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Integrating Morphological and Behavioral Phenotypes in Developing Zebrafish

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The rights and wrongs of zebrafish: Behavioral phenotyping of zebrafish

Abstract

The zebrafish, with its prolific reproduction, rapid development, and genetic homology to humans, provides an ideal model to efficiently characterize behavioral, developmental, and morphological phenotypes in biomedical research. By designing experiments to take advantage of these properties, behavioral phenotypes can be interpreted within the context of relevant neuromorphological phenotypes in order to present a truly integrative analysis of the rich biological data. These experiments may take the form of targeted studies or scaled up to a discovery mode that can keep pace with in vitro high-throughput screening (HTS) experimental systems. Achieving the goal of data integration will require appropriate application and adaptation of traditional statistical approaches, as well as the development of novel methods implemented in concert with new experimental approaches. In this chapter, we survey experimental designs and statistical methods for behavioral studies in developing zebrafish, then highlight experimental factors and analysis strategies that facilitate integration of morphological and behavioral phenotypes. We conclude that appropriate design and analysis of integrated morphological and behavioral studies using zebrafish can elucidate new chemical bioactivity pathways, identify compounds eliciting a broad range of effects, and paint a more comprehensive picture of development than either class of phenotype alone.

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References

  1. Wambaugh JF, Setzer RW, Reif DM, Gangwal S, Mitchell-Blackwood J, Arnot JA, Joliet O, Frame A, Rabinowitz J, Knudsen TB, Judson RS, Egeghy P, Vallero D, Cohen Hubal EA. High-throughput models for exposure-based chemical prioritization in the ExpoCast project. Environ Sci Technol. 2013;47(15):8479–88.

    CAS  PubMed  Google Scholar 

  2. Collins FS, Gray GM, Bucher JR. Toxicology: transforming environmental health protection. Science. 2008;319(5865):906–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Judson RS, Houck KA, Kavlock RJ, Knudsen TB, Martin MT, Mortensen HM, Reif DM, Rotroff DM, Shah I, Richard AM, Dix DJ. In vitro screening of environmental chemicals for targeted testing prioritization: the ToxCast project. Environ Health Perspect. 2010;118(4):485–92.

    Article  CAS  PubMed  Google Scholar 

  4. Burns CJ, McIntosh LJ, Mink PJ, Jurek AM, Li AA. Pesticide exposure and neurodevelopmental outcomes: review of the epidemiologic and animal studies. J Toxicol Environ Health B Crit Rev. 2013;16(3–4):127–283.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Selderslaghs IW, Hooyberghs J, Blust R, Witters HE. Assessment of the developmental neurotoxicity of compounds by measuring locomotor activity in zebrafish embryos and larvae. Neurotoxicol Teratol. 2013;37:44–56.

    Article  CAS  PubMed  Google Scholar 

  6. Cao Y, Semanchik N, Lee SH, Somlo S, Barbano PE, Coifman R, Sun Z. Chemical modifier screen identifies HDAC inhibitors as suppressors of PKD models. Proc Natl Acad Sci U S A. 2009;106(51):21819–24.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Makris N, Biederman J, Monuteaux MC, Seidman LJ. Towards conceptualizing a neural systems-based anatomy of attention-deficit/hyperactivity disorder. Dev Neurosci. 2009;31(1–2):36–49.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Padilla S, Corum D, Padnos B, Hunter DL, Beam A, Houck KA, Sipes N, Kleinstreuer N, Knudsen T, Dix DJ, Reif DM. Zebrafish developmental screening of the ToxCast Phase I chemical library. Reprod Toxicol. 2012;33(2):174–87.

    Article  CAS  PubMed  Google Scholar 

  9. Sanes DH, Reh TA, Harris WA, editors. Development of the nervous system. New York: Elsevier Science; 2005.

    Google Scholar 

  10. Truong L, Reif DM, St Mary L, Geier MC, Truong HD, Tanguay RL. Multidimensional in vivo hazard assessment using zebrafish. Toxicol Sci. 2014;137(1):212–33.

