Knockdown of the thyroid hormone transporter MCT8 in chicken retinal precursor cells hampers early retinal development and results in a shift towards more UV/blue cones at the expense of green/red cones.

Thyroid hormones (THs) play a crucial role in coordinating brain development in vertebrates. They fine-tune processes like cell proliferation, migration, and differentiation mainly by regulating the transcriptional activity of many essential genes. Regulators of TH availability thereby define the cellular concentration of the bioactive 3,5,3'-triiodothyronine, which binds to nuclear TH receptors. One important regulator, the monocarboxylate transporter 8 (MCT8), facilitates cellular TH uptake and is known to be necessary for correct brain development, but data on its potential role during retinal development is lacking. The retinal cyto-architecture has been conserved throughout vertebrate evolution, and we used the chicken embryo to study the need for MCT8 during retinal development. Its external development allows easy manipulation, and MCT8 is abundantly expressed in the retina from early stages onwards. We induced MCT8 knockdown by electroporating a pRFP-MCT8-RNAi vector into the retinal precursor cells (RPCs) at embryonic day 4 (E4), and studied the consequences for early (E6) and late (E18) retinal development. The empty pRFP-RNAi vector was used as a control. RPC proliferation was reduced at E6. This resulted in cellular hypoplasia and a thinner retina at E18 where mainly photoreceptors and horizontal cells were lost, the two predominant cell types that are born around the stage of electroporation. At E6, differentiation into retinal ganglion cells and amacrine cells was delayed. However, since the proportion of a given cell type within the transfected cell population at E18 was similar in knockdown and controls, the partial loss of some cell types was most-likely due to reduced RPC proliferation and not impaired cell differentiation. Photoreceptors displayed delayed migration at first, but had successfully reached the outer nuclear layer at E18. However, they increasingly differentiated into short wavelength-sensitive cones at the expense of medium/long wavelength-sensitive cones, while the proportion of rods was unaltered. Improperly formed sublaminae in the inner plexiform layer additionally suggested defects in synaptogenesis. Altogether, our data echoes effects of hypothyroidism and the loss of some other regulators of TH availability in the developing zebrafish and rodent retina. Therefore, the expression of MCT8 in RPCs is crucial for adequate TH uptake during cell type-specific events in retinal development.

Insights from zebrafish deficiency models to understand the impact of local thyroid hormone regulator action on early development.

Thyroid hormones (THs) stimulate and coordinate a wide range of processes to ensure normal development, mainly by binding of the most active TH 3,5,3'-triiodothyronine (T3) to nuclear receptors resulting in changes in gene transcription. Local TH action is monitored at three distinct levels by different types of regulators: transmembrane transporters (TH influx and efflux), deiodinases (TH activation and inactivation) and nuclear receptors (TH signalling). Since TH regulators are strongly conserved among vertebrate species, the externally and rapidly developing zebrafish (Danio rerio) has become one of the favourite models to study their role in TH-dependent development. Most regulators are expressed in zebrafish from early stages in development in a dynamic and tissue-specific pattern. Transient or permanent disruption of a given regulator severely perturbs development of multiple organs. These zebrafish deficiency models help to explain why, next to overall hypo-/hyperthyroidism, inactivating mutations in the genes encoding TH regulators such as MCT8 and THRA/B have irreversible adverse effects on human development. Zebrafish are also increasingly used as a high-throughput model to assess the toxicity of various xenobiotics and their impact on development. While adverse effects on TH metabolism and gene expression have been shown, information on direct interaction with TH regulators is scarce, albeit essential to fully understand their mechanism of action. For the future, the combination of novel gene silencing tools, fluorescent reporter lines and (single-cell) transcriptomics holds promise for new zebrafish models to further elucidate the role of each TH regulator in vertebrate development.

Intracellular thyroid hormone metabolism as a local regulator of nuclear thyroid hormone receptor-mediated impact on vertebrate development.

