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

Mutation in proteins containing polyglutamine (polyQ) tracts has been shown to underlie a number of severe human neurodegenerative disorders such as Huntington's Disease and Spinocerebellar Ataxia. In this study, we identify and describe FAM171B as a novel polyQ protein containing fourteen consecutive glutamine residues in its National Center for Biotechnology Information (NCBI) referenced sequence. Utilizing western blotting, in situ hybridization, and immunohistochemistry, we demonstrate that FAM171B is widely expressed in mouse brain with pronounced localization in the hippocampus, cerebellum, and cerebral cortex. Furthermore, immunofluorescence experiments reveal that FAM171B predominantly localizes to vesicle-like structures in the cytoplasm of neurons. Finally, bioinformatic analysis suggests that FAM171B is robustly expressed in human brain, and (similar to other polyQ disease genes) its polyQ tract is polymorphic within the general human population. Thus, as a polyQ protein that is expressed in brain, FAM171B should be considered a candidate gene for an as yet molecularly uncharacterized neurodegenerative disease.

Abstract Thyroid hormone (TH) is essential for cochlear development and normal auditory function. Considering the importance of TH in mediating cochlear development, understanding the degree to which developing tissues can adapt to perturbations in thyroid hormone signaling is extremely important. The deiodinases (Dio2 and Dio3) are enzymes that tightly control TH availability at the tissue level and have been proposed to function as adaptive mechanisms that maintain tissue TH homeostasis. Dio2 converts thyroxine (T4) to a biologically active ligand triiodothyronine (T3); locally amplifying a T3 signal. Conversely, Dio3 inactivates T3 and T4 by converting these iodothyronines to the inactive metabolites diiodothyronine (T2) and reverse T3 (rT3), respectively. During cochlear development, Dio3 expression is high prenatally while Dio2 expression is low. During the first postnatal week, the expression levels of these enzymes invert, resulting in high expression of Dio2 and low expression of Dio3. Together, the deiodinases control the timing of postnatal cochlear remodeling; suggesting a genetic developmental clock controls TH-mediated cochlear development. Considering the role deiodinases as adaptive mechanisms, it is important to understand whether such a developmental clock can be negated during times of developmental thyroid hormone insufficiency. We hypothesize that the perinatal change in deiodinase expression is controlled by a developmental clock rather than environmental clues and therefore have limited capacity function as compensatory mechanisms in response to low TH during development. To test this, timed-pregnant mice were treated with thyroid gland inhibitors to induced hypothyroidism from gestational day 12.5 until pup sacrifice. A parallel set of untreated timed-pregnant mice served as controls. Cochlea were harvest from control and hypothyroid mice at postnatal (P) ages P1, P5, P10 and P15 for qRT-PCR, and P7 for in situ hybridization. In situ hybridization for Dio2 mRNA demonstrated a striking reduction in Dio2 mRNA in hypothyroid animals compared to euthyroid animals. Additionally, we observed a significant effect of age (p<0.001) and an interaction between age and treatment (p=0.0035) on Dio2 mRNA levels analyzed by qRT-PCR. Specifically, Dio2 mRNA levels were similar or reduced in hypothyroid animals compared to controls at P1, P5, and P10. This finding indicates that Dio2 mRNA at these ages did not respond in manner consistent with compensation. However, at P15, Dio2 mRNA levels were increased in hypothyroid animals compared to controls; a finding that is consistent with a compensatory mechanism. Taken together, our results suggest that developmental programs in tissues may be dominant over potential compensatory mechanisms and that developing tissues may be more susceptible to perturbations in tissue TH levels due to a reduced capacity to compensate.

