Retraction for Fusco et al., "A mos Oncogene-Containing Retrovirus, Myeloproliferative Sarcoma Virus, Transforms Rat Thyroid Epithelial Cells and Irreversibly Blocks Their Differentiation Pattern".

[This retracts the article DOI: 10.1128/JVI.56.1.284-292.1985.].

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.

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.

Intronic elements in the Na+/I- symporter gene (NIS) interact with retinoic acid receptors and mediate initiation of transcription.

Activity of the sodium/iodide symporter (NIS) in lactating breast is essential for iodide (I(-)) accumulation in milk. Significant NIS upregulation was also reported in breast cancer, indicating a potential use of radioiodide treatment. All-trans-retinoic acid (tRA) is a potent ligand that enhances NIS expression in a subset of breast cancer cell lines and in experimental breast cancer models. Indirect tRA stimulation of NIS in breast cancer cells is very well documented; however, direct upregulation by tRA-activated nuclear receptors has not been identified yet. Aiming to uncover cis-acting elements directly regulating NIS expression, we screened evolutionary-conserved non-coding genomic sequences for responsiveness to tRA in MCF-7. Here, we report that a potent enhancer in the first intron of NIS mediates direct regulation by tRA-stimulated nuclear receptors. In vitro as well as in vivo DNA-protein interaction assays revealed direct association between retinoic acid receptor-alpha (RARalpha) and retinoid-X-receptor (RXR) with this enhancer. Moreover, using chromatin immunoprecipitation (ChIP) we uncovered early events of NIS transcription in response to tRA, which require the interaction of several novel intronic tRA responsive elements. These findings indicate a complex interplay between nuclear receptors, RNA Pol-II and multiple intronic RAREs in NIS gene, and they establish a novel mechanistic model for tRA-induced gene transcription.

In vivo role of different domains and of phosphorylation in the transcription factor Nkx2-1.

BACKGROUND: The transcription factor Nkx2-1 (also known as TTF-1, Titf1 or T/EBP) contains two apparently redundant activation domains and is post-translationally modified by phosphorylation. We have generated mouse mutant strains to assess the roles of the two activation domains and of phosphorylation in mouse development and differentiation. RESULTS: Mouse strains expressing variants of the transcription factor Nkx2-1 deleted of either activation domain have been constructed. Phenotypic analysis shows for each mutant a distinct set of defects demonstrating that distinct portions of the protein endow diverse developmental functions of Nkx2-1. Furthermore, a mouse strain expressing a Nkx2-1 protein mutated in the phosphorylation sites shows a thyroid gland with deranged follicular organization and gene expression profile demonstrating the functional role of phosphorylation in Nkx2-1. CONCLUSIONS: The pleiotropic functions of Nkx2-1 are not all due to the protein as a whole since some of them can be assigned to separate domains of the protein or to specific post-translational modifications. These results have implication for the evolutionary role of mutations in transcription factors.

Comparative genomics reveals a functional thyroid-specific element in the far upstream region of the PAX8 gene.

BACKGROUND: The molecular mechanisms leading to a fully differentiated thyrocite are still object of intense study even if it is well known that thyroglobulin, thyroperoxidase, NIS and TSHr are the marker genes of thyroid differentiation. It is also well known that Pax8, TTF-1, Foxe1 and Hhex are the thyroid-enriched transcription factors responsible for the expression of the above genes, thus are responsible for the differentiated thyroid phenotype. In particular, the role of Pax8 in the fully developed thyroid gland was studied in depth and it was established that it plays a key role in thyroid development and differentiation. However, to date the bases for the thyroid-enriched expression of this transcription factor have not been unraveled yet. Here, we report the identification and characterization of a functional thyroid-specific enhancer element located far upstream of the Pax8 gene. RESULTS: We hypothesized that regulatory cis-acting elements are conserved among mammalian genes. Comparison of a genomic region extending for about 100 kb at the 5'-flanking region of the mouse and human Pax8 gene revealed several conserved regions that were tested for enhancer activity in thyroid and non-thyroid cells. Using this approach we identified one putative thyroid-specific regulatory element located 84.6 kb upstream of the Pax8 transcription start site. The in silico data were verified by promoter-reporter assays in thyroid and non-thyroid cells. Interestingly, the identified far upstream element manifested a very high transcriptional activity in the thyroid cell line PC Cl3, but showed no activity in HeLa cells. In addition, the data here reported indicate that the thyroid-enriched transcription factor TTF-1 is able to bind in vitro and in vivo the Pax8 far upstream element, and is capable to activate transcription from it. CONCLUSIONS: Results of this study reveal the presence of a thyroid-specific regulatory element in the 5' upstream region of the Pax8 gene. The identification of this regulatory element represents the first step in the investigation of upstream regulatory mechanisms that control Pax8 transcription during thyroid differentiation and are relevant to further studies on Pax8 as a candidate gene for thyroid dysgenesis.

