Imai Yuta, Ozaki Satoru, Noda Taiki, Kobayashi Isao, Sugitani Kayo, Kasashima Satomi, Morishita Eriko, Araiso Yuhei
Department of Clinical Laboratory Science, Division of Health Sciences, Graduate School of Medical Sciences, Kanazawa University, 5-11-80 Kodatsuno, Kanazawa, Ishikawa, 920-0942, Japan.
Faculty of Biological Science and Technology, Institute of Science and Engineering, Kanazawa University, Kanazawa, Ishikawa, Japan.
Sci Rep. 2025 May 27;15(1):18538. doi: 10.1038/s41598-025-01658-z.
Severe type I antithrombin (AT) deficiency is considered to cause embryonic lethality. Although several pathological analyses using mice or zebrafish have been attempted, the previous studies did not unveil the detailed mechanism leading to lethality in the early developmental stage. In order to solve this problem, we established type I AT deficient zebrafish by the CRISPR/Cas9 system into Tg(gata1:dsRed) and Tg(fli1a:GFP) lines, so that we could conduct real-time imaging of thrombosis and angiogenesis using fluorescence stereo zoom microscopy. The established zebrafish AT (zAT) mutants harbored frameshift mutations which resulted to be type I AT deficient, unable to secrete zAT protein into blood. Both heterozygous (zAT) and homozygous (zAT) mutants showed reduced survival rate and diverse thrombosis up to 9 days post fertilization. In addition, blood vessel formation was delayed at 30 hpf in zAT, which was recovered normally by 5 dpf and had little effect on survival. Notably, we analyzed the differences in gene expression profiles under AT-depleted conditions by real-time quantitative PCR, and zAT juvenile zebrafish showed increased PLG gene expression and decreased F2 gene expression. Our in vivo study revealed the effects of AT deficiency on embryos during development from the aspects of coagulation and vascular formation.
严重的I型抗凝血酶（AT）缺乏被认为会导致胚胎致死。尽管已经尝试使用小鼠或斑马鱼进行了多项病理分析，但先前的研究并未揭示导致早期发育阶段致死的详细机制。为了解决这个问题，我们通过CRISPR/Cas9系统在Tg(gata1:dsRed)和Tg(fli1a:GFP)品系中建立了I型AT缺陷斑马鱼，以便我们能够使用荧光立体变焦显微镜对血栓形成和血管生成进行实时成像。所建立的斑马鱼AT（zAT）突变体携带移码突变，导致I型AT缺乏，无法将zAT蛋白分泌到血液中。杂合子（zAT）和纯合子（zAT）突变体在受精后9天内均显示存活率降低和多种血栓形成。此外，zAT在30 hpf时血管形成延迟，在5 dpf时正常恢复，对存活影响不大。值得注意的是，我们通过实时定量PCR分析了AT耗尽条件下基因表达谱的差异，zAT幼体斑马鱼显示PLG基因表达增加，F2基因表达降低。我们的体内研究从凝血和血管形成方面揭示了AT缺乏对胚胎发育的影响。