BACKGROUND: Thyroid hormones (THs) play an essential role in vertebrate development, acting predominantly via nuclear TH receptors (TRs) which are ligand-dependent transcription factors. Binding of the ligand (predominantly T3) induces a switch from gene activation to gene repression or vice versa. Iodothyronine deiodinases (Ds) and TH transporters are important regulators of intracellular T3 availability and therefore contribute to the control of TR-dependent development. FOCUS: The present review discusses the possible roles of Ds and TH transporters in regulating embryonic and larval (pre-juvenile) TR-dependent development in vertebrates. It focuses mainly on well-known model species for direct and indirect vertebrate development, including zebrafish, Xenopus, chicken and mouse. Data are provided on stage- and tissue/cell-specific changes in expression of Ds and TH transporters. This information is combined with functional data obtained from gain-and-loss of function studies. CONCLUSION: Knockout/knockdown of each type of D has provided strong evidence for their implication in the control of important developmental processes and several D expression patterns and functions have been conserved throughout vertebrate evolution. Knockout/knockdown of the inactivating D3 enzyme indicates that a premature switch from unliganded to liganded TR action is often more detrimental than a delayed one. The majority of ontogenetic studies on TH transporter distribution and function have focused on brain development, showing variable impact of knockout/knockdown depending on the species. Future research in different models using conditional silencing will hopefully further improve our understanding on how TH transporters, Ds and TRs cooperate to regulate TR-mediated impact on vertebrate development. This article is part of a Special Issue entitled: Nuclear receptors in animal development.

Zebrafish as a model to study peripheral thyroid hormone metabolism in vertebrate development.

To unravel the role of thyroid hormones (THs) in vertebrate development it is important to have suitable animal models to study the mechanisms regulating TH availability and activity. Zebrafish (Danio rerio), with its rapidly and externally developing transparent embryo has been a widely used model in developmental biology for some time. To date many of the components of the zebrafish thyroid axis have been identified, including the TH transporters MCT8, MCT10 and OATP1C1, the deiodinases D1, D2 and D3, and the receptors TRα and TRß. Their structure and function closely resemble those of higher vertebrates. Interestingly, due to a whole genome duplication in the early evolution of ray-finned fishes, zebrafish possess two genes for D3 (dio3 and dio3a) and for TRα (thraa and thrab). Transcripts of all identified genes are present during embryonic development and several of them show dynamic spatio-temporal distribution patterns. Transient morpholino-knockdown of D2, D3 or MCT8 expression clearly disturbs embryonic development, confirming the importance of each of these regulators during early life stages. The recently available tools for targeted stable gene knockout will further increase the value of zebrafish to study the role of peripheral TH metabolism in pre- and post-hatch/post-natal vertebrate development.

Disruption of deiodinase type 2 in zebrafish disturbs male and female reproduction.

Thyroid hormones are crucial mediators of many aspects of vertebrate life, including reproduction. The key player is the biologically active 3,5,3'-triiodothyronine (T3), whose local bio-availability is strictly regulated by deiodinase enzymes. Deiodinase type 2 (Dio2) is present in many tissues and is the main enzyme for local T3 production. To unravel its role in different physiological processes, we generated a mutant zebrafish line, completely lacking Dio2 activity. Here we focus on the reproductive phenotype studied at the level of offspring production, gametogenesis, functioning of the hypothalamic-pituitary-gonadal axis and sex steroid production. Homozygous Dio2-deficient zebrafish were hypothyroid, displayed a delay in sexual maturity, and the duration of their reproductive period was substantially shortened. Fecundity and fertilization were also severely reduced. Gamete counts pointed to a delay in oogenesis at onset of sexual maturity and later on to an accumulation of oocytes in mutant ovaries due to inhibition of ovulation. Analysis of spermatogenesis showed a strongly decreased number of spermatogonia A at onset of sexual maturity. Investigation of the hypothalamic-pituitary-gonadal axis revealed that dysregulation was largely confined to the gonads with significant upregulation of igf3, and a strong decrease in sex steroid production concomitant with alterations in gene expression in steroidogenesis/steroid signaling pathways. Rescue of the phenotype by T3 supplementation starting at 4 weeks resulted in normalization of reproductive activity in both sexes. The combined results show that reproductive function in mutants is severely hampered in both sexes, thereby linking the loss of Dio2 activity and the resulting hypothyroidism to reproductive dysfunction.

