Morpholino Gene Knockdown in Adult Fundulus heteroclitus: Role of SGK1 in Seawater Acclimation

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

SGK1 protein and mRNA levels in gill of <i>Fundulus heteroclitus</i> injected with the SGK1 translation blocking vivo-morpholino.

<p><b>A</b>: Representative Western blot of SGK1. <b>B</b>: Summary of SGK1 Western blot experiments. <b>C</b>: SGK1 mRNA abundance. Freshwater acclimated fish were injected with 14 µg/g SGK1 vivo-morpholino, or control vivo-morpholino. Four hours after injection fish were transferred to seawater for 1 h. n = 5. Different letters indicate statistically significant treatment means p<0.05. FW-Control: Freshwater control vivo-morpholino, FSW-Control: Freshwater to seawater control vivo-morpholino, FSW SGK1: Freshwater to seawater SGK1 vivo-morpholino.</p

Validation of plasma membrane preparation.

<p>Representative Western blots of whole cell lysates (WCL) and plasma membrane preparations (PM) from freshwater or seawater acclimated killifish for: <b>A</b>: Rab4a. <b>B</b>: Na⁺, K⁺-ATPase and <b>C</b>: CFTR. FW: freshwater, SW: seawater acclimated fish. Equal volumes of WCL and PM were loaded in each lane.</p

SGK1 mRNA and protein levels in gills of adult <i>Fundulus heteroclitus</i> injected with splice blocking SGK1 vivo-morpholino.

<p><b>A</b>: SGK1 mRNA abundance <b>B</b>: Representative Western blot of SGK1. <b>C</b>: Summary of SGK1 Western blot experiments. Freshwater acclimated fish were injected with 20 µg/g SGK1 vivo-morpholino, control vivo-morpholino or PBS control. Four hours after injection fish were transferred to seawater for 1 h. n = 5. Different letters indicate statistically significant treatment means p<0.05. FW-Control: Freshwater control vivo-morpholino, FSW-Control: Freshwater to seawater control vivo-morpholino, FSW SGK1: Freshwater to seawater SGK1 vivo-morpholino.</p

SGK1 protein levels in liver of <i>Fundulus heteroclitus</i> injected with the translational blocking vivo-morpholino.

<p><b>A</b>: Representative Western blot of SGK1 in liver. <b>B</b>: Summary of SGK1 Western blot experiments in liver. Freshwater acclimated fish were injected with 14 µg/g SGK1 vivo-morpholino, or control vivo-morpholino. Eight hours after injection fish were transferred to seawater for 1 h. n = 5. Different letters indicate statistically significant treatment means p<0.05. FW-Control: Freshwater control vivo-morpholino, FSW-Control: Freshwater to seawater control vivo-morpholino, FSW SGK1: Freshwater to seawater SGK1 vivo-morpholino.</p

SGK1 protein levels in intestine of <i>Fundulus heteroclitus</i> injected with the translational blocking vivo-morpholino.

<p><b>A</b>: Representative Western blot of SGK1 in intestine. <b>B</b>: Summary of SGK1 Western blot experiments in intestine. Freshwater acclimated fish were injected with 14 µg/g SGK1 vivo-morpholino, or control vivo-morpholino. Eight hours after injection fish were transferred to seawater for 1 h. n = 5. Different letters indicate statistically significant treatment means p<0.05. FW-Control: Freshwater control vivo-morpholino, FSW-Control: Freshwater to seawater control vivo-morpholino, FSW SGK1: Freshwater to seawater SGK1 vivo-morpholino.</p

Na⁺, K⁺-ATPase protein levels in gill of fish injected with SGK1 vivo-morpholino.

<p>Freshwater acclimated fish were injected with 14 µg/g SGK1 or control vivo-morpholino. Four hours after injection fish were transferred to seawater for 1 h. The increase in plasma membrane Na⁺, K⁺-ATPase in seawater fish compared to freshwater fish was not due to differences in contamination of the membrane preparation by cytosolic proteins, since the amount of Rab4a in each preparation was minimal, and equivalent in all membrane preparations (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029462#pone-0029462-g004" target="_blank">Figure 4A</a>). <b>A</b>: Representative Western blot of Na⁺, K⁺-ATPase. <b>B</b>: Summary of Na⁺, K⁺-ATPase WCL protein levels. <b>C</b>: Summary of Na⁺, K⁺-ATPase PM protein levels. n = 4. Different letters indicate statistically significant treatment means p<0.05. FW-C: Freshwater control vivo-morpholino, FSW-C: Freshwater to seawater control vivo-morpholino, FSW-M: Freshwater to seawater SGK1 vivo-morpholino, WCL: whole cell lysate, PM: plasma membrane.</p

CFTR protein levels in gill of fish injected with SGK1 vivo-morpholino.

<p>Freshwater acclimated fish were injected with 14 µg/g SGK1 or control vivo-morpholino. Four hours after injection fish were transferred to seawater for 1 h. The increase in plasma membrane CFTR in seawater fish compared to freshwater fish was not due to differences in contamination of the membrane preparation by cytosolic proteins (i.e., CFTR), since the amount of Rab4a in each preparation was minimal, and equivalent in all membrane preparations (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029462#pone-0029462-g004" target="_blank">Figure 4A</a>). <b>A</b>: Representative Western blot of CFTR. <b>B</b>: Summary of CFTR WCL protein levels. <b>C</b>: Summary of CFTR PM protein levels. n = 4. Different letters indicate statistically significant treatment means p<0.05. FW-C: Freshwater control vivo-morpholino, FSW-C: Freshwater to seawater control vivo-morpholino, FSW-M: Freshwater to seawater SGK1 vivo-morpholino, WCL: whole cell lysate, PM: plasma membrane.</p

Primer sets used for amplification of Killifish SGK1 intron and exon boundaries and for qPCR.

<p>Primer sets used for amplification of Killifish SGK1 intron and exon boundaries and for qPCR.</p