PUBLICATION
Use of a carbonic anhydrase ca17a knockout to investigate mechanisms of ion uptake in zebrafish (Danio rerio)
- Authors
- 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.
- ID
- ZDB-PUB-201022-13
- Date
- 2020
- 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
- MeSH Terms
-
- Acid-Base Equilibrium*
- Animals
- Animals, Genetically Modified
- CRISPR-Cas Systems
- Carbonic Anhydrases/deficiency*
- Carbonic Anhydrases/genetics
- Chlorides/metabolism*
- Gene Knockout Techniques*
- Hydrogen-Ion Concentration
- Ion Transport
- Mutation
- Sodium/metabolism*
- Zebrafish/embryology
- Zebrafish/genetics
- Zebrafish/metabolism*
- Zebrafish Proteins/deficiency*
- Zebrafish Proteins/genetics
- PubMed
- 33085911 Full text @ Am. J. Physiol. Regul. Integr. Comp. Physiol.
Citation
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.
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 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.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping