The retina is crucial for converting light into neuronal signals for visual perception. Understanding the retina’s structure, function, and development is essential for vision research. It is known that the thyroid hormone (TH) receptor type beta 2 (TRβ2) is a key element in the regulation of cone differentiation in the retina, but other elements of TH signaling, such as transporters and enzyme deiodinases, have also been implicated in retinal cell development and survival. In the present study, we investigated the expression profile of genes involved in TH signaling and analyzed the impact of thyroidal status on retinal morphology, opsin expression, cell death/proliferation profile, as well as color preference behavior during the early retina development of zebrafish larvae. mRNA expression analysis on dissected whole eyes revealed that TH signaling elements gradually increase during eye development, with dio3b being the component that shows the most dramatic change. Mutations generated by CRISPR/CAS9 in the dio3b gene, but not in the thrb gene, modifies the structure of the retina. Disruption in TH level reduces the cell number of the ganglion cell layer, increases cell death, and modifies color preference, emphasizing the critical importance of precise TH regulation by its signaling elements for optimal retinal development and function.

Background: Thyroid hormone signaling is essential for development, metabolism, and response to stress but declines during aging, the cause of which is unknown. DNA damage accumulating with time is a main cause of aging, driving many age-related diseases. Previous studies in normal and premature aging mice, due to defective DNA repair, indicated reduced hepatic thyroid hormone signaling accompanied by decreased type 1 deiodinase (DIO1) and increased DIO3 activities. We investigated whether aging-related changes in deiodinase activity are driven by systemic signals or represent cell- or organ-autonomous changes. Methods: We quantified liver and plasma thyroid hormone concentrations, deiodinase activities and expression of T3-responsive genes in mice with a global, liver-specific and for comparison brain-specific inactivation of Xpg, one of the endonucleases critically involved in multiple DNA repair pathways. Results: Both in global and liver-specific Xpg knockout mice, hepatic DIO1 activity was decreased. Interestingly, hepatic DIO3 activity was increased in global, but not in liver-specific Xpg mutants. Selective Xpg deficiency and premature aging in the brain did not affect liver or systemic thyroid signaling. Concomitant with DIO 1 inhibition, Xpg−/− and Alb-Xpg mice displayed reduced thyroid hormone-related gene expression changes, correlating with markers of liver damage and cellular senescence. Conclusions: Our findings suggest that DIO1 activity during aging is predominantly modified in a tissue-autonomous manner driven by organ/cell-intrinsic accumulating DNA damage. The increase in hepatic DIO3 activity during aging largely depends on systemic signals, possibly reflecting the presence of circulating cells rather than activity in hepatocytes.

Thyroid hormones clearly play a role in the seasonal regulation of reproduction, but any role they might play in song behavior and the associated seasonal neuroplasticity in songbirds remains to be elucidated. To pursue this question, we first established seasonal patterns in the expression of thyroid hormone regulating genes in male European starlings employing in situ hybridization methods. Thyroid hormone transporter LAT1 expression in the song nucleus HVC was elevated during the photosensitive phase, pointing toward an active role of thyroid hormones during this window of possible neuroplasticity. In contrast, DIO3 expression was high in HVC during the photostimulated phase, limiting the possible effect of thyroid hormones to maintain song stability during the breeding season. Next, we studied the effect of hypothyroidism on song behavior and neuroplasticity using in vivo MRI. Both under natural conditions as with methimazole treatment, circulating thyroid hormone levels decreased during the photosensitive period, which coincided with the onset of neuroplasticity. This inverse relationship between thyroid hormones and neuroplasticity was further demonstrated by the negative correlation between plasma T3 and the microstructural changes in several song control nuclei and cerebellum. Furthermore, maintaining hypothyroidism during the photostimulated period inhibited the increase in testosterone, confirming the role of thyroid hormones in activating the hypothalamic-pituitary-gonadal (HPG) axis. The lack of high testosterone levels influenced the song behavior of hypothyroid starlings, while the lack of high plasma T4 during photostimulation affected the myelination of several tracts. Potentially, a global reduction of circulating thyroid hormones during the photosensitive period is necessary to lift the brake on neuroplasticity imposed by the photorefractory period, whereas local fine-tuning of thyroid hormone concentrations through LAT1 could activate underlying neuroplasticity mechanisms. Whereas, an increase in circulating T4 during the photostimulated period potentially influences the myelination of several white matter tracts, which stabilizes the neuroplastic changes. Given the complexity of thyroid hormone effects, this study is a steppingstone to disentangle the influence of thyroid hormones on seasonal neuroplasticity.

