The thyroid gland is an organ composed primarily of endoderm-derived follicular cells and is responsible for thyroid hormone production in all vertebrates. The main regulator of thyroid function is thyrotropin (thyroid-stimulating hormone, TSH), which is synthesized and secreted from the pituitary gland and is under the control of thyrotropin-releasing hormone (TRH), secreted by the hypothalamus. Thyroid secretion and serum concentrations of thyroxine (T4) and 3,5,3′-triiodothyronine (T3) are maintained by a negative feedback loop involving inhibition of TSH and TRH secretion by T4 and T3, and by tissue-specific and hormone-regulated expression of the three iodothyronine deiodinase enzymes that metabolize thyroid hormones. Thus, the regulation of thyroid function depends on the normal development of the hypothalamic–pituitary–thyroid axis, which occurs independently but coordinately during embryonic and neonatal life.
This chapter will focus on the development of the thyroid gland, with emphasis on recent studies of the morphogenesis and differentiation of the gland, as well as the hypothalamic–pituitary axis, which is required for normal thyroid function. The recent identification of several transcription factors expressed specifically in the thyroid has helped to define the molecular events responsible for thyroid development. Most of the recent advances in the understanding in the formation of the hypothalamus, pituitary, and thyroid have come from studies in animals, but where possible this information will be integrated with what is known about the anatomy, morphology, and development of the axis in humans.
THE HYPOTHALAMIC–PITUITARY AXIS
The vertebrate embryo relies on complex inductive interactions to orchestrate pattern formation, organogenesis, and ultimately, the development of integrated organ systems. The hypothalamic–pituitary axis is a prime example of such a system. The regulated functions of the hypothalamus and pituitary are intrinsically linked throughout life, and they result from the coordinated development of the two organs. Experimental embryology and molecular genetic studies have yielded evidence that the determination, development, and differentiation of the pituitary gland and hypothalamus are intimately coupled. How both organs, of different embryonic origin, codevelop introduces a level of complexity still not well understood.
The hypothalamus emerges from the ventral-caudal prosencephalon (diencephalon) during the sixth week of gestation in humans, and between embryonic (E) days E-11 and E-18 in rats. The progenitor of the central and peripheral nervous systems is the neural plate, which is first seen as a thickening of the midline ectoderm that overlies the notochord. Ten days later, this thickened ectoderm forms neural folds that fuse to form the neural tube; some cells in the dorsal region of the neural tube, the neural crest, migrate and give rise to neurons, glia, melanocytes, connective tissue of the head, and numerous endocrine derivatives, such as the parafollicular cells of the thyroid. The rest of the neural tube becomes the central nervous system. The primitive hypothalamic region is initially localized at the most rostral region of the neural tube, and later has a more caudal and ventral position. During development of the hypothalamus, neurosecretory cells are organized into several nuclei, including the paraventricular, supraoptic, and arcuate nuclei. Two different neurosecretory systems are organized in the hypothalamus, one formed of magnocellular neurons, whose axons migrate directly into the posterior lobe of the pituitary gland, and the other formed of parvocellular neurons, which synthesize, among other neuropeptides, TRH for regulating the TSH–thyroid axis (Fig. 2.1).
FIGURE 2.1. The hypothalamic–pituitary–thyroid axis. Thyrotropin-releasing hormone (TRH) is synthesized in the hypothalamus and secreted into the hypothalamic–pituitary portal venous system, in which it is carried to the pituitary, where it stimulates the synthesis and secretion of thyrotropin (TSH). TSH binds to its receptor in the thyroid gland, stimulating the synthesis and secretion of thyroxine (T4) and triiodothyronine (T3). Precise control of the axis is maintained by the inhibitory actions of T4 and T3 on both TRH and TSH secretion.
In the ventral hypothalamus, the infundibulum forms as an evagination that gives rise to multiple structures, including the posterior pituitary gland, the median eminence, and the pituitary stalk, which connects the hypothalamus and the pituitary gland. The median eminence is the site where nerves, whose cell bodies originate in the hypothalamus, release neurosecretory peptides into the hypothalamic–pituitary portal venous system (1). The anterior pituitary is derived from Rathke's pouch, an invagination of an ectodermal layer of cells beneath the diencephalon that, in mice, appears at around day E-8.5 and detaches from the ectoderm by E-12.5 (2,3,4). Even before the pouch is visible, certain cells are committed to develop into the anterior pituitary. Thus, the pituitary gland originates from two embryonic tissues; the anterior and intermediate lobes (adenohypophysis) are derived from the oral ectoderm and the posterior lobe (neurohypophysis) from the neural ectoderm (5). During this process there is a direct association between the neuroectoderm of the diencephalon and Rathke's pouch, and it is a unique region in the developing head in which there is no intervening mesoderm between neuroectoderm and ectoderm. The close apposition of these tissues suggests that cell–cell contact and inductive tissue interactions may be important for their determination and differentiation. Many early studies focused on the role of neural contact in the determination and differentiation of the adenohypophysis (6). Recently, it has become clear that several genes expressed in the ventral diencephalon are involved in the formation and development of Rathke's pouch, providing evidence that the infundibulum acts as a key organizing center (7). For example, mice with deletion of the gene for the homeodomain protein, thyroid transcription factor-1 (TTF-1; also called T/EBP or Nkx2.1, see later in this chapter), which is expressed in the ventral diencephalon (but not Rathke's pouch), lack some ventral regions of the brain, including the infundibulum, and have no pituitary gland (8). These data are consistent with the critical role of the infundibular signals in pituitary organogenesis.
The role of the pituitary primordium in the development of the hypothalamus has been less extensively studied, but contact between the two organs is important for proper formation of the ventral hypothalamus and median eminence, and for differentiation and proliferation of the different types of pituitary cells.
Signaling and Transcriptional Mechanisms Involved in Pituitary Organogenesis
The development of the pituitary gland is a model system in which to study the complex processes involved in intercellular signaling, because it contains different cell types that are derived from a common ectodermal primordium and arise in a distinct spatial and temporal fashion in response to intrinsic and extrinsic signals.
Each cell type of the anterior pituitary gland is characterized by the secretion of different trophic hormones that regulate a diverse range of biological processes in response to signals from the hypothalamus and peripheral organs. Two cell types synthesize proopiomelanocortin (POMC), which is cleaved by proteolytic processing to generate melanocyte-stimulating hormone in melanotrophs and corticotropin, or adrenocorticotropic hormone (ACTH), in corticotrophs. The somatotrophs secrete growth hormone, lactotrophs secrete prolactin, thyrotrophs secrete TSH, and gonadotrophs secrete follicle-stimulating hormone and luteinizing hormone. The last three hormones are heterodimeric glycoproteins consisting of a common α subunit and a specific β subunit (see section on chemistry and biosynthesis of thyrotropin in Chapter 10). Recent studies have identified some of the molecules (morphogens) that signal differentiation of these different cells from the common population of progenitor cells (3). These signals have a central role in early organogenesis and are especially important in development of the pituitary gland (see Chapter 3).
Morphogenic Signals Involved in Early Steps of Pituitary Development
The signaling molecules called bone-morphogenic proteins (BMPs) are members of the transforming-growth factor-β superfamily, which play critical roles in patterning and cell-type specification in several species (9). In the case of the pituitary, multiple BMPs play different roles in morphogenesis of the pituitary gland.
BMP-4 is expressed in the ventral diencephalon where the infundibulum makes contact with Rathke's pouch at E-8.5 to E-9.0 in mice, and it is one of the early signals required for the initial commitment of some cells of the oral ectoderm to form the pituitary gland (Fig. 2.2). However, the signals required for the initial invagination of Rathke's pouch are still unclear, although the notochord may have a role in this process. Blockade of the BMP-4 signal results in arrest of pituitary gland development and absence of all pituitary endocrine cell types (10,11,12).
FIGURE 2.2. Schematic diagram of the development of the hypothalamic–pituitary axis showing the main transcription factors involved in pituitary organogenesis in mice. At embryonic day E-7, the anterior neural ridge (ANR), primordial endocrine hypothalamus, and primordial anterior pituitary are present. The signals originating on E-9.0 in the ventral diencephalon include Wnt-5a, bone-morphogenic protein (BMP)-4, and fibroblast growth factors (FGFs)-8 and -10. At E-10.5, the ectoderm provides the ventral signal Shh and the mesenchyme signal BMP-2, both of which signal ventrally to dorsally in Rathke's pouch (RP). All these signals control the expression of specific transcription factors involved in the commitment of cell lineages at E-11.5. Cell types later arise in a temporal and spatial fashion, starting on E-12.5 for corticotrophs (C) and rostral thyrotrophs (Tr), and subsequently from E-15.5 to E-17.5 for somatotrophs (S), lactotrophs (L), thyrotrophs (T), gonadotrophs (G), and melanotrophs (M). The temporal expression of the main transcription factors controlling specification of pituitary cell types and organogenesis are shown in the table below the diagram. (From Scully KM, Rosenfeld MG. Pituitary development: regulatory codes in mammalian organogenesis. Science 2002;295:2231, modified with permission.)
Several members of the fibroblast growth factor (FGF) family (FGF-8 and FGF-10) are expressed in the infundibulum and control pituitary proliferation and positional-restricted determination of pituitary cell lineage (10,13). The requirement for FGF-8 has been further suggested by studies in mice lacking TTF-1, in which FGF-8 is not expressed in the ventral diencephalon. This absence is linked to the absence of Rathke's pouch-specific transcription factors (Rpx) (14). FGF receptors are also important in the process of pituitary development. In addition to BMP-4 and FGF-8 and -10, signals that originate in the dorsal region of the infundibulum, ventral signals, and intrinsic pouch signals [such as BMP-2 and sonic hedgehog (Shh)], contribute to the establishment of the positional identity of pituitary cells at days E-9.5 through E-10.5 in mice (13). BMP-2 controls the expression of different, ventrally expressed pituitary transcription factors, and also expression of the gene for the α subunit of the glycoprotein hormones, which is an early marker of cells destined to be thyrotrophs and gonadotrophs. Together, these data suggest that BMP-2 signaling specifies the progenitors that will later give rise to the ventral pituitary cell types.
