Introduction
Primary congenital hypothyroidism (CH) is the most frequent endocrine-metabolic disease in infancy, with an incidence of about 1/3-4000 newborns. In about 85% of the cases, CH is caused by an alteration in the morphogenesis of the thyroid (thyroid dysgenesis, TD) (2). In 5-16% of cases TD it is associated with other major birth defects, mostly cardiac (Table 1) (3).
Most of the critical events in thyroid morphogenesis take place in the first 60 days of gestation in man or the first 15 days in mice. For this reason, thyroid developmental abnormalities result from morphogenetic errors during this period.
The regulation of formation, migration and proliferation of the thyroid gland are still largely unknown. Several genes, including those encoding thyroid specific transcription factors (TITF1, TITF2, PAX8), thyrotropin (TSH) and its receptor (TSHR), and/or other genes, have been demonstrated to play a role (1). Alterations in any of these genes can be responsible for thyroid dysgenesis.
Mutations in the genes involved in thyroid development give rise to animal models with TD, and mutations in the same genes have been identified also in a small number of patients with congenital hypothyroidism associated with TD.

In this review we will briefly describe the role of thyroid transcription factors and their involvement in the pathogenesis of TD.

NKX2-1/TITF1
NKX2-1, also known as TITF1 (Thyroid Transcription Factor–1) is a homeodomain transcription factor that was initially identified in a rat thyroid cell as a nuclear protein able to bind to specific sequences in the Tg promoter. TITF1 belongs to the Nkx2 class of transcription factors and is encoded by a gene, located on chromosome 14q13 (Table 1). The gene is formed by at least 3 exons and encodes for 42 kDa protein that is phosphorylated. During human development, the gene is expressed in the ventral diencephalon and in the telencephalon; in the lung bud and in the thyroid primordium (1, 4).
Studies in mice demonstrated that Titf1 is required for the survival and subsequent differentiation of the cells.

TITF1/NKX2-1 disease
TITF1/NKX2-1 disease is characterized by a variable spectrum of neurological, thyroid and lung abnormalities with incomplete penetrance and the variability of the phenotype (Table 1).
A heterozygous homozygous deletion and a loss of function mutations in TITF1/NKX2-1 gene were initially identified in an isolated infant (5) and in two siblings (6) respectively. All the patients were affected by respiratory failure, hypotonia and thyroid dysfunction, without apparent TD. Later reports (7, 8) have demonstrated the association between mutations in TITF1/NKX2-1 genes and a syndrome characterized by choreoathetosis, respiratory distress and a thyroid phenotype ranging from a normal gland to athyreosis.
When tested in vitro, the mutated forms of Titf1/Nkx2-1 show neither functional activity nor a dominant negative effect on the wild type form, suggesting that the haploinsufficiency is responsible for the pathological phenotype.
After these reports, several other mutations in TITF1/NKX2-1 have been shown to be responsible for this syndrome characterized by the presence of TD, benign familial chorea with or without pulmonary distress (1).

PAX8
Pax8 (Paired Box gene 8) is a member of a family of transcription factors characterized by the presence of the paired domain (Prd), a 128 aminoacid-long domain that recognizes and binds to specific DNA sequences. The gene encoding Pax8 (called PAX8 in humans) is located on chromosome 2 (Table 1). It consists of 12 exons encoding for a 450 aminoacids protein. Pax8 is expressed in the adult and developing thyroid from the early stages of morphogenesis. In addition, during embryonic life Pax8 is transiently expressed in the myelecephalon and in the neural tube. Expression is also present in the developing and adult kidney (1, 4).
Experiments in Pax8 null mice (9) demonstrated that, during morphogenesis, Pax8 is required for the survival of the thyroid precursor cells and to maintain the tissue-specific gene expression program.
In adult thyroid cells Pax8 promotes transcription from the TPO and the Tg promoters (10).

PAX8 disease
The involvement of PAX8 has been described in sporadic and familial cases of CH with TD (1, 11). All affected individuals are heterozygous for the mutations and in the familial cases transmission is autosomal dominant with a variable penetrance and expressivity. In humans, both PAX8 alleles are necessary for correct thyroid morphogenesis and a reduced dosage of the gene product (haploinsufficiency) causes dysgenesis (Table 1); in contrast, the Pax8+/- mice display a normal phenotype (9).
Of note, in mice the combination of partial deficiencies in the Titf1 and Pax8 genes results in a small thyroid gland, elevated TSH, reduced thyroglobulin biosynthesis, and high occurrence of hemiagenesis (12).

Foxe1/TITF2
Foxe1 (also called TTF-2 for Thyroid Transcription Factor–2) was originally identified as a thyroid specific nuclear protein that bind to a sequence present on both Tg and TPO promoters (1).
Foxe1 belongs to the winged helix/forkhead family of transcription factors. The gene encoding Foxe1 (called FOXE1) in humans is located on chromosome 9q22 and consists of a single exon encoding for a 42 kDa protein that is phoshorylated and contains an alanine stretch of variable length (13-15).
During development, Foxe1 is expressed in the thyroid, in the tongue, in the epiglottis palate and in the esophagus as well as in the Rathke's pouch, which gives rise to the anterior pituitary. In adult, Foxe1 is expressed in the thyroid, in the tongue, in the secondary palate, in the choanae, and in the whiskers and hair follicles.
Analysis of Foxe1 null mice revealed that, during embryonic life, Foxe1 has a specific role in controlling the migration of thyroid follicular cell precursors.
The role of Foxe1 in adult thyroid follicular cells was only partially clarified, and functional studies in cell culture systems have shown that Foxe1 can act as a promoter-specific transcriptional repressor. The transcription of the Foxe1 gene is regulated by TSH and insulin or IGF-1 (reviewed in (1)). These data suggest that Foxe1 plays an important role in the hormonal control of gene expression in thyroid cells.