Cortical interneuron neurogenesis is strictly regulated and depends on the presence of thyroid hormone (TH). In particular, inhibitory interneurons expressing the calcium binding protein Parvalbumin are highly sensitive toward developmental hypothyroidism. Reduced numbers of Parvalbumin-positive interneurons are observed in mice due to the combined absence of the TH transporters Mct8 and Oatp1c1. To unravel if cortical Parvalbumin-positive interneurons depend on cell-autonomous action of Mct8/Oatp1c1, we compared Mct8/Oatp1c1 double knockout (dko) mice to conditional knockouts with abolished TH transporter expression in progenitors of Parvalbumin-positive interneurons. These conditional knockouts exhibited a transient delay in the appearance of Parvalbumin-positive interneurons in the early postnatal somatosensory cortex while cell numbers remained permanently reduced in Mct8/Oatp1c1 dko mice. Using fluorescence in situ hybridization on E12.5 embryonic brains, we detected reduced expression of sonic hedgehog signaling components in Mct8/Oatp1c1 dko embryos only. Moreover, we revealed spatially distinct expression patterns of both TH transporters at brain barriers at E12.5 by immunofluorescence. At later developmental stages, we uncovered a sequential expression of first Oatp1c1 in individual interneurons and then Mct8 in Parvalbumin-positive subtypes. Together, our results point to multiple cell-autonomous and noncell-autonomous mechanisms that depend on proper TH transport during cortical interneuron development.

Thyroid hormones are messengers that bind to specific nuclear receptors and regulate a wide range of physiological processes in the early stages of vertebrate embryonic development, including neurodevelopment and myelogenesis. We here tested the effects of reduced T3 availability upon the myelination process by treating zebrafish embryos with low concentrations of iopanoic acid (IOP) to block T4 to T3 conversion. Black Gold II staining showed that T3 deficiency reduced the myelin density in the forebrain, midbrain, hindbrain and the spinal cord at 3 and 7 dpf. These observations were confirmed in 3 dpf mbp:egfp transgenic zebrafish, showing that the administration of IOP reduced the fluorescent signal in the brain. T3 rescue treatment restored brain myelination and reversed the changes in myelin-related gene expression induced by IOP exposure. NG2 immunostaining revealed that T3 deficiency reduced the amount of oligodendrocyte precursor cells in 3 dpf IOP-treated larvae. Altogether, the present results show that inhibition of T4 to T3 conversion results in hypomyelination, suggesting that THs are part of the key signaling molecules that control the timing of oligodendrocyte differentiation and myelin synthesis from very early stages of brain development.

Traditionally, research unraveling seasonal neuroplasticity in songbirds has focused on the male song control system and testosterone. We longitudinally monitored the song behavior and neuroplasticity in male and female starlings during multiple photoperiods using Diffusion Tensor and Fixel-Based techniques. These exploratory data-driven whole-brain methods resulted in a population-based tractogram confirming microstructural sexual dimorphisms in the song control system. Furthermore, male brains showed hemispheric asymmetries in the pallium, whereas females had higher interhemispheric connectivity, which could not be attributed to brain size differences. Only females with large brains sing but differ from males in their song behavior by showing involvement of the hippocampus. Both sexes experienced multisensory neuroplasticity in the song control, auditory and visual system, and cerebellum, mainly during the photosensitive period. This period with low gonadal hormone levels might represent a 'sensitive window' during which different sensory and motor systems in the cerebrum and cerebellum can be seasonally re-shaped in both sexes.

