Thyroid Precursor Research Articles

Mesodermal FGF and BMP govern the sequential stages of zebrafish thyroid specification.

Thyroid tissue, the site of de novo thyroid hormone biosynthesis, is derived from ventral pharyngeal endoderm and defects in morphogenesis are a predominant cause of congenital thyroid diseases. The first molecularly recognizable step of thyroid development is the specification of thyroid precursors in anterior foregut endoderm. Recent studies have identified crucial roles of FGF and BMP signaling in thyroid specification, but the interplay between signaling cues and thyroid transcription factors remained elusive. By analyzing Pax2a and Nkx2.4b expression dynamics in relation to endodermal FGF and BMP signaling activities in zebrafish embryos, we identified a Pax2a-expressing thyroid progenitor population that shows enhanced FGF signaling but lacks Nkx2.4b expression and BMP signaling. Concurrent with upregulated BMP signaling, a subpopulation of these progenitors subsequently differentiates into lineage-committed thyroid precursors co-expressing Pax2a and Nkx2.4b. Timed manipulation of FGF/BMP activities suggests a model in which FGF signaling primarily regulates Pax2a expression, whereas BMP signaling regulates both Pax2a and Nkx2.4b expression. Our observation of similar expression dynamics of Pax8 and Nkx2-1 in mouse embryos suggests that this refined model of thyroid cell specification is evolutionarily conserved in mammals.

Sox9 is involved in the thyroid differentiation program and is regulated by crosstalk between TSH, TGF\u03b2 and thyroid transcription factors

While the signaling pathways and transcription factors involved in the differentiation of thyroid follicular cells, both in embryonic and adult life, are increasingly well understood, the underlying mechanisms and potential crosstalk between the thyroid transcription factors Nkx2.1, Foxe1 and Pax8 and inductive signals remain unclear. Here, we focused on the transcription factor Sox9, which is expressed in Nkx2.1-positive embryonic thyroid precursor cells and is maintained from embryonic development to adulthood, but its function and control are unknown. We show that two of the main signals regulating thyroid differentiation, TSH and TGFβ, modulate Sox9 expression. Specifically, TSH stimulates the cAMP/PKA pathway to transcriptionally upregulate Sox9 mRNA and protein expression, a mechanism that is mediated by the binding of CREB to a CRE site within the Sox9 promoter. Contrastingly, TGFβ signals through Smad proteins to inhibit TSH-induced Sox9 transcription. Our data also reveal that Sox9 transcription is regulated by the thyroid transcription factors, particularly Pax8. Interestingly, Sox9 significantly increased the transcriptional activation of Pax8 and Foxe1 promoters and, consequently, their expression, but had no effect on Nkx2.1. Our study establishes the involvement of Sox9 in thyroid follicular cell differentiation and broadens our understanding of transcription factor regulation of thyroid function.

From Endoderm to Progenitors: An Update on the Early Steps of Thyroid Morphogenesis in the Zebrafish.

The mechanisms underlying thyroid gland development have a central interest in biology and this review is aimed to provide an update on the recent advancements on the early steps of thyroid differentiation that were obtained in the zebrafish, because this teleost fish revealed to be a suitable organism to study the early developmental stages. Physiologically, the thyroid precursors fate is delineated by the appearance among the endoderm cells of the foregut of a restricted cell population expressing specific transcription factors, including pax2a, nkx2.4b, and hhex. The committed thyroid primordium first appears as a thickening of the pharyngeal floor of the anterior endoderm, that subsequently detaches from the floor and migrates to its final location where it gives rise to the thyroid hormone-producing follicles. At variance with mammalian models, thyroid precursor differentiation in zebrafish occurs early during the developmental process before the dislocation to the eutopic positioning of thyroid follicles. Several pathways have been implicated in these early events and nowadays there is evidence of a complex crosstalk between intrinsic (coming from the endoderm and thyroid precursors) and extrinsic factors (coming from surrounding tissues, as the cardiac mesoderm) whose organization in time and space is probably required for the proper thyroid development. In particular, Notch, Shh, Fgf, Bmp, and Wnt signaling seems to be required for the commitment of endodermal cells to a thyroid fate at specific developmental windows of zebrafish embryo. Here, we summarize the recent findings produced in the various zebrafish experimental models with the aim to define a comprehensive picture of such complicated puzzle.

