THYROID FOLLICULAR CELLS

When observed under the light microscope, thyroid follicular cells (TFCs) show a neutrophilic cytoplasm, a basal nucleus, and para-aminosalicylic acid (PAS)-positive vacuoles (phagosomes).⁵¹ Follicular cells appear as cuboidal epithelial cells whose height is approximately 15 µm. Cells become flatter (squamous) or higher (columnar) depending on whether or not they undergo TSH stimulation.

Electron microscopy (Fig. 72-2) reveals the characteristic features of cells actively engaged in protein synthesis.⁵² The rough endoplasmic reticulum and the Golgi apparatus are the dominant organelles in the cell. The apical surface of the cell is covered with thin microvilli or pseudopods that protrude into the follicular lumen. A distinctive feature of follicular cells engaged in protein synthesis is the presence of several vesicles localized in the apical or subapical cytoplasm. The smaller (150 to 200 nm) vesicles are exocytotic vesicles containing newly synthesized thyroglobulin. The fusion of these vesicles with the apical plasma membrane leads to the delivery of thyroglobulin into the follicle lumen. TPO and hydrogen peroxide–producing enzymes are localized to the luminal side of the follicular cell membrane,¹⁸ thus allowing the iodination process to occur. The larger vesicles (500 to 4000 nm) are filled with dense material called colloid droplets that are the result of the uptake of the iodinated thyroglobulin stored in the follicular lumen. TSH induces the uptake of thyroglobulin from the follicle lumen, increasing the number of colloid droplets.⁵³ The reabsorption of the colloid involves a macropinocytosis mechanism whose first step is the formation of pseudopods at the apical pole. The pseudopods close, and a portion of the colloid is internalized into the cell.⁵⁴

FIGURE 72-2. Electron micrograph of a rat follicular cell. AV, Apical vesicles; ER, endoplasmic reticulum; G, Golgi apparatus; L, lumen; Ly, lysosome; M, microvilli; N, nucleus; Tg, thyroglobulin; TJ, tight junction.

(Courtesy Professor L. Nitsch.)

At the molecular level (Fig. 72-3), a follicular cell can be identified by the presence of a specific set of proteins—and corresponding mRNAs—indispensable for its specialized functions.⁵⁵ Among such proteins, thyroglobulin (Tg) and TPO are remarkably specific, being detectable exclusively in thyroid follicular cells. Other proteins, such as TSHR, NIS, pendrin, and Duox, though expressed in thyroid follicular cells, are also present in other tissues. The exclusive or prevalent expression of genes necessary for thyroid hormone biosynthesis in thyroid follicular cells appears to be due to a combination of transcription factors unique to this cell type.⁵⁵ These transcription factors (Nkx2-1/Ttf-1, Pax8, Foxe1) have subsequently been found to be important in controlling not only the differentiation but also the morphogenesis of the gland. The molecular features and the functions of these factors will be described further on (see “Molecular Genetics of Thyroid Gland Development”).

FIGURE 72-3. Thyroid follicular cell. A, Schematic structural representation. B, Schematic molecular representation. CD, Colloid droplets; ER, endoplasmic reticulum; G, Golgi apparatus; M, microvilli; MI, mitochondrion; N, nucleus; P, pseudopod.

C CELLS

C cells represent the other endocrine cellular type present in the thyroid gland of mammals. They synthesize and secrete calcitonin,⁵⁶ a polypeptide hormone involved in calcium metabolism. In lower vertebrates, C cells form an organ called the ultimobranchial gland that is separate from the thyroid gland. Quail chick chimera experiments have demonstrated that C cells of avian species derive from neural crest cells,⁵⁷ which during embryonic development colonize the ultimobranchial body, a transient organ in mammals, to finally disperse into the thyroid gland.⁵⁸

C cells are known as parafollicular cells because of their distribution among follicular cells. However, in spite of their name, not all C cells are located between follicular cells and the basement membrane (a real parafollicular position); they are also found among the follicles (interfollicular) or in an intrafollicular position. In fact, C cells are found dispersed as individual cells in small groups closely associated to follicular cells, and in more complex structures consisting of both follicular and C cells. The number of parafollicular cells in the thyroid differs among species.⁵⁹ In humans, the number of C cells decreases with age: the neonatal thyroid has 10 times more C cells than the adult thyroid, where they are fewer than 1% of the follicular cells. These cells are usually distributed in the upper two thirds of the lateral lobe in intrafollicular and parafollicular positions.⁶⁰

