The influence of hyproxia an GATA-1 and Epo expression levels in developing zebrafish

MA-Thesis / Master von Markus Holotta

Abstract:

The transcription factor GATA-1 is essential for the development of the erythroid cell lineage in vertebrates. In this article we introduce a method to easily determine the approximately development status of red blood cells and the progression of blood formation by intensity of fluorescence in GATA-1/Ds-Red marked zebrafish. We classified the blood cells on the basis of their fluorescence intensity in 5 intensity stages (IS) with the brightest in IS 1. The comparison with our erythropoietin (Epo) data showed a noticeable correlation between GATA-1, Epo mRNA and EPO protein level. Between 2 and 3 dpf we observed a major increase in blood cell concentration to circa 1200 cells*nl-1, until 15 dpf this value decreased to about the half.

The appearance of IS 1 cells correspond approximately to the peaks in Epo cRNA copies and the highest values in EPO protein emerged about 1 day later. Our data show that the blood cell concentration, Epo and Gata-1 expression in zebrafish larvae is subjected to large fluctuations in the first few days of development.

The zebrafish Danio rerio, also known as Brachidanio rerio, Cyprinus rerio and others, is a omnivorous, tropical fish of the family Cyprinidae and was at fist described by Hamilton in 1822. Its natural range within Asia are slow moving or stagnant water bodies in India, Bangladesh, Nepal and Pakistan.

In the past few decades the zebrafish became an important model organism for genetical, developmental and physiological studies. Fish are vertebrates and thus the genetic program is more similar to that of mammals than invertebrate models like the fruity (Drosophila melanogaster). Because of this relationship most of the zebrafish genes have human orthologs.

Due to its short generation time of approximately 3 month, the rapid development and the transparency up to adulthood this tropical teleost is predestined for in vivo hematologic studies and digital imaging techniques. Especially digital imaging is a gentle, non-invasive and thereby seminal method for researcher who are working with transparent animals.

The combination with fluorescent reporter genes allows real time imaging of gene expression and cell migration studies over their whole lifespan.

Table of Contents:

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Chapter 2.1, Animals:

To start our own breeding colony GATA-1/DsRed transgenic zebrafish were obtained from Tim J. Chico, Shefield University, United Kingdom. For breeding a male and a female fish has been kept in a separate plastic beaker over night. A mesh above the bottom of the beaker protects the eggs against being eaten. The female fish lay about 100 eggs in the morning which immediately get fertilised by the male. The larvae were reared in small beakers (9x6x6cm) hung in an aquarium at 28 at 16: 8h light-dark conditions. To ensure adequate water circulation the beakers were equipped with two lateral meshes and water dropped into the beakers. They have been fed after 5 dpf with Zmsystems Advanced Fry Feed ZM-000 (ZM Ltd) 2 times a day.

Zebrafish eggs used for hypoxic experiments have been incubated at 12% Oxygen saturation (0,88 mg l -1) immediately (<1 h) after fertilisation. This value was chosen to set a high stimulus without the risk of high mortality. For incubation a self-made hypoxic chamber containing about 7,5 l water equipped with a Clark oxygen electrode and a temperature sensor connected to a Twin-Flow Respirometer control unit (Cyclobios) was used. Nitrogen was injected to the chamber in order to replace the oxygen in the water. The accuracy of the oxygen electrode was verified by the Winkler Method.