Micall2a基因表达下调抑制斑马鱼血管发育

doi:10.12300/j.issn.1674-5817.2022.166

实验动物与比较医学 ›› 2023, Vol. 43 ›› Issue (3): 282-287.DOI: 10.12300/j.issn.1674-5817.2022.166

Micall2a基因表达下调抑制斑马鱼血管发育

杨晋娴¹^,²(), 王淑娟¹, 翟金云¹, 朱顺星¹^,²()()

^1.南通大学杏林学院, 南通 226001
^2.南通大学实验动物中心, 南通 226001

收稿日期:2022-10-31 修回日期:2023-04-17 出版日期:2023-06-25 发布日期:2023-07-18
通讯作者: 朱顺星（1968—），男，博士，副教授，研究方向：人类疾病与动物模型。E-mail：zsx@ntu.edu.cn。ORCID： 0000-0001-8011-4099
作者简介:杨晋娴（1990—），女，硕士，讲师，研究方向：人类疾病与动物模型。E-mail：yjx0815@ntu.edu.cn
基金资助:
江苏省高等学校自然科学研究面上项目“LncRNA SLC25A34-AS1调控斑马鱼动静脉分化的机制研究”(20KJB310028);南通市科技计划项目“利用斑马鱼模型研究miR-22a调控血管芽生导向的机制”(JC2020104);南通大学杏林学院大学生创新创业训练计划项目“Micall2a基因调控斑马鱼胚胎血管发育”(201813993021xl)

Downregulation of Micall2a Gene Expression Inhibited Vascular Development in Zebrafish

Jinxian YANG¹^,²(), Shujuan WANG¹, Jinyun ZHAI¹, Shunxing ZHU¹^,²()()

^1.Xinglin College of Nantong University, Nantong 226001, China
^2.Laboratory Animal Center of Nantong University, Nantong 226001, China

Received:2022-10-31 Revised:2023-04-17 Published:2023-06-25 Online:2023-07-18
Contact: ZHU Shunxing (ORCID: 0000-0001-8011-4099), E-mail: zsx@ntu.edu.cn

摘要/Abstract

摘要：

目的探究斑马鱼胚胎早期发育过程中Micall2a基因的表达模式，以及该基因对斑马鱼血管发育的影响。方法使用Tg（fli：GFP）转基因斑马鱼（用绿色荧光蛋白标记血管）和野生型斑马鱼（AB），采用全胚胎原位杂交技术检测其早期胚胎不同发育阶段的Micall2a基因表达水平。通过显微注射吗啉反义寡核苷酸下调Micall2a基因，并采用实时荧光定量PCR技术检测该基因在斑马鱼胚胎不同发育阶段mRNA表达水平。采用激光共聚焦显微成像技术，观察并分析Micall2a基因下调后斑马鱼的血管表型变化。结果受精后24 h（24 hours post-fertilization，24 hpf）、36 hpf和48 hpf的斑马鱼早期胚胎的脑、心脏、血管系统中均有Micall2a基因表达。显微注射吗啉反义寡核苷酸后Micall2a基因的mRNA水平增加，抑制斑马鱼胚胎的血管发育，导致斑马鱼节间血管发育缺陷。结论 Micall2a基因表达下调可以抑制斑马鱼血管的发育。

关键词: Micall2a基因, 原位杂交, 胚胎发育, 血管发育, 斑马鱼, 血管依赖性疾病

Abstract:

Objective To explore the expression pattern of Micall2a gene during the early development of zebrafish embryos and the effect of this gene on zebrafish vascular development. Methods Whole embryo in situ hybridization was used to detect Micall2a expression levels at different stages of early embryo development of Tg (fli:GFP) transgenic (labeled with green fluorescent protein) and wild type zebrafish (AB). Micall2a gene expression was downregulated by microinjection of a morpholine antisense oligonucleotide, and real-time fluorescent quantitative PCR was used to detect mRNA expression of the gene at different developmental stages of zebrafish embryos. Laser confocal microscopy was used to observe and analyze vascular phenotypic changes in zebrafish after the downregulation of Micall2a. Results Micall2a was expressed in the brain, heart, and vascular system of zebrafish embryos at the 24th, 36th, and 48th hours post fertilization. The mRNA level of Micall2a increased after microinjection of morpholine antisense oligonucleotides, inhibiting vascular development in zebrafish embryos, resulting in internode angiogenesis defects in zebrafish. Conclusion Downregulation of Micall2a expression inhibits the development of blood vessels in zebrafish.

Key words: Micall2a gene, In situ hybridization, Vascular development, Embryonic development, Zebrafish, Vascular dependent diseases

中图分类号:

R-332

杨晋娴, 王淑娟, 翟金云, 朱顺星. Micall2a基因表达下调抑制斑马鱼血管发育[J]. 实验动物与比较医学, 2023, 43(3): 282-287.

Jinxian YANG, Shujuan WANG, Jinyun ZHAI, Shunxing ZHU. Downregulation of Micall2a Gene Expression Inhibited Vascular Development in Zebrafish[J]. Laboratory Animal and Comparative Medicine, 2023, 43(3): 282-287.

图/表 3

图 1 Micall2a基因在斑马鱼胚胎血管系统中的表达注：A～C分别为受精后24、36和48 h。ISV为节间血管，PCV为后主静脉，DA为背主动脉。Br为大脑，He为心脏（红色箭头指示位置）。

Figure 1 Expression of the Micall2a gene in the vascular system of zebrafish embryosNote： A-C, Micall2a expression levels at the 24th, 36th, and 48th hours post fertilization in the zebrafish. ISV, intersegmental vessel; DA, dorsal aorta; PCV, posterior cardinal vein; Br, brain; He, heart. Red arrow indicates the location.

图2 下调Micall2a基因表达后24和50 hpf斑马鱼胚胎中节间血管的发育情况注：A为显微注射吗啉反义寡核苷酸下调转基因斑马鱼Tg（fli：GFP）Micall2a基因（Micall2a-Mo组）和注射相同体积PBS转基因斑马鱼（Control组），并培养至受精后24 h（24 hpf）和50 h（50 hpf），斑马鱼胚胎内节间血管形态的激光共聚焦显微镜成像图片；背侧纵向吻合口血管（DLAV）由红色实线表示，水平肌间隔用黄色虚线表示。B为各组斑马鱼胚胎中节间血管（ISV）生长的表型占比（红色为血管生长至背侧纵向吻合口血管，橘色为穿过肌隔膜，黄色为到达横向肌隔膜，绿色为肌隔膜下）。每组10个胚胎，每个胚胎统计7个ISV。

Figure 2 Morphology of zebrafish embryos larval internodal vascular after the knock-down of Micall2a gene at 24 and 50 hpfNote：(A) Laser confocal microscope image of internode vascular morphology in zebrafish embryos with microinjection of morpholine antisense oligonucleotide knockdown of the expression of Micall2a in Tg (fli :GFP) transgenic lines (Micall2a-Mo group) and injection of the same volume of PBS in transgenic zebrafish (control group) cultured to 24 and 50 hpf. The dorsal longitudinal anastomosis vessel (DLAV) is indicated by a solid red line and the horizontal septum by a yellow dashed line. (B) Phenotypic proportion of internodal vascular (ISV) growth in zebrafish embryos (red indicates blood vessel growth to the dorsal longitudinal anastomotic blood vessel, orange indicates growth through the myseptum, yellow indicates growth to the transverse myodiam, and green indicates submyspheric growth). 10 embryos per group and 7 ISVs per embryo were counted.

图3 实时荧光定量PCR分析注射Micall2a-Mo后斑马鱼胚胎体内Micall2a基因表达变化注：Micall2a-Mo组为显微注射吗啉反义寡核苷酸下调转基因斑马鱼Tg（fli：GFP）中Micall2a基因并培养至受精后24 h（24 hpf）和50 hpf；对照组为注射相同体积PBS的转基因斑马鱼。与对照组比较，??P＜0.01，???P＜0.001；与24 hpf相比，##P＜0.01。每组15个胚胎，实验重复3次。

Figure 3 Real-time PCR analysis of Micall2a gene expression changes in zebrafish embryos after injection of Micall2a-MoNote：The Micall2a-Mo group was microinjected with morpholine antisense oligonucleotide and the expression of Micall2a in Tg (fli:GFP) transgenic lines and cultured to 24 and 50 hpf. The control group was injected with an equal volume of PBS. Compared with the control group, ??P<0.01, ???P<0.001. Compared with 24 hpf, ##P<0.01. The experiment was repeated three times for each group of 15 embryos.

