Expression dynamics of genes in the hypothalamic-pituitary-thyroid (HPT) cascade and their responses to 3,3′,5-triiodo-l-thyronine (T3) highlights potential vulnerability to thyroid-disrupting chemicals in zebrafish (Danio rerio) embryo-larvae

Highlights

• Genes in the HPT axis were dynamically expressed in zebrafish early life stages.

• Exposure to T3 disrupted the thyroid axis of zebrafish at multiple mRNA transcripts.

• The effect of T3 on examined mRNA transcripts was tissue- and life stage-specific.

• Environmental chemicals may disrupt the development of TH-dependent tissues.

Abstract

Some chemicals in the environment disrupt thyroid hormone (TH) systems leading to alterations in organism development, but their effect mechanisms are poorly understood. In fish, this has been limited by a lack of fundamental knowledge on thyroid gene ontogeny and tissue expression in early life stages. Here we established detailed expression profiles for a suite of genes in the hypothalamic-pituitary-thyroid (HPT) axis of zebrafish (Danio rerio) between 24–120 h post fertilisation (hpf) and quantified their responses following exposure to 3,3’,5-triiodo-L-thyronine (T3) using whole mount in situ hybridisation (WISH) and qRT-PCR (using whole-body extracts). All of the selected genes in the HPT axis demonstrated dynamic transcript expression profiles across the developmental stages examined. The expression of thyroid receptor alpha (thraa) was observed in the brain, gastrointestinal tract, craniofacial tissues and pectoral fins, while thyroid receptor beta (thrb) expression occurred in the brain, otic vesicles, liver and lower jaw. The TH deiodinases (dio1, dio2 and dio3b) were expressed in the liver, pronephric ducts and brain and the patterns differed depending on life stage. Both dio1 and dio2 were also expressed in the intestinal bulb (96–120 hpf), and dio2 expression occurred also in the pituitary (48–120 hpf). Exposure of zebrafish embryo-larvae to T3 (30 and 100 μg L⁻¹) for periods of 48, 96 or 120 hpf resulted in the up-regulation of thraa, thrb, dio3b, thyroid follicle synthesis proteins (pax8) and corticotropin-releasing hormone (crhb) and down-regulation of dio1, dio2, glucuronidation enzymes (ugt1ab) and thyroid stimulating hormone (tshb) (assessed via qRT-PCR) and responses differed across life stage and tissues. T3 induced thraa expression in the pineal gland, pectoral fins, brain, somites, gastrointestinal tract, craniofacial tissues, liver and pronephric ducts. T3 enhanced thrb expression in the brain, jaw cartilage and intestine, while thrb expression was suppressed in the liver. T3 exposure suppressed the transcript levels of dio1 and dio2 in the liver, brain, gastrointestinal tract and craniofacial tissues, while dio2 signalling was also suppressed in the pituitary gland. Dio3b expression was induced by T3 exposure in the jaw cartilage, pectoral fins and brain. The involvement of THs in the development of numerous body tissues and the responsiveness of these tissues to T3 in zebrafish highlights their potential vulnerability to exposure to environmental thyroid-disrupting chemicals.

Introduction

The ability of xenobiotic compounds to alter endocrine function has been reported widely over the last two decades, with attention largely focused on chemicals that disrupt the reproductive system of humans and wildlife (Tyler et al., 1998). Growing awareness of the role of thyroid hormones (TH) during development has led to increasing concern over environmental contaminants which act as thyroid-disrupting chemicals (TDCs), such as polychlorinated biphenyls (PCB), dichlorodiphenyltrichloroethane (DDT), hexachlorobenzene (HCB), perchlorates, phthalates and brominated flame retardants (BFR) (Boas et al., 2006). These TDCs act via a wide variety of mechanisms to disrupt TH homeostasis in vertebrates. For example, some compounds can alter the function of the thyroid gland itself, by inhibiting the uptake of iodide and/or inhibiting the activity of thyroid peroxidase and subsequently decreasing TH synthesis. Other compounds act by altering the biliary elimination of TH via the induction of the metabolising enzymes, altering the activity of blood and cellular TH transporters, interfering with hepatic, serum and target tissue deiodinase activity and/or altering TH-responsive genomic signalling in target tissue (reviewed in (Crofton, 2008)). Aquatic and semi-aquatic species, including fish and amphibians, are especially vulnerable to TDCs, with uptake occuring via the skin and gills, via the diet and they can even be transferred to the offspring of exposed adults (Brown et al., 2002; Kim et al., 2011; Wu et al., 2009; Yu et al., 2011).

