Immunolocalization patterns of cytokeratins during salivary acinar cell development in mice

Embryonic development of the mouse salivary glands begins with epithelial thickening and continues with sequential changes from the pre-bud to terminal bud stages. After birth, morphogenesis proceeds, and the glands develop into a highly branched epithelial structure that terminates with saliva-producing acinar cells at the adult stage. Acinar cells derived from the epithelium are differentiated into serous, mucous, and seromucous types. During differentiation, cytokeratins, intermediate filaments found in most epithelial cells, play vital roles. Although the localization patterns and developmental roles of cytokeratins in different epithelial organs, including the mammary glands, circumvallate papilla, and sweat glands, have been well studied, their stage-specific localization and morphogenetic roles during salivary gland development have yet to be elucidated. Therefore, the aim of this study was to determine the stage and acinar cell type-specific localization pattern of cytokeratins 4, 5, 7, 8, 13, 14, 18, and 19 in the major salivary glands (submandibular, sublingual, and parotid glands) of the mouse at the E15.5, PN0, PN10, and adult stages. In addition, cell physiology, including cell proliferation, was examined during development via immunostaining for Ki67 to understand the cellular mechanisms that govern acinar cell differentiation during salivary gland morphogenesis. The distinct localization patterns of cytokeratins in conjunction with cell physiology will reveal the roles of epithelial cells in salivary gland formation during the differentiation of serous, mucous or seromucous salivary glands.     

Early markers of regeneration following ductal ligation in rat submandibular gland

Rat submandibular glands can recover their function and secretory protein content following ductal ligation-induced atrophy. Morphological studies have established that following ligation, deligation of the gland allows the regeneration of new salivary gland tissue. However, little is known about changes happening during early regeneration following intra-oral duct ligation, which does not damage the parasympathetic nerves. Glands that had been 2 weeks ligated or 2 weeks ligated + 3 days deligated were compared. Tissue was prepared for histological, immunohistochemical (SMG-B and Ki-67) and immunocytochemical analyses (smooth muscle actin, aquaporin 5). Haematoxylin and eosin staining of deligated glands showed that some acini regained their cytoplasmic volume; moreover, the loss of Alcian blue/periodic acid-Schiff’s staining from the lumen of ducts suggested successful deligation. The deligated gland was characterized by atypical acinar-ductal branched structures, which were less frequent in the ligated gland and rarely seen in normal unoperated tissue. Myoepithelial cells were also investigated since changes in their morphology reflected changes in the acini morphology not readily detected by conventional staining. Actin staining revealed the presence of some shrunken acini in the atrophic tissue, whereas they had regained their normal morphology in the deligated gland suggesting that the acini were recovering. Some acini during deligation regained aquaporin 5 expression, which had decreased during atrophy. SMG-B protein, located in the pro-acinar cell during gland development and usually found in the intercalated duct cells in the adult, was detected in the newly formed acini of the deligated gland. This study suggests that morphological markers of regeneration appear as early as 3 days following ligation removal. The authors thank the Wellcome Trust for funding.     

Pharmacological Activation of the EDA/EDAR Signaling Pathway Restores Salivary Gland Function following Radiation-Induced Damage

Radiotherapy of head and neck cancers often results in collateral damage to adjacent salivary glands associated with clinically significant hyposalivation and xerostomia. Due to the reduced capacity of salivary glands to regenerate, hyposalivation is treated by substitution with artificial saliva, rather than through functional restoration of the glands. During embryogenesis, the ectodysplasin/ectodysplasin receptor (EDA/EDAR) signaling pathway is a critical element in the development and growth of salivary glands. We have assessed the effects of pharmacological activation of this pathway in a mouse model of radiation-induced salivary gland dysfunction. We report that post-irradiation administration of an EDAR-agonist monoclonal antibody (mAbEDAR1) normalizes function of radiation damaged adult salivary glands as determined by stimulated salivary flow rates. In addition, salivary gland structure and homeostasis is restored to pre-irradiation levels. These results suggest that transient activation of pathways involved in salivary gland development could facilitate regeneration and restoration of function following damage.     

