Developing Mouse Submandibular Gland Research Articles

Expression of Grhl2 and Its Target Gene Products in Developing Mouse Submandibular Gland

Expression of Grhl2 and Its Target Gene Products in Developing Mouse Submandibular Gland

Identification of genes encoding apical membrane proteins up-regultaed during lumen formation and of prominin-1 as a maker of the initial luminal cell surfaces in the developing mouse submandibular gland

Identification of genes encoding apical membrane proteins up-regultaed during lumen formation and of prominin-1 as a maker of the initial luminal cell surfaces in the developing mouse submandibular gland

Assembly of tight junction molecules in the developing mouse submandibular gland

Assembly of tight junction molecules in the developing mouse submandibular gland

Salivary gland organogenesis

Our understanding of vertebrate salivary gland organogenesis has been largely informed by the study of the developing mouse submandibular gland (SMG), which will be the major focus of this review. The mouse SMG has been historically used as a model system to study epithelial-mesenchymal interactions, growth factor-extracellular matrix (ECM) interactions, and branching morphogenesis. SMG organogenesis involves interactions between a variety of cell types and their stem/progenitor cells, including the epithelial, neuronal, and mesenchymal cells, and their ECM microenvironment, or niche. Here, we will review recent literature that provides conceptual advances in understanding the molecular mechanisms of salivary gland development. We will describe SMG organogenesis, introduce the model systems used to study development, and outline the key signaling pathways and cellular processes involved. We will also review recent research focusing on the identification of stem/progenitor cells in the SMG and how they are directed along a series of cell fate decisions to form a functional gland. The mechanisms that drive SMG organogenesis provide a template to regenerate functional salivary glands in patients who suffer from salivary hypofunction due to irreversible glandular damage after irradiation or removal of tumors. Additionally, these mechanisms may also control growth and development of other organ systems.

Histological and Ultrastructural Characterization of Developing Miniature Pig Salivary Glands

Salivary glands are a classic model of organ development and differentiation. Miniature pigs are considered as a unique animal model for salivary gland researchers in the fields of gene transfer, radiation damage, and functional reconstruction. However, there is little information about the development of miniature pig salivary glands. The present article was designed to study the developmental stages of salivary glands in miniature pigs using histological and ultrastructural methods. Sections from E40, E60, E80, E95 embryos, and P0 pups were stained with hematoxylin-eosin, Alcian blue, or periodic acid-schiff. Selected specimens were also processed for electron microscopy. The development of the miniature pig salivary glands can be divided into five different stages that refer to the stages of the developing mouse submandibular gland. The histological characteristics of the miniature pig salivary glands at different developmental stages were synchronously verified at the ultrastructural level. Interestingly, the development of the miniature pig parotid gland trailed that of the submandibular gland by approximately 15 days. Our study provides first-hand data regarding the morphological organogenesis of salivary glands in the miniature pig and provides a foundation for further research on this model.

Aquaporin 11 in the developing mouse submandibular gland

Several aquaporins (AQPs) have been detected in mature and embryonic mammalian salivary glands (AQP1 and AQP3-AQP8). However, AQP11 has, to our knowledge, never before been described in salivary glands, but is known to be important in, for example, kidney development in mice. We therefore thought it relevant to investigate if AQP11 was present during salivary organogenesis. The submandibular salivary gland (SMG) from CD1 mice was studied during prenatal development and early postnatal development, and also in young adult male and female mice. The expression trend of the AQP11 transcript was detected using the reverse transcription-polymerase chain reaction (RT-PCR), and the temporal-spatial pattern was observed using in situ hybridization. The AQP11 transcript was first detected at embryonic day 13.5 and showed a more or less constitutive expression trend during the prenatal and early postnatal SMG development. Spatial studies demonstrated that the AQP11 transcript was present in the developing and mature duct structures at all stages studied. In the end pieces, the AQP11 transcript was reduced during glandular development. Our results point to an important role for AQP11 during salivary gland development.

Tube formation in developing mouse submandibular gland

Tube formation in developing mouse submandibular gland

Temporary accumulation of glycogen in the epithelial cells of the developing mouse submandibular gland

Temporary accumulation of glycogen in the epithelial cells of the developing mouse submandibular gland was examined under light microscopic histochemistry and electron microscopy. To avoid loss of water-soluble glycogen during histological tissue preparation, fixation with ethanol and embedding in hydrophilic glycol methacrylate resin was used for light microscopy, and high-pressure freezing/freeze substitution for electron microscopy. Glycogen was detected on periodic acid-Schiff stain, periodic acid-thiosemicarbazide-silver proteinate reaction, and the digestion test with alpha-amylase. On embryonic day 14, glycogen began to accumulate in the proximal portions of the developing epithelial cords. On embryonic day 17, marked glycogen particles were seen at the basal portion of the ductal epithelial cells and an abrupt increase of glycogen accumulation occurred in the secretory cells in the terminal bulbs. Ultrastructural observation indicated large clumps of glycogen particles localized in the basal portion of the terminal bulb cells. The initiation of glycogen accumulation preceded the formation of lumens in the ducts and terminal bulbs. Furthermore, proliferation analysis by bromodeoxyuridine labeling showed that this glycogen accumulation followed the cessation of the epithelial cell proliferation. Postnatally, glycogen accumulation in the terminal bulbs became gradually inconspicuous and completely disappeared by postnatal day 3, but that in the ducts was retained until around postnatal day 12. Temporary glycogen accumulation after the cell proliferation and before/during the lumen formation and secretory granule formation suggests significant involvement of the carbohydrate metabolism in the organogenesis of the submandibular gland.

