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Fig. 2

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ZDB-IMAGE-200520-25
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Figures for Marchi et al., 2020
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Fig. 2 HIF signalling inversely correlate with GC transcriptional activity and cortisol biosynthesis. A. Schematic view of RTqPCR analysis on fkbp5 expression performed on the vhl+/-(phd3:eGFP) and the arnt1+/-(phd3:eGFP) mutant lines at 5 dpf:. Upregulated (in vhl-/-) HIF signalling repressed Gr activity, whereas arnt1 loss of function derepressed it. Statistical analysis was performed on ΔΔCt values, whereas data are shown as fold change values for RTqPCR analysed samples; ordinary Two-way ANOVA followed by Dunnett’s multiple comparison test (*P < 0.05; **P < 0.01; ***P <0.001; ****P < 0.0001). B-C’. Representative pictures of WISH performed on DMSO and BME [30 μM] treated arnt1 mutant line, at 5 dpf, using pomca as probe. arnt1 wt DMSO treated (n = 30/30 larvae) showed normal pomca expression; arnt1 wt BME treated (n = 29/30 larvae) showed downregulated pomca expression. In contrast, arnt1-/- DMSO treated (n = 28/30) and arnt1-/- BME treated (n = 30/30) larvae showed downregulated pomca expression. Chi-square test (****P < 0.0001). Scale bar 50 μm. D-E’. Representative pictures of WISH performed on DMSO and BME [30 μM] treated vhl mutant line, at 5 dpf, using pomca as probe. DMSO treated vhl siblings (n = 26/28) showed normal pomca expression; BME treated vhl siblings (n = 28/30) showed downregulated pomca expression. In contrast, vhl-/- DMSO (n = 28/29) and BME (n = 28/28) treated larvae showed downregulated pomca expression. Chi-square test (****P < 0.0001). Scale bar 50 μm. F-F’. Steroid quantification results showed a significantly reduced cortisol concentration (P value <0;0028) in vhl mutants (92.7 fg/larva, in triplicate), compared to vhl siblings (321 fg/larva, in triplicate) at 5 dpf (F). Moreover, a significantly increased cortisol concentration (P value <0;0001) was measured in arnt1 mutants (487.5 fg/larva, in triplicate), compared to arnt1 wild-types (325 fg/larva, in triplicate) at 5 dpf (F’); unpaired t-test (**P < 0.01; ***P <0.001). G. DMSO: Speculative scheme of how the putative HIF-GC crosstalk occurs in wildtypes and how it is affected both in arnt1-/- and in vhl-/- larvae at 5 dpf. In wildtype scenario HIF signalling helps the GC-GR negative feedback to protect the body from an uncontrolled stress response. In particular, we speculate that HIF transcriptional activity is able to inhibit pomca expression when cortisol levels arise over a certain threshold in order to maintain both HIF and GC basal levels. However, in arnt1-/- scenario, the HIF-mediated negative feedback is compromised by the lack of a functional Arnt1. This triggers an initial uncontrolled pomca expression, which increases cortisol levels and subsequently downregulate pomca expression itself. Vice versa, in vhl-/- scenario, the HIF-mediated negative feedback can exert a stronger inhibition of pomca due to the presence of upregulated HIF signalling. This results both in downregulated cortisol levels and in a suppressed GR responsiveness. However, the presence of alternative mechanisms cannot be completely excluded (i.e HIF might interact more directly with GC/GR to impair its function). Finally, the combination high cortisol/low pomca is very rare and this combination may change over the course of development. G. BME: Speculative scheme of how the putative HIF-GC crosstalk occurs in wildtypes and how it is affected both in arnt1-/- and in vhl-/- larvae at 5 dpf, after BME[30μM] treatment. In all the cases, because of betamethasone acts downstream of the HPI axis, by binding directly to Gr, it is able to upregulate glucocorticoid target genes expression. Consequently, since GC are able to stimulate HIF signalling, as expected, we observed an increase phd3:eGFP-related brightness both in wildtypes and in vhl-/-. However, the fact that we did not observed any HIF upregulation both in arnt1-/- and in arnt1-/-;vhl-/-, highlighted the fact that the BME-induced HIF signalling activation is an Arnt1 dependent mechanism.

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