    Article  CAS  PubMed  Google Scholar 

  11. Barbazuk WB, Korf I, Kadavi C, Heyen J, Tate S, Wun E, Bedell JA, McPherson JD, Johnson SL. The syntenic relationship of the zebrafish and human genomes. Genome Res. 2000;10(9):1351–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Howe K, Clark MD, Torroja CF, Torrance J, Berthelot C, Muffato M, Collins JE, Humphray S, McLaren K, Matthews L, McLaren S, Sealy I, Caccamo M, Churcher C, Scott C, Barrett JC, Koch R, Rauch GJ, White S, Chow W, Kilian B, Quintais LT, Guerra-Assuncao JA, Zhou Y, Gu Y, Yen J, Vogel JH, Eyre T, Redmond S, Banerjee R, Chi J, Fu B, Langley E, Maguire SF, Laird GK, Lloyd D, Kenyon E, Donaldson S, Sehra H, Almeida-King J, Loveland J, Trevanion S, Jones M, Quail M, Willey D, Hunt A, Burton J, Sims S, McLay K, Plumb B, Davis J, Clee C, Oliver K, Clark R, Riddle C, Eliott D, Threadgold G, Harden G, Ware D, Mortimer B, Kerry G, Heath P, Phillimore B, Tracey A, Corby N, Dunn M, Johnson C, Wood J, Clark S, Pelan S, Griffiths G, Smith M, Glithero R, Howden P, Barker N, Stevens C, Harley J, Holt K, Panagiotidis G, Lovell J, Beasley H, Henderson C, Gordon D, Auger K, Wright D, Collins J, Raisen C, Dyer L, Leung K, Robertson L, Ambridge K, Leongamornlert D, McGuire S, Gilderthorp R, Griffiths C, Manthravadi D, Nichol S, Barker G, Whitehead S, Kay M, Brown J, Murnane C, Gray E, Humphries M, Sycamore N, Barker D, Saunders D, Wallis J, Babbage A, Hammond S, Mashreghi-Mohammadi M, Barr L, Martin S, Wray P, Ellington A, Matthews N, Ellwood M, Woodmansey R, Clark G, Cooper J, Tromans A, Grafham D, Skuce C, Pandian R, Andrews R, Harrison E, Kimberley A, Garnett J, Fosker N, Hall R, Garner P, Kelly D, Bird C, Palmer S, Gehring I, Berger A, Dooley CM, Ersan-Urun Z, Eser C, Geiger H, Geisler M, Karotki L, Kirn A, Konantz J, Konantz M, Oberlander M, Rudolph-Geiger S, Teucke M, Osoegawa K, Zhu B, Rapp A, Widaa S, Langford C, Yang F, Carter NP, Harrow J, Ning Z, Herrero J, Searle SM, Enright A, Geisler R, Plasterk RH, Lee C, Westerfield M, de Jong PJ, Zon LI, Postlethwait JH, Nusslein-Volhard C, Hubbard TJ, Roest Crollius H, Rogers J, Stemple DL. The zebrafish reference genome sequence and its relationship to the human genome. Nature. 2013;496(7446):498–503.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Tropepe V, Sive HL. Can zebrafish be used as a model to study the neurodevelopmental causes of autism? Genes Brain Behav. 2003;2(5):268–81.

    Article  CAS  PubMed  Google Scholar 

  14. Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF. Stages of embryonic development of the zebrafish. Dev Dyn. 1995;203(3):253–310.

    Article  CAS  PubMed  Google Scholar 

  15. Kalueff AV, Gebhardt M, Stewart AM, Cachat JM, Brimmer M, Chawla JS, Craddock C, Kyzar EJ, Roth A, Landsman S, Gaikwad S, Robinson K, Baatrup E, Tierney K, Shamchuk A, Norton W, Miller N, Nicolson T, Braubach O, Gilman CP, Pittman J, Rosemberg DB, Gerlai R, Echevarria D, Lamb E, Neuhauss SC, Weng W, Bally-Cuif L, Schneider H, C. Zebrafish Neuroscience Research. Towards a comprehensive catalog of zebrafish behavior 1.0 and beyond. Zebrafish. 2013;10(1):70–86.

    Article  PubMed  PubMed Central  Google Scholar 

  16. Kokel D, Bryan J, Laggner C, White R, Cheung CY, Mateus R, Healey D, Kim S, Werdich AA, Haggarty SJ, Macrae CA, Shoichet B, Peterson RT. Rapid behavior-based identification of neuroactive small molecules in the zebrafish. Nat Chem Biol. 2010;6(3):231–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Noyes PD, Haggard DE, Gonnerman GD, Tanguay RL. Advanced morphological—behavioral test platform reveals neurodevelopmental defects in embryonic zebrafish exposed to comprehensive suite of halogenated and organophosphate flame retardants. Toxicol Sci. 2015;145(1):177–95.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Kokel D, Dunn TW, Ahrens MB, Alshut R, Cheung CY, Saint-Amant L, Bruni G, Mateus R, van Ham TJ, Shiraki T, Fukada Y, Kojima D, Yeh JR, Mikut R, von Lintig J, Engert F, Peterson RT. Identification of nonvisual photomotor response cells in the vertebrate hindbrain. J Neurosci. 2013;33(9):3834–43.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Raftery TD, Isales GM, Yozzo KL, Volz DC. High-content screening assay for identification of chemicals impacting spontaneous activity in zebrafish embryos. Environ Sci Technol. 2014;48(1):804–10.

