ZFIN ID: ZDB-PUB-191220-6
Reassessing the contribution of the Na+/H+ exchanger Nhe3b to Na+ uptake in zebrafish (Danio rerio) using CRISPR/Cas9 gene editing
Zimmer, A.M., Shir-Mohammadi, K., Kwong, R.W.M., Perry, S.F.
Date: 2020
Source: The Journal of experimental biology   223(Pt 2): (Journal)
Registered Authors: Perry, Steve F.
Keywords: Compensatory regulation, H+-ATPase, Ion regulation, Ionocyte, Na+-Cl−-cotransporter (Ncc), Rhesus glycoprotein Rhcgb (formerly Rhcg1)
MeSH Terms:
  • Animals
  • CRISPR-Cas Systems*
  • Zebrafish/genetics*
  • Zebrafish/metabolism
PubMed: 31852755 Full text @ J. Exp. Biol.
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ABSTRACT
Freshwater fishes absorb Na+ from their dilute environment using ion-transporting cells. In larval zebrafish (Danio rerio), Na+ uptake is coordinated by (1) Na+/H+-exchanger 3b (Nhe3b) and (2) H+-ATPase-powered electrogenic uptake in H+-ATPase-rich (HR) cells and by (3) Na+-Cl----cotransporter (Ncc) expressed in NCC cells. The present study aimed to better understand the roles of these 3 proteins in Na+ uptake by larval zebrafish under 'normal' (800 µmol/L) and 'low' (10 µmol/L) Na+ conditions. We hypothesized that Na+ uptake would be reduced by CRISPR/Cas9 knockout (KO) of slc9a3.2 (encoding Nhe3b), particularly in low Na+ where Nhe3b is believed to play a dominant role. Contrary to this hypothesis, Na+ uptake was sustained in nhe3b KO larvae under both Na+ conditions, which led to the exploration of whether compensatory regulation of H+-ATPase or Ncc was responsible for maintaining Na+ uptake in nhe3b KO larvae. mRNA expression of the genes encoding H+-ATPase and Ncc were not altered in nhe3b KO. Moreover, morpholino knockdown of H+-ATPase, which significantly reduced H+ flux by HR cells, did not reduce Na+ uptake in nhe3b KO larvae, nor did rearing larvae in chloride-free conditions, thereby eliminating any driving force for Na+-Cl--cotransport via Ncc. Finally, simultaneously treating nhe3b KO larvae with H+-ATPase morpholino and chloride-free conditions did not reduce Na+ uptake under normal or low Na+ These findings highlight the flexibility of the Na+ uptake system and demonstrate that Nhe3b is expendable to Na+ uptake in zebrafish and that our understanding of Na+ uptake mechanisms in this species is incomplete.
ADDITIONAL INFORMATION
ERRATA and NOTES
Corrected by ZDB-PUB-200904-2