参考文献 22

1	HUNG R J, YAZDANI U, YOON J, et al. Mical links semaphorins to F-actin disassembly[J]. Nature, 2010, 463(7282):823-827. DOI: 10.1038/nature08724 .
2	BEUCHLE D, SCHWARZ H, LANGEGGER M, et al. Drosophila MICAL regulates myofilament organization and synaptic structure[J]. Mech Dev, 2007, 124(5):390-406. DOI: 10.1016/j.mod.2007.01.006 .
3	WEIDE T, TEUBER J, BAYER M, et al. MICAL-1 isoforms, novel rab1 interacting proteins[J]. Biochem Biophys Res Commun, 2003, 306(1):79-86. DOI: 10.1016/s0006-291x(03)00918-5 .
4	XUE Y L, KUOK C XIAO A,et al. Identification and expression analysis of mical family genes in zebrafish[J]. J Genet Genom, 2010, 37(10):685-693. DOI: 10.1016/S1673-8527(09)60086-2 .
5	YANG Y X, YE F W, XIA T X, et al. High MICAL-L2 expression and its role in the prognosis of colon adenocarcinoma[J]. BMC Cancer, 2022, 22(1):487. DOI: 10.1186/s12885-022-09614-0 .
6	WU H, YESILYURT H G, YOON J, et al. The MICALs are a family of F-actin dismantling oxidoreductases conserved from drosophila to humans[J]. Sci Rep, 2018, 8(1):937. DOI: 10.1038/s41598-017-17943-5 .
7	WANG Y, DENG W, ZHANG Y, et al. MICAL2 promotes breast cancer cell migration by maintaining epidermal growth factor receptor (EGFR) stability and EGFR/P38 signalling activation[J]. Acta Physiol (Oxf), 2018, 222(2):10.1111/apha.12920. DOI: 10.1111/apha.12920 .
8	BAKKERS J. Zebrafish as a model to study cardiac development and human cardiac disease[J]. Cardiovasc Res, 2011, 91(2):279-288. DOI: 10.1093/cvr/cvr098 .
9	HOGAN B M, SCHULTE-MERKER S. How to plumb a Pisces: understanding vascular development and disease using zebrafish embryos[J]. Dev Cell, 2017, 42(6):567-583. DOI: 10.1016/j.devcel.2017.08.015 .
10	江霞, 钱豪杰, 魏迅, 等. 斑马鱼糖尿病模型的构建及应用进展[J]. 实验动物与比较医学, 2020, 40(6):547-552. DOI: 10.3969/j.issn.1674-5817.2020.06.016 .
	JIANG X, QIAN H J, WEI X, et al. Research progress in construction and application of diabetes model in zebrafish[J]. Lab Anim Comp Med, 2020, 40(6):547-552. DOI: 10.3969/j.issn.1674-5817.2020.06.016 .
11	Folkman J. Angiogenesis:an organizing principle for drug discovery[J].Nat Rev Drug Discov, 2007,6(4):273-286.DOI:10.1038/nrd2115 .
12	WANG J L, ZHANG X H, XU X Y, et al. Pro-angiogenic activity of Tongnao Decoction on HUVECs in vitro and zebrafish in vivo [J]. J Ethnopharmacol, 2020, 254:112737. DOI: 10.1016/j.jep.2020.112737 .
13	CASEY M J, STEWART R A. Zebrafish as a model to study neuroblastoma development[J]. Cell Tissue Res, 2018, 372(2):223-232. DOI: 10.1007/s00441-017-2702-0 .
14	CHITRAMUTHU B P, BENNETT H P J. High resolution whole mount in situ hybridization within zebrafish embryos to study gene expression and function[J]. J Vis Exp, 2013(80): e50644. DOI: 10.3791/50644 .
15	张晶晶, 王新, 刘东. C型利钠肽基因调控斑马鱼胚胎血管发育[J]. 生理学报, 2017, 69(1): 11-16. DOI: 10.13294/j.aps.2016.0098 .
	ZHANG J J, WANG X, LIU D. C-type natriuretic peptide gene regulates the vascular development of zebrafish embryos[J]. Acta Physiol Sin, 2017, 69(1): 11-16. DOI: 10.13294/j.aps.2016.0098 .
16	SUMMERTON J E. Morpholino, siRNA, and S-DNA compared: impact of structure and mechanism of action on off-target effects and sequence specificity[J]. Curr Top Med Chem, 2007, 7(7):651-660. DOI: 10.2174/156802607780487740 .
17	ZHOU Y P, GUNPUT R A F, ADOLFS Y, et al. MICALs in control of the cytoskeleton, exocytosis, and cell death[J]. Cell Mol Life Sci, 2011, 68(24):4033-4044. DOI: 10.1007/s00018-011-0787-2 .
18	RAHAJENG J, GIRIDHARAN S S P, CAI B S, et al. Important relationships between Rab and MICAL proteins in endocytic trafficking[J]. World J Biol Chem, 2010, 1(8):254-264. DOI: 10.4331/wjbc.v1.i8.254 .
19	TERAI T, NISHIMURA N, KANDA I, et al. JRAB/MICAL-L2 is a junctional Rab13-binding protein mediating the endocytic recycling of occludin[J]. Mol Biol Cell, 2006, 17(5):2465-2475. DOI: 10.1091/mbc.e05-09-0826 .
20	杨晋娴, 王新, 刘东, 等. Clec14a基因调控斑马鱼胚胎血管发育[J]. 交通医学, 2018, 32(6):535-539. DOI: 10.1016/j.bbrc.2016.12.118 .
	YANG J X, WANG X, LIU D, et al. Clec14a gene contributes to zebrafish angiogenesis[J]. Med J Commun, 2018, 32(6): 535-539. DOI: 10.1016/j.bbrc.2016.12.118 .
21	DIOMEDE F, DILETTA MARCONI G, FONTICOLI L, et al. Functional relationship between osteogenesis and angiogenesis in tissue regeneration[J]. Int J Mol Sci, 2020, 21(9):3242. DOI: 10.3390/ijms21093242 .
22	王晓航, 陈洋, 戚中田, 等. 血脑屏障动物模型的研究进展[J]. 海军军医大学学报, 2022(3):314-319.
	WANG X H, CHEN Y, QI Z T, et al. Animal models of blood-brain barrier: research progress[J]. Acad J Nav Med Univ, 2022(3):314-319.

