Evaluation of whole-mount in situ hybridization as a tool for pathway-based toxicological research with early-life stage fathead minnows

J. E. Cavallin; A. L. Schroeder; K. M. Jensen; D. L. Villeneuve; B. R. Blackwell; K. Carlson; M. D. Kahl; Carlie A LaLone; E. C. Randolph; G. T. Ankley

doi:10.1016/j.aquatox.2015.10.002

Evaluation of whole-mount in situ hybridization as a tool for pathway-based toxicological research with early-life stage fathead minnows

J. E. Cavallin, A. L. Schroeder, K. M. Jensen, D. L. Villeneuve, B. R. Blackwell, K. Carlson, M. D. Kahl, Carlie A LaLone, E. C. Randolph, G. T. Ankley

Water Resources Center

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6 Scopus citations

Abstract

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 6.7. nM) 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.

Original language	English (US)
Pages (from-to)	19-26
Number of pages	8
Journal	Aquatic Toxicology
Volume	169
DOIs	https://doi.org/10.1016/j.aquatox.2015.10.002
State	Published - Dec 1 2015

Bibliographical note

Funding Information:
We thank Krysta Nelson (EPA), Joe Korte (EPA), Kevin Lott (Badger Technical Services), and Dalma Martinović-Weigelt (University of St. Thomas) for additional technical support and assistance. We also thank Jon Haselman for reviewing an earlier draft of this manuscript. J. Cavallin was supported in part by the University of Minnesota-U.S. Environmental Protection Agency Cooperative Training Partnership. This article has been reviewed in accordance with official U.S. EPA policy. Mention of products or trade names does not indicate endorsement or recommendation for use. Conclusions drawn in this study neither constitute nor reflect the view or policies of the U.S. EPA. Appendix A

Publisher Copyright:
© 2015 Elsevier B.V.

Keywords

Adverse outcome pathway
Development
Endocrine disruption
Fathead minnow

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Cavallin, J. E., Schroeder, A. L., Jensen, K. M., Villeneuve, D. L., Blackwell, B. R., Carlson, K., Kahl, M. D., LaLone, C. A., Randolph, E. C., & Ankley, G. T. (2015). Evaluation of whole-mount in situ hybridization as a tool for pathway-based toxicological research with early-life stage fathead minnows. Aquatic Toxicology, 169, 19-26. https://doi.org/10.1016/j.aquatox.2015.10.002

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abstract = "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 6.7. nM) 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.",

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AU - Schroeder, A. L.

AU - Jensen, K. M.

AU - Villeneuve, D. L.

AU - Blackwell, B. R.

AU - Carlson, K.

AU - Kahl, M. D.

AU - LaLone, Carlie A

AU - Randolph, E. C.

AU - Ankley, G. T.

N1 - Funding Information: We thank Krysta Nelson (EPA), Joe Korte (EPA), Kevin Lott (Badger Technical Services), and Dalma Martinović-Weigelt (University of St. Thomas) for additional technical support and assistance. We also thank Jon Haselman for reviewing an earlier draft of this manuscript. J. Cavallin was supported in part by the University of Minnesota-U.S. Environmental Protection Agency Cooperative Training Partnership. This article has been reviewed in accordance with official U.S. EPA policy. Mention of products or trade names does not indicate endorsement or recommendation for use. Conclusions drawn in this study neither constitute nor reflect the view or policies of the U.S. EPA. Appendix A Publisher Copyright: © 2015 Elsevier B.V.

PY - 2015/12/1

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N2 - 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 6.7. nM) 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.

AB - 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 6.7. nM) 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.

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ER -

Evaluation of whole-mount in situ hybridization as a tool for pathway-based toxicological research with early-life stage fathead minnows

Abstract

Bibliographical note

Keywords

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