Dual Sympathetic Input into Developing Salivary Glands.

Neuronal signaling is known to be required for salivary gland development, with parasympathetic nerves interacting with the surrounding tissues from early stages to maintain a progenitor cell population and control morphogenesis. In contrast, postganglionic sympathetic nerves arrive late in salivary gland development to perform a secretory function; however, no previous report has shown their role during development. Here, we show that a subset of neuronal cells within the parasympathetic submandibular ganglion (PSG) express the catecholaminergic marker tyrosine hydroxylase (TH) in developing murine and human submandibular glands. This sympathetic phenotype coincided with the expression of transcription factor Hand2 within the PSG from the bud stage (E12.5) of mouse embryonic salivary gland development. Hand2 was previously associated with the decision of neural crest cells to become sympathetic in other systems, suggesting a role in controlling neuronal fate in the salivary gland. The PSG therefore provides a population of TH-expressing neurons prior to the arrival of the postganglionic sympathetic axons from the superior cervical ganglion at E15.5. In culture, in the absence of nerves from the superior cervical ganglion, these PSG-derived TH neurons were clearly evident forming a network around the gland. Chemical ablation of dopamine receptors in explant culture with the neurotoxin 6-hydroxydopamine at early stages of gland development resulted in specific loss of the TH-positive neurons from the PSG, and subsequent branching was inhibited. Taken altogether, these results highlight for the first time the detailed developmental time course of TH-expressing neurons during murine salivary gland development and suggest a role for these neurons in branching morphogenesis.

Apoptosis in Early Salivary Gland Duct Morphogenesis and Lumen Formation.

Salivary glands are essential for the maintenance of oral health by providing lubrication and antimicrobial protection to the mucosal and tooth surfaces. Saliva is modified and delivered to the oral cavity by a complex multifunctional ductal system. During development, these ducts form as solid tubes, which undergo cavitation to create lumens. Apoptosis has been suggested to play a role in this cavitation process along with changes in cell polarity. Here, we show that apoptosis occurs from the very earliest stages of mouse salivary gland development, much earlier than previously reported. Apoptotic cells were observed in the center of the first epithelial stalk at early-stage embryonic day 12.5 (E12.5) according to both TUNEL staining and cleaved caspase 3 immunofluorescence. The presumptive lumen space was highlighted by the colocalization of a predictive lumen marker, cytokeratin 7. At E14.5, as lumens start to form throughout the glands, apoptotic expression decreased while cytokeratin 7 remained positive. In vitro inhibition of all caspases in E12.5 and E13.5 salivary glands resulted in wider ducts, as compared with the controls, and a defect in lumen formation. In contrast, no such defect in lumen formation was observed at E14.5. Our data indicate that apoptosis is involved during early stages of gland formation (E12.5 onward) and appears important for shaping the forming ducts.

Distribution of small Rho GTPases in the developing rat submandibular gland.

During the rat submandibular gland (SMG) development, organogenesis and cytodifferentiation depend on the actin cytoskeleton, which is regulated by small Rho GTPases. These proteins link cell surface receptors to pathways that regulate cell motility, polarity, gene expression, vesicular trafficking, proliferation and apoptosis. The aim of this study was to evaluate, by immunohistochemistry, the distribution pattern of RhoA, RhoB, RhoC, Rac1 and Cdc42 during cytodifferentiation of the rat SMG and in male adults. All GTPases were found in epithelial and mesenchymal tissues throughout gland development. Rac1 appeared to be important for parenchyma expansion at the beginning of cytodifferentiation, while RhoC, Cdc42 and the inactive phosphorylated form of Rac1 seemed associated with lumen formation and cell polarization in terminal tubules. RhoA and RhoB labeling was evident throughout development. All GTPases were differentially expressed in the adult gland, suggesting that they play specific roles during differentiation and function of the rat SMG.

Efecto de la fenitoína prenatal en fetos de rata: estudio morfológico y morfométrico de la glándula submandibular / Effect of prenatal phenytoin on rat fetuses: and morphometrical study of the submandibular gland

