The THRB thyroid hormone receptor gene has attracted growing attention for its role in retinal development and disease. This gene has particularly critical roles in cone photoreceptors, the specialized cells that mediate color vision and high acuity vision. THRB controls the diversity of cone types that are required for color vision and influences cone survival in mammalian model species and human retinal organoid cultures. This central role of THRB in the cone life history prompts an expectation of equally critical roles in the human retina in vivo. Puzzlingly, overt retinal phenotypes have gone unnoticed for most known human THRB mutations. However, upon closer inspection, retinal impairment is now increasingly recognized. Mutations in 3’-exons of THRB (encoding the receptor ligand-binding domain) are known in resistance to thyroid hormone, typically a dominant syndrome with endocrine and other impairments but generally without mention of retinal disorders. However, a few specific investigations have revealed variable, usually moderate cone impairment. Recently, non-syndromic macular dystrophy cases have been found with sequence variants in a THRB 5’-exon encoding the N-terminus of one of the receptor isoforms expressed by the gene, suggesting a surprisingly wider involvement in retinal disease. We discuss this intriguing receptor gene and its emerging role in human retinal disorders.

Thyroid hormone is required for brain development but under constraint because its unregulated action can damage target tissues. Type 3 deiodinase, a thyroid hormone-degrading enzyme encoded by Dio3, constrains thyroid hormone exposure in the brain and has primarily been described in neuronal populations. However, detection is difficult because of low, transient expression, often in specialized cell populations. Here, using Dio3Cre-mediated cell labeling with a sensitive fluorescent reporter, we uncovered Dio3 expression in non-neuronal cell types in blood-cerebrospinal fluid (CSF) and blood-brain barriers during embryonic and neonatal development in mice. These barriers control solute transport, suggesting that Dio3 regulates thyroid hormone availability at cellular barriers between hormone-bearing fluids and the brain. Dio3 expression was detected in the arachnoid barrier layer in the meninges, the membranes surrounding the brain, and in the epithelial layer of the choroid plexus, which regulates solute exchange with CSF. RNA analyses corroborated early Dio3 expression in these tissues, beginning even before barrier function matures. Dio3 expression was also detected in tanycytes lining the third ventricle in the mediobasal hypothalamus, in a restricted portion of the glia limitans at the ventral hypothalamus, and in pericytes at the neurovascular interface. These findings suggest a role for Dio3 in regulating thyroid hormone availability at cellular interfaces with fluids that transport thyroid hormone in the immature brain.

Thyroid hormone (triiodothyronine, T3) promotes neurodevelopment but under strict control because unconstrained exposure to T3 impairs brain and sensory functions. Thyroid hormone-inactivating type 3 deiodinase, encoded by Dio3, critically limits T3 signaling and controls diverse neural functions. Accordingly, understanding the cellular basis of T3 action requires identification of Dio3-expressing cell types but this is difficult because of low level, transient expression within the complexity of the nervous system. Here, we derived a knock-in Dio3Cre driver that sensitively labels Dio3-expressing cells in male and female mice. In this anatomical study, we identified Dio3 expression in the immature amygdala and other brain regions associated with emotion and motivation, and in serotonergic raphe nuclei, which influence many behavioral and physiological systems. Notably, expression in circumventricular organs, including the chemosensory subfornical organ and organum vasculosum laminae terminalis, suggested regulation of centers that lack a blood-brain barrier and directly sense signaling factors in the circulation. Expression in trigeminal, dorsal root, cochleovestibular, and other sensory ganglia highlighted contributions to sensory pathways. Although Dio3 expression declines during maturation, a conditional Dio3CreERt2 driver revealed neurons with T3-inducible expression in the adult brain, suggesting ongoing homeostatic functions. These Cre drivers indicate strategically located neuronal groups for control of T3 signaling in behavioral, chemosensory and sensory systems.

