Collagenase inhibitor stimulates cleft formation during early morphogenesis of mouse salivary gland

A collagenase inhibitor obtained from the culture medium of bovine dental pulp markedly enhanced the cleft formation of mouse embryonic salivary gland epithelium when the inhibitor was included in the culture medium for 12-day and 13-day salivary glands. Determination of collagenase activity using [3H]collagen as substrate indicated that there was a latent collagenase activity in 12-day glands. In addition, a highly purified Clostridial collagenase freed from protease and hyaluronidase activities, strongly inhibited initiation of the cleft formation of the 12-day epithelium. Scanning electron microscopic observation showed that abundant collagen-like fibrils were seen on the epithelium in the collagenase-inhibitor-treated glands compared to those in the control. These findings suggest that during early morphogenesis tissue collagenase may regulate the cleft formation in the epithelium.     

Cell proliferation is not required for the initiation of early cleft formation in mouse embryonic submandibular epithelium in vitro

An X-ray irradiation method was employed to analyse the role of cell proliferation in vitro in the cleft formation of mouse embryonic submandibular epithelium at early stages. When the mid 12-day gland was exposed to 200 rad of X-rays, the growth was severely retarded. In contrast, late 12-day and early 13-day glands grew apparently in a normal fashion, as did the control gland, for up to 40 h. In either case, they formed shallow clefts within 10 h of culture. With 1000 rad irradiation, the mid 12-day gland did not grow at all, but formed clefts within 20 h of culture followed by a rapid degeneration. Under the same conditions, the growth of the late 12-day gland, which was at the stage just before branching, was retarded until 10 h of culture, followed by a slight increase in epithelial size, but cleft formation was also observed within 6-10 h, as in the control gland. When exposed to a dose of 1000 rad of X-rays, the early 13-day and the late 12-day glands exhibited similar radiosensitivity; the initial narrow clefts in the epithelium deepened and new clefts began to form within 6-10 h of culture. [3H]thymidine incorporation studies revealed that a dose of 1000 rad reduced DNA synthesis of mid and late 12-day glands by 72 and 65%, respectively. Histological examination of X-irradiated late 12-day gland showed that mitotic figures were rarely seen in the epithelium at 6 h of culture. Aphidicolin, a specific inhibitor of DNA synthesis, could not halt the cleft formation of the late 12-day gland. In this experiment 89% of DNA synthesis was inhibited. Treatment of an X-ray irradiated late 12-day gland with aphidicolin blocked 92% of the DNA synthesis, but did not prevent cleft formation taking place. These results indicate that neither cell division nor DNA synthesis, is required for the initiation process of the cleft formation of the mouse embryonic submandibular epithelium at early morphogenetic stages in vitro.     

Developmental expression of survivin during embryonic submandibular salivary gland development

Background  

The regulation of programmed cell death is critical to developmental homeostasis and normal morphogenesis of embryonic tissues. Survivin, a member of the inhibitors of apoptosis protein (IAP) family primarily expressed in embryonic cells, is both an anti-apoptosis and a pro-survival factor. Since our previous studies have demonstrated the importance of apoptosis during embryonic submandibular salivary gland (SMG) development, we postulated that survivin is a likely mediator of SMG epithelial cell survival.     

Acinar ultrastructure of the submandibular gland of Mus musculus during embryonic development

Summary The development of the submandibular gland of the mouse was studied by means of electron microscopy, from the 14th day of gestation up to birth. In the first two days the acini are solid and their cells contain polyribosomes and a few lamellae of endoplasmic reticulum. Beginning with the 16th day secretory granules appear and rapidly fill an increasing number of cells. The different electron density of the granules makes it possible to distinguish 1. two types of granules, dense and pale, and sometimes intermediate ones, 2. polymorphic granules. The latter consists of electron dense and electron pale parts combined in different configurations. The possible significance of the various types of granules is discussed.     

Epithelial shape change in mouse embryonic submandibular gland: modulation by extracellular matrix components

Early morphogenesis of mouse submandibular gland provides an excellent model for the formation of epithelial lobules as a consequence of epithelial-mesenchymal interactions. Both proteoglycans and a glycosaminoglycan, high molecular weight components which contain amino-sugars and hexuronic acids, seem to be important in maintaining the lobular structure through the formation of epithelial basal lamina. Collagen also appears to play a crucial role in this morphogenesis. By visualizing the distribution of collagen fibrils and by changing the concentration of collagen in the gland, we have developed a new hypothesis which emphasizes the mechanical role of mesenchyme in epithelial cleft formation. Precise mechanisms for the involvement of these molecules have not been elucidated, yet it is now clear that knowledge of the function of the extracellular matrix components is a prerequisite for understanding the epithelial-mesenchymal interactions.     

