Bidirectional crosstalk between HIF and Glucocorticoid signalling in zebrafish larvae

In the last decades few in vitro studies highlighted the potential for cross-talk between hypoxia inducible factor-(HIF) and glucocorticoid-(GC) signalling pathways. However, how this interplay precisely occurs in vivo is still debated. Here, we use zebrafish larvae (Danio rerio) to elucidate how and to what degree hypoxic signalling affects the endogenous glucocorticoid pathway and vice versa, in vivo. Firstly, our results demonstrate 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, our data show that it already impedes glucocorticoid activity and levels. Secondly, we further analysed the in vivo contribution of glucocorticoids to HIF signalling. Interestingly, our results show that both glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) play a key role in enhancing the HIF response. Finally, we found indications that glucocorticoids promote HIF signalling via multiple routes. Cumulatively, our findings allowed us to suggest a model for how this cross-talk occurs in vivo.


Introduction
Glucocorticoids constitute a well-characterized class of lipophilic steroid hormones produced by the adrenal glands in humans and by the interrenal tissue in teleosts. The circadian production of glucocorticoids in teleosts is regulated by the hypothalamuspituitary-interrenal (HPI) axis, which is the equivalent of the mammalian hypothalamuspituitary-adrenal (HPA) axis. Both are central to stress adaptation (Alsop and Vijayan, 2009;Griffiths et al., 2012;Tokarz et al., 2013;Faught and Vijayan, 2018). Glucocorticoids exert their function via direct binding to the intracellular glucocorticoid receptor (GR) (Bamberger, Schulte and Chrousos, 1996), and together act as a transcription factor, which can function either in a genomic or in non-genomic way ( Hypoxia-inducible factor (HIF) transcription factors are key regulators of the cellular response to hypoxia, which coordinate a metabolic shift from aerobic to anaerobic metabolism in the presence of low oxygen availability in order to assure homeostasis (Semenza, 2011). Hypoxia, is a common pathophysiological condition (Bertout, Patel and Simon, 2008;Semenza, 2013) to which cells must promptly respond in order to avert metabolic shutdown and subsequent death (Elks et al., 2015). In the presence of normal oxygen levels, a set of prolyl hydroxylases (PHD1, 2 and 3) use the available molecular oxygen directly to hydroxylate HIF-a subunit. Hydroxylated HIF-a is then recognised by the Von Hippel Lindau (VHL) protein, which acts as the substrate recognition part of a E3-ubiquitin ligase complex. This leads to HIF-a proteasomal degradation to avoid HIF pathway activation under normoxic conditions. On the other hand, low O2 levels impair the activity of the PHDs enzymes leading to HIF-a stabilisation and subsequent translocation in the nucleus. Here, together with HIF-b subunit, HIF-a forms a functional transcription complex, which drives the hypoxic response (Semenza, 2012). Although the HIF response is aimed to restore tissue oxygenation and perfusion, it can sometimes be maladaptive and can contribute to a variety of pathological conditions including inflammation, tissue ischemia, stroke and growth of solid tumours (Cummins and Taylor, 2005). Finally, it is important to note for this study that HIF signalling is able to regulate its own activation via negative feedback, by inducing the expression of PHD genes, in particular prolyl hydroxylase 3 (PHD3) (Pescador et al., 2005;Santhakumar et al., 2012).
However, due to the presence of adverse effects (Moghadam-Kia and Werth, 2010) and glucocorticoid resistance (Barnes and Adcock, 2009;Barnes, 2011), their use has been limited. Therefore, extending the research on how precisely this interplay occurs in vivo, may have a wide physiological significance in health and disease.