Thyroid hormone receptors (TRs, nomenclature as agreed by the NC-IUPHAR Subcommittee on Nuclear Hormone Receptors [13, 2]) are nuclear hormone receptors of the NR1A family, with diverse roles regulating macronutrient metabolism, cognition and cardiovascular homeostasis. TRs are activated by thyroxine (T4) and thyroid hormone (triiodothyronine). Once activated by a ligand, the receptor acts as a transcription factor either as a monomer, homodimer or heterodimer with members of the retinoid X receptor family. NH-3 has been described as an antagonist at TRs with modest selectivity for TRβ [43].

A growing body of evidence has established that thyroid hormone (triiodothyronine, T3) is a key factor in the differentiation and survival of the light-sensing photoreceptors in the retina. These functions include a critical role in generating the cone photoreceptor diversity that is required for color vision. Here, we review some of these functions of T3 and the critical mechanisms that regulate the T3 signal in the mammalian retina. The provision of T3, the active form of thyroid hormone, is determined by developmentally rising levels of T3 and its precursor T4 (thyroxine) in the circulation and by intrinsic control within the retina itself by deiodinase enzymes that deplete or amplify the available level of T3. Dynamic profiles of inactivating (DIO3) and activating (DIO2) deiodinases suggest that the T3 signal is progressively calibrated throughout early development, maturation and later functional maintenance of the retina. However, the benefits of T3 come at a cost: photoreceptors are susceptible to impairment and cell death when T3 signaling becomes imbalanced. These findings have implications regarding the influence of T3 in retinal diseases.

Abstract Disclosure: E.A. Gregersen: None. R.P. Peeters: None. D. Forrest: None. D.S. Sharlin: None. Thyroid hormone (TH) is essential for auditory system development. In rodent models, low levels of TH during the early postnatal period delay cochlear tissue remodeling and alter ion channel function required for normal auditory function. TH receptors are ligand-dependent transcription factors that regulate gene expression. Considering this, auditory deficits observed in hypothyroidism are thought to be largely the result of altered expression of specific TH target genes. However, few TH-regulated genes in the developing cochlea have been reported. TH is reported to impact calcium signaling in several tissues, but whether regulators of calcium signaling are altered following developmental hypothyroidism in the cochlea is largely unexplored. In this report, we investigated the spatial and temporal expression of the ATPase, Ca++ transporting, plasma membrane 2 (Atp2b2) gene in a mouse model of developmental hypothyroidism. Timed-pregnant dams were treated with thyroid gland inhibitors to induce developmental TH insufficiency from embryonic day 12.5 (E12.5) through post-natal day 15 (P15). Untreated timed-pregnant dams served as euthyroid controls. Control and experimental cochleae were collected over a developmental period ranging from embryonic day 18.5 (E18.5) through P15. Cochleae were then processed to detect Atp2b2 mRNA by in situ hybridization or quantitative real-time PCR. In situ hybridization showed that Atp2b2 localizes to inner and outer hair cells as expected, but Atp2b2 was also observed at lower levels in the greater epithelial ridge and spiral ganglion. Quantification of Atp2b2 mRNA revealed an effect of postnatal day and treatment on Atp2b2. In euthyroid animals, Atp2b2 expression gradually increased until P10, then decreased slightly by P15. In hypothyroid animals, Atp2b2 mRNA was consistently decreased (2-2.5 fold). Hypothyroidism similarly impacted expression regardless of sex. The reduction in Atp2b2 mRNA expression in the hypothyroid animals suggests that Atp2b2 is developmentally regulated in part by thyroid hormone signaling. This study characterizes the relationship between TH and Atp2b2 expression during early cochlear development while providing insight into abnormal cochlear development due to hypothyroidism. Presentation: 6/3/2024

The key role of a thyroid hormone receptor in determining the maturation and diversity of cone photoreceptors reflects a profound influence of endocrine signaling on the cells that mediate color vision. However, the route by which hormone reaches cones remains enigmatic as cones reside in the retinal photoreceptor layer, shielded by the blood-retina barrier. Using genetic approaches, we report that cone differentiation is regulated by a membrane transporter for thyroid hormone, MCT8 (SLC16A2), in the retinal pigment epithelium (RPE), which forms the outer blood-retina barrier. Mct8- deficient mice display hypothyroid-like cone gene expression and compromised electroretinogram responses. Mammalian color vision is typically facilitated by cone types that detect medium- long (M) and short (S) wavelengths of light but Mct8- deficient mice have a partial shift of M to S cone identity, resembling the phenotype of thyroid hormone receptor deficiency. RPE- specific ablation of Mct8 results in similar shifts in cone identity and hypothyroid-like gene expression whereas reexpression of MCT8 in the RPE in Mct8- deficient mice partly restores M cone identity, consistent with paracrine-like control of thyroid hormone signaling by the RPE. Our findings suggest that in addition to transport of essential solutes and homeostatic support for photoreceptors, the RPE regulates the thyroid hormone signal that promotes cone- mediated vision.

