Establishment and transcriptome analysis of single blastomere-derived cell lines from zebrafish

Jia Xu; Siqi Liu; Yirui Ai; Yunbin Zhang; Shifeng Li; Yiping Li

doi:10.1016/j.jgg.2024.07.018

Volume 51 Issue 9

Sep. 2024

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Article Navigation > Journal of Genetics and Genomics > 2024 > 51(9): 957-969

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Establishment and transcriptome analysis of single blastomere-derived cell lines from zebrafish

doi: 10.1016/j.jgg.2024.07.018

Key Laboratory of Multi-Cell Systems, Shanghai Key Laboratory of Molecular Andrology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai 200031, China

Funds:

This work was partly supported by the National Key Research and Development Program of China (2018YFA0801003 and 2019YFA0109900) and the Center for Excellence in Molecular Cell Science (2021DF06). This project was supported by the Shanghai Municipal Science and Technology Major Project.

Received Date: 2024-04-09
Accepted Date: 2024-07-29
Rev Recd Date: 2024-07-29

Available Online: 2025-06-06

Publish Date: 2024-08-02

Abstract

Abstract

Maintaining chromosome euploidy in zebrafish embryonic cells is challenging because of the degradation of genomic integrity during cell passaging. In this study, we report the derivation of zebrafish cell lines from single blastomeres. These cell lines have a stable chromosome status attributed to BMP4 and exhibit continuous proliferation in vitro. Twenty zebrafish cell lines are successfully established from single blastomeres. Single-cell transcriptome sequencing analysis confirms the fidelity of gene expression profiles throughout long-term culturing of at least 45 passages. The long-term cultured cells are specialized into epithelial cells, exhibiting similar expression patterns validated by integrative transcriptomic analysis. Overall, this work provides a protocol for establishing zebrafish cell lines from single blastomeres, which can serve as valuable tools for in vitro investigations of epithelial cell dynamics in terms of life-death balance and cell fate determination during normal homeostasis.
- Zebrafish,
- Single embryo,
- Cell lines,
- Sngle-cell RNA sequencing (scRNA-Seq),
- Embryonic epidermis,
- Epithelium

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References(53)

References

Bejar, J., Hong, Y., Alvarez, M.C., 2002. An ES-like cell line from the marine fish Sparus aurata: characterization and chimaera production. Transgenic Res. 11, 279-289.

Chen, S.L., Sha, Z.X., Ye, H.Q., 2003. Establishment of a pluripotent embryonic cell line from sea perch (Lateolabrax japonicus) embryos. Aquaculture 218, 141-151.

Chen, S.L., Ye, H.Q., Sha, Z.X., Hong, Y., 2003. Derivation of embryonic cell lines from red sea bream blastulas. Journal of Fish Biology 63, 795-805.

Ciarlo, C., Kaufman, C.K., Kinikoglu, B., Michael, J., Yang, S., D'Amato, C., Blokzijl-Franke, S., den Hertog, J., Schlaeger, T.M., Zhou, Y., et al., 2017. A chemical screen in zebrafish embryonic cells establishes that Akt activation is required for neural crest development. Elife 6, e29145.

Ciarlo, C.A., Zon, L.I., 2016. Embryonic cell culture in zebrafish. Methods Cell Biol. 133, 1-10.

Cokus, S.J., De La Torre, M., Medina, E.F., Rasmussen, J. P., Ramirez-Gutierrez, J., Sagasti, A., Wang, F., 2019. Tissue-specific transcriptomes reveal gene expression trajectories in two maturing skin epithelial layers in zebrafish embryos. G3 (Bethesda) 9, 3439-3452.

Collodi, P., Kamei, Y., Sharps, A., Weber, D., Barnes, D., 1992. Fish embryo cell cultures for derivation of stem cells and transgenic chimeras. Mol. Mar. Biol. Biotechnol. 1, 257-265.

Duan, C., Korzh, V., Li, Y., Zhou, J., Liu, Y., Lu, L., Dai, W., Jiao, S., 2011. The conserved clusterin gene is expressed in the developing choroid plexus under the regulation of notch but not IGF signaling in zebrafish. Endocrinology 152, 1860-1871.

Etchin, J., Kanki, J.P., Look, A.T., 2011. Zebrafish as a model for the study of human cancer. Methods Cell Biol. 105, 309-337.

Fan, L., Collodi, P., 2006. Zebrafish embryonic stem cells. Methods Enzymol. 418, 64-77.

Fan, L., Crodian, J., Collodi, P. 2004. Culture of embryonic stem cell lines from zebrafish. Methods Cell Biol. 76, 151-160.

Fan, L., Crodian, J., Liu, X., Alestrom, A., Alestrom, P., Collodi, P., 2004. Zebrafish embryo cells remain pluripotent and germ-line competent for multiple passages in culture. Zebrafish 1, 21-26.

