs. e, f, Quantification of shape changes in mother and daughter cells, as determined by aspect ratio (e) and circularity (f). g, Schematic timeline of time-lapse imaging over a 9.5-day period with a 12-hr interval.h, Representative images of a single SEC that gives rise to four progeny cells. i, Percentages of each type of division event (n = 116 cell trajectories). j, Percentages of each type of shedding event (n = 40 cell trajectories). Data are shown as mean ± s.d. (e, f). Scale bars, 20 µm (b, h); 100 µm (c). n = number of SECs (d–f). Significance was examined by two-tailed Mann-Whitney test (e) or two-tailed Student’s t-test (f). P-values are shown above the horizontal lines indicating intergroup comparisons.
Extended Data Fig. 4 SECs division is reversible at low frequency.
a, Time-lapse images of a single SEC that fuses after division. Yellow dashed line outlines cell boundaries. b, palmskin images captured at 8 and 10 dpf. SEC nuclei were visualized by histone-tagged BFP2. White arrows point to binucleated SECs. c, Binucleated SEC ratio at 8 and 10 dpf. The ratio is determined by the number of binucleated SECs over total number of examined SECs. Data are shown as mean ± s.d. (c). Scale bar, 20 µm (a); 50 µm (b). n = number of SECs (c).
Extended Data Fig. 5 No detectable DNA replication in the SEC population.
a, b, Schematic timeline of EdU assay conducted at 6, 8, 10, and 14 dpf. The entire BEC and SEC populations were visualized by Tg(krt19:H2A-mCherry) and Tg(krt4:NLS-EGFP), respectively (b). Yellow dashed line marks body margins. c, Whole-mount images of EdU+ cells in the BEC and SEC populations. White arrows point to the EdU+ BECs. Yellow arrows point to non-BEC or non-SEC EdU+ cells. No EdU+ cells were detected in the SEC population. d, Quantification of EdU+ cells. e, Schematic timeline of HU treatment. EdU assay was conducted at 10 dpf to visualize EdU+ BECs. f, g, Whole-mount images (f) and quantification of EdU+ BECs (g) upon HU treatment. White arrows point to the EdU+ BECs. h, Schematic timeline of HU treatment. i, palmskin images were captured at 9 and 10 dpf to visualize divided SECs. j, Percentage of each clone size. k, Quantification of the divided pool (sum of 2-cell, 3-cell, and 4-cell clones / total number of SEC clone). Data are shown as mean ± s.d. (d, g, k). Stitched images (b). Scale bar, 500 µm (b); 50 µm (c, f); 100 µm (i). n = number of BECs or SECs (d); n = number of BECs (g); n = number of SEC clones (j); n = number of larvae (k). Significance was examined by two-tailed Student’s t-test (g) or ordinary one-way ANOVA with Tukey correction for multiple comparisons (k). Percent differences and P-values are shown above the horizontal lines for the indicated intergroup comparisons.
Extended Data Fig. 6 SEC division is sensitive to the tubulin polymerization inhibitor Nocodazole.
a–e, Whole mount images (a–d) and quantification of EdU+ cells (e) at 10 dpf, after 12-hr chemical treatments. f–i, palmskin images were captured at 10 dpf, after 12-hr chemical treatments. SEC nuclei were visualized by histone-tagged BFP2. White arrows point to binucleated SECs. j–n, Quantification of changes in clone size (j), the divided pool (sum of 2-cell, 3-cell, and 4-cell clones / total number of SEC clones) (k), the binucleated SEC ratio (sum of binucleated SECs / total number of SECs) (l), Standard length (m), and Trunk surface area (n). Data are shown as mean ± s.d. (e, k–n). Scale bar, 50 µm. n = number of SEC clones (j); n = number of SECs (l); n = number of larvae (e, k, m, n). Significance was examined by two-tailed Student’s t-test or two-tailed Mann-Whitney test (e, k–n). Percent differences and P-values are shown above the horizontal lines for the indicated intergroup comparisons.