In vertebrates, the Shh protein is expressed in several organizing centers during embryogenesis, during which it exerts crucial roles in patterning (15). During the early development of the pituitary gland, Shh is expressed in the oral ectoderm, although not in the region that forms Rathke's pouch (16). It may act in a signaling cascade with BMP-2 in the determination of ventral pituitary cell lineages (Fig. 2.2).
Other morphogenic factors have also been implicated in the formation of different cell types within the pituitary. The Wnt proteins are a family of secreted proteins involved in a variety of early embryonic events that function through transcriptional mediators such as β-catenin and T-cell factor lymphoid-enhancing factor (17,18). In the pituitary, Wnt-4 and Wnt-5 are expressed in the ventral diencephalon and within the cells of Rathke's pouch, respectively. Mice lacking Wnt-4 have a hypocellular pituitary gland with differentiated but incompletely expanded ventral cells (18). In cultured Rathke's pouch cells, Wnt-5 and BMP-4 induce expression of the α subunit gene (2,19,20), suggesting that both signals may act in synergy to expand pituitary cell lineages and induce cell determination. All the morphogens that contribute to pituitary development are important for thyroid development and function, because pituitary TSH is the main regulator of the thyroid gland function.
Transcription Factors Controlling the Early Phases of Pituitary Development
Many transcription factors responsible for the differentiation of pituitary cells have been identified (4,5,21,22,23), establishing a hierarchy of expression during pituitary development (Fig. 2.2). Thus, multiple members of the LIM homeodomain family of transcription factors are expressed in Rathke's pouch, including Lhx-3, Lhx-4, and Isl-1. Lhx-3 is specifically expressed in Rathke's pouch on E-9.5 in mice, and it plays a decisive role, together with Lhx-4, in the earliest phases of pituitary organogenesis. Lhx-3 knockout mice have a rudimentary Rathke's pouch, but the ectoderm fails to continue to proliferate and all pituitary cell types are absent, with the exception of a few corticotrophs (24). Isl-1 is initially expressed in Rathke's pouch during early pituitary development, but is later (E-10.5) restricted to the ventral cell population. The expression of this gene is regulated by both BMP-4 and BMP-2.
Two pituitary homeobox (Pitx) genes (previously referred to as P-Otx and Pitx-1) are expressed throughout the pituitary, with distinct overlapping patterns of expression. Pitx-1 interacts with the amino-terminal domain of the pituitary-specific POU-domain protein Pit-1 (see later in this chapter), and is expressed, in the earliest stages of pituitary organogenesis, in the anterior region of the neural plate, the oral ectoderm. Targeted disruption of Pitx-1 leads to decreased expression of terminal differentiation markers for the gonadotrophs and thyrotrophs (25). The Pitx-2 gene was identified through positional cloning of the gene responsible for the Rieger's syndrome in humans (26). Both Pitx genes, Pitx-1 and -2, are expressed in most pituitary cell types from early to late pituitary ontogeny, and also in other developing organs. Pitx-2-null mice have many severe developmental defects, including arrest of the early stage of pituitary development after establishment of the contact between the infundibulum and Rathke's pouch, and arrest of the subsequent signaling gradients; the pituitary gland fails to develop after E-10.5, which results in marked hypoplasia of the gland. The similarity of the phenotypes of Pitx-2-null and Lhx-3-null mice suggests that these two classes of homeodomain factors collaborate to regulate the same pituitary-specific genes. Indeed, both factors act synergistically to activate the expression of the α subunit gene (27). In summary, the induction of Lhx-3 expression in response to infundibular FGF signals is the critical step in the selection of oral ectoderm for development into the pituitary gland, and it acts synergistically with Pitx-2 to direct pituitary-specific gene expression.
The paired homeodomain factors Prop-1 and Rpx are expressed in a sequential overlapping temporal pattern and are required for Rathke's pouch cell types to populate the anterior lobe of the pituitary. By positional cloning in Ames dwarf mice, a gene named Prophet of Pit-1 (Prop-1) was identified. Its expression is coincident with the closure of Rathke's pouch at E-10.5, and it is down-regulated on days E-15.5 through E-16.5, at the time of terminal differentiation of pituitary-specific cells. Prop-1-deficient mice do not express Pit-1, and consequently lack somatotrophs, lactotrophs, and thyrotrophs. Prop-1 is also required for the generation of gonadotrophs and corticotrophs, indicating that it is important for the expansion of all anterior pituitary cell lineages. Mutations in the Prop-1 gene are the cause of combined pituitary hormone deficiency in humans (see Chapter 48) (28,29,30,31).
The Rathke's pouch factor Rpx is the homologue of the Hesx-1 gene, a homeodomain protein that contains a conserved repressor domain named eh1. Rpx expression is restricted to the oral ectoderm and pouch. The attenuation of Rpx/Hesx-1 expression by different corepressors coincides with the appearance of terminal differentiation markers for anterior pituitary cell types, suggesting that down-regulation of this gene is required for progression of pituitary development. This gene can dimerize with Prop-1 and inhibit Prop-1 activity, suggesting that Rpx/Hesx-1 acts to antagonize Prop-1 function (32).
Another paired-domain factor important in the early development of Rathke's pouch is Pax-6. This gene is transiently expressed in the dorsal part of the pouch, and is down-regulated when cell-type differentiation starts. The pituitary glands of Pax-6-null mice have expansion of the ventral cell types that express α subunits, predominantly thyrotrophs, with a corresponding loss of the more dorsal somatotrophs (33,34). Thus, Pax-6 is required for delineating the dorsal/ventral boundaries between the thyrotroph/ gonadotroph and the somatotroph/lactotroph progenitor regions of the pituitary gland.
Transcription Factors Controlling Specification of Different Pituitary Cell Types
Pit-1 is a member of the family of POU domain–containing transcription factors, which have a paired DNA-binding domain. The POU domain consists of an amino-terminal POU-specific domain separated by a short linker to a carboxy-terminal POU homeodomain. Both subdomains contain helix-turn-helix motifs that directly associate with two DNA binding-sites (35). Referred to also as GHF-1, Pit-1 was originally identified through analysis of the nuclear proteins regulating the transcription of growth hormone and prolactin (36,37). Later, Pit-1 was found to be required for generation and cell-type specification of three pituitary cell lineages: somatotrophs, lactotrophs, and thyrotrophs (38). Pit-1 binds to the promoter region of the genes for growth hormone, prolactin, the β subunit of TSH, the receptor for growth hormone-releasing hormone, the type-1 somatostatin receptor 1, and the TRH receptor (35), where it interacts with other transcription factors to form functionally active heterodimers. Pit-1 also interacts with various members of the nuclear-receptor family including, thyroid hormone receptors (TRs) and retinoic acid receptors (RARs) (39).
An interaction between Pit-1 and the zinc finger protein GATA-2 is a critical determinant of the development of both thyrotrophs and gonadotrophs. In the thyrotrophs, this interaction leads to synergistic activation of thyrotroph-specific genes such as the gene for the β subunit of TSH. In gonadotrophs, Pit-1 inhibits GATA-2 binding to promoters not containing an adjacent Pit-1 site (40). In Snell dwarf mice, in which the Pit-1 gene is mutated, Pit-1 and GATA-2 do not interact, and the thyrotrophs assume the fate of gonadotrophs.
Taken together, these studies indicate that there are extrinsic and intrinsic signaling mechanisms that govern the early and late aspects of the development of the hypothalamus and the pituitary gland (Fig. 2.2). Coordination between signal molecules and transcription factors is necessary for the early patterning, proliferation, and positional determination of pituitary cell types, including the thyrotrophs.
THE THYROID GLAND
The thyroid, like the anterior pituitary gland, develops from a diverticulum of the pharynx. It consists of two lobes on either side of larynx, in the middle of the neck, connected by an isthmus that normally overlies the second or third cartilaginous rings of the trachea in humans; this gland also includes cells (the parafollicular or C-cells) derived from the ultimobranchial bodies. Its name comes from its topographic relationship to the laryngeal thyroid cartilage, whose shape resembles a Greek shield. Differential growth of the neck structures is fundamental for the development and anatomy of the thyroid gland (41). The thyroid is the largest endocrine gland in humans; it weighs 1 to 2 g at birth and 10 to 20 g in adults.
The thyroid gland of vertebrates has a dual embryonic origin, and its several cell types derive from all three germ layers. The most abundant cells, the thyroid follicular cells (thyrocytes), arise from the embryonic endoderm as a thickening in the floor of the primitive pharynx (Fig. 2.3). It is the first endocrine structure that becomes recognizable in humans. This thickening, the so-called median anlagen, is located between the first and second branchial arches and is adjacent to the newly differentiating myocardium (Fig. 2.4A). The anlagen appear as a visible bud on E-16 or E-17 in humans and E-8.5 or E-9.0 in mice. The bud expands ventrally as a diverticulum, with rapid proliferation of the cells at its distal end (Fig. 2.3), but it remains attached to the pharyngeal floor by a tubular stalk called the thyroglossal duct (Fig. 2.4B). The thyrocyte progenitor cells continue to proliferate distally and then begin to proliferate laterally, which leads to the formation of the characterized bilobed structure connected by an isthmus (Fig. 2.3). Because of its close association with the embryonic heart, the thyroid can be viewed as being pulled downward by the heart during its descent. This caudal migration is accompanied by rapid elongation of the thyroglossal duct, which eventually fragments and degenerates. This migration occurs from E-24 to E-32 in humans and E-10.5 to E-14.5 in mice.
