Development of human minor salivary glands: expression of mucins according to stage of morphogenesis.

The formation of salivary glands entails the proliferation of epithelial cells from the stomatodeum into the underlying ectomesenchyme, culminating in a complex network of ducts and acinar bulbs. The extent to which mucins regulate this process is unknown, but they appear to mediate luminal space formation and maturation. Our aim was to examine mucin expression patterns during the morphogenesis of human salivary glands. Mucin expression - MUC1, 2, 3, 4, 5AC, 5B, 6, and 16 - was analyzed in specimens of developing human salivary glands, obtained from fetuses at 4-24 weeks' gestation, and fully developed salivary glands by immunohistochemistry. Expression patterns were analyzed qualitatively according to the development stage of the salivary glands. Mucins 1, 3, 4, 5B, and 16 were expressed during salivary gland development - being stronger in all ductal segments by the final phases of branching morphogenesis and in mature glands. Acinar cells were negative for most mucins, including MUC1 in mature salivary glands. Mucins 2, 5AC, and 6 were not expressed. Mucins MUC1, 3, 4, 5B, and 16 are expressed in developing human salivary glands and in mature glands, suggesting important roles in the maturation and maintenance of the ductal network.

Defects and rescue of the minor salivary glands in Eda pathway mutants.

Despite their importance to oral health, the mechanisms of minor salivary gland (SG) development are largely unexplored. Here we present in vivo and in vitro analyses of developing minor SGs in wild type and mutant mice. Eda, Shh and Fgf signalling pathway genes are expressed in these glands from an early stage of development. Developing minor SGs are absent in Eda pathway mutant embryos, and these mice exhibit a dysplastic circumvallate papilla with disrupted Shh expression. Supplementation of Eda pathway mutant minor SG explants with recombinant EDA rescues minor SG induction. Supplementation with Fgf8 or Shh, previously reported targets of Eda signalling, leads to induction of gland like structures in a few cases, but these fail to develop into minor SGs.

Developing human minor salivary glands: morphological parallel relation between the expression of TGF-beta isoforms and cytoskeletal markers of glandular maturation.

Morphogenesis of salivary glands involves complex coordinated events. Synchronisation between cell proliferation, polarisation and differentiation, which are dependent on epithelial-mesenchymal interactions and on the microenvironment, is a requirement. Growth factors mediate many of these orchestrated biological processes and transforming growth factor-beta (TGF-beta) appear to be relevant. Using immunohistochemistry and immunofluorescence, we have mapped the distribution of TGF-beta 1, 2 and 3 and compared it with the expression of maturation markers in human salivary glands obtained from foetuses ranging from weeks 4 to 24 of gestation. TGF-beta 1 first appeared during canalisation stage in the surrounding mesenchyme and, in the more differentiated stages, was expressed in the cytoplasm of acinar cells throughout the adult gland. TGF-beta 2 was detected since the bud stage of the salivary gland. Its expression was observed in ductal cells and increased along gland differentiation, TGF-beta 3 was detected from the canalisation stage of the salivary gland, being weakly expressed on ductal cells, and it was the only factor detected on myoepithelial cells. The data suggest that TGF-beta have a role to play in salivary gland development and differentiation.

Association of Shh and Ptc with keratin localization in the initiation of the formation of circumvallate papilla and von Ebner's gland.

The development of gustatory papillae in mammalian embryos requires the coordination of a series of morphological events, such as proliferation, differentiation and innervation. In mice, the circumvallate papilla (CVP) is a specialized structure that develops in a characteristic spatial and temporal pattern in the posterior region of the tongue dorsal surface. The distinct expression patterns of Shh and Ptc, which play important roles in the development of other epithelial appendages, have been localized in the trench wall that gives rise to von Ebner's gland (VEG). To define the cellular mechanisms responsible for morphogenesis and differentiation during early development of CVP and VEG, the localization patterns of keratins (cytokeratins) K7, K8, K18, K19, K14 and connexin-43, which are dependent on Shh expression in other developmental systems, have been examined in detail. The distinct localization of keratins K7, K8, K18, K19, K14 and connexin-43 in the epithelium giving rise to the CVP and VEG suggests that cytodifferentiation is established prior to morphological changes. Interestingly, the localization of proliferating cell nuclear antigen, a marker for cell proliferation, is similar to that of Shh. An understanding of the regulatory roles of cell-cell interactions and signalling molecules in orchestrating a mutual network will bring us nearer to defining the molecular and cellular mechanisms underlying morphogenesis in mammalian taste bud development.

