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Pleomorphic adenoma gene 1 (PLAG1) belongs to the PLAG family of zinc finger transcription factors along with PLAG-like 1 and PLAG-like 2. The PLAG1 gene is best known as an oncogene associated with certain types of cancer, most notably pleomorphic adenomas of the salivary gland. While the mechanisms of PLAG1-induced tumorigenesis are reasonably well understood, the role of PLAG1 in normal physiology is less clear. It is known that PLAG1 is involved in cell proliferation by directly regulating a wide array of target genes, including a number of growth factors such as insulin-like growth factor 2. This is likely to be a central mode of action for PLAG1 both in embryonic development and in cancer. The phenotype of Plag1 knockout mice suggests an important role for PLAG1 also in postnatal growth and reproduction, as PLAG1 deficiency causes growth retardation and reduced fertility. A role for PLAG1 in growth and reproduction is further corroborated by genome-wide association studies in humans and domestic animals in which polymorphisms in the PLAG1 genomic region are associated with body growth and reproductive traits. Here we review the current evidence for PLAG1 as a regulator of growth and fertility and discuss possible endocrine mechanisms involved., (© 2016 Society for Endocrinology.)

A major life stage transition in birds and other oviparous sauropsids is the hatching of the cleidoic egg. Not unlike amphibian metamorphosis, hatching in these species can be regarded as a transition from a relatively well-protected "aqueous" environment to a more hazardous and terrestrial life outside the egg, a transition in which thyroid hormones (THs) (often in concert with glucocorticoids) play an important role. In precocial birds such as the chicken, the perihatch period is characterized by peak values of THs. THs are implicated in the control of muscle development, lung maturation and the switch from chorioallantoic to pulmonary respiration, yolk sac retraction, gut development and induction of hepatic genes to accommodate the change in dietary energy source, initiation of thermoregulation, and the final stages of brain maturation as well as early post-hatch imprinting behavior. There is evidence that, at least for some of these processes, THs may have similar roles in non-avian sauropsids. In altricial birds such as passerines on the other hand, THs do not rise significantly until well after hatching and peak values coincide with the development of endothermy. It is not known how hatching-associated processes are regulated by hormones in these animals or how this developmental mode evolved from TH-dependent precocial hatching.

The last trimester of the embryonic life of chickens is marked by a steady increase in circulating thyroxine (T(4)) levels, reaching a maximum around hatching. We have measured thyroidal mRNA expression levels of several genes involved in the biosynthesis of T(4), namely sodium/iodine symporter (NIS), thyroglobulin (Tg), thyroid peroxidase (TPO), thyrotropin receptor (TSHR), and thyroid transcription factor 1 (TTF-1), during this period. Subsequently, we measured the expression of these genes in more detail during the entire hatching process and compared the gene expression profiles with concomitant changes in intrathyroidal and circulating thyroid hormone levels. We found that NIS and TPO mRNA expression increased significantly in the perinatal period, whereas Tg mRNA expression rose gradually throughout the last week of embryogenesis but was stable during hatching. TSHR and TTF-1 mRNA levels did not change significantly during the last week of embryonic development and hatching. Our results suggest that the elevated plasma T(4) levels observed in the developmental period studied are caused by an increased synthesis and secretion of T(4) by the thyroid gland. Augmented expression of Tg may play an important role in the increasing T(4) production during the last week of embryonic development, whereas increased NIS and TPO expression around hatching allows the thyrocytes to boost T(4) synthesis even further., (Copyright © 2011 Elsevier Inc. All rights reserved.)

We investigated the presence of thyrotropin receptor (TSHR) mRNA in chicken pituitary and brain, and quantified the changes in its expression during the last week of embryonic development. We found that in the pituitary gland, TSHR mRNA co-localizes with folliculo-stellate cells but not with thyrotropic cells, suggesting the existence of a paracrine ultra-short thyrotropin feedback loop. TSHR mRNA was also present throughout the diencephalon and various other brain regions, which implies a more general function for thyrotropin in the avian brain. During late embryogenesis, when the activity of the hypothalamo-pituitary-thyroidal axis increases markedly, a significant rise in TSHR mRNA expression was observed in pituitary, which may signify an important change in pituitary ultra-short thyrotropin feedback regulation around the period of hatching.

The ease of in vivo experimental manipulation is one of the main factors that have made the chicken embryo an important animal model in developmental research, including developmental endocrinology. This review focuses on the development of the thyrotropic, corticotropic and somatotropic axes in the chicken, emphasizing the central role of the pituitary gland in these endocrine systems. Functional maturation of the endocrine axes entails the cellular differentiation and acquisition of cell function and responsiveness of the different glands involved, as well as the establishment of top-down and bottom-up anatomical and functional communication between the control levels. Extensive cross-talk between the above-mentioned axes accounts for the marked endocrine changes observed during the last third of embryonic development. In a final paragraph we shortly discuss how genomic resources and new transgenesis techniques can increase the power of the chicken embryo model in developmental endocrinology research.

