Early Thyroid Development Research Articles

GWAS of thyroid dysgenesis identifies a risk locus at 2q33.3 linked to regulation of Wnt signaling.

Congenital hypothyroidism due to thyroid dysgenesis (TD), presented as thyroid aplasia, hypoplasia or ectopia, is one of the most prevalent rare diseases with an isolated organ malformation. The pathogenesis of TD is largely unknown, although a genetic predisposition has been suggested. We performed a genome-wide association study (GWAS) with 142 Japanese TD cases and 8380 controls and found a significant locus at 2q33.3 (top single nucleotide polymorphism, rs9789446: P = 4.4 × 10-12), which was replicated in a German patient cohort (P = 0.0056). A subgroup analysis showed that rs9789446 confers a risk for thyroid aplasia (per allele odds ratio = 3.17) and ectopia (3.12) but not for hypoplasia. Comprehensive epigenomic characterization of the 72-kb disease-associated region revealed that it was enriched for active enhancer signatures in human thyroid. Analysis of chromosome conformation capture data showed long-range chromatin interactions of this region with promoters of two genes, FZD5 and CCNYL1, mediating Wnt signaling. Moreover, rs9789446 was found to be a thyroid-specific quantitative trait locus, adding further evidence for a cis-regulatory function of this region in thyroid tissue. Specifically, because the risk rs9789446 allele is associated with increased thyroidal expression of FDZ5 and CCNYL1 and given the recent demonstration of perturbed early thyroid development following overactivation of Wnt signaling in zebrafish embryos, an enhanced Wnt signaling in risk allele carriers provides a biologically plausible TD mechanism. In conclusion, our work found the first risk locus for TD, exemplifying that in rare diseases with relatively low biological complexity, GWAS may provide mechanistic insights even with a small sample size.

Glis3 as a critical regulator of thyroid primordium specification

Background: GLIS3 (GLI-Similar protein 3) is a transcription factor involved in several cellular processes. Homozygous mutations in the GLIS3 gene have been typically associated with neonatal diabetes and congenital hypothyroidism (CH) in a syndrome called NDH. NDH patients present developmental abnormalities including endocrine pancreas defects and a spectrum of thyroid abnormalities, mainly including thyroid dysgenesis (TD). The mouse models revealed a key role of Glis3 in pancreatic islets but not in early thyroid development, as Glis3 was described to retain a role in regulating thyroid hormone synthesis downstream the thyrotropin (TSH)/TSHR signaling pathway and in postnatal follicle proliferation. Hence, in this study, we have been taking advantage of the zebrafish model to gain insights on the Glis3 activity during thyroid organogenesis. Methods: Transient glis3-knockdown zebrafish embryos (called glis3 morphants) were generated by the microinjection of specific glis3 morpholinos at one- to two-cell stage to analyze the thyroid phenotype in vivo. Several additional analyses (in situ hybridization, immunohistochemistry, and pharmacological treatments) were performed for further molecular characterization. Results: The analysis of thyroid embryonic development revealed that Glis3 is involved in early steps of thyroid specification. glis3 morphants exhibited a reduced expression of the early transcription factors nkx2.4 and pax2a at the thyroid primordium level, which is not caused by changes in proliferation or apoptosis of the pharyngeal endoderm. As a result, the differentiated thyroid tissue in morphants appeared reduced in size with decreased expression of tg and slc5a5, a low number of thyroxine (T4)-producing follicles, associated with an elevation of tshba (homologous of the human TSHβ), thus resembling the clinical and biochemical manifestations of patients with TD. Interestingly, glis3 morphants have pancreatic β-cell defects, but not liver defects. In vitro and in vivo data also demonstrated that Glis3 is an effector of the Sonic Hedgehog (SHH) pathway. Molecular and pharmacological inhibition of SHH reproduced the thyroid defects observed in glis3 morphant. Conclusions: Our results demonstrate that glis3, within the SHH pathway, appears to determine the number of endodermal cells committed to a thyroid fate. This is the first evidence of the involvement of Glis3 in TD, thereby expanding the understanding of the genetic basis of thyroid development and CH.

Glis3 as a Critical Regulator of Thyroid Primordium Specification.