FIGURE 2.3. Frontal view of the stages of development of the median thyroid anlagen. The anlagen first appears as a group of cells located on the floor of the primitive pharynx (upper panel), which then form as a visible bud on embryonic day (E)-16 or E-17 in humans, and E-8.5 or E-9.0 in mice. The bud proliferates ventrally, then expands laterally, forming the characteristic bilobed structure of the thyroid gland. Caudal migration occurs between E-24 to E-32 in humans and E-10.5 to E-14.5 in mice; the thyroid reaches its final position on about E-40 to E-50 in humans and E-15.5 in mice. The association between the medial and lateral (C-cells) thyroid anlagen with an isthmus connecting the two lateral lobes appears on E-50 to E-51 in humans. (From Van Vliet G. Development of the thyroid gland: lessons from congenitally hypothyroid mice and men. Clin Genetics 2003; 63:445, modified with permission.)
FIGURE 2.4. Sections of human embryos showing thyroid development. A: Section through a 2-somite embryo (150× magnification). B: Section through the thyroid anlagen of a 10-somite embryo (150× magnification).
The thyroid reaches its final position at about E-40 to E-50 in humans and about E-15.5 in mice. At that time it connects with the two lateral anlagen, the ultimobranchial bodies, resulting in the incorporation of C-cells into the thyroid. These lateral anlagen are derived from the endoderm of the fourth pharyngeal pouch and the ectoderm of the abortive fifth pouch (41) (Fig. 2.5). In the mature thyroid gland, the C-cells occur either singly or in small groups, and their contribution to the total thyroid mass is minimal (10%). They are more concentrated in the central parts of the lobes, but can be found throughout the gland (42). The origin of the C-cells is still debated [for more details, see the previous edition of this book (43)].
FIGURE 2.5. Schematic diagrams showing the neural crest origin of C-cells and their incorporation into the thyroid gland. Primordial cells arising from the neural crest migrate ventrally during embryonic life to become incorporated in the last (ultimobranchial) pharyngeal pouch. The ultimobranchial body fuses with the primordial thyroid gland, and the C-cells are distributed throughout the thyroid gland. (From Foster GV, Byfield PGH, Gudmundsson TV. Calcitonin. Clin Endocrinol Metab 1972;1:93, modified with permission.)
The association between the medial and lateral thyroid anlagen is complete by about E-50 in humans, at which time the thyroid gland exhibits its definitive external form, with an isthmus connecting the two lateral lobes (Figs. 2.3 and 2.5). The foramen cecum at the base of the tongue is the remnant of the origination of the gland in the floor of the primitive pharynx. The development and migration of the medial and lateral thyroid anlagen should be viewed in conjunction with the development and migration of other structures of the head and neck, such as the parathyroid glands and thymus (Fig. 2.6).
FIGURE 2.6. Frontal view of the structures derived from the pharyngeal organ. The thyroid gland forms from the posterior migration of the thyroid anlagen, which, as shown in Figure 2.3, is derived from the ventral floor of the pharynx. As it migrates downward, it is joined by the ultimobranchial bodies (derived from the fourth branchial pouch), which contains C-cells, to form the mature thyroid gland. The foramen cecum is an opening formed by the evagination of the thyroid anlagen that closes later in development. The parathyroid glands and thymus are derived from the third branchial pouch. The tympanic cavity and external auditory meatus are formed from the first pouch. (From Manley NR, Capecchi MR. The role of Hoxa-3 in mouse thymus and thyroid development. Development 1995;121:1989, modified with permission.)
Most of the critical events in thyroid morphogenesis take place in the first 60 days of gestation in humans or the first 15 days of gestation in mice. For that reason, most developmental thyroid abnormalities result from morphogenetic errors during this period, which lead to displacement of cells derived from the medial anlagen. Ectopic thyroid tissue can result from abnormal thyroid migration that is secondary to abnormal morphogenesis of the heart, or from abnormal interactions between the thyroid primordium and the heart. As a result, ectopic thyroid cells have been found throughout the regions through which the developing thyroid migrates, including sublingual, high cervical, mediastinal, and even intracardiac locations. In the first three cases, interaction between the heart and median anlagen is abrogated earlier in development than normal, whereas in the last, thyroid tissue differentiates within the cardiac endothelium. Finally, the thyroglossal duct may not degenerate, but instead persist as a fistulous tract, which may include some thyroid follicular cells and in which thyroid carcinoma may arise. Recent studies have related mutations in thyroid transcription factors to abnormalities in thyroid development resulting in congenital hypothyroidism (see later in this chapter and Chapter 48; also see section on congenital hypothyroidism in Chapter 75) [reviewed in (44,45)].
Histology and Ultrastructure
The structure of the thyroid is unique among endocrine glands in that it is the only endocrine gland in which the hormonal products are stored in an extracellular location. It is composed of follicles of varying size that consist of an outer layer of thyroid follicular cells, which enclose a lumen that contains thyroglobulin-rich colloid. The follicular cells are surrounded by a basement membrane. Thyroid folliculogenesis is a complex process that begins with the proliferation of irregularly arranged cell cords derived from the endoderm of the thyroglossal duct. The follicles are formed by proliferation of follicular cells, coalescence of colloid-containing droplets in individual cells, and extrusion of the droplets into the follicular lumen. Studies in rats showed that the volumetric fractions of the different histologic components (follicular cells, C-cells, colloid, and interstitial tissue) change considerably during development (birth to 120 days of age). The fraction of follicular cells decreased from 61% at birth to 37% at 120 days. The fraction of C-cells increased from 3% at birth to 4% at 15 days, with no further change at 120 days. Colloid and stroma together represented 36% of the volume at birth and 59% at 120 days. During this period, the absolute volumes occupied by follicular cells, C-cells, colloid, and stroma increased 13-, 31-, 39-, and 34-fold, respectively (46).
Follicular structure is maintained by the integrity of the cytoskeleton, including microtubules and microfilaments (47), through which TSH and intercellular contact may regulate adhesion of follicular cells to each other and to the extracellular matrix, and also influence thyroid-cell behavior. The extracellular matrix plays a role in the adhesion, proliferation, differentiation, and migration of thyroid follicular cells. Molecules involved in these processes include type I and type IV collagen, fibronectin, laminin (48), and cadherin (49). Type IV collagen and laminin (major components of the basement membrane surrounding each follicle) play an important positive role in the proliferation and differentiation of follicular cells, and E-cadherin plays an important role in the maintenance of the thyroid epithelial cell phenotype throughout organogenesis (50).
Thyroid follicular cells have long profiles of rough endoplasmic reticulum and a large Golgi apparatus in their cytoplasm for synthesis and packaging of thyroglobulin that is then transported into the follicular lumen (see Chapter 5). The cytoplasm also contains numerous electron-dense lysosomal bodies, which are important in the secretion of thyroid hormones. The surface characteristics of the apical (luminal) and the basolateral sides of the cells are different, according to the role of the particular surface in thyroid hormonogenesis (see Chapter 4) (51). The secretory polarity of the cells is directed toward the lumen of the follicles; this polarity is important for iodine uptake. The apical (luminal) surfaces of the follicular cells have numerous microvilli that protrude into the follicular lumen, greatly increasing the surface area in contact with the colloid (Fig. 2.7) (46). The follicular structure seems to be required for normal thyroid hormone synthesis and secretion (52).
FIGURE 2.7. Electron micrograph of normal thyroid follicular cells with long microvilli (V) that extend into the luminal colloid (C). Pseudopods from the apical plasma membrane surround a portion of the colloid to form an intracellular colloid droplet (CD). Numerous lysosomes (L) are present in the apical cytoplasm in proximity to the colloid droplets. An intrafollicular capillary is visible in the lower left.
An extensive network of interfollicular and intrafollicular capillaries provides the follicular cells with an abundant blood supply. There is also a network of lymphatics in the gland. The stroma also contains nerve fibers, some of which are parasympathetic, but most are sympathetic. These nerves terminate on blood vessels or in apposition to the follicular cells. Growth factors and vasoactive factors are produced in the thyroid. They include fibroblast growth factor and vascular-endothelial growth factor, which are potent angiogenic proteins, and which, in cooperation with TSH, may regulate the growth and function of follicular cells (53). The role of these growth factors in thyroid development during embryonic life has not been explored, but they are likely to be important, by analogy with their effects on other organs derived from endoderm such as the liver (54) and pancreas (55).
The follicular cells vary in height, depending on degree of stimulation by TSH—between low cuboidal and tall columnar (Fig. 2.8). In humans, the cells are more cuboidal, whereas in rats, in which the rates of production and clearance of T4 and T3 are more rapid, the cells are more columnar. In the normal human thyroid, the size of the follicles is quite variable, and there is no discernible pattern in the distribution of small and large follicles within the gland. The histologic appearance of the thyroid is dramatically influenced by TSH (56). When TSH secretion increases, the first response is formation of numerous cytoplasmic pseudopods, which result in increased endocytosis of thyroglobulin-rich colloid from the follicular lumen (57). If the increase in secretion of TSH is sustained, thyroid follicular cells become more columnar, and the lumens of the follicles become smaller (even to the point of collapse) because of the increase in endocytosis of colloid (53,55,56), and numerous periodic acid-Schiff (PAS)–positive colloid droplets are present along the luminal side of the hypertrophied follicular cells. Whether caused by iodine deficiency or goitrogens, a sustained increase in TSH secretion results in thyroid follicular-cell hyperplasia and enlargement of the entire thyroid gland (see section on biological actions of TSH in Chapter 10). The hyperplasia is accompanied by the formation of papillary projections of follicular cells into the lumens of follicles or of multiple layers of cells lining follicles (Fig. 2.9). The opposite changes occur when TSH secretion is inhibited. In this situation, thyroid follicular cells become flat (atrophic), their microvilli disappear, and the follicles become greatly enlarged, due to accumulation of colloid (Fig. 2.10).
