<i>tbx2b</i> Affects Atrioventricular Canal Development in Zebrafish

doi:10.13523/j.cb.2103060

China Biotechnology

2021, Vol. 41

Issue (8): 1-7 DOI: 10.13523/j.cb.2103060

tbx2b Affects Atrioventricular Canal Development in Zebrafish

ZHAO Xia^1,^2,³,ZHU Zhe^1,^2,³,ZU Yao^1,^2,^3,^**(

)

1 International Research Center for Marine Biosciences, Ministry of Science and Technology, College of Fisheries and Life Sciences, Shanghai Ocean University, Shanghai 201306, China
2 Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education, College of Fisheries and Life Sciences, Shanghai Ocean University, Shanghai 201306, China
3 National Pathogen Collection Center for Aquatic Animals, Shanghai Ocean University, Shanghai 201306, China

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Abstract

tbx2 plays an important role in heart development. To further investigate the gene function in the atrioventricular canal (AVC) development, tbx2b^-/- mutation zebrafish was successfully established using the CRISPR/ Cas9 mediated gene knockout technique. The knockout efficiency of F₀ was detected by T7E1 assay, and the result showed that the average knockout efficiency was about 57.5%. Sanger sequencing confirmed that tbx2b F₁ mutant had a-11bp-base deletion and caused frameshift mutation. Homozygous mutations were lethal in 5 dpf and early embryos suffered from cardiac looping abnormalities. In situ hybridization in 3 dpf tbx2b^-/- showed ectopic expression of nppa and nppb, which are chamber-specific marker genes, and significantly decreased expression of has2, which is AVC marker gene. tbx2b ^-/- mutant was efficiently constructed and effects on downstream genes were discussed, which laid a foundation for further research on the effect of cardiac AVC development and understanding of early cardiac regulatory networks.

Key words： tbx2b Heart development Atrioventricular canal CRISPR

Received: 23 March 2021 Published: 31 August 2021

ZTFLH:

Q812

Corresponding Authors: Yao ZU E-mail: yzu@shou.edu.cn

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Cite this article:

ZHAO Xia,ZHU Zhe,ZU Yao. tbx2b Affects Atrioventricular Canal Development in Zebrafish. China Biotechnology, 2021, 41(8): 1-7.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2103060 OR https://manu60.magtech.com.cn/biotech/Y2021/V41/I8/1

Table 1 Primers of tbx2b

Table 2 Sequences of ISH probe primers

Fig.1 The evolution of tbx2b

Fig.2 CRISPR/ Cas9-mediated tbx2b gene knock out (a)CRISPR/ Cas9 knockout diagram and target location (b)Detection of F₀ knockout efficiency with T7E1 (c)F₁ sequencing results and mutation sites

Fig.3 Amino acids sequence and protein structure of Tbx2b mutation

Fig.4 Phenotypes of tbx2b homozygous mutant and ISH of tbx2b (a)The lateral view of WT and tbx2b^-/- under bright field at 3 dpf (b)The phenotypes of WT and tbx2b^-/- imaged with cmlc2∶EGFP at 3 dpf (c)tbx2b expression pattern in WT and tbx2b^-/- at 3 dpf

Fig.5 In situ hybridization of nppa, nppb and has2


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