Abstract
The Atlantic killifish (Fundulus heteroclitus) is an environmental sentinel organism used extensively for studies on environmental toxicants and salt (NaCl) homeostasis. Previous research in our laboratory has shown that rapid acclimation of killifish to seawater is mediated by trafficking of CFTR chloride channels from intracellular vesicles to the plasma membrane in the opercular membrane within the first hour in seawater, which enhances chloride secretion into seawater, thereby contributing to salt homeostasis. Acute transition to seawater is also marked by an increase in both mRNA and protein levels of serum glucocorticoid kinase 1 (SGK1) within 15 minutes of transfer. Although the rise in SGK1 in gill and its functional analog, the opercular membrane, after seawater transfer precedes the increase in membrane CFTR, a direct role of SGK1 in elevating membrane CFTR has not been established in vivo. To test the hypothesis that SGK1 mediates the increase in plasma membrane CFTR we designed two functionally different vivo-morpholinos to knock down SGK1 in gill, and developed and validated a vivo-morpholino knock down technique for adult killifish. Injection (intraperitoneal, IP) of the splice blocking SGK1 vivo-morpholino reduced SGK1 mRNA in the gill after transition from fresh to seawater by 66%. The IP injection of the translational blocking and splice blocking vivo-morpholinos reduced gill SGK1 protein abundance in fish transferred from fresh to seawater by 64% and 53%, respectively. Moreover, knock down of SGK1 completely eliminated the seawater induced rise in plasma membrane CFTR, demonstrating that the increase in SGK1 protein is required for the trafficking of CFTR from intracellular vesicles in mitochondrion rich cells to the plasma membrane in the gill during acclimation to seawater. This is the first report of the use of vivo-morpholinos in adult killifish and demonstrates that vivo-morpholinos are a valuable genetic tool for this environmentally relevant model organism.
Highlights
The Atlantic killifish (Fundulus heteroclitus) is an environmental sentinel organism used extensively for studies of environmental toxicants and salt (NaCl) homeostasis [1,2,3,4,5,6,7]
The goal of this study was two-fold; to develop a method using IP injection of vivo-morpholinos to selectively knock down target genes in adult killifish, and to use this method to test the hypothesis that serum glucocorticoid kinase 1 (SGK1) mediates the rapid (1 hour) increase in plasma membrane CFTR in the gill when killifish are transferred from freshwater to seawater
The SGK1 translational blocking vivo-morpholino elicited a significant, 64% reduction (p,0.05) in gill SGK1 protein in fish transferred from freshwater to seawater compared to SGK1 in adult killifish injected with the same concentration of control vivo-morpholino, which had a 1.5 fold increase (p,0.05) in SGK1 protein, a result comparable to a previous study (Figure 1A and 1B) [5]
Summary
The Atlantic killifish (Fundulus heteroclitus) is an environmental sentinel organism used extensively for studies of environmental toxicants and salt (NaCl) homeostasis [1,2,3,4,5,6,7]. Acclimation to increased salinity involves the remodeling of mitochondrion rich cells in the opercular membrane and gill from ion absorption to ion (NaCl) secretion in order to maintain osmotic balance [8]. In the seawater gill chloride (Cl2) secretion is a two-step process; uptake across the basolateral membrane via the Na+, K,+ 2Cl2 co-transporter, followed by secretion of Cl2 across the apical membrane through the cystic fibrosis transmembrane conductance regulator (CFTR) [9,10]. Na+, K+-ATPase is necessary to maintain a low intracellular sodium concentration, which facilitates Cl2 uptake through the Na+, K+, 2Cl2 co-transporter [6]. CFTR is a cAMP regulated Cl2 channel that belongs to the ATPbinding cassette family of proteins, whose mutations result in cystic fibrosis the most common lethal autosomal recessive disorder in Caucasians [11]
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