C cells are characterized by clear cytoplasm and small, compact nuclei. Electron microscopy reveals that these cells contain cytoplasmic secretory granules 100 to 200 nm in diameter.⁶¹ At the molecular level, C cells are identified by the presence of calcitonin. The calcitonin/calcitonin gene-related peptide (CGRP) gene encodes four proteins: calcitonin, CGRP, katacalcin I and katacalcin II. The splicing of the first three exons to the fourth gives rise to an mRNA that encodes for a protein precursor which is subsequently processed to give calcitonin and a peptide, katacalcin I. CGRP and katacalcin II are the products of alternative splicing and are by far less abundant than calcitonin in C cells. CGRP is a 37-amino-acid vasoactive peptide with unknown effect on calcium metabolism.⁶² Interestingly, C cells, although different from follicular cells in function and embryologic derivation, express Nkx2-1/Ttf-1,⁶³ which is a distinctive marker of thyroxine-producing cells. Nkx2-1/Ttf-1 is also present in immature C cells, in the migrating ultimobranchial body, and in the cells of the fourth pharyngeal pouch.^14,64,65

Parafollicular cells share several biological features with other neuroendocrine cells that originate from the neural crest. Indeed, parafollicular cells express neuroendocrine markers such as neurospecific enolase and chromogranin A and a large number of regulatory peptides and their receptors,⁶⁶ including somatostatin, serotonin, cholecystokinin₂-receptor (CCK₂R), gastrin releasing peptide, thyrotropin-releasing hormone (TRH), and helodermin. Whether different subpopulations of parafollicular cells synthesize different sets of regulatory factors has not yet been demonstrated. However, there is some evidence of functional heterogeneity within mammalian parafollicular cells.⁷ In neonatal rats, 90% of calcitonin-producing cells coexpress somatostatin, while in adults this factor is detected in only 1% of parafollicular cells.⁶⁶ Also, in humans calcitonin and somatostatin are co-localized in few parafollicular cells.⁶⁷ However, somatostatin is detected in almost all C-cell carcinomas in rats.⁶⁶ Similarly, in many human medullary thyroid carcinomas, C cells can express somatostatin.⁶⁸ The functional relevance of the production of these regulatory peptides by C cells is still unclear. These biologically active peptides might regulate thyroid function in a paracrine pathway, because parafollicular cells are distributed among follicular cells and often tightly adhered to them. Somatostatin, calcitonin, CGRP, and katacalcin inhibit thyroid hormone secretion, while gastrin-releasing peptide and helodermin stimulate this process.⁷ The expression of CCK₂R, which binds cholecystokinin and gastrin,⁶⁹ and the observation that gastrin induces calcitonin secretion suggest an interrelationship between calcium homeostasis and gastrointestinal hormones. In addition, the presence of CCK₂ in thyroid tissues allows one to hypothesize that CCK₂ and its receptor are both involved in an autocrine loop required for C-cell function.⁶⁹

ULTIMOBRANCHIAL BODY–DERIVED EPITHELIAL CELLS

In addition to the common thyroid follicles, other epithelial structures are evident in the mammalian thyroid. These structures, described as a “second kind of thyroid follicles,”⁷⁰ rarely display a clear follicular organization. The finding that the second kind of thyroid follicles are absent in the avian thyroid (where ultimobranchial bodies never merge with the thyroid) has suggested that these structures represent remnants of the ultimobranchial body of endodermal origin.⁷¹

In humans, ultimobranchial-body remnants known as solid cell nests (SCN)⁷² are frequently present in the thyroid gland and are preferentially located in the middle and upper third of the lobes.⁷² SCN appear as para- or intrafollicular clusters and cords of epithelial cells clearly separated from the follicles by a basal lamina.

