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

Authors: 
Zimmer AM, Mandic M, Yew HM, Kunert E, Pan YK, Ha J, Kwong RWM, Gilmour KM, Perry SF
Citation: 
Am J Physiol Regul Integr Comp Physiol. 2021;320(1):R55-R68. doi:10.1152/ajpregu.00215.2020
Abstract: 
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, O2 consumption, CO2 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.
Organism or Cell Type: 
zebrafish
Delivery Method: 
microinjection