Department of Biological Sciences and.
Department of Chemistry, University of North Texas, Denton, TX.
Blood Adv. 2019 May 14;3(9):1406-1415. doi: 10.1182/bloodadvances.2018023960.
Zebrafish thrombocytes are similar to mammalian platelets. Mammals have young platelets (also called reticulated platelets) and mature platelets. Likewise, zebrafish have 2 populations of thrombocytes; one is DiI-C (DiI) (DP), and the other is DiI (DN). However, the mechanism of selective thrombocyte labeling by DiI is unknown. Furthermore, there is no transgenic zebrafish line where DP and DN thrombocytes are differentially labeled with fluorescent proteins. In this study, we found that Glo fish, in which the myosin light chain 2 promoter drives the gene, have a population of thrombocytes that are red fluorescent protein (RFP) labeled. We also generated transgenic GloFli fish in which DP and DN thrombocytes are labeled with RFP and green fluorescent protein (GFP), respectively. Single-cell lipid analysis showed a twofold increase in phosphatidylethanolamine (PE) and a twofold decrease in phosphatidylcholine (PC) in RFP thrombocytes compared with GFP thrombocytes, suggesting that lipid composition may be important for DiI differential labeling. Therefore, we tested liposomes prepared with different ratios of PC and PE and observed that liposomes prepared with higher amounts of PE favor DiI labeling, whereas the PC concentration had a modest effect. In liposomes prepared using only PE or PC, increased concentrations of PE resulted in increased DiI binding. These results suggest that because RFP thrombocytes have higher PE concentrations, DiI may bind to them efficiently, thus explaining the selective labeling of thrombocytes by DiI. This work also provides GloFli fish that should be useful in understanding the mechanism of thrombocyte maturation.
斑马鱼的血小板类似于哺乳动物的血小板。哺乳动物有年轻的血小板（也称为网织血小板）和成熟的血小板。同样，斑马鱼有 2 种血小板群体；一种是 DiI-C（DiI）（DP），另一种是 DiI（DN）。然而，DiI 选择性标记血小板的机制尚不清楚。此外，没有转基因斑马鱼品系可以用荧光蛋白对 DP 和 DN 血小板进行差异标记。在这项研究中，我们发现肌球蛋白轻链 2 启动子驱动基因的 Glo 鱼有一种血小板，其被红色荧光蛋白（RFP）标记。我们还生成了转基因 GloFli 鱼，其中 DP 和 DN 血小板分别用 RFP 和绿色荧光蛋白（GFP）标记。单细胞脂质分析显示，与 GFP 血小板相比，RFP 血小板中的磷脂酰乙醇胺（PE）增加了两倍，而磷脂酰胆碱（PC）减少了两倍，这表明脂质组成可能对 DiI 差异标记很重要。因此，我们测试了用不同比例的 PC 和 PE 制备的脂质体，并观察到含有较高量 PE 的脂质体有利于 DiI 标记，而 PC 浓度的影响较小。在仅用 PE 或 PC 制备的脂质体中，PE 浓度的增加导致 DiI 结合增加。这些结果表明，由于 RFP 血小板中 PE 浓度较高，DiI 可能与之有效结合，从而解释了 DiI 对血小板的选择性标记。这项工作还提供了 GloFli 鱼，这应该有助于理解血小板成熟的机制。