The first evidence of interaction between HIF and GR was provided by Kodama et al. 2003, who discovered that ligand-dependent activation of glucocorticoid receptor enhances hypoxia-dependent gene expression and hypoxia response element (HRE) activity in HeLa cells. Leonard et al. 2005 subsequently revealed that GR is transcriptionally upregulated by hypoxia in human renal proximal tubular epithelial cells. Furthermore, the hypoxic upregulation of GR was confirmed by Zhang et al 2015. In contrast, a dexamethasone-mediated inhibition of HIF-1α target genes expression in hypoxic HEPG2 cells was demonstrated by Wagner et al. 2008. In addition to that, they showed retention of HIF-1α in the cytoplasm, suggesting a blockage in nuclear import.
Finally, Gaber et al., 2011 indicated the presence of dexamethasone-induced suppression of HIF-1α protein expression, which resulted in reduced HIF-1 target gene expression.
From these in vitro results it has become clear that HIF-GCs cross-talk is complex and may depend on cell type. In the present study, we have used the zebrafish (Danio rerio) as an in vivo model organism to study how and to what degree hypoxic signalling affects the endogenous glucocorticoids' response and vice versa. The use of whole animals allows us to show how these signals interact at a more global level than in cell culture, where interactions between different tissues and cell types are not easily modelled. The zebrafish offers an excellent genetic vertebrate model system for endocrine studies, and similar to humans, they are diurnal and use cortisol as the main glucocorticoid hormone (Weger et al., 2016). Importantly, unlike other teleosts, zebrafish have only a single glucocorticoid (zGr) and mineralocorticoid receptor (Mr) (zMr) isoform (Faught and Vijayan, 2018). Moreover, zGr shares high structural and functional similarities to its human equivalent, making zebrafish a reliable model for studying glucocorticoids activity in vivo (Alsop and Vijayan, 2008;Chatzopoulou et al., 2015;Xie et al., 2019). Additionally, zebrafish share all the components of the human HIF signalling pathway and it has been proved to be a very informative and genetically tractable organism for studying hypoxia and HIF pathway both in physiological and pathophysiological conditions (van Rooijen et al., 2011;Santhakumar et al., 2012;Elks et al., 2015).
In our previous work, we identified new activators of the HIF pathway, e.g. betamethasone, a synthetic glucocorticoid drug (Vettori et al., 2017 In the present study, we utilised both a genetic and pharmacological approach to alter these two pathways during the first 120 hours post fertilisation of zebrafish embryos. In particular, we took advantage of two different mutant lines we have generated (hif1β sh544 (Arnt1) and gr sh543 (nr3c1) respectively), coupled to an already existing vhl hu2117/+ ;phd3::EGFP i144/i144 hypoxia reporter line (Santhakumar et al., 2012), to study the effect of HIF response on GCs signalling and vice-versa, via a "gain-offunction/loss-of-function" approach. Phenotypic and molecular analyses of these mutants have been accompanied by optical and fluorescence microscope imaging. Importantly, we not only confirm that betamethasone is able to increase the expression of phd3:eGFP, a marker of HIF activation in our zebrafish HIF-reporter line, but we also show that BME-driven HIF response requires Hif1b/Arnt1 action to occur. Furthermore, our results also demonstrate that both Gr and Mr loss of function are able to partially rescue vhl phenotype, allowing us to confirm the importance of glucocorticoids in assuring a proper HIF response.
Our results also demonstrate that in the presence of upregulated HIF pathway (by mutating vhl), both glucocorticoid response and the endogenous cortisol levels are repressed in 5 dpf larvae, whereas when HIF pathway is suppressed (by mutating hif1b) they are significantly increased. Finally, qPCR analysis on GCs target genes, in situ hybridisation on the expression of steroidogenic genes and cortisol quantification on the aforementioned mutant lines confirmed our hypothesis.
Taken together, these results allow us to deepen the knowledge of how the crosstalk between HIF and glucocorticoid pathway occurs in vivo and to underscore a new model of interaction between these two major signalling pathways.