    Article  CAS  PubMed  Google Scholar 

  20. Reif DM, Truong L, Mandrell D, Marvel S, Zhang G, Tanguay RL. High-throughput characterization of chemical-associated embryonic behavioral changes predicts teratogenic outcomes. Arch Toxicol. 2016;90(6):1459–70.

    Google Scholar 

  21. Lockwood B, Bjerke S, Kobayashi K, Guo S. Acute effects of alcohol on larval zebrafish: a genetic system for large-scale screening. Pharmacol Biochem Behav. 2004;77(3):647–54.

    Article  CAS  PubMed  Google Scholar 

  22. Baraban SC, Taylor MR, Castro PA, Baier H. Pentylenetetrazole induced changes in zebrafish behavior, neural activity and c-fos expression. Neuroscience. 2005;131(3):759–68.

    Article  CAS  PubMed  Google Scholar 

  23. MacPhail RC, Brooks J, Hunter DL, Padnos B, Irons TD, Padilla S. Locomotion in larval zebrafish: influence of time of day, lighting and ethanol. Neurotoxicology. 2009;30(1):52–8.

    Article  CAS  PubMed  Google Scholar 

  24. Mandrell D, Truong L, Jephson C, Sarker MR, Moore A, Lang C, Simonich MT, Tanguay RL. Automated zebrafish chorion removal and single embryo placement: optimizing throughput of zebrafish developmental toxicity screens. J Lab Autom. 2012;17(1):66–74.

    Article  PubMed  PubMed Central  Google Scholar 

  25. Westerfield M. The zebrafish book: a guide for the laboratory use of zebrafish (Danio rerio). Eugene: University of Oregon Press; 2007.

    Google Scholar 

  26. Truong L, Zaikova T, Richman EK, Hutchison JE, Tanguay RL. Media ionic strength impacts embryonic responses to engineered nanoparticle exposure. Nanotoxicology. 2012;6(7):691–9.

    Article  CAS  PubMed  Google Scholar 

  27. Jarema KA, Hunter DL, Shaffer RM, Behl M, Padilla S. Acute and developmental behavioral effects of flame retardants and related chemicals in zebrafish. Neurotoxicol Teratol. 2015;52(Pt B):194–209.

    Google Scholar 

  28. Legradi J, El Abdellaoui N, van Pomeren M, Legler J. Comparability of behavioural assays using zebrafish larvae to assess neurotoxicity. Environ Sci Pollut Res Int. 2015;22(21):16277–89.

    Article  CAS  PubMed  Google Scholar 

  29. Parker MO, Ife D, Ma J, Pancholi M, Smeraldi F, Straw C, Brennan CH. Development and automation of a test of impulse control in zebrafish. Front Syst Neurosci. 2013;7:65.

    Article  PubMed  PubMed Central  Google Scholar 

  30. Rihel J, Prober DA, Arvanites A, Lam K, Zimmerman S, Jang S, Haggarty SJ, Kokel D, Rubin LL, Peterson RT, Schier AF. Zebrafish behavioral profiling links drugs to biological targets and rest/wake regulation. Science. 2010;327(5963):348–51.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Selderslaghs IW, Hooyberghs J, De Coen W, Witters HE. Locomotor activity in zebrafish embryos: a new method to assess developmental neurotoxicity. Neurotoxicol Teratol. 2010;32(4):460–71.

    Article  CAS  PubMed  Google Scholar 

  32. Box GEP, Hunter JS, Hunter WG, editors. Statistics for experimenters: design, innovation, and discovery. Hoboken, NJ: Wiley-Interscience; 2005.

    Google Scholar 

  33. Noble WS. How does multiple testing correction work? Nat Biotechnol. 2009;27(12):1135–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Armstrong RA. When to use the Bonferroni correction. Ophthalmic Physiol Opt. 2014;34(5):502–8.

    Article  PubMed  Google Scholar 

  35. Schulz KF, Grimes DA. Multiplicity in randomised trials I: endpoints and treatments. Lancet. 2005;365(9470):1591–5.

    Article  PubMed  Google Scholar 

  36. Benjamini Y, Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc Ser B Methodol. 1995;57(1):289–300.

    Google Scholar 

  37. Perneger TV. What’s wrong with Bonferroni adjustments. BMJ. 1998;316(7139):1236–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Rothman KJ. No adjustments are needed for multiple comparisons. Epidemiology. 1990;1(1):43–6.

    Article  CAS  PubMed  Google Scholar 

  39. Nakagawa S. A farewell to Bonferroni: the problems of low statistical power and publication bias. Behav Ecol. 2004;15(6):1044–5.

    Article  Google Scholar 

  40. Fox J, editor. Applied regression analysis and generalized linear models. London: Sage; 2008.

    Google Scholar 

  41. Higgins JJ, editor. Introduction to modern nonparametric statistics. Belmont, CA: Duxbury Press; 2003.

    Google Scholar 

  42. Krishnamoorthy K, Lu F, Mathew T. A parametric bootstrap approach for ANOVA with unequal variances: fixed and random models. Comput Stat Data Anal. 2007;51(12):5731–42.

