Yaqoob Nadeem, Holotta Markus, Prem Caroline, Kopp Renate, Schwerte Thorsten
University of Innsbruck, Institute of Zoology, Technikerstrasse 25, A-6020 Innsbruck, Austria.
Comp Biochem Physiol A Mol Integr Physiol. 2009 Oct;154(2):270-8. doi: 10.1016/j.cbpa.2009.06.024. Epub 2009 Jul 5.
For the erythroid cell lineage development in vertebrates, GATA-1 transcription factor is essential. In our report, we have demonstrated that the approximate developmental status of erythrocytes and the progression of blood formation can be studied non-invasively in GATA-1:DsRed transgenic zebrafish (Danio rerio) embryo and larva by characterization of fluorescence luminance spectra. The study was carried out for animals maintained under normoxic and hypoxic (152 and 20 torr PO(2) respectively) conditions up to 10 days post-fertilization (dpf) and total blood cell concentrations and fluorescent cells' percentage were determined for this purpose. The erythroids were classified into five intensity stages (IS) on the basis of their fluorescence intensity. The luminescent cells with medium intensities (IS3) in normoxic animals were found throughout 2 to 10 dpf although in lower quantity while in hypoxic group they appeared from 5 dpf to 10 dpf showing a maximum of 15% of the total luminescent cells at 8 dpf. The total blood cell concentration dropped after 8 dpf in contrast to hypoxic group which showed further increasing trend. The fluorescent cells' percentage in normoxic group was generally higher as compared to the hypoxic ones. Our method successfully defined various stages of erythroid development. An effort was also made to correlate our luminance data (GATA-1 expression) and total blood cell concentrations with Epo mRNA production. Quantitative RT-PCR of 2-15 dpf old zebrafish was carried out for this purpose. Normoxic animals showed 1-3 Epo mRNA copies per ng RNA in contrast to the hypoxic larvae that showed remarkable fluctuation of 1 to 12 Epo mRNA copies per ng RNA during development. The blood volume (aortic diameter) and production time scale proved to be important factors to define the relationship of Epo mRNA with total blood cell concentration and GATA-1 protein expression respectively.
对于脊椎动物的红细胞系发育而言，GATA-1转录因子至关重要。在我们的报告中，我们已经证明，通过对荧光亮度光谱进行表征，可以在GATA-1:DsRed转基因斑马鱼（Danio rerio）胚胎和幼体中对红细胞的大致发育状态和造血进程进行非侵入性研究。该研究针对在常氧和低氧（分别为152和20托PO₂）条件下饲养至受精后10天（dpf）的动物进行，为此测定了全血细胞浓度和荧光细胞百分比。根据荧光强度，将红细胞分为五个强度阶段（IS）。在常氧动物中，中等强度（IS3）的发光细胞在整个2至10 dpf期间都有发现，尽管数量较少，而在低氧组中，它们从5 dpf出现至10 dpf，在8 dpf时占总发光细胞的最大比例为15%。与显示出进一步上升趋势的低氧组相比，全血细胞浓度在8 dpf后下降。常氧组中荧光细胞的百分比通常高于低氧组。我们的方法成功地定义了红细胞发育的各个阶段。我们还努力将我们的亮度数据（GATA-1表达）和全血细胞浓度与促红细胞生成素（Epo）mRNA产生相关联。为此对2至15 dpf的斑马鱼进行了定量逆转录聚合酶链反应（RT-PCR）。常氧动物每纳克RNA显示1至3个Epo mRNA拷贝，相比之下，低氧幼体在发育过程中每纳克RNA显示出1至12个Epo mRNA拷贝的显著波动。血容量（主动脉直径）和产生时间尺度被证明是分别定义Epo mRNA与全血细胞浓度和GATA-1蛋白表达之间关系的重要因素。
