斑馬魚是迄今為止研究脊椎動物發育生物學最熱門的模式生物。先前的研究發現，磷脂絲胺酸受體 (PSR) 與早期胚胎發育有關，可辨識凋亡細胞並將其吞噬。而原腸胚形成階段，只有一層細胞的囊胚重組形成含有三個胚層 (即外胚層、中胚層和內胚層) 細胞的原腸胚，並再各自分化成不同器官。本研究利用CRISPR/Cas9 system將斑馬魚的PSR敲除，並發現PSR基因剔除的子代在原腸胚時期 (8hpf) 時，胚胎中都可以檢測到ROS的表現量相較於野生種有提升的現象。還發現在PSR剔除後的受精後8小時的胚胎，發現相對於野生種胚胎，有epiboly延遲約1~2小時的現象發生。而本研究還新做了代謝體學分析，發現三羧酸循環 (TCA cycle) 中的檸檬酸跟蘋果酸有顯著下調。並從過去8hpf的轉錄組學數據中發現PSR剔除子代胚胎中的Zeb1a, Zeb1b和Mxtx1等細胞遷移相關調控基因有顯著下調，而添加了檸檬酸跟蘋果酸後，則有上調的趨勢；也使得抗氧化轉錄因子、抗氧化酶相關基因表現量上調，進而清除過多的ROS，而這跟抗氧化劑N-acetylcysteine (NAC) 的處理後情況一致，間接證明了檸檬酸、蘋果酸的抗氧化功效。並用焦磷酸定序 (Pyrosequencing) 去證實了檸檬酸很可能是透過降低zeb1b DNA的啟動子CpG島的甲基化程度，來回復其表現量。

Zebrafish are by far the most popular model organisms for studying vertebrate developmental biology. Previous studies have found that phosphatidylserine receptors (PSR) are involved in early embryonic development and can recognize apoptotic cells and engulf them. Gastrulation is a stage in the embryonic development of most animals during which the blastocyst, which has only one layer of cells, reorganizes to form a gastrula containing cells of the three germ layers (ectoderm, mesoderm, and endoderm), which then differentiate into different organs. This study found that when the PSR gene was knocked out at the gastrula stage (8 hpf), the expression level of ROS could be detected in embryos (F3), which was higher than that of wild-type embryos. It was also found that after PSR knockout, epiboly was delayed by about 1 to 2 hours compared with wild-type embryos at 8 hpf, and these results are consistent with previous laboratory research data. This study also conducted a new metabolome analysis between the groups and found that citric acid and malic acid, which are closely related to ATP production and cell development in the tricarboxylic acid cycle (TCA cycle), were significantly downregulated. According to the previous transcriptomics data, cell migration-related genes such as Zeb1b, Zeb2b, and Mxtx1 were found to be significantly downregulated in PSR knockout embryos. However, after the addition of citric acid, malic acid or N-acetylcysteine (NAC), an upregulation trend was observed. The restoration of Zeb1b gene expression is likely due to the removal of ROS in PSR gene knockout embryos by citric acid treatment, which in turn leads to a decrease in the methylation level of its DNA promoter region.

白世中，證明磷脂醯絲胺酸受體基因能經由調控氧活性分子信號及細胞自噬作用來調節斑馬魚腦部發育，國立成功大學生物科技研究所碩士論文，2021。
白植友，斑馬魚磷脂醯絲胺酸受器啟動子之選殖與功能分析，國立成功大學生物科技研究所碩士論文，2007。
唐婉倫，利用CRISPR/Cas9系統建立斑馬魚磷脂醯絲胺酸受體及Bad基因剔除魚隻及其胚胎發育功能分析，國立成功大學生物科技研究所碩士論文，2017。
陳富琪，利用CRISPR/Cas9系統建立斑馬魚磷脂醯絲胺酸受體基因剔除之斑馬魚來觀察神經系統早期之發育，國立成功大學生物科技研究所碩士論文，2022。
黃培軒，探討斑馬魚之磷脂醯絲胺酸受器其吞噬死亡細胞能力與否對早期胚胎發育之影響，國立成功大學生物科技研究所碩士論文，2015。
Anglada-Girotto, M., Handschin, G., Ortmayr, K., Campos, A.I., Gillet, L., Manfredi, P., Mulholland, C.V., Berney, M., Jenal, U., Picotti, P., and Zampieri, M. Combining CRISPRi and metabolomics for functional annotation of compound libraries. Nat Chem Biol 18, 482-491, 2022.
Balachandran, A., Siyumbwa, S.N., Froemming, G.R.A., Beata, M.M., Małgorzata, J., Lavilla, C.A., Jr., Billacura, M.P., and Okechukwu, P.N. In Vitro Antioxidant and Fibroblast Migration Activities of Fractions Eluded from Dichloromethane Leaf Extract of Marantodes pumilum. Life (Basel) 13, 2023.
Balciunas, D., and Ronne, H. Evidence of domain swapping within the jumonji family of transcription factors. Trends Biochem Sci 25, 274-276, 2000.
Barrangou, R., Fremaux, C., Deveau, H., Richards, M., Boyaval, P., Moineau, S., Romero, D.A., and Horvath, P. CRISPR provides acquired resistance against viruses in prokaryotes. Science 315, 1709-1712, 2007.
Bird, A. Perceptions of epigenetics. Nature 447, 396-398, 2007.
Boeckel, J.N., Guarani, V., Koyanagi, M., Roexe, T., Lengeling, A., Schermuly, R.T., Gellert, P., Braun, T., Zeiher, A., and Dimmeler, S. Jumonji domain-containing protein 6 (Jmjd6) is required for angiogenic sprouting and regulates splicing of VEGF-receptor 1. Proc Natl Acad Sci U S A 108, 3276-3281, 2011.
Böse, J., Gruber, A.D., Helming, L., Schiebe, S., Wegener, I., Hafner, M., Beales, M., Köntgen, F., and Lengeling, A. The phosphatidylserine receptor has essential functions during embryogenesis but not in apoptotic cell removal. J Biol 3, 15, 2004.
Bretscher, M.S. Asymmetrical lipid bilayer structure for biological membranes. Nat New Biol 236, 11-12, 1972.
Chang, B., Chen, Y., Zhao, Y., and Bruick, R.K. JMJD6 is a histone arginine demethylase. Science 318, 444-447, 2007.
Cikala, M., Alexandrova, O., David, C.N., Pröschel, M., Stiening, B., Cramer, P., and Böttger, A. The phosphatidylserine receptor from Hydra is a nuclear protein with potential Fe(II) dependent oxygenase activity. BMC Cell Biol 5, 26, 2004.
Clissold, P.M., and Ponting, C.P. JmjC: cupin metalloenzyme-like domains in jumonji, hairless and phospholipase A2beta. Trends Biochem Sci 26, 7-9, 2001.
Cui, P., Qin, B., Liu, N., Pan, G., and Pei, D. Nuclear localization of the phosphatidylserine receptor protein via multiple nuclear localization signals. Exp Cell Res 293, 154-163, 2004.
Esteller, M. Epigenetics in Cancer. New England Journal of Medicine 358, 1148-1159,
Fadok, V.A., Bratton, D.L., Konowal, A., Freed, P.W., Westcott, J.Y., and Henson, P.M. Macrophages that have ingested apoptotic cells in vitro inhibit proinflammatory cytokine production through autocrine/paracrine mechanisms involving TGF-beta, PGE2, and PAF. J Clin Invest 101, 890-898, 1998.
Fadok, V.A., Voelker, D.R., Campbell, P.A., Cohen, J.J., Bratton, D.L., and Henson, P.M. Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J Immunol 148, 2207-2216, 1992.
Feil, R., and Fraga, M.F. Epigenetics and the environment: emerging patterns and implications. Nature Reviews Genetics 13, 97-109, 2012.
Feissner, R.F., Skalska, J., Gaum, W.E., and Sheu, S.-S. Crosstalk signaling between mitochondrial Ca2+ and ROS. FBL 14, 1197-1218, 2009.
Flekna, G., Schneeweiss, W., Smulders, F.J., Wagner, M., and Hein, I. Real-time PCR method with statistical analysis to compare the potential of DNA isolation methods to remove PCR inhibitors from samples for diagnostic PCR. Mol Cell Probes 21, 282-287, 2007.
Fragou, D., Fragou, A., Kouidou, S., Njau, S., and Leda, K. Epigenetic mechanisms in metal toxicity. Toxicology mechanisms and methods 21, 343-352, 2011.
Franco, R., Sánchez-Olea, R., Reyes-Reyes, E.M., and Panayiotidis, M.I. Environmental toxicity, oxidative stress and apoptosis: ménage à trois. Mutat Res 674, 3-22, 2009.
Hong, J.R., Lin, G.H., Lin, C.J., Wang, W.P., Lee, C.C., Lin, T.L., and Wu, J.L. Phosphatidylserine receptor is required for the engulfment of dead apoptotic cells and for normal embryonic development in zebrafish. Development 131, 5417-5427, 2004.
