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

In fishes, branchial cytosolic carbonic anhydrase (CA) plays an important role in ion and acid-base regulation. The Ca17a isoform in zebrafish () 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 knockout is lethal with 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 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 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 consumption, CO excretion, and ammonia excretion were generally unaltered in 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.