Fan, Z., Liu, L., Huang, X., Zhao, Y., Zhou, L., Wang, D., Wei, J., 2017. Establishment and growth responses of Nile tilapia embryonic stem-like cell lines under feeder-free condition. Dev. Growth Differ. 59, 83-93.

Farnsworth, D.R., Saunders, L.M., Miller, A.C., 2020. A single-cell transcriptome atlas for zebrafish development. Dev. Biol. 459, 100-108.

Fukazawa, C., Santiago, C., Park, K.M., Deery, W.J., Gomez de la Torre Canny, S., Holterhoff, C.K., Wagner, D.S., 2010. poky/chuk/ikk1 is required for differentiation of the zebrafish embryonic epidermis. Dev. Biol. 346, 272-283.

Ghosh, C., Collodi, P., 1994. Culture of cells from zebrafish (Brachydanio rerio) blastula-stage embryos. Cytotechnology 14, 21-26.

Gistelinck, C., Gioia, R., Gagliardi, A., Tonelli, F., Marchese, L., Bianchi, L., Landi, C., Bini, L., Huysseune, A., Witten, P.E., et al., 2016. Zebrafish collagen type I: molecular and biochemical characterization of the major structural protein in bone and skin. Sci. Rep. 6, 21540.

Gong, Z., Ju, B., Wang, X., He, J., Wan, H., Sudha, P.M., Yan, T., 2002. Green fluorescent protein expression in germ-line transmitted transgenic zebrafish under a stratified epithelial promoter from keratin8. Dev. Dyn. 223, 204-215.

Harding, M.J., McGraw, H.F., Nechiporuk, A., 2014. The roles and regulation of multicellular rosette structures during morphogenesis. Development 141, 2549-2558.

He, M., Zhang, R., Jiao, S., Zhang, F., Ye, D., Wang, H., Sun, Y., 2020. Nanog safeguards early embryogenesis against global activation of maternal β-catenin activity by interfering with TCF factors. PLoS Biol. 18, e3000561.

Helmrich, A., Barnes, D. 1998. Zebrafish embryonal cell culture. Methods Cell Biol. 59, 29-37.

Hemmati Brivanlou, A., Melton, D., 1997. Vertebrate embryonic cells will become nerve cells unless told otherwise. Cell 88, 13-17.

Ho, S.Y., Goh, C.W., Gan, J.Y., Lee, Y.S., Lam, M.K., Hong, N., Hong, Y., Chan, W. K., Shu-Chien, A.C., 2014. Derivation and long-term culture of an embryonic stem cell-like line from zebrafish blastomeres under feeder-free condition. Zebrafish 11, 407-420.

Hong, N., Schartl, M., Hong, Y., 2014. Derivation of stable zebrafish ES-like cells in feeder-free culture. Cell Tissue Res. 357, 623-632.

Howe, K., Clark, M.D., Torroja, C.F., Torrance, J., Berthelot, C., Muffato, M., 2013. The zebrafish reference genome sequence and its relationship to the human genome. Nature 496, 498-503.

Huang, H., Lindgren, A., Wu, X., Liu, N.A., Lin, S., 2012. High-throughput screening for bioactive molecules using primary cell culture of transgenic zebrafish embryos. Cell Rep. 2, 695-704.

Klein, A.M., Mazutis, L., Akartuna, I., Tallapragada, N., Veres, A., Li, V., Peshkin, L., Weitz, D.A., Kirschner, M.W., 2015. Droplet barcoding for single-cell transcriptomics applied to embryonic stem cells. Cell 161, 1187-1201.

Le Guellec, D., Morvan-Dubois, G., Sire, J.Y., 2004. Skin development in bony fish with particular emphasis on collagen deposition in the dermis of the zebrafish (Danio rerio). Int. J. Dev. Biol. 48, 217-231.

Lin, S., Long, W., Chen, J., Hopkins, N., 1992. Production of germ-line chimeras in zebrafish by cell transplants from genetically pigmented to albino embryos. Proc. Natl. Acad. Sci. U. S. A. 89, 4519-4523.

Liu, H., Duncan, K., Helverson, A., Kumari, P., Mumm, C., Xiao, Y., Carlson, J.C., Darbellay, F., Visel, A., Leslie, E., et al., 2020. Analysis of zebrafish periderm enhancers facilitates identification of a regulatory variant near human KRT8/18. Elife 9, e51325 9.

Liu, S., Xu, J., Ai, Y., Zhang, Y., Li, S., Li, J., Li, Y., 2024. Derivation of zebrafish heart-related haploid cells. J. Mol. Cell Biol. 15, mjad077.

Ma, C., Fan, L., Ganassin, R., Bols, N., Collodi, P., 2001. Production of zebrafish germ-line chimeras from embryo cell cultures. Proc. Natl. Acad. Sci. U. S. A. 98, 2461-2466.

