Temporal and spatial requirements for Hoxa3 in mouse embryonic development.

Hoxa3(null) mice have severe defects in the development of pharyngeal organs including athymia, aparathyroidism, thyroid hypoplasia, and ultimobranchial body persistence, in addition to defects of the throat cartilages and cranial nerves. Some of the structures altered in the Hoxa3(null) mutant embryos are anterior to the described Hoxa3 gene expression boundary: the thyroid, soft palate, and lesser hyoid horn. All of these structures develop over time and through the interactions of multiple cell types. To investigate the specific cellular targets for HOXA3 function in these structures across developmental time, we performed a comprehensive analysis of the temporal and tissue-specific requirements for Hoxa3, including a lineage analysis using Hoxa3(Cre). The combination of these approaches showed that HOXA3 functions in both a cell autonomous and non-cell autonomous manner during development of the 3rd and 4th arch derivatives, and functions in a neural crest cell (NCC)-specific, non-cell autonomous manner for structures that were Hoxa3-negative by lineage tracing. Our data indicate that HOXA3 is required for tissue organization and organ differentiation in endodermal cells (in the tracheal epithelium, thymus, and parathyroid), and contributes to organ migration and morphogenesis in NCCs. These data provide a detailed picture of where and when HOXA3 acts to promote the development of the diverse structures that are altered in the Hoxa3(null) mutant. Data presented here, combined with our previous studies, indicate that the regionally restricted defects in Hoxa3 mutants do not reflect a role in positional identity (establishment of cell or tissue fate), but instead indicate a wider variety of functions including controlling distinct genetic programs for differentiation and morphogenesis in different cell types during development.

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.

Development of thyroid gland and ultimobranchial body cyst is independent of p63.

The ultimobranchial body (UBB) and thyroid primordium are the origins of the thyroid gland that fuse around embryonic day 14.5 of mouse gestation, ultimately giving rise to calcitonin-producing C cells and thyroglobulin-producing follicular cells, respectively. A homeodomain transcription factor NKX2-1 is expressed both in the UBB and the thyroid primordium, and is critical for development of the thyroid gland. In this study, the role of p63 in development of UBB and the thyroid gland was analyzed by histological, immunohistochemical, and electron microscopic analyses using mice with various combinations of Nkx2-1 and p63 wild-type, heterozygous, and null alleles. In the absence of p63, a normal thyroid gland develops, as revealed by expression of thyroglobulin and calcitonin, thus showing that p63 is not required for thyroid development. However, in mice carrying the Nkx2-1-null allele, the UBB remains as a cystic vesicular structure and/or in nested patterns consisting of p63-positive cells surrounding the vesicle and undifferentiated immature cells with occasional cilia lying inside. The cystic UBB was present even in the Nkx2-1;p63 double-null mice. The structure and p63 expression pattern of the UBB cyst strikingly resemble the solid cell nest. These results show that in the absence of NKX2-1, UBB becomes cystic independent of p63, which is likely the origin of SCN.

Origin of the ultimobranchial body cyst: T/ebp/Nkx2.1 expression is required for development and fusion of the ultimobranchial body to the thyroid.

The ultimobranchial body (UBB) is an outpocketing of the fourth pharyngeal pouch that fuses with the thyroid diverticulum, giving rise to calcitonin-producing C-cells. In this study, we demonstrate that the UBB is composed of two types of cells: one expressing T/ebp/Nkx2.1 and the other expressing p63. The former cell type, accounting for a majority of the UBB, requires T/ebp/Nkx2.1 for their survival. In contrast, the p63-positive cells, even in the absence of T/ebp/Nkx2.1 expression, can proliferate and give rise to a vesicular structure that is lined by a monolayer of p63-negative cells, surrounded by a cluster and/or single layer of p63-positive cells, displaying the basal/stem cell phenotype. T/ebp/Nkx2.1 haploinsufficiency causes abnormal fusion of the UBB with the thyroid diverticulum, which stays as a cluster of C-cells around the vesicular structure, similar to the one observed in mice null for T/ebp/Nkx2.1 expression. These results demonstrate that T/ebp/Nkx2.1 plays a role in the survival of UBB cells, their dissemination into the thyroid diverticulum, and the formation of UBB-derived vesicular structure.