FIGURE 2.8. Microscopic section of the thyroid gland of a normal rat showing colloid-filled (C) follicles of varying size (arrows) lined by cuboidal follicular cells. An extensive network of capillaries is present between the thyroid follicles (periodic acid–Schiff-stained section)
FIGURE 2.9. Microscopic section of thyroid tissue from a rat showing diffuse hyperplasia induced by a chronic increase in thyrotropin secretion. The follicles are small and lined by columnar thyroid follicular cells, and there are projections of the cells (arrows) into the follicular lumens. The small size and partial collapse of some follicles are due to increased endocytosis of colloid.
FIGURE 2.10. Microscopic section of thyroid tissue of a rat in which thyrotropin secretion was chronically inhibited. The follicular cells (arrows) are low-cuboidal, and the thyroid follicles are distended with dense colloid (C).
Thyroid Follicular Cells
Thus, functional unit of the mature thyroid gland is the thyroid follicle enclosed by a basement membrane. C-cells are found within this basement membrane in contact with follicular cells, but they do not abut the lumen.
During thyroid development, there are three stages of histologic differentiation of the thyroid follicular cells: the precolloid stage (7 to 13 weeks in humans), the beginning colloid stage (13 to 14 weeks), and the follicular stage (> 14 weeks). The three stages are characterized by the appearance and enlargement of canaliculi, which are extensions of the smooth endoplasmic reticulum (Fig. 2.11). The coalescence of the canaliculi, and finally their extrusion through the apical membrane, forms the lumen that is surrounded by the follicular cells. The major component within these canaliculi, and ultimately the colloid, is thyroglobulin.
FIGURE 2.11. Photomicrograph of thyroid tissue from a human fetus of 50 mm crown-to-rump length. The arrows indicate two intracellular canaliculi (2,400× magnification). (From Shepard TH. Onset of function in the human fetal thyroid: biochemical and radioautographic studies from organ culture. J Clin Endocrinol Metab 1967;27:945, with permission.)
Multiple proteins are involved in the process of synthesis and secretion of T4 and T3 by the thyroid follicles (Fig. 2.12). In addition to thyroglobulin (60), they include the sodium/iodide symporter (NIS), located on the basolateral membrane of the cells, which transports iodide into the cells (see section on thyroid iodine transport in Chapter 4) (61,62). Once within the cells, the iodide is transported through the apical membrane into the follicular lumen by anion transporters, among which is pendrin, an iodide/ chloride transporter (63,64). Iodide oxidation and binding to the tyrosine residues of thyroglobulin, as well as the coupling of iodotyrosines to form T4 and T3, is catalyzed by thyroid peroxidase (TPO) in the presence of hydrogen peroxide (H2O2) (65). The H2O2-generating system of the thyroid is a membrane system composed of at least two NADPH thyroid oxidases, THOX-1 and THOX-2, localized in the apical membrane (66). For production of individual molecules of T4 and T3, thyroglobulin in the follicular lumen is absorbed across the apical membrane in the form of colloid droplets by endocytosis. These droplets then fuse with lysosomes, and most of the thyroglobulin is hydrolyzed by proteolytic enzymes, forming T4and T3 for release into the circulation. This proteolysis also releases mono- and diiodotyrosine (MIT and DIT), the precursors of T4 and T3, which then are deiodinated by a dehalogenase (67) (see section on thyroid hormone synthesis in Chapter 4).
FIGURE 2.12. Schematic diagram of a thyroid follicular cell showing the major intracellular structures and the main proteins and other substances involved in the biosynthesis and secretion of the thyroid hormones thyroxine (T4) and triiodothyronine (T3). Iodide ions (I-) are concentrated by follicular cells from the circulation by the sodium-iodide symporter (NIS) (left), then rapidly transported into the follicular lumen by pendrin and other channels (right). Amino acids (tyrosine and others) and sugars are assembled into thyroglobulin (Tg) in apical vesicles that are released into the follicular lumen. Iodination of tyrosine residues occurs within Tg, after the I- is oxidized by thyroid peroxidase (TPO), using H2O2 generated by two oxidases, THOX-1 and THOX-2. The iodinated Tg is taken up by endocytosis of colloid, and the colloid droplets fuse with lysosomes, after which Tg is enzymatically cleaved with the release of monoiodotyrosine (MIT), diiodotyrosine (DIT), T4, and T3. The MIT and DIT are deiodinated by a deshalogenase, whereas the T4 and T3 are released into the extracellular fluid. The cell nucleus contains the genes for NIS, TPO, Tg, and TSH receptors, the transcription factors TTF-1 and TTF-2, and Pax-8, which augments the transcription of these and other genes within the thyroid.
TSH is the main regulator of thyroid hormone synthesis and secretion; it acts through its thyroid-specific G protein–coupled receptor in the cell surface of thyroid cells (see section on the thyrotropin receptor in Chapter 10) (68,69). The action of TSH is largely mediated by an increase in intracellular cyclic adenosine monophosphate (cAMP), which activates multiple signaling pathways to increase thyroid hormone synthesis and release, and also contributes to thyroid differentiation and proliferation (see section on biological actions of thyrotropin in Chapter 10) (70,71).
The genes encoding these proteins and enzymes are expressed either specifically in thyroid follicular cells, for example, thyroglobulin and TPO, or in a very restricted number of tissues, such as NIS and the TSH receptor (TSHR). Fully differentiated thyroid follicular cells express all these genes; they become expressed in a coordinate way during thyroid morphogenesis. Thyroglobulin has been detected in human thyroid tissue as early as the fifth gestational week, when morphogenesis is still occurring. Iodine uptake and T4 production occur by the 10th to 14th weeks, during a period that corresponds to the final stages of formation of the lumen of follicles (see Chapter 74). In mice thyroglobulin and TSHR expression occur at E-15, and other markers of thyroid-cell differentiation such as TPO or NIS are detected at E-16 during the process of formation of thyroid follicles (72).
Thyroid-Specific Transcription Factors
The promoter regions of the genes for thyroglobulin, TPO, the TSHR, and NIS are well characterized, and transcription factors that bind to these genes and regulate their activity have been identified (Fig. 2.13). Three thyroid-specific transcription factors—TTF-1, TTF-2, and Pax-8—that have decisive roles in thyroid gland morphogenesis have been cloned (73,74,75).
FIGURE 2.13. Schematic diagram showing interactions of multiple transcription factors with the promoter regions of the genes for thyroglobulin (Tg), thyroid peroxidase (TPO), thyrotropin receptors (TSHR), and sodium-iodide symporter (NIS). The structures of the promoter regions of the Tg and TPO genes are similar. NUE denotes the NIS upstream enhancer element, which is the main promoter region of the NIS gene.
Thyroid Transcription Factor-1
Thyroid transcription factor-1 (TTF-1, also called T/EBP or Nkx2.1) is a homeodomain transcription factor encoded by the genetic locus named Titf-1. Genomic regions and complementary DNAs for TTF-1 have been cloned from various species (76,77). In humans, the Titf-1 gene, located on chromosome 14q13, contains three exons and two introns of 150 and 850 bp, respectively; its structure is well conserved among mammals. It encodes a 42 kDa protein that binds DNA through its homeodomain sequence of 61 amino acids that consist of three helical regions (I, II, and III) folded in a globular structure (78). Helix I is preceded by an N-terminal arm and separated by a loose loop from helix II, which together with helix III forms a helix-turn-helix motif. Helix III is the recognition helix that binds to DNA. In addition to the DNA-binding domain, TTF-1 contains two transcriptional activation domains in its N- and C-terminal regions (79).
Thyroid Transcription Factor-1 Expression during Development
In mice, TTF-1 is expressed in the endodermal cells of the primitive pharynx, in the thyroid anlagen (8,80), as well as in the fourth branchial pouch and the ultimobranchial body (76,77) between days E-8.5 to E-10. Its expression coincides with the proliferation of cells that give rise to the primitive thyroid bud, before the start of migration of the precursor follicular cells (Fig. 2.14). Consistent with this time of expression, Titf-1-null mice have only a rudimentary thyroid gland that is later eliminated by apoptosis (8,81). Therefore, TTF-1 is necessary for survival and proliferation of primitive thyroid cells, but not for initial specification of endodermal cells to give rise to the primitive thyroid cells.
FIGURE 2.14. Schematic representation of the stages of development of the thyroid gland. The upper boxes are the names of the different steps of thyroid organogenesis and differentiation. The middle boxes show representative diagrams of these steps. The arrows in the lower boxes show the thyroid transcription factors and their target genes. TFC denotes thyroid follicular cells. (From Macchia PE. Recent advances in understanding the molecular basis of primary congenital hypothyroidism. Mol Med Today 2000;6:36, modified with permission.)
