A mouse model for hereditary thyroid dysgenesis and cleft palate.

Alteration of thyroid gland morphogenesis (thyroid dysgenesis) is a frequent human malformation. Among the one in three to four thousand newborns in which congenital hypothyroidism is detected, 80% have either an ectopic, small and sublingual thyroid, or have no thyroid tissue. Most of these cases appear sporadically, although a few cases of recurring familial thyroid dysgenesis have been described. The lack of evidence for hereditary thyroid dysgenesis may be due to the severity of the hypothyroid phenotype. Neonatal screening and early thyroid hormone therapy have eliminated most of the clinical consequences of hypothyroidism such that the heritability of this condition may become apparent in the near future. We have recently cloned cDNA encoding a forkhead domain-containing transcription factor, TTF-2, and have located the position of the gene, designated Titf2, to mouse chromosome 4 (ref. 3). Titf2 is expressed in the developing thyroid, in most of the foregut endoderm and in craniopharyngeal ectoderm, including Rathke's pouch. Expression of Titf2 in thyroid cell precursors is down-regulated as they cease migration, suggesting that this factor is involved in the process of thyroid gland morphogenesis. Here we show that Titf2-null mutant mice exhibit cleft palate and either a sublingual or completely absent thyroid gland. Thus, mutation of Titf2-/- results in neonatal hypothyroidism that shows similarity to thyroid dysgenesis in humans.

PAX8 mutations associated with congenital hypothyroidism caused by thyroid dysgenesis.

Permanent congenital hypothyroidism (CH) is a common disease that occurs in 1 of 3,000-4,000 newborns. Except in rare cases due to hypothalamic or pituitary defects, CH is characterized by elevated levels of thyroid-stimulating hormone (TSH) resulting from reduced thyroid function. When thyroid hormone therapy is not initiated within the first two months of life, CH can cause severe neurological, mental and motor damage. In 80-85% of cases, CH is associated with and presumably is a consequence of thyroid dysgenesis (TD). In these cases, the thyroid gland can be absent (agenesis, 35-40%), ectopically located (30-45%) and/or severely reduced in size (hypoplasia, 5%). Familial cases of TD are rare, even though ectopic or absent thyroid has been occasionally observed in siblings. The pathogenesis of TD is still largely unknown. Although a genetic component has been suggested, mutations in the gene encoding the receptor for the thyroid-stimulating hormone (TSHR) have been identified in only two cases of TD with hypoplasia. We report mutations in the coding region of PAX8 in two sporadic patients and one familial case of TD. All three point mutations are located in the paired domain of PAX8 and result in severe reduction of the DNA-binding activity of this transcription factor. These genetic alterations implicate PAX8 in the pathogenesis of TD and in normal thyroid development.

Regional expression of the homeobox gene Nkx-2.2 in the developing mammalian forebrain.

A novel mouse homeobox-containing gene, Nkx-2.2, has been isolated. Nkx-2.2 is a member of a family of genes whose homeodomains are homologous to that of the Drosophila NK-2 gene. Nkx-2.2 transcripts are found in localized domains of the brain during mouse embryogenesis. Nkx-2.2 expression in the brain abuts and partially overlaps with the expression domains of two other related homeobox-containing genes, TTF-1 and Dlx. The expression domains of the three genes in the developing prosencephalon coincide with anatomical boundaries, particularly apparent in the diencephalon. This result raises the possibility that these genes may specify regional differentiation of the developing diencephalon into its anatomically and functionally defined subregions. Nkx-2.2 may be involved in specifying diencephalic neuromeric boundaries.

Molecular genetics of congenital hypothyroidism.

Congenital thyroid gland defects - resulting in reduced production of the hormones triiodothyronine (T3) and thyroxine (T4) - can be a consequence of either reduced or absent thyroid tissue (thyroid dysgenesis) or, less frequently, of impairment in the biochemical mechanisms responsible for hormone biosynthesis (thyroid dyshormonogenesis). Recent studies have revealed how mutations in the genes encoding either transcription factors or the thyroid stimulating hormone receptor cause, in humans or in mouse models, thyroid dysgenesis. This demonstrates, for the first time, the heritability of this condition. New genes responsible for thyroid dyshormonogenesis have also been discovered.

