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 approximately 12 days post-fertilization (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 to wild-type individuals at 4 and 9 dpf, however whole-body Na+ content remained constant. In contrast, Cl- uptake was significantly reduced in ca17a-/- mutants, while Cl- content also 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, O2 consumption, CO2 excretion, and ammonia excretion were generally unaltered in ca17a-/- mutants. In conclusion, while loss of Ca17a caused marked changes in ion uptake rates, providing strong evidence for a Ca17a-depedent Cl- uptake mechanism, the underlying causes of the lethality of this mutation in zebrafish remain unclear.