Abstract
The hypoxia inducible transcription factor (HIF) has been shown to coordinate the hypoxic response of vertebrates and is expressed in three different isoforms, HIF-1α, HIF-2α and HIF-3α. Knock down of either Hif-1α or Hif-2α 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-1α antibody we expressed the whole zfHif-2α protein and used it for immunization and antibody generation. Similarly, fragments of the zfHif-3α protein were used for immunization and generation of a zfHif-3α 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-1α protein was present under normoxic conditions in all developmental stages, but no significant differences between the different developmental stages could be detected. Hif-2α 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-3α was expressed from 1 dpf onwards, and the expression level significantly increased until 5 dpf, suggesting that Hif-2α and Hif-3α 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-1α protein even at 1 dpf and in later stages, while neither Hif-2α nor Hif-3α protein level were affected. In these early developmental stages Hif-1α therefore appears to be more important for the coordination of hypoxic responsiveness.
Highlights
The response of tissues of eukaryotic organisms to reduced oxygen availability is by and large coordinated by hypoxia inducible transcription factors (HIF proteins) [1,2,3,4,5]
While the specific antibody directed against zfHif-1α protein has been used in previous studies [23,24], for this study additional antibodies had to be generated against zfHif-2α and zfHif-3α protein
Our results show for the first time that at least one paralog of all three Hif- α proteins is present in the doi:10.1371/journal.pone.0128938.g003
Summary
The response of tissues of eukaryotic organisms to reduced oxygen availability is by and large coordinated by hypoxia inducible transcription factors (HIF proteins) [1,2,3,4,5]. Under normoxic conditions two specific proline residues within the so-called oxygen dependent degradation domain (ODDD) of HIF-1α or of HIF-2α are hydroxylated, inducing an ubiquitination through interaction with the von Hippel-Lindau tumor suppressor protein (VHL), which is the recognition site of the E3 ubiquitination-ligase complex [2,5,8,9]. The lack of oxygen inhibits PHD activity and HIF-α proteins accumulate, dimerize with HIF-β, enter the nucleus and act as a transcription factor controlling hypoxia responsive genes
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