The lung-specific surfactant protein B gene promoter is a target for thyroid transcription factor 1 and hepatocyte nuclear factor 3, indicating common factors for organ-specific gene expression along the foregut axis.

We used the lung epithelial cell-specific surfactant protein B (SPB) gene promoter as a model with which to investigate mechanisms involved in transcriptional control of lung-specific genes. In a previous study, we showed that the SPB promoter specifically activated expression of a linked reporter gene in the continuous H441 lung cell line and that H441 nuclear proteins specifically protected a region of this promoter from bp -111 to -73. In this study, we further show that this region is a complex binding site for thyroid transcription factor 1 (TTF-1) and hepatocyte nuclear factor 3 (HNF-3). Whereas TTF-1 bound two highly degenerate and closely spaced sites, HNF-3 proteins bound a TGT3 motif (TGTTTGT) that is also found in several liver-specific gene regulatory regions, where it appears to be a weak affinity site for HNF-3. Point mutations of these binding sites eliminated factor binding and resulted in significant decreases in transfected SPB promoter activity. In addition, we developed a cotransfection assay and showed that a family of lung-specific gene promoters that included the SPB, SPC, SPA, and Clara cell secretory protein (CCSP) gene promoters were specifically activated by cotransfected TTF-1. We conclude that TTF-1 and HNF-3 are major activators of lung-specific genes and propose that these factors are involved in a general mechanism of lung-specific gene transcription. Importantly, these data also show that common factors are involved in organ-specific gene expression along the mammalian foregut axis.

Multiple ras downstream pathways mediate functional repression of the homeobox gene product TTF-1.

Expression of oncogenic Ras in thyroid cells results in loss of expression of several thyroid-specific genes and inactivation of TTF-1, a homeodomain-containing transcription factor required for normal development of the thyroid gland. In an effort to understand how signal transduction pathways downstream of Ras may be involved in suppression of the differentiated phenotype, we have tested mutants of the Ras effector region for their ability to affect TTF-1 transcriptional activity in a transient-transfection assay. We find that V12S35 Ras, a mutant known to interact specifically with Raf but not with RalGDS or phosphatidylinositol 3-kinase (PI3 kinase) inhibits TTF-1 activity. Expression of an activated form of Raf (Raf-BXB) also inhibits TTF-1 function to a similar extent, while the MEK inhibitors U0126 and PD98059 partially relieve Ras-mediated inactivation of TTF-1, suggesting that the extracellular signal-regulated kinase (ERK) pathway is involved in this process. Indeed, ERK directly phosphorylates TTF-1 at three serine residues, and concomitant mutation of these serines to alanines completely abolishes ERK-mediated phosphorylation both in vitro and in vivo. Since activation of the Raf/MEK/ERK pathway accounts for only part of the activity elicited by oncogenic Ras on TTF-1, other downstream pathways are likely to be involved in this process. We find that activation of PI3 kinase, Rho, Rac, and RalGDS has no effect on TTF-1 transcriptional activity. However, a poorly characterized Ras mutant, V12N38 Ras, can partially repress TTF-1 transcriptional activity through an ERK-independent pathway. Importantly, concomitant expression of constitutive activated Raf and V12N38 Ras results in almost complete loss of TTF-1 activity. Our data indicate that the Raf/MEK/ERK cascade may act in concert with an as-yet-uncharacterized signaling pathway activated by V12N38 Ras to repress TTF-1 function and ultimately to inhibit thyroid cell differentiation.

Cell-type-specific expression of the rat thyroperoxidase promoter indicates common mechanisms for thyroid-specific gene expression.