Materials and methods
Zebrafish husbandry and maintenance: Experiments performed on zebrafish embryos conformed to UK Home Office regulations.

Zebrafish strains:
The following zebrafish lines were used: wild-type (wt) strain AB (ZDB-GENO-960809- were selected and crossed to obtain homozygous mutant embryos (F2 generation).

Generation of CRISPR/Cas9-mediated mutants (CRISPANTs):
To generate G0 knockout embryos we used the method developed by Wu et al., 2018. In short, a pool of four guide-RNAs (25µM each, Sigma Aldrich) were co-injected with 0,5 µl

Cortisol extraction and quantification:
Cortisol quantification was carried out according to the protocol published by Eachus et al., 2017 . Three biological replicates of 150 larvae at 5dpf each of hif1b sh544 mutants, hif1b sh544 siblings, vhl hu2117 mutants and vhl hu2117 siblings, respectively, were used for steroid hormone extraction and quantification.

RNA isolation, cDNA synthesis and qPCR analysis:
Transcript abundance of target genes was measured by quantitative real-time PCR (Dr03432748_m1) and/or rps29 (Dr03152131_m1) and fold change values were generated relative to wild-type DMSO treated control levels, according to ΔΔCT method (Livak and Schmittgen, 2001). All data were expressed as fold change mean ± s.e.m and P ⩽ 0.05 was considered statistically significant.
Quantifying phd3:eGFP-related brightness: Images were acquired using Leica Application Suite version 4.9, which allowed the capture of both bright-field and GFP fluorescent images. To quantify the phd3:eGFP- Unpaired t tests were used to test for significant differences between two sample groups (i.e cortisol quantification). One-way ANOVA was used for assessing mean grey values data quantification, whereas two-way ANOVA was used to evaluate qPCR data. As posthoc correction tests, Sidak's method for multiple comparisons was used on normally distributed populations following one-way ANOVA, while Dunnett's correction was used for comparing every mean to a control mean, on normally distributed populations following two-way ANOVA.

Generating arnt1 and arnt1;vhl knockout in zebrafish:
To study the interplay between HIF and GC signalling in vivo, using a genetic approach, we required an Hif1β/Arnt1 mutant line (in a phd3:eGFP;vhl +/background) to enable the downregulation HIF signalling. Hif-1β (hypoxia-inducible factor 1 beta, Arnt1) is a nuclear receptor that is targeted by and bound to Hif-α subunits, when the latter migrate into the nucleus after its stabilization in the cytoplasm. It represents the most downstream protein in the HIF pathway and for this reason it is the most suitable target.
Using CRISPR mutagenesis we obtained a 7 bp insertion in exon 5 (coding bHLH DNA binding domain (DBD) of the Hif-1β protein; allele name sh544) in vhl heterozygote embryos (Fig.1a) Initial analysis performed on arnt1 +/-;vhl +/incross-derived 5 dpf larvae (F1 generation) confirmed the suppressive effect that arnt1 mutation was expected to have on vhl mutants. Overall, arnt1 -/-;vhl -/larvae showed a substantially attenuated vhl phenotype, characterized by a reduced phd3:eGFP related brightness, especially in the liver, with the absence of pericardial edema, excessive caudal vasculature and normal yolk usage compared to vhl -/larvae (Fig.1b). In particular, this was quantified as a 39% downregulation (P<0.0017) at the level of the head, a 75% downregulation (P<0.0001) in liver and a 58% downregulation (P<0.0001) in the rest of the body (from the anus to the caudal peduncle), in terms of phd3:eGFP-related brightness, compared to vhl -/larvae ( Fig.1c and Fig.EV1a). in the rest of the body (equals to 46%, P<0.0001), compared to uninjected vhl mutant larvae (Fig.1d,e).
Furthermore, when both arnt1 and arnt2 isoforms were simultaneously knockedout, the downregulation was even stronger at the level of the head (equals to 74%, P<0.0001), liver (equals to 86%, P<0.0001) and in the rest of the body (equals to 83%, P<0.0001) (Fig. 1d,e). Overall, these data allow to confirm that Arnt1, even if not fundamental for survival, is the main isoform required for HIF signaling at the hepatic level in zebrafish larvae, whereas Arnt2 is more expressed in the developing central nervous system (CNS), as reported by Hill et., al 2009. Of note, since both isoforms can form a functional complex with HIF-a and appear to function in the same organs, this allows us to confirm that they have partially overlapping functions in vivo and to show that they synergistically contribute to the HIF response.
To further examine the ability of HIF in repressing GCs response, we performed betamethasone (BME) treatment [30 µM] on the aforementioned mutant lines, followed by RTqPCR analysis. Interestingly, both in the presence of upregulated and partially attenuated HIF levels (vhl -/and arnt1 -/-;vhl -/-, respectively), BME was not able to significantly increase the expression of all the four glucocorticoid target genes analysed ( Fig. 2a and Fig. EV2). In contrast, when the HIF pathway was suppressed (arnt1 -/-), BME was able to further upregulate mainly the expression of fkbp5 (fold change=14,5; P<0,0001), pck1 (fold change=11,1; P=0,0040) and il6st (fold change=5,73; P=0,0041) ( Fig. 2a and Fig. EV2). Collectively, these results indicate that upregulated HIF is somehow able to repress glucocorticoid response and can strongly blunt or abolish the response to an exogenous GR agonist. Interestingly, although HIF activity is expected to be low in wild-type larvae in a normoxic environment, its function is detectable with respect to suppression of GR activity.

