Follicular cell lineage in persistent ultimobranchial remnants of mammals.

It has been a subject of much debate whether thyroid follicular cells originate from the ultimobranchial body, in addition to median thyroid primordium. Ultimobranchial remnants are detected in normal dogs, rats, mice, cattle, bison and humans and also in mutant mice such as Eya1 homozygotes, Hox3 paralogs homozygotes, Nkx2.1 heterozygotes and FRS2α2F/2F. Besides C cells, follicular cell lineages immunoreactive for thyroglobulin are located within these ultimobranchial remnants. In dogs, the C cell complexes, i.e., large cell clusters consisting of C cells and undifferentiated cells, are present together with parathyroid IV and thymus IV in or close to the thyroid lobe. In addition, follicular cells in various stages of differentiation, including follicular cell groups and primitive and minute follicles storing colloid, are intermingled with C cells in some complexes. This review elaborates the transcription factors and signaling molecules involved in folliculogenesis and it is supposed why the follicular cells in the ultimobranchial remnants are sustained in immature stages. Pax8, a transcription factor crucial for the development of follicular cells, is expressed in the fourth pharyngeal pouch and the ultimobranchial body in human embryos. Pax8 expression is also detected in the ultimobranchial remnants of Eya1 and Hes1 null mutant mice. To determine whether the C cells and follicular cells in the ultimobranchial remnants consist of dual lineage cells or are derived from the common precursor, the changes of undifferentiated cells in dog C cell complexes are examined after chronically induced hypercalcemia or antithyroid drug treatment.

Restricted localization of ultimobranchial body remnants and parafollicular cells in the one-humped camel (Camelus dromedarius).

Parafollicular cells (C-cells) exist within the thyroid glands and display different distributions within the glands among mammalian species. In the one-humped camel (Camelus dromedarius), localization of the C-cells remains under debate. We herein investigated appearance of C-cells and the remnants of the ultimobranchial body, origin of C-cells, in the thyroid glands of one-humped camels. Macroscopically, a white mass was present at one-third the length from the cranial end of the thyroid glands where the cranial thyroid artery entered. In addition, large fossae were frequently found adjacent to the white mass. Histologically, the mass was mainly composed of connective tissues, thyroid follicles, and two types of cell clusters: one was composed of cells with clear cytoplasm and the other was composed of non-keratinized epidermoid cells. The mass and the fossae contained p63-positive cells, indicating that they consisted of ultimobranchial body remnants. Calcitonin was expressed in cells with clear cytoplasm, which were localized just beneath the fossae and in the cell clusters of the white mass. C-cells also resided in both subfollicular and interfollicular spaces adjacent to the white mass, but gradually decreased toward the periphery. C-cells tended to display round shapes in the ultimobranchial body remnants and subfollicular spaces, and spindle shapes in interfollicular spaces. In conclusion, we demonstrated that the ultimobranchial body remnants were limited to the region around the entrance of cranial thyroid artery and vein, and C-cells were mainly concentrated within and around the ultimobranchial body remnants.

Morphological and molecular evolution of the ultimobranchial gland of nonmammalian vertebrates, with special reference to the chicken C cells.

This review summarizes the current understanding of the nonmammalian ultimobranchial gland from morphological and molecular perspectives. Ultimobranchial anlage of all animal species develops from the last pharyngeal pouch. The genes involved in the development of pharyngeal pouches are well conserved across vertebrates. The ultimobranchial anlage of nonmammalian vertebrates and monotremes does not merge with the thyroid, remaining as an independent organ throughout adulthood. Although C cells of all animal species secrete calcitonin, the shape, cellular components and location of the ultimobranchial gland vary from species to species. Avian ultimobranchial gland is unique in several phylogenic aspects; the organ is located between the vagus and recurrent laryngeal nerves at the upper thorax and is densely innervated by branches emanating from them. In chick embryos, TuJ1-, HNK-1-, and PGP 9.5-immunoreactive cells that originate from the distal vagal (nodose) ganglion, colonize the ultimobranchial anlage and differentiate into C cells; neuronal cells give rise to C cells. Like C cells of mammals, the cells of fishes, amphibians, reptiles, and also a subset of C cells of birds, appear to be derived from the endodermal epithelium forming ultimobranchial anlage. Thus, the avian ultimobranchial C cells may have dual origins, neural progenitors and endodermal epithelium. Developmental Dynamics 246:719-739, 2017. © 2017 Wiley Periodicals, Inc.