TTF-1 is also expressed in the embryonic ventral forebrain, certain hypothalamic areas, the posterior pituitary, and in the lung. Furthermore, it has been detected in several tissues, including thyroid C-cells and parathyroid cells in adult rats (82). In the brain of mice, TTF-1 is expressed in the median ganglionic eminence that gives rise to the pallidal component of the basal ganglia at E-10.5. Consistent with this expression, Titf-1-null mice do not form the pallidal structures due to a ventral to dorsal transformation of the pallidal primordium into a striatum-like structure (8,83). The expression of TTF-1 in the early developing diencephalon (E-10.5) is restricted to the hypothalamic areas and to the infundibulum, which gives rise to the posterior lobe of the pituitary gland (see previous section on the hypothalamic–pituitary axis). TTF-1 expression continues during all stages of development of the posterior pituitary, whereas expression has not been detected in Rathke's pouch, which gives rise to the anterior pituitary gland. In Titf-1-null mice the pituitary gland is completely missing, both in its epithelial (from Rathke's pouch) and diencephalic (from infundibulum) components (8). A rudimentary Rathke's pouch is initially formed from the diencephalon, but is eliminated by apoptosis before definitive formation of the gland (8,14). Altogether, these data suggest that the presence of TTF-1 in the diencephalon is essential to induce full development of Rathke's pouch, and therefore to form the anterior and intermediate lobes of the pituitary gland. FGF-8 expression is not detectable in the diencephalon of mutant mice, suggesting that this factor is involved in the signaling events.
In the lung, TTF-1 expression is detected at E-9.5 and is restricted to the ventral migrating edge of the lung bud in which the tracheal diverticulum forms. Later, TTF-1 is expressed in all pulmonary epithelial cells. Titf-1-null mice die at birth because they have severely hypoplastic lungs, with dilated sac-like structures and only rudimentary bronchial branching (8). These findings suggest that TTF-1 is not required for initial commitment of endodermal cells to originate the lung, but is required for differentiation of lung cells (84).
In conclusion, the phenotypic analysis of Titf-1-null mice indicates that TTF-1 is necessary for development of the thyroid, lung, and ventral brain structures, but not for the selection of precursor cells for these structures.
Thyroid Transcription Factor-2
TTF-2 is another transcription factor, also called FOXE-1, due to a recent change in the nomenclature of this family of transcription factors (85). The gene, named Titf-2/Foxe1, is located on chromosome 9q22.3 in humans and chromosome 4–4C2 in mice. It encodes a 42-kDa protein that binds DNA through its forkhead domain (fkh), a well-conserved domain described initially in hepatocyte nuclear factor-3, a factor involved in the development of organs derived from the primitive endoderm. The fkh domain is 100 amino acids long with three α-helices in its N-terminal half, forming a compact structure that presents the third helix to DNA. These proteins bind to DNA as monomers, and are able to bend it; their main property as transcriptional activators is in the remodeling of chromatin structure (86). These factors are expressed early in development, and they appear to alter the chromatin structure in such a way that allow the other specific transcription factors to bind and transactivate the DNA (87).
Thyroid Transcription Factor-2 Expression during Development
In mice, TTF-2 mRNA is detected at E-8.5 in the endoderm lining the foregut, the developing thyroid, and in the ectoderm that gives rise to the anterior pituitary (75). In addition, TTF-2 is expressed at E-9 in the palate, nasal choanae, and hair follicles (88). The expression at the beginning of development of the anterior pharynx is restricted to a group of cells in the midline that give rise to the thyroid anlagen, whereas expression is more widespread in the posterior region of the primitive pharynx, where TTF-1 is not expressed. Both TTF-1 and TTF-2 transcripts are detectable in the migrating thyroid anlagen at E-9.5 and thereafter (Fig. 2.14). Hence, both transcription factors seem to be coexpressed in the same cells during a defined temporal window at the time of early thyroid development. The expression pattern of TTF-2 suggests that this factor has only a marginal role in the specification of thyroid follicular-cell precursors. This was confirmed by the thyroid phenotype of Titf-2-null mice; at E-8.5 these mice have normal budding of the thyroid anlagen, but at E-9.5 the thyroid precursor cells are unable to migrate downward, and remain contiguous to the pharyngeal endoderm (89). Therefore, the main function of TTF-2 in thyroid development is control of the migration of the thyroid to its position alongside of the trachea. In these mice two phenotypes have been observed. In half, the precursor cells remain in their initial position (thyroid ectopia), and in the other half the cells are not detected (agenesis) (89).
On E-15.5, Titf-2-null mice have a small ectopic thyroid that is able to complete the differentiation program because it expresses thyroglobulin, indicating that terminal differentiation of thyroid follicular cells is independent of the position of the cells. In these null mice the shelves of the palate do not fuse with each other, resulting in a cleft palate (89). This anomaly correlates with the expression of TTF-2 in the posterior region of the primitive pharynx (88).
TTF-2 is also expressed in ectodermal cells close to the pharyngeal membrane that give rise to the anterior lobe of the pituitary gland. At E-10.5 in mice, it can be detected in the migrating Rathke's pouch, which is moving upward, but its expression disappears at E-12 to E-12.5, when Rathke's pouch is no longer connected to the oral cavity and the cells of the pouch begin to proliferate. The pattern of expression of TTF-2 in the developing pituitary is similar to that of Rpx. Both factors are down-regulated before the expression of pituitary terminal differentiation markers, suggesting that both could be a repressor of late differentiation events (4). The onset of TTF-2 expression precedes that of the α subunit, and cessation of its expression correlates with the expression of proopiomelanocortin.
This factor is a member of the Pax family of transcription factors defined by a common element, the Paired (Prd) domain, by which they bind to specific DNA sequences (90). Based on the similarity of this domain, these proteins are grouped in several classes, such as Pax-2, Pax-5, and Pax-8. In addition to the Prd domain, these three proteins have a truncated homeodomain, a conserved octapeptide sequence, and sequence homology in the transcriptional activation domain located at the carboxy-terminal end of the molecules. The Pax-8 gene is located on human chromosome 2q12-q14 and on mouse chromosome 4, and encodes a protein of approximately 46 kDa. The molecular structure of Pax genes is known (91). The Prd is 128 amino acids long and consists of two different domains, each containing a helix-turn-helix motif, joined by a linker region. The amino-terminal domain is named PAI and the carboxy terminal domain RED. Because of this independent subdomain structure, each Pax protein binds to different DNA sequences. The binding sites for Pax-8 and TTF-1 in the promoter regions of the thyroglobulin and TPO genes partially overlap (Fig. 2.13), and the two factors therefore compete for binding. In the case of the thyroglobulin promoter, both subdomains of Pax-8 are required for efficient binding (92). In addition, Pax-8 interacts with TTF-1 for full expression of the thyroglobulin promoter (93).
Pax-8 Expression during Development
Pax genes display dynamic patterns of expression during ontogenesis and are involved in pattern formation during organogenesis (94). In mice, the Pax-8gene is expressed during development in thyroid, kidney, and neural tissue.
In the thyroid, it is expressed on E-8.5 in the endoderm from the primitive pharynx (Fig. 2.14), coincident with Titf-1 expression, and its expression in the thyroid continues until adult life. In the kidney, Pax-8 is expressed together with Pax-2 in the nephrogenic cords and the mesonephric tubules on E-10.5. Subsequently, both are expressed in mesenchymal condensations and the epithelial structures of the nephric ducts. In the nervous system, both Pax-8 and Pax-2 are expressed at the midbrain/hindbrain boundary and in the spinal cord on E-11.5.
The role of Pax-8 in thyroid development has been elucidated from the phenotype of Pax-8-null mice. These mice are growth retarded, likely due to thyroid deficiency (95), and they die at an early age unless given thyroid hormone. At birth, they have low serum T4 concentrations, and their thyroid glands are very small and lack follicles; most of the cells are C-cells. The cells express TTF-1 but do not contain thyroglobulin or TPO, as determined by immunohistochemistry. These findings indicate that Pax-8 plays a role in the development of the follicular cells but not C-cells. In these null mice, at E-10.5 to E-11.0, the thyroid anlagen that derives from the endodermal cells of the primitive pharynx is present, but at E-11.5 they do not expand laterally, as occurs in wild type mice, and the lobar structure is completely missing. The absence of Pax-8 is compatible with the initial events of thyroid development but not later stages, such as formation of thyroid follicles and functional differentiation in thyroid cells (96).
The presence of Pax-8, Pax-5, and Pax-2 in fish suggests that these genes originated from a common ancestral gene. Pax-2.1 is required for the development of thyroid follicles in zebrafish (97), animals in which it should be easy to study the molecular mechanisms involved in thyroid organogenesis.
From the prior information, the temporal expression of the thyroid-specific transcription factors during thyroid development take place before specific thyroid genes are expressed. A schematic representation of the initial mechanisms that govern thyroid gland formation (i.e., budding, specification of cells, cell migration, and follicle formation) is shown in Figure 2.14.
Other Transcription Factors Involved in Thyroid Development
The identification of other transcription factors controlling thyroid development has been elucidated from the detailed study of mice in which different genes were knocked out. Some of these factors bind to the promoter region of the Titf-1 gene, and therefore regulate transcription of that gene.
The Hox genes encode a class of transcription factors that contain a highly conserved DNA-binding motif, the Antennapedia homeodomain. Humans and mice have multiple Hox genes distributed in four groups, named Hox-A, Hox-B, Hox-C, and Hox-D, located on four separate chromosomes. These genes are thought to specify positional identity according to the Hox code (98). The genes of the third group are expressed in the anterior neuroectoderm, branchial arches, and their derivatives, including the thyroid bud. Hox-3 is expressed in a region in which thyroid is included, and it binds and activates TTF-1; thus, it is possible that this gene carries positional information contributing to turning on the expression of the Titf-1 gene (99). Mice in which the Hox-3 gene is knocked out have a poorly developed thyroid gland and abnormal development of the thymus and parathyroid glands (100,101). Hox-5 has a role in thyroid organogenesis; Hox-5-null mice have manifestations of hypothyroidism, including transient growth retardation and delayed eye opening and ear elevation. Thyroid gland morphogenesis begins normally, but formation of thyroid follicles and processing of thyroglobulin are impaired (102).
