|ZFIN ID: ZDB-PUB-201022-13|
Use of a carbonic anhydrase ca17a knockout to investigate mechanisms of ion uptake in zebrafish (Danio rerio)
Zimmer, A.M., Mandic, M., Yew, H.M., Kunert, E., Pan, Y.K., Ha, J., Kwong, R.W.M., Gilmour, K.M., Perry, S.F.
|Source:||American journal of physiology. Regulatory, integrative and comparative physiology 320(1): R55-R68 (Journal)|
|Registered Authors:||Kunert, Emma, Perry, Steve F., Yew, Hong Meng|
|Keywords:||Acid-base balance, CRISPR/Cas9, Chloride uptake, H+-ATPase-rich cell, Sodium uptake|
|PubMed:||33085911 Full text @ Am. J. Physiol. Regul. Integr. Comp. Physiol.|
Zimmer, A.M., Mandic, M., Yew, H.M., Kunert, E., Pan, Y.K., Ha, J., Kwong, R.W.M., Gilmour, K.M., Perry, S.F. (2020) Use of a carbonic anhydrase ca17a knockout to investigate mechanisms of ion uptake in zebrafish (Danio rerio). American journal of physiology. Regulatory, integrative and comparative physiology. 320(1):R55-R68.
ABSTRACTIn 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.