Effects of rapamycin on <i>pten</i> dko embryos.

<p>(<b>A</b>) A total of 41 embryos obtained from <i>ptena</i>^+/−<i>ptenb</i>^−/− parents was treated with 5 μM rapamycin for 96 h beginning at 26 hpf. (<b>B</b>) At 7 dpf, the embryos were photographed and genotyped. Eight of the 41 embryos had the <i>ptena</i>^−/−<i>ptenb</i>^−/− genotype (images 1–8), nine were <i>ptena</i>^+/−<i>ptenb</i>^−/−(image 9), and 10 were <i>ptena</i>^+/+<i>ptenb</i>^−/−(image 10). Partial Cuvierian ducts developed in seven of the eight dko embryos. X, no duct. (<b>C</b>) Snapshots from movies of the dko embryos shown in images 2 (upper) and 4 (lower) in (B). Blood cells can be seen flowing through the Cuvierian duct (upper) and the tail vascular duct (lower) indicated by the arrows. Times are in seconds.</p

Mutation efficiency for <i>pten</i> TALEN constructs revealed by analysis of G0 medaka embryos.

<p>Mutation efficiency for <i>pten</i> TALEN constructs revealed by analysis of G0 medaka embryos.</p

Establishment of <i>pten</i> knockout medaka with transcription activator–like effector nucleases (TALENs) as a model of PTEN deficiency disease

<div><p>Phosphatase and tensin homolog (PTEN) is a lipid and protein phosphatase that antagonizes signaling by the phosphatidylinositol 3-kinase (PI3K)–AKT signaling pathway. The <i>PTEN</i> gene is a major tumor suppressor, with mutations of this gene occurring frequently in tumors of humans and mice. We have now developed mutant medaka deficient in PTEN with the use of transcription activator–like effector nuclease (TALEN) technology. Medaka possesses two <i>pten</i> genes, <i>ptena</i> and <i>ptenb</i>, similar to zebrafish. We established 16 <i>ptena</i> mutant lines and two <i>ptenb</i> mutant lines. Homozygous single <i>pten</i> mutants were found to be viable and fertile. In contrast, <i>pten</i> double-knockout (dko) embryos manifested severe abnormalities in vasculogenesis, eye size, and tail development at 72 hours post fertilization(hpf) and died before hatching. Immunoblot analysis revealed that the ratio of phosphorylated to total forms of AKT (pAKT/AKT) in <i>pten</i> dko embryos was four times that in wild-type embryos, indicative of up-regulation of signaling by the PI3K-AKT pathway. Treatment of <i>pten</i> dko embryos with the PI3K inhibitor LY294002 reduced the pAKT/AKT ratio by about one-half and partially rescued the defect in vasculogenesis. Additional inhibitors of the PI3K-AKT pathway, including rapamycin and <i>N</i>-α-tosyl-L-phenylalanyl chloromethyl ketone, also partially restored vasculogenesis in the dko embryos. Our model system thus allows <i>pten</i> dko embryos to be readily distinguished from wild-type embryos at an early stage of development and is suitable for the screening of drugs able to compensate for PTEN deficiency.</p></div

Characterization of medaka PTEN genes and design of TALEN constructs.

<p><b>(A</b>) RT-PCR analysis of total RNA isolated from whole embryos of medaka was performed with two sets of primers designed to amplify almost the entire open reading frames of <i>ptena</i> (lane 1) and <i>ptenb</i> (lane 2). The arrow indicates the specific amplification products, with the faster-migrating bands being found to represent technical artifacts by sequencing analysis. Lane M, molecular size markers. (<b>B</b>) Products of medaka <i>ptena</i> and <i>ptenb</i> predicted from the sequences of the amplified cDNAs and database information. Arrowheads labeled #1 and #2 indicate the localization of the TALEN target sequences shown in (C). (<b>C</b>) TALEN target sites in <i>ptena</i> and <i>ptenb</i>.</p

Effects of LY294002 on vascular development and blood cell flow in <i>pten</i> dko embryos.

<p>(<b>A</b>) Wild-type or <i>pten</i> dko embryos were exposed (or not) to 15 μM LY294002 for 48 h beginning at 30 or 48 hpf. (<b>B</b>) At 7 dpf, the embryos were photographed and genotyped. The dko embryos treated with LY294002 developed partial Cuvierian ducts (4 of 4 treated at 30 hpf, and 2 of 3 treated at 48 hpf). The dko embryos not exposed to the drug (2/2) did not manifest vasculogenesis. X, no duct. (<b>C</b>) Snapshots from a movie of a 4-dpf dko embryo that had been treated with LY294002 for 48 h beginning at 30 hpf. Blood cells can be seen flowing through the Cuvierian duct (arrow). Times are indicated in seconds.</p

PI3K-AKT signaling pathway activity in adult <i>pten</i> mutant medaka.