Precursor and borderline lesions of the thyroid (indolent lesions of epithelial origin): from theory to practice.

The precursor lesions of thyroid neoplasms can arise from either C cells or follicular cells. Although MTC has an established and recognized precursor lesion, the C-cell hyperplasia, the same does not occur for the follicular cell-derived tumors, which are much more frequent. One of the significant obstacles to recognizing follicular cell-derived precursor lesions (FCPL) is the lack of refinement of the morphological spectrum and biology of these putative premalignant lesions. What are the "gold standard" histological criteria in thyroid pathology to identify the progression of dysplasia to cancer? Diagnostical irreproducibility and misnomer in the terminology of some FCPLs have been lying behind the answer to this question. The last past decades' advances in molecular pathology allow us to transform the knowledge in thyroid pathology to cancer prevention and early detection, which will only be possible by improving our understanding of the nature of thyroid precursor and borderline lesions. This review, amassed with the augmented expertise of thyroid pathology documented in the literature, is an attempt at underlining the present understanding of precursor and borderline lesions of the thyroid, with a particular highlight on practice differences in Asian and Western geographies.

Enhanced Canonical Wnt Signaling During Early Zebrafish Development Perturbs the Interaction of Cardiac Mesoderm and Pharyngeal Endoderm and Causes Thyroid Specification Defects.

Background: Congenital hypothyroidism due to thyroid dysgenesis is a frequent congenital endocrine disorder for which the molecular mechanisms remain unresolved in the majority of cases. This situation reflects, in part, our still limited knowledge about the mechanisms involved in the early steps of thyroid specification from the endoderm, in particular the extrinsic signaling cues that regulate foregut endoderm patterning. In this study, we used small molecules and genetic zebrafish models to characterize the role of various signaling pathways in thyroid specification. Methods: We treated zebrafish embryos during different developmental periods with small-molecule compounds known to manipulate the activity of Wnt signaling pathway and observed effects in thyroid, endoderm, and cardiovascular development using whole-mount in situ hybridization and transgenic fluorescent reporter models. We used the antisense morpholino (MO) technique to create a zebrafish acardiac model. For thyroid rescue experiments, bone morphogenetic protein (BMP) pathway induction in zebrafish embryos was obtained by manipulation of heat-shock inducible transgenic lines. Results: Combined analyses of thyroid and cardiovascular development revealed that overactivation of Wnt signaling during early development leads to impaired thyroid specification concurrent with severe defects in the cardiac specification. When using a model of MO-induced blockage of cardiomyocyte differentiation, a similar correlation was observed, suggesting that defective signaling between cardiac mesoderm and endodermal thyroid precursors contributes to thyroid specification impairment. Rescue experiments through transient overactivation of BMP signaling could partially restore thyroid specification in models with defective cardiac development. Conclusion: Collectively, our results indicate that BMP signaling is critically required for thyroid cell specification and identify cardiac mesoderm as a likely source of BMP signals.

Functional Characterization of Xenopus Thyroid Hormone Transporters mct8 and oatp1c1.

