Analysis of thrombocyte development in CD41-GFP transgenic zebrafish
Analysis of thrombocyte development in CD41-GFP transgenic zebrafish
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DOI：
10.1182/blood-2005-01-0179
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发表时间：
2005-12-01
期刊：
影响因子：
20.3
通讯作者：
Handin, RI
中科院分区：
文献类型：
--
作者：
Lin, HF;Traver, D;Handin, RI
在非哺乳类脊椎动物（如斑马鱼）中，血栓细胞相当于有核的血小板。我们已经克隆了斑马鱼CD41 cDNA（α(IIb)，糖蛋白IIb[GPIIb]）及其启动子，并培育出了带有绿色荧光蛋白（GFP）标记血栓细胞的转基因斑马鱼品系。通过逆转录 - 聚合酶链反应（RT - PCR），在受精后42小时（hpf）可检测到CD41 mRNA转录本，在48 hpf时可在循环造血细胞中检测到。对成年CD41 - GFP斑马鱼中肾的血栓细胞进行流式分选，显示出一个GFP（高）亚群，其具有成熟血栓细胞的形态外观，以及一个GFP（低）亚群，外观不成熟，这表明它们可能是血栓细胞前体。对40和48 hpf的胚胎进行共聚焦激光显微镜检查，还发现在背主动脉和尾静脉之间的一个离散区域存在一群不移动的GFP（+）细胞。通过注射针对干细胞转录因子scl和血小板生成素受体c - mpl的反义吗啉代寡核苷酸，可抑制循环血栓细胞的产生。scl基因敲低可消除不移动的GFP（+）细胞群，c - mpl基因敲低可部分抑制该细胞群。这些研究表明，在斑马鱼胚胎中识别血栓细胞、血栓细胞前体以及可能的早期造血干细胞，并追踪它们的增殖和成熟是可能的。
Thrombocytes are the nucleated equivalent of platelets in nonmarnmalian vertebrates such as the zebrafish, Danio rerio. We have cloned zebrafish CD41 cDNA (alpha(IIb), glycoprotein IIb [GPIIb]) and its promoterandhavegeneratedtransgeniczebra- fish lines with green fluorescent protein (GFP)-tagged thrombocytes. CD41 mRNA transcripts appeared 42 hours after fertilization (hpf) by reverse-transcriptase-polymerase chain reaction (RT-PCR) and at 48 hpf in circulating hematopoietic cells. Flow sorting of thrombocytes from the mesonephros of adult CD41-GFP zebrafish showed a GFP(high) subset, which had the morphologic appearance of mature thrombocytes, and a GFP(low) subset with an immature appearance, suggesting that they may be thrombocyte precursors. Confocal laser microscopy of embryos 40 and 48 hpf also showed a nonmobile population of GFP(+) cells in a discrete area between the dorsal aorta and caudal vein. Production of circulating thrombocytes was inhibited by the injection of antisense morpholinos for the stem-cell transcription factor scl and c-mpl, the receptor for thrombopoietin. The nonmobile pool of GFP(+) cells was abolished by scl knockdown and partially inhibited by c-mpl knockdown. These studies have shown that it is possible to identify thrombocytes, thrombocyte precursors, and, possibly, early hematopoietic stem cells in zebrafish embryos and track their proliferation and maturation.