Concomitant Nrf2- and ATF4-activation by Carnosic Acid Cooperatively Induces Expression of Cytoprotective Genes.

: Carnosic acid (CA) is a phytochemical found in some dietary herbs, such as Rosmarinus officinalis L., and possesses antioxidative and anti-microbial properties. We previously demonstrated that CA functions as an activator of nuclear factor, erythroid 2 (NF-E2)-related factor 2 (Nrf2), an oxidative stress-responsive transcription factor in human and rodent cells. CA enhances the expression of nerve growth factor (NGF) and antioxidant genes, such as HO-1 in an Nrf2-dependent manner in U373MG human astrocytoma cells. However, CA also induces NGF gene expression in an Nrf2-independent manner, since 50 µM of CA administration showed striking NGF gene induction compared with the classical Nrf2 inducer tert-butylhydroquinone (tBHQ) in U373MG cells. By comparative transcriptome analysis, we found that CA activates activating transcription factor 4 (ATF4) in addition to Nrf2 at high doses. CA activated ATF4 in phospho-eIF2α- and heme-regulated inhibitor kinase (HRI)-dependent manners, indicating that CA activates ATF4 through the integrated stress response (ISR) pathway. Furthermore, CA activated Nrf2 and ATF4 cooperatively enhanced the expression of NGF and many antioxidant genes while acting independently to certain client genes. Taken together, these results represent a novel mechanism of CA-mediated gene regulation evoked by Nrf2 and ATF4 cooperation.

Chronic subcutaneous infusion of neurosecretory protein GM increases body mass gain in chicks.

Recently we discovered a small hypothalamic protein in the chicken, named neurosecretory protein GL (NPGL), which is associated with body growth and energy metabolism in birds and rodents. Genome database analysis suggested that the NPGL gene has a paralogous gene in vertebrates, named neurosecretory protein GM (NPGM). However, the biological action of NPGM remains unclear. In this study, we investigated whether NPGM affects body growth in chicks. We found that subcutaneous infusion of NPGM for six days increased body mass gain in a dose-dependent manner. Despite the observed increase in body mass, infusion of NPGM did not alter food and water intake. Of note, we observed tendency of mass increase of several peripheral tissues, specifically. When we compared several tissue types, NPGM seemed to induce the largest growth increase in white adipose tissue mass. These results suggest that NPGM may accelerate fat accumulation and body growth. In addition, we analyzed whether NPGM increases body growth through the action of pituitary hormones. However, we observed no significant changes in mRNA expression of pituitary hormones or plasma levels of growth hormone in NPGM-treated chicks. This is the first report describing the biological action of NPGM in vertebrates.

Identification of a cDNA encoding a novel small secretory protein, neurosecretory protein GL, in the chicken hypothalamic infundibulum.

To find novel neuropeptide and/or peptide hormone precursors in the avian brain, we performed a cDNA subtractive screen of the chicken hypothalamic infundibulum, which contains one of the feeding and neuroendocrine centers. After sequencing 596 clones, we identified a novel cDNA encoding a previously unknown protein. The deduced precursor protein consisted of 182 amino acid residues, including one putative small secretory protein of 80 amino acid residues. This small protein was flanked at the N-terminus by a signal peptide and at the C-terminus by a glycine amidation signal and a dibasic amino acid cleavage site. Because the predicted C-terminal amino acids of the small protein were Gly-Leu-NH2, the small protein was named neurosecretory protein GL (NPGL). Quantitative RT-PCR analysis demonstrated specific expression of the NPGL precursor mRNA in the hypothalamic infundibulum. Furthermore, the mRNA levels in the hypothalamic infundibulum increased during post-hatching development. In situ hybridization analysis showed that the cells containing the NPGL precursor mRNA were localized in the medial mammillary nucleus and infundibular nucleus within the hypothalamic infundibulum of 8- and 15-day-old chicks. Subcutaneous infusion of NPGL in chicks increased body weight gain without affecting food intake. To our knowledge, this is the first report to describe the identification and localization of the NPGL precursor mRNA and the function of its translated product in animals. Our findings indicate that NPGL may participate in the growth process in chicks.

Identification of neurotensin and LANT-6 and localization of mRNA encoding their precursor in the chicken brain.