La fenitoína (difenil hidantoína) es una droga anticonvulsiva y antiarrítmica, que posee acción teratógena en animales y humanos, causando importantes alteraciones, aún cuando se administra en dosis no teratógenas. Con el objetivo de estudiar morfológica y morfométricamente los efectos de la droga sobre la glándula submandibular fetal, ratas Wistar fueron inyectadas el 10ª día de preñez, con 70, 100 ó 150 mg/kg de fenitoína. Las dosis más altas provocaron la muerte intrauterina de todos los fetos. El peso de los fetos de las ratas inyectadas con 70 mg/kg de fenitoína fue menor que el de los controles. El examen histopatológico de la glándulas submandibular reveló una glándula inmadura, constituida por acinos y conductos mayores y con luz disminuida. Las células de los acinos y conductos eran mayores, con núcleos más grandes. El tejido conjuntivo era más escaso en los fetos del grupo tratado. Morfométricamente, fue posible confirmar los hallazgos histopatológicos. Los resultados sugieren que la exposición prenatal a la fenitoína, en dosis no teratógenas, actúa en la embriogénesis, causando alteraciones en el desarrollo (menor peso, cordón umbilical más corto y glándula submandibular menos diferenciada), mostrando el feto tratado aspectos de inmaturidad

Field emission SEM, conventional TEM and HVTEM study of submandibular gland in prenatal and postnatal aging mouse.

The development of acinar and ductal cells of the mouse submandibular gland was studied using field emission SEM, conventional TEM and HVTEM methods. The specimens, at 15 and 18 days of gestation and 1, 3, 7, 14, 21, 30, 90 and 180 postnatal days were fixed in 2.5% glutaraldehyde solution in 0.1 M sodium phosphate buffer (pH 7.3). At 15 and 18 days of gestation, the structure of mouse submandibular gland contains acinar and ductal cells in proliferation. The cytoplasmic organelles such as mitochondria, granular endoplasmic reticulum and Golgi apparatuses are scattered in the cytoplasm. At 18 prenatal days only several acinar cells present immature secretory granules in the apical portion. In this stage the acinar and ductal cells are enveloped by bundles of fine collagen fibrils disposed in several directions. There are also numerous capillaries located closely to the acinar cell membranes. In the aging stages of 1, 3, 7, 14, 21, and 30 postnatal days, the histo-differentiation of acinar, intercalated and ductal cell components are observed. At newborn day one the cytoplasmic organelles start to place themselves around the nucleus. Several immature secretory granules are observed at day one, however, they increase in the aging days. At postnatal day 30, the cytoplasms of acinar and ductal cells are filled with a large number of secretory granules of different sizes. The stacks of granular endoplasmic reticulum and Golgi apparatus and some vesicles and free ribosomes are noted. The intercellular membranes are attached by desmosomes and cytoplasmic interdigitations. The luminal surface shows several small projections of microvilli. An electron-dense line of basement membranes followed by fine collagen fibrils are recognized. Delicate capillaries are found in the outer surface of acinar cells. At postnatal day 90 and 180 the acinar, intercalated and striated ductal cells reveal numerous secretory granules in the apical portion. The acinar cells showed basal nuclei and the parallel arrangement of granular endoplasmic reticulum. The mitochondria are located at the base of ductal cells showing a typical pattern of cristae. In these stages the intercellular digitations of cytoplasmic protrusions and desmosomes are also noted. The cytoplasm of myoepithelial cells are seen along the cell membranes. The spongy-like structures constituting the basement membrane are followed by bundles of fine collagen fibers.

Development of mouse submandibular gland studied by field emission scanning electron microscopy.

The ultrastructural organization of mouse submandibular gland from 15 days gestation to 180 days of age was studied by field emission scanning electron microscopy. At 15 days of gestation several groups of acini, intercalated and striated ducts were present. They consisted of numerous pyramidal and/or polyhedral cells with spherical or elongated nuclei and short microvilli. At 3 days after birth different shapes of acinar structures covered by a network of fine collagen bundles were observed. The acinar portions corresponded to the terminal tubules and showed the acinar cells containing immature secretory granules. Fractured specimens of ducts revealed a flat surface with large central nuclei. At 14 days after birth acinar terminal portions possessed a round shape; branches of either intercalated and striated ducts were also observed. Gap junctions and interdigitations were numerous at the base of acinar cells. At 30 days after birth the acinar terminal portions and striated ducts revealed a large number of secretory granules. Acinar cells showed a pyramidal shape and basal nuclei. Freeze-cracked surfaces of the striated ductal cells evidenced also a pyramidal shape. Secretory granules ranged from 0.3 to 1.2 microns in size and were clearly observed by infoldings of the basement membranes. At 90 days after birth the ultrastructural features were more differentiated when compared with the previous ages. The freeze-cracked specimens showed very numerous secretory granules in all acinar and striated ductal cells. The granules occupied almost all the apical region of the cells. The outer surface of the basement membrane of acinar and myoepithelial cells was constituted by a spongy-like material covered by fine collagen fibrils. At 180 days after birth numerous secretory granules were seen either in acinar and ductal cells. A morphofunctional polarization of the cell was finally clearly observed in that the cytoplasmic organelles were concentrated in the basal portion whereas the secretory granules were located in the apical region of the cells.