HIF signalling acts as negative regulator of steroidogenesis:
To investigate the relationship between HIF signaling and steroidogenesis, we initially performed in situ hybridization on embryos from our arnt1 +/mutant line, using We found that 5 dpf arnt1 -/larvae, which were characterized by an upregulated glucocorticoid response, showed upregulated cyp17a2 expression coupled to downregulated pomca. As expected, arnt1 siblings showed normally expressed pomca and cyp17a2, which were observed to be downregulated only as a consequence of BME treatment ( Fig. 2b and 2d). Therefore, we speculate that in the absence of arnt1, pomca downregulation is most likely to occur as a consequence of GC-GR induced negative feedback loop, triggered by an upregulated glucocorticoid response (Fig. 2b').
We subsequently examined both pomca and cyp17a2 expression in the opposite -HIF upregulated-scenario, by performing in situ hybridization on the vhl mutant line.
Interestingly, 5 dpf vhl -/larvae, which were characterized by a downregulated glucocorticoid response, displayed downregulated cyp17a2 expression, coupled to downregulated pomca expression. On the other hand, vhl siblings showed normally expressed pomca, which was observed to be downregulated after BME treatment, as expected ( Fig. 2c and 2e).
As both fkbp5 and pck1 (GCs target genes) are downregulated in vhl mutants (Fig.   EV2a), we speculate that by upregulating HIF (vhl knock-out larvae), glucocorticoid response is effectively repressed as a consequence of HIF-mediated downregulation of pomca expression (Fig. 2c'). Cumulatively, if this is true, we predict to observe reduced levels of endogenous cortisol in vhl -/and normal or even increased levels in arnt1 -/at 5 dpf.

Generating gr and gr;vhl knockout in zebrafish:
To further investigate the reverse role of glucocorticoids on HIF response, we ligand binding domain and is predicted to be a true null (Fig. 3a). The homozygous gr/nr3c1 mutants, characterized during the first 5dpf, were morphologically similar to control siblings and adult fish were viable and fertile, as predicted (Facchinello et al., 2017).
To confirm loss-of-function, we initially subjected larvae to a visual background adaptation (VBA) test, as VBA is linked to impaired glucocorticoid biosynthesis and action (Griffiths et al., 2012;Muto et al., 2013). Larvae derived from gr +/incross were VBA analyzed and sorted according to melanophore size at 5 dpf. PCR-based genotyping on negative VBA-response sorted samples revealed that most larvae were homozygous for the gr allele, whereas positive VBA-response samples were always gr siblings (Fig.3b).
Furthermore, WISH analysis performed on 5 dpf DMSO and BME treated gr +/incross derived larvae, using pomca as probe, showed the presence of upregulated pomca expression at the level of the anterior part of the pituitary gland, compared to wild-type siblings (Fig. 3b'). Of note, BME treatment was not able to downregulate pomca levels of gr -/-, via negative feedback loop, due to the absence of a functional gr allele. Finally, the loss of function was also determined in 5 dpf gr mutants by the strong downregulation of fkbp5 mRNA levels quantified via RTqPCR, both in the presence (fold change=0,01; P<0,0001) and in the absence of BME treatment (DMSO treated, fold change=0,01; P<0,0001) (Fig. 3b").
gr mutation partially rescues vhl phenotype: We next analyzed the effect of gr loss of function on vhl phenotype. Phenotypic analysis carried out on 5dpf larvae, derived from gr +/-;vhl +/incross, revealed that nr3c1 mutation was able to cause an efficient, but not complete rescue of vhl phenotype, in a way which resembled arnt1 mutation (Fig. 3c).
Rescue was also apparent by morphology. Indeed, even if gr -/-;vhl -/showed reduced yolk usage, they displayed a reduction in ectopic vessel formation at the level of the dorsal tailfin, no pericardial edema, and developed air-filled swim bladders (Fig. 3c).
Of note, whereas vhl single mutants are inevitably deceased by 10 dpf (van Rooijen et al., 2009), we were able to raise all selected double mutants beyond 15 dpf, but then (similarly to arnt1 -/-;vhl -/-) they failed to grow and thrive when compared to their siblings.
This led us to euthanise them due to health concerns at 21 dpf (Fig. EV3b). Together, these data indicate for the first time, in our in vivo animal model, that GR function is essential to assure a proper HIF response in zebrafish larvae, in particular at the level of the head and the liver.