Iodothyronine deiodinases are enzymes capable of activating and inactivating thyroid hormones (THs) and have an important role in regulating TH action in tissues throughout the body. Three types of deiodinases (D1, D2, and D3) were originally defined based on their biochemical characteristics. Cloning of the first complementary DNAs in the 1990s (Dio1 in rat and dio2 and dio3 in frog) allowed to confirm the existence of 3 distinct enzymes. Over the years, increasing genomic information revealed that deiodinases are present in all chordates, vertebrates, and nonvertebrates and that they can even be found in some mollusks and annelids, pointing to an ancient origin. Research in nonmammalian models has substantially broadened our understanding of deiodinases. In relation to their structure, we discovered for instance that biochemical properties such as inhibition by 6-propyl-2-thiouracil, stimulation by dithiothreitol, and temperature optimum are subject to variation. Data from fish, amphibians, and birds were key in shifting our view on the relative importance of activating and inactivating deiodination pathways and in showing the impact of D2 and D3 not only in local but also whole body T3 availability. They also led to the discovery of new local functions such as the acute reciprocal changes in D2 and D3 in hypothalamic tanycytes upon photostimulation, involved in seasonal rhythmicity. With the present possibilities for rapid and precise gene silencing in any species of interest, comparative research will certainly further contribute to a better understanding of the importance of deiodinases for adequate TH action, also in humans.

Traditionally, research unraveling seasonal neuroplasticity in songbirds has focused on the male song control system and testosterone. We longitudinally monitored the song and neuroplasticity in male and female starlings during multiple photoperiods using Diffusion Tensor and Fixel-Based techniques. These exploratory data-driven whole-brain methods resulted in a population-based tractogram uncovering microstructural sexual dimorphisms in the song control system and beyond. Male brains showed microstructural hemispheric asymmetries, whereas females had higher interhemispheric connectivity, which could not be attributed to brain size differences. Only females with large brains sing but differ from males in their song behavior by showing involvement of the hippocampus. Both sexes experienced multisensory neuroplasticity in the song control, auditory and visual system, and the cerebellum, mainly during the photosensitive period. This period with low gonadal hormones might represent a ‘sensitive window’ during which different sensory and motor systems in telencephalon and cerebellum can be seasonally re-shaped in both sexes.

In vertebrates, the embryonic environment is known to affect the development and the health of individuals. In broiler chickens, the thermal-manipulation (TM) of eggs during the incubation period was shown to improve heat tolerance at slaughter age (35 days of age) in association with several modifications at the molecular, metabolic and physiological levels. However, little is known about the Japanese quail (Coturnix japonica), a closely related avian species widely used as a laboratory animal model and farmed for its meat and eggs. Here we developed and characterized a TM procedure (39.5°C and 65% relative humidity, 12 h/d, from days 0 to 13 of incubation) in quails by analyzing its short and long-term effects on zootechnical, physiological and metabolic parameters. Heat-tolerance was tested by a heat challenge (36°C for 7h) at 35 days of age. TM significantly reduced the hatching rate of the animals and increased mortality during the first four weeks of life. At hatching, TM animals were heavier than controls, but lighter at 25 days of age for both sexes. Thirty-five days after hatching, TM decreased the surface temperature of the shank in females, suggesting a modulation of the blood flow to maintain the internal temperature. TM also increased blood partial pressure and oxygen saturation percentage at 35 days of age in females, suggesting a long-term modulation of the respiration physiology. Quails physiologically responded to the heat challenge, with a modification of several hematologic and metabolic parameters, including an increase in plasma corticosterone concentration. Several physiological parameters such as beak surface temperature and blood sodium concentration revealed that TM birds responded differently to the heat challenge compared to controls. Altogether, this first comprehensive characterization of TM in Japanese quail showed durable effects that may affect the response of TM quails to heat.

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 beta and alpha 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 beta and alpha 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.

•Adequate thyroid hormone (TH) signalling is essential from early development.•Transporters, deiodinases and receptors regulate TH action at the peripheral level.•Zebrafish regulator deficiency models show widespread impact on organ development.•Zebrafish are useful to study impact of mutations/xenobiotics on human development.

Adult neuroplasticity in the song control system of seasonal songbirds is largely driven by photoperiod-induced increases in testosterone. Prior studies of the relationships between testosterone, song performance and neuroplasticity used invasive techniques, which prevent analyzing the dynamic changes over time and often focus on pre-defined regions-of-interest instead of examining the entire brain. Here, we combined (i) in vivo diffusion tensor imaging (DTI) to assess structural neuroplasticity with (ii) repeated monitoring of song and (iii) measures of plasma testosterone concentrations in thirteen female photosensitive starlings (Sturnus vulgaris) who received a testosterone implant for 3 weeks. We observed fast (days) and slower (weeks) effects of testosterone on song behavior and structural neuroplasticity and determined how these effects correlate on a within-subject level, which suggested separate contributions of the song motor and anterior forebrain pathways in the development of song performance. Specifically, the increase in testosterone correlated with a rapid increase of song rate and RA volume, and with changes in Area X microstructure. After implant removal, these variables rapidly reverted to baseline levels. In contrast, the more gradual improvement of song quality was positively correlated with the fractional anisotropy values (DTI metric sensitive to white matter changes) of the HVC-RA tract and of the lamina mesopallialis, which contains fibers connecting the song control nuclei. Thus, we confirmed many of the previously reported testosterone-induced effects, like the increase in song control nuclei volume, but identified for the first time a more global picture of the spatio-temporal changes in brain plasticity.