Background: GLIS3 (GLI-Similar protein 3) is a transcription factor involved in several cellular processes. Homozygous mutations in the GLIS3 gene have been typically associated with neonatal diabetes and congenital hypothyroidism (CH) in a syndrome called NDH. NDH patients present developmental abnormalities including endocrine pancreas defects and a spectrum of thyroid abnormalities, mainly including thyroid dysgenesis (TD). The mouse models revealed a key role of Glis3 in pancreatic islets but not in early thyroid development, as Glis3 was described to retain a role in regulating thyroid hormone synthesis downstream the thyrotropin (TSH)/TSHR signaling pathway and in postnatal follicle proliferation. Hence, in this study, we have been taking advantage of the zebrafish model to gain insights on the Glis3 activity during thyroid organogenesis. Methods: Transient glis3-knockdown zebrafish embryos (called glis3 morphants) were generated by the microinjection of specific glis3 morpholinos at one- to two-cell stage to analyze the thyroid phenotype in vivo. Several additional analyses (in situ hybridization, immunohistochemistry, and pharmacological treatments) were performed for further molecular characterization. Results: The analysis of thyroid embryonic development revealed that Glis3 is involved in early steps of thyroid specification. glis3 morphants exhibited a reduced expression of the early transcription factors nkx2.4 and pax2a at the thyroid primordium level, which is not caused by changes in proliferation or apoptosis of the pharyngeal endoderm. As a result, the differentiated thyroid tissue in morphants appeared reduced in size with decreased expression of tg and slc5a5, a low number of thyroxine (T4)-producing follicles, associated with an elevation of tshba (homologous of the human TSHβ), thus resembling the clinical and biochemical manifestations of patients with TD. Interestingly, glis3 morphants have pancreatic β-cell defects, but not liver defects. In vitro and in vivo data also demonstrated that Glis3 is an effector of the Sonic Hedgehog (SHH) pathway. Molecular and pharmacological inhibition of SHH reproduced the thyroid defects observed in glis3 morphant. Conclusions: Our results demonstrate that glis3, within the SHH pathway, appears to determine the number of endodermal cells committed to a thyroid fate. This is the first evidence of the involvement of Glis3 in TD, thereby expanding the understanding of the genetic basis of thyroid development and CH.

Small-Molecule Screening in Zebrafish Embryos Identifies Signaling Pathways Regulating Early Thyroid Development.

Background: Defects in embryonic development of the thyroid gland are a major cause for congenital hypothyroidism in human newborns, but the underlying molecular mechanisms are still poorly understood. Organ development relies on a tightly regulated interplay between extrinsic signaling cues and cell intrinsic factors. At present, however, there is limited knowledge about the specific extrinsic signaling cues that regulate foregut endoderm patterning, thyroid cell specification, and subsequent morphogenetic processes in thyroid development. Methods: To begin to address this problem in a systematic way, we used zebrafish embryos to perform a series of in vivo phenotype-driven chemical genetic screens to identify signaling cues regulating early thyroid development. For this purpose, we treated zebrafish embryos during different developmental periods with a panel of small-molecule compounds known to manipulate the activity of major signaling pathways and scored phenotypic deviations in thyroid, endoderm, and cardiovascular development using whole-mount in situ hybridization and transgenic fluorescent reporter models. Results: Systematic assessment of drugged embryos recovered a range of thyroid phenotypes including expansion, reduction or lack of the early thyroid anlage, defective thyroid budding, as well as hypoplastic, enlarged, or overtly disorganized presentation of the thyroid primordium after budding. Our pharmacological screening identified bone morphogenetic protein and fibroblast growth factor signaling as key factors for thyroid specification and early thyroid organogenesis, highlighted the importance of low Wnt activities during early development for thyroid specification, and implicated drug-induced cardiac and vascular anomalies as likely indirect mechanisms causing various forms of thyroid dysgenesis. Conclusions: By integrating the outcome of our screening efforts with previously available information from other model organisms including Xenopus, chicken, and mouse, we conclude that signaling cues regulating thyroid development appear broadly conserved across vertebrates. We therefore expect that observations made in zebrafish can inform mammalian models of thyroid organogenesis to further our understanding of the molecular mechanisms of congenital thyroid diseases.

Molecular defects in thyroid dysgenesis.