Immunohistochemical profiling of the ultimobranchial remnants in the rat postnatal thyroid gland.

Ultimobranchial (UB) remnants are a constant presence in the thyroid throughout rat postnatal life; however, the difficulty in identifying the most immature forms from the surrounding thyroid tissue prompted us to search for a specific marker. With that objective, we applied a panel of antibodies reported to be specific for their human counterpart, solid cell nests (SCNs), using double immunohistochemistry and immunofluorescence. Our results demonstrated that cytokeratin 34ßE12 and p63 are highly sensitive markers for the immunohistologic screening of UB-remnants, independently of their maturity or size. Furthermore, rat UB-follicles (UBFs) coincided with human SCNs in the immunohistochemical pattern exhibited by both antigens. In contrast, the pattern displayed for calcitonin and thyroglobulin differs considerably but confirm the hypothesis that rat UB-cells can differentiate into both types of thyroid endocrine cells. This hypothesis agrees with recent findings that thyroid C-cells share an endodermic origin with follicular cells in rodents. We suggest that the persistence of p63-positive undifferentiated cells in UB-remnants may constitute a reservoir of basal/stem cells that persist beyond embryogenesis from which, in certain unknown conditions, differentiated thyroid cells or even unusual tumors may arise.

Ultimobranchial gland respond in a different way in male and female fresh water teleost Mastacembelus armatus (Lacepede) during reproductive cycle.

The present study was carried out to analyze the differences in the activity of ultimobranchial gland (UBG) between male and female fresh water teleost Mastacembelus armatus during reproductive cycle. Considerable variations in the nuclear diameter of UBG cells and plasma calcitonin (CT) levels during different reproductive phases of testicular and ovarian cycle suggested that the activity of the UBG depends upon the sexual maturity of fishes. A positive correlation was observed between plasma CT and sex steroid levels and the gonadosomatic index in both sexes which further confirmed the involvement of UBG in the processes related to gonadal development in fishes irrespective of the sex. Sudden increase in the level of plasma CT and nuclear diameter of UBG cells after administration of 17 α-methyltestosterone in males and 17 ß-estradiol in females during resting phase of the reproductive cycle clearly showed that UBG becomes hyperactive with increases in the level of sex steroids. Plasma calcium level was also found to be positively correlated with gonadal maturation in females. However no such change in plasma calcium level in relation to testicular cycle was observed. Thus it can be concluded that UBG becomes hyperactive during gonadal maturation but its role differs between male and female fishes. In females it may involved in both gonadal maturation and plasma calcium regulation while in males its involvement in calcium regulation was not justified. Variations in the level of CT during various phases of testicular cycle evidenced its involvement in gonadal maturation only.

Hes1 is required for the development of pharyngeal organs and survival of neural crest-derived mesenchymal cells in pharyngeal arches.