Xenopus is an excellent model for studying thyroid hormone signaling as it undergoes thyroid hormone-dependent metamorphosis. Despite the fact that receptors and deiodinases have been described in Xenopus, membrane transporters for these hormones are yet to be characterized. We cloned Xenopus monocarboxylate transporter 8 (mct8) and organic anion-transporting polypeptide 1C1 (oatpc1c1), focusing on these two transporters given their importance for vertebrate brain development. Protein alignment and bootstrap analysis showed that Xenopus mct8 and oatp1c1 are closer to their mammalian orthologs than their teleost counterparts. We functionally characterized the two transporters using a radiolabeled hormones in vitro uptake assay in COS-1 cells. Xenopus mct8 was found to actively transport both T3 and T4 bidirectionally. As to the thyroid precursor molecules, diiodotyrosine (DIT) and monoiodotyrosine (MIT), both human and Xenopus mct8, showed active efflux, but no influx. Again similar to humans, Xenopus oatp1c1 transported T4 but not T3, MIT, or DIT. We used reverse transcription quantitative polymerase chain reaction and in situ hybridization to characterize the temporal and spatial expression of mct8 and oatp1c1 in Xenopus. Specific expression of the transporter was observed in the brain, with increasingly strong expression as development progressed. In conclusion, these results show that Xenopus thyroid hormone transporters are functional and display marked spatiotemporal expression patterns. These features make them interesting targets to elucidate their roles in determining thyroid hormone availability during embryonic development.

Thyrospheres From Normal or Malignant Thyroid Tissue Have Different Biological, Functional, and Genetic Features.

Cancer stem cells from several human malignancies, including poorly differentiated thyroid carcinoma and thyroid cancer cell lines, have been cultured in vitro as sphere-forming cells. These thyroid cancer stem cells were proven to be able to reproduce the original tumor in a xenograft orthotopic model. The objective of the study was to characterize papillary thyroid carcinoma (PTC) spheres from well-differentiated thyroid cancer and normal thyroid (NT) spheres obtained from the contralateral thyroid tissue of the same patient. Thyrospheres from PTCs and NTs were isolated. Gene expression analysis by real-time PCR, immunofluorescence studies, and fluorescence-activated cell sorter analysis in thyrospheres from PTCs and NTs have been evaluated. Compared with NT spheres, PTC spheres are larger, more irregular, and more clonogenic and have a higher rate of symmetric division. Moreover, PTC spheres express higher levels of stem cell markers and lower levels of thyroid-specific genes compared with NT spheres. Under appropriate conditions, NT spheres differentiated into thyrocytes, whereas PTC spheres did not, displaying a defect in the differentiation potential. Immunofluorescence experiments indicated that, in NT spheres, progenitor cells are mainly present in the sphere core, and the sphere periphery contains thyroid precursor cells already committed to differentiation. PTC spheres are not polarized like NT spheres. Unlike cells differentiated from NT spheres, TSH did not significantly stimulate cAMP production in cells differentiated from PTC spheres. A microarray analysis performed in paired samples (NT and PTC spheres from the same patient) indicated that NT and PTC spheres display a gene expression pattern typical of stem/progenitor cells; however, compared with NT spheres, PTC spheres display a unique gene expression pattern that might be involved in PTC progression.

Zebrafish bcl2l is a survival factor in thyroid development

Regulated cell death, defined in morphological terms as apoptosis, is crucial for organ morphogenesis. While differentiation of the thyroid gland has been extensively studied, nothing is yet known about the survival mechanisms involved in the development of this endocrine gland. Using the zebrafish model system, we aim to understand whether genes belonging to the Bcl-2 family that control apoptosis are implicated in regulation of cell survival during thyroid development. Evidence of strong Bcl-2 gene expression in mouse thyroid precursors prompted us to investigate the functions played by its zebrafish homologs during thyroid development. We show that the bcl2-like (bcl2l) gene is expressed in the zebrafish thyroid primordium. Morpholino-mediated knockdown and mutant analyses revealed that bcl2l is crucial for thyroid cell survival and that this function is tightly modulated by the transcription factors pax2a, nk2.1a and hhex. Also, the bcl2l gene appears to control a caspase-3-dependent apoptotic mechanism during thyroid development. Thyroid precursor cells require an actively maintained survival mechanism to properly proceed through development. The bcl2l gene operates in the inhibition of cell death under direct regulation of a thyroid specific set of transcription factors. This is the first demonstration of an active mechanism to ensure survival of the thyroid primordium during morphogenesis.

A High Prevalence of Dual Thyroid Ectopy in Congenital Hypothyroidism: Evidence for Insufficient Signaling Gradients during Embryonic Thyroid Migration or for the Polyclonal Nature of the Thyroid Gland?

