Abstract
The sphk1 gene plays a crucial role in cell growth and signal transduction. However, the developmental functions of the sphk1 gene during early vertebrate zebrafish embryo remain not completely understood. In this study, we constructed zebrafish sphk1 mutants through CRISPR/Cas9 to investigate its role in zebrafish embryonic development. Knockout of the sphk1 gene was found to cause abnormal development in zebrafish embryos, such as darkening and atrophy of the head, trunk deformities, pericardial edema, retarded yolk sac development, reduced heart rate, and premature death. The acetylcholinesterase activity was significantly increased after the knockout of sphk1, and some of the neurodevelopmental genes and neurotransmission system–related genes were expressed abnormally. The deletion of sphk1 led to abnormal expression of immune genes, as well as a significant decrease in the number of hematopoietic stem cells and neutrophils. The mRNA levels of cardiac development–related genes were significantly decreased. In addition, cell apoptosis increases in the sphk1 mutants, and the proliferation of head cells decreases. Therefore, our study has shown that the sphk1 is a key gene for zebrafish embryonic survival and regulation of organ development. It deepened our understanding of its physiological function. Our study lays the foundation for investigating the mechanism of the sphk1 gene in early zebrafish embryonic development.
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References
Avni D, Harikumar KB, Sanyal AJ, Spiegel S (2021) Deletion or inhibition of SphK1 mitigates fulminant hepatic failure by suppressing TNFα-dependent inflammation and apoptosis. FASEB J 35(3):e21415. https://doi.org/10.1096/fj.202002540R
Bassett AR, Tibbit C, Ponting CP, Liu JL (2013) Highly efficient targeted mutagenesis of Drosophila with the CRISPR/Cas9 system. Cell Rep 4(1):220–228. https://doi.org/10.1016/j.celrep.2013.06.020
Cao Z, Wang H, Mao X, Luo L (2016) Noncanonical function of threonyl-tRNA synthetase regulates vascular development in zebrafish. Biochem Biophys Res Commun 473(1):67–72. https://doi.org/10.1016/j.bbrc.2016.03.051
Cao Z, Su M, Wang H, Zhou L, Meng Z, Xiong G, Liao X, Lu H (2021) Carboxyl graphene oxide nanoparticles induce neurodevelopmental defects and locomotor disorders in zebrafish larvae. Chemosphere 270:128611. https://doi.org/10.1016/j.chemosphere.2020.128611
Cazaméa-Catalan D, Besseau L, Falcón J, Magnanou E (2014) The timing of timezyme diversification in vertebrates. PLoS One 9(12):e112380. https://doi.org/10.1371/journal.pone.0112380
Cheah FS, Jabs EW, Chong SS (2005) Genomic, cDNA, and embryonic expression analysis of zebrafish transforming growth factor beta 3 (tgfbeta3). Dev Dyn 232(4):1021–1030. https://doi.org/10.1002/dvdy.20282
Chen M, Mao A, Xu M, Weng Q, Mao J, Ji J (2019) CRISPR-Cas9 for cancer therapy: opportunities and challenges. Cancer Lett 447:48–55. https://doi.org/10.1016/j.canlet.2019.01.017
Cheng B, Jiang F, Su M, Zhou L, Zhang H, Cao Z, Liao X, Xiong G, Xiao J, Liu F, Lu H (2020a) Effects of lincomycin hydrochloride on the neurotoxicity of zebrafish. Ecotoxicol Environ Safety 201:110725. https://doi.org/10.1016/j.ecoenv.2020.110725
Cheng B, Zhang H, Jia K, Li E, Zhang S, Yu H, Cao Z, Xiong G, Hu C, Lu H (2020b) Effects of spinetoram on the developmental toxicity and immunotoxicity of zebrafish. Fish Shellfish Immunol 96:114–121. https://doi.org/10.1016/j.fsi.2019.11.066
Cui C, Benard EL, Kanwal Z, Stockhammer OW, van der Vaart M, Zakrzewska A, Spaink HP, Meijer AH (2011) Infectious disease modeling and innate immune function in zebrafish embryos. Methods Cell Biol 105:273–308. https://doi.org/10.1016/b978-0-12-381320-6.00012-6