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.

Salivary heterotopia, cysts, and the parathyroid gland: branchial pouch derivatives and remnants.

Five cases of periparathyroid salivary heterotopia associated with cysts were studied. The specimens were obtained from three men and two women age 36 to 62 years who underwent surgery for primary hyperparathyroidism (four patients) and thyroid nodule (one patient). The heterotopia-cyst combination occurred with normal and abnormal parathyroid glands (four inferior and one of unknown location). Review of histologic slides of all parathyroid glands excised from 258 patients during a 1-year period at the Mayo Clinic revealed two similar salivary gland-cyst units. Seven more cases featured one or more periparathyroid cysts, five with other nonsalivary-type epithelial accompaniments. One of the latter additionally had a focus of parathyroid cells in the cyst wall, and associated thyroid parenchyma with C cells, and cartilage.

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.

Electron microscopic study on the development of the chicken ultimobranchial glands, with special reference to innervation of C cells.

The development of chicken ultimobranchial glands was studied by electron microscopy. As early as at 8 days of incubation, some cells contained a few secretory granules, although most of the ultimobranchial cells were undifferentiated. Single axons or small bundles of axons were occasionally detected in close contact with the ultimobranchial cells. Subsequently, immature C cells gradually increased in number with age. At 12 days of incubation, the developing C cells, which contained some secretory granules from 60 to 200 nm in diameter, occupied the greater part of the gland. The cells were oval, elongated or irregular in shape and frequently gave rise to long cytoplasmic processes that touched other C cells. Numerous axons enveloped with Schwann cell processes occurred in close vicinity to C cells. At 14 days of incubation, the cytoplasmic processes of C cells reached their maximum number and size. Desmosome-like membrane specialization was observed at the contact between the processes and cell bodies of other C cells, while numerous microtubules were arranged in parallel to the long axes of the processes, and secretory granules were distributed along them. Thus, the C cells at these stages seem to regulate other homologous cells by direct contact. Axon terminals, which contained small, clear and large, dense-cored vesicles, were first found in direct contact with the surface of C cells in 14-day-old embryos. Subsequently, the cytoplasmic processes of C cells progressively decreased, while nerve fibers continued to increase in the ultimobranchial glands. At the late stages of embryonic development, many C cells displayed an oval outline and increased number and size of secretory granules. At hatching, many C cells were filled with large secretory granules ranging from 200 to 700 nm in diameter (average 300 nm). Some cells were still elongated or irregular in shape and contained small secretory granules, 60-200 nm in diameter.

Immunohistochemical localization of a neuron-specific beta-tubulin isotype in the developing chicken ultimobranchial glands.

The localization of a neuron-specific beta-tubulin isotype in the ultimobranchial glands from chickens at various stages of development was studied by means of light- and electron microscopic immunohistochemistry with a monoclonal antibody (TuJ1) against the beta-tubulin isotype, c beta 4. At 8 days of incubation, many C cells in the ultimobranchial glands showed immunoreactivity for TuJ1 invariable degrees, weak to intense. At 12 days of incubation, a vast majority of C cells were intensely immunoreactive for TuJ1, and further TuJ1-immunoreactive nerve fibers were distributed in the ultimobranchial glands. At 14 and 16 days of incubation, intense immunoreactivity for TuJ1 was sustained in the C cells. Electron microscopic analyses revealed that TuJ1 immunoreactivity was diffusely distributed throughout the cytoplasm and also localized on the secretory granules of C cells at these stages. TuJ1 immunoreactivity in the C cells started to decrease at late stages of embryonic development. At the hatching period, dense distributions of TuJ1-immunoreactive nerve fibers were observed in the ultimobranchial glands, whereas TuJ1 immunoreactivity of the C cells became very weak. In 10-day-old chickens, TuJ1 immunoreactivity was restricted to the nerve fibers.

