合子基因mcm3在斑马鱼肝早期发育中的作用
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摘要
目的研究斑马鱼合子基因mcm3是否参与了斑马鱼肝早期发育的调控。方法运用原位杂交分析mcm3在斑马鱼发育早期表达谱;运用mcm3 MO敲降mcm3基因功能,然后用原位杂交及转基因鱼胚胎分析肝的发育状况。结果 mcm3为合子表达基因,在中囊胚期后到尾牙期广泛表达,而在体节发生期及以后分别在体节、头部及内胚层组织高表达。进一步研究发现在mcm3功能敲降后肝变小,而中胚层器官血管心脏的发育并未受到明显影响。mcm3 mRNA回救实验表明mcm3特异的调控了肝的发育。结论斑马鱼合子基因mcm3参与了肝早期发育的调控。
Objective MCM3, a component of the MCM complex(MCM2-MCM7 complex), plays a critical role in the initiation of DNA replication and is a marker overexpressed in most malignant tumors such as liver cancer. However, whether it is involved in the early development of zebrafish liver is largely unknown. Our current study addressed whether zygotic mcm3 contributes to the regulation of liver development in zebrafish. Methods Expression of zygotic mcm3 was examined using in situ hybridization. mcm3 was then knocked down by injecting a mcm3 MO, and the liver developmental phenotype was analyzed in transgenic line(fabp10: GFP) and wild type embryos. Results Being as a zygotic gene, mcm3 was expressed ubiquitously before the bud stage, whereas it was highly restricted in somite, head and endoderm-related tissues from the somite stage. After mcm3 loss of function, the liver was smaller than that in the control embryos, but no other distinct developmental defects were observed. In addition, mcm3 mRNA injection rescued the defect of liver development in the mcm3 morphants. Conclusion Zygotic mcm3 is involved in the regulation of early development of zebrafish lever.
Objective MCM3, a component of the MCM complex(MCM2-MCM7 complex), plays a critical role in the initiation of DNA replication and is a marker overexpressed in most malignant tumors such as liver cancer. However, whether it is involved in the early development of zebrafish liver is largely unknown. Our current study addressed whether zygotic mcm3 contributes to the regulation of liver development in zebrafish. Methods Expression of zygotic mcm3 was examined using in situ hybridization. mcm3 was then knocked down by injecting a mcm3 MO, and the liver developmental phenotype was analyzed in transgenic line(fabp10: GFP) and wild type embryos. Results Being as a zygotic gene, mcm3 was expressed ubiquitously before the bud stage, whereas it was highly restricted in somite, head and endoderm-related tissues from the somite stage. After mcm3 loss of function, the liver was smaller than that in the control embryos, but no other distinct developmental defects were observed. In addition, mcm3 mRNA injection rescued the defect of liver development in the mcm3 morphants. Conclusion Zygotic mcm3 is involved in the regulation of early development of zebrafish lever.
引文
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[19] 黄超,张冲,蒋发明,等.斑马鱼消化器官发育缺陷突变体的遗传筛选 [J].中国实验动物学报,2015,23(05):441-445.Huang C,Zhang C,Jiang F,et al.Genetic screening of zebrafish mutants with developmental defects in the digestive organs [J].Acta Lab Anim Sci Sin,2015,23(05):441-445.
[20] 王政,李金鹏,闫一芳,等.isthmin 1 对斑马鱼胚胎汇聚延伸运动的影响 [J].中国比较医学杂志,2018,28(2):40-45.Wang Z,Li J,Yan Y,et al.ism1 regulates convergence and extension movements during zebrafish gastrulation [J].Chin J Comp Med,2018,28(2):40-45.
[21] Kimmel CB,Ballard WW,Kimmel SR,et al.Stages of embryonic development of the zebrafish [J].Dev Dyn,1995,203(3):253-310.
[2] Shinya M,Machiki D,Henrich T,et al.Evolutionary diversification of MCM3 genes in Xenopus laevis and Danio rerio [J].Cell Cycle,2014,13(20):3271-3281.
[3] Takara TJ,Bell SP.Putting two heads together to unwind DNA [J].Cell,2009,139(4):652-654.
[4] Remus D,Beuron F,Tolun G,et al.Concerted loading of Mcm2-7 double hexamers around DNA during DNA replication origin licensing [J].Cell,2009,139(4):719-730.