Hes genes are required to maintain diverse progenitor cell populations during embryonic development. Loss of Hes1 results in a spectrum of malformations of pharyngeal endoderm-derived organs, including the ultimobranchial body (progenitor of C cells), parathyroid, thymus and thyroid glands, together with highly penetrant C-cell aplasia (81%) and parathyroid aplasia (28%). The hypoplastic parathyroid and thymus are mostly located around the pharyngeal cavity, even at embryonic day (E) 15.5 to E18.5, indicating the failure of migration of the organs. To clarify the relationship between these phenotypes and neural crest cells, we examine fate mapping of neural crest cells colonized in pharyngeal arches in Hes1 null mutants by using the Wnt1-Cre/R26R reporter system. In null mutants, the number of neural crest cells labeled by X-gal staining is markedly decreased in the pharyngeal mesenchyme at E12.5 when the primordia of the thymus, parathyroid and ultimobranchial body migrate toward their destinations. Furthermore, phospho-Histone-H3-positive proliferating cells are reduced in number in the pharyngeal mesenchyme at this stage. Our data indicate that the development of pharyngeal organs and survival of neural-crest-derived mesenchyme in pharyngeal arches are critically dependent on Hes1. We propose that the defective survival of neural-crest-derived mesenchymal cells in pharyngeal arches directly or indirectly leads to deficiencies of pharyngeal organs.

Ultimobranchial and parathyroid glands of the pigeon Columba livia in response to 1,25OH2D3 administration.

Columba livia were given daily intraperitoneal injections of 100 pmol of 1,25(OH)(2)D(3)/100 g body wt. for 15 days. Ultimobranchial and parathyroid glands were fixed on 1st, 3rd, 5th, 10th and 15th day of the experiment. Following 1,25(OH)(2)D(3) treatment, the plasma calcium levels of pigeon remain unchanged on day 1. The levels increase significantly after day 3 which progresses up to day 10. The plasma calcium level becomes normal at day 15. The plasma inorganic phosphate levels of Columba livia injected with 1,25(OH)(2)D(3) remain unaffected up to day 3. The levels exhibit a progressive increase from day 5 to day 10. The levels become normophosphatemic at day 15. Up to day 3 following 1,25(OH)(2)D(3) treatment, there is no change in the ultimobranchial gland of Columba livia. The gland exhibits an increased activity after 5 days 1,25(OH)(2)D(3) treatment which is evident by the increased nuclear volume and weak staining response of the cytoplasm of ultimobranchial cells. After day 10, the nuclear volume depicts a further increase and a few completely exhausted cells are discerned. Following 15-day 1,25(OH)(2)D(3) treatment the nuclear volume records an increase and degenerating cells have been observed at certain places. The parathyroid glands of 1,25(OH)(2)D(3)-treated Columba livia remain unaffected up to day 5. After day 10 and day 15, there is a progressive decrease in the nuclear volume of parathyroidal cells and reduced chromaticity of nuclei has been noticed. Moreover, after 15 days few degenerating cells are observed.

Calcitonin is expressed in the chicken pituitary gland: influence of gonadal steroids and sexual maturation.

Calcitonin (CT) is primarily produced by the thyroid C cells in mammals or by the ultimobranchial gland in chickens. CT is also expressed by the pituitary gland in rats in which it functions as a paracrine factor causing decreased lactotroph proliferation and prolactin (PRL) secretion. Gonadal steroids influence CT expression in the rat pituitary gland. However, the expression of the CT gene in the pituitary gland of chickens or of any other avian species has not previously been reported. We have tested the hypotheses that CT is expressed in the chicken pituitary gland, and that its expression is influenced by sexual maturation or in response to ovarian steroid administration. We have detected robust expression of CT cDNA in the chicken pituitary gland by reverse transcription/polymerase chain reaction (PCR). The sequence of the pituitary-derived CT cDNA is identical to that of the ultimobranchial gland. CT-immunoreactive (ir) cells have been observed throughout the anterior pituitary gland by confocal microscopy. Many of the PRL-ir cells show co-localization with CT-ir cells. Quantitative real-time PCR analysis has revealed an inverse relationship between the quantities of PRL mRNA and CT mRNA in the pituitary gland: sexually mature hens contain lower amounts of CT mRNA but larger quantities of PRL mRNA compared with sexually immature chickens. Estradiol and/or progesterone treatment of sexually immature chickens leads to a significant decrease in the quantity of pituitary CT mRNA relative to that in the vehicle-treated chickens. We conclude that pituitary CT plays an important paracrine/autocrine role in the control of lactotroph function and PRL secretion in the chicken.

