arnt1 and arnt2 have partially overlapping functions and synergistically contribute to HIF signalling. A. Schematic representation of zebrafish hif1β (arnt1) gene. Exons are shown as black boxes, whereas introns as lines. The red arrowhead shows the position of a +7 bp insertion in exon 5 (encoding the bHLH DNA binding domain). In the arnt1 wt and mutant sequence. CRISPR target site: bold. Protospacer-adjacent-motif (PAM) sequence: red. B-B’. Magnified picture of a representative 5 dpf vhl-/- larva compared to 5dpf arnt1-/-;vhl-/- (C-C’). Among the 120 GFP+ embryos derived from arnt1+/-;vhl+/-(phd3:eGFP) x arnt1-/-;vhl+/-(phd3:eGFP), 15 larvae were characterized by the absence of pericardial oedema, no ectopic extra vasculature at the level of the tail, no bright liver and a reduced brightness in the rest of the body (black and white arrowheads). Genotyping post phenotypic analysis on sorted larvae confirmed genotype-phenotype correlation. Fluorescence, exposure = 2 seconds. Scale bar 200 μm. D-G. Representative picture of phenotypic analysis performed on DMSO and BME [30 μM] treated 5 dpf larvae, derived from arnt1+/-;vhl+/-(phd3:eGFP) x arnt1-/-;vhl+/-(phd3:eGFP) (n = 540). All the genotype combinations observed are represented in the figure. Among the 405 GFP+ larvae, all the 25 arnt1-/-;vhl-/- showed the aforementioned partially rescued vhl phenotype (D). Fluorescence, exposure = 2 seconds. Scale bar 500 μm. H-J. Representative pictures of 5 dpf CRISPANT mutants created by redundantly targeting arnt2 gene via co-injection of 4x gRNAs in arnt1+/-;vhl+/-(phd3:eGFP) x arnt1-/-; vhl+/-(phd3:eGFP) derived embryos (n = 300). Uninjected embryos were used as control (n = 120). White asterisks: head, liver and tail ragions. Fluorescence, exposure = 991,4 ms. Scale bar 500 μm. K. Statistical analysis performed on mean grey values quantification (at the level of the head, liver and tail), after phenotypic analysis on 5 dpf arnt2 4x gRNAs injected and uninjected larvae. vhl-/- uninjected n = 8 larvae: head 93.1 ± 2.33 (mean ± s.e.m); liver 99.65 ± 3.49 (mean ± s.e.m); tail 29.58 ± 0.73 (mean ± s.e.m). arnt1-/-;vhl-/- uninjected n = 10 larvae: head 56.49 ± 3.36 (mean ± s.e.m); liver 24,7 ± 2.36 (mean ± s.e.m); tail 12.39 ± 0,75 (mean ± s.e.m). vhl-/- injected n = 12 larvae: head 43.69 ± 3.25 (mean ± s.e.m); liver 45.54 ± 4.57 (mean ± s.e.m); tail 16.09 ± 1.37 (mean ± s.e.m). arnt1-/-;vhl-/- injected n = 11 larvae: head 24.66 ± 1.63 (mean ± s.e.m); liver 13.88 ± 0.66 (mean ± s.e.m); tail 5.16 ± 0.33 (mean ± s.e.m). Ordinary One-way ANOVA followed by Sidak’s multiple comparison test (*P < 0.05; **P < 0.01; ***P <0.001; ****P < 0.0001).
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