gr loss of function effect is stronger when HIF-response is attenuated:
The similarity of gr and arnt1 mutations could mean they work in a single linear  (Fig. 4a). Of note, 7 putative very weak GFP + larvae were selected and genotypic analysis confirmed that 5 out of 7 were indeed gr -/-; arnt1 -/-;vhl -/-. In particular, these triple mutants showed a 54% downregulation at the level of the head, a 71% downregulation in the liver and a 72% downregulation in the tail region, in terms of phd3:eGFP-related brightness compared to vhl -/- (Fig.4a and EV4). Thus, these data suggest that GCs are likely to interfere with both Arnt1 and Arnt2 mediated HIF signalling pathway.
This suggests that in vhl -/larvae, BME treatment can increase HIF response by overriding HIF-mediated pomca negative regulation. However, in arnt1 -/and arnt1 -/-;vhl -/larvae, even if BME can act downstream of pomca, it is not able to trigger HIF response due to arnt1 loss of function.
These data suggest that in gr -/-;vhl -/the uncontrolled upregulation of pomca, triggered by gr loss of function, cannot be counteracted with the same efficiency by HIF action. Furthermore, even if steroidogenesis is upregulated, endogenous cortisol cannot act via Gr to stimulate the HIF response any longer. Nevertheless, since there is still a clear upregulation of HIF signaling in gr -/-;vhl -/larvae compared to wild-types (Fig.3d), we considered that the Mr may also able to promote HIF activity.

Both Gr and Mr are directly required for assuring proper HIF response:
Cortisol has high affinity both for Gr and Mr and they have been recently shown to be differentially involved in the regulation of stress axis activation and function in zebrafish (Faught and Vijayan, 2018), Therefore, we analysed the role of mr in the HIF response. To achieve this, we knocked-out mr in gr +/-;vhl +/-;phd3:eGFP incrossed derived embryos using CRISPant technology (Wu et al., 2018). Interestingly, phenotypic analysis performed on 5 dpf injected and uninjected larvae revealed that mr CRISPR injected vhl mutants were characterized by a significant downregulation of phd3:eGFP-related brightness at the level of the head (equals to 49%, P<0.0001), in the liver (equals to 56%, P<0.0001) and in the rest of the body (equals to 47%, P<0.0001), compared to vhl -/mutant uninjected larvae ( Fig.6a and 6b). Moreover, when both gr and mr were knocked-out, the downregulation was even stronger at the level of the head (equals to 62%, P<0.0001), in the liver (equals to 77%, P<0.0001) and in the rest of the body (equals to 63%, P<0.0001) ( Fig.6a and 6b).
To confirm the specificity of Wu et al., 2018 method, we chose to target a gene which was not involved in the HIF pathway. Laminin, beta 1b (lamb1b), which codes for an extracellular matrix glycoprotein, was injected as CRISPR-injection control in vhl +/incross derived embryos at 1 cell stage. Genotypic analysis carried out on these larvae confirmed that these guides were effective. Finally, quantification of phd3:eGFP-related brightness performed on 5 dpf injected and uninjected larvae, showed no significant differences between the two groups ( Fig.6c and 6d). Overall, these data indicate that both glucocorticoid and mineralocorticoid receptor play a pivotal role in assuring HIF response in vivo in zebrafish. for cross-talk between HIF and glucocorticoid pathways, however there are still controversial data on how the interaction between these two major signalling pathways occurs in vivo. In this regard, we have presented a novel in vivo study using zebrafish larvae, focusing on elucidation of genetic control of one pathway over the other. In contrast to in vitro cell culture studies, a whole animal study allows us to take into account the interactions that occur between various tissues and provide novel insights. To do this, we generated arnt1 and gr null mutants to downregulate HIF and GR signalling respectively, as a basis for a genetic analysis of this crosstalk.

Discussion
Comparison between arnt1 and arnt2 in the overall HIF response: As a prelude to this, we had to establish the relative importance of arnt1 and arnt2 in the overall HIF response. To achieve this, a discriminative test was devised to place The effect of HIF overexpression on glucocorticoid response: We next investigated the interplay between HIF and glucocorticoid response, by performing RTqPCR analysis on 5 dpf larvae. Collectively, we show that the presence of strongly activated (vhl -/-) and partially attenuated HIF response (arnt1 -/-;vhl -/-) appears to blunt glucocorticoid signaling, whereas arnt1 loss of function increased endogenous glucocorticoid response. Furthermore, betamethasone treatment on the aforementioned mutant lines was unable to significantly increase glucocorticoid target genes expression both in vhl -/and in arnt1 -/-;vhl -/-, whereas it was able to do it in arnt1 -/larvae. Together, these results indicate that upregulated HIF levels are somehow able to repress glucocorticoid response and that low normoxic HIF activity nevertheless suffices to attenuate GR activity.
To test whether this was due to any potential effect of overexpressed HIF response  Cumulatively, if this is true, we predicted to observe reduced levels of endogenous glucocorticoids in vhl -/and normal or even increased levels in arnt1 -/-. Indeed, we found that cortisol levels were significantly reduced in vhl mutant larvae (where HIF pathway is overexpressed), whereas they were significantly increased in arnt1 mutants (where HIF pathway is suppressed).
Together, these data allow us to show the presence of a HIF-induced negative feedback, aimed to blunt steroidogenesis in order to regulate HIF activity itself. By the way, HIF-mediated negative feedback seems to be a logic homeostatic response aimed to avoid a further GCs-induced upregulation of HIF pathway, since hypoxia has been shown to trigger glucocorticoid response (Leonard et al., 2005) and consequently, cortisol was shown to increase HIF expression (Vettori et al., 2017).

