Reassessing the contribution of the Na+/H+ exchanger Nhe3b to Na+ uptake in zebrafish (Danio rerio) using CRISPR/Cas9 gene editing
| Autor: | Alex M. Zimmer, Steve F. Perry, Raymond W. M. Kwong, Khatereh Shir-Mohammadi |
|---|---|
| Rok vydání: | 2019 |
| Předmět: |
0106 biological sciences
animal structures Morpholino Physiology 030310 physiology Mrna expression Danio Aquatic Science 010603 evolutionary biology 01 natural sciences 03 medical and health sciences parasitic diseases CRISPR 14. Life underwater Molecular Biology Zebrafish Ecology Evolution Behavior and Systematics 0303 health sciences biology Chemistry Na uptake fungi biology.organism_classification Molecular biology Sodium–hydrogen antiporter Insect Science embryonic structures Animal Science and Zoology Flux (metabolism) |
| Zdroj: | Journal of Experimental Biology. |
| ISSN: | 1477-9145 0022-0949 |
| DOI: | 10.1242/jeb.215111 |
| Popis: | 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 three proteins in Na+ uptake by larval zebrafish under ‘normal’ (800 µmol l−1) and ‘low’ (10 µmol l−1) 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 was not altered in nhe3b KO larvae. 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. |
| Databáze: | OpenAIRE |
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