Hong, X., Zang, J., White, J., Wang, C., Pan, C.H., Zhao, R., Murphy, R.C., Dai, S., Henson, P., Kappler, J.W., Hagman, J., and Zhang, G. Interaction of JMJD6 with single-stranded RNA. Proc Natl Acad Sci U S A 107, 14568-14572, 2010.
Howe, K., Clark, M.D., Torroja, C.F., Torrance, J., Berthelot, C., Muffato, M., Collins, J.E., Humphray, S., McLaren, K., Matthews, L., McLaren, S., Sealy, I., Caccamo, M., Churcher, C., Scott, C., Barrett, J.C., Koch, R., Rauch, G.-J., White, S., Chow, W., Kilian, B., Quintais, L.T., Guerra-Assunção, J.A., Zhou, Y., Gu, Y., Yen, J., Vogel, J.-H., Eyre, T., Redmond, S., Banerjee, R., Chi, J., Fu, B., Langley, E., Maguire, S.F., Laird, G.K., Lloyd, D., Kenyon, E., Donaldson, S., Sehra, H., Almeida-King, J., Loveland, J., Trevanion, S., Jones, M., Quail, M., Willey, D., Hunt, A., Burton, J., Sims, S., McLay, K., Plumb, B., Davis, J., Clee, C., Oliver, K., Clark, R., Riddle, C., Elliott, D., Threadgold, G., Harden, G., Ware, D., Begum, S., Mortimore, B., Kerry, G., Heath, P., Phillimore, B., Tracey, A., Corby, N., Dunn, M., Johnson, C., Wood, J., Clark, S., Pelan, S., Griffiths, G., Smith, M., Glithero, R., Howden, P., Barker, N., Lloyd, C., Stevens, C., Harley, J., Holt, K., Panagiotidis, G., Lovell, J., Beasley, H., Henderson, C., Gordon, D., Auger, K., Wright, D., Collins, J., Raisen, C., Dyer, L., Leung, K., Robertson, L., Ambridge, K., Leongamornlert, D., McGuire, S., Gilderthorp, R., Griffiths, C., Manthravadi, D., Nichol, S., Barker, G., Whitehead, S., Kay, M., Brown, J., Murnane, C., Gray, E., Humphries, M., Sycamore, N., Barker, D., Saunders, D., Wallis, J., Babbage, A., Hammond, S., Mashreghi-Mohammadi, M., Barr, L., Martin, S., Wray, P., Ellington, A., Matthews, N., Ellwood, M., Woodmansey, R., Clark, G., Cooper, J.D., Tromans, A., Grafham, D., Skuce, C., Pandian, R., Andrews, R., Harrison, E., Kimberley, A., Garnett, J., Fosker, N., Hall, R., Garner, P., Kelly, D., Bird, C., Palmer, S., Gehring, I., Berger, A., Dooley, C.M., Ersan-Ürün, Z., Eser, C., Geiger, H., Geisler, M., Karotki, L., Kirn, A., Konantz, J., Konantz, M., Oberländer, M., Rudolph-Geiger, S., Teucke, M., Lanz, C., Raddatz, G., Osoegawa, K., Zhu, B., Rapp, A., Widaa, S., Langford, C., Yang, F., Schuster, S.C., Carter, N.P., Harrow, J., Ning, Z., Herrero, J., Searle, S.M.J., Enright, A., Geisler, R., Plasterk, R.H.A., Lee, C., Westerfield, M., de Jong, P.J., Zon, L.I., Postlethwait, J.H., Nüsslein-Volhard, C., Hubbard, T.J.P., Crollius, H.R., Rogers, J., and Stemple, D.L. The zebrafish reference genome sequence and its relationship to the human genome. Nature 496, 498-503, 2013.
Huang, W., Mo, J., Li, J., and Wu, K. Exploring developmental toxicity of microplastics and nanoplastics (MNPS): Insights from investigations using zebrafish embryos. Sci Total Environ 933, 173012, 2024.
Irahara, N., Nosho, K., Baba, Y., Shima, K., Lindeman, N.I., Hazra, A., Schernhammer, E.S., Hunter, D.J., Fuchs, C.S., and Ogino, S. Precision of pyrosequencing assay to measure LINE-1 methylation in colon cancer, normal colonic mucosa, and peripheral blood cells. J Mol Diagn 12, 177-183, 2010.
Ishaq, O., Sadanandan, S.K., and Wählby, C. Deep Fish: Deep Learning–Based Classification of Zebrafish Deformation for High-Throughput Screening. SLAS Discovery 22, 102-107, 2017.