[Immunohistochemical and morphometric studies on the development of the thyroid, parathyroid and ultimobranchial body in Xenopus laevis Daudin].

The development of the thyroid, parathyroid and ultimobranchial body was examined by immunohistochemistry and morphometric analysis in the larvae of Xenopus laevis Daudin from the time immediately after hatch to the end of metamorphosis. The thyroid appeared at stage 43 and began to secrete thyroglobulin from stage 47/48. Both the volume of the thyroid and the height of the follicular epithelium reached the peak at stage 61 and decreased thereafter towards the end of metamorphosis at stage 66. The parathyroid first appeared at stage 43 as the thickening of the wall of the third and fourth visceral pouch. The volume of the parathyroid gradually increased with the progress of development until the end of metamorphosis. The ultimobranchial body appeared at stage 45 as a recess of the epithelium lining the floor of the pharynx. It became follicular in the structure at stage 53. Its volume reached the peak at stage 61 and decreased thereafter towards the end of metamorphosis. Calcitonin-immunoreactive cells first appeared in the ultimobranchial body at stage 47/48, showed the most intense reaction at stage 59, and rapidly decreased in number thereafter towards the end of metamorphosis.

[Embryonic induction and development of the ultimobranchial body in the embryogenesis of amphibia]. / Zakladka i razvitie u'ltimobrakhia'lnoi zhelezy v émbriogeneze amfibii.

At early embryonal and larval stages of development 7 species of amphibia have been studied. The ultimobranchial anlage and processes resulted in formation of the secretory follicle are investigated. Dynamics on changes of amount of the gland cells and the first appearance of capillaries are analyzed. In Anura and Urodela the anlage of the ultimobranchial gland develops from the epithelial lining of the pharynx behind the last branchial pocket rudiment. The gland is asymmetric and can be laid either in the right or in the left side of the body. Death of calcitonin-secreting cells is compensated at the expense of repeated anlage of follicles from the pharyngeal epithelium. The newly formed follicles can either incorporate into the existing gland, or form independent follicles. For amphibia formation of the capillary network around the gland after beginning of the follicular secretion is specific. Owing to these data, it is possible to conclude that the stimulus for the gland to become overgrown with capillaries is the beginning of calcitonin secretion.

Persistent truncus arteriosus in the Splotch mutant mouse.

The Splotch mutant mouse shows defects in neural crest-derived cell populations. The septation of the truncus arteriosus and the development of the aortic arch-derived blood vessels was studied in homozygotes of the Splotch mutant allele Sp1H. It is shown that in homozygous mutant embryos, the septation of the truncus arteriosus does not proceed normally, resulting in persistent truncus arteriosus. The ostium of the persistent truncus arteriosus opens to the right ventricle. Frequently, variations of the aortic arch-derived blood vessels are observed. The development of the thymus, the parathyroid and the ultimobranchial bodies are also variably affected in mutants. These results provide indirect evidence, that cells contributing to the aortic arches and the septum of the truncus arteriosus in mice are derived from the neural crest. The Splotch mutant mouse is proposed to be an animal model for persistent truncus arteriosus. The implications of the vascular malformations for the midgestational death of this mutant are discussed.

Origin of the ultimobranchial body and its colonizing cells in human embryos.

The early development of the ultimobranchial body and its colonizing cells was studied in human embryos (O'Rahilly's stages 14 and 15). In our studies we have obtained evidence that permits us to propose a new hypothesis on the origin of both the ultimobranchial body and its colonizing cells. Based on our interpretation of the morphogenetic features in human development, we think that the ultimobranchial body derives from the fifth endodermal pharyngeal pouch, which is colonized, from O'Rahilly's stage 14 on, by cellular material of ectodermal placodial nature that originates in the most caudal portion of the epicardiac branchial placode.

Morphogenesis of the ultimobranchial body and its colonizing cells in the chick embryo.