Diverse functions for the semaphorin receptor PlexinD1 in development and disease

Plexins are a family of single-pass transmembrane proteins that serve as cell surface receptors for Semaphorins during the embryonic development of animals. Semaphorin–Plexin signaling is critical for many cellular aspects of organogenesis, including cell migration, proliferation and survival. Until recently, little was known about the function of PlexinD1, the sole member of the vertebrate-specific PlexinD (PlxnD1) subfamily. Here we review novel findings about PlxnD1's roles in the development of the cardiovascular, nervous and immune systems and salivary gland branching morphogenesis and discuss new insights concerning the molecular mechanisms of PlxnD1 activity.     

Neurosteroids: trophic effects in the nervous system]

Some steroids, named "neurostero?ds", can be synthesized from cholesterol within both the central and peripheral nervous systems. Thus, pregnenolone and progesterone persist in the brain and in peripheral nerves long after removal of the steroidogenic endocrine glands by castration and adrenalectomy. The role of neurosteroids during the development of the nervous system is not well known, although they are synthesized by glial cells and some populations of neurons already during embryonic life. Cell culture experiments suggest that neurosteroids may influence the survival and differentiation of neurons and glial cells. In the adult nervous system, neurosteroids play an important role during regeneration. Progesterone is indeed synthesized by Schwann cells in peripheral nerves, where it plays an important role in the formation of new myelin sheaths after lesion. This is the first demonstration of a vital role for a neurosteroid in the nervous system.     

Rac function in epithelial tube morphogenesis

Epithelial cell migration and morphogenesis require dynamic remodeling of the actin cytoskeleton and cell-cell adhesion complexes. Numerous studies in cell culture and in model organisms have demonstrated the small GTPase Rac to be a critical regulator of these processes; however, little is known about Rac function in the morphogenic movements that drive epithelial tube formation. Here, we use the embryonic salivary glands of Drosophila to understand the role of Rac in epithelial tube morphogenesis. We show that inhibition of Rac function, either through loss of function mutations or dominant-negative mutations, disrupts salivary gland invagination and posterior migration. In contrast, constitutive activation of Rac induces motile behavior and subsequent cell death. We further show that Rac regulation of salivary gland morphogenesis occurs through modulation of cell-cell adhesion mediated by the E-cadherin/beta-catenin complex and that shibire, the Drosophila homolog of dynamin, functions downstream of Rac in regulating beta-catenin localization during gland morphogenesis. Our results demonstrate that regulation of cadherin-based adherens junctions by Rac is critical for salivary gland morphogenesis and that this regulation occurs through dynamin-mediated endocytosis.     

Identification of factors that function in Drosophila salivary gland cell death during development using proteomics

Proteasome inhibitors induce cell death and are used in cancer therapy, but little is known about the relationship between proteasome impairment and cell death under normal physiological conditions. Here, we investigate the relationship between proteasome function and larval salivary gland cell death during development in Drosophila. Drosophila larval salivary gland cells undergo synchronized programmed cell death requiring both caspases and autophagy (Atg) genes during development. Here, we show that ubiquitin proteasome system (UPS) function is reduced during normal salivary gland cell death, and that ectopic proteasome impairment in salivary gland cells leads to early DNA fragmentation and salivary gland condensation in vivo. Shotgun proteomic analyses of purified dying salivary glands identified the UPS as the top category of proteins enriched, suggesting a possible compensatory induction of these factors to maintain proteolysis during cell death. We compared the proteome following ectopic proteasome impairment to the proteome during developmental cell death in salivary gland cells. Proteins that were enriched in both populations of cells were screened for their function in salivary gland degradation using RNAi knockdown. We identified several factors, including trol, a novel gene CG11880, and the cop9 signalsome component cop9 signalsome 6, as required for Drosophila larval salivary gland degradation.     

