| Summary: | Hypoxic signaling is involved in homeostatic and developmental angiogenic and neurogenic proliferation and differentiation as well as modulating many disease processes that result in or from restricted blood flow. A better understanding of this pathway and how other pathways interact with it is important for progression towards targeted therapeutics. Von Hippel Lindau (vhl-/-) zebrafish embryos demonstrated hypoxic phenotypes such as hyperventilation, polycythemia and an altered vascular network (van Rooijen et al., 2009). The fih-/- embryos showed no morphological phenotype and the adults are fully viable and fertile. By combining the two lines it was possible to investigate the effect of a double knock out of these genes and the effect this had on downstream targets. The first hypothesis to test, the first observation to make, was to assess the effect of the loss of fih on both wild type embryos and on vhl null embryos. Given the roles of fih and vhl are independent of each other in regulating HIF function (FIH regulating HIF function, while VHL regulates HIF turnover), the hypothesis in this case would be that the loss of fih alone may not show dramatic phenotypes, however in the context of the loss of vhl it may alter or exacerbate phenotypes observed in the organisms where vhl was lost. This was proved to be correct when it came to assessment of global and morphological phenotypes. The loss fih resulted in no significant changes compared with wild type embryos in initial observations; however the loss of both fih and vhl altered the known vhl-/- phenotype, which indicated a role for fih, under conditions of de-regulated hypoxia inducible factor (HIF), in enhancing hypoxic signaling. Expression levels of Hif targets, phd3 and vegfa, were assessed and showed that these were elevated in the vhl-/- zebrafish line and further elevated in fih-/-;vhl-/- embryos, by in situ hybridisation. Further analyses of the downstream effects of the loss of fih were assessed using microarray analysis and further validated using qRT-PCR. Preliminary results suggested that on a transcriptional level, fih in isolation was not influencing expression and that fih functioned mainly through HIF. Vascular branching was altered in the vhl-/- zebrafish line compared to wild-type siblings, and this was exacerbated in the fih-/-;vhl-/- embryos. This correlated with increased vegf expression and a corresponding increase in endothelial cell numbers. The fih-/-;vhl-/- embryos exhibited decreased blood flow along the aorta, which ceased at 5dpf despite continuation of the heart beat. The fih-/- embryos exhibited an increase in blood velocity along the aorta with a corresponding increase in heart rate at the same stage. Our data suggested that Fih had an essential role in the regulation of heart rate in the zebrafish and, in combination with vhl; it influenced formation and functionality of blood vessels by affecting the hypoxic signaling pathway. Also investigated were correlations between the zebrafish model of fih loss and those published in the mouse, and several connections and corroborations were made. However there was also scope, in the zebrafish, for the proposal of a potential mechanism of action which was not proposed in the mouse model. Microarray analysis revealed that, specifically in the fih null embryos, two key genes in the renin angiotensin system were down regulated. Since this is a mechanism by which fluid dynamics are regulated, and since there have been observations of altered blood flow dynamics in double mutants, further investigation as to the potential role of this homeostatic mechanism and its potential regulation by fih are needed to further investigate these findings. A second hypothesis that was proposed was that lead by a study by Lee et al, which proposed a model whereby the three proteins, FIH, VHL and HIF are required to bind together with VHL acting as a functional bridge in order to result in downstream effects (Lee et al., 2003). In this case a loss of vhl would disrupt the hydroxylation of HIF-a by fih as well. The hypothesis in this case would therefore be that the loss of fih as well, in an individual that had lost vhl function, would result in no alteration in the phenotypes observed. This hypothesis was shown not to be supported by the data and further investigation into possible interactions between the proteins would need to be assessed in order to ascertain the discrepancy.
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