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.     

Sulpholipid formation in rat submandibular gland.

Comparisons of nuclease digestions of chromatin in nuclei from rye embryos and rat liver show that their nucleosomal DNA is similar, i.e. DNA subunits consist of 200 base-pair repeats with 140 base-pair cores of identical substructure. The identical nucleosome structure is present in nuclei from cells of rye embryos that have been non-viable (i.e. dead) for more than 7 years. These findings indicate a high degree of stability of the DNA-histone complex and are consistent with conservation of the nucleosomal structure of chromatin during evolution.     

Smad1 expression and function during mouse embryonic lung branching morphogenesis

Bone morphogenetic protein (BMP) 4 plays very important roles in regulating developmental processes of many organs, including lung. Smad1 is one of the BMP receptor downstream signaling proteins that transduce BMP4 ligand signaling from cell surface to nucleus. The dynamic expression patterns of Smad1 in embryonic mouse lungs were examined using immunohistochemistry. Smad1 protein was predominantly detected in peripheral airway epithelial cells of early embryonic lung tissue [embryonic day 12.5 (E12.5)], whereas Smad1 protein expression in mesenchymal cells increased during mid-late gestation. Many Smad1-positive mesenchymal cells were localized adjacent to large airway epithelial cells and endothelial cells of blood vessels, which colocalized with a molecular marker of smooth muscle cells (alpha-smooth muscle actin). The biological function of Smad1 in early lung branching morphogenesis was then studied in our established E11.5 lung explant culture model. Reduction of endogenous Smad1 expression was achieved by adding a Smad1-specific antisense DNA oligonucleotide, causing approximately 20% reduction of lung epithelial branching. Furthermore, airway epithelial cell proliferation and differentiation were also inhibited when endogenous Smad1 expression was knocked down. Therefore, these data indicate that Smad1, acting as an intracellular BMP signaling pathway component, positively regulates early mouse embryonic lung branching morphogenesis.     

Localization of AQP5 during development of the mouse submandibular salivary gland

Aquaporin 5 (AQP5) is known to be central for salivary fluid secretion. A study of the temporal-spatial distribution of AQP5 during submandibular gland (SMG) development and in adult tissues might offer further clues to its unknown role during development. In the present work, SMGs from embryonic day (E) 14.5–18.5 and postnatal days (P) 0, 2, 5, 25, and 60 were immunostained for AQP5 and analyzed using light microscopy. Additional confocal and transmission electron microscopy were performed on P60 glands. Our results show that AQP5 expression first occurs in a scattered pattern in the late canalicular stage and becomes more prominent and organized in the terminal tubuli/pro-acinar cells towards birth. Additional apical membrane staining in the entire intralobular duct is found just prior to birth. During postnatal development, AQP5 is expressed in both the luminal and lateral membrane of pro-acinar/acinar cells. AQP5 is also detected in the basal membrane of acinar cells at P25 and P60. In the intercalated ducts at P60, the male glands show apical staining in the entire segment, while only the proximal region is positive in the female glands. These results demonstrate an evolving distribution of AQP5 during pre- and postnatal development in the mouse SMGs.     

The postnatal development of the submandibular gland of the mouse

Summary The postnatal development of the submandibular gland was investigated in male mice of the Swiss-Webster strain, which were killed at 1, 2, 3, 4, 5, 6, 8, 10, 12, 16 and 20 weeks of age, while the older mice had been weaned at 3 weeks of age. The mean weight of the submandibular gland increases from 9.5 mg at 1 week to 232.9 mg at 20 weeks of age, and the rate of increase is rapid between 3 and 10 weeks of age. The gland's contents of DNA, RNA and protein increase in a similar manner.The changes in the constituent cell types of the gland were studied in radioautographs prepared from Epon-embedded sections of mice given ³H-thymidine and stained with toluidine blue. At 1 week of age, the gland consists of acinar cells (36%), intercalated duct cells (26%), juxta-acinar cells (13%), striated duct cells (12%) and others. The cellular composition of the gland changes little before weaning, but the absolute number of all types of cells increases with age. Between 3 and 4 weeks, juxta-acinar cells disappear and granular convoluted tubule cells appear and increase rapidly in number with age. The rapid expansion of the population size of granular convoluted tubule cells after weaning coincides with the second peak of increased proliferative activity of intercalated duct cells, whereas all the other cell types show a progressive decrease in their proliferative activity with age. In spite of the burst in proliferative activity, there is no corresponding increase in the absolute number of intercalated duct cells. The number of striated duct cells peak at 5 weeks of age and then declines. These findings indicate that the mitoses of intercalated duct cells give rise to granular convoluted tubule cells through a stage of striated duct cells. At 20 weeks of age, the gland consists of granular convoluted tubule cells (47%), acinar cells (28%), intercalated duct cells (12%), striated duct cells (1%) and others.Supported by Public Health Service Research Grant AMDE 19753 from the National Institute of Health. The authors are indebted to Mr. I. Borcsanyi for technical assistance     