García-Moreno D, Tyrkalska SD, Valera-Pérez A, Gómez-Abenza E, Pérez-Oliva AB, Mulero V (2019) The zebrafish: a research model to understand the evolution of vertebrate immunity. Fish Shellfish Immunol 90:215–222. https://doi.org/10.1016/j.fsi.2019.04.067
Gupta M, Shin DM, Ramakrishna L, Goussetis DJ, Platanias LC, Xiong H, Morse HC 3rd, Ozato K (2015) IRF8 directs stressinduced autophagy in macrophages and promotes clearance of Listeria monocytogenes. Nat Commun 6:6379. https://doi.org/10.1038/ncomms7379
Harrington JK, Sorabella R, Tercek A, Isler JR, Targoff KL (2017) Nkx2.5 is essential to establish normal heart rate variability in the zebrafish embryo. Am J Physiol Reg Integr Compar Physiol 313(3):R265–r271. https://doi.org/10.1152/ajpregu.00223.2016
He B, Chou J, Brandimarti R, Mohr I, Gluzman Y, Roizman B (1997) Suppression of the phenotype of gamma(1)34.5- herpes simplex virus 1: failure of activated RNA-dependent protein kinase to shut off protein synthesis is associated with a deletion in the domain of the alpha47 gene. J Virol 71(8):6049–6054. https://doi.org/10.1128/jvi.71.8.6049-6054.1997
Hisano Y, Inoue A, Okudaira M, Taimatsu K, Matsumoto H, Kotani H, Ohga R, Aoki J, Kawahara A (2015) Maternal and zygotic sphingosine kinase 2 are indispensable for cardiac development in zebrafish. J Biol Chem 290(24):14841–14851. https://doi.org/10.1074/jbc.M114.634717
Hryhorowicz M, Lipiński D, Zeyland J, Słomski R (2017) CRISPR/Cas9 immune system as a tool for genome engineering. Arch Immunol Ther Exp 65(3):233–240. https://doi.org/10.1007/s00005-016-0427-5
Huang Y, Ma J, Meng Y, Wei Y, Xie S, Jiang P, Wang Z, Chen X, Liu Z, Zhong K, Cao Z, Liao X, Xiao J, Lu H (2020) Exposure to oxadiazon-butachlor causes cardiac toxicity in zebrafish embryos. Environ Pollut 265(Pt A):114775. https://doi.org/10.1016/j.envpol.2020.114775
Jiang W, Zhou H, Bi H, Fromm M, Yang B, Weeks DP (2013) Demonstration of RISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice. Nuc Acids Res 41(20):e188. https://doi.org/10.1093/nar/gkt780
Jozefczuk E, Szczepaniak P, Guzik TJ, Siedlinski M (2021) Silencing of sphingosine kinase 1 affects maturation pathways in mouse neonatal cardiomyocytes. Int J Mol Sci 22(7). https://doi.org/10.3390/ijms22073616
Katsouda A, Peleli M, Asimakopoulou A, Papapetropoulos A, Beis D (2020) Generation and characterization of a CRISPR/Cas9 - induced 3-mst deficient zebrafish. Biomolecules 10(2). https://doi.org/10.3390/biom10020317
Khoei SG, Sadeghi H, Samadi P, Najafi R, Saidijam M (2021) Relationship between Sphk1/S1P and microRNAs in human cancers. Biotechnol Appl Biochem 68(2):279–287. https://doi.org/10.1002/bab.1922
Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF (1995) Stages of embryonic development of the zebrafish. Dev Dyn 203(3):253–310. https://doi.org/10.1002/aja.1002030302
Lan T, Li C, Yang G, Sun Y, Zhuang L, Ou Y, Li H, Wang G, Kisseleva T, Brenner D, Guo J (2018) Sphingosine kinase 1 promotes liver fibrosis by preventing miR-19b-3p-mediated inhibition of CCR2. Hepatology 68(3):1070–1086. https://doi.org/10.1002/hep.29885
Lebrun AH, Moll-Khosrawi P, Pohl S, Makrypidi G, Storch S, Kilian D, Streichert T, Otto B, Mole SE, Ullrich K, Cotman S, Kohlschütter A, Braulke T, Schulz A (2011) Analysis of potential biomarkers and modifier genes affecting the clinical course of CLN3 disease. Mol Med 17(11-12):1253–1261. https://doi.org/10.2119/molmed.2010.00241
Li H, Yu S, Cao F, Wang C, Zheng M, Li X, Qiu L (2018) Developmental toxicity and potential mechanisms of pyraoxystrobin to zebrafish (Danio rerio). Ecotoxicol Environ Safety 151:1–9. https://doi.org/10.1016/j.ecoenv.2017.12.061
Li L, Wang H, Zhang J, Sha Y, Wu F, Wen S, He L, Sheng L, You Q, Shi M, Liu L, Zhou H (2020) SPHK1 deficiency protects mice from acetaminophen-induced ER stress and mitochondrial permeability transition. Cell Death Diff 27(6):1924–1937. https://doi.org/10.1038/s41418-019-0471-x