Thyroid ectopy results from the failure of the thyroid precursor cells to migrate from the primordial pharynx to the anterior part of the neck. Most ectopic thyroids are revealed by congenital hypothyroidism and present as a single round mass at the base of the tongue, with no other thyroid tissue. However, some cases have dual ectopy, with part of the tissue having partially migrated. We hypothesized that this occurs more frequently than previously reported. To determine the prevalence of dual ectopy, we reviewed the pertechnetate scintigraphies of 81 patients with congenital hypothyroidism from thyroid ectopy diagnosed between 2002 and 2011 at our institution. We report a series of seven cases (9%) of dual ectopy, representing an incidence ranging from 1:50,000 to 1:70,000. Almost one in 10 cases with congenital hypothyroidism due to thyroid ectopy has dual ectopy. This suggests that two populations of cells diverged at an early stage of development, which may arise from insufficient signaling gradients in surrounding tissues during early organogenesis or may indirectly support the polyclonal nature of the thyroid.

Evidence for Calcitonin-Producing Cells in Human Lingual Thyroids

The thyroid contains two types of cells, the thyroid follicular cells and the calcitonin-producing cells. The site of origin of the thyroid follicular cells is the median thyroid anlage, an endothelial diverticulum in the midline of the ventral pharynx between the first and the second pharyngeal pouches. The ultimobranchial bodies (UBB), a pair of transient embryonic structures evaginated from the fourth pharyngeal pouch and located symmetrically on the sides of the developing neck, are the source of calcitonin-producing cells. In human embryos, the thyroid bud starts its migration at embryonic day 24 and reaches its final location in front of the trachea at embryonic day 45-50. The UBB fuse with the primitive thyroid when thyroid migration is completed. Lingual thyroids result from the failure of the thyroid precursor cells to migrate from the primordial pharynx to the anterior part of the neck. Therefore, calcitonin-producing cells are not expected to be present in lingual thyroids. Our objective was to determine whether calcitonin-producing C cells are present in ectopic lingual thyroids. We performed calcitonin immunolabeling and transcript detection on four flash-frozen ectopic lingual thyroids. Additional calcitonin immunolabeling was performed on two other paraffin-embedded ectopic lingual thyroids. We report evidence of calcitonin-producing cells in six independent cases of ectopic lingual thyroids. The UBB are not the only source of calcitonin-producing cells in humans. Interactions between calcitonin-producing and thyroid follicular cells occur earlier than previously accepted.

Insulin Receptor Isoforms and Insulin-Like Growth Factor Receptor in Human Follicular Cell Precursors from Papillary Thyroid Cancer and Normal Thyroid

Factors involved in the biology of normal and cancer stem/precursor cells from the thyroid are unknown. Thyroid cancer cells are responsive to insulin and IGF-I and IGF-II and often overexpress the insulin receptor (IR) and the IGF-I receptor (IGF-IR). We investigated the role of IR isoforms (IR-A and IR-B), IGF-IR, and their ligands in thyroid follicular cell precursors both normal and malignant. We established cultures of follicular cell precursors as thyrospheres from three papillary thyroid cancers and the corresponding nonaffected tissues. The expression of IR, IGF-IR, and their ligands was evaluated by quantitative RT-PCR and, in one case, also by Western blot. The effects of insulin and IGFs on thyrosphere growth and self-renewal were evaluated. Thyrospheres were characterized by the expression of stem cell markers and low/absent thyroid specific markers. Thyrospheres from normal tissue, but not from cancer tissue, could be induced to differentiate. Both IR isoforms, IGF-IR, IGF-I and IGF-II, were expressed at high levels in thyrospheres and markedly decreased in differentiating cells. IR-A was the predominant isoform in thyrospheres, especially from cancer, while IR-B was predominant in differentiating cells. Cancer thyrosphere growth was stimulated by insulin and IGFs. Our data suggest that IR isoforms and IGF-IR play a role in the biology of follicular thyroid precursors. Cell differentiation is associated with marked changes in the expression of these receptors and cognate ligands. These data may provide insight for future differentiation therapies in thyroid cancer.