Maternal hormones are often considered a mediator of anticipatory maternal effects; namely, mothers adjust maternal hormone transfer to prepare the offspring for the anticipated environment. The flexibility for mothers to adjust hormone transfer is therefore a prerequisite for such anticipatory maternal effects. Nevertheless, previous studies have focused only on the average differences of maternal hormone transfer between groups and neglected the substantial individual variation, despite the fact that individual plasticity in maternal hormone transfer is actually the central assumption. In this study, we studied the between- and within-individual variation of maternal thyroid hormones (THs) in egg yolk of wild great tits (Parus major) and estimated the individual plasticity of maternal yolk THs across environmental temperature, clutch initiation dates, and egg laying order using linear mixed effects models. Interestingly, our models provide statistical evidence that the two main THs-the main biologically active hormone T3 and T4, which is mostly considered a prohormone-exhibited different variation patterns. Yolk T3 showed significant between-individual variation on the average levels, in line with its previously reported moderate heritability. Yolk T4, however, showed significant between-clutch variation in the pattern over the laying sequence, suggesting a great within-individual plasticity. Our findings suggest that the role and function of the hormone within the endocrine axis likely influences its flexibility to respond to environmental change. Whether the flexibility of T4 deposition brings a fitness advantage should be examined along with its potential effects on offspring, which remain to be further investigated.

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.

Context: Thyroid hormones are indispensable for normal fetal development.Since the fetus depends to a large extent on maternal thyroid hormone supply through the placenta, this challenges maternal thyroid economy.Several molecular mechanisms are involved in placental thyroid hormone transport and metabolism.Chronic pregnancy complications, associated with utero-placental hypoxia, trigger the development of accelerated placental maturation in order to improve fetal-placental exchange to strengthen the offspring's chance of survival.This review provides an overview of normal maternal-fetal thyroid hormone supply and explores the presence of placental adaptive mechanisms in complicated pregnancies with chronical utero-placental hypoxia to improve the thyroid hormone supply to the fetus under pressure, to end with reflections about the long term health consequences.Evidence acquisition: This work is based on a comprehensive literature review of the Pubmed and Embase database, including relevant articles from 1969 to June 2018.Conclusions:The placenta is actively involved in fetal thyroid hormone delivery through a combination of stimulatory and inhibitory mechanisms.Parallel with histological adaptations to improve trans-placental fetal-maternal exchange, there are indications of placental adaptive mechanisms in thyroid hormone transport and metabolism in case of complicated pregnancies, from animal models and in-vitro experiments.Evidence from human in-vivo studies is limited due to heterogeneity in study populations, small study samples and technical limitations.Further research is necessary to reveal the role of the placenta in pathological circumstances.The placenta might thus be considered as the infants' black box of pregnancy.Results will contribute to more insights in the concept of fetal programming, which lays the foundations of optimum health, growth, and neurodevelopment across the lifespan.

