Evaluation of whole-mount in situ hybridization as a tool for pathway-based toxicological research with early-life stage fathead minnows.
Early-life stage fish can be more sensitive to toxicants than adults, so delineating mechanisms of perturbation of biological pathways by chemicals during this life stage is crucial. Whole-mount in situ hybridization (WISH) paired with quantitative real-time polymerase chain reaction (QPCR) assays can enhance pathway-based analyses through determination of specific tissues where changes in gene expression are occurring. While WISH has frequently been used in zebrafish (Danio rerio), this technology has not previously been applied to fathead minnows (Pimephales promelas), another well-established small fish model species. The objective of the present study was to adapt WISH to fathead minnow embryos and larvae, and use the approach to evaluate the effects of estrone, an environmentally-relevant estrogen receptor (ER) agonist. Embryos were exposed via the water to 0, 18 or 1800 ng estrone/L (0, 0.067 and 6.7nM) for 3 or 6 days in a solvent-free, flow-through test system. Relative transcript abundance of three estrogen-responsive genes, estrogen receptor-α (esr1), cytochrome P450-aromatase B (cyp19b), and vitellogenin (vtg) was examined in pooled whole embryos using QPCR, and the spatial distribution of up-regulated gene transcripts was examined in individual fish using WISH. After 3 days of exposure to 1800 ng estrone/L, esr1 and cyp19b were significantly up-regulated, while vtg mRNA expression was not affected. After 6 days of exposure to 1800 ng estrone/L, transcripts for all three genes were significantly up-regulated. Corresponding WISH assays revealed spatial distribution of esr1 and vtg in the liver region, an observation consistent with activation of the hepatic ER. This study clearly demonstrates the potential utility of WISH, in conjunction with QPCR, to examine the mechanistic basis of the effects of toxicants on early-life stage fathead minnows.
University of Minnesota-Duluth, Integrated Biosciences Graduate Program, 1035 University Drive, Duluth, MN 55812, USA. Electronic address: cavallin.jenna@epa.gov.
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Cavallin
University of Minnesota-Water Resources Center, U.S. Environmental Protection Agency, Office of Research and Development, National Health and Environmental Effects Research Laboratory, Mid-Continent Ecology Division, 6201 Congdon Blvd., Duluth, MN 55804, USA.
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Schroeder
U.S. Environmental Protection Agency, Office of Research and Development, National Health and Environmental Effects Research Laboratory, Mid-Continent Ecology Division, 6201 Congdon Blvd., Duluth, MN 55804, USA.
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Jensen
U.S. Environmental Protection Agency, Office of Research and Development, National Health and Environmental Effects Research Laboratory, Mid-Continent Ecology Division, 6201 Congdon Blvd., Duluth, MN 55804, USA.
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Villeneuve
ORISE Research Participation Program, U.S. Environmental Protection Agency, Office of Research and Development, National Health and Environmental Effects Research Laboratory, Mid-Continent Ecology Division, 6201 Congdon Blvd., Duluth, MN 55804, USA.
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Blackwell
University of St. Thomas, Department of Biology, 2115 Summit Ave., St. Paul, MN 55105, USA.
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Carlson
U.S. Environmental Protection Agency, Office of Research and Development, National Health and Environmental Effects Research Laboratory, Mid-Continent Ecology Division, 6201 Congdon Blvd., Duluth, MN 55804, USA.
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Kahl
U.S. Environmental Protection Agency, Office of Research and Development, National Health and Environmental Effects Research Laboratory, Mid-Continent Ecology Division, 6201 Congdon Blvd., Duluth, MN 55804, USA.
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LaLone
U.S. Environmental Protection Agency, Office of Research and Development, National Health and Environmental Effects Research Laboratory, Mid-Continent Ecology Division, 6201 Congdon Blvd., Duluth, MN 55804, USA.
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Randolph
U.S. Environmental Protection Agency, Office of Research and Development, National Health and Environmental Effects Research Laboratory, Mid-Continent Ecology Division, 6201 Congdon Blvd., Duluth, MN 55804, USA.
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Ankley
D000818:Animals; D001141:Aromatase / Q000235:genetics / Q000378:metabolism; D001681:Biological Assay; D003530:Cyprinidae / Q000196:embryology*; D004625:Embryo, Nonmammalian / Q000187:drug effects* / Q000378:metabolism; D004784:Environmental Monitoring / Q000379:methods*; D047628:Estrogen Receptor alpha / Q000235:genetics / Q000378:metabolism; D004967:Estrogens / Q000235:genetics / Q000378:metabolism; D017403:In Situ Hybridization; D007814:Larva; D008099:Liver / Q000187:drug effects / Q000196:embryology / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D015854:Up-Regulation; D014819:Vitellogenins / Q000235:genetics / Q000378:metabolism; D014874:Water Pollutants, Chemical / Q000633:toxicity*
D047628:Estrogen Receptor alpha; D004967:Estrogens; D014819:Vitellogenins; D014874:Water Pollutants, Chemical; D001141:Aromatase
Adverse outcome pathway; Development; Endocrine disruption; Fathead minnow