SNC are composed of two cell types: C cells and “main” cells, the most important cell population of these structures. The presence of C cells is consistent with the common ultimobranchial derivation of both SNC and C cells. In most cases, the SNC are found mixed with another structure known as a mixed follicle, in which follicular cells and main cells underline a lumen filled with colloid-like material.^72,73 Main cells, polygonal in shape, show squamoid features, with oval nuclei and eosinophilic cytoplasm lacking intercellular bridges. The molecular phenotype of main cells is peculiar. These cells express p63,⁷³ Bcl2 and telomerase.⁷⁴ They do not express markers of differentiation such as Nkx2-1/Ttf-1, Tg, calcitonin, and CGRP. It is worth noting that recent studies in mouse embryos have revealed that Nkx2-1/Ttf-1–negative, p63-positive cells are present both in the epithelium of the fourth pharyngeal pouch and in the ultimobranchial body, confirming the SNC are ultimobranchial-body remnants.⁶⁴ The presence in the main cells of both telomerase and p63, a transcription factor present in basal/stem cells of several multilayered epithelia and absent in differentiated cells, has suggested the hypothesis that these cells could be a source of multipotent cells able to differentiate towards either Tg- or calcitonin-producing cells. In addition, it has been suggested that main cells could be the cells of origin of a subset of papillary thyroid carcinoma.⁷⁵

SPECIFICATION OF THE THYROID FOLLICULAR CELLS: THE THYROID ANLAGE

Gastrulation involves dramatic changes in the overall structure of the embryo, converting it into a complex three-dimensional structure. The anterior and posterior endoderm invaginates, forming two pockets which extend and fuse, generating a primitive gut tube that runs along the anterior-posterior axis of the embryo. The foregut is the most anterior (cranial) region of this tube. The endoderm of the foregut gives rise to a number of cell lineages which eventually form the epithelial components of thyroid, thymus, lungs, stomach, liver, and pancreas. All organs derived from the gut tube undergo a rather similar process. First, a restricted group of cells differentiate from their neighbors, as can be visualized both by the appearance of specific molecular markers (usually transcription factors) and by the almost simultaneous appearance of a multilayered structure. Subsequently, all organs undergo a morphogenetic process that entails, depending on the organ, proliferation, branching morphogenesis, and/or migration or a combination of these processes. Finally, each organ forms a specific cellular organization that is directly correlated to the organ function (i.e., follicular organization in the case of the thyroid). The first differentiation step in this process will be called throughout this chapter specification, even though the term usually refers to a reversible process; in the case of all organs derived from the gut, it is at present unknown whether the first differentiation event is either reversible or irreversible.

While the inductive mechanisms by which endodermal cells produce liver, pancreas, and lung cell lineage begin to be detailed, the events that commit a group of multipotent endodermal cells to a thyroid fate are almost unknown. The first morphologic consequence of thyroid specification is the appearance of the thyroid anlage (also called thyroid placode). In the mouse (19.5-day-long gestation) it is visible at embryonic day (E)8 to 8.5 and in humans at E22.⁷⁷ In both the species, the thyroid anlage appears as a midline endodermal thickening in the ventral wall of the primitive pharynx caudal to the region of the first branchial arch that forms the tuberculum impar⁷⁸ (see Fig. 72-4). The cells of the thyroid anlage simultaneously express four transcription factors: Hhex,Nkx2-1/Ttf-1, Pax8 and Foxe1.⁷⁹ This unique molecular signature hallmarks these cells, which can be defined as the precursors of the TFCs.

The genetic program directing the specification of thyroid precursor cells is rather obscure. Any genes relevant in the patterning process of the foregut, such as Nodal, members of the Gata family, or Sox genes could play a role in thyroid specification. As yet, there have been no reports of either cell-fate mapping studies or genetically modified mice clarifying the early steps of the thyroid specification. Actually, mouse embryos carrying targeted inactivation of genes involved in foregut patterning usually show developmental arrest at stages that preclude the assessment of the thyroid anlage. In zebrafish, Bon and Gata5, two transcription factors that are downstream effectors of Nodal activity, seem to be specifically involved in the commitment of thyroid fate. Indeed, in the absence of these factors, endodermal cells form a reduced gut tissue but do not contribute to the thyroid.⁸⁰

The study of the developing thyroid in mice has shown that at E8.5, the earliest identifiable thyroid anlage appears in close apposition to the aortic sac, the cardiac region that gives rise to the embryonic heart outflow and pharyngeal arteries. This observation suggests that short-range inductive signals from cardiac mesoderm or from the endothelial lining of the aortic sac could have an inductive role to specify undifferentiated endodermal cells towards a thyroid fate. In mice, alterations in the foregut have been observed as a consequence of an impaired heart development⁸¹; furthermore, it has been reported that signals from cardiac tissue adjacent to the endodermal layer are crucial in the early stages of liver, pancreas, and lung development.^82–84