Congenital hypothyroidism (CH) is a neonatal endocrine disorder that might occur as itself or be associated to congenital extra-thyroidal defects. About 85% of affected subjects experience thyroid dysgenesis (TD), characterized by defect in thyroid gland development. In vivo experiments on null mice paved the way for the identification of genes involved thyroid morphogenesis and development, whose mutation has been strongly associated to TD. Most of them are thyroid-specific transcription factors expressed during early thyroid development. Despite the arduous effort in unraveling the genetics of TD in animal models, up to now these data have been discontinuously confirmed in humans and only 5% of TD have associated with known null mice-related mutations (mainly PAX8 and TSHR). Notwithstanding, the advance in genetic testing represented by the next-generation sequencing (NGS) approach is steadily increasing the list of genes whose highly penetrant mutation predisposes to TD. In this review we intend to outline the molecular bases of TD, summarizing the current knowledge on thyroid development in both mice and humans and delineating the genetic features of its monogenetic forms. We will also highlight current strategies to enhance the insight into the non-Mendelian mechanisms of abnormal thyroid development.

Thyroid Progenitors Are Robustly Derived from Embryonic Stem Cells through Transient, Developmental Stage-Specific Overexpression of Nkx2-1.

SummaryThe clinical importance of anterior foregut endoderm (AFE) derivatives, such as thyrocytes, has led to intense research efforts for their derivation through directed differentiation of pluripotent stem cells (PSCs). Here, we identify transient overexpression of the transcription factor (TF) NKX2-1 as a powerful inductive signal for the robust derivation of thyrocyte-like cells from mouse PSC-derived AFE. This effect is highly developmental stage specific and dependent on FOXA2 expression levels and precise modulation of BMP and FGF signaling. The majority of the resulting cells express thyroid TFs (Nkx2-1, Pax8, Foxe1, Hhex) and thyroid hormone synthesis-related genes (Tg, Tpo, Nis, Iyd) at levels similar to adult mouse thyroid and give rise to functional follicle-like epithelial structures in Matrigel culture. Our findings demonstrate that NKX2-1 overexpression converts AFE to thyroid epithelium in a developmental time-sensitive manner and suggest a general methodology for manipulation of cell-fate decisions of developmental intermediates.

DYRK1A BAC transgenic mouse: a new model of thyroid dysgenesis in Down syndrome.

The most common thyroid abnormality among Down syndrome (DS) children corresponds to a mildly elevated TSH, with T4 decreased or in the normal range and thyroid hypoplasia, from the neonatal period onward, which aggravate their mental impairment. Transgenic Dyrk1A mice, obtained by bacterial artificial chromosome engineering (mBACTgDyrk1A), have 3 copies of the Dyrk1A gene. The objective is to determine whether this transgenic Dyrk1A (Dyrk1A(+/++)) mouse is an adequate murine model for the study of thyroid dysgenesis in DS. Embryonic thyroid development from embryonic day 13.5 (E13.5) to E17.5 was analyzed in wild-type (WT) and Dyrk1A(+/++) mice by immunofluorescence with anti-Nkx2-1, anti-thyroglobulin, and anti-T4 antibodies, markers of early thyroid development, hormonogenesis, and final differentiation, respectively. The expression of transcription factors Nkx2-1, Pax8, and Foxe1 involved in thyroidogenesis were studied by quantitative RT-PCR at the same embryonic stages. We then compared the adult phenotype at 8 to 12 weeks in Dyrk1A(+/++) and WT mice for T4 and TSH levels, thyroidal weight, and histological analysis. Regarding thyroidal development, at E15.5, Dyrk1A(+/++) thyroid lobes are double the size of WT thyroids (P = .01), but the thyroglobulin stained surface in Dyrk1A(+/++) thyroids is less than a third as large at E17.5 (P = .04) and their differentiated follicular surface half the size (P = .004). We also observed a significant increase in Nkx2-1, Foxe1, and Pax8 RNA levels in E13.5 and E17.5 Dyrk1A(+/++) embryonic thyroids. Dyrk1A(+/++) young adult mice have significantly lower plasma T4 (2.4 ng/mL versus WT, 3.7 ng/mL; P = 0.019) and nonsignificantly higher plasma TSH (114 mUI/L versus WT, 73mUI/L; P = .09). In addition, their thyroids are significantly heavier (P = .04) and exhibit large disorganized regions. Dyrk1A overexpression directly leads to thyroidal embryogenetic, functional and morphological impairment. The young adult thyroid phenotype is probably a result of embryogenetic impairment. The Dyrk1A(+/++) mouse can be considered a suitable study model for thyroid dysgenesis in DS.

