Zebrafish etv2 knock-in line labels vascular endothelial and blood progenitor cells
Zebrafish etv2 knock-in line labels vascular endothelial and blood progenitor cells
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DOI：
10.1002/dvdy.130
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发表时间：
2020-02-01
影响因子：
2.5
通讯作者：
Sumanas, Saulius
中科院分区：
文献类型：
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作者：
Chestnut, Brendan;Sumanas, Saulius
研究背景ETS转录因子ETV2/ETSRP是最早识别血管和造血祖细胞的标志物之一，是包括斑马鱼在内的多种脊椎动物血管发育的关键调节因子。因此，转基因ETV2报告系为研究血管生成和造血提供了有价值的工具。结果利用CRISPR/Cas9介导的同源非依赖的DNA修复方法，将Gal4转录激活子插入斑马鱼ETV2基因组，构建了ETV2(Ci32Gt)基因陷阱系。ETV2(Ci32Gt)；UAS：GFP胚胎在血管内皮细胞、髓系细胞和红细胞中均有GFP表达。由于Gal4插入阻断了ETV2基因座，纯合子ETV2(Ci32Gt)胚胎在血管生成和骨髓生成方面表现出缺陷，并能够在活体胚胎中显示ETV2缺陷的血管祖细胞。此外，我们对杂合子和纯合子ETV2(Ci32Gt)胚胎中分离的GFP阳性细胞进行了差异转录组分析。在ETV2(Ci32Gt)纯合子胚胎中发现了约500个表达下调的基因，其中包括在血管内皮细胞和髓系细胞中表达的多个基因。结论ETV2(Ci32Gt)基因陷阱系和错误调控基因数据集将为研究造血和血管发育提供有价值的资源。
Background ETS transcription factor Etv2/Etsrp is one of the earliest markers for vascular and hematopoietic progenitors and functions as a key regulator of hematovascular development in multiple vertebrates, including zebrafish. Therefore, transgenic etv2 reporter lines provide a valuable tool to study vasculogenesis and hematopoiesis. However, previously generated zebrafish reporter lines do not fully recapitulate the endogenous pattern of etv2 expression.Results Here we used CRISPR/Cas9-mediated homology-independent DNA repair approach to knock-in a Gal4 transcriptional activator into the zebrafish etv2 genomic locus, thus generating etv2 (ci32Gt) gene trap line. etv2 (ci32Gt); UAS:GFP embryos show GFP expression in vascular endothelial, myeloid and red blood cells. Because gal4 insertion interrupts the etv2 locus, homozygous etv2 (ci32Gt) embryos display defects in vasculogenesis and myelopoiesis, and enable visualizing etv2-deficient hematovascular progenitors in live embryos. Furthermore, we performed differential transcriptome analysis of sorted GFP-positive cells from heterozygous and homozygous etv2 (ci32Gt) embryos. Approximately 500 downregulated genes were identified in etv2 (ci32Gt) homozygous embryos, which include multiple genes expressed in vascular endothelial and myeloid cells.Conclusions The etv2 (ci32Gt) gene trap line and the data sets of misregulated genes will be valuable resources to study hematopoietic and vascular development.