TH dynamics are primarily under the control of the hypothalamic-pituitary-thyroid (HPT) axis, a complex regulatory network which coordinates TH synthesis, secretion, transport and metabolism (Zoeller et al., 2007). L-3,5,3',5'-Tetraiodothyronine (Thyroxine; T4) is the main TH secreted by the thyroid follicles of teleost fish, but 3,3′,5-triiodo-L-thyronine (T3) is the biologically active form that is under the control of peripheral tissues (Power et al., 2001). The genomic actions of THs depend on the binding of T3 with nuclear thyroid hormone receptors (TRs) and the subsequent interaction with specific thyroid response elements (TREs) in the promoters of target genes, which either enhance or repress their transcription (Power et al., 2001). The iodothyronine deiodinase enzymes type I, II and III (D1, D2 and D3) regulate the activity of TH by removing iodine moieties from T3 or T4. D2 generates T3 via deiodination of T4, while D3 produces the inactive metabolites, 3,5',3'-triiodothyronine (rT3) and 3,3’-diiodothyronine (3,3′-T2) by deiodination of T4 and T3, respectively. D1 is kinetically inefficient and can deiodinate both the inner and outer rings of T4, and therefore has activating and inactivating abilities (Power et al., 2001).

THs are involved in a variety of physiological and developmental processes in vertebrates. In teleost fish, developmental roles include mediating the metamorphic transition from larval to adult stages and influencing the maturation of tissues including bone, gonads, intestine and the central nervous system (Campinho et al., 2014; Matta et al., 2002; Power et al., 2001). In adults, they modulate growth, energy homeostasis, cardiac rhythm, the smolting process, osmoregulation and the behaviours/physiology associated with rheotaxis and migration (Boeuf et al., 1989; Eric et al., 2004; Godin et al., 1974). Consequently, even minor alterations in TH levels, particularly during sensitive developmental windows, can have significant acute and potentially long-term health effects. In recent years, the expression profiles of several genes in the HPT axis of teleost fish have been used as indicators for thyroid disruption by different environmental pollutants (Parsons et al., 2019; Shi et al., 2009). However, the spatial and temporal expression of many of these genes during zebrafish early life stages and their regulation by THs have not been thoroughly evaluated. A greater understanding of the transcriptional dynamics of TH-related genes in teleost fish would greatly facilitate the identification of target genes, tissues and developmental stages which may be particularly sensitive to TDCS.

In this study, we studied the ontogeny of expression of several genes in the HPT axis of zebrafish (Danio rerio), and both identified their tissue localisations and measured their regulatory responses to THs. We first characterised the expression dynamics of genes of the HPT axis from 24 to 120 h post fertilisation (hpf), and subsequently examined the responses of these genes to T3 at key developmental stages. The overall objective of this work was to establish whether T3 differentially regulated the ontogenic expression of target genes and by doing so identify some of the potential mechanisms and thyroid targets related to TDCs. We used a combination of both whole-mount in situ hybridisation (WISH) assays to identify tissue-specific gene expression patterns in whole zebrafish embryo-larvae, and quantitative reverse transcription polymerase chain reaction (qRT-PCR) assays to quantify changes in gene transcript levels in whole body extracts.