Thyroid development and its disorders: genetics and molecular mechanisms.

Thyroid gland organogenesis results in an organ the shape, size, and position of which are largely conserved among adult individuals of the same species, thus suggesting that genetic factors must be involved in controlling these parameters. In humans, the organogenesis of the thyroid gland is often disturbed, leading to a variety of conditions, such as agenesis, ectopy, and hypoplasia, which are collectively called thyroid dysgenesis (TD). The molecular mechanisms leading to TD are largely unknown. Studies in murine models and in a few patients with dysgenesis revealed that mutations in regulatory genes expressed in the developing thyroid are responsible for this condition, thus showing that TD can be a genetic and inheritable disease. These studies open the way to a novel working hypothesis on the molecular and genetic basis of this frequent human condition and render the thyroid an important model in the understanding of molecular mechanisms regulating the size, shape, and position of organs.

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.

Conditional inactivation of the E-cadherin gene in thyroid follicular cells affects gland development but does not impair junction formation.

We have conditionally inactivated the E-cadherin gene in the thyroid follicular cells of mouse embryo to unravel its role in thyroid development. We used the Cre-loxP system in which the Cre-recombinase was expressed under the control of the tissue-specific thyroglobulin promoter that becomes active at embryonic d 15. At postnatal d 7, thyroid follicle lumens in the knockout mice were about 30% smaller with respect to control mice and had an irregular shape. E-cadherin was almost completely absent in thyrocytes, beta-catenin was significantly reduced, whereas no change in gamma-catenin was detected. alpha-Catenin was also reduced on the cell plasma membrane. Despite the dramatic loss of E-cadherin and beta-catenin, cell-cell junctions were not affected, the distribution of tight junction proteins was unaltered, and no increase of thyroglobulin circulating in the blood was observed. In addition, we found that other members of the cadherin family, the R-cadherin and the Ksp-cadherin, were expressed in thyrocytes and that their membrane distribution was not altered in the E-cadherin conditional knockout mouse. Our results indicate that E-cadherin has a role in the development of the thyroid gland and in the expression of beta-catenin, but it is not essential for the maintenance of follicular cell adhesion.

Duox expression and related H2O2 measurement in mouse thyroid: onset in embryonic development and regulation by TSH in adult.

In the thyroid, H(2)O(2) is produced at the apical pole of thyrocytes by one or two NADPH oxidases (NOX), Duox1/2 proteins. The onset of Duox expression was analysed by immunohistochemistry in the developing mouse thyroid in parallel with thyroglobulin (Tg) iodination and the expression of other thyroid differentiation markers. Duox proteins were found at embryonic day (E) 15.5 and were mainly localised at the apical pole of thyrocytes. Tg was detected 1 day before (E14.5) and Tg iodination was concomitant with the expression of both Duox and Na(+)/I(-) symporter (NIS; E15.5). The role of TSH in regulating Duox expression and H(2)O(2) accumulation was evaluated in thyroids of adult mice with reduced (Tshr(hyt/hyt) or mice treated with thyroxine) or increased (methimazole or perchlorate treatment) TSH/Tshr activity. In mice with suppressed TSH/Tshr activity, Duox expression was only partially decreased when compared with wild-type, as observed by western blot. In Tshr(hyt/hyt) strain, Duox was still expressed at the apical pole and H(2)O(2) measurements were normal. On the other hand, chronic TSH stimulation of the gland led to a decrease of H(2)O(2) measurements without affecting Duox expression. The onset of Duox protein expression is compatible with their proposed function in thyroid hormone synthesis and it can be considered as a functional marker of the developing thyroid. However, Duox expression in adult is much less regulated by TSH than NIS and thyroperoxidase. It is not always correlated with the overall thyroid H(2)O(2) accumulation, highlighting the importance of additional regulatory mechanisms which control either the production or H(2)O(2) degradation.

