Separate Na,K-ATPase genes are required for otolith formation and semicircular canal development in zebrafish.
We have investigated the role of Na,K-ATPase genes in zebrafish ear development. Six Na,K-ATPase genes are differentially expressed in the developing zebrafish inner ear. Antisense morpholino knockdown of Na,K-ATPase alpha1a.1 expression blocked formation of otoliths. This effect was phenocopied by treatment of embryos with ouabain, an inhibitor of Na,K-ATPase activity. The otolith defect produced by morpholinos was rescued by microinjection of zebrafish alpha1a.1 or rat alpha1 mRNA, while the ouabain-induced defect was rescued by expression of ouabain-resistant zebrafish alpha1a.1 or rat alpha1 mRNA. Knockdown of a second zebrafish alpha subunit, alpha1a.2, disrupted development of the semicircular canals. Knockdown of Na,K-ATPase beta2b expression also caused an otolith defect, suggesting that the beta2b subunit partners with the alpha1a.1 subunit to form a Na,K-ATPase required for otolith formation. These results reveal novel roles for Na,K-ATPase genes in vestibular system development and indicate that different isoforms play distinct functional roles in formation of inner ear structures. Our results highlight zebrafish gene knockdown-mRNA rescue as an approach that can be used to dissect the functional properties of zebrafish and mammalian Na,K-ATPase genes.
Department of Pharmacology, Penn State University College of Medicine, H078, Hershey, PA 17033, USA.
Brian
B
Blasiole
Victor A
VA
Canfield
Melissa A
MA
Vollrath
David
D
Huss
Manzoor-Ali P K
MA
Mohideen
J David
JD
Dickman
Keith C
KC
Cheng
Donna M
DM
Fekete
Robert
R
Levenson
D000818:Animals; D004423:Ear / Q000254:growth & development; D004625:Embryo, Nonmammalian; D007527:Isoenzymes; D009024:Morphogenesis; D010037:Otolithic Membrane / Q000201:enzymology*; D021122:Protein Subunits; D051381:Rats; D012665:Semicircular Canals / Q000201:enzymology*; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics* / Q000502:physiology; D015027:Zebrafish
D007527:Isoenzymes; D021122:Protein Subunits; D000254:Sodium-Potassium-Exchanging ATPase