The NR superfamily comprises 48 transcription factors in humans that control a plethora of gene network programs involved in a wide range of physiologic processes. This review will summarize and discuss recent progress in NR biology and drug development derived from integrating various approaches, including biophysical techniques, structural studies, and translational investigation. We also highlight how defective NR signaling results in various diseases and disorders and how NRs can be targeted for therapeutic intervention via modulation via binding to synthetic lipophilic ligands. Furthermore, we also review recent studies that improved our understanding of NR structure and signaling. SIGNIFICANCE STATEMENT: Nuclear receptors (NRs) are ligand-regulated transcription factors that are critical regulators of myriad physiological processes. NRs serve as receptors for an array of drugs, and in this review, we provide an update on recent research into the roles of these drug targets.

The retina is increasingly recognized as a target of thyroid hormone. We previously reported critical functions for thyroid hormone receptor TRβ2, encoded by Thrb, in cones, the photoreceptors that mediate color vision. TRβ1, another Thrb receptor isoform, is widely expressed in other tissues but little studied in the retina. Here, we investigate these N-terminal isoforms by RNA-sequencing analysis and reveal a striking biphasic profile for TRβ1 in mouse and human retina. In contrast to the early TRβ2 peak, TRβ1 peaks later during retinal maturation or later differentiation of human retinal organoids. This switch in receptor expression profiles was confirmed using lacZ reporter mice. TRβ1 localized in cones, amacrine cells and ganglion cells in contrast to the restricted expression of TRβ2 in cones. Intriguingly, TRβ1 was also detected in the retinal pigmented epithelium and in anterior structures in the ciliary margin zone, ciliary body and iris, suggesting novel functions in non-retinal eye tissues. Although TRβ1 was detected in cones, TRβ1-knockout mice displayed only minor changes in opsin photopigment expression and normal electroretinogram responses. Our results suggest that strikingly different temporal and cell-specific controls over TRβ1 and TRβ2 expression may underlie thyroid hormone actions in a range of ocular cell types. The TRβ1 expression pattern suggests novel functions in retinal and non-neural ocular tissues.

<p>Figure S1. TRβ2 and TRβ1 inversely regulate retinoblastoma cell proliferation and cell cycle progression.Figure S2. Pituitary anterior lobe and intermediate lobe markers and Trβ isoforms expressed in pituitary tumors in Rb1+/- mice of different Trβ genetic backgrounds.Figure S3. EMI1 mediates TRβ2 regulation of SKP2.Figure S4. Rescue of Rb knockdown-induced cell cycle arrest and SKP2 down-regulation by ectopic Rb expression.Figure S5. Gene expression changes in response to RB1-KD in synchronized SKN-BE(2) neuroblastoma cells.Figure S6. TRβ1 and TRβ2 regulate proliferation and SKP2 and p27 expression in Rb-deficient MXF8000 myxofibrosarcoma cells.</p>

Supplementary Information. This file contains Extended Experimental Procedures including Table S1 (pLKO lentiviral shRNAs), Table S2 (Antibodies used in this study), and Table S3 (qPCR primers).

Thyroid hormone receptors (TRs, nomenclature as agreed by the NC-IUPHAR Subcommittee on Nuclear Hormone Receptors [12, 2]) are nuclear hormone receptors of the NR1A family, with diverse roles regulating macronutrient metabolism, cognition and cardiovascular homeostasis. TRs are activated by thyroxine (T4) and thyroid hormone (triiodothyronine). Once activated by a ligand, the receptor acts as a transcription factor either as a monomer, homodimer or heterodimer with members of the retinoid X receptor family. NH-3 has been described as an antagonist at TRs with modest selectivity for TRβ [42].