Neurotensin (NT) and neurotensin-related peptide (Lys(8), Asn(9), NT(8-13): LANT-6) have previously been purified from chicken intestine. However, the presence of these peptides and the localization of their precursor mRNA in the brain were not well understood. In the present study, through a comprehensive analysis of bioactive substances, NT and LANT-6 were identified in the chicken brain using tandem mass spectrometry combined with a bioassay of the colon contraction. The effect of NT and LANT-6 on the colon contraction was assessed, and NT was found to be 10 times more potent than LANT-6. Furthermore, the sites of NT/LANT-6 precursor mRNA expression in the brain were investigated using quantitative RT-PCR. The result showed that the mRNA was expressed most in the telencephalon, followed by the diencephalon. In situ hybridization analysis revealed that cells containing NT/LANT-6 precursor mRNA were widely distributed throughout the brain except for the cerebellum. Additionally, these were highly concentrated in the frontal telencephalon, including the nidopallium, hyperpallium, and hippocampus. Collectively, these results indicate that NT and LANT-6 are produced in the chicken brain, and they may participate in multiple functions.

IGF-1 gene expression is differentially regulated by estrogen receptors α and ß in mouse endometrial stromal cells and ovarian granulosa cells.

Insulin-like growth factor 1 (IGF-1) is involved in regulations of reproductive functions in rats and mice. IGF-1 expression is regulated by estrogen in several reproductive organs including the uterus and ovary. Two types of estrogen receptor (ERα and ERß) are expressed in mouse uteri and ovaries, and it is unclear whether they differently mediate IGF-1 gene transcription. To clarify the roles of ERα and ERß, mouse endometrial stromal cells and ovarian granulosa cells were treated with ligands specific for individual estrogen receptors. In endometrial stromal cells, propyl-pyrazole-triol (PPT; ERα-selective agonist) increased Igf1 mRNA expression, which was suppressed by methyl-piperidino-pyrazole (MPP, ERα-selective antagonist), while diarylpropionitrile (DPN, ERß-potency selective agonist) increased Igf1 mRNA expression, which was inhibited by MPP but not by 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-α]pyrimidin-3-yl]phenol (PHTPP; ERß antagonist). PHTPP enhanced the DPN-induced increase in Igf1 mRNA expression. In ovarian granulosa cells, E2 and DPN decreased Igf1 mRNA expression, whereas PPT did not affect Igf1 mRNA levels. In these cells, PHTPP inhibited the DPN-induced decrease in Igf1 mRNA expression. These results suggest that ERα facilitates Igf1 transcription, whereas ERß appears to inhibit Igf1 gene transcription in mouse endometrial stromal cells and ovarian granulosa cells.

Transforming growth factor-α mRNA expression and its possible roles in mouse endometrial stromal cells.

Transforming growth factor-α (TGFα) is thought to be involved in the regulation of endometrial cells. We investigated Tgfa mRNA expression, and the effects of TGFα on DNA-synthesis and gene expression of insulin-like growth factor 1 (IGF1), IGF binding protein-3 (IGFBP3) and IGF1 receptor in the mouse endometrial cells, because IGF1 is involved in estrogen-induced growth of endometrial cells. We also investigated the role of TGFα on matrix metalloproteinase (MMP) expression, as MMPs are involved both in tissue remodeling during cell proliferation and in enhancement of IGF1 signaling through the degradation of IGFBP3. Tgfa mRNA expression was detected in endometrial luminal and glandular epithelial cells, and stromal cells. Tgfa mRNA signals did not appear to change in endometrial luminal epithelial cells, but signals in glandular epithelial cells were higher at diestrus 1, 2 and proestrus, and the number of stromal cells showing strong signals appeared to increase at diestrus 1 and 2. Endometrial epithelial and stromal cells were treated with estradiol-17ß (E2) or progesterone (P4). E2 or P4 stimulated Tgfa mRNA expression in stromal cells. TGFα stimulated DNA synthesis in endometrial epithelial and stromal cells, while E2 and P4 stimulated DNA synthesis in stromal cells. In stromal cells, TGFα, at as low as 1 ng/ml, decreased Igfbp3 and Mmp9 mRNA levels, while high dose (10 ng/ml) of TGFα decreased Igf1 mRNA level and increased Mmp3 mRNA level. These results imply that TGFα stimulates proliferation of endometrial stromal cells through multiple mechanisms, including its regulation of Igfbp3 and Mmp3 transcription.