[5] Ilves I,Petojevic T,Pesavento JJ,et al.Activation of the MCM2-7 helicase by association with Cdc45 and GINS proteins [J].Mol Cell,2010,37(2):247-258.
[6] Riera A,Tognetti S,Speck C.Helicase loading:how to build a MCM2-7 double-hexamer [J].Semin Cell Dev Biol,2014,30:104-109.
[7] Han X,Mayca Pozo F,Wisotsky JN,et al.Phosphorylation of minichromosome maintenance 3 (MCM3) by checkpoint kinase 1 (Chk1) negatively regulates DNA replication and checkpoint activation [J].Biol Chem,2015,290(19):12370-12378.
[8] Davuluri G,Gong W,Yusuff S,et al.Mutation of the zebrafish nucleoporin elys sensitizes tissue progenitors to replication stress [J].PLoS Genet,2008,4(10):e1000240.
[9] Gan N,Du Y,Zhang W,et al.Increase of Mcm3 and Mcm4 expression in cervical squamous cell carcinomas [J].Eur J Gynaecol Oncol,2010,31(3):291-294.
[10] Zhuang L,Yang Z,Meng Z.Upregulation of BUB1B,CCNB1,CDC7,CDC20,and MCM3 in tumor tissues predicted worse overall survival and disease-free survival in hepatocellular carcinoma patients [J].Biomed Res Int,2018,2018(163):1-8.
[11] Rezvani G,Andisheh-Tadbir A,Ashraf MJ,et al.Evaluation of minichromosome maintenance-3 (MCM3) in oral squamous cell carcinoma [J].J Dent Shiraz Univ Med Sci.2015,16(2):87-92.
[12] Zhong H,Chen B,Neves H,et al.Expression of minichromosome maintenance genes in renal cell carcinoma [J].Cancer Manag,2017,9:637-647.
[13] Woodward AM,Gohler T,Luciani MG,et al.Excess Mcm2-7 license dormant origins of replication that can be used under conditions of replicative stress [J].Cell Biol,2006,173(5):673-683.
[14] Ge XQ,Jackson DA,and Blow JJ.Dormant origins licensed by excess Mcm2-7 are required for human cells to survive replicative stress [J].Genes Dev,2007,21(24):3331-3341.
[15] Ma D,Wang L,Wang S,et al.Foxn1 maintains thymic epithelial cells to support T-cell development via mcm2 in zebrafish [J].Proc Natl Acad Sci U S A,2012,109(51):21040-21045.
[16] 张雨,吴永梅,刘敏,等.斑马鱼mcm5基因的表达分析及在体节发生中的功能 [J].中国比较医学杂志,2018,28(10):8-14.Zhang Y,Wu Y,Liu M,et al.The expression pattern of zebrafish mcm5 and its role in somitogenesis [J].Chin J Comp Med,2018,28(10):8-14.
[17] Alvarez S,Diaz M,Flach J,et al.Replication stress caused by low MCM expression limits fetal erythropoiesis and hematopoietic stem cell functionality [J].Nat Commun,2015,6,8548.
[18] Shinya M,Machiki D,Henrich T,et al.Evolutionary diversification of MCM3 genes in Xenopus laevis and l.Danio rerio [J].Cell Cycle,2014,13(20):3271-3281.
[19] 黄超,张冲,蒋发明,等.斑马鱼消化器官发育缺陷突变体的遗传筛选 [J].中国实验动物学报,2015,23(05):441-445.Huang C,Zhang C,Jiang F,et al.Genetic screening of zebrafish mutants with developmental defects in the digestive organs [J].Acta Lab Anim Sci Sin,2015,23(05):441-445.
[20] 王政,李金鹏,闫一芳,等.isthmin 1 对斑马鱼胚胎汇聚延伸运动的影响 [J].中国比较医学杂志,2018,28(2):40-45.Wang Z,Li J,Yan Y,et al.ism1 regulates convergence and extension movements during zebrafish gastrulation [J].Chin J Comp Med,2018,28(2):40-45.
[21] Kimmel CB,Ballard WW,Kimmel SR,et al.Stages of embryonic development of the zebrafish [J].Dev Dyn,1995,203(3):253-310.