Rhes is involved in striatal function.

The development and the function of central nervous system depend on thyroid hormones. In humans, the lack of thyroid hormones causes cretinism, a syndrome of severe mental deficiency. It is assumed that thyroid hormones affect the normal development and function of the brain by activating or suppressing target gene expression because several genes expressed in the brain have been shown to be under thyroid hormone control. Among these, the Rhes gene, encoding a small GTP-binding protein, is predominantly expressed in the striatal region of the brain. To clarify the role of Rhes in vivo, we disrupted the Rhes gene by homologous recombination in embryonic stem cells and generated mice homozygous for the Rhes null mutation (Rhes(-/-)). Rhes(-/-) mice were viable but weighed less than wild-type mice. Furthermore, they showed behavioral abnormalities, displaying a gender-dependent increase in anxiety levels and a clear motor coordination deficit but no learning or memory impairment. These results suggest that Rhes disruption affects selected behavioral competencies.

Functional inactivation of the transcription factor Pax8 through oligomerization chain reaction.

Among the approaches used to provide a functional inactivation of a target protein, we have chosen the recently described oligomerization chain reaction (OCR) strategy to functionally inactivate the transcription factor Pax8, a member of the Pax gene family expressed in thyroid cells. The OCR strategy is based on the fusion of the self-associating coiled-coil (CC) domain of the nuclear factor promyelocytic leukemia (PML) to target proteins that are able to self-associate naturally or that form heterocomplexes. In the thyroid tissue, the transcription factor Pax8 is involved in the morphogenesis of the gland and in the transcriptional regulation of thyroid-expressed genes. We have recently demonstrated that in thyroid cells Pax8 interacts biochemically and functionally with the transcription factor TTF-1 (thyroid transcription factor 1), and that such interaction leads to the synergistic activation of thyroglobulin (Tg) gene expression. Fusion of the CC domain to Pax8 leads to the formation of aberrant, nonfunctional high-molecular mass complexes to which TTF-1 is also recruited. The CC-Pax8 chimera inhibits the transcriptional activity of Pax8 and of TTF-1 on both synthetic and physiological promoters and prevents the synergistic activation of the Tg promoter mediated by these two transcription factors. Furthermore, the expression of the CC-Pax8 chimera in differentiated thyroid cells leads to the down-regulation of the endogenous expression of several differentiation markers such as Tg, sodium/iodide symporter, Foxe1, TTF-1, and thyroid oxidase 2. These results demonstrate that the OCR is a useful tool to functionally inactivate a transcription factor. Moreover, by this approach, we identified Foxe1, TTF-1, and thyroid oxidase 2 as new direct targets of Pax8 or TTF-1.

Missense mutation in the transcription factor NKX2-5: a novel molecular event in the pathogenesis of thyroid dysgenesis.