Martorana, M.L., Tawk, M., Lapointe, T., Barre, N., Imboden, M., Joulie, C., Geraudie, J., Vriz, S., 2001. Zebrafish keratin 8 is expressed at high levels in the epidermis of regenerating caudal fin. Int. J. Dev. Biol. 45, 449-452.

Mazutis, L., Gilbert, J., Ung, W.L., Weitz, D.A., Griffiths, A.D., Heyman, J.A., 2013. Single-cell analysis and sorting using droplet-based microfluidics. Nat. Protoc. 8, 870-891.

Mushtaq, M.Y., Verpoorte, R., Kim, H.K., 2013. Zebrafish as a model for systems biology. Biotechnol. Genet. Eng. Rev. 29, 187-205.

Na, H., Park, J., Jeon, H., Jin, S., Choe, C.P., 2021. Pharyngeal endoderm expression of nanos1 is dispensable for craniofacial development. Gene Expr. Patterns 41, 119202.

Onichtchouk, D., 2012. Pou5f1/oct4 in pluripotency control: insights from zebrafish. Genesis 50, 75-85.

Padmanabhan, K., Grobe, H., Cohen, J., Soffer, A., Mahly, A., Adir, O., Zaidel-Bar, R., Luxenburg, C., 2020. Thymosin β4 is essential for adherens junction stability and epidermal planar cell polarity. Development 147, dev193425.

Sanchez-Sanchez, A.V., Camp, E., Garcia-Espana, A., Leal-Tassias, A., Mullor, J.L., 2010. Medaka Oct4 is expressed during early embryo development, and in primordial germ cells and adult gonads. Dev. Dyn. 239, 672-679.

Schaffeld, M., Knappe, M., Hunzinger, C., Markl, J., 2003. cDNA sequences of the authentic keratins 8 and 18 in zebrafish. Differentiation 71, 73-82.

Smart, N., Risebro, C.A., Melville, A.A.D., Moses, K., Schwartz, R.J., Chien, K.R., Riley, P.R., 2007. Thymosin β4 induces adult epicardial progenitor mobilization and neovascularization. Nature 445, 177-182.

Son, M.J., Gong, S.P., 2022. Feeder cell-dependent primary culture of single blastula-derived embryonic cell lines from marine medaka (Oryzias dancena). In Vitro Cell Dev. Biol. Anim. 58, 840-850.

Sun, J., Yan, L., Shen, W., Meng, A., 2018. Maternal Ybx1 safeguards zebrafish oocyte maturation and maternal-to-zygotic transition by repressing global translation. Development 145.

Sun, L., Bradford, C.S., Barnes, D.W., 1995. Feeder cell cultures for zebrafish embryonal cells in vitro. Mol. Mar. Biol. Biotechnol. 4, 43-50.

Sun, L., Bradford, C.S., Ghosh, C., Collodi, P., Barnes, D.W., 1995. ES-like cell cultures derived from early zebrafish embryos. Mol. Mar. Biol. Biotechnol. 4, 193-199.

Tat, J., Liu, M., Wen, X.Y., 2013. Zebrafish cancer and metastasis models for in vivo drug discovery. Drug Discov. Today Technol. 10, e83-89.

Wagner, D.E., Weinreb, C., Collins, Z.M., Briggs, J.A., Megason, S.G., Klein, A.M., 2018. Single-cell mapping of gene expression landscapes and lineage in the zebrafish embryo. Science 360, 981-987.

Wang, H., Liu, Y., Ye, D., Li, J., Liu, J., Deng, F., 2016. Knockdown of zebrafish Nanog increases primordial germ cells during early embryonic development. Dev. Growth Differ. 58, 355-366.

Wang, M., Zhao, K., Liu, M., Wang, M., Qiao, Z., Yi, S., Jiang, Y., Kou, X., Zhao, Y., Yin, J., et al., 2022. BMP4 preserves the developmental potential of mESCs through Ube2s- and Chmp4b-mediated chromosomal stability safeguarding. Protein Cell 13, 580-601.

Wang, X., Zhu, J., Wang, H., Deng, W., Jiao, S., Wang, Y., He, M., Zhang, F., Liu, T., Hao, Y., et al., 2023. Induced formation of primordial germ cells from zebrafish blastomeres by germplasm factors. Nat. Commun. 14, 7918.

White, R.J., Collins, J.E., Sealy, I.M., Wali, N., Dooley, C.M., Digby, Z., Stemple, D. L., Murphy, D.N., Billis, K., Hourlier, T., et al., 2017. A high-resolution mRNA expression time course of embryonic development in zebrafish. Elife 6, e30860.

Xu, C., Tabebordbar, M., Iovino, S., Ciarlo, C., Liu, J., Castiglioni, A., Price, E., Liu, M., Barton, E.R., Kahn, C.R., et al., 2013. A zebrafish embryo culture system defines factors that promote vertebrate myogenesis across species. Cell 155, 909-921.

Yi, M., Hong, N., Hong, Y., 2009. Generation of medaka fish haploid embryonic stem cells. Science 326, 430-433.

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