Bidirectional crosstalk between Hypoxia-Inducible Factor and glucocorticoid signalling in zebrafish larvae

Hypoxia inducible factor (HIF) and Glucocorticoids (GCs) transcriptional responses play a pivotal role in tissue homeostasis, glucose metabolism and in the regulation of cellular responses to various forms of stress and inflammation. In the last decades few in vitro studies highlighted the potential for crosstalk between these two major signalling pathways. However, how this interplay precisely takes place in vivo is still unclear.
To this end, the aim of this project is to elucidate how and to what degree hypoxic signalling affects the endogenous glucocorticoid pathway and vice versa, using zebrafish larvae (Danio rerio) as an in vivo model organism. Indeed, the use of whole animals allows us to show how these pathways interact at a more systemic and complex level than in cell culture, where interactions between different tissues and cell types cannot be easily modelled. Previous work from my lab identified glucocorticoids as activators of the HIF pathway (Vettori et al., 2017). Counterintuitively, despite their anti-inflammatory
action, it was shown that GR loss of function prevents the transcriptional activity linked to immune response (Facchinello et al., 2017). Finally, the glucocorticoid receptor has been also observed to synergistically activate proinflammatory genes by interacting with other signalling pathways (Langlais et al., 2008, 2012; Dittrich et al., 2012; Xie et al., 2019). Nevertheless, how the crosstalk between hypoxic and glucocorticoid signalling occurs in vivo is still debated. By taking advantage of both a genetic and a pharmacological approach I altered these two pathways during the first 120 hours post fertilisation of zebrafish embryos. In particular, I used two different mutant lines I have generated (hif1βsh544 (arnt1) and grsh543 (nr3c1)), coupled to an already existing vhlhu2117/+;phd3:eGFPi144/i144 hypoxia reporter line (Santhakumar et al., 2012), to study the effect of HIF signalling on glucocorticoid response and vice-versa, through a “gain-of-function/loss-of-function” approach. Phenotypic and molecular analyses of these mutant lines were accompanied by optical and fluorescence microscope imaging.
Firstly, I have demonstrated that in the presence of upregulated HIF signalling, both glucocorticoid response and endogenous cortisol levels are repressed in 5 days post fertilisation larvae. In addition, despite HIF activity being low at normoxia, my
data show that it already impedes glucocorticoid activity and levels. Secondly, I further analysed the in vivo contribution of glucocorticoids to HIF signalling. Interestingly, my results show that both glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) play a key role in enhancing the HIF response. Finally, I found indications that glucocorticoids promote HIF signalling via multiple routes. Cumulatively, this study suggests a model for how this crosstalk occurs in vivo and, more broadly, paves the way for further in vivo analysis on the extensive interaction behind these two major signalling pathways.