Hedgehog peptide promotes cell polarization and lumen formation in developing mouse submandibular gland

Tube formation of the developing mouse submandibular salivary gland (SMG) begins at embryonic day (E) 14. The SMG of Sonic hedgehog ( Shh) null mice was recently shown to fail to progress to stages beyond around E14. Here, we examined the effects of Shh peptide on tube formation of SMG explants. When the SMG rudiments from E14 mice were cultured, terminal buds of glands treated with Shh peptide formed the acini-like structure with a lumen whereas those of control glands remained as cell masses. In the acini-like terminal buds of the treated glands, tight junction proteins of ZO-1 and claudin-3 delineated the lumen and the apical membrane protein aquaporin-5 accumulated at the luminal cell surfaces. Moreover, laminin-5 deposition at the basal lamina region of terminal buds was accelerated in treated glands. It is suggested that hedgehog signaling promotes lumen formation and cell polarization of developing SMG epithelium.

Expression patterns of claudin family of tight junction membrane proteins in developing mouse submandibular gland.

Here, we investigated the expression of the claudin family of tight junction transmembrane proteins in the developing mouse submandibular gland. Data obtained by reverse transcriptase-polymerase chain reaction, Western blot, and immunofluorescence microscopy showed the expression and localization of claudin-3 to -8, -10, and -11 at epithelial tight junctions. Examination of the glands taken from embryonic day (E) 14, E16, and newborn mice revealed differential expression patterns of these claudins in the developing epithelium. Claudin-3, -5, and -7 were expressed in all of the luminal epithelial cells of the ducts at all of the developmental stages examined and in those of terminal tubules at E16 and later. Claudin-4 was expressed mainly in the ducts at all the developmental stages. The expression of claudin-6 and -8 was also restricted to the ducts at E14 and E16; but after birth, the former was undetectable, whereas the latter was expressed in both the ducts and terminal tubules. Claudin-10 and -11 were detectable mainly in the terminal tubules at E16 and later. In addition to being found in the epithelium, claudin-5 was also expressed in certain mesenchymal cells, probably endothelial cells. These results will provide a valuable resource for further investigation of tubulogenesis and physiological regulation of claudin-based tight junctions.

Acidic β-galactosidase in the developing mouse submandibular gland

Acidic β-galactosidase (β-gal) is expressed at high concentrations in the salivary glands, where its function is unknown but probably related to lysosomal catabolism of glycolipids or glycoproteins such as mucin. Here, the developmental pattern of the enzyme activity in the submandibular gland of B10A/SGSNJ mice was investigated in relation to the expression of mucin. Biochemical studies indicated that the activity of acidic β-gal increases significantly from birth to 3 weeks of age ( P<0.05). Histochemical staining of the submandibular glands of embryonic day 15 (E15), E18, 3-week-old and adult mice showed a trend of increasing staining intensity accompanied by a change in the cellular distribution of enzyme activity from E15 to adulthood. Acidic β-gal activity was first evident at E18, around the time mucin production starts, primarily in terminal-bud epithelial cells. The spatial and temporal coordination between the appearance of β-gal and embryonic mucin in the terminal buds of acinar cells might be related to a role of this enzyme in lysosomal degradation of embryonic mucin. In contrast, in adult mice, β-gal activity was primarily found in granular-duct cells. The function of β-gal in duct cells is unclear, but the change in the cellular distribution of β-gal in the mouse submandibular gland could reflect a change in the substrate specificity of this enzyme between E18 and adulthood.

Involvement of Heparin-Binding EGF-like Growth Factor and Its Processing by Metalloproteinases in Early Epithelial Morphogenesis of the Submandibular Gland

In the present study, the role of a member of the epidermal growth factor (EGF) family, heparin-binding EGF-like growth factor (HB-EGF), in organ development was investigated by using developing mouse submandibular gland (SMG), in which the EGF receptor signaling and heparan sulfate chains have been implicated. HB-EGF mRNA was detected in developing SMG by RT-PCR analysis and was expressed mainly in epithelium and weakly in mesenchyme of the embryonic SMG. Epithelial morphogenesis was inhibited by a synthetic peptide corresponding to the heparin-binding domain of HB-EGF and by anti-HB-EGF neutralizing antibody. An in vitro assay using an EGF receptor ligand-dependent cell line, EP170.7 cells, allowed us to detect the growth factor activity in SMG-conditioned media, which was significantly reduced by anti-HB-EGF antibody. Furthermore, treatment of SMG rudiments with the hydroxamate-based metalloproteinase inhibitor OSU8-1, which inhibits processing of EGFR ligands including HB-EGF, markedly diminished the growth factor activity in conditioned media and resulted in almost complete inhibition of SMG morphogenesis. The inhibitory effects on morphogenesis were reversed, though partially, by adding the soluble form of HB-EGF. Our results provide the first evidence that HB-EGF is a crucial regulator of epithelial morphogenesis during organ development, highlighting the importance of its processing by metalloproteinases.