Liang P, Xu Y, Zhang X, Ding C, Huang R, Zhang Z, Lv J, Xie X, Chen Y, Li Y, Sun Y, Bai Y, Songyang Z, Ma W, Zhou C, Huang J (2015) CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes. Protein Cell 6(5):363–372. https://doi.org/10.1007/s13238-015-0153-5
Liu CX, Li CY, Hu CC, Wang Y, Lin J, Jiang YH, Li Q, Xu X (2018) CRISPR/Cas9-induced shank3b mutant zebrafish display autism-like behaviors. Mol Autism 9:23. https://doi.org/10.1186/s13229-018-0204-x
Liu J, Huang L, Wan M, Chen G, Su M, Han F, Liu F, Xiong G, Liao X, Lu H, Li W, Cao Z (2022) Lenvatinib induces cardiac developmental toxicity in zebrafish embryos through regulation of Notch mediated-oxidative stress generation. Environ Toxicol 37(6):1310–1320. https://doi.org/10.1002/tox.23485
Luo S, Li W, Xie Y, Wu B, Sun Y, Tian Q, Wang Z, Han F (2021) A molecular insight into the resistance of yellow drum to Vibrio harveyi by genome-wide association analysis. Aquaculture 543:736998. https://doi.org/10.1016/j.aquaculture.2021.736998
Ma Y, Zhang L, Huang X (2014) Genome modification by CRISPR/Cas9. FEBS J 281(23):5186–5193. https://doi.org/10.1111/febs.13110
Ma Y, Xing X, Kong R, Cheng C, Li S, Yang X, Li S, Zhao F, Sun L, Cao G (2021) SphK1 promotes development of non-small cell lung cancer through activation of STAT3. IntJ Mol Med 47(1):374–386. https://doi.org/10.3892/ijmm.2020.4796
Matrone G, Meng S, Gu Q, Lv J, Fang L, Chen K, Cooke JP (2017) Lmo2 (LIM-domain-only 2) modulates Sphk1 (sphingosine kinase) and promotes endothelial cell migration. Arterioscl Thromb Vasc Biol 37(10):1860–1868. https://doi.org/10.1161/atvbaha.117.309609
Meng H, Shang Y, Cheng Y, Wang K, Yu J, Cao P, Fan S, Li Y, Cui J (2021) Knockout of zebrafish colony-stimulating factor 1 receptor by CRISPR/Cas9 affects metabolism and locomotion capacity. Biochem Biophys Res Commun 551:93–99. https://doi.org/10.1016/j.bbrc.2021.02.122
Mu X, Liu J, Yuan L, Yang K, Huang Y, Wang C, Yang W, Shen G, Li Y (2019) The mechanisms underlying the developmental effects of bisphenol F on zebrafish. Sci Total Environ 687:877–884. https://doi.org/10.1016/j.scitotenv.2019.05.489
Ni W, Qiao J, Hu S, Zhao X, Regouski M, Yang M, Polejaeva IA, Chen C (2014) Efficient gene knockout in goats using CRISPR/Cas9 system. PLoS One 9(9):e106718. https://doi.org/10.1371/journal.pone.0106718
Park KH, Ye ZW, Zhang J, Hammad SM, Townsend DM, Rockey DC, Kim SH (2019) 3-ketodihydrosphingosine reductase mutation induces steatosis and hepatic injury in zebrafish. Sci Rep 9(1):1138. https://doi.org/10.1038/s41598-018-37946-0
Plano D, Amin S, Sharma AK (2014) Importance of sphingosine kinase (SphK) as a target in developing cancer therapeutics and recent developments in the synthesis of novel SphK inhibitors. J Med Chem 57(13):5509–5524. https://doi.org/10.1021/jm4011687
Platt RJ, Chen S, Zhou Y, Yim MJ, Swiech L, Kempton HR, Dahlman JE, Parnas O, Eisenhaure TM, Jovanovic M, Graham DB, Jhunjhunwala S, Heidenreich M, Xavier RJ, Langer R, Anderson DG, Hacohen N, Regev A, Feng G et al (2014) CRISPR-Cas9 knockin mice for genome editing and cancer modeling. Cell 159(2):440–455. https://doi.org/10.1016/j.cell.2014.09.014
Schröder M, Richter C, Juan MH, Maltusch K, Giegold O, Quintini G, Pfeilschifter JM, Huwiler A, Radeke HH (2011) The sphingosine kinase 1 and S1P1 axis specifically counteracts LPS-induced IL-12p70 production in immune cells of the spleen. Mol Immunol 48(9-10):1139–1148. https://doi.org/10.1016/j.molimm.2011.02.007
Seruggia D, Fernández A, Cantero M, Pelczar P, Montoliu L (2015) Functional validation of mouse tyrosinase non-coding regulatory DNA elements by CRISPR-Cas9-mediated mutagenesis. Nucl Acids Res 43(10):4855–4867. https://doi.org/10.1093/nar/gkv375