Perfluorooctane sulfonate (PFOS) affects hormone receptor activity, steroidogenesis, and expression of endocrine‐related genes in vitro and in vivo

Perfluorooctane sulfonate (PFOS) is a widespread and persistent chemical in the environment. We investigated the endocrine-disrupting effects of PFOS using a combination of in vitro and in vivo assays. Reporter gene assays were used to detect receptor-mediated (anti-)estrogenic, (anti-)androgenic, and (anti-)thyroid hormone activities. The effect of PFOS on steroidogenesis was assessed both at hormone levels in the supernatant and at expression levels of hormone-induced genes in the H295R cell. A zebrafish-based short-term screening method was developed to detect the effect of PFOS on endocrine function in vivo. The results indicate that PFOS can act as an estrogen receptor agonist and thyroid hormone receptor antagonist. Exposure to PFOS decreased supernatant testosterone (T), increased estradiol (E2) concentrations in H295R cell medium and altered the expression of several genes involved in steroidogenesis. In addition, PFOS increased early thyroid development gene (hhex and pax8) expression in a concentration-dependent manner, decreased steroidogenic enzyme gene (CYP17, CYP19a, CYP19b) expression, and changed the expression pattern of estrogen receptor production genes (esr1, esr2b) after 500 µg/L PFOS treatment in zebrafish embryos. These results indicate that PFOS has the ability to act as an endocrine disruptor both in vitro and in vivo by disrupting the function of nuclear hormone receptors, interfering with steroidogenesis, and altering the expression of endocrine-related genes in zebrafish embryo.

Transgenic zebrafish illuminate the dynamics of thyroid morphogenesis and its relationship to cardiovascular development

Among the various organs derived from foregut endoderm, the thyroid gland is unique in that major morphogenic events such as budding from foregut endoderm, descent into subpharyngeal mesenchyme and growth expansion occur in close proximity to cardiovascular tissues. To date, research on thyroid organogenesis was missing one vital tool—a transgenic model that allows to track the dynamic changes in thyroid size, shape and location relative to adjacent cardiovascular tissues in live embryos. In this study, we generated a novel transgenic zebrafish line, tg(tg:mCherry), in which robust and thyroid-specific expression of a membrane version of mCherry enables live imaging of thyroid development in embryos from budding stage throughout formation of functional thyroid follicles. By using various double transgenic models in which EGFP expression additionally labels cardiovascular structures, a high coordination was revealed between thyroid organogenesis and cardiovascular development. Early thyroid development was found to proceed in intimate contact with the distal ventricular myocardium and live imaging confirmed that thyroid budding from the pharyngeal floor is tightly coordinated with the descent of the heart. Four-dimensional imaging of live embryos by selective plane illumination microscopy and 3D-reconstruction of confocal images of stained embryos yielded novel insights into the role of specific pharyngeal vessels, such as the hypobranchial artery (HA), in guiding late thyroid expansion along the pharyngeal midline. An important role of the HA was corroborated by the detailed examination of thyroid development in various zebrafish models showing defective cardiovascular development. In combination, our results from live imaging as well es from 3D-reconstruction of thyroid development in tg(tg:mCherry) embryos provided a first dynamic view of late thyroid organogenesis in zebrafish—a critical resource for the design of future studies addressing the molecular mechanisms of these thyroid-vasculature interactions.

Gene expression profiling at early organogenesis reveals both common and diverse mechanisms in foregut patterning

The thyroid and lungs originate as neighboring bud shaped outgrowths from the midline of the embryonic foregut. When and how organ specific programs regulate development into structures of distinct shapes, positions and functions is incompletely understood. To characterize, at least in part, the genetic basis of these events, we have employed laser capture microdissection and microarray analysis to define gene expression in the mouse thyroid and lung primordia at E10.5. By comparing the transcriptome of each bud to that of the whole embryo as well as to each other, we broadly describe the genes that are preferentially expressed in each developing organ as well as those with an enriched expression common to both. The results thus obtained provide a valuable resource for further analysis of genes previously unrecognized to participate in thyroid and lung morphogenesis and to discover organ specific as well as common developmental mechanisms. As an initial step in this direction we describe a regulatory pathway involving the anti-apoptotic gene Bcl2 that controls cell survival in early thyroid development.