It is well known that thyroid hormone plays a key role in amphibian metamorphosis and mammalian tissue remodeling. We and others have reported that treatment with thyroid hormone will prevent or postpone the regression of the adrenal inner cortex (X-zone) and leads to X-zone hypertrophy in mice. The expression of thyroid hormone receptor beta 1 (TRβ1) in the adrenal inner cortex indicates that thyroid hormone could elicit its function directly on this cell population. To support this idea, we examined the expression and the function of major thyroid hormone transporters Mct8, Mct10, and Oatp1c1 in mouse adrenal glands. RNA-seq data showed that the expression levels of Mct10 and Oatp1c1 are relatively low in the adrenal gland (with RPKM less than 5 and 1, respectively) whereas Mct8 is the most abundant transporter (with RPKM around 20). Quantitative RT-PCR showed that Mct8 in the adrenal gland has an expression profile similar to X-zone marker genes and TRβ1 throughout developmental stages. LacZ staining showed similarities between Mct8 and TRβ1 expression, with both specifically expressed in the most inner cortex of the adrenal gland next to the medulla. However, by using thyroid hormone (T3) treatment, we found that the T3-mediated effect in the adrenal gland was not fully blocked in Mct8 knock-out mice. The expression of thyroid hormone transporter Mct8 in the adrenal inner cortex supports our claim that transporters mediate the direct action of thyroid hormone in the adrenal inner cortex. The blunted response to thyroid hormone in Mct8 knockout mice suggests that Mct8 is responsible for the thyroid hormone action in the adrenal gland. However, the deletion of Mct8 does not completely block thyroid hormones effect in the adrenal gland. Our finding suggests other thyroid hormone transporter(s) in the adrenal gland are also responsible for thyroid hormone signaling in the adrenal gland.

Transmembrane proteins that mediate the cellular uptake or efflux of thyroid hormone potentially provide a key level of control over neurodevelopment. In humans, defects in one such protein, solute carrier SLC16A2 (MCT8) are associated with psychomotor retardation. Other proteins that transport the active form of thyroid hormone triiodothyronine (T3) or its precursor thyroxine (T4) have been identified in vitro but the wider significance of such transporters in vivo is unclear. The development of the auditory system requires thyroid hormone and the cochlea is a primary target tissue. We have proposed that the compartmental anatomy of the cochlea would necessitate transport mechanisms to convey blood-borne hormone to target tissues. We report hearing loss in mice with mutations in Slc16a2 and a related gene Slc16a10 (Mct10, Tat1). Deficiency of both transporters results in retarded development of the sensory epithelium similar to impairment caused by hypothyroidism, compounded with a progressive degeneration of cochlear hair cells and loss of endocochlear potential. Administration of T3 largely restores the development of the sensory epithelium and limited auditory function, indicating the T3-sensitivity of defects in the sensory epithelium. The results indicate a necessity for thyroid hormone transporters in cochlear development and function.

More than a century ago, associations between deaf-mutism, cretinism, and goiter were reported. Since then, our understanding for the role of T3 and its receptors in auditory system development has greatly advanced. Much of current understanding is due to the creation of genetic mouse models that have allowed for the dissection of individual components of thyroid hormone action, including the thyroid hormone receptors. This chapter highlights our existing knowledge for the role of T3 and the thyroid hormone receptors, TRα and TRβ, in cochlear development and auditory function that have obtained from these important genetic models. It also discusses auditory deficits in humans with the syndrome of resistance to thyroid hormone caused by mutations in the thyroid hormone receptors. It concludes by briefly discussing where the deficiencies in our understanding of thyroid hormone action in cochlear development remain.

Thyroid function and action in tissues is maintained by dynamic interrelationship between the function of the hypothalamus–pituitary–thyroid axis, serum transport, cellular uptake and metabolism, and elimination via liver conjugation. Disruption of this tight balance during development results in permanent neurological deficits that can be attributed to defects in nervous system development such as neuronal migration in the developing cerebral cortex, gliogenesis and myelination, cerebellar maturation, and cochlear maturation and auditory function. Considering this, there is concern that environmental chemicals can target the thyroid gland and perturb thyroid hormone-mediated development. Over the past few decades, studies in humans and experimental animals have provided strong evidence that chemicals released into the environment can perturb the thyroid system. Therefore, the goal of this chapter is to articulate the complexities of thyroid hormone (TH) action in development and assimilate our current understanding of how thyroid toxicants may disrupt brain development. It begins by discussing the regulation of TH action and then addresses the potential points of thyroid disruption. Finally, it concludes by discussing the consequences of disruption of TH action by thyroid toxicants on the developing brain, by focusing on well-understood TH-mediated developmental processes.

BACKGROUND An in-depth understanding of the fundamental principles that regulate thyroid hormone homeostasis is critical for the development of new diagnostic and treatment approaches for patients with thyroid disease. SUMMARY Important clinical practices in use today for the treatment of patients with hypothyroidism, hyperthyroidism, or thyroid cancer are the result of laboratory discoveries made by scientists investigating the most basic aspects of thyroid structure and molecular biology. In this document, a panel of experts commissioned by the American Thyroid Association makes a series of recommendations related to the study of thyroid hormone economy and action. These recommendations are intended to promote standardization of study design, which should in turn increase the comparability and reproducibility of experimental findings. CONCLUSIONS It is expected that adherence to these recommendations by investigators in the field will facilitate progress towards a better understanding of the thyroid gland and thyroid hormone dependent processes.