Early developmental specification of the thyroid gland depends onhan-expressing surrounding tissue and on FGF signals

The thyroid is an endocrine gland in all vertebrates that develops from the ventral floor of the anterior pharyngeal endoderm. Unravelling the molecular mechanisms of thyroid development helps to understand congenital hypothyroidism caused by the absence or reduction of this gland in newborn humans. Severely reduced or absent thyroid-specific developmental genes concomitant with the complete loss of the functional gland in the zebrafish hands off (han, hand2) mutant reveals the han gene as playing a novel, crucial role in thyroid development. han-expressing tissues surround the thyroid primordium throughout development. Fate mapping reveals that, even before the onset of thyroid-specific developmental gene expression, thyroid precursor cells are in close contact with han-expressing cardiac lateral plate mesoderm. Grafting experiments show that han is required in surrounding tissue, and not in a cell-autonomous manner, for thyroid development. Loss of han expression in the branchial arches and arch-associated cells after morpholino knock-down of upstream regulator genes does not impair thyroid development, indicating that other han-expressing structures, most probably cardiac mesoderm, are responsible for the thyroid defects in han mutants. The zebrafish ace (fgf8) mutant has similar thyroid defects as han mutants, and chemical suppression of fibroblast growth factor (FGF) signalling confirms that this pathway is required for thyroid development. FGF-soaked beads can restore thyroid development in han mutants, showing that FGFs act downstream of or in parallel to han. These data suggest that loss of FGF-expressing tissue in han mutants is responsible for the thyroid defects.

The developing mouse thyroid: Embryonic vessel contacts and parenchymal growth pattern during specification, budding, migration, and lobulation

Normal mouse thyroid development has been revised to identify critical morphogenetic events. The early thyroid primordium associates with the aortic sac endothelium at the time of specification and budding. The vascular contact is lost after the thyroid buds from the pharyngeal endoderm, but is resumed before the gland divides to form two lobes. Lateral expansion of parenchyma takes place along the course of the third pharyngeal arch arteries. Thyroid precursor cells expressing Titf1/Nkx2.1 do not proliferate until the migration stage, implicating that progenitors likely are recruited from outside the thyroid placode. Early lobulation involves engulfment of the entire ultimobranchial bodies by the growing midline thyroid. At the same time, proliferation of the ultimobranchial body epithelium is silenced preceding the differentiation of C cells. Before folliculogenesis, thyroid lobe enlargement is reminiscent of a budding-branching-like growth pattern. It is suggested that thyroid inductive signals arise from embryonic vessels, and that this provides ideas to conceptually new pathogenetic mechanisms of thyroid dysgenesis.

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.

An integrated regulatory network controlling survival and migration in thyroid organogenesis

The thyroid gland originates from the ventral floor of the foregut as a thickening of the endodermal cell layer. The molecular mechanisms underlying the early steps of thyroid morphogenesis are not known. Gene targeting experiments have contributed to the identification of several transcription factors, in general playing a role in the proliferation, survival, and migration of the thyroid cell precursors. The experiments reported here analyze the expression of the transcription factors Titf1, Hhex, Pax8, and Foxe1 in the thyroid primordium of null mutants of each of them. We found that most of these transcription factors are linked in an integrated regulatory network, each of them controlling the presence of other members of the network. The expression of Foxe1 is regulated in an intriguing fashion as it is strongly dependent on the presence of Pax8 in thyroid precursor cells, while the expression of the same gene in the pharyngeal endoderm surrounding the primordium is dependent on Sonic hedgehog (Shh)-derived signaling. Moreover, by the generation of mouse mutants expressing Foxe1 exclusively in the thyroid primordium, we provide a better understanding of the role of Foxe1 in these cells in order to acquire the competence to migrate into the underlying mesenchyme. In conclusion, we provide the first evidence of gene expression programs, controlled by a hierarchy of transcription factors expressed in the thyroid presumptive gut domain and directing the progression of thyroid morphogenesis.