Age-Dependent Changes in Glucose Homeostasis in Male Deiodinase Type 2 Knockout Zebrafish.

Thyroid hormones (THs) are crucial regulators of glucose metabolism and insulin sensitivity. Moreover, inactivating mutations in type 2 deiodinase (DIO2), the major TH-activating enzyme, have been associated with type 2 diabetes mellitus in both humans and mice. We studied the link between Dio2 deficiency and glucose homeostasis in fasted males of two different Dio2 knockout (KO) zebrafish lines. Young adult Dio2KO zebrafish (6 to 9 months) were hyperglycemic. Both insulin and glucagon expression were increased, whereas ß and α cell numbers in the main pancreatic islet were similar to those in wild-types. Insulin receptor expression in skeletal muscle was decreased at 6 months, accompanied by a strong downregulation of hexokinase and pyruvate kinase expression. Blood glucose levels in Dio2KO zebrafish, however, normalized around 1 year of age. Older mutants (18 to 24 months) were normoglycemic, and increased insulin and glucagon expression was accompanied by a prominent increase in pancreatic islet size and ß and α cell numbers. Older Dio2KO zebrafish also showed strongly decreased expression of glucagon receptors in the gastrointestinal system as well as decreased expression of glucose transporters GLUT2 and GLUT12, glucose-6-phosphatase, and glycogen synthase 2. This study shows that Dio2KO zebrafish suffer from transient hyperglycemia, which is counteracted with increasing age by a prominent hyperplasia of the endocrine pancreas together with decreases in hepatic glucagon sensitivity and intestinal glucose uptake. Further research on the mechanisms allowing compensation in older Dio2KO zebrafish may help to identify new therapeutic targets for (TH deficiency-related) hyperglycemia.

Permanent Deiodinase Type 2 Deficiency Strongly Perturbs Zebrafish Development, Growth, and Fertility.

Iodothyronine deiodinases are selenocysteine-containing enzymes that activate or inactivate thyroid hormones (THs). Deiodinase type 2 (Dio2) catalyzes the conversion of the prohormone T4 into the transcriptionally active T3 and is the predominant activating deiodinase in zebrafish. Using zinc finger nucleases, we generated two different dio2(-/-) mutant zebrafish lines to investigate the physiological function of this TH activator. The first line contains a deletion of 9 bp, resulting in an in-frame elimination of three conserved amino acids. The other line is characterized by an insertion of 4 bp, leading to the introduction of a premature stop-codon. Both lines completely lack Dio2 activity, resulting in a strong reduction of T3 abundancy in all tissues tested. Early development is clearly perturbed in these animals, as shown by a diverse set of morphometric parameters, defects in swim bladder inflation, and disturbed locomotor activity tested between 1 and 7 days after fertilization. Permanent Dio2 deficiency also provokes long-term effects because growth and especially fertility are severely hampered. Possible compensatory mechanisms were investigated in adult dio2(-/-) mutants, revealing a down-regulation of the inactivating deiodinase Dio3 and TH receptor transcript levels. As the first nonmammalian model with permanent Dio2 deficiency, these mutant zebrafish lines provide evidence that Dio2 is essential to assure normal development and to obtain a normal adult phenotype.

Deiodinase knockdown affects zebrafish eye development at the level of gene expression, morphology and function.

