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Evaluation of whole mount in situ hybridization as a tool for pathway-based toxicological research in early-life stage fathead minnows
Cavallin, J., A. Schroeder, B. Blackwell, K. Carlson, K. Jensen, M. Kahl, E. Randolph, Dan Villeneuve, AND G. Ankley. Evaluation of whole mount in situ hybridization as a tool for pathway-based toxicological research in early-life stage fathead minnows. SETAC North America, Salt Lake City, UT, November 01 - 05, 2015.
Early-life stage fish can be more sensitive to chemical exposure than mature, adult fish. Therefore, defining adverse outcome pathways (AOPs) relevant to early-life stages is critical for linking perturbations of key events during fish development to potential adverse outcomes of chemical exposure. To determine chemical effects and/or mechanisms of action in exposed fish embryos and larvae, whole mount in situ hybridization (WISH) paired with quantitative polymerase chain reaction (QPCR) assays hold excellent promise. While WISH has frequently been used in zebrafish (Danio rerio) early-life stage developmental work, this technology has not previously been applied to fathead minnows (Pimephales promelas), another well-established laboratory small fish model. In the present study, WISH was implemented in fathead minnow embryos and larvae as a tool to aid in the development of AOPs associated with early-life stages. As a proof of concept, fathead minnow embryos were exposed to the known estrogen receptor agonist, estrone (0, 18, and 1800 ng/L), for 3 and 6 days in a solvent-free, flow-through test system. Relative transcript abundance of three estrogen-responsive genes, estrogen receptor- (esr1), vitellogenin (vtg), and cytochrome P450-aromatase B (cyp19b) was examined in pooled whole embryos using QPCR, and spatial distribution of significantly up-regulated gene transcripts was further examined using WISH in individual fish. After 3 d of exposure to estrone, relative transcript abundance of esr1 and cyp19b was significantly up-regulated, while vtg mRNA expression was not significantly affected. Transcripts for all three genes were significantly up-regulated after the 6 d exposure to 1800 ng estrone/L. Subsequently, WISH assays revealed spatial distribution of esr1 and vtg in the liver region, suggesting that estrone is activating estrogen receptors in the liver of exposed embryos after 6 d of exposure. Using WISH (as a complement to QPCR) to determine which specific tissues are targeted during chemical exposure has potential to lend insight relative to those biological pathways perturbed by chemicals of interest. Consequently, WISH may be particularly useful for further investigation of AOP development in fathead minnows, notably identification of tissue-specific alterations in key molecular initiating events (e.g., ER activation in the liver in this study) that may lead to subsequent effects on early-life stage development.