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.

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.

Immuno-electron-microscopic localization of enkephalin in the secretory granules of C cells in the chicken ultimobranchial glands.

In the chicken, enkephalin-immunoreactive cells and nerve fibers are distributed in the ultimobranchial glands, which consist of C-cell groups and cyst structures. Ultrastructural features of the enkephalin cells and nerve fibers were examined by immuno-electron microscopy using both the streptavidin-biotin-peroxidase method and the protein A-colloidal gold method. Immunoreactivity for enkephalin was located on the secretory granules of C cells. In 1-day-old chickens, three types of C cells were distinguished on the basis of their granule size. Type-I cells were filled with large secretory granules (200-600 nm in diameter). These elements represented a majority of the C-cell population. Type-II cells contained medium-sized granules (100-280 nm in diameter). Type-III cells displayed small secretory granules (60-200 nm in diameter). The latter cells were elongate or irregular in shape and frequently extended cytoplasmic processes into the connective tissue stroma or contacted other C cells. Enkephalin-immunoactivity was revealed by dense deposits of immunogold particles on the secretory granules of type-II and type-III cells. There were only a few type-I cells showing immunoreactivity for enkephalin. A double immunogold labeling procedure demonstrated that calcitonin and enkephalin were colocalized in the same secretory granules of type-I and type-II cells. Type-III cells were devoid of immunoreactivity for calcitonin. Enkephalin-immunoreactive nerve fibers were characterized by the presence of granular vesicles, 60-160 nm in diameter, and frequently established direct contact with the surface of C cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunocytochemical localization and development of multiple kinds of neuropeptides and neuroendocrine proteins in the chick ultimobranchial gland.

The ultimobranchial gland is an endocrine organ consisting of C cell groups. In chickens, the glands are richly supplied by nerve fibers immunoreactive for neurofilaments. It was found by immunocytochemical staining that C cells of chick ultimobranchial glands showed immunoreactivities for multiple kinds of neuropeptides and neuroendocrine proteins in addition to calcitonin, i.e., calcitonin gene-related peptide (CGRP), somatostatin, neurotensin, chromogranin A, and tyrosine hydroxylase. Furthermore, enkephalin-immunoreactive cells that showed long cytoplasmic processes and large cell bodies, being distinct from the C cell feature, were detected. The densities of these cells per unit area of ultimobranchial gland were assessed using computer-assisted image analysis system; calcitonin cells were 42.9 +/- 10.0%; CGRP cells 26.9 +/- 5.6%; neurotensin cells 8.6 +/- 6.9%; somatostatin cells 3.1 +/- 1.4%; chromogranin A cells 11.8 +/- 1.8%; tyrosine hydroxylase cells 10.0 +/- 5.2%; enkephalin cells 2.9 +/- 1.3%. Dense distributions of peptidergic nerve fibers were also detected in chick ultimobranchial glands. Numerous varicose fibers immunoreactive for substance P were distributed in the close vicinity to C cell clusters and blood vessels. Enkephalin-immunoreactive fibers were also prominent around C cell clusters. Galanin-, vasoactive intestinal peptide (VIP)-, and tyrosine hydroxylase-immunoreactive fibers were distributed around blood vessels only. Subsequently, the ontogeny of these neuropeptides, neuroendocrine proteins, and peptidergic innervations was examined in chickens at various developmental stages. In 10-day-old embryos, weak to moderately intense immunoreactivity for calcitonin was already present in almost all C cells. Immunoreactivities for somatostatin, CGRP, and tyrosine hydroxylase began to appear at this age. At 12 days of incubation, substance P-immunoreactive fibers were first detected in the parenchyma of ultimobranchial glands. Considerable numbers of enkephalin-immunoreactive fibers and cells were also observed. At 14 days of incubation, the largest populations of somatostatin- and enkephalin-immunoreactive cells were attained; the densities of somatostatin- and enkephalin-immunoreactive cells per unit area were 21.2 +/- 3.2% and 12.9 +/- 3.1%, respectively. Substance P-immunoreactive fibers became numerous throughout the gland at this age. Thereafter, calcitonin-, CGRP-, tyrosine hydroxylase-immunoreactive cells progressively increased in number with embryonic age, whereas somatostatin- and enkephalin-immunoreactive cells started to decrease. Chromogranin A- and neurotensin-immunoreactive cells began to appear at 16 days and 18 days of incubation, respectively. Galanin-, VIP-, and tyrosine hydroxylase-immunoreactive fibers were inconspicuous during embryonic life.

Innervation of the C cells of chicken ultimobranchial glands studied by immunohistochemistry, fluorescence microscopy, and electron microscopy.