Data obtained from zebrafish are proving invaluable for the study of thyroid specification. The close association between thyroid anlage and aortic sac is maintained also in zebrafish.⁸⁵ In this species it has been demonstrated that in the absence of the basic Helix-loop-helix (bHLH) transcription factor Hand2, expressed in the cardiac mesoderm surrounding the site of initiation of thyroid development, the endoderm appears to be normal, but thyroid precursor cells are not present. Experiments strongly suggest both that Hand2 has a non–cell-autonomous role in thyroid specification and that FGF proteins participate in this pathway.⁸⁵ Interestingly, in mice it has been proved that FGF signals are required for correct thyroid development, but the role of these factors in thyroid specification has never been demonstrated.⁸⁶

Consistent with the hypothesis of a role of cardiac tissue in thyroid development are the findings that cardiac malformations represent the most frequent birth defects associated with thyroid dysgenesis in humans,⁸⁷ and DiGeorge syndrome is characterized by both congenital heart defects and an increased risk of congenital hypothyroidism.⁸⁸

EARLY STAGES OF THYROID MORPHOGENESIS: BUDDING, MIGRATION, AND LOBULATION OF THE THYROID PRIMORDIUM

After the specification stage, the developing thyroid undergoes very rapid changes in its appearance. In mice, by E8.5 the thyroid anlage first appears as a multilayered epithelium. Shortly after, it forms a bud that, evaginating from the floor of the pharynx, invades the surrounding mesenchyma as an endodermal extroflexion close to the aortic sac. In these early stages of morphogenesis, the expansion of the thyroid primordium does not seem to be due to the proliferation of cells of the primitive thyroid anlage; indeed, these cells show a very low cell proliferation rate as compared to that of the cells of the surrounding endoderm. Other cells from the pharyngeal endoderm could be recruited into the developing thyroid, thus contributing to the increase of the number of thyroid progenitor cells.⁸⁹ At E10.5, the thyroid bud has a flask-shaped structure still connected to the floor of the pharynx by a thin cord, the thyroglossal duct, a transient narrow channel. One day later, the thyroid bud is visible as a caplike shape that has migrated caudally into the mesenchyme, losing all connections with the floor of the pharynx (see Fig. 72-4). At this stage, the developing thyroid begins to expand laterally, the first step of the process that eventually leads to the formation of the two lobes. At E12.5, the thyroid primordium appears as an elongated structure, extended laterally, in contact with the third pharyngeal arch arteries that will participate in the formation of the definitive carotid vessels.

By E13, the thyroid bud continues its downward relocation into the mesenchyme and approaches the ultimobranchial bodies that are accomplishing their ventro-caudal migration from the fourth pharyngeal pouch. By E13.5, the developing thyroid, already formed by rudimental lobes connected by a thin central portion, reaches its definitive pretracheal position where it merges with the ultimobranchial bodies containing the precursors of C cells derived from the neural crest.⁷⁸ Once the gland reaches its final location, the two rudimentary paratracheal lobes expand, and by E15 and 16, the thyroid gland assumes its definitive shape (see Fig. 72-4). In the last stages of thyroid organogenesis, the gland increases further in size, probably due to the high proliferation of TFCs.

In humans, as described in mice, at an early stage of embryonic life, the thyroid anlage appears as a bud invading the mesenchyma at E26; in a few days it appears as a migrating primordium, connected to the pharynx by the thyroglossal duct that disappears around E37. At this stage, the thyroid bud acquires a bilobed shape. The developing thyroid merges with the ultimobranchial bodies by the sixth week and reaches its final position in front of the trachea around the seventh week.⁹⁰