TTLL5/STAMP (tubulin tyrosine ligase-like family member 5) has multiple activities in cells. TTLL5 is one of 13 TTLLs, has polyglutamylation activity, augments the activity of p160 coactivators (SRC-1 and TIF2) in glucocorticoid receptor-regulated gene induction and repression, and displays steroid-independent growth activity with several cell types. To examine TTLL5/STAMP functions in whole animals, mice were prepared with an internal deletion that eliminated several activities of the Stamp gene. This mutation causes both reduced levels of STAMP mRNA and C-terminal truncation of STAMP protein. Homozygous targeted mutant (Stamp(tm/tm)) mice appear normal except for marked decreases in male fertility associated with defects in progressive sperm motility. Abnormal axonemal structures with loss of tubulin doublets occur in most Stamp(tm/tm) sperm tails in conjunction with substantial reduction in alpha-tubulin polyglutamylation, which closely correlates with the reduction in mutant STAMP mRNA. The axonemes in other structures appear unaffected. There is no obvious change in the organs for sperm development of WT versus Stamp(tm/tm) males despite the levels of WT STAMP mRNA in testes being 20-fold higher than in any other organ examined. This defect in male fertility is unrelated to other Ttll genes or 24 genes previously identified as important for sperm function. Thus, STAMP appears to participate in a unique, tissue-selective TTLL-mediated pathway for alpha-tubulin polyglutamylation that is required for sperm maturation and motility and may be relevant for male fertility.

Thyroid hormone serves many functions throughout brain development, but the mechanisms that control the timing of its actions in specific brain regions are poorly understood. In the cerebellum, thyroid hormone controls formation of the transient external germinal layer, which contains proliferative granule cell precursors, subsequent granule cell migration, and cerebellar foliation. We report that the thyroid hormone-inactivating type 3 deiodinase (encoded by Dio3) is expressed in the mouse cerebellum at embryonic and neonatal stages, suggesting a need to protect cerebellar tissues from premature stimulation by thyroid hormone. Dio3(-/-) mice displayed reduced foliation, accelerated disappearance of the external germinal layer, and premature expansion of the molecular layer at juvenile ages. Furthermore, Dio3(-/-) mice exhibited locomotor behavioral abnormalities and impaired ability in descending a vertical pole. To ascertain that these phenotypes resulted from inappropriate exposure to thyroid hormone, thyroid hormone receptor α1 (TRα1) was removed from Dio3(-/-) mice, which substantially corrected the cerebellar and behavioral phenotypes. Deletion of TRα1 did not correct the previously reported small thyroid gland or deafness in Dio3(-/-) mice, indicating that Dio3 controls the activation of specific receptor isoforms in different tissues. These findings suggest that type 3 deiodinase constrains the timing of thyroid hormone action during cerebellar development.

Here we present the Transcription Factor Encyclopedia (TFe), a new web-based compendium of mini review articles on transcription factors (TFs) that is founded on the principles of open access and collaboration. Our consortium of over 100 researchers has collectively contributed over 130 mini review articles on pertinent human, mouse and rat TFs. Notable features of the TFe website include a high-quality PDF generator and web API for programmatic data retrieval. TFe aims to rapidly educate scientists about the TFs they encounter through the delivery of succinct summaries written and vetted by experts in the field. TFe is available at http://www.cisreg.ca/tfe .

