Using Flow Cytometry to Detect and Quantitate Altered Blood Formation in the Developing Zebrafish.
Using Flow Cytometry to Detect and Quantitate Altered Blood Formation in the Developing Zebrafish.
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DOI：
10.3791/61035
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发表时间：
2021-04-29
期刊：
影响因子：
--
通讯作者：
Stachura DL
中科院分区：
文献类型：
--
作者：
Rueb KF;Stachura DL
脊椎动物成熟血液中细胞谱系的多样性源于造血干细胞和祖细胞 (HSPC) 的分化。这是在生物体整个生命周期中发生的一个关键过程，胚胎发生过程中涉及的分子途径的破坏可能会产生灾难性的长期后果。由于多种原因，斑马鱼（Danio rerio）已成为研究造血作用的模式生物。斑马鱼胚胎在体外发育，并在受精后 7 天 (dpf) 时产生了大多数终生血细胞亚型。评估造血细胞数量的测定方法已经开发出来，主要利用特定的组织学染色、原位杂交技术和利用驱动荧光蛋白表达的血细胞特异性启动子的转基因动物的显微镜检查。然而，大多数染色测定和原位杂交技术不能准确定量存在的血细胞数量；只有细胞数量的巨大差异才容易被观察到。可以利用转基因动物并通过荧光或共聚焦显微镜分析个体，但这些测定的定量依赖于手动计数或使用昂贵的成像软件，这两种方法都可能出错。开发额外的方法来评估血细胞数量将是经济的、更快的，甚至可以自动化，以快速评估 CRISPR 介导的基因修饰、吗啉代介导的转录减少的效果，以及大规模影响造血的药物化合物的效果。这种定量血细胞的新颖测定方法是通过分离整个斑马鱼胚胎并分析存在的荧光标记血细胞的量来进行的。这些测定应该能够阐明胚胎发生过程中负责血细胞生成、扩增和调节的分子途径，这将使研究人员能够进一步发现血液疾病期间改变的新因素，以及脊椎动物造血进化过程中必不可少的途径。该测定是定量发育胚胎斑马鱼中造血细胞的简单方法。对分离的斑马鱼的血细胞进行流式细胞术分析。这使得可以检测突变动物和基因改造后的血液缺陷。
The diversity of cell lineages that comprise mature blood in vertebrate animals arise from the differentiation of hematopoietic stem and progenitor cells (HSPCs). This is a critical process that occurs throughout the lifespan of organisms, and disruption of the molecular pathways involved during embryogenesis can have catastrophic long-term consequences. For a multitude of reasons, zebrafish (Danio rerio) has become a model organism to study hematopoiesis. Zebrafish embryos develop externally, and by 7 days post-fertilization (dpf) have produced most of the subtypes of definitive blood cells that will persist for their lifetime. Assays to assess the number of hematopoietic cells have been developed, mainly utilizing specific histological stains, in situ hybridization techniques, and microscopy of transgenic animals that utilize blood cell-specific promoters driving the expression of fluorescent proteins. However, most staining assays and in situ hybridization techniques don’t accurately quantitate the number of blood cells present; only large differences in cell numbers are easily visualized. Utilizing transgenic animals and analyzing individuals with fluorescent or confocal microscopy can be performed, but the quantitation of these assays relies on either counting manually or utilizing expensive imaging software, both of which can make errors. Development of additional methods to assess blood cell numbers would be economical, faster, and could even be automated to quickly assess the effect of CRISPR-mediated genetic modification, morpholino-mediated transcript reduction, and the effect of drug compounds that affect hematopoiesis on a large scale. This novel assay to quantitate blood cells is performed by dissociating whole zebrafish embryos and analyzing the amount of fluorescently labelled blood cells present. These assays should allow elucidation of molecular pathways responsible for blood cell generation, expansion, and regulation during embryogenesis, which will allow researchers to further discover novel factors altered during blood diseases, as well as pathways essential during the evolution of vertebrate hematopoiesis. This assay is a simple method to quantitate hematopoietic cells in developing embryonic zebrafish. Blood cells from dissociated zebrafish are subjected to flow cytometry analysis. This allows the detection of blood defects in mutant animals and after genetic modification.
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20.3
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