CONTEXT: Congenital hypothyroidism (CH) is a common endocrine disorder with an incidence of 1:3000-4000 at birth. In 80-85% of cases, CH is caused by defects in thyroid organogenesis, resulting in absent, ectopically located, and/or severely reduced gland [thyroid dysgenesis (TD)]. Mutations in genes controlling thyroid development have demonstrated that in a few cases, TD is a Mendelian trait. However, accumulating evidence supports the view that the genetics of TD are complex, possibly with a polygenic/multifactorial basis. A higher prevalence of congenital heart disease has been documented in children with CH than in the general population. Such an association suggests a possible pathogenic role of genes involved in both heart and thyroid development. NKX2-5 encodes a homeodomain-containing transcription factor with a major role in heart development, and mutations affecting this gene have been reported in individuals with congenital heart disease. OBJECTIVE: In the present work we investigated the possible involvement of NKX2-5 mutations in TD. RESULTS: Our results indicate that Nkx2-5(-/-) embryos exhibit thyroid bud hypoplasia, providing evidence that NKX2-5 plays a role in thyroid organogenesis and that NKX2-5 mutations contribute to TD. NKX2-5 mutational screening in 241 patients with TD allowed the identification of three heterozygous missense changes (R25C, A119S, and R161P) in four patients with TD. Functional characterization of the three mutations demonstrated reduced DNA binding and/or transactivation properties, with a dominant-negative effect on wild-type NKX2-5. CONCLUSION: Our results suggest a previously unknown role of NKX2-5 in the pathogenesis of TD.

A physiological mechanism to regulate D-aspartic acid and NMDA levels in mammals revealed by D-aspartate oxidase deficient mice.

Free D-aspartic acid and NMDA are present in the mammalian central nervous system and endocrine glands at significant concentrations, but their physiological role is still matter of debate. The only enzyme known to metabolize in vitro selectively these D-amino acids is D-aspartate oxidase (DDO). To clarify the role in vivo of the enzyme, we generated mice with targeted deletion of Ddo gene by homologous recombination. Mutated animals showed increased amounts of both D-aspartic acid and NMDA in all tissues examined demonstrating a physiological role of DDO in the regulation of their endogenous levels.

PAX8 expression in human bladder cancer.

PAX8 is a transcription factor with a role in ontogenesis of the urinary tract. The aim of the present investigation was to investigate PAX8 expression in normal bladder and in non-invasive urothelial tumours. Nine cases of normal urothelial mucosa, 2 cases of papillary urothelial neoplasia of low malignant potential, 12 cases of low grade non-invasive papillary urothelial carcinoma and 16 cases of high grade non-invasive papillary urothelial carcinoma were investigated by immunohistochemistry. PAX8 mRNA expression was evaluated by RT-PCR in a different set of normal bladder mucosas and tumours. In addition, PAX8 expression was evaluated by RT-PCR in bladder from 2 human embryos and in several continuous cell lines derived from bladder tumours (5637, RT-112, TCC-SUP, HT 1376). In immunohistochemical studies, PAX8 was expressed in 28 out of 30 non-invasive urothelial tumours, but not in the normal adult bladders. In RT-PCR studies, PAX8 was expressed in 13 out of 13 bladder tumours but not in the 6 normal bladder mucosa. Contrary to that in adults, PAX8 was expressed in 2 cases of bladder mucosa from 16-week-old embryos. PAX8 was expressed in all the cell lines from bladder tumours. Both in the bladder tumours and cell lines PAX8 expression was highly heterogeneous in terms of the splicing isoforms. Treatment of cell lines with sodium butyrate (NaB) induced several changes of the splicing isoforms. Therefore, only subsets of molecular events that determine the PAX8 mRNA splicing heterogeneity in bladder tumours are sensitive to NaB treatment.

Dose-dependent inhibition of thyroid differentiation by RAS oncogenes.

Activating mutations in RAS protooncogenes are associated with several different histotypes of thyroid cancer, including anaplastic thyroid carcinoma. The latter is the most aggressive cancer of the thyroid gland, showing little or no expression of the differentiated phenotype. Likewise, expression of viral RAS oncogenes in FRTL-5 rat thyroid cells mimics such loss of differentiation. We established FRTL-5 cell lines stably expressing constitutively active forms of RAS, either of viral (v-Ha-RAS or v-Ki-RAS) or cellular (H-RAS(V12)) origin and generated a tamoxifen-inducible RAS oncoprotein to analyze the timing of RAS effects on thyroid differentiation. In RAS-transformed FRTL-5 cells, we measured the expression of many thyroid-specific genes by real-time PCR and observed that a clear loss of differentiation was only obtained in the presence of high RAS oncogene expression. In contrast, TSH-independent growth appeared to be induced in the presence of both low and high levels of oncogenic RAS expression. We also showed that inhibition of differentiation is an early RAS-induced phenomenon. Finally, we demonstrated that only high doses of RAS oncogenes are able to inhibit the activity of Titf1 and Pax8, two transcription factors essential for the maintenance of thyroid differentiation, and that the homeodomain of Titf1 is a target of the inhibitory action of RAS. Our results represent the first evidence of a dose-dependent effect of RAS oncogenes on thyroid epithelial differentiation.

