Enabling comparative gene expression studies of thyroid hormone action through the development of a flexible real-time quantitative PCR assay for use across multiple anuran indicator and sentinel species

Nik Veldhoen, Catherine R Propper, Caren C. Helbing

Research output: Contribution to journalArticle

20 Citations (Scopus)

Abstract

Studies performed across diverse frog species have made substantial contributions to our understanding of basic vertebrate development and the natural or anthropogenic environmental factors impacting sensitive life stages. Because, anurans are developmental models, provide ecosystems services, and act as sentinels for the identification of environmental chemical contaminants that interfere with thyroid hormone (TH) action during postembryonic development, there is demand for flexible assessment techniques that can be applied to multiple species. As part of the "thyroid assays across indicator and sentinel species" (TAXISS) initiative, we have designed and validated a series of cross-species real time quantitative PCR (qPCR) primer sets that provide information on transcriptome components in evolutionarily distant anurans. Validation for fifteen gene transcripts involved a rigorous three-tiered quality control within tissue/development-specific contexts. Assay performance was confirmed on multiple tissues (tail fin, liver, brain, and intestine) of Rana catesbeiana and Xenopus laevis tadpoles enabling comparisons between tissues and generation of response profiles to exogenous TH. This revealed notable differences in TH-responsive gene transcripts including thra, thrb, thibz, klf9, col1a2, fn1, plp1, mmp2, timm50, otc, and dio2, suggesting differential regulation and susceptibility to contaminant effects. Evidence for the applicability of the TAXISS anuran qPCR assay across seven other species is also provided with five frog families represented and its utility in defining genome structure was demonstrated. This novel validated approach will enable meaningful comparative studies between frog species and aid in extending knowledge of developmental regulatory pathways and the impact of environmental factors on TH signaling in frog species for which little or no genetic information is currently available.

Original languageEnglish (US)
Pages (from-to)162-173
Number of pages12
JournalAquatic Toxicology
Volume148
DOIs
StatePublished - Mar 2014

Fingerprint

multiple use
thyroid hormones
Thyroid Hormones
Anura
gene expression
hormone
frogs
Real-Time Polymerase Chain Reaction
quantitative polymerase chain reaction
assay
Gene Expression
frog
assays
Rana catesbeiana
Lithobates catesbeianus
environmental factors
Xenopus laevis
tadpoles
Transcriptome
transcriptome

Keywords

  • Anuran
  • Frog tadpole
  • Metamorphosis
  • Multispecies assay
  • Normalizer
  • Quantitative PCR
  • Thyroid hormone

ASJC Scopus subject areas

  • Aquatic Science
  • Health, Toxicology and Mutagenesis

Cite this

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title = "Enabling comparative gene expression studies of thyroid hormone action through the development of a flexible real-time quantitative PCR assay for use across multiple anuran indicator and sentinel species",
abstract = "Studies performed across diverse frog species have made substantial contributions to our understanding of basic vertebrate development and the natural or anthropogenic environmental factors impacting sensitive life stages. Because, anurans are developmental models, provide ecosystems services, and act as sentinels for the identification of environmental chemical contaminants that interfere with thyroid hormone (TH) action during postembryonic development, there is demand for flexible assessment techniques that can be applied to multiple species. As part of the {"}thyroid assays across indicator and sentinel species{"} (TAXISS) initiative, we have designed and validated a series of cross-species real time quantitative PCR (qPCR) primer sets that provide information on transcriptome components in evolutionarily distant anurans. Validation for fifteen gene transcripts involved a rigorous three-tiered quality control within tissue/development-specific contexts. Assay performance was confirmed on multiple tissues (tail fin, liver, brain, and intestine) of Rana catesbeiana and Xenopus laevis tadpoles enabling comparisons between tissues and generation of response profiles to exogenous TH. This revealed notable differences in TH-responsive gene transcripts including thra, thrb, thibz, klf9, col1a2, fn1, plp1, mmp2, timm50, otc, and dio2, suggesting differential regulation and susceptibility to contaminant effects. Evidence for the applicability of the TAXISS anuran qPCR assay across seven other species is also provided with five frog families represented and its utility in defining genome structure was demonstrated. This novel validated approach will enable meaningful comparative studies between frog species and aid in extending knowledge of developmental regulatory pathways and the impact of environmental factors on TH signaling in frog species for which little or no genetic information is currently available.",
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