Sieger D, Stein C, Neifer D, van der Sar AM, Leptin M (2009) The role of gamma interferon in innate immunity in the zebrafish embryo. Dis Models Mechan 2(11-12):571–581. https://doi.org/10.1242/dmm.003509
Singleman C, Holtzman NG (2012) Analysis of postembryonic heart development and maturation in the zebrafish. Danio rerio. Dev Dyn 241(12):1993–2004. https://doi.org/10.1002/dvdy.23882
Staudt D, Stainier D (2012) Uncovering the molecular and cellular mechanisms of heart development using the zebrafish. Ann Rev Gen 46:397–418. https://doi.org/10.1146/annurev-genet-110711-155646
Sun Q, Kim HE, Cho H, Shi S, Kim B, Kim O (2018) Red light-emitting diode irradiation regulates oxidative stress and inflammation through SPHK1/NF-κB activation in human keratinocytes. J Photochem Photobiol Biol 186:31–40. https://doi.org/10.1016/j.jphotobiol.2018.05.015
Sun Y, Cao Y, Tong L, Tao F, Wang X, Wu H, Wang M (2020) Exposure to prothioconazole induces developmental toxicity and cardiovascular effects on zebrafish embryo. Chemosphere 251:126418. https://doi.org/10.1016/j.chemosphere.2020.126418
Tsarouchas TM, Wehner D, Cavone L, Munir T, Keatinge M, Lambertus M, Underhill A, Barrett T, Kassapis E, Ogryzko N, Feng Y, van Ham TJ, Becker T, Becker CG (2018) Dynamic control of proinflammatory cytokines Il-1β and Tnf-α by macrophages in zebrafish spinal cord regeneration. Nat Commun 9(1):4670. https://doi.org/10.1038/s41467-018-07036-w
Welsch R, Zhou X, Koschmieder J, Schlossarek T, Yuan H, Sun T, Li L (2019) Characterization of cauliflower or mutant variants. Front Plant Sci 10:1716. https://doi.org/10.3389/fpls.2019.01716
Xiong Y, Yang P, Proia RL, Hla T (2014) Erythrocyte-derived sphingosine 1-phosphate is essential for vascular development. J Clin Investig 124(11):4823–4828. https://doi.org/10.1172/jci77685
Zhang X, Wang W, Ji XY, Ritter JK, Li N (2019) Knockout of sphingosine kinase 1 attenuates renal fibrosis in unilateral ureteral obstruction model. Am J Nephrol 50(3):196–203. https://doi.org/10.1159/000502448
Zhang K, Yuan G, Werdich AA, Zhao Y (2020) Ibuprofen and diclofenac impair the cardiovascular development of zebrafish (Danio rerio) at low concentrations. Environ Pollut 258:113613. https://doi.org/10.1016/j.envpol.2019.113613
Zhu XY, Wu SQ, Guo SY, Yang H, Xia B, Li P, Li CQ (2018) A zebrafish heart failure model for assessing therapeutic agents. Zebrafish 15(3):243–253. https://doi.org/10.1089/zeb.2017.1546
Funding
This work was supported by the National Natural Science Foundation (31900597, 81860282, 32072969), the Jiangxi Province’s major academic and technical leaders training plan for young talents (20204BCJL23043), the Jiangxi Natural Science Foundation for Distinguished Young Scholars (20224ACB215001), and the Jiangxi Provincial Department of Education Science and Technology Program Project (GJJ211004).
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Zigang Cao, Wanbo Li and Ling Huang designed the experiments; Zigang Cao and Wanbo Li supervised the study; Ling Huang performed all of experiments; Ling Huang, Wanbo Li, Fang Han and Ying Huang wrote the first draft of the manuscript; Ling Huang, Jieping Liu and Xinjun Liao analyzed data. All authors read and approved the final manuscript.
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All experimental procedures on zebrafish were performed according to the guiding principles for the care and use of laboratory animals and were approved by the Independent Animal Care Committee (IACC) of Jinggangshan University.
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Huang, L., Han, F., Huang, Y. et al. Sphk1 deficiency induces apoptosis and developmental defects and premature death in zebrafish. Fish Physiol Biochem 49, 737–750 (2023). https://doi.org/10.1007/s10695-023-01215-3
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DOI: https://doi.org/10.1007/s10695-023-01215-3