Genetic Deletion of Sonic Hedgehog Causes Hemiagenesis and Ectopic Development of the Thyroid in Mouse

Thyroid dysgenesis encountered in 85% of patients with congenital hypothyroidism is a morphologically heterogeneous condition with primarily unknown pathogenesis. Here we identify sonic hedgehog (Shh) as a novel regulator of thyroid development. In Shh knockout mice the thyroid primordium is correctly specified in the pharyngeal endoderm, but budding and dislocation are slightly delayed. In late development the thyroid fails to form a bilobed gland. Instead a single thyroid mass is found unilaterally and mostly to the left of the midline. Thyroid-specific transcription factors (TTF-1 and TTF-2) and thyroglobulin are expressed indicating terminal differentiation. Strikingly, TTF-1- and TTF-2-positive cells aberrantly develop in the presumptive trachea of Shh-/- embryos. The ectopic tissue buds ventrolaterally into the adjacent mesenchyme, and less extensively into the tracheal lumen, forming follicle-like structures that accumulate thyroglobulin. Shh mRNA is not expressed in the thyroid precursor cells at any developmental stage. The results indicate that Shh signaling indirectly governs the symmetric bilobation of the thyroid during late organogenesis. Shh also seems to repress inappropriate thyroid differentiation in nonthyroid embryonic tissues. This study provides clues to the molecular mechanisms that might be dysregulated in thyroid hemiagenesis and development of ectopic thyroid tissue outside the thyroglossal duct.

Expression of nk2.1a during early development of the thyroid gland in zebrafish

We show here that a zebrafish orthologue of the Thyroid Transcription Factor-1 (TTF-1), nk2.1a, is expressed in the developing thyroid gland. Using a fate mapping approach we found that an early nk2.1a expression domain in the endoderm adjacent to the heart follows morphogenetic movements of the lower jaw, ending up in the region in which the mature thyroid gland is located. We therefore suggest that nk2.1a labels the thyroid precursor cells from somitogenesis stages onwards.

Comparison of high-performance liquid chromatographic detection methods for thyronine and tyrosine residues in toxicological studies of the thyroid

Four high-performance liquid chromatographic methods for the detection of thyroid hormones (iodinated thyronines) and precursors (iodinated tyrosines) have been developed and evaluated. Two methods consist of direct determination of the parent compounds with detection at ultraviolet wavelength (230 nm) and with electrochemical detection. The two other methods consist of a pre-column derivatization (with fluorenylmethyl chloroformate and dabsyl chloride) prior to high-performance liquid chromatographic analysis. The various methods were evaluated based on their practical use and sensitivity. The method with direct ultraviolet detection turned out to be the most practical method. With this method analyses of thyroid homogenates have been performed from rats from a toxicological experiment.

Pathogenesis of endemic goitre in Eastern Nigeria.

1. A survey in Eastern Nigeria revealed an area of endemic goitre with a marked variation in incidence form village to village not accounted for by iodine deficiency alone.2. As dried unfermented cassava was consumed in large quantities in the highly goitrous areas, experiments with rats were undertaken to assess the significance of this factor.3. Groups of rats were fed (a) cassava, (b) equal parts cassava and standard diet, (c) cassava with added iodine and (d) standard diet, and each rat received and intraperitioneal injection of 20 μc iodine 24 h before being killed.4. The iodine content and hardness of the water in the various areas were estimated and the chemical and bacteriological indices of pollution determined.5. The following effects were observed: thyroid weight, iodine uptake and plasma-protein-bound iodine were all increased in the cassava-fed animals, the last markedly so. the thyroid's precursor and hormone iodine stores were severely depleted in these animals, which also showed an impaired transfer of iodine from monoiodotyrosine to diiodotyrosine and a high proportion the iodine present as iodothyronine. The giving of iodine with the cassava did not prevent these changes from taking place.6. Many of the observed effects suggest that cassava was acting like the thionamide group of an antithyroid drug.7. These findings are discussed.