Pit-1w may regulate prolactin gene expression in mouse testis.

Pit-1 is a POU-domain transcription factor that promotes growth hormone (GH), prolactin (PRL), and thyroid-stimulating hormone ß subunit (TSHß) gene expression in the pituitary gland. Alternative splicing of Pit-1 gene transcripts has been shown to give rise to several variants with discrete transactivation properties. Recently, we identified a mouse Pit-1 w that is generated by alternative promoter usage and is expressed in a variety of tissues including the testis. Using a combination of reverse-transcription polymerase chain reaction analyses and luciferase reporter gene assays, we investigated the possible role of Pit-1 w in the mouse testis. In postnatal testicular development, the expression of Pit-1 w mRNA was significantly up-regulated between 18 and 20 days after birth when the numbers of secondary spermatocytes and spermatids have been reported to increase in mice. The PRL mRNA, but not the mRNAs for GH or TSHß, showed intratesticular expression patterns that were similar to those of the Pit-1 w mRNA. In experimental unilaterally cryptorchid testes of adult mice, spermatid numbers were extremely low and the expression levels of both the Pit-1 w and PRL mRNAs dropped dramatically. Furthermore, in the luciferase reporter gene assays, we found that Pit-1 w specifically transactivated the PRL promoter but had no effect on the promoters of GH or TSHß. These results suggested that Pit-1 w could be involved in the paracrine/autocrine system in mice and may be necessary for normal testicular function via its possible role in regulating PRL expression in testicular germ cells. This is the first report demonstrating the possible role of Pit-1 w in mammals.

Elaborate color patterns of individual chicken feathers may be formed by the agouti signaling protein.

Hair and feather pigmentation is mainly determined by the distribution of two kinds of melanin, eumelanin and pheomelanin, which produce brown to black and yellow to red colorations, respectively. The agouti signaling protein (ASIP) acts as an antagonist or an inverse agonist of the melanocortin 1 receptor (MC1R), a G protein-coupled receptor for α-melanocyte-stimulating hormone (α-MSH). This antagonism of the MC1R by ASIP on melanocytes initiates a switch of melanin synthesis from eumelanogenesis to pheomelanogenesis in mammals. In the present study, we isolated multiple ASIP mRNA variants generated by alternative splicing and promoters in chicken feather follicles. The mRNA variants showed a discrete tissue distribution. However, mRNAs were expressed predominantly in the feather pulp of follicles. Paralleling mRNA distribution, ASIP immunoreactivity was observed in feather pulp. Interestingly, ASIP was stained with pheomelanin but not eumelanin in pulp areas that face developing barbs. We suggest that the elaborate color pattern of individual feathers is formed in part by the antagonistic action of ASIP that is produced by multiple mRNA variants in chicken feather follicles.

Runx3 expression and its roles in mouse endometrial cells.

Runx3 is a transcription factor that belongs to the Runx family. We studied the localization of Runx3 mRNA in the mouse uterus, and its function in the mouse endometrium using Runx3 knockout (Runx3(-/-)) mice. Runx3 mRNA was detected in the endometrial luminal epithelial cells, glandular epithelial cells and stromal cells below the epithelial cell layer on the luminal side. The uteri of Runx3(-/-) mice were smaller than those of wt mice. The endometrial layer and uterine glands of Runx3(-/-) mice were less developed than those of wild-type mice, and the endometrial stromal layer was thinner. Transforming growth factor ß1 and ß3 (TGFß1 and ß3) mRNA levels in endometrial stromal cells of Runx3(-/-) mice were low compared with those of wild-type mice. Estradiol-17ß (E2) increased Tgfb2 mRNA levels in endometrial stromal cells of Runx3(-/-) mice, but not in those of wild-type mice. E2 increased epidermal growth factor (EGF) mRNA levels in endometrial stromal cells of wild-type mice, but did not increase those of Runx3(-/-) mice. The diminished Tgfb1 and Tgfb3 mRNA expressions may lead to the reduced proliferation of endometrial stromal cells. Alterations of E2-associated expressions of Tgfb2 and Egf mRNA in endometrial stromal cells of Runx3(-/-) mice may be associated with suppression of E2-dependent endometrial epithelial cell proliferation in Runx3(-/-) mice. Thus, Runx3 is likely to be a regulatory factor responsible for endometrial growth.

