Downregulation of Micall2a Gene Expression Inhibited Vascular Development in Zebrafish

doi:10.12300/j.issn.1674-5817.2022.166

Abstract

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

CLC Number:

R-332

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.

Figures/Tables 3

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.

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.

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.

References 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.

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