Immunoexpression of extracellular matrix proteins in human salivary gland development.

Immunoexpression of the extracellular matrix (ECM) proteins laminin, fibronectin, tenascin and types I, III and IV collagen was analyzed in the major and minor salivary glands of seven human fetuses at different gestational ages. The results showed the presence and localization of laminin, collagen IV and fibronectin around glandular structures at all stages of development. Tenascin was only detectable around excretory ducts. In the earliest stages of development, type I and type III collagen were presented as fine fibers delineating the glandular structures and delimiting the extension of the future lobule. As glandular development proceeded, the lobule was gradually filled with collagens and glandular tissue.

Prenatal development of the palatine gland of rats.

The present study aimed to elucidate the prenatal development of the rat palatine gland. Parasagittal 5 microm thick serial sections made from Wistar rats at embryonic days (E) 17 to 22 were stained with haematoxylin-eosin (HE), Alcian blue-Kernechtrot or immunohistochemistry for 5-bromo-2'-deoxyuridine (BrdU) as a marker of proliferating cells. Additionally, three-dimensional images of developing glandular parenchyma were reconstructed from serial HE sections with a personal computer. At E 17, several thickenings of the palatal epithelium had appeared which thereafter became the epithelial cords. Branching and lumenization commenced at E 20, and immature acini were observed at E 21. Three-dimensional reconstruction showed that the proximal part of the epithelial cord differentiated into the duct, and the distal part of the epithelial cord differentiated into the acinus. In immunohistochemical staining, there were many BrdU-positive cells in the epithelial cords including thickenings of the palatal epithelium, ducts, and acini. The BrdU labeling index of the cells of the epithelial cord was the highest (statistically significant) of the three in the primitive palatine gland. In conclusion, during the development of the rat palatine gland, epithelial cords with very high proliferative activity arise from the palatal epithelium, and then the proximal part of the epithelial cord differentiates into the duct, and the distal part of the epithelial cord differentiates into the acinus. Proliferation of these glandular parenchyma contributes to the growth of the developing palatine gland.

Expression of cytoskeletal proteins in developing human minor salivary glands.

The presence of an epithelium at different stages of proliferation and differentiation raises interesting questions concerning the histogenesis, cell turnover and differentiation of normal salivary glands. In order to expand knowledge of these aspects, we investigated the expression of cytokeratins (CKs) 7,8,10,13,14,16,18 and 19, vimentin (VIM), and smooth muscle actin (SMA) in developing human minor salivary glands using monoclonal antibodies. Labial, buccal, palatine, and lingual salivary glands and those from the floor of the mouth were obtained from human fetuses (forensic postmortem) ranging in age from gestational weeks 10 to 29. Serial sections, 3 microm thick, were immunostained using a strepto-avidin-biotin technique. Reactivity for all antibodies was negative in the salivary gland epithelium during the developmental stages of bud formation, cord growth, and branching of cord. During canalization and cytodifferentiation, the glandular epithelial cells showed a positive reaction to some CKs and SMA. Cytokeratins 7, 8, 18, and 19 showed strong labeling in luminal duct cells that exhibited some degree of morphological differentiation. Myoepithelial cellc were recognized by antibodies to SMA. Cytoskeletal protein expression changes according to the cell type, degree of differentiation, and stage of morphological development of the glandular structure. These changes occur independently of the localization of the gland.

Postnatal growth of the rat palatine gland.

To elucidate how the palatine glands grow postnatally, the palatine glands of rats from 0 to 8 weeks of age were investigated histologically and immunohistochemically. Under light microscope, three dimensions of the right part of the palatine glands were measured and the total number of excretory ducts of the glands was counted from the parasagittal serial sections. Immunohistochemistry with anti-5-bromo-2'-deoxyuridine (BrdU) monoclonal antibody was also employed to detect the cellular proliferative activity. At birth (0 weeks), the palatine glands consisted of ducts and immature acini. The ducts in the glands were connected with excretory ducts. After 2 weeks, there was no duct in the glands. Most acinar cells became mature as mucous cells and took the form of tubulo-acini connected directly with excretory ducts. In the posterior region of the glands, serous acinar cells forming demilunes were occasionally seen. All three dimensions of the palatine glands became longer, and the number of excretory ducts tended to increase. Immunohistochemistry showed acinar and duct cells were highly proliferative in early stage of postnatal life and their proliferative activity decreased thereafter. This study demonstrated that immature rat palatine glands of newborn rats grow three-dimensionally during maturation, and that the parenchymal cell proliferation contributes to the growth of the rat palatine glands. In addition, it is suggested that the glandular tissue arises from the excretory ducts formed postnatally.