The homeobox Hex gene is expressed in the anterior visceral endoderm and rostral endoderm in early mouse embryos. Later, Hex transcripts can be detected in the liver, thyroid, and endothelial precursor cells (103). Hex-/- embryos, which die during mid-gestation, have variable truncations of the forebrain, liver, and thyroid dysplasia. Unlike Hox genes, which are barely expressed in the thyroid gland of adult animals, the Hex gene is expressed in thyroid follicular cells of adult mice, in which it acts as a repressor of thyroglobulin gene expression (92). Hex expression falls in thyroid cell lines transformed with oncogenes as well as in human thyroid cancer cells (104). TTF-1 increases Hex promoter activity. Moreover, Hex and TTF-1 interact to increase Hex gene expression, demonstrating the existence of direct crossregulation between thyroid transcription factors.
Hepatocyte Nuclear Factor-3 (FOXE-2) Gene
The fork head transcription factor hepatocyte nuclear factor (HNF)-3 (also known as FOXE-2) is expressed early in the primitive endoderm and is decisive for organogenesis of the liver (58,105). It binds in vitro to the TPO promoter at the same binding site at which TTF-2 binds (106). However, whether this factor is important in thyroid development is not known. This factor is also expressed in the lungs, in which it binds to the promoter region of the Titf-1 gene (84).
The Eya (Eyes absent) gene regulates organogenesis in both vertebrates and invertebrates, and has an important role in the morphogenesis of organs derived from the pharyngeal region, including the thymus, parathyroid glands, and thyroid gland. Eya-1 is expressed in the third and fourth pharyngeal regions and ultimobranchial bodies. Eya-1-null mice have thyroid hypoplasia, with severe reduction in the number of C-cells and the size of the thyroid lobes, and the C-cells and thyroid cells lobes do not fuse together. These results suggest that Eya-1 controls critical early inductive events involved in the morphogenesis of the thyroid gland and other organs derived from the pharyngeal region.
Mutations in the TTF-1, TTF-2, and Pax-8 genes have been described in humans with congenital hypothyroidism (see section on congenital hypothyroidism in Chapter 75) (44,107). Thyroid malformations or dysfunction have not been described in humans with Hox, Hex, or Eya gene mutations.
Other Signals Involved in Early Steps of Thyroid Development
TSH is the main regulator of thyroid function in adults, in whom it stimulates almost every aspect of thyroid hormone biosynthesis, including increases in thyroglobulin synthesis and TPO and NIS activity (see Chapter 4, and section on biological actions of TSH in Chapter 10). TSH also stimulates thyroid proliferation, aggregation of thyroid cells into follicles, and maintenance of follicular architecture. In rats, there is a temporal correlation between expression of TSHRs and the formation of thyroid follicles.
In mouse embryos, TSHR messenger RNA is expressed by E-15 (80), and its expression increases on E-17 or E-18. At this stage, thyroid-specific genes (thyroglobulin, TPO, and NIS) are up-regulated and colloid formation begins (108). Studies of mice in which the genes for TSH or TSHR were knocked out have revealed that TSH signaling is not a global regulator of thyroid function during development (72). TSH and TSHR are required for expression of some of the genes involved in thyroid hormone biosynthesis, such as TPO and NIS, but not for the onset of thyroglobulin expression. Knock-in mice, in which there is early expression of a constitutively activated form of TSHR, do not express TPO or NIS at an earlier stage, suggesting that additional mechanisms are involved in TSH-controlled gene expression. Thyroid gland size is not altered in TSH-null or TSHR-null mice, indicating that different signals control thyroid gland size and growth during embryogenesis and adult life. In fact, other growth factors such as insulin-like growth factor-1 and epidermal growth factor can promote thyroid-cell proliferation in culture (70,71). Moreover, these two growth factors are expressed during embryonic life (109,110) and could be the primary regulators of thyroid growth at that time.
In TSHR-null mice the thyroid glands are small, and consist of small follicles filled with colloid, suggesting that TSH signaling maintains rather than initiates folliculogenesis (72), although other studies suggests a role for TSH in the initiation of folliculogenesis (111). If activation of the TSH/TSHR pathway occurs in mice at E-15, and the expression of thyroid transcription factors TTF-1, TTF-2, and Pax-8, starts at E-8.5, then signals different from TSH must be involved in early thyroid development as well as in thyroid migration and final organogenesis. Despite the considerable information that is available about the morphogenic signals involved in pituitary development, there is little information about the inductive signals responsible for formation and growth of the thyroid anlagen and the signals that control migration and final organogenesis. These signals may involve a stimulus from the adjacent mesenchyme, because transplants of the presumptive thyroid region form typical thyroid tissue only when mesenchyme is present (112). The fact that fibroblast growth factor and its receptor regulate patterning of the pharyngeal region suggests that this pathway could be one of the initial steps directing the formation of the thyroid anlagen (113). Shh may also be involved, because Shh-null mice have thyroid ectopia or hemiagenesis (114).
INFLUENCE OF THE MATERNAL ENVIRONMENT ON FETAL THYROID FUNCTION
The maturation of the human fetal hypothalamic–pituitary–thyroid unit is complex, and the role of the maternal environment is still somewhat uncertain. Neither TSH nor TRH crosses the placenta in sufficient amounts to increase thyroid function, although high doses of exogenous TRH administered to pregnant women can increase fetal serum TSH concentrations (115). For years it was thought that very little maternal T4 and T3 reached the fetus during most of development, because the placenta contains a high level of type 3 deiodinase, which catalyzes the conversion of T4 to reverse T3 and T3 to 3,3′-diiodothyronine (T2), both of which are biologically inactive (see Chapters 7 and 74) (116). Nonetheless, T4 and T3 can be detected in embryonic and fetal fluids very early in the first trimester (6 weeks gestation), and the concentrations steadily increase thereafter (117). Therefore, fetal tissues are exposed to concentrations of T4 and T3 that are biologically relevant in adults long before the onset of active T4 and T3 secretion by the fetal thyroid gland at about mid-gestation. Furthermore, some maternal T4 and T3 reaches the fetus until delivery, because at birth athyreotic infants have serum T4 and T3 concentrations that are 20% to 50% of the concentrations in normal infants (118). In rats, T4 and T3 are found on E-9, well before the onset of thyroid function at E-18, and maternal thyroidectomy delays fetal development, including slowing the increase in T4 and T3 content in the fetal thyroid (119). Thus, in rats, maternal T4 and T3 affect the fetus at early stages, and it is likely that this is true for humans as well. Indeed, serum-free T4 concentrations are lower in premature neonates than in normal fetuses in utero (120), and postnatal maturation of thyroid function is delayed in comparison with term infants who were not prematurely cut off from maternal T4.
Another important aspect of fetal thyroid development that depends on the placenta is the provision of sufficient iodine for T4 and T3 production. Iodine is actively transported across the placenta, and fetal serum iodide concentrations at mid-gestation are considerably higher than those in maternal serum. Maternal iodine deficiency leads to neurological cretinism (see Chapter 49).
1. Treier M, Rosenfeld MG. The hypothalamic-pituitary axis: co-development of two organs. Curr Opin Cell Biol 1996;8:833.
2. Rosenfeld MG, Briata P, Dasen J, et al. Multistep signaling and transcriptional requirements for pituitary organogenesis in vivo. Recent Prog Horm Res 2000;55:1.
3. Scully KM, Rosenfeld MG. Pituitary development: regulatory codes in mammalian organogenesis. Science 2002;295:2231.
4. Dasen JS, Rosenfeld MG. Signaling and transcriptional mechanisms in pituitary development. Annu Rev Neurosci 2001;24:327.
5. Sheng HZ, Moriyama K, Yamashita T, et al. Multistep control of pituitary organogenesis. Science 1997;278:1809.
6. Gleiberman AS, Fedtsova NG, Rosenfeld MG. Tissue interactions in the induction of anterior pituitary: role of the ventral diencephalon, mesenchyme, and notochord. Dev Biol 1999; 213:340.
7. Kawamura K, Kikuyama S. Induction from posterior hypothalamus is essential for the development of the pituitary proopiomelacortin (POMC) cells of the toad (Bufo japonicus). Cell Tissue Res 1995;279:233.
8. Kimura S, Hara Y, Pineau T, et al. The T/ebp null mouse: thyroid-specific enhancer-binding protein is essential for the organogenesis of the thyroid, lung, ventral forebrain, and pituitary. Genes Dev 1996;10:60.
9. Hogan BL. Bone morphogenetic proteins in development. Curr Opin Genet Dev 1996;6:432.
10. Ericson J, Norlin S, Jessell TM, et al. Integrated FGF and BMP signaling controls the progression of progenitor cell differentiation and the emergence of pattern in the embryonic anterior pituitary. Development 1998;125:1005.
11. Winnier G, Blessing M, Labosky PA, et al. Bone morphogenetic protein-4 is required for mesoderm formation and patterning in the mouse. Genes Dev 1995;9:2105.
12. Olson LE, Rosenfeld MG. Perspective: genetic and genomic approaches in elucidating mechanisms of pituitary development. Endocrinology 2002;143:2007.
13. Treier M, Gleiberman AS, O'Connell SM, et al. Multistep signaling requirements for pituitary organogenesis in vivo. Genes Dev 1998;12:1691.
14. Takuma N, Sheng HZ, Furuta Y, et al. Formation of Rathke's pouch requires dual induction from the diencephalon. Development 1998;125:4835.