A 420-bp fragment from the 5' end of the rat thyroperoxidase (TPO) gene was fused to a luciferase reporter and shown to direct cell-type-specific expression when transfected into rat thyroid FRTL-5 cells. Analysis of this DNA fragment revealed four regions of the promoter which interact with DNA-binding proteins present in FRTL-5 cells. Mutation of the DNA sequence within any of these regions reduced TPO promoter activity. The trans-acting factors binding to these sequences were compared with thyroid transcription factor 1 (TTF-1) and TTF-2, previously identified as transcriptional activators of another thyroid-specific gene, the thyroglobulin (Tg) gene. Purified TTF-1 binds to three regions of TPO which are protected by FRTL-5 proteins. Two of the binding sites overlap with recognition sites for other DNA-binding proteins. One TTF-1 site can also bind a protein (UFB) present in the nuclei of both expressing and nonexpressing cells. TTF-1 binding to the proximal region overlaps with that for a novel protein present in FRTL-5 cells which can also recognize the promoter-proximal region of Tg. Using a combination of techniques, the factor binding to the fourth TPO promoter site was shown to be TTF-2. We conclude, therefore, that the FRTL-5-specific expression of two thyroid restricted genes, encoding TPO and Tg, relies on a combination of the same trans-acting factors present in thyroid cells.

Pax-8, a paired domain-containing protein, binds to a sequence overlapping the recognition site of a homeodomain and activates transcription from two thyroid-specific promoters.

The Pax-8 gene, a member of the murine family of paired box-containing genes (Pax genes), is expressed in adult thyroid and in cultured thyroid cell lines. The Pax-8 protein binds, through its paired domain, to the promoters of thyroglobulin and thyroperoxidase, genes that are exclusively expressed in the thyroid. In both promoters, the binding site of Pax-8 overlaps with that of TTF-1, a homeodomain-containing protein involved in the activation of thyroid-specific transcription. Pax-8 activates transcription from cotransfected thyroperoxidase and thyroglobulin promoters, indicating that it may be involved in the establishment, control, or maintenance of the thyroid-differentiated phenotype. Thus, the promoters of thyroglobulin and thyroperoxidase represent the first identified natural targets for transcriptional activation by a paired domain-containing protein.

The paired-domain transcription factor Pax8 binds to the upstream enhancer of the rat sodium/iodide symporter gene and participates in both thyroid-specific and cyclic-AMP-dependent transcription.

The gene encoding the Na/I symporter (NIS) is expressed at high levels only in thyroid follicular cells, where its expression is regulated by the thyroid-stimulating hormone via the second messenger, cyclic AMP (cAMP). In this study, we demonstrate the presence of an enhancer that is located between nucleotides -2264 and -2495 in the 5'-flanking region of the NIS gene and that recapitulates the most relevant aspects of NIS regulation. When fused to either its own or a heterologous promoter, the NIS upstream enhancer, which we call NUE, stimulates transcription in a thyroid-specific and cAMP-dependent manner. The activity of NUE depends on the four most relevant sites, identified by mutational analysis. The thyroid-specific transcription factor Pax8 binds at two of these sites. Mutations that interfere with Pax8 binding also decrease transcriptional activity of the NUE. Furthermore, expression of Pax8 in nonthyroid cells results in transcriptional activation of NUE, strongly suggesting that the paired-domain protein Pax8 plays an important role in NUE activity. The NUE responds to cAMP in both protein kinase A-dependent and -independent manners, indicating that this enhancer could represent a novel type of cAMP responsive element. Such a cAMP response requires Pax8 but also depends on the integrity of a cAMP responsive element (CRE)-like sequence, thus suggesting a functional interaction between Pax8 and factors binding at the CRE-like site.

Multiple mechanisms of interference between transformation and differentiation in thyroid cells.

Transformation of the thyroid cell line FRTL-5 results in loss or reduction of differentiation as measured by the expression of thyroglobulin and thyroperoxidase, two proteins whose genes are exclusively expressed in thyroid follicular cells. The biochemical mechanisms leading to this phenomenon were investigated in three cell lines obtained by transformation of FRTL-5 cells with Ki-ras, Ha-ras, and polyomavirus middle-T oncogenes. With the ras oncogenes, transformation leads to undetectable expression of the thyroglobulin and thyroperoxidase genes. However, the mechanisms responsible for the extinction of the differentiated phenotype seem to be different for the two ras oncogenes. In Ki-ras-transformed cells, the mRNA encoding TTF-1, a transcription factor controlling thyroglobulin and thyroperoxidase gene expression, is severely reduced. On the contrary, nearly wild-type levels of TTF-1 mRNA are detected in Ha-ras-transformed cells. Furthermore, overexpression of TTF-1 can activate transcription of the thyroglobulin promoter in Ki-ras-transformed cells, whereas it has no effect on thyroglobulin transcription in the Ha-ras-transformed line. Expression of polyoma middle-T antigen in thyroid cells leads to only a reduction of differentiation and does not severely affect either the activity or the amount of TTF-1. Another thyroid cell-specific transcription factor, TTF-2, is more sensitive to transformation, since it disappears in all three transformed lines, and probably contributes to the reduced expression of the differentiated phenotype.