Fate Map of Serotonin Transporter‐Expressing Cells in Developing Mouse Thyroid

A Cre/loxP-based fate mapping approach was used to follow the regions of the mouse thyroid labeled by the serotonin transporter SERT. Reporter gene expression (lacZ) is activated by Cre expression from the SERT locus in SERT(Cre/+) ;ROSA26R compound mouse embryos. Cell labeling, first detected in the thyroid primordium at the E10.5 prenatal stage, was followed until the postnatal day P30. The co-localization of lacZ staining in the same cells that express the transcription factors Nkx2.1 and Pax8 at the E12.5 stage confirms their identity as thyroid cell precursors. SERT immunohistochemistry on thyroid sections of E18.5 embryos showed SERT expression in thyroid follicular cells. Western blotting analysis confirmed the expression of the protein in adult thyroid tissue and cultured FRTL-5 cells. These results describe the fate of SERT-expressing cells during thyroid development, suggesting an active role of SERT in the development and functions of mammalian thyroid. They also highlight the possibility to use the SERT-Cre mouse line as a good Cre driver in early thyroid development.

Morphogenetics of early thyroid development

The thyroid develops from the foregut endoderm. Yet uncharacterized inductive signals specify endoderm progenitors to a thyroid cell fate that assembles in the pharyngeal floor from which the primordium buds and migrates to the final position of the gland. The morphogenetic process is regulated by both cell-autonomous (e.g. activated by NKX2-1, FOXE1, PAX8, and HHEX) and mesoderm-derived (e.g. mediated by TBX1 and fibroblast growth factors) mechanisms acting in concert to promote growth and survival of progenitor cells. The developmental role of TSH is limited to thyroid differentiation set to work after the gross anatomy of the gland is already sculptured. This review summarizes recent advances on the molecular genetics of thyroid morphogenesis put into context of endoderm developmental traits and highlights established and novel mechanisms of thyroid dysgenesis of potential relevance to congenital hypothyroidism in man.

The UV-absorber benzophenone-4 alters transcripts of genes involved in hormonal pathways in zebrafish ( Danio rerio) eleuthero-embryos and adult males

Benzophenone-4 (BP-4) is frequently used as UV-absorber in cosmetics and materials protection. Despite its frequent detection in the aquatic environment potential effects on aquatic life are unknown. In this study, we evaluate the effects of BP-4 in eleuthero-embryos and in the liver, testis and brain of adult male fish on the transcriptional level by focusing on target genes involved in hormonal pathways to provide a more complete toxicological profile of this important UV-absorber. Eleuthero-embryos and males of zebrafish were exposed up to 3 days after hatching and for 14 days, respectively, to BP-4 concentrations between 30 and 3000 μg/L. In eleuthero-embryos transcripts of vtg1, vtg3, esr1, esr2b, hsd17ß3, cyp19b cyp19a, hhex and pax8 were induced at 3000 μg/L BP-4, which points to a low estrogenic activity and interference with early thyroid development, respectively. In adult males BP-4 displayed multiple effects on gene expression in different tissues. In the liver vtg1, vtg3, esr1 and esr2b were down-regulated, while in the brain, vtg1, vtg3 and cyp19b transcripts were up-regulated. In conclusion, the transcription profile revealed that BP-4 interferes with the expression of genes involved in hormonal pathways and steroidogenesis. The effects of BP-4 differ in life stages and adult tissues and point to an estrogenic activity in eleuthero-embryos and adult brain, and an antiestrogenic activity in the liver. The results indicate that BP-4 interferes with the sex hormone system of fish, which is important for the risk assessment of this UV-absorber.

Exposure to DE-71 alters thyroid hormone levels and gene transcription in the hypothalamic–pituitary–thyroid axis of zebrafish larvae