SPM Receptor Expression and Localization in Irradiated Salivary Glands

Radiation therapy–mediated salivary gland destruction is characterized by increased inflammatory cell infiltration and fibrosis, both of which ultimately lead to salivary gland hypofunction. However, current treatments (e.g., artificial saliva and sialagogues) only promote temporary relief of symptoms. As such, developing alternative measures against radiation damage is critical for restoring salivary gland structure and function. One promising option for managing radiation therapy–mediated damage in salivary glands is by activation of specialized proresolving lipid mediator receptors due to their demonstrated role in resolution of inflammation and fibrosis in many tissues. Nonetheless, little is known about the presence and function of these receptors in healthy and/or irradiated salivary glands. Therefore, the goal of this study was to detect whether these specialized proresolving lipid mediator receptors are expressed in healthy salivary glands and, if so, if they are maintained after radiation therapy–mediated damage. Our results indicate that specialized proresolving lipid mediator receptors are heterogeneously expressed in inflammatory as well as in acinar and ductal cells within human submandibular glands and that their expression persists after radiation therapy. These findings suggest that epithelial cells as well as resident immune cells represent potential targets for modulation of resolution of inflammation and fibrosis in irradiated salivary glands.     

Integrin expression in developing human salivary glands

The development and complete differentiation of salivary glands is a complex process that involves a large number of co-ordinated events. Little is known about the molecular basis for salivary gland development. However, we have reported previously that integrins appear to play a role. Integrins are heterodimeric transmembrane receptors consisting of one α and one β subunit that play a pivotal role in the interaction of cells with the extracellular matrix. Such interactions regulate the organisation of cells of tissues and organs during development as well as cell proliferation and differentiation. Using immunohistochemistry and Western and Northern blot analysis, we mapped the localisation and expression of integrins β1, β3 and β4 in human salivary glands obtained from foetuses ranging from weeks 4–24 of gestation and compared it with adult salivary glands. Integrin β1 first appeared during the canalisation stage and during the differentiation stage. A message first appeared at week 6 of development. The expression of β4 integrin protein and message was observed only in the late stage of differentiation. Integrin β3 was not detected in the developing glands; however, integrins β1, β3 and β4 were all expressed in adult salivary gland tissues. The data suggest that integrins, particularly β1, have a role to play in salivary gland development and differentiation.     

Different Wnt signals act through the Frizzled and RYK receptors during Drosophila salivary gland migration

Guided cell migration is necessary for the proper function and development of many tissues, one of which is the Drosophila embryonic salivary gland. Here we show that two distinct Wnt signaling pathways regulate salivary gland migration. Early in migration, the salivary gland responds to a WNT4-Frizzled signal for proper positioning within the embryo. Disruption of this signal, through mutations in Wnt4, frizzled or frizzled 2, results in misguided salivary glands that curve ventrally. Furthermore, disruption of downstream components of the canonical Wnt pathway, such as dishevelled or Tcf, also results in ventrally curved salivary glands. Analysis of a second Wnt signal, which acts through the atypical Wnt receptor Derailed, indicates a requirement for Wnt5 signaling late in salivary gland migration. WNT5 is expressed in the central nervous system and acts as a repulsive signal, needed to keep the migrating salivary gland on course. The receptor for WNT5, Derailed, is expressed in the actively migrating tip of the salivary glands. In embryos mutant for derailed or Wnt5, salivary gland migration is disrupted; the tip of the gland migrates abnormally toward the central nervous system. Our results suggest that both the Wnt4-frizzled pathway and a separate Wnt5-derailed pathway are needed for proper salivary gland migration.     

Adrenergic and cholinergic nerves of bovine,guinea pig and hamster salivary glands

Summary The distribution of formaldehyde-induced fluorescence and acetylcholine-esterase (AChE) activity was histochemically investigated in certain salivary glands of the cow (submandibular gland), guinea pig and hamster (submandibular and sublingual glands). Adrenergic nerves occur around the secretory acini of the bovine, guinea pig and hamster submandibular glands, as well as around those of the hamster sublingual gland. The mucous secretory acini of the guinea pig sublingual gland, however, seem to be devoid of adrenergic nerve supply. Except in the sublingual gland of the hamster, no adrenergic nerves occur in relation to duct cells.The pattern of AChE activity is similar to that of adrenergic nerves. Thus, AChE-positive nerves form a network around secretory acini of all the five glands examined. Furthermore, AChE activity was also observed in nerve fibres in close proximity to striated duct cells.Both adrenergic and AChE-containing fibres were observed around blood vessels of different sizes. Ganglionic cells are occasionally to be seen; they all display AChE-activity. No adrenergic ganglionic cells were observed in any of the glands examined.All glands were also studied in the electron microscope. Interest was focussed on the fine structure of the autonomic nerves with special reference to their contents and type of storage vesicles.The content of noradrenaline was chemically determined in each type of salivary gland studied.This work was supported by grants from the University of Umeå and from the Swedish Society for Medical Research and was also carried out within a research organization supported by the Swedish Medical Research Council (projects B73-04X-712-08C and B73-04X-56-09C). The authors are indebted to Miss Kristina Karlsson and Miss Marianne Borg for valuable technical assistance.     