Histochemical study of lectin binding in the human fetal minor salivary glands.

The emerging synthesis of glycoconjugates containing specific oligosaccharides in developing human fetal labial and lingual salivary glands has been investigated by lectin histochemistry. An avidin-biotin technique was used to study the binding of lectins from Ulex europeus I (UEA-I), Dolichos biflorus (DBA), Glycine maximus (SBA), Helix pomatia (HPA), Arachis hypogaea (PNA) and Triticum vulgare (WGA) to specific sugars on sections of tissue from labial glands, glands of Blandin and Nuhn, glands of von Ebner and the dorsoposterior lingual salivary glands. Incipient synthesis of glycoconjugates in early glands and their presence in the cells and ducts of the later glands was shown. The study also showed a time-related increase in both staining intensity and binding sites of serous acinar cells from all glands and for all lectins used. For mucous cells, peak intensity of staining was reached by the middle phase of development. During later gland development this intensity was maintained in dorsoposterior lingual glands but tended to decline in labial glands. The various lectins showed different degrees of binding but UEA-I lectin generally bound the L-fucose sugar group in all salivary glands at all gestational ages. The results showed that lectins appear to bind to the oligosaccharides on epithelial cell surfaces of fetal salivary glands at all stages of development. The degree of change depends upon the stage of differentiation and maturation of the glands.

Stereological and immunohistochemical study of development of human fetal labial salivary glands and their S-100 protein reactivity.

Stereological and certain histochemical aspects of fetal growth and development of human labial salivary glands are reported. Stereological analysis showed a highly significant progressive increase in proportional gland volume occupied by acini from 27% at 20 weeks to 56% at 38 weeks (P < 0.0001), and a comparable having of the relative gland volume occupied by connective tissue in the same period (P < 0.0001). Linear regression fitted the data well (r2 = 0.59 and 0.47 respectively, n = 46). The change in relative volume occupied by ducts or by vascular tissue was small and did not reach significance. S-100 protein reactivity was demonstrated in the cytoplasm of cells of the labial gland primordia from their origin. As gland differentiation progressed, the S-100 reactivity became localized in basophil acinar cells and in proximal (intercalated and intralobular), but not in distal, duct cells. A gradual increase in intensity of S-100 protein activity at these sites during salivary gland development was observed. Morphological maturity seems to be complete before 29 weeks but myoepithelial cells could not be identified with certainty.

Incidence and histology of human accessory parotid glands.

The parotid glands of 228 Japanese human cadavers were examined to determine the incidence and histological features of accessory parotid glands. The incidence was found to be 56% with no differences between right and left sides or between sexes. Thirty parotid glands and their associated accessory glands were examined histologically: eight of these accessory glands were found to be mixed secretory glands (i.e., containing both serous and mucous acini). Thus, the pattern of differentiation of a significant fraction of accessory glands differs from that of the main parotid gland: it appears that mixed acini, present in the early stages of development, persist into later life, and their presence may be related to tumors developing at these sites.

Prenatal development of human palatine glands: a structural and cytochemical study.

The histogenesis of the salivary glands was structurally and cytochemically studied in human embryos and fetuses during the period of 8 to 32 weeks of intrauterine life. Glandular buds appeared at about 12 weeks of embryonic development. The rounded distal ends of the epithelial cords and neighbouring mesenchyma showed small and abundant PAS positive and alcianophilic granules. At age 14 weeks the secretory end pieces and the duct system were seen at different morphologic and structural stages of a differentiation. Mucous acini with scanty mixed acini predominated and serous acini appeared occasionally. From 20 to 24 weeks the mucous acini stained with toluidine blue featured different degrees of metachromasia even in the case of cells of the same acinus. In the ducts it was also possible to identify metachromatic cells intermingled with basophilic cells in the epithelial coat. These findings suggest that the palatine glands present typical histophysiological material from 14 to 20 weeks. The presence of PAS positive, alcianophilic and metachromatic secretory substance in the acinar lumen and the luminal content of ducts suggests that mucin secretion begins during intrauterine life.