Postnatal development of trypsin-like esteroproteases in mouse submandibular gland

The development of trypsin-like esteroproteases in the submandibular gland of mice was studied, using a newly synthesized naphthyl ester (tosyl-L-lysine alpha-naphthyl ester) for the preparation of zymograms and for histochemical demonstration of the enzyme. Esteroprotease activities were first detected spectrophotometrically on day 15 after birth; then increased markedly after day 20. A sex difference in esteroprotease activity appeared on day 25. Zymograms prepared after isoelectric focusing in polyacrylamide slab gels showed that the glands of neonatal mice contained esteroproteases with a rather different composition from that of adult mice. The adult type isozymes appeared first on day 15, and their activities increased markedly after day 20. Histochemical studies revealed that the isozymes of neonatal mice were derived from mast cells. A few striated ducts were first stained on day 15, and the sex difference of the granular tubules became obvious on day 25. These results indicate that the development of trypsin-like esteroproteases faithfully reflects the differentiation of granular tubules in the mouse submandibular gland, except in the neonatal period.     

Postnatal development of trypsin-like esteroproteases in mouse submandibular gland

Summary The development of trypsin-like esteroproteases in the submandibular gland of mice was studied, using a newly synthesized napthyl ester (tosyl-l-lysine -naphthyl ester) for the preparation of zymograms and for histochemical demonstration of the enzyme. Esteroprotease activities were first detected spectrophotometrically on day 15 after birth; then increased markedly after day 20. A sex difference in esteroprotease activity appeared on day 25. Zymograms prepared after isoelectric focusing in polyacrylamide slab gels showed that the glands of neonatal mice contained esteroproteases with a rather different composition from that of adult mice. The adult type isozymes appeared first on day 15, and their activities increased markedly after day 20. Histochemical studies revealed that the isozymes of neonatal mice were derived from mast cells. A few striated ducts were first stained on day 15, and the sex difference of the granular tubules became obvious on day 25. These results indicate that the development of trypsin-like esteroproteases faithfully reflects the differentiation of granular tubules in the mouse submandibular gland, except in the neonatal period.This work was supported in part by Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan     

Development of secretory units of mouse submandibular gland

Summary The fine structure of the secretory units of the mouse submandibular gland was studied according to the developmental sequence. The embryonic submandibular gland consists of terminal tubules and ducts. Myoepithelium is associated only with the terminal tubules, and the cells of the primary intercalated ducts show characteristics of the young striated duct cells. The major changes shortly after birth consist of: 1) opening of the secretory lumina, 2) increasing rough ER and its altered configuration, 3) dilatation of Golgi cisternae and 4) changes in the granular structure. These findings suggest that the salivary secretion first occurs after birth, and acinar differentiation or transformation of the secretory cells of the terminal tubules is induced and profoundly affected by the commencement of the secretory activity. In the intercalated ducts this process is somehow inhibited, and the granular cells found in the adult can be considered as the remnants of the secretory cells of the terminal tubules.     

Role of PI 3-kinase and PIP3 in submandibular gland branching morphogenesis

The mouse submandibular gland (SMG) epithelium undergoes extensive morphogenetic branching during embryonic development as the first step in the establishment of its glandular structure. However, the specific signaling pathways required for SMG branching morphogenesis are not well understood. Using E13 mouse SMG organ cultures, we showed that inhibitors of phosphatidylinositol 3-kinase (PI 3-kinase), wortmannin and LY294002, substantially inhibited branching morphogenesis in SMG. Branching morphogenesis of epithelial rudiments denuded of mesenchyme was inhibited similarly, indicating that PI 3-kinase inhibitors act directly on the epithelium. Immunostaining and Western analysis demonstrated that the p85 isoform of PI 3-kinase is expressed in epithelium at levels higher than in the mesenchyme. A target of PI 3-kinase, Akt/protein kinase B (PKB), showed decreased phosphorylation at Ser(473) by Western analysis in the presence of PI 3-kinase inhibitors. The major lipid product of PI 3-kinase, phosphatidylinositol 3,4,5-trisphosphate (PIP(3)), was added exogenously to SMG via a membrane-transporting carrier in the presence of PI 3-kinase inhibitors and was found to stimulate cleft formation, the first step of branching morphogenesis. Together, these data indicate that PI 3-kinase plays a role in the regulation of epithelial branching morphogenesis in mouse SMG acting through a PIP(3) pathway.