Extended Data Fig. 7 Long-term tracking of the whole 1-to-4 division trajectories reveals collective apical surface increase but not the collective volume.
a–e, Quantification of total apical surface area and total cell volume. The entire 1-to-4-cell division trajectories of five clones were analyzed. White dashed line outlines cell boundaries for a dividing SEC (top). Data from each progeny cell are color-coded. f, g, Quantification of changes in total apical surface area (f) and total cell volume (g) between 1-cell and 4-cell stages. All measurements from each stage were averaged for comparison. h, Quantification of changes in median cell height after the first division. i–k, Quantification of changes in apical surface area (j) and cell volume (k) prior to the first and the second division. In contrast to the first division, the second division readily occurs in cells with a smaller size. l, m, Relative changes in apical surface area (l) and cell volume (m) in 3-cell clones. Notably, one cell is markedly larger than the other two cells in a 3-cell clone, as predicted by the simple geometric model (Fig. 4). Data are shown as mean ± s.d. (h, j, k). n = number of SECs (h). Significance was examined by paired two-tailed Student’s t-test (f, g) or unpaired two-tailed Student’s t-test (h, j, k). Percent differences and P-values are shown above the horizontal lines for the indicated intergroup comparisons.
Extended Data Fig. 8 Local surface demand directs asynthetic fission.
a, Schematic timeline of fin fold amputation and imaging. b, palmskin images were captured at 10 dpf (2 dpa) to visualize SEC behavior. All SEC clones within 500 µm proximal to the amputation plane were included in the analysis. Red dashed line indicates amputation plane (a, b). Yellow dashed line outlines the margin of fin fold (b). c–e, Quantification of each clone size (c), the divided pool (sum of 2-cell, 3-cell, and 4-cell clones / total number of SEC clones) (d), and distribution of clone division axis (e). f, Schematic shows timeline of adult tailfin amputation-regeneration experiments. Red dashed line indicates amputation plane. g, h, Flow cytometry analysis of DNA content in live SECs, as determined by EGFP+/PI-/Hoechst. Red-shaded zones mark sub-2N populations (f, g). Stitched images (b). Data are shown as mean ± s.d. (d, h). Scale bar, 100 µm (b). n = number of SEC clones (c, e). n = number of larvae (d). Significance was examined by two-tailed Student’s t-test (d, h). Percent difference and P-value are shown above the horizontal lines for intergroup comparison.
Extended Data Fig. 9 Tension dynamics and Piezo1 regulate asynthetic fission.
a, Time-lapse images of laser-incisions. White dashed line indicates incision sites. White solid lines mark cell edges. Double-headed arrow indicates the total displacement. b, Using Tg(krt19:Lifeact-mScarlet), skin tension was determined by real-time monitoring of tissue displacement upon incision. Measurements from the first 0.8 s (red-shaded zone) were used to estimate recoil velocity, whereas displacements from 30 to 50 s (blue-shaded zone) were average as maximum recoil distance. c, d, Quantification of recoil velocity (c) and maximum recoil distance (d). e, palmskin images were captured at 10 dpf, after two days of chemical treatments. f-i, Quantification of changes in clone size (f), the divided pool (sum of 2-cell, 3-cell, and 4-cell clones / total number of SEC clone) (g), Standard length (h) and Trunk surface area (i). j, k, CRISPR-Cas9-mediated deletion of the piezo 1 locus. The wild-type and the deletion alleles were examined by PCR amplification of genomic DNA (j) and Sanger sequencing (k). The sgRNA pair removes the entire genomic region (~78 kb) spanning Exon 6 and Exon 54. For gel source data, see Supplementary Figure 1. l, m, CRISPR-Cas9-mediated knockdown of the piezo1 expression in palmskin (l) and RT-qPCR analysis (m). n, palmskin images were captured at 8 dpf in the sgRNAs-injected larvae. o-r, Quantification of changes in clone size (o), the divided pool (sum of 2-cell, 3-cell, and 4-cell clones / total number of SEC clone) (p), Standard length (q) and Trunk surface area (r). Data are shown as mean (b); mean ± s.d. (c, d, g–i, p–r); mean ± s.e.m. (m). Scale bars, 10 µm (a); 50 µm (e, n). n = number of larvae (c, d, g–i, p–r); n = number of SEC clones (f, o); n = number of biological replicates (m). Significance was examined by two-tailed Student’s t-test (c, d, g, i, m, q, r) or two-tailed Mann-Whitney test (h, p). Percent differences and P-values are shown above the horizontal lines indicating intergroup comparisons.