Nicotinamide promotes long-term survival and extensive neurite outgrowth in ultimobranchial C cells cultured from chick embryos.

In avian species, the ultimobranchial anlage is populated with neuronal cells derived from the distal vagal ganglion. We found that ultimobranchial C cells of chick embryos cultured in the presence of nicotinamide continued to grow for at least 60 days and exhibited profound morphological changes, resulting in the formation of dense networks of neuronal fibers. Nicotinamide, thus, facilitated the manifestation of neuronal features in C cells. The neuronal phenotypes of cultured C cells were analyzed in detail by both scanning and transmission electron microscopy. Their neural nature was also positively established by immunostaining with monoclonal antibodies to the neuronal markers neuron-specific class III beta-tubulin (TuJ1), microtubule-associated protein (MAP) 2, and synaptophysin. Confocal laser scanning microscopy confirmed that these neuron-specific proteins are colocalized with calcitonin in both the somata and the neuronal processes of C cells. Furthermore, reverse transcriptase-polymerase chain reaction analyses, performed at various times up to 30 days in culture, indicated that the C cells have persistent gene expression of calcitonin, the catecholamine-synthesizing enzyme tyrosine hydroxylase, proenkephalin, proopiomelanocortin, neuron-specific beta-tubulin (cbeta4), SCG10, and Bcl-2. The morphological responses of C cells to nicotinamide treatment were analyzed quantitatively over a period of 60 days. The area of C-cell colonies, number of processes per colony, and length of processes continued to increase until culture day 45. In conclusion, nicotinamide stimulates long-term survival and neuronal differentiation of chick embryo C cells, and this culture system may provide a useful model for studying neuronal differentiation mechanisms.

Possible direct induction by estrogen of calcitonin secretion from ultimobranchial cells in the goldfish.

The plasma level of calcitonin (CT), a calcium (Ca)-regulating hormone, is known to increase in female teleosts during the reproductive period. In the present study, a correlation between plasma CT and Ca and one between plasma CT and the gonad somatic index were demonstrated in the female goldfish but not in the male. To clarify the relationship between CT and Ca, we examined the plasma CT and Ca levels after injecting immature goldfish with estrogen. At day 1, the plasma CT level significantly increased, whereas the plasma Ca level was not changed from its initial level. This result suggests that the trigger of CT secretion is estrogen and that estrogen directly acts on the ultimobranchial gland (UBG), a CT-secreting organ. To determine whether the UBG is equipped with estrogen receptor (ER), an ER binding assay and immunohistochemical staining of UBG cells with an antibody against ER were conducted. As a result, estrogen-specific binding (Kd, 18.52 nM; Bmax, 1.35 pmol/mg protein) and ER-immunoreactivity in the UBG were demonstrated. Furthermore, the expression of alpha, beta, and gamma types of ER in the UBG was also detected by use of the reverse-transcription polymerase chain reaction. Thus, we concluded that estrogen acts on the UBG to induce the release of CT, which in turn plays an important role in reproduction directly and/or indirectly through Ca. This is the first report on the existence of ERs in a teleost UBG and the occurrence of CT secretion caused by estrogen.

Expression and localization of prohormone convertase PC1 in the calcitonin-producing cells of the bullfrog ultimobranchial gland.

We examined the expression and localization of the prohormone convertases, PC1 and PC2, in the ultimobranchial gland of the adult bullfrog using immunohistochemical (IHC) and in situ hybridization (ISH) techniques. In the ultimobranchial gland, PC1-immunoreactive cells were columnar, and were present in the follicular epithelium. When serial sections were immunostained with anti-calcitonin, anti-CGRP, anti-PC1, and anti-PC2 sera, PC1 was found only in the calcitonin/CGRP-producing cells. No PC2-immunopositive cells were detected. In the ISH, PC1 mRNA-positive cells were detected in the follicle cells in the ultimobranchial gland. No PC2 mRNA-positive cells were detected. RT-PCR revealed expression of the mRNAs of PC1 and the PC2 in the ultimobranchial gland. However, very little of the PC2 mRNA is probably translated because no PC2 protein was detected either by IHC staining or by Western blotting analysis. We conclude that the main prohormone convertase that is involved in the proteolytic cleavage of procalcitonin in the bullfrog is PC1.