Genes Cluster ID name T6/T3 T8/T3 T11/T3 T24/T3 gene name function CIBE_0313 6up++ 5.3 6.2 7.2 5.8 conserved protein of unknown function CIBE_0312 6up++ 5.0 6.3 7.4 5.6 conserved protein of unknown function CIBE_0311 6up++ 5.0 6.4 7.0 5.3 conserved protein of unknown function CIBE_0314 6up++ 4.1 5.9 7.0 5.8 conserved protein of unknown function CIBE_0315 6up++ 5.1 5.9 6.1 5.0 Radical SAM-family protein CIBE_0310 6up++ 4.7 6.3 5.9 4.6 putative ABC transporter ATP-binding protein CIBE_3477 6up++ 5.5 5.6 5.4 4.4 Accessory gene regulator B CIBE_3478 6up++ 5.3 5.8 5.0 4.3 conserved membrane protein of unknown function CIBE_0316 6up++ 4.5 5.5 5.6 4.2 conserved protein of unknown function CIBE_4430 6up++ 4.4 6.4 5.4 3.6 putative permease CIBE_4429 6up++ 3.7 5.7 4.8 3.5 putative permease CIBE_0280 6up++ 4.5 4.9 4.3 3.9 conserved exported protein of unknown function CIBE_3828 6up++ 4.4 5.6 4.4 3.2 Glycosyl transferase CIBE_4431 6up++ 2.7 5.4 5.5 3.6 conserved exported protein of unknown function CIBE_4419 6up++ 3.7 5.5 4.4 3.1 conserved exported protein of unknown function CIBE_4428 6up++ 3.1 5.4 4.7 3.2 glr Transporter CIBE_4418 6up++ 3.4 5.4 4.4 2.4 conserved exported protein of unknown function CIBE_3476 6up+ 4.4 4.6 4.3 3.6 conserved protein of unknown function CIBE_3751 6up+ 3.8 5.2 3.8 3.1 conserved protein of unknown function CIBE_3750 6up+ 3.9 4.6 3.7 3.4 conserved protein of unknown function CIBE_4789 6up+ 3.7 4.3 3.9 3.5 cfg putative AgrB-like protein CIBE_0824 6up+ 4.8 3.9 3.8 2.7 Type 11 methyltransferase CIBE_3749 6up+ 4.1 4.4 3.5 2.8 Peptide synthetase CIBE_5303 6up+ 4.6 4.0 3.1 2.7 conserved protein of unknown function CIBE_2622 6up+ 2.8 3.7 3.9 4.0 adh NADPH-dependent butanol dehydrogenase CIBE_4040 6up+ 3.0 3.3 4.1 3.7 Aliphatic sulfonates family ABC transporter, periplsmic ligand-binding protein CIBE_3079 6up+ 4.3 3.5 3.3 2.8 panD aspartate 1-decarboxylase CIBE_4039 6up+ 2.9 3.2 3.9 4.1 tauB taurine transporter subunit ; ATP-binding component of ABC superfamily CIBE_3082 6up+ 4.2 3.4 3.0 2.9 conserved protein of unknown function CIBE_4610 6up+ 2.1 4.1 4.1 3.3 Peptidase C1A, papain CIBE_4417 6up+ 3.1 4.7 3.5 2.2 conserved membrane protein of unknown function CIBE_3475 6up+ 3.5 3.7 3.4 2.8 Histidine kinase CIBE_4036 6up+ 2.7 3.0 3.9 3.7 Thioredoxin reductase CIBE_3355 6up+ 3.8 3.0 3.1 3.3 RNA polymerase sigma factor SigI CIBE_3081 6up+ 4.1 3.4 2.9 2.8 panB ketopantoate hydroxymethyltransferase CIBE_4038 6up+ 2.8 2.8 3.8 3.7 Type I phosphodiesterase/nucleotide pyrophosphatase CIBE_2623 6up+ 2.6 3.3 3.8 3.3 conserved protein of unknown function CIBE_5304 6up+ 4.3 3.6 2.7 2.4 conserved protein of unknown function CIBE_3537 6up+ 2.9 4.5 2.5 3.1 putative transcriptional regulator CIBE_5707 6up+ 3.0 3.4 3.6 2.9 conserved protein of unknown function CIBE_0782 6up+ 2.2 2.8 3.7 4.1 Nitrogenase CIBE_4041 6up+ 2.3 3.1 3.8 3.6 tauC taurine transporter subunit ; membrane component of ABC superfamily CIBE_2624 6up+ 2.2 3.3 3.8 3.5 sudA Sulfide dehydrogenase subunit alpha CIBE_3080 6up+ 4.0 3.3 3.1 2.3 panC pantothenate synthetase CIBE_3353 6up+ 3.5 2.7 2.7 3.4 Beta-glucanase (fragment) CIBE_4861 6up+ 1.9 3.8 3.2 3.4 conserved protein of unknown function CIBE_3700 6up+ 1.2 3.3 3.9 3.9 YIEGIA protein CIBE_3753 6up+ 3.1 3.6 2.4 3.1 protein of unknown function CIBE_3354 6up+ 3.8 2.9 2.6 2.8 bglA Beta-glucanase CIBE_3748 6up+ 3.7 3.7 2.7 2.0 putative cyclic peptide transporter CIBE_4416 6up+ 3.2 4.3 3.2 1.2 conserved membrane protein of unknown function CIBE_1176 6up+ 4.3 3.3 2.6 1.7 rplY 50S ribosomal protein L25 CIBE_4788 6up+ 3.2 3.5 2.6 2.4 CHAP domain containing protein CIBE_3357 6up+ 4.2 2.8 2.3 2.5 Carbohydrate-binding family V/XII CIBE_2891 6up+ 3.7 3.1 2.3 2.6 Electron transport complex, RnfABCDGE type, D subunit CIBE_6013 6up+ 4.4 3.5 1.9 1.7 conserved protein of unknown function CIBE_0281 6up+ 3.7 3.9 3.3 0.7 conserved membrane protein of unknown function CIBE_4862 6up+ 1.8 3.3 3.1 3.3 2-hydroxyglutaryl-CoA dehydratase CIBE_5605 6up+ 2.2 2.8 3.5 3.0 conserved protein of unknown function CIBE_2892 6up+ 3.8 3.0 2.2 2.3 Electron transport complex, RnfABCDGE type, G subunit CIBE_5208 6up+ 3.0 2.9 3.1 2.4 Nucleotidyl transferase CIBE_0725 6up+ 2.5 2.9 3.1 2.5 cstA carbon starvation-induced membrane protein CIBE_3837 6up+ 3.0 3.7 2.1 2.2 yqaJ putative nuclease; skin element CIBE_4414 6up+ 2.7 3.7 2.8 1.7 Cell wall-associated hydrolase-like protein CIBE_4037 6up+ 2.2 2.3 3.5 2.8 Thioredoxin domain CIBE_5606 6up+ 2.2 2.7 3.2 2.8 Membrane spanning protein CIBE_3761 6up+ 3.7 2.9 2.4 1.4 Multidrug transporter MatE CIBE_3441 6up+ 1.6 3.4 3.1 2.4 ABC transporter substrate-binding protein CIBE_2221 6up+ 1.1 3.7 3.5 2.0 ytfJ Uncharacterized spore protein YtfJ CIBE_2951 6up+ 1.4 3.5 2.8 2.3 conserved protein of unknown function CIBE_2222 6up+ 1.4 3.5 3.0 1.6 conserved membrane protein of unknown function CIBE_3534 6up+ 1.4 3.3 2.3 2.0 putative DnaD-like protein, phage replisome organizer functional annotation genes

