Non‐invasive characterization of red blood cell population under normoxic and hypoxic conditions in developing zebrafish (Danio rerio)

Abstract

For erythroid cell lineage development in vertebrates, GATA‐1 transcription factor is essential. We demonstrated that the approximate developmental status of red blood cells and the progression of blood formation can be studied non‐invasively in GATA‐1:DsRed transgenic zebrafish larvae by characterization of fluorescence luminance spectra. Study was carried out for the animals maintained under normoxic and hypoxic (5.8 and 0.8mg of O2 l−1) conditions up to 10 days post‐fertilization (dpf). The blood cells were classified into five Intensity Stages (IS) on the basis of their fluorescence intensity. We evaluated Epo‐expression by carrying out RT‐PCR of wild type zebrafish up to 15 dpf old larvae in order to determine its correlation with the luminance data. Significant relationship was observed between Epo‐mRNA production, GATA‐1 expression and blood cell concentration. In contrast to normoxic animals 1‐3 Epo mRNA copies that was maintained throughout the development, the hypoxic animals showed remarkable fluctuation of 1 to 12 Epo‐mRNA copies per ng RNA during animal development. Fluorescent 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 till 10 dpf showing a maximum of 15% of the total luminescent cells at 8 dpf. The fluorescent cells concentration in normoxic group was generally higher as compared to the hypoxic one. A major observable feature of the hypoxic group was its high blood volume and increased dorsal aorta vessel diameter compared to controls.