The translocation of TFC precursors to reach the sublaryngeal position is a process that lasts for almost 4 days in the mouse embryo and almost 4 weeks in the human embryo. Budding and translocation from the gut tube is a developmental process shared by many endoderm-derived organs.⁹¹ In the case of the thyroid, since the definitive location is rather distant from the site of primitive specification, this process mostly involves active migration of the precursors, even if other morphogenetic events occurring in the neck region and in the mouth⁹² could contribute to the definitive location of the thyroid. The molecular mechanisms involved in the movement of the thyroid primordium are still a matter of debate. In many processes of embryogenesis, such as gastrulation, neural-crest migration, and heart formation, cell migration is involved. However, in these processes, migrating cells lose the epithelial phenotype and acquire mesenchymal features.⁹³ This phenomenon, called epithelial-mesenchymal transition, is hallmarked by an increased expression of N-cadherin and the down-regulation of E-cadherin. In contrast, TFC precursors seem to use a different and yet unidentified pathway to move, because they maintain their epithelial phenotype throughout the entire translocation process and never acquire a mesenchymal identity.⁹⁴ The expression of transcription factors such as either Hhex or Pax8 or Nkx2-1/Ttf-1 is not sufficient for thyroid migration, while Foxe1 plays a crucial role because the presence of this factor in the thyroid bud is required to allow the cells to move.^79,95

The genetic basis of the formation of two symmetrical lateral lobes (lobulation) is beginning to be understood. When the lobulation process begins at E11.5, the developing thyroid is in contact with the third pharyngeal arch arteries that will participate in the formation of the definitive carotid vessels located most closely to the mature thyroid lobes. Signals originating from adjacent vessels or factors regulating vasculogenesis could instruct the process of lobulation by non–cell-autonomous mechanisms. The study of animal models seems to confirm this hypothesis. Mice deficient in either Shh (a key regulator of embryogenesis)⁹⁶ or TBX (a factor regulated by Shh itself)⁸⁸ display a disturbed morphogenetic patterning of vessels adjacent to the developing thyroid. In these mutated embryos, as a consequence of the absence of caudal pharyngeal arch arteries, the thyroid bud is never in close contact with vessels, and the lobulation process is impaired. The thyroid fails to separate into two distinct lobes, maintaining the shape of a single tissue mass throughout development. In line with these findings is the observation that thyroid dysgenesis is not uncommon in patients affected by DiGeorge syndrome,⁹⁷ characterized by congenital anomalies of the heart and great vessels. However, the inductive signals from adjacent tissues must interact with events restricted to the thyroid cells to accomplish the lobulation process. Indeed, thyroid hemiagenesis is very frequent in mice double heterozygous for the null allele of Nkx2-1/Ttf-1 and Pax8, genes expressed in thyroid precursor cells and absent in other structures close to the developing thyroid.⁹⁸

LATE STAGES OF THYROID MORPHOGENESIS: FUNCTIONAL DIFFERENTIATION AND HISTOGENESIS

When the thyroid primordium reaches the sublaryngeal position, TFC precursors accomplish their functional differentiation. Notably, the normal final location of thyroid follicular cells in front of the trachea is not an essential requirement for functional differentiation, since an ectopic, sublingual thyroid expresses thyroglobulin in both human patients⁹⁹ and mutated mice.⁹⁵

The functional differentiation of TFCs is hallmarked by the expression of a series of proteins essential for thyroid hormone biosynthesis, such as Tg, TPO, TSHR, NIS, Duox, and pendrin. This program requires almost 3 days, between E14 and E16.5, and results in the differentiation of the thyroid primordium in a functional thyroid gland that is able to produce and release hormones. Tg and Tshr genes are expressed by E14¹⁰⁰; TPO and NIS, the two key enzymes involved in the process of Tg iodination appear between E15 and E15.5,¹⁰¹ probably because their expression is absolutely dependent on the pathway activated by the binding of TSH to its receptor, TSHR.¹⁰¹ Duox appears at E15.5¹⁰² and finally, thyroxine by E16.5.¹⁴

Alongside functional differentiation, the thyroid gland accomplishes its peculiar histologic organization. An inductive role of the stromal component surrounding follicular cells can be hypothesized in histogenesis. Accordingly, follicular cells, when explanted from a developing chick thyroid, can organize a correct histologic pattern in vitro only if co-cultured in the presence of fibroblasts obtained from the capsule of a thyroid gland.¹⁰³ By E15.5, TFCs start to form small rudimentary follicles, as revealed by the expression of ZO-1, a tight-junction marker. At E16.5, the gland displays an evident follicular organization. The histogenesis is complete in late fetal life between E17 and E18: the thyroid parenchyma is organized into small follicles surrounded by a capillary network, enclosing thyroglobulin in their lumen.⁸⁹ At birth, the thyroid gland is able to produce and release thyroid hormones, though the regulation of its growth and function by the hypothalamic-pituitary axis is fully active only after birth.¹⁰⁴