Jiang, L.H., Mousawi, F., Yang, X., and Roger, S. ATP-induced Ca(2+)-signalling mechanisms in the regulation of mesenchymal stem cell migration. Cell Mol Life Sci 74, 3697-3710, 2017.
Kim, H.Y., Huang, B.X., and Spector, A.A. Phosphatidylserine in the brain: metabolism and function. Prog Lipid Res 56, 1-18, 2014.
Kimmel, C.B., Ballard, W.W., Kimmel, S.R., Ullmann, B., and Schilling, T.F. Stages of embryonic development of the zebrafish. Dev Dyn 203, 253-310, 1995.
Kouzarides, T. Chromatin Modifications and Their Function. Cell 128, 693-705, 2007.
Krieser, R.J., Moore, F.E., Dresnek, D., Pellock, B.J., Patel, R., Huang, A., Brachmann, C., and White, K. The Drosophila homolog of the putative phosphatidylserine receptor functions to inhibit apoptosis. Development 134, 2407-2414, 2007.
Kunisaki, Y., Masuko, S., Noda, M., Inayoshi, A., Sanui, T., Harada, Ｍ., Sasazuki, T., and Fukui, Y. Defective fetal liver erythropoiesis and T lymphopoiesis in mice lacking the phosphatidylserine receptor. Blood 103, 3362-3364, 2004.
Lavrik, I., Golks, A., and Krammer, P.H. Death receptor signaling. J Cell Sci 118, 265-267, 2005.
Li, M.O., Sarkisian, M.R., Mehal, W.Z., Rakic, P., and Flavell, R.A. Phosphatidylserine receptor is required for clearance of apoptotic cells. Science 302, 1560-1563, 2003.
Lo, K.H., Hui, M.N., Yu, R.M., Wu, R.S., and Cheng, S.H. Hypoxia impairs primordial germ cell migration in zebrafish (Danio rerio) embryos. PLoS One 6, e24540, 2011.
Londoño Gentile, T., Lu, C., Lodato, P.M., Tse, S., Olejniczak, S.H., Witze, E.S., Thompson, C.B., and Wellen, K.E. DNMT1 is regulated by ATP-citrate lyase and maintains methylation patterns during adipocyte differentiation. Mol Cell Biol 33, 3864-3878, 2013.
Marlow, F., Gonzalez, E.M., Yin, C., Rojo, C., and Solnica-Krezel, L. No tail co-operates with non-canonical Wnt signaling to regulate posterior body morphogenesis in zebrafish. Development 131, 203-216, 2004.
Martin, E.M., and Fry, R.C. Environmental Influences on the Epigenome: Exposure- Associated DNA Methylation in Human Populations. Annu Rev Public Health 39, 309-333, 2018.
Meyer, A., Biermann, C.H., and Ortí, G. The phylogenetic position of the zebrafish (Danio rerio), a model system in developmental biology: an invitation to the comparative method. Proc Biol Sci 252, 231-236, 1993.
Nunnari, J., and Suomalainen, A. Mitochondria: in sickness and in health. Cell 148, 1145-1159, 2012.
O'Hagan, H.M., Wang, W., Sen, S., Destefano Shields, C., Lee, S.S., Zhang, Y.W., Clements, E.G., Cai, Y., Van Neste, L., Easwaran, H., Casero, R.A., Sears, C.L., and Baylin, S.B. Oxidative damage targets complexes containing DNA methyltransferases, SIRT1, and polycomb members to promoter CpG Islands. Cancer Cell 20, 606-619, 2011.
Pang, H., and Hu, Z. Metabolomics in drug research and development: The recent advances in technologies and applications. Acta Pharm Sin B 13, 3238-3251, 2023.
Poon, I.K., Lucas, C.D., Rossi, A.G., and Ravichandran, K.S. Apoptotic cell clearance: basic biology and therapeutic potential. Nat Rev Immunol 14, 166-180, 2014.
Prasanth, M.I., Sivamaruthi, B.S., Cheong, C.S.Y., Verma, K., Tencomnao, T., Brimson, J.M., and Prasansuklab, A. Role of Epigenetic Modulation in Neurodegenerative Diseases: Implications of Phytochemical Interventions. Antioxidants (Basel) 13, 2024.
Ran, F.A., Hsu, P.D., Wright, J., Agarwala, V., Scott, D.A., and Zhang, F. Genome engineering using the CRISPR-Cas9 system. Nature Protocols 8, 2281-2308, 2013.