Polybrominated diphenyl ethers (PBDEs) have the potential to disrupt thyroid hormone homeostasis, but the molecular mechanisms underlying this process have not yet been clarified. In the present study, zebrafish ( Danio rerio) embryos were exposed to a low concentration (0, 1, 3, and 10 μg/L) of DE-71 from fertilization to 14 days thereafter. The whole-body content of thyroid hormone and transcription of genes in the hypothalamic–pituitary–thyroid (HPT) axis were analyzed. Exposure to up to 10 μg/L of DE-71 significantly reduced thyroxine (T4) levels and significantly upregulated the transcription of corticotrophin-releasing hormone (CRH) and thyroid-stimulating hormone (TSHβ) genes in a concentration-dependent manner. The transcription of genes involved in the synthesis of TH proteins, sodium/iodide symporter (Slc5a5), and thyroglobulin (TG) and the transcription of marker genes associated with early thyroid development (Pax8 and Nkx2.1) were significantly upregulated upon DE-71 exposure. The expression of thyronine deiodinase (Deio1 and Deio2) mRNAs was also significantly upregulated, possibly as a compensatory response to the decreased T4 levels. However, DE-71 exposure resulted in the downregulation of transthyretin (TTR) gene transcription and did not affect the transcription of thyroid hormone receptors (TRs). Exposure to DE-71 significantly induced the transcription of the uridinediphosphate-glucuronosyltransferase (UGT1ab) gene. The results of our study confirmed the reliability of the zebrafish larvae as models for assessment of the developmental toxicity of PBDEs and transcription of genes of the HPT axis can evaluate the potential mechanisms of thyroid disruption.

Early thyroid development requires a Tbx1– Fgf8 pathway

The thyroid develops within the pharyngeal apparatus from endodermally-derived cells. The many derivatives of the pharyngeal apparatus develop at similar times and sometimes from common cell types, explaining why many syndromic disorders express multiple birth defects affecting different structures that share a common pharyngeal origin. Thus, different derivatives may share common genetic networks during their development. Tbx1, the major gene associated with DiGeorge syndrome, is a key player in the global development of the pharyngeal apparatus, being required for virtually all its derivatives, including the thyroid. Here we show that Tbx1 regulates the size of the early thyroid primordium through its expression in the adjacent mesoderm. Because Tbx1 regulates the expression of Fgf8 in the mesoderm, we postulated that Fgf8 mediates critical Tbx1-dependent interactions between mesodermal cells and endodermal thyrocyte progenitors. Indeed, conditional ablation of Fgf8 in Tbx1-expressing cells caused an early thyroid phenotype similar to that of Tbx1 mutant mice. In addition, expression of an Fgf8 cDNA in the Tbx1 domain rescued the early size defect of the thyroid primordium in Tbx1 mutants. Thus, we have established that a Tbx1-> Fgf8 pathway in the pharyngeal mesoderm is a key size regulator of mammalian thyroid.

Developmental toxicity and alteration of gene expression in zebrafish embryos exposed to PFOS

Perfluorooctanesulfonate (PFOS) is a persistent organic pollutant, the potential toxicity of which is causing great concern. In the present study, we employed zebrafish embryos to investigate the developmental toxicity of this compound. Four-hour post-fertilization (hpf) zebrafish embryos were exposed to 0.1, 0.5, 1, 3 and 5 mg/L PFOS. Hatching was delayed and hatching rates as well as larval survivorship were significantly reduced after the embryos were exposed to 1, 3 and 5 mg/L PFOS until 132 hpf. The fry displayed gross developmental malformations, including epiboly deformities, hypopigmentation, yolk sac edema, tail and heart malformations and spinal curvature upon exposure to PFOS concentrations of 1 mg/L or greater. Growth (body length) was significantly reduced in the 3 and 5 mg/L PFOS-treated groups. To test whether developmental malformation was mediated via apoptosis, flow cytometry analysis of DNA content, acridine orange staining and TUNEL assay was used. These techniques indicated that more apoptotic cells were present in the PFOS-treated embryos than in the control embryos. Certain genes related to cell apoptosis, p53 and Bax, were both significantly up-regulated upon exposure to all the concentrations tested. In addition, we investigated the effects of PFOS on marker genes related to early thyroid development ( hhex and pax8) and genes regulating the balance of androgens and estrogens ( cyp19a and cyp19b). For thyroid development, the expression of hhex was significantly up-regulated at all concentrations tested, whereas pax8 expression was significantly up-regulated only upon exposure to lower concentrations of PFOS (0.1, 0.5, 1 mg/L). The expression of cyp19a and of cyp19b was significantly down-regulated at all exposure concentrations. The overall results indicated that zebrafish embryos constitute a reliable model for testing the developmental toxicity of PFOS, and the gene expression patterns in the embryos were able to reveal some potential mechanisms of developmental toxicity.