The molecular mechanisms involved in the differentiation of the human thyroid are not much different from those found in mice. Functional differentiation of TFCs requires almost 3 weeks. It begins after the developing thyroid is located in front of the trachea, at E48, when TFCs express Tg and TSHR; T₄ synthesis is detected at the 10th week.⁹⁰ In humans, the establishment of the characteristic histologic organization lasts several weeks and can be divided into three phases: the precolloid, the beginning colloid, and the follicular growth, which occur at 7 to 10, 10 to 11, and after 11 weeks of gestation, respectively.⁷⁶ In the precolloid phase, small intracellular canaliculi develop as an accumulation of colloid material. These small canaliculi enlarge, and the colloid organizes itself into extracellular spaces. In the last phase, primary follicles are clearly visible, and the fetal thyroid is able to concentrate iodide and synthesize thyroid hormones. The human thyroid continues to expand until term, and contrary to mice, the hypothalamic-pituitary-thyroid axis starts functioning at mid-gestation.

It is widely accepted that thyroglobulin-producing cells are derived from the endodermal cells of the thyroid anlage. However, the thyroid is assembled from both thyroid anlage and ultimobranchial bodies. Because of this composite origin, the question arises whether the neural crest–derived cells of ultimobranchial bodies (fated to become calcitonin-producing parafollicular cells) could also differentiate towards thyroglobulin-producing cells. In fish, amphibians, and birds, Tg- and calcitonin-producing cells are found in separate gland organs. In addition, lineage studies in zebrafish suggest that ultimobranchial bodies do not contribute to the development of the thyroid, which derives completely from the endodermal cells of the thyroid anlage.¹⁰⁵

In mammals, where endodermal cells from thyroid anlage and ultimobranchial bodies merge in the definitive gland, the contribution of the different cell lineages to the TFC population is still controversial. In the past, embryologists considered the ultimobranchial bodies as the lateral anlage of the thyroid, whose cells were fated to differentiate towards the typical follicular cells and become a definitive component of the mature gland.¹⁰⁶ This hypothesis is consistent with the report of patients displaying thyroid tissue in the submandibular region and no detectable thyroid tissue in the normal median position.¹⁰⁷ Furthermore, structures appearing as colloid-containing follicles have been observed in ultimobranchial bodies which fail to fuse with the thyroid bud (persistent ultimobranchial body).¹⁰⁸ Data suggesting that thyroid follicular cells could originate from ultimobranchial bodies are supported by the study of some murine models displaying persistent ultimobranchial bodies.^109–112 In mice, the size of the follicular thyroid appears smaller than would be expected if only cells contributed by ultimobranchial bodies were missing. However, it is worth noting that the expression of follicular cell-specific genes (such as Tg or TPO) in the ultimobranchial bodies has not been described. Thus, there is no conclusive evidence that these cells can differentiate in cells producing thyroid hormones.

ULTIMOBRANCHIAL BODIES DEVELOPMENT

In vertebrates, calcitonin-producing cells differentiate from the ultimobranchial body, a structure that derives from the fourth pharyngeal pouch. The ultimobranchial body is a definitive organ in all vertebrates except in placental mammals, where it is an embryonic transient structure fated to join the medial thyroid bud.