Postnatal development of von Ebner's glands: accumulation of a protein of the lipocalin superfamily in taste papillae of rat tongue.

Antibodies produced against rat von Ebner's gland (VEG) protein, a recently characterized member of a lipophilic ligand carrier protein family, detect this protein immunocytochemically in von Ebner's gland acini and show that it is present at high concentrations in the clefts of circumvallate and foliate papillae. During embryonic development, von Ebner's gland anlagen are innervated (as shown immunocytochemically using neuronal specific antibodies) as early as embryonic day 20, before lateral glandular outgrowth and VEG protein can be observed. Expression of the VEG protein as determined by in situ hybridization and immunocytochemistry begins at postnatal day-2 cells in differentiating and branching off from von Ebner's gland ducts, and sharply increases with further enlargement and maturation of the gland. The close temporal correlation of von Ebner's gland innervation and VEG protein expression with papilla innervation and taste-bud development suggests a functional relationship of both structures. VEG protein might control access of lipophilic sapid molecules, such as bitter substances, to the gustatory receptors.

Embryology and secretory activity of labial salivary glands.

This study was carried out on the heads of 20 foetuses aged between 11 and 26 weeks. Four stages were distinguished in the development of labial salivary glands, each stage corresponding to gestational age. Stage I is characterized by a localized, rounded thickening of the stomodial epiblast into the mesenchyma of the mucosal side of the lower lip. This occurs at the 9th-10th week of gestation. Stage II is reached when the epiblastic thickening assumes the form of a single cord. It is oval when sectioned transversally and is the result of the proliferation of epiblastic cells into the underlying mesenchyma during the 11th-12th week. Stage III corresponds to a branching process which takes place at the 13th week. The single cord branches to form the future secretory lobes, which rapidly assume a grape-like appearance. The number of rudimentary glands increases during the first three stages of development, i.e. as long as the first formed glands have not developed ducts. Stage IV corresponds to the process of duct formation at the 18th week. Simultaneous differentiation of acinal and duct cells is observed and this procedes the onset of secretory activity.

Cytochemical variations of human von Ebner's glands.

Human von Ebner's glands were analyzed during fetal and childhood development, and adultness, with the purpose of observing possible age-related cytochemical changes. Tongues from 8-38 weeks-old fetuses, samples of the lingual V from newborns, 8 to 14 years-old children and adults aged 20 years and older were used. H/E, and techniques for mucosubstances (PAS, PAS/amylase, PAS/sialidase, methenamine-silver, Alcian blue at pH 2.5 and 1.0, and Toluidine blue at pH 3.8) were employed. Between 16 and 20 weeks acini and ducts in the process of formation were identified, being the parenchyma completely developed at 24 weeks. The morphology of glands from newborns consisted of basophilic and periodate-negative serous acini. In infants secretory cells contained PAS positive apical granules that were sensitive to sialidase, periodate-reactive and slightly metachromatic. These characteristics were increased with age. Besides, in adults, the apical cytoplasm of the adenomerous and the luminal content were also alcianophilic. The cytochemical variations observed indicates that human von. Ebner's glands are composed of cells of the seromucous type that contain sialomucins and sulphomucins.

[Morphohistochemical development of mesenchyme in human fetal labial glands]. / Evolución morfohistoquímica del mesénquima en glándulas labiales fetales humanas.

The structural and histochemical patterns of the salivary mesenchyma were analysed in relation to the epithelium of the labial glands during the embryonic development to correlate the structural and histochemical characteristics in both tissues during the histogenesis. Samples of human fetal lips were analysed using H/E, PAS, Cason, Alcian blue, Toluidine blue and Methenamine/silver. The process of glandular histogenesis begun between 8 to 10 weeks. The mesenchyma surrounding the glandular buds had PAS positive granulations which were also alcianophilic, metachromatic and periodatoreactive. Periodatoreactive collagenous fibrillae, reticular cells and nervous fibers of considerable diameter were observed. Basement membranes were PAS positive, alcianophilic and discontinuous. At 12 weeks the mesenchyma differentiated as loose connective tissue which produced a delicate periglandular capsule with fibroblast and collagenous fibrillae. From 20 to 24 weeks the acini were structurally and histochemically differentiated as serous, mucous and mixed. It was postulated that the periglandular mesenchyma would play and important role in the morphogenetic process in relation to the histochemical identification of molecules which have a specific biological functions in the epithelium-mesenchyma interactions during organogenesis.