Pendrin Is a Novel In Vivo Downstream Target Gene of the TTF-1/Nkx-2.1 Homeodomain Transcription Factor in Differentiated Thyroid Cells

Thyroid transcription factor gene 1 (TTF-1) is a homeobox-containing gene involved in thyroid organogenesis. During early thyroid development, the homeobox gene Nkx-2.5 is expressed in thyroid precursor cells coincident with the appearance of TTF-1. The aim of this study was to investigate the molecular mechanisms underlying thyroid-specific gene expression. We show that the Nkx-2.5 C terminus interacts with the TTF-1 homeodomain and, moreover, that the expression of a dominant-negative Nkx-2.5 isoform (N188K) in thyroid cells reduces TTF-1-driven transcription by titrating TTF-1 away from its target DNA. This process reduced the expression of several thyroid-specific genes, including pendrin and thyroglobulin. Similarly, down-regulation of TTF-1 by RNA interference reduced the expression of both genes, whose promoters are sensitive to and directly associate with TTF-1 in the chromatin context. In conclusion, we demonstrate that pendrin and thyroglobulin are downstream targets in vivo of TTF-1, whose action is a prime factor in controlling thyroid differentiation in vivo.

Pax2.1 is required for the development of thyroid follicles in zebrafish.

The thyroid gland is an organ primarily composed of endoderm-derived follicular cells. Although disturbed embryonic development of the thyroid gland leads to congenital hypothyroidism in humans and mammals, the underlying principles of thyroid organogenesis are largely unknown. In this study, we introduce zebrafish as a model to investigate the molecular and genetic mechanisms that control thyroid development. Marker gene expression suggests that the molecular pathways of early thyroid development are essentially conserved between fish and mammals. However during larval stages, we find both conserved and divergent features of development compared with mammals. A major difference is that in fish, we find evidence for hormone production not only in thyroid follicular cells, but also in an anterior non-follicular group of cells. We show that pax2.1 and pax8, members of the zebrafish pax2/5/8 paralogue group, are expressed in the thyroid primordium. Whereas in mice, only Pax8 has a function during thyroid development, analysis of the zebrafish pax2.1 mutant no isthmus (noi(-/-)) demonstrates that pax2.1 has a role comparable with mouse Pax8 in differentiation of the thyroid follicular cells. Early steps of thyroid development are normal in noi(-/-), but later expression of molecular markers is lost and the formation of follicles fails. Interestingly, the anterior non-follicular site of thyroid hormone production is not affected in noi(-/-). Thus, in zebrafish, some remaining thyroid hormone synthesis takes place independent of the pathway leading to thyroid follicle formation. We suggest that the noi(-/-) mutant serves as a new zebrafish model for hypothyroidism.

Xnkx-2.1: a homeobox gene expressed during early forebrain, lung and thyroid development in Xenopus laevis.

Nkx-2.1 is a member of the vertebrate Nkx family of homeobox genes; it was originally identified as a tissue-specific regulator of thyroglobulin and thyroperoxidase gene transcription. Here we report on the embryonic expression of Xnkx-2.1, which is expressed in the presumptive forebrain from early neurulation onwards. In tadpole stage embryos Xnkx-2.1 transcripts are primarily detected in ventral forebrain, lung buds and thyroid anlage. Therefore, Xnkx-2.1 may be part of the genetic network that controls the early development of these organs.

A unique combination of transcription factors controls differentiation of thyroid cells

The thyroid follicular cell type is devoted to the synthesis of thyroid hormones. Several genes, whose protein products are essential for efficient hormone biosynthesis, are uniquely expressed in this cell type. A set of transcriptional regulators, unique to the thyroid follicular cell type, has been identified as responsible for thyroid specific gene expression; it comprises three transcription factors, named TTF-1, TTF-2, and Pax8, each of which is expressed also in cell types different from the thyroid follicular cells. However, the combination of these factors is unique to the thyroid hormone producing cells, strongly suggesting that they play an important role in differentiation of these cells. An overview of the molecular and biological features of these transcription factors is presented here. Data demonstrating that all three play also an important role in early thyroid development, at stages preceding expression of the differentiated phenotype, are also reviewed. The wide temporal expression, from the beginning of thyroid organogenesis to the adult state, is suggestive of a recycling of the thyroid-specific transcription factors, that is, the control of different sets of target genes at diverse developmental stages. The identification of molecular mechanisms leading to specific gene expression in thyroid cells renders this cell type an interesting model in which to address several aspects of cell differentiation and organogenesis.