Distribution of Tight Junction Proteins in Adult Human Salivary Glands

Tight junctions (TJs) are an essential structure of fluid-secreting cells, such as those in salivary glands. Three major families of integral membrane proteins have been identified as components of the TJ: claudins, occludin, and junctional adhesion molecules (JAMs), plus the cytosolic protein zonula occludens (ZO). We have been working to develop an orally implantable artificial salivary gland that would be suitable for treating patients lacking salivary parenchymal tissue. To date, little is known about the distribution of TJ proteins in adult human salivary cells and thus what key molecular components might be desirable for the cellular component of an artificial salivary gland device. Therefore, the aim of this study was to determine the distribution of TJ proteins in human salivary glands. Salivary gland samples were obtained from 10 patients. Frozen and formalin-fixed paraffin-embedded sections were stained using IHC methods. Claudin-1 was expressed in ductal, endothelial, and ∼25% of serous cells. Claudins-2, -3, and -4 and JAM-A were expressed in both ductal and acinar cells, whereas claudin-5 was expressed only in endothelial cells. Occludin and ZO-1 were expressed in acinar, ductal, and endothelial cells. These results provide new information on TJ proteins in two major human salivary glands and should serve as a reference for future studies to assess the presence of appropriate TJ proteins in a tissue-engineered human salivary gland. (J Histochem Cytochem 56:1093–1098, 2008)     

The innervation of the salivary gland in the american cockroach: Light and electron microscopic observations

The salivary gland of Periplaneta americana (L.) is innervated from both the stomatogastric nervous system (SNS) and subesophageal ganglion (SEG). Methylene-blue preparations, histological sections and electron microscopy revealed a pair of nerves from the SEG, each of which contains two axons 5–7 μ in diameter, and these are accompanied by several smaller ones. The nerves going to the salivary glands from the SNS contain a dozen or more axons, each less than 2 μ thick. Axons from two sources innervate the efferent salivary ducts, the acini, the anterior ends of the salivary reservoirs, and the reservoir suspensory muscles. A nerve which has reached an acinus forms a plexus upon its surface. Electron micrographs disclose penetration of axons with or without glial wrappings, into the intercellular spaces between gland cells. Axons without glial wrappings have been observed in intimate contact with gland-cell membranes, and several areas which resemble synaptic junctions have been seen.     

Laminin-immunoreactive glia distinguish regenerative adult CNS systems from non-regenerative ones.

Most regions of the adult mammalian central nervous system (CNS) do not support axonal growth and regeneration. Laminin, expressed by cultured astrocytes and known to promote neurite outgrowth of cultured neurons, is normally present in brain basement membranes, and only transiently induced in adult brain astrocytes by injury. Here I provide three lines of evidence which suggest that the continued expression of laminin by astrocytes may be a prerequisite for axonal growth and regeneration in adult CNS. Firstly, laminin is continuously present in astrocytes of adult rat olfactory bulb apparently in close association with the olfactory nerve axons. Secondly, laminin is continuously expressed by astrocytes in adult frog brain, and sectioning of the optic tract further increases laminin immunoreactivity in astrocytes of the optic tectum during the period of axonal regeneration. Lastly, laminin appears normally in astrocytes of the frog and goldfish optic nerves which regenerate, but not in astrocytes of the rat or chick optic nerves which do not regenerate. The selective association of laminin with axons that undergo growth and regeneration in vivo is consistent with the possibility that astrocytic laminin provides these central nervous systems with their regenerative potential.     