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.

Expression of thyroid-specific transcription factors TTF-1 and PAX-8 in human thyroid neoplasms.

TTF-1 and PAX-8 are tissue-specific transcription factors expressed in the thyroid follicular cells, contributing to the maintenance of the differentiated phenotype. In fact, it has been demonstrated that TTF-1 and PAX-8 are able to activate transcription from thyroglobulin and thyroperoxidase (TPO) promoters, the transcriptional activity of which is in vivo restricted only to the thyroid follicular cell. In order to gain insight into how these transcription factors control in vivo the differentiation of the thyroid cell and to have a better molecular characterization of human thyroid tumors, TTF-1, PAX-8, thyroglobulin, and TPO mRNA levels were measured in nonmalignant and malignant human thyroid tissues. Results indicate that the expression of TTF-1 and PAX-8 is not sufficient per se for the expression of the thyroid-differentiated phenotype. Furthermore, in follicular adenomas, PAX-8 mRNA levels are strictly related to TPO mRNA levels, suggesting that the amount of PAX-8 could play a role in the modulation of TPO gene expression. TTF-1 mRNA is always well detectable in papillary carcinomas and, in contrast, always absent in anaplastic carcinomas. Identical results were obtained when the expression of TTF-1 protein was investigated using immunohistochemistry. Thus, TTF-1 gene expression could be a molecular marker in order to distinguish these two types of thyroid neoplasms.

Reexpression of thyroid peroxidase in a derivative of an undifferentiated thyroid carcinoma cell line by introduction of wild-type p53.

Loss of function of p53 is believed to result in transformation through impairment of its properties as a transcription factor, which interferes with the regulation of the cell cycle and under certain conditions, with programmed cell death. We report that stable transfection of clonal undifferentiated thyroid carcinoma cell lines harboring endogenous p53 mutations with a wild-type p53 expression vector only rarely yields transfectants expressing authentic wild-type p53. Among these, most exhibited an increase in doubling time and an impairment of colony formation in soft agar. Only one clonal wild-type p53-overexpressing derivative of the NPA papillary carcinoma cell line was obtained, and these cells were found to reexpress thyroid peroxidase (TPO). This clone also demonstrated reexpression of the paired box domain transcription factor Pax-8, which specifically activates transcription of TPO. Wild-type p53 did not directly stimulate transcriptional activity of a TPO promoter construct. Although the low frequency of authentic wild-type p53 stable transfectants limits the power of this analysis, these data suggest that in addition to its role in malignant transformation, p53 may be significant in the determination or maintenance of cell differentiation in thyroid neoplasms.

p53 genes mutated in the DNA binding site or at a specific COOH-terminal site exert divergent effects on thyroid cell growth and differentiation.

Expression of mutated versions of the p53 gene deranged the differentiation program of thyroid cells and resulted in deregulated growth. Specifically, p53 mutants in several residues of the DNA-binding region induced thyrotropin (TSH) -independent growth and inhibition of the expression of thyroid-specific genes. The loss of the differentiated phenotype invariably correlated with the blockage of the expression of the genes coding for the thyroid transcriptional factors PAX-8 and TTF2. Conversely, thyroid cells transfected with a p53 gene mutated at codon 392, located outside the DNA-binding region, stimulated the expression of differentiation genes in the absence of the TSH, and induced TSH-independent growth. cAMP intracellular levels were higher in thyroid cells transfected with the p53 gene mutated at the 392 site than in the untransfected thyroid cells, but lower in the cells transfected with the other mutated p53 genes. Fra-1 and c-jun were induced by p53, resulting in increased AP-1 levels. The results of this study suggest that p53 exerts effects on cAMP transduction pathway in thyroid cells, which are exquisitely sensitive to cAMP.