    Article  Google Scholar 

  43. Liu Y, Carmer R, Zhang G, Venkatraman P, Brown SA, Pang CP, Zhang M, Ma P, Leung YF. Statistical analysis of zebrafish locomotor response. PLoS One. 2015;10(10), e0139521.

    Article  PubMed  PubMed Central  Google Scholar 

  44. Ducharme NA, Peterson LE, Benfenati E, Reif D, McCollum CW, Gustafsson JA, Bondesson M. Meta-analysis of toxicity and teratogenicity of 133 chemicals from zebrafish developmental toxicity studies. Reprod Toxicol. 2013;41:98–108.

    Article  CAS  PubMed  Google Scholar 

  45. Pandey UB, Nichols CD. Human disease models in Drosophila melanogaster and the role of the fly in therapeutic drug discovery. Pharmacol Rev. 2011;63(2):411–36.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Sipes NS, Martin MT, Kothiya P, Reif DM, Judson RS, Richard AM, Houck KA, Dix DJ, Kavlock RJ, Knudsen TB. Profiling 976 ToxCast chemicals across 331 enzymatic and receptor signaling assays. Chem Res Toxicol. 2013;26(6):878–95.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Beker van Woudenberg A, Snel C, Rijkmans E, de Groot D, Bouma M, Hermsen S, Piersma A, Menke A, Wolterbeek A. Zebrafish embryotoxicity test for developmental (neuro)toxicity: demo case of an integrated screening approach system using anti-epileptic drugs. Reprod Toxicol. 2014;49:101–16.

    Article  CAS  PubMed  Google Scholar 

  48. Lumaret JP, Errouissi F, Floate K, Rombke J, Wardhaugh K. A review on the toxicity and non-target effects of macrocyclic lactones in terrestrial and aquatic environments. Curr Pharm Biotechnol. 2012;13(6):1004–60.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Gerlai R, Lahav M, Guo S, Rosenthal A. Drinks like a fish: zebra fish (Danio rerio) as a behavior genetic model to study alcohol effects. Pharmacol Biochem Behav. 2000;67(4):773–82.

    Article  CAS  PubMed  Google Scholar 

  50. Kalueff AV, Stewart AM, Gerlai R. Zebrafish as an emerging model for studying complex brain disorders. Trends Pharmacol Sci. 2014;35(2):63–75.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. MacRae C, Peterson R. Zebrafish-based small molecule discovery. Chem Biol. 2003;10:901–8.

    Article  CAS  PubMed  Google Scholar 

  52. Vinken M. The adverse outcome pathway concept: a pragmatic tool in toxicology. Toxicology. 2013;312:158–65.

    Article  CAS  PubMed  Google Scholar 

  53. Kleinstreuer NC, Yang J, Berg EL, Knudsen TB, Richard AM, Martin MT, Reif DM, Judson RS, Polokoff M, Dix DJ, Kavlock RJ, Houck KA. Phenotypic screening of the ToxCast chemical library to classify toxic and therapeutic mechanisms. Nat Biotechnol. 2014;32(6):583–91.

    Article  CAS  PubMed  Google Scholar 

  54. Meisner M, Reif DM. Computational methods used in systems biology. In: Fry RC, editor. Systems biology in toxicology and environmental health. 1st ed. Amsterdam: Academic; 2015. p. 85–115.

    Chapter  Google Scholar 

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Authors and Affiliations

  1. Department of Biological Sciences, Bioinformatics Research Center, North Carolina State University, Raleigh, NC, USA

    Guozhu Zhang & David M. Reif

  2. Department of Environmental and Molecular Toxicology, The Sinnhuber Aquatic Research Laboratory and the Environmental Health Sciences Center at Oregon State University, Corvallis, OR, USA

    Lisa Truong & Robert L. Tanguay

Authors
  1. Guozhu Zhang
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  2. Lisa Truong
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  3. Robert L. Tanguay
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  4. David M. Reif
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Corresponding author

Correspondence to David M. Reif .

Editor information

Editors and Affiliations

  1. The International Zebrafish Neurosceince Research Consortium (ZNRC) and ZENEREI Research Center, Slidell, Louisiana, USA

    Allan V. Kalueff

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Zhang, G., Truong, L., Tanguay, R.L., Reif, D.M. (2017). Integrating Morphological and Behavioral Phenotypes in Developing Zebrafish. In: Kalueff, A. (eds) The rights and wrongs of zebrafish: Behavioral phenotyping of zebrafish. Springer, Cham. https://doi.org/10.1007/978-3-319-33774-6_12

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