Innervation of the ultimobranchial glands in the chicken was investigated by immunohistochemistry, fluorescence microscopy and electron microscopy. The nerve fibers distributed in ultimobranchial glands were clearly visualized by immunoperoxidase staining with antiserum to neurofilament triplet proteins (200K-, 150K- and 68K-dalton) extracted from chicken peripheral nerves. The ultimobranchial glands received numerous nerve fibers originating from both the recurrent laryngeal nerves and direct vagal branches. The left and right sides of the ultimobranchial region were asymmetrical. The left ultimobranchial gland had intimate contact with the vagus nerve trunk, especially with the distal vagal ganglion, but was somewhat separated from the recurrent nerve. The right gland touched the recurrent nerve, the medial edge being frequently penetrated by the nerve, but the gland was separated from the vagal trunk. The left gland was innervated mainly by the branches from the distal vagal ganglion, whereas the right gland received mostly the branches from the recurrent nerve. The carotid body was located cranially near to the ultimobranchial gland. Large nerve bundles in the ultimobranchial gland ran toward and entered into the carotid body. By fluorescence microscopy, nerve fibers in ultimobranchial glands were observed associated with blood vessels. Only a few fluorescent nerve fibers were present in close proximity to C cell groups; the C cells of ultimobranchial glands may receive very few adrenergic sympathetic fibers. By electron microscopy, numerous axons ensheathed with Schwann cell cytoplasm were in close contact with the surfaces of C cells. In addition, naked axons regarded as axon terminals or "en passant" synapses came into direct contact with C cells. The morphology of these axon terminals and synaptic endings suggest that ultimobranchial C cells of chickens are supplied mainly with cholinergic efferent type fibers. In the region where large nerve bundles and complex ramifications of nerve fibers were present, Schwann cell perikarya investing the axons were closely juxtaposed with C cells; long cytoplasmic processes of Schwann cells encompassed large portions of the cell surface. All of these features suggest that C-cell activity, i.e., secretion of hormones and catecholamines, may be regulated by nerve stimuli.

Autonomic and sensory innervation of anuran ultimobranchial glands: a horseradish peroxidase study.

The extrinsic innervation of the endocrine ultimobranchial glands in the frog were identified by retrograde labeling with horseradish peroxidase. Labeled neurons were seen ipsilateral to the side injected in the caudal region of the vagal motor nucleus, the jugular sensory ganglion and first and second cervical sympathetic ganglia. These studies demonstrate a dual autonomic and vagal sensory innervation of the gland that are compatible with previous ultrastructural studies, but indicate a more complex innervation than previously shown.

Histology, innervation and histochemistry of the UB gland in the Mexican cave fish Anoptichthys jordani Hubbs et Innes (Teleostei: Characidae).

The histomorphological investigations carried out on the ultimobranchial body of the Teleost Anoptichthys jordani Hubbs and Innes have enabled us to establish that it is an epithelial multifollicular body which lies within the transverse septum, beneath the musculature of the oesophagus and caudal to the Sinus venosus. Its parenchyma is surrounded by an extensive nerve network revealed by the Bodian silver technique. This nerve network and the occurrence of MAO activity in the ultimobranchial tissue indicate that it possesses a sympathetic innervation. The follicular epithelium sometimes acquire a pseudostratified condition in some follicles and sometimes in multistratified in others. In this latter case the presence of two main types of cell was noticed (light cells and dark cells). In these cells by the fluorescence microscopic analysis was revealed an intensely green fluorescence due to the presence of an aromatic monoamine, dopamine. This conclusion was further confirmed by the agreement between the argyrophilic cells and the fluorescent cells after silver staining. The possible partecipation of dopamine in the elaboration of polypeptide factor (calcitonin) is suggested. A notable analogy from the structural point of view to the calcitonin-producing cells of higher Vertebrates is also confirmed by the markedly argyrophilia of the main cells.

Observations on the fine structure, enzyme histochemistry, and innervation of parathyroid gland and ultimobranchial body of Chthonerpeton indistinctum (Gymnophiona, Amphibia).

Fine structural and enzyme histochemical observations on ultimobranchial body and parathyroid gland of the caecilian Chthonerpeton are presented. The cell clusters and follicles of the ultimobranchial body consist mainly of granulated cells which are termed C-cells and obviously belong to the APUD cell series. In the larger follicles additional possibly exhausted degranulated cells and replacement cells occur. A rich supply of nerve fibres has been found in this gland. Frequently nerve terminals were observed to come into synaptic contact with the C-cells. Two categories of nerve fibres occur: a) fibres containing large polymorphic electron dense granules (probably purinergic fibres), b) fibres containing small electron transparent vesicles and a few electron dense granules (probably cholinergic fibres). The parathyroid gland consists of elongated cells (one cell type) poor in organelles and often containing fields of glycogen and lipid droplets. The cells are further characterized by fair amounts of lysosomal enzymes; they are interconnected by maculae adhaerentes and occludentes. No nerves and blood vessels have been found in the parathyroid gland of Chthonerpeton.