Concomitant activation of MEK-1 and Rac-1 increases the proliferative potential of thyroid epithelial cells, without affecting their differentiation.

Activating point mutations in the Ras oncogene occur in a large number of human tumors, especially of epithelial origin. In thyroid follicular cells, ectopic expression of oncogenic H-Ras results in growth factor-independent proliferation, loss of differentiation and tumor formation in nude mice. In fibroblasts concomitant activation of the MAP kinase cascade and the small GTPase Rac-1 leads to full malignant transformation. We have tested the effects of these key downstream mediators of Ras in thyroid epithelial cells, by stably expressing either a constitutively active form of MEK-1 (MEK(deltaN3/S218E/S222D)), a constitutively active form of Rac-1 (Val12-Rac), or both. While the activation of one molecule or the other results in a weak phenotype, concomitant activation of both MEK-1 and Rac-1 in thyroid cells leads to growth factor-independent proliferation, morphological transformation and anchorage-independent growth. However, in contrast to Ras-transformed thyroid cells, the ones expressing the constitutively active forms of MEK-1 and Rac-1 maintain their differentiate phenotype and fail to form tumors when injected into nude mice. Thus, in thyroid epithelial cells, concomitant activation of MEK-1 and Rac-1 can reproduce only a subset of the Ras-induced effects and is not sufficient to cause full malignant transformation. Significantly, Ras-mediated increased proliferation and loss of differentiation can be dissociated in these cells.

Immediate early genes induced by H-Ras in thyroid cells.

Expression of oncogenic v-H-Ras in the thyroid cell line FRTL-5 (FRTL-5(Ras)) results in uncontrolled proliferation, loss of thyroid-specific gene expression and tumorigenicity. Concomitant expression of constitutively activated MEK and Rac, two major H-Ras downstream effectors, in FRTL-5 (FRTL-5(MEK/Rac)) recapitulates H-Ras effects on proliferation and morphology. In contrast to FRTL-5(Ras), however, FRTL-5(MEK/Rac) cells remain differentiated and are not tumorigenic. To find H-Ras induced genes potentially responsible for tumorigenicity and loss of differentiation, we have used subtractive suppression hybridization (SSH), a PCR-based cDNA subtraction technique, between de-differentiated and tumorigenic FRTL-5(Ras) cells and differentiated and non-tumorigenic FRTL-5(MEK/Rac) cells. We examined 800 of the cDNA clones obtained after subtraction and verified their levels of expression in the two cell lines by reverse northern, identifying 337 H-Ras induced genes. By sequence analysis, we clustered 57 different genes. Among these, 39 were known genes (involved in diverse signal transduction processes regulating mitogenic activity, cell survival, cytoskeletal reorganization, stress response and invasion) while the remaining 18 clones were novel genes. Among the 57 H-Ras specific clones, we identified those genes whose expression is induced early by H-Ras. We suggest that these immediate-early genes may play a crucial role in H-Ras-mediated transformation in thyroid epithelial cells.

Transfection of TTF-1 gene induces thyroglobulin gene expression in undifferentiated FRT cells.

The thyroglobulin gene, the substrate for thyroid hormone biosynthesis, is not expressed in the FRT cell line, which, even though it manifests the polarised epithelial phenotype, does not express any of the thyroid functional properties. Two transcription factors, TTF-1 and Pax-8, have been implicated in thyroid specific expression of the thyroglobulin gene. FRT cells contain Pax-8 but they lack TTF-1. In this paper, we show that transfection of TTF-1 expression vectors in FRT cells results in activation of thyroglobulin gene expression. If the expression vector encoded for TTF-1-ER, a fusion gene coding for the entire TTF-1 protein fused to the hormone-binding domain of the steroid receptor, under the control of the RSV promoter, thyroglobulin gene expression was controlled by estrogen. These data provide a direct demonstration that TTF-1 activates the chromosomal thyroglobulin promoter. Since transfection of TTF-1 expression vectors in non-thyroid cell types did not result in thyroglobulin gene expression, it is suggested that Pax-8, in addition, perhaps, to a specific cellular environment, might be required for thyroid specific expression of the thyroglobulin gene.