FOXE1 disease
Bamforth syndrome (16) is characterized by cleft palate, bilateral choanal atresia, spiky hair and athyreosis. The observation that Foxe1-/- mice display thyroid defects and cleft palate (Table 1) (17) has led to the hypothesis that FOXE1 could be a candidate gene for this syndrome. Indeed, so far three mutations in FOXE1 gene have been identified in patients affected by this syndrome (18-20), The patient described in the last report presented the Bamforth syndrome phenotype, and congenital hypothyroidism without athyreosis (20). All the affected members carry homozygous missense mutations within the FoxE1 forkhead domain. The mutant proteins were tested in vitro and have shown a reduction in both DNA binding and transcriptional activity. In all the patients thyroid tissue is undetectable, while in the mice the absence of this factor causes either athyreosis or defects in thyroid migration. In humans ectopic thyroid associated with FOXE1 mutations has not yet described.

Nkx2-6, Nkx2-3 and Nkx2-5
In addition to Nkx2-1, other genes of the Nkx2 family are present in the primitive pharynx and the thyroid anlage.
Nkx2-6 is transiently expressed in the endodermal layer of the midline region of the pharynx (21). Nkx2-3 is strongly expressed in the developing thyroid and disappears at birth (21).
Nkx2-5 is expressed in the ventral region of the pharynx and in thyroid bud later Nkx2-5 transcript disappears from the thyroid bud, persisting in the heart region (21-23). The gene encoding Nkx2-5 (called NKX2-5) in humans is located on chromosome 5q34 and consists of two exons encoding for a 324 aminoacids protein (Table 1). In vitro studies indicate that Nkx2-5 is a potent inducer of the NIS promoter (24), that Nkx2-5 C-terminus interacts with the TTF-1 homeodomain and, moreover, that the expression of a dominant-negative Nkx2-5 isoform (N188K) in thyroid cells reduces TTF-1-driven transcription of several thyroid-specific genes, including pendrin and thyroglobulin (25).

NKX2-5 disease
NKX2-5 is essential for normal heart morphogenesis, myogenesis, and function (26), and several loss of function mutation in NKX2-5 have been described in patients with congenital heart diseases (Table 1) (27). Three heterozygous mutations (A119S, R161P, R25C) were found in four subjects with TD (three patients with thyroid ectopy and one with athyreosis) (23). Functional studies demonstrated that these mutants exhibited a significant functional impairment, with reduction of transactivation properties and dominant negative effect.

Hhex
Hhex (hematopoietically expressed homeobox) is a homeodomain-containing transcription factor. The gene (called HHEX in humans and located on chromosome 10q23.32 encodes for a 270 aminoacids protein that is expressed, in adults thyroid, liver and lung. Hhex is necessary for thyroid morphogenesis: studies in Hhex null embryos thyroid precursor cells demonstreated that, at early stages, Hhex is required to maintain the expression of these genes in the thyroid primordium. In vitro experiments demonstrate that Hhex is regulated by Titf1 and its overexpression partly inhibits Tg promoter activity. These data suggest that Hhex act as transcriptional repressor in thyroid cells (Reviewed in (1)).

CONCLUSIONS
NKX2-1, FOXE1, PAX8 and NKX2-5 are transcription factors involved in thyroid development. Mutations in the genes encoding for those transcription factors cause alterations in thyroid morphogenesis with or without other congenital defects. However, despite the several studies completed to address the role of transcription factors in thyroid morphogenesis, mutations have been identified only in no more of the 5% of the cases. Such low frequency of mutation can be an underestimate because the molecular analyses to search for mutations in TITF1, FOXE1, PAX8 and NKX2-5 it has been limited to the coding region of these genes, and therefore alterations in regulatory non-coding regions can lead to a disease phenotype.
In addition, it should be considered that TTIF1, FOXE1, PAX8 and NKX2-5 are transcription factors able to modulate target downstream genes that ultimately activate the organogenesis of the thyroid and that, in order to produce their biological effects, they may require the presence of cofactors. Some cases of TD could be due to mutations in the not yet identified gene targets for these transcription factors as well as in factors involved in the modulation of their action.
An other possibility is that TD is the consequence of combined defects (multigenic disease) as recently demonstrated in animal models (12), that make the identification of the candidate genes a much more complex process.
Finally, other genes seem to be important candidate genes in controlling of the thyroid development and therefore can be responsible for TD. In this group should be included Hoxa3 and Hoxa5 that have not been investigated as possible cause of TD in humans.

Table 1: Chromosomal localization, molecular features and phenotype produced by mutations of the genes described in the text.