Retinal development in vertebrates relies extensively on thyroid hormones. Their local availability is tightly controlled by several regulators, including deiodinases (Ds). Here we used morpholino technology to explore the roles of Ds during eye development in zebrafish. Transcriptome analysis at 3 days post fertilization (dpf) revealed a pronounced effect of knockdown of both T4-activating Ds (D1D2MO) or knockdown of T3-inactivating D3 (D3bMO) on phototransduction and retinoid recycling. This was accompanied by morphological defects (studied from 1 to 7 dpf) including reduced eye size, disturbed retinal lamination and strong reduction in rods and all four cone types. Defects were more prominent and persistent in D3-deficient fish. Finally, D3-deficient zebrafish larvae had disrupted visual function at 4 dpf and were less sensitive to a light stimulus at 5 dpf. These data demonstrate the importance of TH-activating and -inactivating Ds for correct zebrafish eye development, and point to D3b as a central player.

Deiodinase knockdown during early zebrafish development affects growth, development, energy metabolism, motility and phototransduction.

Thyroid hormone (TH) balance is essential for vertebrate development. Deiodinase type 1 (D1) and type 2 (D2) increase and deiodinase type 3 (D3) decreases local intracellular levels of T3, the most important active TH. The role of deiodinase-mediated TH effects in early vertebrate development is only partially understood. Therefore, we investigated the role of deiodinases during early development of zebrafish until 96 hours post fertilization at the level of the transcriptome (microarray), biochemistry, morphology and physiology using morpholino (MO) knockdown. Knockdown of D1+D2 (D1D2MO) and knockdown of D3 (D3MO) both resulted in transcriptional regulation of energy metabolism and (muscle) development in abdomen and tail, together with reduced growth, impaired swim bladder inflation, reduced protein content and reduced motility. The reduced growth and impaired swim bladder inflation in D1D2MO could be due to lower levels of T3 which is known to drive growth and development. The pronounced upregulation of a large number of transcripts coding for key proteins in ATP-producing pathways in D1D2MO could reflect a compensatory response to a decreased metabolic rate, also typically linked to hypothyroidism. Compared to D1D2MO, the effects were more pronounced or more frequent in D3MO, in which hyperthyroidism is expected. More specifically, increased heart rate, delayed hatching and increased carbohydrate content were observed only in D3MO. An increase of the metabolic rate, a decrease of the metabolic efficiency and a stimulation of gluconeogenesis using amino acids as substrates may have been involved in the observed reduced protein content, growth and motility in D3MO larvae. Furthermore, expression of transcripts involved in purine metabolism coupled to vision was decreased in both knockdown conditions, suggesting that both may impair vision. This study provides new insights, not only into the role of deiodinases, but also into the importance of a correct TH balance during vertebrate embryonic development.

Knockdown of type 3 iodothyronine deiodinase severely perturbs both embryonic and early larval development in zebrafish.

Exposure to appropriate levels of thyroid hormones (THs) at the right time is of key importance for normal development in all vertebrates. Type 3 iodothyronine deiodinase (D3) is the prime TH-inactivating enzyme, and its expression is highest in the early stages of vertebrate development, implying that it may be necessary to shield developing tissues from overexposure to THs. We used antisense morpholino knockdown to examine the role of D3 during early development in zebrafish. Zebrafish possess 2 D3 genes, dio3a and dio3b. Here, we show that both genes are expressed during development and both contribute to in vivo D3 activity. However, dio3b mRNA levels in embryos are higher, and the effects of dio3b knockdown on D3 activity and on the resulting phenotype are more severe. D3 knockdown induced an overall delay in development, as determined by measurements of otic vesicle length, eye and ear size, and body length. The time of hatching was also severely delayed in D3-knockdown embryos. Importantly, we also observed a severe disturbance of several aspects of development. Swim bladder development and inflation was aberrant as was the development of liver and intestine. Furthermore, D3-knockdown larvae spent significantly less time moving, and both embryos and larvae exhibited perturbed escape responses, suggesting that D3 knockdown affects muscle development and/or functioning. These data indicate that D3 is essential for normal zebrafish embryonic and early larval development and show the value of morpholino knockdown in this model to further elucidate the specific role of D3 in some aspects of vertebrate development.