Here we present the Transcription Factor Encyclopedia (TFe), a new web-based compendium of mini review articles on transcription factors (TFs) that is founded on the principles of open access and collaboration. Our consortium of over 100 researchers has collectively contributed over 130 mini review articles on pertinent human, mouse and rat TFs. Notable features of the TFe website include a high-quality PDF generator and web API for programmatic data retrieval. TFe aims to rapidly educate scientists about the TFs they encounter through the delivery of succinct summaries written and vetted by experts in the field. TFe is available at http://www.cisreg.ca/ tfe.

The typical mammalian visual system is based upon three photoreceptor types: rods for dim light vision and two types of cones (M and S) for color vision in daylight. However, the process that generates photoreceptor diversity and the cell type in which diversity arises remain unclear. Mice deleted for thyroid hormone receptor β2 (TRβ2) and neural retina leucine zipper factor (NRL) lack M cones and rods, respectively, but gain S cones. We therefore tested the hypothesis that NRL and TRβ2 direct a common precursor to a rod, M cone, or S cone outcome using Nrlb2/b2 “knock-in” mice that express TRβ2 instead of NRL from the endogenous Nrl gene. Nrlb2/b2 mice lacked rods and produced excess M cones in contrast to the excess S cones in Nrl−/− mice. Notably, the presence of both factors yielded rods in Nrl+/b2 mice. The results demonstrate innate plasticity in postmitotic rod precursors that allows these cells to form three functional photoreceptor types in response to NRL or TRβ2. We also detected precursor cells in normal embryonic retina that transiently coexpressed Nrl and TRβ2, suggesting that some precursors may originate in a plastic state. The plasticity of the precursors revealed in Nrlb2/b2 mice suggests that a two-step transcriptional switch can direct three photoreceptor fates: first, rod versus cone identity dictated by NRL, and second, if NRL fails to act, M versus S cone identity dictated by TRβ2.