Neuronal properties in cultured ultimobranchial C cells of chick embryos: process outgrowth and expression of TuJ1 and enkephalin.

We have analyzed the neuronal properties in cultured ultimobranchial C cells isolated from embryonic chicks at different developmental stages (12--16 days of incubation) by immunohistochemistry and electron microscopy. The ultimobranchial glands mostly consist of C cell solids. In 13-day-old embryos, many C cells cultured for 7 days on laminin-coated slides extended long neurite-like processes, reaching 300 microm in length. Neuritic outgrowth of C cells was regulated developmentally and virtually unaffected by nerve growth factor (NGF). The cultured C cells expressed intense immunoreactivity for calcitonin and enkephalin. It was also confirmed by confocal laser-scanning microscopy that almost all C cells were intensely immunostained by both the calcitonin antiserum and the monoclonal antibody TuJ1, a neuron-specific marker. Scanning electron microscopy identified the outgrowth of long, branching neuritic processes emerging from C cell soma. The processes had numerous varicosities along their course and ended in growth cones. The C cells with processes were usually monopolar and less frequently bipolar or multipolar. Transmission electron microscopy revealed the presence of membrane-bounded secretory granules in the cultured C cells. The neuritic processes of C cells contained aggregations of microfilaments, intermediate filaments and microtubules arranged in parallel to the long axis. In addition, synaptic-like contacts showing desmosome-like membrane-thickenings and accumulations of small clear vesicles and dense-cored vesicles were formed between the endings of the processes and the surface of C cells. These results indicate that the C cells cultured from early chick embryos (12- and 13-day-old) maintain the neuronal characteristics for long periods in vitro.

Expression and development of the proenkephalin mRNA in the C cells of chicken ultimobranchial glands.

A large number of enkephalin-immunoreactive cells transiently appear in chick ultimobranchial glands during embryonic development. The expression and development of proenkephalin mRNA were examined in the ultimobranchial glands by in situ hybridization with digoxigenin (DIG)-labeled oligonucleotide probes, in comparison with those of calcitonin mRNA and enkephalin peptide. Proenkephalin mRNA, as well as calcitonin mRNA, appeared in some C cells at embryonal day 14 (E 14), and in many cells at E 16. Subsequently, there is a marked increase in the level of calcitonin mRNA around E 18-19; all C cells exhibited intense reaction for calcitonin mRNA. After hatching, intensity of calcitonin mRNA expression was more and more increased. Northern blot analysis with the calcitonin probe also indicated that calcitonin synthesis of the C cells progressively increased with developmental gradient, and reached to the adult level at 1 month after hatching. On the other hand, intensity of hybridization signal of proenkephalin mRNA was maintained moderately during development. In contrast to enkephalin immunoreactivity, which is markedly decreased after hatching, proenkephalin mRNA expression was consistently detected in many C cells of 1- and 2-month-old chickens. Reverse transcription-polymerase chain reaction (RT-PCR) analysis confirmed that proenkephalin mRNA was obtained in the ultimobranchial glands of not only embryos but also 1-day- and 1-month-old chickens. Furthermore, Northern blot analysis demonstrated that a single band for proenkephalin mRNA was obtained in the poly (A)+RNA isolated from the ultimobranchial gland of 1-day-old chicks. Thus, the present study evidences that proenkephalin mRNA is synthesized in almost all C cells of chicken ultimobranchial glands throughout life. Enkephalin may be essential for C cell function.

Ultrastructural features of "solid cell nest" of the human thyroid gland: a study of 8 cases.