Identification of mammalian Pit-1w, possibly involved in spermatogenesis in mice.

Pit-1 is a pituitary-specific transcription factor responsible for pituitary development and hormone expression in mammals. Alternative splicing of Pit-1 gene transcripts has been shown to give rise to several variants with discrete transactivation properties; however, those arising from alternative promoters such as avian Pit-1 w have not yet been identified in mammals. Here, comparative genomics analysis followed by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of 5' cDNA ends (5'RACE) were used in identifying Pit-1 w mRNA in the mouse pituitary. The mouse Pit-1 w mRNA is generated by using an alternative promoter located in the first intron, as with chicken Pit-1 w, and is expressed in a wide variety of tissues besides the pituitary. In the testis, Pit-1 w is expressed as the predominant variant and a protein of 33 kDa. During the first wave of spermatogenesis, expression of Pit-1 w mRNA at substantial levels was observed from 3 weeks, but not at 1 or 2 weeks after birth. A combination of immunohistochemistry and in situ hybridization detected Pit-1 mRNA and Pit-1 immunoreactivity in the spermatogonia, spermatocytes, and spermatids in the testis of adult mice. Because secondary spermatocytes and haploid spermatids increase in number between 18 and 20 days after birth in mice, it is possible that mouse Pit-1 w plays a role in spermatogenesis. This is the first report demonstrating the expression of Pit-1 variants arising from alternative promoters in mammals.

Role of chicken Pit-1 isoforms in activating growth hormone gene.

In the present study, we expressed chicken (ch) Pit-1α (chPit-1α) and chPit-1γin vitro to compare the roles of chPit-1s in the transcription of the chicken growth hormone (chGH) gene. Both green fluorescence protein (GFP)-fused chPit-1γ and GFP-fused chPit-1α were localized in the nuclei of COS-7 cells. In a luciferase reporter gene assay, both chPit-1α and chPit-1γ transactivated the chGH promoter, and chPit-1α showed a more potent effect than chPit-1γ. On the other hand, an increase of cellular cAMP induced by forskolin promoted transactivation of the chGH gene with chPit-1α and chPit-1γ to similar extents. These results suggest that chPit-1γ may modulate the basal promoter activity of the chGH gene to the same degree as chPit-1α; however, a structural difference observed at the N-terminus transactivation domains in chPit-1α and chPit-1γ could be associated with the efficiency of basal activation of the chGH promoter.

Feather follicles express two classes of pro-opiomelanocortin (POMC) mRNA using alternative promoters in chickens.

Feather coloration in chickens mainly depends on melanin produced by melanocytes located in the feather follicles. The melanocortin 1 receptor (MC1R) on follicular melanocytes regulates melanin synthesis; however, the source of the melanocortins that interact with the receptors remains unclear. In this study, we examine the potential expression of melanocortins and characterize the mRNAs for the precursor pro-opiomelanocortin (POMC) in chicken feather follicles. Reverse transcription-polymerase chain reaction (RT-PCR) revealed the expression of mRNAs for POMC, prohormone convertase 1 (PC1) and PC2, and western blotting detected adrenocorticotropic hormone (ACTH)-related products of POMC processing in feather follicles, suggesting that melanocortins are produced locally in the tissues of chickens. A combination of 5'RACE (rapid amplification of cDNA 5' end), 3'RACE and RT-PCR analyzes identified two classes of POMC mRNA, class a and class b, which encode the same full-length POMC protein but have different non-coding leader exons. Class a mRNAs were expressed specifically in feather follicles, whereas class b mRNAs were expressed in the pituitary, hypothalamus, and various peripheral tissues that we examined. Within the feather follicles, the class a mRNAs were distributed in epidermal layers from middle to distal locations, whereas the class b mRNAs were mainly expressed in pulp at proximal locations. Our findings suggest that feather pigmentation is regulated by locally produced melanocortins, and indicate that the melanocortins encoded by the different classes of POMC mRNAs may play different intra-follicular roles in chickens. This is the first report that demonstrates alternative promoter usage generating different full-length POMC mRNAs in vertebrates.

The ATF6ß-calreticulin axis promotes neuronal survival under endoplasmic reticulum stress and excitotoxicity.