By E10 in mice, the fourth pharyngeal pouch is evident for the first time. It appears as a lateral extroflexion of the primitive foregut expressing both the transcription factor Islet1 (Isl)¹¹³ and protein gene product (PGP)9.5.^114,115 Shortly after, the caudal portion of the pouch grows, and at E11.5 the fourth pharynx-branchial duct is pinched off, forming an ultimobranchial body primordium visible as an ovoid vesicle with a lumen lined by a columnar epithelium identified by the simultaneous presence of Isl1, PGP9.5, and Nkx2-1/Ttf-1.^64,114,115 By E11.5 ultimobranchial body primordia migrate, and at E13 they appear as solid clusters of cells in contact with the midline primordium of the thyroid. By E14.5, ultimobranchial body cells begin to disperse within the thyroid parenchyma, and 1 day later only remnants of ultimobranchial bodies can be distinguished in the thyroid gland. C cells complete their differentiation program through the expression of a series of proteins according to a precise temporal pattern: the basic helix-loop-helix transcription factor Mash1 is expressed by E12.5; the neuronal markers TuJ1, CGRP, and somatostatin by E14.5. One day later, the expression of Mash1 disappears, and calcitonin-producing cells can be detected between follicular cells. During the late stages of thyroid morphogenesis, the expression of Isl1 decreases,¹¹³ while calcitonin-producing cells gradually increase in number.^114,115

In humans, the development of ultimobranchial bodies is similar to that of mice. At E24 the ultimobranchial body primordia appear as an outpouching of the ventral component of the fourth pharyngeal pouch. At this stage, the primordium of parathyroid IV is visible as a dorsal evagination of the same pouch. Some authors describe a transient fifth pouch as the endodermal origin of the ultimobranchial body.¹¹⁶ Probably the shape of the ultimobranchial body anlage itself, which appears as an incomplete pouch, has generated these different interpretations. By E35 the ventral extroflexion is a long-necked flask still attached to the pharynx; a few days later, the ultimobranchial body primordium loses its connection with the pharyngeal cavity, starts its migration, and at E40 reaches the posterior surface of the median thyroid. A connective layer separates these two buds, which display a different histologic organization: the lateral bud is composed of a compact mass of cells, while the median bud is composed of interconnecting sheets of epithelium. Finally, at E55 the ultimobranchial bodies are incorporated with the lateral lobes of the thyroid, and the cells from both structures mix with each other.

The genetic mechanisms that allow the developing thyroid and ultimobranchial body to recognize each other and fuse are beginning to be understood. Ultimobranchial bodies are absent in Splotch mutant mice,¹¹⁷ a strain characterized by an impaired migration of neural crest cells due to a loss-of-function mutation in Pax3, a gene expressed in the migrating neural crest cells.¹¹⁸ The absence of ultimobranchial bodies is also reported in Pax9 null mice.¹¹⁹ Pax9 is expressed in the entire pharyngeal endoderm and has been reported to be involved in the regulation of epithelial-mesenchymal interactions that are crucial for the correct morphogenesis of both teeth¹¹⁹ and thymus.¹²⁰ Ultimobranchial body defects have been reported in mice carrying mutations in Hox3 paralog genes. In Hoxa-3 null mice,¹¹⁰ the migration of neural crest cells is not impaired and C cells differentiate correctly, but their number is significantly reduced. In many cases, ultimobranchial bodies fail to fuse with the thyroid bud and remain as bilateral vesicles composed exclusively of calcitonin-producing cells (persistent ultimobranchial bodies). The phenotype appears more severe in mice carrying various mutant combinations in Hoxa3 and its paralogs Hoxb3 and Hoxd3.¹¹¹ These data indicate that Hox3 paralogs do not play a direct role in the migration and differentiation of C cells but control the correct development of the ultimobranchial body and its fusion to the ventral thyroid primordium. Another gene, Eya1, expressed in the pharyngeal arches mesenchyme and in the endoderm pouches, could control the interactions between ultimobranchial bodies and the thyroid primordium. Indeed, mice in which the Eya1 gene has been inactivated show fewer calcitonin-producing cells and persistent ultimobranchial bodies.¹¹² Thus both Hoxa3 and Eya1 seem to control the merging of ultimobranchial bodies and thyroid primordium, a step in thyroid gland organogenesis that only occurs in mammals. Recently it has been demonstrated that Nkx2-1/Ttf-1is required for the survival of ultimobranchial body cells during migration, but it is not necessary for ultimobranchial body formation.⁶⁴ Nkx2-1/Ttf-1 functions are in part dosage sensitive. Indeed, Nkx2-1/Ttf-1^+/− mice display an abnormal fusion of the ultimobranchial bodies with the thyroid diverticulum. Ultimobranchial body cells are incompletely incorporated into the thyroid parenchyma and remain at the dorsal part of the thyroid lobe.⁶⁴ A phenotype similar to that has been reported in mice defective for Hox3 genes.