The development of the ultimobranchial body and its colonizing cells was studied in chick embryos in Hamburger-Hamilton's stages 21 to 46. The most significant observations included the following: 1.-The ultimobranchial body is a separate morphological entity with particular characteristics distinguishing it from the fourth pharyngeal pouch in all developmental stages. 2.-The fifth pharyngeobranchial ducts disappear in Hamburger-Hamilton's stage 27. 3.-From Hamburger-Hamilton's stage 27 on, the left ultimobranchial body is in contact with the caudal end of the left parathyroid IV primordium. 4.-The group made up of the ultimobranchial body and its colonizing cells at no time fuses with the thyroid gland.

Localization of immunoreactive keratins in cyst epithelium of chick ultimobranchial glands.

The cyst structures of chick ultimobranchial glands were studied by electron microscopy and immunocytochemistry to characterize the type of intermediate-sized filaments present in the cells lining cyst lumina. Electron microscopy showed that the majority of the lumen-bordering cells contained extensive meshworks of intermediate-sized (7-11 nm) filaments, many of which were arranged in bundles. Apical regions of C cells directly bordering on cyst lumina were also filled with thinner (5-6 nm) filaments. Immunoperoxidase staining showed that the majority of cyst epithelial cells were stained intensely with anti-keratin antiserum, but not with anti-neurofilament antiserum, which is a specific marker for neuronal differentiation. The cyst epithelium also showed moderate-to-weak immunoreactivity for actin. Subsequently, the differentiation and maturation of cyst structures related to intermediate filament expression were studied. In 18-day-old chick embryos, keratin immunoreactivity began to appear in the cell clusters destined to form cysts and in the primordial cysts with small cavities. At this time, fine networks of intermediate filaments were already detected in the cells lining the cystic cavities. At 1 day after hatching, the cysts became a consistent feature of ultimobranchial glands. Intermediate filaments associated in bundles were observed, and the intensity of immunostaining for keratins increased. Thereafter, with progressive enlargement of cysts, numbers of intermediate filaments and intensity of keratin immunoreactivity gradually increased with age. Thus, the data indicate that in cyst epithelium keratin filaments are highly organized and may confer the structural strength necessary for cells lining cyst lumina.

The role of ultimobranchial bodies in the modulation of the response of chick embryos to 1,25-dihydroxy-cholecalciferol.

Ultimobranchial bodies (UBBs) were dissected from 17-day-old chick embryos and grafted onto the chorioallantoic membrane of 8-day-old embryos. The embryos with UBB grafts as well as sham-grafted controls were injected on the 10th day of incubation with 100 ng 1,25(OH)2D3 dissolved in ethyl alcohol or with an equal volume of ethyl alcohol alone; embryos were sacrificed on the 13th day. Grafted UBBs showed ultrastructural characteristics typical of actively secreting glands. A histological study of the tibiae from all embryos showed that while the grafted embryos responded to the injection of 1,25(OH)2D3 with a peripheral rim of undermineralized bone trabeculae, sham-grafted embryos never did so. These results confirm the original hypothesis that the presence of differentiated UBBs is a precondition for the production of undermineralized bone (osteoid) by 1,25(OH)2D3. In a second series of experiments, similarly treated embryos were sacrificed on the 10th, 11th, 12th and 13th day; the levels of calcium and inorganic phosphate were determined in their blood. The injection of 1,25(OH)2D3 produced in all embryos hypercalcaemia and hypophosphataemia. However, the hypophosphataemic response was more prolonged in the embryos with UBB grafts than in sham-grafted ones. These results suggest that the grafted UBBs prolonged the hypophosphataemic response, probably by secreting calcitonin and thus reducing the rate of bone resorption. It is also probable that the prolonged hypophosphataemia produced or contributed to the undermineralization of the peripheral (subperiosteal) trabeculae.

A study on the relationship between solid cell nests and mucoepidermoid carcinoma of the thyroid.

