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Evaluation of whole-mount in situ hybridization as a tool for pathway-based toxicological research with early-life stage fathead minnows
Citation:
Cavallin, J., A. Schroeder, K. Jensen, Dan Villeneuve, B. Blackwell, K. Carlson, M. Kahl, C. LaLone, E. Randolph, AND G. Ankley. Evaluation of whole-mount in situ hybridization as a tool for pathway-based toxicological research with early-life stage fathead minnows. AQUATIC TOXICOLOGY. Elsevier Science Ltd, New York, NY, 169:19-26, (2015).
Impact/Purpose:
To determine chemical effects and support adverse outcome pathway (AOP) development in 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 work, this technology has not previously been applied to fathead minnows (Pimephales promelas), another well-established small fish model. In the present study, WISH was implemented in fathead minnow embryos and larvae in studies with estrone, an environmentally-relevant estrogen receptor (ER) agonist. Estrone increased relative transcript abundance (measured using QPCR) of three estrogen-responsive genes, estrogen receptor-a (esr1), vitellogenin (vtg), and cytochrome P450-aromatase B (cyp19b) in pooled whole embryos. Examination of spatial distribution of up-regulated gene transcripts examined using WISH in individual fish revealed spatial upregulation of esr1 and vtg in the liver region, an observation consistent with activation of the hepatic ER. Using WISH as a complement to QPCR to determine specific tissues targeted by chemicals in fish early-life stages provides important insights relative to biological pathways perturbed by chemicals of interest, thereby supporting the development of AOPs.
Description:
Early-life stage fish can be more sensitive to chemical exposure than adult fish. Therefore, determining possible adverse outcome pathways (AOPs) for early-life stages is crucial. 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 work, this technology has not previously been applied to fathead minnows (Pimephales promelas), another well-established small fish model. In the present study, WISH was implemented in fathead minnow embryos and larvae in studies with estrone, an environmentally-relevant estrogen receptor (ER) agonist. Embryos were exposed to estrone (0, 18, and 1,800 ng/L) for 3 or 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 up-regulated gene transcripts was examined using WISH in individual fish. After 3 d of exposure to estrone esr1 and cyp19b were significantly up-regulated, while vtg mRNA expression was not affected. Transcripts for all three genes were significantly up-regulated after the 6 d exposure to 1,800 ng estrone/L. Corresponding WISH assays revealed spatial distribution of esr1 and vtg in the liver region, an observation consistent with activation of the hepatic ER. Using WISH as a complement to QPCR to determine which specific tissues targeted by chemicals in fish early-life stages provides important insights relative to biological pathways perturbed by chemicals of interest, thereby supporting the development of AOPs.