The ultrastructural features of solid cell nests (SCN), made of squamous cells, and associated calcitonin cells (C cells), of the thyroid gland were studied in only a few cases in humans. A study was performed on 8 paraffin-embedded SCN, postembedded in Epon, to look for their ultrastructural features. Immunohistochemical analysis using calcitonin antibody was performed on semithin sections of SCN to explore the presence of C cells. Three cases (37.5%) of SCN were positive for calcitonin, and electron-dense secretory granules were observed in the cytoplasm. In two of these cases, an increased number of C cells in the adjacent thyroid parenchyma was observed. The presence of ciliated and lymphoid cells, in addition to intracytoplasmic microvacuolar and microfollicular (microglandular) structures, was noticed. Ciliated cells have already been reported in embryonic rests of human and animals, but ultrastructurally for the first time in human SCN. The presence of microfollicular structures, intracytoplasmic microvacuolar, secretory granules features, and ciliated cells, in addition to lymphoid cell, suggests the existence of a common ultimobranchial stem cell for C cells or for one or more cell types of the thyroid gland.

Parathyroids and ultimobranchial bodies in monotremes.

Only scant information is available in the scientific literature on the parathyroids and ultimobranchial bodies in the primitive mammals, the echidna (Tachyglossus aculeatus) and platypus (Ornithorhynchus anatinus). The major aim of this paper is to describe the morphology of the monotreme parathyroid gland and to compare it with parathyroids in mammals and reptiles. The gross anatomy and light microscopic structure of the ultimobranchial body, thymus, and thyroid are also given. Animals were dissected and routine light and electron microscopic techniques used to examine the microscopic morphology. The locations of parathyroid hormone, calcitonin and calcitonin gene-related peptide in tissue sections were identified by immunostaining. Monotremes have one pair of parathyroid glands located in the thorax and they are often associated with thymic tissue but never with the thyroid which is also present in the mediastinum. Ultimobranchial bodies are ventrolateral to the commencement of the trachea. Thymic lobules with Hassall's corpuscles are scattered in the fibrofatty tissue of the mediastinum and the ventral surface of the pericardium. Histologically, principal cells, water-clear cells, and non-secretory cells were identified in the parathyroid glands. Principal cells showed polarity and had microlamellar projections that formed intercellular canaliculi. Non-secretory cells had features similar to those of thymic epithelial reticular cells. Immunostaining of parathyroid hormone showed a diffuse distribution in parathyroid principal cells and none in ultimobranchial bodies. Identification of the ultimobranchial bodies was confirmed by immunostaining. The monotreme parathyroid gland, ultimobranchial bodies and thyroid show reptilian as well as mammalian features.

Morphological and functional aspects of reptilian ultimobranchial gland.

The intention of this review is to compare studies on the morphology and histology (light and electron microscopic) of ultimobranchial glands of various groups of reptiles. Moreover, experiments (including our investigations) on suppression or stimulation of the ultimobranchial gland are included. Adult reptiles possess one (on the left side) or two ultimobranchial glands (UBG). The UBG lie just anterior to the heart. Light as well as electron microscopically, the gland has been shown to contain follicles and cell cords (cell aggregates). The follicular epithelium is lined by simple cuboidal or pseudostratified columnar cells. Ciliated and goblet cells may be present in the follicular epithelia in some groups. The lumen may contain a colloid-like substance with desquamated cells or debris. The UBG of reptiles seem to be an active secretory organ with influence on calcium regulation. Other functions of calcitonin have also been suggested in reptiles for example in neurotransmission, in volume regulation, phosphate balance and promotion of bone calcification (at least in juveniles).

Immunocytochemical study of parafollicular cells of the thyroid and ultimobranchial remnants of the European bison.