Caspases function in autophagic programmed cell death in Drosophila

Self-digestion of cytoplasmic components is the hallmark of autophagic programmed cell death. This auto-degradation appears to be distinct from what occurs in apoptotic cells that are engulfed and digested by phagocytes. Although much is known about apoptosis, far less is known about the mechanisms that regulate autophagic cell death. Here we show that autophagic cell death is regulated by steroid activation of caspases in Drosophila salivary glands. Salivary glands exhibit some morphological changes that are similar to apoptotic cells, including fragmentation of the cytoplasm, but do not appear to use phagocytes in their degradation. Changes in the levels and localization of filamentous Actin, alpha-Tubulin, alpha-Spectrin and nuclear Lamins precede salivary gland destruction, and coincide with increased levels of active Caspase 3 and a cleaved form of nuclear Lamin. Mutations in the steroid-regulated genes beta FTZ-F1, E93, BR-C and E74A that prevent salivary gland cell death possess altered levels and localization of filamentous Actin, alpha-Tubulin, alpha-Spectrin, nuclear Lamins and active Caspase 3. Inhibition of caspases, by expression of either the caspase inhibitor p35 or a dominant-negative form of the initiator caspase Dronc, is sufficient to inhibit salivary gland cell death, and prevent changes in nuclear Lamins and alpha-Tubulin, but not to prevent the reorganization of filamentous Actin. These studies suggest that aspects of the cytoskeleton may be required for changes in dying salivary glands. Furthermore, caspases are not only used during apoptosis, but also function in the regulation of autophagic cell death.     

The intact parasympathetic nerve promotes submandibular gland regeneration through ductal cell proliferation

ObjectivesSalivary gland regeneration is closely related to the parasympathetic nerve; however, the mechanism behind this relationship is still unclear. The aim of this study was to evaluate the relationship between the parasympathetic nerve and morphological differences during salivary gland regeneration.Materials and MethodsWe used a duct ligation/deligation‐induced submandibular gland regeneration model of Sprague‐Dawley (SD) rats. The regenerated submandibular gland with or without chorda lingual (CL) innervation was detected by haematoxylin–eosin staining, real‐time PCR (RT‐PCR), immunohistochemistry and Western blotting. We counted the number of Ki67‐positive cells to reveal the proliferation process that occurs during gland regeneration. Finally, we examined the expression of the following markers: aquaporin 5, cytokeratin 7, neural cell adhesion molecule (NCAM) and polysialyltransferases.ResultsIntact parasympathetic innervation promoted submandibular gland regeneration. The process of gland regeneration was significantly repressed by cutting off the CL nerve. During gland regeneration, Ki67‐positive cells were mainly found in the ductal structures. Moreover, the expression of NCAM and polysialyltransferases‐1 (PST) expression in the innervation group was significantly increased during early regeneration and decreased in the late stages. In the denervated submandibular glands, the expression of NCAM decreased during regeneration.ConclusionsOur findings revealed that the regeneration of submandibular glands with intact parasympathetic innervation was associated with duct cell proliferation and the increased expression of PST and NCAM.     

The parasite invasion marker SRPN6 reduces sporozoite numbers in salivary glands of Anopheles gambiae

For malaria transmission to occur, Plasmodium sporozoites must infect the salivary glands of their mosquito vectors. This study reports that Anopheles gambiae SRPN6 participates in a local salivary gland epithelial response against the rodent malaria parasite, Plasmodium berghei . We showed previously that SRPN6, an immune inducible midgut invasion marker, influences ookinete development. Here we report that SRPN6 is also specifically induced in salivary glands with the onset of sporozoite invasion. The protein is located in the basal region of epithelial cells in proximity to invading sporozoites. Knockdown of SRPN6 during the late phase of sporogony by RNAi has no effect on oocyst rupture but significantly increases the number of sporozoites present in salivary glands. Despite several differences between the passage of Plasmodium through the midgut and the salivary glands, this study identifies a striking overlap in the molecular responses of these two epithelia to parasite invasion.