Intracellular signalling cascades activated by the EGF receptor and/or by integrins, with potential relevance for branching morphogenesis of the fetal mouse submandibular gland.

The fetal mouse submandibular gland grown in vitro is a valuable model system to study epithelio-mesenchymal interactions vital for branching morphogenesis. This basic developmental process is dependent upon simultaneous engagement of receptors for growth factors and components of the extracellular matrix. Here we review intracellular signalling cascades mediated by activation of the EGFR in the developing mouse submandibular gland.

Distribution of alpha 6 integrin subunit in developing mouse submandibular gland.

We examined the development of mouse submandibular gland by light and electron microscopy and determined the distribution of the alpha 6 integrin subunit and laminin in this process by immunofluorescence and immunoelectron microscopy. At Days 13.5 and 14 of gestation alpha 6 was localized over the entire cell surface of undifferentiated epithelial cells of the terminal cluster. On Day 15 the expression of alpha 6 could no longer be detected over central cells in the proximal portion of branched terminal cluster, whereas peripheral cells were stained over the entire surface. On Day 17 of gestation to day of birth, alpha 6 expression was restricted to the basolateral surface of the differentiated acinotubular structure. Its expression on acinar cells was uniformly distributed throughout the basolateral membrane, but on duct cells stronger staining towards the basal surface was noted. Similar expression was observed in adult acinar and duct cells. Expression of the alpha 6-subunit at the cell-cell contact in an early stage and its expression at the basolateral surface in an advanced stage indicate that integrin containing alpha 6 plays a significant role in cell-cell and cell-substrate interaction. Stage and cell type-specific change of integrin expression may be significant for submandibular development.

Three-dimensional computer images of the alignment of collagen III fibrils at epithelial-mesenchymal interfaces of the developing mouse submandibular gland.

The three-dimensional computer images of the alignment of collagen III fibrils at epithelial-mesen-chymal interfaces during critical stages of submandibular gland morphogenesis in the 12- to 13-day-old mouse embryos were reconstructed from serial paraffin sections stained with an anti-collagen III antibody. The computer program used for processing section data was Cosmozone 2S, which required only a personal computer and a coordinate digitizer interfaced to the computer graphics system. The mid 12-day gland with a round lobule showed a rather randomly scattered distribution of the collagen on the surfaces of the lobule and stalk. The interface at the joining portion of the lobule and stalk showed a significant accumula-tion. In the late 12-day gland with signs of cleft initiation, a specific accumulation of the collagen was clearly seen on basal epithelial surface in shallow clefts. This tendency was much more evident as cleft formation proceeded. The images reconstructed from both frontal and transverse section data of the 13-day gland indicated that epithelial surface of the cleft was surrounded by continuous collagen bundles, suggesting the involvement of the collagen during the epithelial branching.

Immunocytochemical localization of renin in the submandibular gland of the mouse during postnatal development

The localization of renin in the developing mouse submandibular gland was studied immunocytochemically using the unlabelled antibody-enzyme method of Sternberger ('74). Bouin-fixed submandibular glands of mice of both sexes were examined at 5-day-intervals from birth (day 0) to 50 days of age. At all stages studied, only granular convoluted tubule (GCT) cells stained immunocytochemically for renin; such cells were first seen in glands of 30-day-old males and of 30-day-old females. The size and number of renin-containing GCT cells increased rapidly in males, attaining adult status by 50 days of age. In females, differentiation of GCT cells immunoreactive for renin was slower and less regular than in males, and at 50 days of age the GCT segment had not yet reached adult conditions with respect to the distribution of renin. Renin appears in GCT cells at later ages than other GCT cell products (e.g., EGF and amylase), suggesting the existence of independent developmental control for the expression of various biologically active substances in the GCTs.

Target organ stimulation of parasympathetic nerve growth in the developing mouse submandibular gland

Parasympathetic nerve growth from the mouse submandibular ganglion is stimulated and directed by the glandular epithelium. Cultured alone, the submandibular ganglion shows little axon extension, but in the presence of salivary epithelium, either cisfilter or transfilter, stimulation of axon outgrowth occurs. The capacity to stimulate such outgrowth from the ganglion is restricted, but not completely specific to salivary epithelium. Stimulation of directed outgrowth occurs even through a 0.1 μm pore size filter and over distances of up to 0.5 mm. Preliminary studies with the parasympathetic ganglia of the pelvic plexus show that axon outgrowth in this case is dependent on a target issue, with salivary epithelium being capable of directing nerve outgrowth from this source.