The effect of glucocorticoids on the HIF response:
To investigate the role of glucocorticoids on the HIF response, we initially analyzed the effect of gr loss of function on vhl phenotype. Surprisingly, we observed that gr mutation was able to cause an efficient, but not complete rescue of the vhl phenotype, which resembled arnt1 mutation. Notably, gr -/-;vhl -/survived much longer than vhl -/-(>=21 dpf compared to max. 10 dpf), but then similarly to arnt1 -/-;vhl -/-, they failed to grow and thrive when compared to siblings.
Together, these data indicate for the first time in an in vivo animal model that Gr is essential to assure a proper HIF response. Of note, our model based on the negative regulatory effect played by HIF on pomca expression, may provide a reliable explanation of this phenotype. In particular, we speculate that in gr -/-;vhl -/-, the absence of GC-GR negative feedback loop triggers a strong upregulation of pomca (Fig.4b), which cannot be counteracted with the same efficiency by upregulated HIF levels (via HIF-mediated negative feedback). In this scenario, the elevated endogenous cortisol levels cannot interact with Gr any longer. Consequently, since cortisol acts via glucocorticoid receptor as an enhancer of the HIF response (Kodama et al., 2003;Vettori Andrea et al., 2016), gr loss of function results in an attenuated HIF pathway activation.
Cumulatively, we suggest that HIF-mediated negative feedback on pomca expression can occur both via Arnt1 and Arnt2, whilst BME-induced HIF upregulation mainly requires functional Arnt1.

Evaluation of mineralocorticoid receptor contribution to HIF response:
Previous work published by Faught and Vijayan, 2018 showed that both Gr and Mr are differentially involved in the regulation of zebrafish stress axis activation and function. In addition, our results suggest that both glucocorticoids and Gr exert a pivotal role in the HIF response. Since nothing was known about mineralocorticoid receptor contribution to the HIF response, we tested the effect of mr knock-out in gr +/-;vhl +/-;phd3:EGFP incrossed derived embryos. Both in mr injected-vhl -/and gr -/-;vhl -/-5 dpf larvae, we showed a significant reduction of phd3:eGFP-related brightness. These data suggest that in fish not only the glucocorticoid receptor, but also the mineralocorticoid receptor is involved in promoting HIF pathway activation, as a consequence of cortisol stimulation. Indeed, in contrast to mammals, teleosts lack aldosterone and cortisol is the primary glucocorticoid hormone which can interact with both Gr and Mr to assure the correct HPI axis functioning (Cruz et al., 2013;Baker and Katsu, 2017). Of note, our hypothesis is supported by Faught and Vijayan, 2018 recent work, showing that both Gr and Mr signalling is involved in the negative feedback regulation of cortisol biosynthesis during stress.
In conclusion, although Mr contribution to HIF response in other organisms remains unclear, our work suggests research into its function is warranted.

Conclusion
Our present study stresses the importance of the glucocorticoid pathway in driving HIF response. In addition, we uncovered a negative regulatory role played by overexpression of HIF in regulating steroidogenesis as demonstrated via RTqPCR and steroid hormone quantification. We also report a novel key role for Arnt1 in regulating BME-induced HIF response and identify a possible mineralocorticoid receptor contribution to HIF-GC crosstalk. Finally, we presented novel gr +/-;vhl +/-, arnt1 +/-;vhl +/and arnt1 +/-;gr +/-;vhl +/zebrafish mutant lines which helped to better understand how the interplay between HIF and glucocorticoids occur in vivo. For these reasons, we believe that this work could pave the way for further in vivo analysis to precisely identify the extensive crosstalk behind these two major signalling pathways.

Data availability
All data are available on request. Writing -Review and Editing: all authors contributed equally.

Conflict of interest
The authors declare that they have no conflict of interest.