[1]	林金杏, 王新栋, 白雪兵, 冯丽萍, 谢淑武, 陈秋生. 成年斑马鱼体肾的微细结构及其外泌体的分布鉴定[J]. 实验动物与比较医学, 2023, 43(5): 531-540.
[2]	谭志刚, 刘锦信, 郑楚雅, 廖文峰, 冯露平, 彭红丽, 严秀, 卓振建. 神经母细胞瘤动物模型研究进展与应用[J]. 实验动物与比较医学, 2023, 43(3): 288-296.
[3]	金仕容, 华叶, 訾化星, 杜旭飞, 卜纪雯. 一种显著提高实验用斑马鱼繁殖效率和使用寿命的优化养殖方案[J]. 实验动物与比较医学, 2023, 43(3): 297-306.
[4]	江霞, 钱豪杰, 魏迅, 郑羽旋, 周正宇. 斑马鱼糖尿病模型的构建及应用进展[J]. 实验动物与比较医学, 2020, 40(6): 547-552.
[5]	邸亚男, 朱丽英, 钱雯, 潘卫. 斑马鱼作为模式动物在人类眼睛疾病研究中的应用[J]. 实验动物与比较医学, 2020, 40(5): 440-.
[6]	王雪, 刘可春, 杨学亮, 马玉奎, 张云. 冈田酸对斑马鱼幼鱼神经行为功能的影响[J]. 实验动物与比较医学, 2020, 40(3): 190-.
[7]	李英娘, 戴玮, 盛健. 斑马鱼甲状腺肿大病例分析[J]. 实验动物与比较医学, 2019, 39(6): 462-466.
[8]	王俐梅, 张建, 刘忠华, 杨威. 视网膜母细胞瘤斑马鱼模型及rb1基因研究进展[J]. 实验动物与比较医学, 2019, 39(3): 244-248.
[9]	周斌, 盛哲津, 冯琛卓, 李利妹. 一种体内实时观察黑色素瘤的斑马鱼模型[J]. 实验动物与比较医学, 2018, 38(1): 22-28.
[10]	田芳, 王玉柱, 夏敏杰, 孙冰, 丁训城, 李卫华, 许慧慧, 胡晶莹. 实验用斑马鱼成鱼内脏组织病理学检查方法的优化[J]. 实验动物与比较医学, 2017, 37(6): 465-469.
[11]	金璐, 李燕, 李志操, 吴西军, 周艳华, 何志旭, 舒莉萍. Irx5a基因过表达对斑马鱼胚胎早期造血的影响[J]. 实验动物与比较医学, 2017, 37(4): 309-314.
[12]	林金杏, 冯丽萍, 胡建华, 高诚. 斑马鱼鳍和鳞片色素细胞的显微观察[J]. 实验动物与比较医学, 2017, 37(2): 94-101.
[13]	张城达, 张丽丽, 张立将, 陈云祥. 阿霉素致斑马鱼心血管毒性模型的建立[J]. 实验动物与比较医学, 2015, 35(5): 362-366.
[14]	张勇, 朱晓宇, 郭胜亚, 李春启. 苯肼诱发建立斑马鱼血栓模型的方法[J]. 实验动物与比较医学, 2015, 35(1): 27-31.
[15]	王美珊, 孔彭成, 朱燕, 朱莲, 陈学进, 李和平, 蒋满喜. 一种改良的小鼠附睾尾常温运输方法[J]. 实验动物与比较医学, 2014, 34(2): 145-148.

Micall2a基因表达下调抑制斑马鱼血管发育

Downregulation of Micall2a Gene Expression Inhibited Vascular Development in Zebrafish

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 3

参考文献 22

相关文章 15

编辑推荐

Metrics

本文评价