We previously described the cloning of the full-length chicken thyrotropin receptor (TSHRa) and two splice variants, lacking exon 3 (TSHRb) or both exons 2 and 3 (TSHRc). Here we report the identification of three novel splice variants of the chicken TSHR, named TSHRd, -e and -f, differing in their C-terminal region and containing unique exonic sequences that are not present in the other TSHR variants. This finding suggests a TSHR gene structure with 13 rather than the previously assumed 10 exons. The three novel exons appear to be chicken-specific, as no equivalents of these exons were found in other vertebrate genomes. Like the full-length receptor, the five TSHR splice variants are most abundantly expressed in thyroid gland. TSHRb, -d, -e and -f mRNA was also present in virtually all extra-thyroidal tissues expressing TSHRa, whereas TSHRc shows a more restricted tissue distribution. Whether these receptor transcripts are translated to functional proteins needs to be verified, but if so, they could be attributed various physiological roles.

In this study, we tried to elucidate the changes in thyroid hormone (TH) receptor beta2 (TRbeta2) expression at the different levels of the hypothalamo-pituitary-thyroidal (HPT) axis during the last week of chicken embryonic development and hatching, a period characterized by an augmented activity of the HPT axis. We quantified TRbeta2 mRNA in retina, pineal gland, and the major control levels of the HPT axis - brain, pituitary, and thyroid gland - at day 18 of incubation, and found the most abundant mRNA content in retina and pituitary. Thyroidal TRbeta2 mRNA content increased dramatically between embryonic day 14 and 1 day post-hatch. In pituitary and hypothalamus, TRbeta2 mRNA expression rose gradually, in parallel with increases in plasma thyroxine concentrations. Using in situ hybridization, we have demonstrated the presence of TRbeta2 mRNA throughout the diencephalon and confirmed the elevation in TRbeta2 mRNA expression in the hypophyseal thyrotropes. In vitro incubation with THs caused a down-regulation of TRbeta2 mRNA levels in embryonic but not in post-hatch pituitaries. The observed expression patterns in pituitary and diencephalon may point to substantial changes in TRbeta2-mediated TH feedback active during the perinatal period. The strong rise in thyroidal TRbeta2 mRNA content could be indicative of an augmented modulation of thyroid development and/or function by THs toward and after hatching. Finally, THs proved to exert an age-dependent effect on pituitary TRbeta2 mRNA expression.

We have studied the involvement of the somatostatin receptor type 5 (SSTR5) in the control of thyrotropin (TSH) release in the chicken. Hypothalamic somatostatin (SS-14) is known to inhibit both thyrotropin-releasing hormone (TRH)- and corticotropin-releasing hormone (CRH)-induced TSH secretion. Studies using receptor-specific agonists have indicated that the inhibitory effect of SS-14 on TRH-induced TSH release is mediated by SSTR2 and SSTR5. Using the same agonists, we were able to demonstrate the involvement of SSTR5 in the inhibition of the in vitro CRH-induced TSH secretion by SS-14. Subsequently, we determined hypophyseal SSTR5 mRNA expression during the last week of embryonic development using real-time PCR. SSTR5 mRNA levels were low until day 19 of incubation, but between day 19 and hatching SSTR5 mRNA expression increased 3-fold. Since this increase coincides with the increasing plasma T(3) levels towards hatching, and a similar ontogenetic expression pattern was found for SSTR2, we quantified hypophyseal SSTR2 and SSTR5 mRNA expression levels in chicken embryos treated with thyroid hormones. Injection of thyroid hormones was indeed found to increase the expression of both mRNAs significantly. We hypothesize that the negative feedback exerted by the increasing plasma T(3) levels towards hatching is at least in part mediated by an increased expression of SSTR2 and SSTR5.

The existence of an interaction between the adrenal/interrenal axis and the thyroidal axis has since long been established in vertebrates, including fish. However, in contrast to mammals, birds and amphibians, no effort was made in fish to expand these studies beyond the level of measuring plasma thyroid hormones. We therefore set out to examine the acute effects of a single dose of dexamethasone (DEX) on plasma thyroxine (T(4)) and 3,5,3'-triiodothyronine (T(3)) levels, as well as on the activity and mRNA expression of the different iodothyronine deiodinases in liver, gills, kidney and brain in Nile tilapia. To take into account the effect of handling stress, this treatment was compared both to a non-treated and to a saline injected group. In general, the observed changes were acute (3 and 6h) while values had returned to control levels by 24h post-injection. Only DEX administration caused an acute drop in circulating T(3) levels compared to non-treated animals, while none of the treatments affected plasma T(4) levels. This indicates that the DEX induced decrease in plasma T(3) levels was not due to a lowered thyroidal hormone production and secretion. DEX injection provoked a decrease in peripheral T(3) production capacity via a decrease in hepatic outer ring deiodination activity (both D1 and D2), whereas T(3) clearance increased by induction of the inner ring deiodinating D3 pathway in liver and in gills. Deiodination activities in kidney and brain were not affected. Effects of saline injection were only observed in liver, where D1 activity decreased and D3 activity increased as in the DEX group, but to a lesser extent. Real-time PCR showed that the changes in hepatic D3 were clearly regulated at the pretranslational level, while this was not confirmed for the other changes. Our results show that both handling stress and DEX injection acutely disturb peripheral deiodination activity in Nile tilapia. However, the effects of the long acting glucocorticoid analogue are more pronounced and result in a decrease in circulating T(3) availability.

