Use of a carbonic anhydrase Ca17a knockout to investigate mechanisms of ion uptake in zebrafish ( Danio rerio ).

In fishes, branchial cytosolic carbonic anhydrase (CA) plays an important role in ion and acid-base regulation. The Ca17a isoform in zebrafish ( Danio rerio ) is expressed abundantly in Na ⁺ -absorbing/H ⁺ -secreting H ⁺ -ATPase-rich (HR) cells. The present study aimed to identify the role of Ca17a in ion and acid-base regulation across life stages using CRISPR/Cas9 gene editing. However, in preliminary experiments, we established that ca17a knockout is lethal with ca17a ^-/- mutants exhibiting a significant decrease in survival beginning at ∼12 days postfertilization (dpf) and with no individuals surviving past 19 dpf. Based on these findings, we hypothesized that ca17a ^-/- mutants would display alterations in ion and acid-base balance and that these physiological disturbances might underlie their early demise. Na ⁺ uptake rates were significantly increased by up to 300% in homozygous mutants compared with wild-type individuals at 4 and 9 dpf; however, whole body Na ⁺ content remained constant. While Cl ^- uptake was significantly reduced in ca17a ^-/- mutants, Cl ^- content was unaffected. Reduction of CA activity by Ca17a morpholino knockdown or ethoxzolamide treatments similarly reduced Cl ^- uptake, implicating Ca17a in the mechanism of Cl ^- uptake by larval zebrafish. H ⁺ secretion, O ₂ consumption, CO ₂ excretion, and ammonia excretion were generally unaltered in ca17a ^-/- mutants. In conclusion, while the loss of Ca17a caused marked changes in ion uptake rates, providing strong evidence for a Ca17a-dependent Cl ^- uptake mechanism, the underlying causes of the lethality of this mutation in zebrafish remain unclear.