Extended Data Fig. 10 Chemical perturbation of stretch-activated ion channels affects SEC cellular features and shedding dynamics.
a, b, Quantification of apical surface area (a) and median cell height (b) of 1-cell stage SECs at 10 dpf, after two days of chemical treatments. c, Percentage distribution of the long cell axis orientation at 10 dpf. d, palmskin images were captured at 10 and 14 dpf. e. f, Quantification of changes in SEC coverage (e) and the binucleated SEC ratio (sum of binucleated SECs / total number of SECs) (f). g, h, Immunostaining of Piezo1 in the sgRNA-injected larvae. The injection of piezo1 sgRNAs markedly reduced the overall Piezo1 signal in some animals (g). Piezo1 is present as cytoplasmic puncta in SECs at 8 and 14 dpf (h). Number of examined animals is shown in the top right corner. Of note. the SEC nuclei were visualized by the nuclear BFP2 expression in palmskin (red; no Dox and Tam treatment). i–l, No detectable apoptotic events in the SEC population. The apoptosis reporter construct and the UV-irradiation assay (i). The stable transgenic line, Tg(ubi:H2A-mCherry-2A-GC3AI), labels the SEC population. Of note, Tg(krt4:NLS-EGFP) allows visualization of the SEC nuclei (j). c, Activation of the GFP signal in SECs upon a single UV pulse. White arrows point to the SECs with the GFP signal (k). Representative images of Tg(ubi:H2A-mCherry-2A-GC3AI) at 8 dpf and 10 dpf. Number of examined animals is shown in the top right corner of the mCherry channel (l). Scale bar, 50 µm (d, j–l); 10 µm (g, h). Data are shown as violin plot (red, median; blue, quartiles; a, b); mean ± s.d. (e, f); n = number of SECs (a–c); n = number of larvae (e, f). Significance was examined by two-tailed Mann-Whitney test (a, b, e, f). Percent differences and P-values are shown above the horizontal lines for the indicated intergroup comparisons.
Supplementary information
Supplementary Fig. 1
This file contains an uncropped gel image of Extended Data Fig. 9j with size markers.
Supplementary Table 1
List of primers used for genotyping, RT–qPCR and sgRNA template synthesis.
Supplementary Video 1
Multicolour cell barcoding of the SEC population in palmskin. A 3D view is shown of a palmskin image captured from a live zebrafish larva.
Supplementary Video 2
Time-lapse images of the SEC population. Serial palmskin images were captured with a 15-min interval over 12 h. White arrows point to dividing SECs. Scale bar, 50 µm.
Supplementary Video 3
Time-lapse, magnified images of a dividing SEC with the microridge pattern. Serial palmskin images were captured at 15-min intervals over the course of 12 h. Scale bar, 20 µm.
Supplementary Video 4
Time-lapse images of a shedding SEC. Serial palmskin images were captured at 15-min intervals over the course of 12 h. Scale bar, 20 µm.
Supplementary Video 5
Long-term tracking of an SEC over its entire surface lifetime. Serial palmskin images were captured at 12-h intervals over the course of 5 d. Scale bar, 20 µm.
Supplementary Video 6
Long-term tracking of an SEC that gives rise to four progeny cells. Serial palmskin images were captured at 12-h intervals over the course of 9.5 d. Scale bar, 20 µm.
Supplementary Video 7
Time-lapse images of a fusion event. Serial palmskin images were captured at 15-min intervals over the course of 10 h. Scale bar, 20 µm.
Supplementary Video 8
Time-lapse images of dividing SECs with anaphase bridges, lagging chromosomes, micronuclei and tripolar segregation. Serial Tg(krt4:H2A-EGFP) images were captured at 5-min intervals over the course of 12 h. A yellow arrow points to the chromosome segregation defects. Scale bar, 10 µm.
Supplementary Video 9
Time-lapse images of a dividing BEC. Serial Tg(krt19:H2A-mCherry) images were captured at 5-min intervals over the course of 12 h. Scale bar, 10 µm.
Supplementary Video 10
Time-lapse images of recoil responses. Using Tg(krt19:Lifeact-mScarlet), serial images were captured at 0.08-s intervals after a laser incision. The first 30 s of images are shown. A white solid line marks cell edges. A double-headed arrow indicates the total displacement. Scale bar, 10 µm.
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Chan, K.Y., Yan, CC.S., Roan, HY. et al. Skin cells undergo asynthetic fission to expand body surfaces in zebrafish. Nature 605, 119–125 (2022). https://doi.org/10.1038/s41586-022-04641-0
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DOI: https://doi.org/10.1038/s41586-022-04641-0
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