Developmental Expression and Hypoxic Induction of Hypoxia Inducible Transcription Factors in the Zebrafish

The hypoxia inducible transcription factor (HIF) has been shown to coordinate the hypoxic response of vertebrates and is expressed in three different isoforms, HIF-1 alpha, HIF-2 alpha and HIF-3 alpha. Knock down of either Hif-1 alpha or Hif-2 alpha in mice results in lethality in embryonic or perinatal stages, suggesting that this transcription factor is not only controlling the hypoxic response, but is also involved in developmental phenomena. In the translucent zebrafish embryo the performance of the cardiovascular system is not essential for early development, therefore this study was designed to analyze the expression of the three Hif-isoforms during zebrafish development and to test the hypoxic inducibility of these transcription factors. To complement the existing zfHif-1 alpha antibody we expressed the whole zfHif-2 alpha protein and used it for immunization and antibody generation. Similarly, fragments of the zfHif-3 alpha protein were used for immunization and generation of a zfHif-3 alpha specific antibody. To demonstrate presence of the Hif-isoforms during development [between 1 day post fertilization (1 dpf) and 9 dpf] affinity-purified antibodies were used. Hif-1 alpha protein was present under normoxic conditions in all developmental stages, but no significant differences between the different developmental stages could be detected. Hif-2 alpha was also present from 1 dpf onwards, but in post hatching stages (between 5 and 9 dpf) the expression level was significantly higher than prior to hatching. Similarly, Hif-3 alpha was expressed from 1 dpf onwards, and the expression level significantly increased until 5 dpf, suggesting that Hif-2 alpha and Hif3 alpha play a particular role in early development. Hypoxic exposure (oxygen partial pressure = 5 kPa) in turn caused a significant increase in the level of Hif-1 alpha protein even at 1 dpf and in later stages, while neither Hif-2 alpha nor Hif-3 alpha protein level were affected. In these early developmental stages Hif-1a therefore appears to be more important for the coordination of hypoxic responsiveness.