Fig 2. Azide chronic and acute inhibition of RC complex IV.

(A–F), Effects of chronic azide exposure beginning at 5 hpf. (A–B) Developmental delay was induced by early azide treatment after 22 hours of treatment, where (A) low-magnification shows that 75 uM azide treated animals had shorter tails (right panel) (B) Higher magnification of 75 uM and 100 uM azide treated embryos clearly demonstrate their developmental delay compared to buffer treated controls after 22 hours, where azide-treated animals had shorter tails and decreased absorption of their yolk. These embryos were manually dechorionated to facilitate observation. (C) After 27 hours of 75 uM or 100 uM azide treatment, delayed pigment development was observed (arrows, left panel). (D) 100 uM azide-treated animals were delayed compared to controls. Control fish (left panels) observed at this time have developed otoliths (white arrows) within their otic capsules (lower right panel) as well as pigmented cells in their flanks (top panel) and eyes (lower left panel). The 100 uM azide treated (right panels) have not developed pigment in their flanks (top panel) or eyes (lower left panel) nor have they developed otoliths (lower right panel). (E) Developmental delay induced by 75 uM azide chronic treatment from 5 hpf (right panels) relative to control fish on 6 dpf (left panel). Azide treatment at 75 uM concentration delayed normal development, but did not arrest it. Azide-treated animals on 6 dpf had delayed inflation of their swim bladder (black arrows) and reduced absorption of yolk (white arrows). (F) Azide chronic exposure from 5hpf and observed after 26 hours of treatment, revealed that cardiac function was delayed. 100 uM azide treated embryos were sufficiently delayed that they did not yet have a heartbeat after azide treatment for 26 hours. n=3 biological replicates with 3 fish per experiment. *p=0.0072. (G–I), Effects of acute azide exposure beginning on 6 dpf. (G) Survival of 7 dpf zebrafish after 16 hr of sodium azide (NaN₃) treatment, n=3 biological replicates with 10 fish per treatment condition. * p=0.0109, **p=0.0001. (H) Effects of acute azide exposure model on zebrafish startle-response after 16 hours azide treatment. Of note, all 125 uM azide treated fish were unresponsive to tapping on the dish. n=4 biological replicates with 10 fish per treatment condition. ** p=0.0033, ***p=0.00001. (I) Treatment with 100 uM azide for 17 hours (acute exposure) resulted in brain necrosis (arrow), which begins at the posterior of the brain and can be observed to move forward (cephalad) as it progresses over time. Error bars represent (±SD).