Mucoepidermoid carcinoma of the thyroid (MECT) has been recently recognized as a pathological entity. The origin of MECT is unknown but the morphology of this tumour closely resembles features seen in the ultimobranchial body (UB) vestiges. Recent studies in man have shown strong evidence that the so-called solid cell nests (SCN) of the thyroid may correspond to the human UB vestiges. To investigate whether these vestiges are the site of origin of this tumour a comparative study on SCN and MECT was undertaken. One hundred autopsied thyroids cut at 2-3 mm intervals were studied for the presence of SCN. Histochemical (H & E, Alcian blue-PAS, Mayer mucicarmine) and immunohistochemical studies (calcitonin, epidermal keratin) were performed in SCN and four cases of MECT. Sixty percent of thyroids were found to have SCN. They were mainly composed of epidermoid-like cells arranged in solid structures or lining cystic cavities, tubular and follicular structures. Solid clusters usually showed lumina containing PAS-positive and mucin-positive cell debris. Mucin stains also revealed mucinous cells placed around lumina filled by mucosubstances. Characteristic PAS-positive rounded bodies were found filling lumina as well as within some apical epidermoid-like cells, mucinous cells and cell debris. An obvious transition between these cells, cell debris and mucosubstances filling the lumina was noticed; suggesting degenerative changes undergone by the epidermoid-like cell. MECT basically presented all histological and histochemical features shown by SCN, furthermore, calcitonin containing cells were observed in 54% of SCN, while a metastatic MECT also showed scattered C cells within solid islands. The presence of epidermal keratin in all SCN and MECT, together with the previous findings, are strong evidence that MECT could originate in the SCN or human UB vestiges.

The development of pharyngeal endocrine organs in mouse and chick embryos.

Some interesting differences exist between development of the pharyngeal endocrine organs in mouse and chicken. In both, the thyroid forms as an evagination of the pharyngeal floor in the midline at the level of pharyngeal arch II and moves caudally to the base of the neck. In the chicken, the thyroid divides to form paired organs whereas in the mouse it forms a moustache-shaped bar that later fuses with the parathyroid and ultimobranchial bodies. The thymi, parathyroids and ultimobranchial bodies form as lateral evaginations of the pharyngeal pouches. The chick forms two pairs of thymi and parathyroids from the third and fourth pouches whereas the mouse forms a single pair of each primordium from the third pouch. Ultimobranchial evaginations form from the caudal wall of the sixth pouch in the chick and from the posterior pharynx in the mouse. In the chick all of these evaginations detach from the pharynx and come to lie along the carotid arteries as separate organs that surround the thyroids. In the mouse the parathyroids and ultimobranchial bodies fuse with the lateral thyroid lobes. The thymi move to the ventral midline and fuse to form a single organ. Positioning of these organs appears to be related to three developmental events. Outgrowth of the original evaginations and their ventral movements conform to the curved shape of the pharyngeal arches. Ventral growth of the primordia should result in their convergence near the origin of the ventral aortic roots. The thyroid appears to be attached to the common carotid arteries. Lateral growth of the carotids is consistent with the change in thyroid shape. In the mouse the thymi lie ventral to the other organs. Their movement appears to be related to ventral closure of ventral ectoderm in the neck region.

Ontogeny of chicken ultimobranchial glands studied by an immunoperoxidase method using calcitonin, somatostatin and 19S-thyroglobulin antisera.

The ontogeny of the ultimobranchial glands in chickens from 9-day-old embryos to adults was investigated by the immunoperoxidase method using anti-calcitonin, anti-somatostatin and anti-19S-thyroglobulin antisera. During embryonic development, the chick ultimobranchial glands consisted of solid cell clusters. Calcitonin immunoreactivity began to appear at 16 days of incubation and rapidly increased at late periods of incubation. At the time of hatching, almost all of the epithelial cells in the ultimobranchial glands exhibited the immunoreaction for calcitonin. Cyst structures showing various sizes, shapes and luminal contents were consistent features of the ultimobranchial glands after hatching. As age proceeded, the cysts and loose connective tissues gradually increased in the glands. In adult chickens, the calcitonin cells came to be interspersed among them and the number of the cells per unit area was very small, compared with that in young animals. No immunoreaction for somatostatin was found in the ultimobranchial glands of chickens of all ages examined. In the glands there were no cells immunoreactive to the 19S-thyroglobulin antiserum. Further, neither cyst epithelium nor luminal contents were stained with the antiserum.