Ontogenetic development of erythropoiesis can be studied non-invasively in GATA-1:DsRed transgenic zebrafish

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.