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.

The hatching process in the chicken is accompanied by dramatic changes in plasma thyroid hormones. The cause of these changes, though crucial for hatching and the onset of endothermy, is not known. One hypothesis is that the pituitary gland becomes more sensitive to hypothalamic stimulation during this period. We have tested whether the responsiveness of the thyrotropes to hypothalamic stimuli changes throughout the last week of embryonic development and hatching by studying the mRNA expression of receptors involved in the control of the secretory activity of this cell type. We used a real-time PCR set-up to quantify whole pituitary mRNA expression of the beta subunit of thyrotrophin (TSH-beta), type 1 thyrotrophin-releasing hormone receptor (TRH-R1), corticotrophin-releasing hormone receptors (CRH-R1 and CRH-R2) and somatostatin subtype receptor 2 (SSTR2) on every day of the last week of embryonic development, including the day of hatch and the first day of posthatch life. The thyrotrope-specific expression was investigated by a combination of in situ hybridization with immunohistochemistry at selected ages. Although TSH-beta mRNA levels increased towards day 19 of incubation (E19), the expression of CRH-R2 and TRH-R1 mRNA by the thyrotropes tended to decrease during this period, suggesting a lower sensitivity of the thyrotropes to the stimulatory factors CRH and TRH. CRH-R1, which is not involved in the control of TSH secretion, increased steadily throughout the period tested. The expression of SSTR2 mRNA by the thyrotropes was low during embryonic development and increased just before hatching. We have concluded that the sensitivity of the pituitary thyrotropes to hypothalamic stimulation decreases throughout the last week of embryonic development, so that the higher expression of TSH-beta mRNA around E16-E19, and hence the increasing plasma thyroxine level, is unlikely to be the result of an increased stimulation by either TRH or CRH.

Thyroid hormones and their receptors (TRs) have critical functions in development and metabolism. In chicken, three TRs are known: TRalpha, TRbeta0, and TRbeta2. The latter was isolated from chicken eye, but its presence in other tissues has not yet been extensively investigated. We therefore developed a real-time PCR assay using a Taqman probe and primers based on the unique amino-terminal region of TRbeta2. We detected a strong TRbeta2 mRNA signal in the pituitary, confirmed with in situ hybridization, and in several other tissues. TRbeta2 mRNA was more abundant in the pituitary of newly hatched chicks than in 15-day-old embryos.

We report the cloning of the complete coding sequence of the putative chicken type 2 corticotropin-releasing hormone receptor (CRH-R2) by rapid amplification of cDNA ends (RACE). The chicken CRH-R2 is a 412-amino acid 7-transmembrane G protein-coupled receptor, showing 87% identity to the Xenopus laevis and Oncorhynchus keta CRH-R2s, and 78-80% to mammalian CRH-R2s. The distribution of CRH-R2 mRNA was studied by RT-PCR analysis and compared to CRH-R1 distribution. Both CRH-R1 and CRH-R2 mRNA are expressed in the main chicken brain parts. In peripheral organs, CRH-R1 mRNA shows a more restricted distribution, whereas CRH-R2 mRNA is expressed in every tissue investigated, indicating that a number of actions of CRH and/or CRH-like peptides remain to be discovered in the chicken as well as in other vertebrates.

Entanglement of functions between the adrenal (or interrenal) and thyroid axis has been well described for all vertebrates and can be tracked down up to the level of gene expression. Both thyroid hormones and corticosteroids may induce morphological changes leading to metamorphosis climax in the neotenic Mexican axolotl (Ambystoma mexicanum). In a first series of experiments, metamorphosis was induced with an injection of 25 microg T(4) on three alternate days as judged by a decrease in body weight and tail height together with complete gill resorption. This injection also resulted in elevated plasma concentrations of T(3) and corticosterone. Previous results have indicated that the same dose of dexamethasone (DEX) is ineffective in this regard (Gen. Comp. Endocrinol. 127 (2002) 157). In a second series of experiments low doses of T(4) (0.5 microg) or DEX (5 microg) were ineffective to induce morphological changes. However, when these submetamorphic doses were injected together, morphological changes were observed within one week leading to complete metamorphosis. It is concluded that thyroid hormones combined with corticosteroids are essential for metamorphosis in the axolotl and that only high doses of either thyroid hormone or corticosteroid can induce morphological changes when injected separately.