The function of a hormone receptor requires mechanisms to control precisely where, when, and at what level the receptor gene is expressed. An intriguing case concerns the selective induction of thyroid hormone receptor β2 (TRβ2), encoded by Thrb, in the pituitary and also in cone photoreceptors, in which it critically regulates expression of the opsin photopigments that mediate color vision. Here, we investigate the physiological significance of a candidate enhancer for induction of TRβ2 by mutagenesis of a conserved intron region in its natural context in the endogenous Thrb gene in mice. Mutation of e-box sites for bHLH (basic-helix-loop-helix) transcription factors preferentially impairs TRβ2 expression in cones whereas mutation of nearby sequences preferentially impairs expression in pituitary. A deletion encompassing all sites impairs expression in both tissues, indicating bifunctional activity. In cones, the e-box mutations disrupt chromatin acetylation, blunt the developmental induction of TRβ2, and ultimately impair cone opsin expression and sensitivity to longer wavelengths of light. These results demonstrate the necessity of studying an enhancer in its natural chromosomal context for defining biological relevance and reveal surprisingly critical nuances of level and timing of enhancer function. Our findings illustrate the influence of noncoding sequences over thyroid hormone functions.

Thyroid hormone (3,5,3’-triiodothyronine, T3) is a key regulator of pituitary gland function. The response to T3 is thought to hinge crucially on interactions of nuclear T3 receptors with enhancers but these sites in pituitary chromatin remain surprisingly obscure. Here, we investigate genome-wide receptor binding in mice using tagged endogenous thyroid hormone receptor β (TRβ) and analyze T3-regulated open chromatin using an anterior pituitary-specific Cre driver ( Thrb b2Cre ). Strikingly, T3 regulates histone modifications and chromatin opening primarily at sites that maintain TRβ binding regardless of T3 levels rather than at sites where T3 abolishes or induces de novo binding. These sites associate more frequently with T3-activated than T3-suppressed genes. TRβ-deficiency blunts T3-regulated gene expression, indicating that TRβ confers transcriptional sensitivity. We propose a model of gene activation in which poised receptor-enhancer complexes facilitate adjustable responses to T3 fluctuations, suggesting a genomic basis for T3-dependent pituitary function or pituitary dysfunction in thyroid disorders.

Pierpont syndrome is a rare disorder characterized mainly by global developmental delay, unusual facial features, altered fat distribution in the limbs and hearing loss. A specific mutation (p.Tyr446Cys) in TBL1XR1, encoding a WD40 repeat-containing protein, which is a component of the SMRT/NCoR (silencing mediator retinoid and thyroid hormone receptors/nuclear receptor corepressors), has been reported as the genetic cause of Pierpont syndrome. Here, we used CRISPR-cas9 technology to generate a mutant mouse with the Y446C mutation in Tbl1xr1, which is also present in Pierpont syndrome. Several aspects of the phenotype were studied in the mutant mice: growth, body composition, hearing, motor behavior, thyroid hormone state and lipid and glucose metabolism. The mutant mice (Tbl1xr1Y446C/Y446C) displayed delayed growth, altered body composition with increased relative lean mass and impaired hearing. Expression of several genes involved in fatty acid metabolism differed in white adipose tissue, but not in liver or muscle of mutant mice compared to wild-type mice (Tbl1xr1+/+). No difference in thyroid hormone plasma concentrations was observed. Tbl1xr1Y446C/Y446C mice can be used as a model for distinct features of Pierpont syndrome, which will enable future studies on the pathogenic mechanisms underlying the various phenotypic characteristics.