The aim of the present study was to compare parafollicular cells in the bison thyroid and its ultimobranchial remnants. The thyroid of 26 European bisons was fixed in Bouin's fluid, 5 microns thick paraffin sections were stained with hematoxylin and eosin. Azan or silver Grimelius methods. For immunocytochemical analysis specific rabbit antisera were used against human calcitonin (CT), human calcitonin gene-related peptide (CGRP), bovine (b) or rat (r) neuron-specific enolase (NSE), human synthetic somatostatin (ST), and porcine chromogranin. Strongly positive reactions in the majority of parafollicular cells were observed after application of antisera against CT, CGRP, bNSE and rNSE only. ST-immunopositive cells were found in small numbers. Immunopositive parafollicular cells were also present outside typical structures of the thyroid within persistent ultimobranchial remnants. In persistent ultimobranchial bodies, parafollicular cells were frequently observed in groups between ultimobranchial follicles in form of solid cell nests. Many of these cells did not react with any of the antisera used and showed features of immature cells. It is concluded that histomorphologic analysis and immunocytochemical examination reveals a heterogeneous population of parafollicular cells in the bison thyroid, and this heterogeneity was particularly clear in persistent ultimobranchial bodies.

Localization of nitrergic neuronal and non-neuronal cells in the ultimobranchial glands of the chicken.

The ultimobranchial glands of 20 chickens, aged 2-3 months, were investigated for their nicotinamide adenine dinucleotide hydrogen phosphate-diaphorase (NADPH-d) reactivity and the distribution of nitric oxide synthase (NOS), using NADPH-d histochemistry and NOS immunocytochemistry respectively. Formazan, the blue reaction product of NADPH-d, was localised in the neuronal cell bodies and nerve fibres. Most of the cell bodies were found in the parenchyma. Some of them occurred in the wall of the ultimobranchial cysts, and a few in the immediate vicinity of the blood vessels. Labelled nerve fibres mostly travelled with blood vessels, while few of them appeared in the cystic lining. In addition to neuronal profiles, some C cells, cystic lining, and vascular endothelium were also labelled. NOS staining was found in neuron-like cells and fibres that were confirmed as neurons in adjacent sections stained with antibodies against neuron-specific enolase. It was also detected in cystic lining and in some C cells, but not in vascular endothelium. The distribution patterns of NADPH-d and NOS suggest that NO may play a role in the regulation of the secretory activity of and the blood flow through the ultimobranchial glands.

Evidence to support the distal vagal ganglion as the origin of C cells of the ultimobranchial gland in the chick.

Formation and development of the ultimobranchial anlage were studied in chicken embryos by immunohistochemistry with the antibodies to class III beta-tubulin, TuJ1, human leukemic cell-line (HNK-1), and protein gene product (PGP) 9.5, all of which recognized neurons. Medial to the fourth aortic arch, the ultimobranchial anlage was formed by the extension of the ventral portion of the fourth pharyngeal pouch at 4.5 days of incubation. At 5 days of incubation, TuJ1-immunoreactive cells with long cell processes began to enter the ultimobranchial anlage, which displayed a follicle structure. At 6 days of incubation, numerous neuronal cells that were continuous with the distal vagal ganglion (nodose ganglion) and expressed immunoreactivity for TuJ1, HNK-1, and PGP 9.5 were found to be in direct contact with the peripheral portion of ultimobranchial anlage. The TuJ1 antibody reacted only with the neuronal cells, whereas the HNK-1 and PGP 9.5 antibodies reacted with both endodermal epithelial cells and the neuronal cells in the ultimobranchial anlage. Subsequently, the ultimobranchial anlage rapidly increased in size; the follicle wall was thickened and a central cavity disappeared. The TuJ1-immunoreactive cells were distributed throughout the ultimobranchial parenchyma in 7-day-old embryos. The neuronal cell streams from the distal vagal ganglion to the ultimobranchial anlage were still prominent at 8 days of incubation. Almost all parenchymal cells of the ultimobranchial glands were intensely immunoreactive for TuJ1, HNK-1, and PGP 9.5 between 10 and 16 days of incubation. These results indicate that the neuronal cells from the distal vagal ganglion enter into the ultimobranchial anlage and give rise to C cells, i.e., C cells differentiate along the neuronal lineage. During embryonic development, C cells of the chick ultimobranchial glands transiently express a number of neuronal properties.