Identification and developmental expression of three Distal-less homeobox containing genes in the ascidian Ciona intestinalis.

Several homeobox-containing genes related to Drosophila Distal-less (Dll) have been isolated from a wide variety of organisms and have been shown to function as developmental regulators. While in Drosophila only one Dll gene has been described so far, in Vertebrates many components of the Dlx multigenic family have been characterized. This suggests that, during the evolution of the Chordate phylum, the Dlx genes arose from an ancestral Dll/Dlx gene via gene duplication. We have previously reported the isolation of two Dll-related homeoboxes from the protochordate Ciona intestinalis, and described their clustered arrangement (Gene 156 (1995) 253). Here we present the detailed genomic organization and spatial-temporal expression of these two genes, Ci-Dll-A and Ci-Dll-B, and describe the isolation and characterization of another member of the ascidian family of Dll-related genes, which we tentatively named Ci-Dll-C.

Identification of a cis-regulatory element and a thyroid-specific nuclear factor mediating the hormonal regulation of rat thyroid peroxidase promoter activity.

The mechanism for hormonal regulation of rat thyroperoxidase (rTPO) gene transcription in rat FRTL-5 thyroid cells has been investigated. Transient transfection experiments demonstrate that the minimal rTPO promoter that confers thyroid-specific expression also confers responsiveness to TSH and insulin. TSH induces a 7-fold increase in promoter activity, and the induction is detected almost immediately after the addition of the hormone. Insulin also stimulates TPO promoter activity, but the effect of this hormone is weaker and slower than that of TSH. The effect of TSH in increasing TPO promoter activity is mimicked by the cAMP agonist forskolin. The calcium-protein kinase C pathway is also involved in the regulation of the rTPO promoter activity, since a calcium ionophore (A23187) and phorbol esters [12-O-tetradecanoyl-phorbol-13-acetate (TPA)] inhibit it quickly. These data indicate that the region of the rTPO promoter used here contains the DNA signals necessary for its hormonal regulation. Protein-DNA binding studies show that the thyroid-specific nuclear protein TTF-2, which binds to the rTPO promoter, is induced by TSH and forskolin, and this effect is clearly observable as early as 5 h post induction. Moreover, the DNA binding activity of TTF-2 is inhibited by both A23187 and TPA. Heterologous promoter constructs containing four, eight, or 12 tandem repeats of an oligonucleotide that includes the TTF-2 binding site increase their activity in response to TSH, forskolin, and insulin, while the the presence of A23187 or TPA inhibits their activity. These data indicate that the TTF-2 protein plays an important role in the hormonal control of thyroid-specific transcription.

Insulin and insulin-like growth factor I regulate a thyroid-specific nuclear protein that binds to the thyroglobulin promoter.

The mechanism responsible for the stimulation of thyroglobulin (Tg) gene expression by insulin and insulin-like growth factor I (IGF-I) in rat thyroid FRTL-5 cells has been investigated. Both insulin and IGF-I stimulate transcription from the Tg promoter in a transient transfection assay demonstrating that the promoter used contains the DNA signals necessary for insulin and IGF-I regulation. Promoter mutations that interfere with the binding of thyroid transcription factor 1 (TTF-1), TTF-2, and the ubiquitous transcription factor abolish the insulin/IGF-I response, indicating that the three factors may be involved in the observed transcriptional control. Protein-DNA binding studies did not reveal any effect of insulin/IGF-I on the ubiquitous transcription factor and the TTF-1 binding capacity. Instead, TTF-2 is absent in nuclear extracts from cells depleted of serum and insulin. Addition of insulin or IGF-I restores the TTF-2 concentration to normal levels and requires ongoing protein synthesis. The insulin effect was maximal at 24 h and at a concentration of 1 microgram/ml. The same effect was observed with a 10-fold lower concentration of IGF-I. These results suggest that insulin (probably through the IGF-I receptor) and IGF-I modulate the levels of TTF-2, which results in an increased expression of the Tg gene.