Cone photoreceptor diversity allows detection of wavelength information in light, the first step in color (chromatic) vision. In most mammals, cones express opsin photopigments for sensitivity to medium/long (M, “green”) or short (S, “blue”) wavelengths and are differentially arrayed over the retina. Cones appear early in retinal neurogenesis but little is understood of the subsequent control of diversity of these postmitotic neurons, because cone populations are sparse and, apart from opsins, poorly defined. It is also a challenge to distinguish potentially subtle differences between cell subtypes within a lineage. Therefore, we derived a Cre driver to isolate individual M and S opsin-enriched cones, which are distributed in counter-gradients over the mouse retina. Fine resolution transcriptome analyses identified expression gradients for groups of genes. The postnatal emergence of gradients indicated divergent differentiation of cone precursors during maturation. Using genetic tagging, we demonstrated a role for thyroid hormone receptor β2 (TRβ2) in control of gradient genes, many of which are enriched for TRβ2 binding sites and TRβ2-regulated open chromatin. Deletion of TRβ2 resulted in poorly distinguished cones regardless of retinal location. We suggest that TRβ2 controls a bipotential transcriptional state to promote cone diversity and the chromatic potential of the species.

Abstract Background: Thyroid hormone promotes many developmental and homeostatic functions. Apart from adequate circulating levels, the concentration of the active hormone T3 within tissues may be amplified by type 2 deiodinase (Dio2) by conversion from the precursor T4. Dio2 is critical in auditory development, bone maturation, brain function and control of the hypothalamic-pituitary-thyroid axis. Despite its crucial role, an obstacle to studying Dio2 is that the protein has a short half-life, is at low levels and is often transiently expressed, making it difficult to identify Dio2 in tissues at cellular resolution. Methods: We derived a Dio2-CreERt2 knockin mouse that expresses tamoxifen-dependent Cre recombinase from the endogenous Dio2 gene. When crossed onto Ai6 reporter mice, following tamoxifen treatment, Dio2-CreERt2 expression is detected as fluorescent signal in specific cells in brain regions, pituitary, and other tissues. We showed previously that Dio2 is essential for hearing with rising expression levels in the cochlea prior to onset of hearing. The Dio2-CreERt2 model identified positive cell types in the cochlear spiral ligament, septal divisions and modiolus around the sensory epithelium. Dio2-positive fibrocytes were adjacent to and extended projections around blood capillary networks, the source of T4 supply. Transcriptome analysis of isolated positive cells revealed bone lineage-related origins for many of these cells. Conclusion: The Dio2-CreERt2 model detects Dio2 expression sensitively at cellular resolution. In the cochlea, Dio2-positive cell types reside in vascularized support tissues, suggesting combined endocrine and paracrine-like control of the T3 supply. Analysis of cell origins suggests novel interactions between endocrine and skeletal systems in promoting T3 action required for hearing.

Abstract Background: Thyroid hormone promotes development, growth and metabolism. The level of thyroid hormone ligand (triiodothyronine, T3) in tissues depends not only on circulating levels but also upon tight regulation by activating and inactivating deiodinases within tissues. Type 3 deiodinase (Dio3) inactivates T3 and its precursor thyroxine (T4) and mediates many functions including in neurodevelopmental, sensory and reproductive systems. Dio3 is subject to genomic imprinting. Despite its critical functions, Dio3 is often expressed transiently and at low levels in restricted cell populations making it difficult to detect in natural tissues. Methods: To visualize Dio3 expression at cellular resolution, we derived a Dio3-CreERt2 knockin allele that expresses tamoxifen-dependent Cre recombinase from the endogenous Dio3 gene. When crossed with Ai6 reporter mice, Dio3-CreERt2-positive cells display fluorescent signals. When tamoxifen-treated at neonatal ages, Dio3-CreERt2 recapitulates endogenous Dio3 expression as previously reported in brain: in the bed nucleus of the stria terminalis and preoptic nuclei. In addition, we uncovered several positive cell groups in the hypothalamus, brain stem, pituitary and other tissues. Drastic differences were observed for Dio3-CreERt2 as a paternally versus maternally inherited allele, revealing imprinting effect in specific cell types. Dio3-CreERT2 activity is enhanced by T3 administration, in accordance with Dio3 as a T3-indicible gene. Conclusion: The Dio3-CreERT2 model sensitively reveals Dio3-expressing cell types in tissues. The model is useful for studying expression patterns, imprinting and lineage tracing of Dio3-positive cells during development and homeostatic challenges.