15. Ruiz I, Altaba A, Palma V, Dahmane N. Hedgehog-Gli signalling and the growth of the brain. Nat Rev Neurosci 2002;3:24.
16. Treier M, O'Connell S, Gleiberman A, et al. Hedgehog signaling is required for pituitary gland development. Development 2001; 128:377.
17. Martinez Arias A. Wnts as morphogens? The view from the wing of Drosophila. Nat Rev Mol Cell Biol 2003;4:321.
18. Eastman Q, Grosschedl R. Regulation of LEF-1/TCF transcription factors by Wnt and other signals. Curr Opin Cell Biol 1999; 11:233.
19. Clevers H. Inflating cell numbers by Wnt. Mol Cell 2002;10: 1260.
20. Kioussi C, Briata P, Baek SH, et al. Identification of a Wnt/Dvl/ beta-catenin–Pitx2 pathway mediating cell-type-specific proliferation during development. Cell 2002;111:673.
21. Watkins-Chow DE, Camper SA. How many homeobox genes does it take to make a pituitary gland? Trends Genet 1998;14:284.
22. Dasen JS, Rosenfeld MG. Combinatorial codes in signaling and synergy: lessons from pituitary development. Curr Opin Genet Dev 1999;9:566.
23. Dosen JS, Rosenfeld MG. Signaling mechanisms in pituitary morphogenesis and cell fate determination. Curr Opin Cell Biol 1999;11:669.
24. Sheng HZ, Zhadanov AB, Mosinger B Jr, et al. Specification of pituitary cell lineages by the LIM homeobox gene Lhx3. Science 1996;272:1004.
25. Szeto DP, Rodriguez-Esteban C, Ryan AK, et al. Role of the bicoid-related homeodomain factor Pitx1 in specifying hindlimb morphogenesis and pituitary development. Genes Dev 1999;13: 484.
26. Semina EV, Reiter R, Leysens NJ, et al. Cloning and characterization of a novel bicoid-related homeobox transcription factor gene, RIEG, involved in Rieger syndrome. Nat Genet 1996; 14:392.
27. Bach I, Rhodes SJ, Pearse RV 2nd, et al. P-Lim, a LIM homeodomain factor, is expressed during pituitary organ and cell commitment and synergizes with Pit-1. Proc Natl Acad Sci USA 1995;92:2720.
28. Wu W, Cogan JD, Pfaffle RW, et al. Mutations in PROP1 cause familial combined pituitary hormone deficiency. Nat Genet 1998;18:147.
29. Deladoey J, Fluck C, Buyukgebiz A, et al. “Hot spot” in the PROP1 gene responsible for combined pituitary hormone deficiency. J Clin Endocrinol Metab 1999;84:1645.
30. Rosenbloom AL, Almonte AS, Brown MR, et al. Clinical and biochemical phenotype of familial anterior hypopituitarism from mutation of the PROP1 gene. J Clin Endocrinol Metab 1999;84:50.
31. Pernasetti F, Toledo SP, Vasilyev VV, et al. Impaired adrenocorticotropin-adrenal axis in combined pituitary hormone deficiency caused by a two-base pair deletion (301–302delAG) in the prophet of Pit-1 gene. J Clin Endocrinol Metab 2000;85:390.
32. Dasen JS, Barbera JP, Herman TS, et al. Temporal regulation of a paired-like homeodomain repressor/TLE corepressor complex and a related activator is required for pituitary organogenesis. Genes Dev 2001;15:3193.
33. Bentley CA, Zidehsarai MP, Grindley JC, et al. Pax6 is implicated in murine pituitary endocrine function. Endocrine 1999; 10:171.
34. Kioussi C, O'Connell S, St-Onge L, et al. Pax6 is essential for establishing ventral-dorsal cell boundaries in pituitary gland development. Proc Natl Acad Sci U S A 1999;96:14378.
35. Andersen B, Rosenfeld MG. POU domain factors in the neuroendocrine system: lessons from developmental biology provide insights into human disease.Endocr Rev 2001;22:2.
36. Bodner M, Castrillo JL, Theill LE, et al. The pituitary-specific transcription factor GHF-1 is a homeobox-containing protein. Cell 1988;55:505.
37. Ingraham HA, Chen RP, Mangalam HJ, et al. A tissue-specific transcription factor containing a homeodomain specifies a pituitary phenotype. Cell 1988;55:519.
38. Li S, Crenshaw EB III, Rawson EJ, et al. Dwarf locus mutants lacking three pituitary cell types result from mutations in the POU-domain gene pit-1. Nature 1990;347:528.
39. Palomino T, Sanchez-Pacheco A, Pena P, et al. A direct protein-protein interaction is involved in the cooperation between thyroid hormone and retinoic acid receptors and the transcription factor GHF-1. FASEB J 1998;12:1201.
40. Dasen JS, O'Connell SM, Flynn SE, et al. Reciprocal interactions of Pit1 and GATA2 mediate signaling gradient-induced determination of pituitary cell types. Cell 1999;97:587.
41. Stone JA, Figueroa RE. Embryology and anatomy of the neck. Neuroimaging Clin N Am 2000;10:55.
42. Hoyes AD, Kershaw DR. Anatomy and development of the thyroid gland. Ear Nose Throat J 1985;64:318.
43. Pintar JE. Normal development of hypothalamic-pituitary-thyroid axis. In: Braverman LE, Utiger RD, eds. Werner's and Ingbar's The thyroid: a fundamental and clinical text, 8th ed. Philadelphia: Lippincott Williams & Wilkins, 2000:7.
44. Macchia PE. Recent advances in understanding the molecular basis of primary congenital hypothyroidism. Mol Med Today 2000;6:36.
45. Van Vliet G. Development of the thyroid gland: lessons from congenitally hypothyroid mice and men. Clin Genet 2003;63: 445.
46. Capen CC. Thyroid anatomy. In: Braverman LE, Utiger RD, eds. Werner's and Ingbar's The thyroid: a fundamental and clinical text, 8th ed. Philaldelphia: Lippincott Williams & Wilkins, 2000:20.
47. Yap AS, Manley SW. Microtubule integrity is essential for apical polarization and epithelial morphogenesis in the thyroid. Cell Motil Cytoskeleton 2001;48:201.
48. Toda S, Matsumura S, Yonemitsu N, et al. Effects of various types of extracellular matrices on adhesion, proliferation, differentiation, and c-fos protein expression of porcine thyroid follicle cells. Cell Struct Funct 1995;20:345.
49. Tepass U. Adherens junctions: new insight into assembly, modulation and function. Bioessays 2002;24:690.
50. Fagman H, Grande M, Edsbagge J, et al. Expression of classical cadherins in thyroid development: maintenance of an epithelial phenotype throughout organogenesis. Endocrinology 2003;144: 3618.
51. Ericson LE, Nilsson M. Structural and functional aspects of the thyroid follicular epithelium. Toxicol Lett 1992;64/65:365.
52. Takasu S, Ohno S, Komiya I, et al. Requirements of follicle structure for thyroid hormone synthesis: cytoskeletons and iodine metabolism in polarized monolayer cells on collagen gel and in double layered, follicle-forming cells. Endocrinology 1992; 131:1143.
53. Ramsdem JD. Angiogenesis in the thyroid gland. J Endocrinol 2000;166: 475.
54. Zaret KS. Liver specification and early morphogenesis. Mech Dev 2000;92:83.
55. Edlund H. Pancreatic organogenesis: developmental mechanisms and implication for therapy. Nat Rev Genet 2002;3:524.
56. Collins WT, Capen CC. Ultrastructural and functional alterations of the rat thyroid gland produced by polychlorinated biphenyls compared with iodide excess and deficiency, and thyrotropin and thyroxine administration. Virchows Arch 1980;33: 213.
57. Nilsson M, Ericson LE. Graded response in the individual thyroid follicle cell to increasing doses of TSH. Mol Cell Endocrinol 1986;44:165.
58. Ericson LE, Engström G. Quantitative electron microscopic studies on exocytosis and endocytosis in the thyroid follicle cell. Endocrinology 1978;103:883.
59. Many M-C, Denef J-F, Haumont S, et al. Morphological and functional changes during thyroid hyperplasia and involution in C3H mice: effects of iodine and 3,5,38-triiodothyronine during involution. Endocrinology 1985;116:798.
60. Vassart G, Bacolla A, Brocas H, et al. Structure, expression and regulation of the thyroglobulin gene. Mol Cell Endocrinol 1985; 40:89.
61. De La Vieja A, Dohan O, Levy O, Carrasco N. Molecular analysis of the sodium/iodide symporter: impact on thyroid and extrathyroid pathophysiology. Physiol Rev 2000;80:1083.
62. Dohan O, De la Vieja A, Paroder V, et al. The sodium/iodide symporter (NIS): characterization, regulation, and medical significance. Endocr Rev 2003;24:48.
63. Yoshida A, Taniguchi S, Hisatome I, et al. Pendrin is an iodide-specific apical porter responsible for iodide efflux from thyroid cells. J Clin Endocrinol Metab 2002;87:3356.
64. Everett LA, Green ED. A family of mammalian anion transporters and their involvement in human genetic diseases. Hum Mol Genet 1999;8:1883.
65. Kimura S, Kotani T, McBride OW, et al. Human thyroid peroxidase: complete cDNA and protein sequence, chromosome mapping, and identification of two alternately spliced mRNAs. Proc Natl Acad Sci USA 1987;84:5555.
66. De Deken X, Wang D, Dumont JE, et al. Characterization of ThOX proteins as components of the thyroid H(2)O(2)–generating system. Exp Cell Res 2002;273:187.
67. Moreno JC, Keijser R, Aarraas, et al. Cloning and characterization of a novel thyroidal gene encoding proteins with a conserved nitroreductase domain. J Endocrinol Invest 2002;25 [Suppl]:23.