While ATF6α plays a central role in the endoplasmic reticulum (ER) stress response, the function of its paralogue ATF6ß remains elusive, especially in the central nervous system (CNS). Here, we demonstrate that ATF6ß is highly expressed in the hippocampus of the brain, and specifically regulates the expression of calreticulin (CRT), a molecular chaperone in the ER with a high Ca2+-binding capacity. CRT expression was reduced to ~ 50% in the CNS of Atf6b-/- mice under both normal and ER stress conditions. Analysis using cultured hippocampal neurons revealed that ATF6ß deficiency reduced Ca2+ stores in the ER and enhanced ER stress-induced death. The higher levels of death in Atf6b-/- neurons were recovered by ATF6ß and CRT overexpressions, or by treatment with Ca2+-modulating reagents such as BAPTA-AM and 2-APB, and with an ER stress inhibitor salubrinal. In vivo, kainate-induced neuronal death was enhanced in the hippocampi of Atf6b-/- and Calr+/- mice, and restored by administration of 2-APB and salubrinal. These results suggest that the ATF6ß-CRT axis promotes neuronal survival under ER stress and excitotoxity by improving intracellular Ca2+ homeostasis.

Nanosecond pulsed electric fields induce the integrated stress response via reactive oxygen species-mediated heme-regulated inhibitor (HRI) activation.

The integrated stress response (ISR) is one of the most important cytoprotective mechanisms and is integrated by phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α). Four eIF2α kinases, heme-regulated inhibitor (HRI), double-stranded RNA-dependent protein kinase (PKR), PKR-like endoplasmic reticulum kinase (PERK), and general control nonderepressible 2 (GCN2), are activated in response to several stress conditions. We previously reported that nanosecond pulsed electric fields (nsPEFs) are a potential therapeutic tool for ISR activation. In this study, we examined which eIF2α kinase is activated by nsPEF treatment. To assess the responsible eIF2α kinase, we used previously established eIF2α kinase quadruple knockout (4KO) and single eIF2α kinase-rescued 4KO mouse embryonic fibroblast (MEF) cells. nsPEFs 70 ns in duration with 30 kV/cm electric fields caused eIF2α phosphorylation in wild-type (WT) MEF cells. On the other hand, nsPEF-induced eIF2α phosphorylation was completely abolished in 4KO MEF cells and was recovered by HRI overexpression. CM-H2DCFDA staining showed that nsPEFs generated reactive oxygen species (ROS), which activated HRI. nsPEF-induced eIF2α phosphorylation was blocked by treatment with the ROS scavenger N-acetyl-L-cysteine (NAC). Our results indicate that the eIF2α kinase HRI is responsible for nsPEF-induced ISR activation and is activated by nsPEF-generated ROS.

Cell-based HTS identifies a chemical chaperone for preventing ER protein aggregation and proteotoxicity.

The endoplasmic reticulum (ER) is responsible for folding secretory and membrane proteins, but disturbed ER proteostasis may lead to protein aggregation and subsequent cellular and clinical pathologies. Chemical chaperones have recently emerged as a potential therapeutic approach for ER stress-related diseases. Here, we identified 2-phenylimidazo[2,1-b]benzothiazole derivatives (IBTs) as chemical chaperones in a cell-based high-throughput screen. Biochemical and chemical biology approaches revealed that IBT21 directly binds to unfolded or misfolded proteins and inhibits protein aggregation. Finally, IBT21 prevented cell death caused by chemically induced ER stress and by a proteotoxin, an aggression-prone prion protein. Taken together, our data show the promise of IBTs as potent chemical chaperones that can ameliorate diseases resulting from protein aggregation under ER stress.

PERK-mediated translational control is required for collagen secretion in chondrocytes.