<p>Extracts (20 μg of protein) derived from the dorsal muscle of 7- to 10-mpf fish were subjected to immunoblot analysis with antibodies to phosphorylated (p) or total forms of AKT as well as with those to α-tubulin (loading control). Each lane corresponds to an individual. The pAKT/AKT ratio for individual fish of the indicated <i>pten</i> genotypes was determined by densitometry. There were another two independent replications that showed much the same results.</p

Effect of sorafenib (0.1 μM) treatment on melanophore hyperplasia and overall survival in <i>Tg</i><i>(</i><i>tyr:HRAS^G12V</i><i>)</i><i>/Tg</i><i>(</i><i>hsp:cre</i><i>)</i> medaka.

<p>(<b>A</b>) Schedule of drug administration. (<b>B</b>) Photos taken from the dorsal side of all fish at 7 days before (day –7) and 29 days after (day +29) the first drug administration. The area of MPLs in the fish body was measured based on the set of captured images on the right, and the average fold change from day –7 to day +29 was calculated for each group. (<b>C</b>) Kaplan-Meier survival curves for the sorafenib-treated and control (DMSO-treated) groups were generated from the experiment shown in (<b>B</b>). Fish with obvious melanophore hyperplasia were divided into two groups, one of which was treated with 0.1 μM sorafenib (<i>n</i> = 14) and the other with DMSO as a control (<i>n</i> = 13). *<i>P</i> = 0.0267 (log-rank test).</p

Hematoxylin-eosin staining of <i>Tg</i><i>(</i><i>tyr:HRAS^G12V</i><i>)</i>/<i>Tg</i><i>(</i><i>hsp:cre</i><i>)</i> medaka.

<p>(<b>A</b>) Overall image of fish #7 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054424#pone-0054424-g003" target="_blank">Figure 3</a>. (<b>B</b>)– (<b>E</b>), Melanophore layers (arrows) of a wild-type adult fish (<b>B</b>) as well as of fish #7 (<b>C</b>), #6 (<b>D</b>), and #4 (<b>E</b>) in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054424#pone-0054424-g003" target="_blank">Figure 3</a>. (<b>F</b>)– (<b>J</b>), Eye, bone and spinal cord, heart, muscle, and gill, respectively, of fish #7 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054424#pone-0054424-g003" target="_blank">Figure 3</a>. (<b>K</b>) Enlarged image of the boxed region in (<b>J</b>). (<b>L</b>) Kidney and digestive duct of fish #7 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054424#pone-0054424-g003" target="_blank">Figure 3</a>. (<b>M</b>) Enlarged image of the boxed region in (<b>L</b>). Scale bars: 1 mm (<b>A</b>) and 0.1 mm (<b>B</b>–<b>M</b>).</p

Establishment of <em>HRAS</em> Transgenic Medaka as a Stable Tumor Model for <em>In Vivo</em> Screening of Anticancer Drugs

<div><p>Most targeted anticancer drugs have been identified by screening at the molecular or cellular level <em>in vitro</em>. However, many compounds selected by such costly and time-consuming screening do not prove effective against tumors <em>in vivo</em>. The development of anticancer drugs would thus be facilitated by the availability of an <em>in vivo</em> screening system based on a multicellular organism. We have now established a transgenic line of the freshwater fish medaka in which melanophores (melanocytes) proliferate in a manner dependent on heat shock–induced signaling by a human RAS oncoprotein. The human <em>HRAS^G12V</em> oncogene was expressed under the control of a melanophore-specific gene promoter in order to allow visualization of tumor growth in live fish maintained in a water tank. The expression of <em>HRAS^G12V</em> was induced as a result of Cre-mediated recombination by exposure of the fish to a temperature of 37°C for 30 min, given that the Cre gene was placed under the control of a medaka heat shock promoter. One of the stable transgenic lines developed abnormal pigment cell proliferation in the eyes and epidermis with 100% penetrance by 6 months postfertilization. Sorafenib, an inhibitor of RAS signaling, was administered to the transgenic fish and was found both to reduce the extent of melanophore proliferation and to improve survival. The transgenic medaka established here thus represents a promising <em>in vivo</em> system with which to screen potential anticancer drugs that target RAS signaling, and this system can readily be adapted for the screening of agents that target other oncogenes.</p> </div

Characteristics of <i>Tg</i><i>(</i><i>tyr:HRAS^G12V</i><i>)</i>/<i>Tg</i><i>(</i><i>hsp:cre</i><i>)</i> medaka.

<p>Age-dependent changes in eight double-transgenic fish were monitored after heat treatment at 1 wpf. Five of the eight fish manifested hyperproliferation of pigment cells around the eyes by 8 wpf. All of the fish had developed MPLs with characteristics of infiltrative tumors by 20 wpf, and all had died by 43 wpf. Arrows indicate the first appearance of metastasis-like lesions.</p