68. Vassart G, Dumont JE. The thyrotropin receptor and the regulation of thyrocyte function and growth. Endocr Rev 1992; 13: 596.
69. Szkudlinski MW, Fremont V, Ronin C, et al. Thyroid-stimulating hormone and thyroid-stimulating hormone receptor structure-function relationships. Physiol Rev 2002;82:473.
70. Medina DL, Santisteban P. Thyrotropin-dependent proliferation of in vitro rat thyroid cell systems. Eur J Endocrinol 2000; 143: 161.
71. Kimura T, Van Keymeulen A, Golstein J, et al. Regulation of thyroid cell proliferation by TSH and other factors: a critical evaluation of in vitro models. Endocr Rev 2001;22:631.
72. Postiglione MP, Parlato R, Rodriguez-Mallon A, et al. Role of the thyroid-stimulating hormone receptor signaling in development and differentiation of the thyroid gland. Proc Natl Acad Sci USA 2002;99:15462.
73. Guazzi S, Price M, De Felice M, et al. Thyroid nuclear factor 1 (TTF-1) contains a homeodomain and displays a novel DNA binding specificity. EMBO J 1990;9:3631
74. Zannini M, Francis-Lang H, Plachov D, et al. 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. Mol Cell Biol 1992; 12: 4230.
75. Zannini M, Avantaggiato V, Biffali E, et al. TTF-2, a new forkhead protein, shows a temporal expression in the developing thyroid which is consistent with a role in controlling the onset of differentiation. EMBO J 1997;16:3185.
76. Damante G, Di Lauro R. Thyroid-specific gene expression. Biochim Biophys Acta 1994;1218:255.
77. Damante G, Tell G, Di Lauro R. A unique combination of transcription factors controls differentiation of thyroid cells. Prog Nucleic Acid Res Mol Biol 2001;66:307.
78. Laughon A. DNA binding specificity of homeodomains. Biochemistry 1991;30:11357.
79. De Felice M, Damante G, Zannini M, et al. Redundant domains contribute to the transcriptional activity of the thyroid transcription factor 1. J Biol Chem 1995;270:26649.
80. Lazzaro D, Price M, de Felice M, et al. The transcription factor TTF-1 is expressed at the onset of thyroid and lung morphogenesis and in restricted regions of the foetal brain. Development 1991;113:1093.
81. Minoo P, Hamdan H, Bu D, et al. TTF-1 regulates lung epithelial morphogenesis. Dev Biol 1995;172:694.
82. Suzuki K, Kobayashi Y, Katoh R, et al. Identification of thyroid transcription factor-1 in C cells and parathyroid cells. Endocrinology 1998;139:3014.
83. Sussel L, Marin O, Kimura S, et al. Loss of Nkx2. 1 homeobox gene function results in a ventral to dorsal molecular respecification within the basal telencephalon: evidence for a transformation of the pallidum into the striatum. Development 1999; 126:3359.
84. Costa RH, Kalinichenko VV, Lim L. Transcription factors in mouse lung development and function. Am J Physiol Lung Cell Mol Physiol 2001;280:L823.
85. Kaestner KH, Knochel W, Martinez DE. Unified nomenclature for the winged helix/forkhead transcription factors. Genes Dev 2000;14:142.
86. Clark KL, Halay ED, Lai E, et al. Co-crystal structure of the HNF-3/fork head DNA-recognition motif resembles histone H5. Nature 1993;364:412.
87. Cirillo LA, Lin FR, Cuesta I, et al. Opening of compacted chromatin by early developmental transcription factors HNF3 (FoxA) and GATA-4. Mol Cell 2002;9:279.
88. Dathan N, Parlato R, Rosica A, et al. Distribution of the titf2/foxe1 gene product is consistent with an important role in the development of foregut endoderm, palate, and hair. Dev Dyn 2002;224:450.
89. De Felice M, Ovitt C, Biffali E, et al. A mouse model for hereditary thyroid dysgenesis and cleft palate. Nat Genet 1998; 19: 395.
90. Walther C, Guenet JL, Simon D, et al. Pax: a murine multigene family of paired box-containing genes. Genomics 1991;11:424.
91. Xu W, Rould MA, Jun S, et al. Crystal structure of a paired domain-DNA complex at 2. 5 A resolution reveals structural basis for Pax developmental mutations. Cell 1995;80:639.
92. Pellizzari L, D'Elia A, Rustighi A, et al. Expression and function of the homeodomain-containing protein Hex in thyroid cells. Nucleic Acids Res 2000;28:2503.
93. Di Palma T, Nitsch R, Mascia A, et al. The paired domain-containing factor Pax8 and the homeodomain-containing factor TTF-1 directly interact and synergistically activate transcription. J Biol Chem 2003;278:3395.
94. Dahl E, Koseki H, Balling R. Pax genes and organogenesis. Bioessays 1997;19:755.
95. Mansouri A, Chowdhury K, Gruss P. Follicular cells of the thyroid gland require Pax8 gene function. Nat Genet 1998;19:87.
96. Pasca di Magliano M, Di Lauro R, Zannini M. Pax8 has a key role in thyroid cell differentiation. Proc Natl Acad Sci USA 2000;97:13144.
97. Wendl T, Lun K, Mione M, et al. Pax2.1 is required for the development of thyroid follicles in zebrafish. Development 2002; 129:3751.
98. Kessel M, Drescher U, Gruss P. The murine homeo domain protein Hox 1.1. Ann N Y Acad Sci 1987;511:88.
99. Guazzi S, Lonigro R, Pintonello L, et al. The thyroid transcription factor-1 gene is a candidate target for regulation by Hox proteins. EMBO J 1994;13:3339.
100. Manley NR, Capecchi MR. The role of Hoxa-3 in mouse thymus and thyroid development. Development 1995;121:1989.
101. Manley NR, Capecchi MR. Hox group 3 paralogs regulate the development and migration of the thymus, thyroid, and parathyroid glands. Dev Biol 1998;195:1.
102. Meunier D, Aubin J, Jeannotte L. Perturbed thyroid morphology and transient hypothyroidism symptoms in Hoxa5 mutant mice. Dev Dyn 2003;227:367–378.
103. Martinez Barbera JP, Clements M, Thomas P, et al. The homeobox gene Hex is required in definitive endodermal tissues for normal forebrain, liver and thyroid formation. Development 2000;127:2433.
104. D'Elia AV, Tell G, Russo D, et al. Expression and localization of the homeodomain-containing protein HEX in human thyroid tumors. J Clin Endocrinol Metab 2002;87:1376.
105. Zaret KS. Regulatory phases of early liver development: paradigms of organogenesis. Nat Rev Genet 2002;3:499.
106. Sato K, Di Lauro R. Hepatocyte nuclear factor 3 beta participates in the transcriptional regulation of the thyroperoxidase promoter. Biochem Biophys Res Commun 1996;220:86.
107. Pohlenz J, Dumitrescu A, Zundel D, et al. Partial deficiency of thyroid transcription factor 1 produces predominantly neurological defects in humans and mice. J Clin Invest 2002;109: 469.
108. Brown RS, Shalhoub V, Coulter S, et al. Developmental regulation of thyrotropin receptor gene expression in the fetal and neonatal rat thyroid: relation to thyroid morphology and to thyroid-specific gene expression. Endocrinology 2000;141: 340.
109. Partanen AM. Epidermal growth factor and transforming growth factor-alpha in the development of epithelial-mesenchymal organs of the mouse. Curr Top Dev Biol 1990;24:31.
110. Bondy CA, Werner H, Roberts CT Jr, et al. Cellular pattern of insulin-like growth factor-I (IGF-I) and type I IGF receptor gene expression in early organogenesis: comparison with IGF-II gene expression. Mol Endocrinol 1990;4:1386.
111. Beamer WG, Cresswell LA. Defective thyroid ontogenesis in fetal hypothyroid (hyt/hyt) mice. Anat Rec 1982;202:387.
112. Dossal WE. Effects of depletion and substitution of perivascular mesenchyme upon the development of the thyroid primordium. J Elisha Mitchell Sci Soc 1957;73:244.
113. Trokovic N, Trokovic R, Mai P, et al. Fgfr1 regulates patterning of the pharyngeal region. Genes Dev 2003;17:141.
114. Fagman H, Grände M, Gritli-Linde A, et al. Genetic deletion of sonic hedgehog causes hemiagenesis and ectopic development of the thyroid in mouse. Am J Pathol 2004;164:1865.
115. Martino E, Grasso S, Bambini G, et al. Ontogenetic development of pancreatic thyrotropin-releasing hormone in human foetuses and in infants. Acta Endocrinol (Copenh) 1986;112: 372.
116. Roti E. Regulation of thyroid-stimulating hormone (TSH) secretion in the fetus and neonate. J Endocrinol Invest 1988; 11:145.
117. Calvo RM, Jauniaux E, Gulbis B, et al. Fetal tissues are exposed to biologically relevant free thyroxine concentrations during early phases of development. J Clin Endocrinol Metab 2002; 87:1768.
118. Vulsma T, Gons MH, de Vijlder JJ. Maternal-fetal transfer of thyroxine in congenital hypothyroidism due to a total organification defect or thyroid agenesis. N Engl J Med 1989;321:13.
119. Morreale de Escobar G, Obregon MJ, Escobar del Rey F. Is neuropsychological development related to maternal hypothyroidism or to maternal hypothyroxinemia? J Clin Endocrinol Metab 2000;85:3975.
120. Morreale de Escobar G, Ares S. The hypothyroxinemia of prematurity. J Clin Endocrinol Metab 1998;83:713.