Ravichandran, K.S. Find-me and eat-me signals in apoptotic cell clearance: progress and conundrums. J Exp Med 207, 1807-1817, 2010.
Reddien, P.W., and Horvitz, H.R. The engulfment process of programmed cell death in caenorhabditis elegans. Annu Rev Cell Dev Biol 20, 193-221, 2004.
Redza-Dutordoir, M., and Averill-Bates, D.A. Activation of apoptosis signalling pathways by reactive oxygen species. Biochim Biophys Acta 1863, 2977-2992, 2016.
Richter, H., Randau, L., and Plagens, A. Exploiting CRISPR/Cas: interference mechanisms and applications. Int J Mol Sci 14, 14518-14531, 2013.
Saeed, K., Ayub, F., Durrani, M.A., and Mujahid, M. CRISPR Cas systems: From bacterial defense mechanisms to revolutionary tools reshaping genetic research and translation therapeutics. The Microbe 7, 100344, 2025.
Savill, J., and Fadok, V. Corpse clearance defines the meaning of cell death. Nature 407, 784-788, 2000.
Schafer, Z.T., Grassian, A.R., Song, L., Jiang, Z., Gerhart-Hines, Z., Irie, H.Y., Gao, S., Puigserver, P., and Brugge, J.S. Antioxidant and oncogene rescue of metabolic defects caused by loss of matrix attachment. Nature 461, 109-113, 2009.
Seth, A., Stemple, D.L., and Barroso, I. The emerging use of zebrafish to model metabolic disease. Dis Model Mech 6, 1080-1088, 2013.
Shiu, W.L., Huang, K.R., Hung, J.C., Wu, J.L., and Hong, J.R. Knockdown of zebrafish YY1a can downregulate the phosphatidylserine (PS) receptor expression, leading to induce the abnormal brain and heart development. J Biomed Sci 23, 31, 2016.
Sies, H., and Jones, D.P. Reactive oxygen species (ROS) as pleiotropic physiological signalling agents. Nat Rev Mol Cell Biol 21, 363-383, 2020.
Tsukada, Y., Fang, J., Erdjument-Bromage, H., Warren, M.E., Borchers, C.H., Tempst, P., and Zhang, Y. Histone demethylation by a family of JmjC domain-containing proteins. Nature 439, 811-816, 2006.
van Steijn, L., Verbeek, F.J., Spaink, H.P., and Merks, R.M.H. Predicting Metabolism from Gene Expression in an Improved Whole-Genome Metabolic Network Model of Danio rerio. Zebrafish 16, 348-362, 2019.
Wang, X., Wu, Y.C., Fadok, V.A., Lee, M.C., Gengyo-Ando, K., Cheng, L.C., Ledwich, D., Hsu, P.K., Chen, J.Y., Chou, B.K., Henson, P., Mitani, S., and Xue, D. Cell corpse engulfment mediated by C. elegans phosphatidylserine receptor through CED-5 and CED-12. Science 302, 1563-1566, 2003.
Widelitz, R., and Chuong, C.-M. Quorum sensing and other collective regenerative behavior in organ populations. Current Opinion in Genetics & Development 40, 138-143, 2016.
Wu, Y.C., and Horvitz, H.R. The C. elegans cell corpse engulfment gene ced-7 encodes a protein similar to ABC transporters. Cell 93, 951-960, 1998.
Yamashita, M. Apoptosis in zebrafish development. Comp Biochem Physiol B Biochem Mol Biol 136, 731-742, 2003.
Yang, Q., Zhang, A.-h., Miao, J.-h., Sun, H., Han, Y., Yan, G.-l., Wu, F.-f., and Wang, X.-j. Metabolomics biotechnology, applications, and future trends: a systematic review. RSC Advances 9, 37245-37257, 2019.
Zhang, R.W., and Du, J.L. In Vivo Whole-Cell Patch-Clamp Recording in the Zebrafish Brain. Methods Mol Biol 1451, 281-291, 2016.
Zhou, Z., Hartwieg, E., and Horvitz, H.R. CED-1 is a transmembrane receptor that mediates cell corpse engulfment in C. elegans. Cell 104, 43-56, 2001.
Zhu, Q.S., Wang, J., He, S., Liang, X.F., Xie, S., and Xiao, Q.Q. Early leucine programming on protein utilization and mTOR signaling by DNA methylation in zebrafish (Danio rerio). Nutr Metab (Lond) 17, 67, 2020.
Zhu, X., Tian, S., and Cai, Z. Toxicity assessment of iron oxide nanoparticles in zebrafish (Danio rerio) early life stages. PLoS One 7, e46286, 2012.