As chondrocytes are highly secretory and they experience a variety of stresses, physiological unfolded protein response (UPR) signalling is essential for extracellular matrix (ECM) secretion and chondrogenesis. In the three branches of the UPR pathway, PERK governs the translational attenuation and transcriptional upregulation of amino acid and redox metabolism and induction of apoptosis. It was previously demonstrated that a defect of the PERK branch of the UPR signalling pathway causes the accumulation of unfolded proteins, leading to cell death without perturbing endoplasmic reticulum (ER)-to-Golgi transport in pancreatic ß cells. However, little is known about the role of PERK in chondrocytes. In this study, we found that PERK signalling is activated in chondrocytes, and inhibition of PERK reduces collagen secretion despite causing excessive collagen synthesis in the ER. Perk -/- mice displayed reduced collagen in articular cartilage but no differences in chondrocyte proliferation or apoptosis compared to the findings in wild-type mice. PERK inhibition increases misfolded protein levels in the ER, which largely hinder ER-to-Golgi transport. These results suggest that the translational control mediated by PERK is a critical determinant of ECM secretion in chondrocytes.

Localization and function of neurosecretory protein GM, a novel small secretory protein, in the chicken hypothalamus.

Recently, we discovered a novel cDNA encoding the precursor of a small secretory protein, neurosecretory protein GL (NPGL), in the hypothalamic infundibulum of chickens. NPGL plays an important role in the regulation of growth and feeding. A database search indicated that the NPGL gene has a paralogous gene: neurosecretory protein GM (NPGM), also in chickens. We identified cDNA encoding the NPGM precursor in chickens. Morphological analysis showed that NPGM-containing cells are specifically localized in the medial mammillary nucleus (MM) and infundibular nucleus (IN) in the hypothalamus. In addition, we found that NPGM and NPGL are co-localized, especially in the MM. The expression levels of NPGM mRNA gradually decreased during post-hatch development, in contrast to those of NPGL mRNA. Moreover, we investigated the relationship between NPGM and other known factors. NPGM was found to be produced in histaminergic neurons in the MM. NPGM and histidine decarboxylase, a histamine-producing enzyme, displayed similar expression patterns during post-hatch development. Acute intracerebroventricular injection of NPGM decreased food intake, similar to the effect of histamine. To our knowledge, this is the first report of the localization and function of NPGM in the brain of vertebrates. These results will further advance the understanding mechanisms underlying energy homeostasis.

Author Correction: Localization and function of neurosecretory protein GM, a novel small secretory protein, in the chicken hypothalamus.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Neurosecretory protein GL stimulates food intake, de novo lipogenesis, and onset of obesity.

Mechanisms underlying the central regulation of food intake and fat accumulation are not fully understood. We found that neurosecretory protein GL (NPGL), a newly-identified neuropeptide, increased food intake and white adipose tissue (WAT) in rats. NPGL-precursor gene overexpression in the hypothalamus caused increases in food intake, WAT, body mass, and circulating insulin when fed a high calorie diet. Intracerebroventricular administration of NPGL induced de novo lipogenesis in WAT, increased insulin, and it selectively induced carbohydrate intake. Neutralizing antibody administration decreased the size of lipid droplets in WAT. Npgl mRNA expression was upregulated by fasting and low insulin levels. Additionally, NPGL-producing cells were responsive to insulin. These results point to NPGL as a novel neuronal regulator that drives food intake and fat deposition through de novo lipogenesis and acts to maintain steady-state fat level in concert with insulin. Dysregulation of NPGL may be a root cause of obesity.

Molecular cloning, tissue distribution, and ontogenic thyroidal expression of the chicken thyrotropin receptor.

TSH and the interaction with its receptor (TSHR) in the thyroid gland play a crucial role in the pituitary-thyroid axis of all vertebrates. Released upon stimulation by TSH, thyroid hormones influence numerous processes in the body and are extremely important during the last week of chicken embryonic development. In this study, we have cloned and functionally characterized the chicken TSHR (cTSHR), which was found to be a G protein-coupled receptor consisting of 10 exons. Besides the full-length cDNA, we detected two splice variants lacking either exon 3, or exons 2 and 3, both part of the extracellular domain of the receptor. Bovine TSH increased intracellular cAMP levels in HEK-239 cells transiently expressing the full-length cTSHR (EC50 = 1.43 nm). In situ hybridization showed the expression of cTSHR mRNA in the thyroidal follicular cells. cTSHR mRNA expression, as determined by real-time PCR, was also found in several other tissues such as brain, pituitary, pineal gland, and retina, suggesting that the TSH-TSHR interaction is not only important in regulating thyroid function. TSHR mRNA expression in the thyroid gland did not change significantly during the last week of embryonic development, which suggests that an increased thyroidal sensitivity is not part of the cause of the concomitant increasing T4 levels.