Neuronal NCX1 overexpression induces stroke resistance while knockout induces vulnerability via Akt.

Three different Na+/Ca2+ exchanger (NCX) isoforms, NCX1, NCX2, and NCX3, are expressed in brain where they play a relevant role in maintaining Na+ and Ca2+ homeostasis. Although the neuroprotective roles of NCX2 and NCX3 in stroke have been elucidated, the relevance of NCX1 is still unknown because of embryonic lethality of its knocking-out, heart dysfunctions when it is overexpressed, and the lack of selectivity in currently available drugs. To overcome these limitations we generated two conditional genetically modified mice that upon tamoxifen administration showed a selective decrease or increase of NCX1 in cortical and hippocampal neurons. Interestingly, in cortex and hippocampus NCX1 overexpression increased, where NCX1 knock-out reduced, both exchanger activity and Akt1 phosphorylation, a neuronal survival signaling. More important, mice overexpressing NCX1 showed a reduced ischemic volume and an amelioration of focal and general deficits when subjected to transient middle cerebral artery occlusion. Conversely, NCX1-knock-out mice displayed a worsening of brain damage, focal and neurological deficits with a decrease in Akt phosphorylation. These results support the idea that NCX1 overexpression/activation may represent a feasible therapeutic opportunity in stroke intervention.

A mouse model demonstrates a multigenic origin of congenital hypothyroidism.

Congenital hypothyroidism with thyroid dysgenesis (TD) is a frequent human condition characterized by elevated levels of TSH in response to reduced thyroid hormone levels. Congenital hypothyroidism is a genetically heterogeneous disease. In the majority of cases studied, no causative mutations have been identified and very often the disease does not show a Mendelian transmission. However, in approximately 5% of cases, it can be a consequence of mutations in genes encoding the TSH receptor or the transcription factors TITF1, FOXE1, or PAX8. We report here that in mouse models, the combination of partial deficiencies in the Titf1 and Pax8 genes results in an overt TD phenotype that is absent in either of the singly deficient, heterozygous mice. The disease is characterized by a small thyroid gland, elevated levels of TSH, reduced thyroglobulin biosynthesis, and high occurrence of hemiagenesis. The observed phenotype is strain specific, and the pattern of transmission indicates that at least two other genes, in addition to Titf1 and Pax8, are necessary to generate the condition. These results show that TD can be of multigenic origin in mice and strongly suggest that a similar pathogenic mechanism may be observed in humans.

Proteins, fatty acids and nutritional value in the muscle of the fish species Mora moro (Risso, 1810).

Deep-water fish are becoming an interesting object of studies and research due to the development of deep fishery activities. This paper analyses the chemical composition and nutritional value of the fish species Mora moro (Risso, 1810) inhabiting deep Mediterranean waters. The fatty acid profile and the principal water-soluble proteins present in the white muscle of this fish species have also been determined. The major fatty acids were 22 : 6n-3, 16 : 0, 18 : 1n-9, 20 : 4n-6 and 20 : 5n-3. The polyunsaturated fatty acid (PUFA) content was higher than that of saturated (SFA) and monounsaturated fatty acids, but the ratio PUFA/SFA was lower than the value reported in other studies. Both the atherogenic index and thrombogenic index were very low. Water-soluble proteins were characterised by monodimensional native PAGE and 2-D SDS-gel electrophoresis. Protein patterns showed the presence of parvalbumins and of the principal myofibrillar proteins. Therefore, the deep-water fish M. moro could represent an interesting target for deep-sea fishery and commercial exploitation.