During avian embryonic development, thyroid hormones (THs) coordinate the expression of a multitude of genes thereby ensuring that the correct sequence of cell proliferation, differentiation and maturation is followed in each tissue and organ. Although THs are needed from the start of development, the embryonic thyroid gland only matures around mid-incubation in precocial birds and around hatching in altricial species. Therefore, maternal THs deposited in the egg yolk play an essential role in embryonic development. They are taken up by the embryo throughout its development, from the first day till hatching, and expression of TH regulators such as distributor proteins, transporters, and deiodinases in the yolk sac membrane provide the tools for selective metabolism and transport starting from this level. TH receptors and regulators of local TH availability are expressed in avian embryos in a dynamic and tissue/cell-specific pattern from the first stages studied, as shown in detail in chicken. Maternal hyperthyroidism via TH supplementation as well as injection of THs into the egg yolk increase TH content in embryonic tissues while induction of maternal hypothyroidism by goitrogen treatment results in a decrease. Both increase and decrease of maternal TH availability were shown to alter gene expression in early chicken embryos. Knockdown of the specific TH transporter monocarboxylate transporter 8 at early stages in chicken cerebellum, optic tectum, or retina allowed to reduce local TH availability, interfering with gene expression and confirming that development of the central nervous system(CNS) is highly dependent on maternal THs. While some of the effects on cell proliferation, migration and differentiation seem to be transient, others result in persistent defects in CNS structure. In addition, a number of studies in both precocial and altricial birds showed that injection of THs into the yolk at the start of incubation influences a number of parameters in posthatch performance and fitness. In conclusion, the data presently available clearly indicate that maternal THs play an important role in avian embryonic development, but how exactly their influence on cellular and molecular processes in the embryo is linked to posthatch fitness needs to be further explored.

Thyroid hormone (TH) transporters are required for the transmembrane passage of TH in target cells. In humans, inactivating mutations in the TH transporter MCT8 cause the Allan-Herndon-Dudley syndrome, characterized by severe neuromuscular symptoms and an abnormal TH serum profile, which is fully replicated in Mct8 knockout mice and Mct8/Oatp1c1 double-knockout (M/O DKO) mice. Analysis of tissue TH content and expression of TH-regulated genes indicate a thyrotoxic state in Mct8-deficient skeletal muscles. Both TH transporters are upregulated in activated satellite cells (SCs). In M/O DKO mice, we observed a strongly reduced number of differentiated SCs, suggesting an impaired stem cell function. Moreover, M/O DKO mice and mice lacking both transporters exclusively in SCs showed impaired skeletal muscle regeneration. Our data provide solid evidence for a unique gate-keeper function of MCT8 and OATP1C1 in SC activation, underscoring the importance of a finely tuned TH signaling during myogenesis.

The adverse outcome pathway (AOP) framework can be used to help support the development of alternative testing strategies aimed at predicting adverse outcomes caused by triggering specific toxicity pathways. In this paper, we present a case-study demonstrating the selection of alternative in chemico assays targeting the molecular initiating events of established AOPs, and evaluate use of the resulting data to predict higher level biological endpoints. Based on two AOPs linking inhibition of the deiodinase (DIO) enzymes to impaired posterior swim bladder inflation in fish, we used in chemico enzyme inhibition assays to measure the molecular initiating events for an array of 51 chemicals. Zebrafish embryos were then exposed to 14 compounds with different measured inhibition potentials. Effects on posterior swim bladder inflation, predicted based on the information captured by the AOPs, were evaluated. By linking the two datasets and setting thresholds, we were able to demonstrate that the in chemico dataset can be used to predict biological effects on posterior chamber inflation, with only two outliers out of the 14 tested compounds. Our results show how information organized using the AOP framework can be employed to develop or select alternative assays, and successfully forecast downstream key events along the AOP. In general, such in chemico assays could serve as a first-tier high-throughput system to screen and prioritize chemicals for subsequent acute and chronic fish testing, potentially reducing the need for long-term and costly toxicity tests requiring large numbers of animals.

The Mexican axolotl (Ambystoma mexicanum) is a salamander species that does not undergo metamorphosis, resulting in the retention of juvenile characteristics in the mature breeding stage (paedomorphosis). Here we review the endocrinological studies investigating the proximate cause of axolotl paedomorphosis with a focus on the hypothalamo-pituitary-thyroid (HPT) axis. It is well established that axolotl paedomorphosis is a consequence of low activity of the HPT axis. The pituitary hormone thyrotropin (TSH) is capable of inducing metamorphosis in the axolotl, which indicates that all processes and interactions in the HPT axis below the pituitary level are functional, but that TSH release is impaired. In metamorphosing species, TSH secretion is largely controlled by the hypothalamic neuropeptide corticotropin-releasing hormone (CRH), which seems to have lost its thyrotropic activity in the axolotl. However, preliminary experiments have not yet confirmed a role for faulty CRH signalling in axolotl paedomorphosis. Other hypothalamic factors and potential pituitary inhibitors need to be investigated to identify their roles in amphibian metamorphosis and axolotl paedomorphosis.