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Development of the salivary gland is characterized by extensive branching morphogenesis. Although various molecules have been implicated in salivary gland development, the role of the mammalian target of rapamycin (mTOR) signalling pathway, including both mTOR complexes 1 and 2 (mTORC1 and 2), in salivary gland development is unknown. Here, we examined protein expression levels related to the mTOR signalling pathway using an ex vivo submandibular salivary gland (SMG) organ culture. We showed that branching buds in the salivary glands were substantially decreased and phosphorylation of mTORC1 signalling pathway related proteins (mTOR, p70 ribosomal protein S6 kinase 1 and eukaryotic initiation factor 4E-binding protein 1) was inhibited by rapamycin (an mTOR inhibitor). In addition, AKT, which is an upstream protein kinase of mTORC1 and is downstream of mTORC2, is inhibited by LY294002 (a phosphatidylinositol 3-kinase inhibitor), but not by rapamycin. Moreover, rapamycin-treated ICR neonatal mice exhibited a reduction in both body weight and salivary glands compared with vehicle-treated neonatal mice. The present data indicate that the mTOR signalling pathway, including both mTORC1 and mTORC2, plays a critical role in salivary gland development both in ex vivo SMG organ culture and ICR neonatal mice in vivo., (© 2019 Federation of European Biochemical Societies.)

Background: Recent advances in tissue regeneration approaches including 3D organoids, were based on various 3D organogenesis models. However, 3D models are generally technique-sensitive and time-consuming. Thus, we utilized an existing model of submandibular salivary gland (SMG) to modify a simple and highly reproducible in vitro 3D culture model of primary SMG cells self-organization into a well-developed cell spheroid inside Matrigel substrate. We used this model to observe the collective multicellular behavior during spheroid formation. Further, we applied various quantitative approaches including real-time live imaging and immune histochemical image analysis to dissect the cellular dynamics during tissue patterning., Results: On a time-scale of hours, we observed marked size and shape transformations in the developed 3D spheroid which resulted in a spatially-controlled growth differential from the canter to the periphery of the formed aggregates. Moreover, we investigated the effect of fibronectin (FN) on SMG cells self-organization using our simplified culture model. Interestingly, we discovered a novel role of FN in inducing duct-like elongation during initial stages of SMG bud formation., Conclusion: This in vitro model provides an excellent tool for analyzing the intercellular dynamics during early SMG tissue development as well as revealing a novel role of FN in SMG ductal expansion., (© 2019 Wiley Periodicals, Inc.)

Hyposalivation and xerostomia create chronic oral complications that decrease the quality of life in head and neck cancer patients who are treated with radiotherapy. Experimental approaches to understanding mechanisms of salivary gland dysfunction and restoration have focused on in vivo models, which are handicapped by an inability to systematically screen therapeutic candidates and efficiencies in transfection capability to manipulate specific genes. The purpose of this salivary gland organotypic culture protocol is to evaluate maximal time of culture viability and characterize cellular changes following ex vivo radiation treatment. We utilized immunofluorescent staining and confocal microscopy to determine when specific cell populations and markers are present during a 30-day culture period. In addition, cellular markers previously reported in in vivo radiation models are evaluated in cultures that are irradiated ex vivo. Moving forward, this method is an attractive platform for rapid ex vivo assessment of murine and human salivary gland tissue responses to therapeutic agents that improve salivary function.

Background: Apert syndrome is characterized by craniosynostosis and bony syndactyly of the hands and feet. The cause of Apert syndrome is a single nucleotide substitution mutation (S252W or P253R) in fibroblast growth factor receptor 2 (FGFR2). Clinical experience suggests increased production of saliva by Apert syndrome patients, but this has not been formally investigated. FGFR2 signaling is known to regulate branching morphogenesis of the submandibular glands (SMGs). With the Apert syndrome mouse model (Ap mouse), we investigated the role of FGFR2 in SMGs and analyzed the SMG pathology of Apert syndrome., Results: Ap mice demonstrated significantly greater SMG and sublingual gland (SMG/SLG complex) mass/body weight and percentage of parenchyma per unit area of the SMG compared with control mice. Furthermore, gene expression of Fgf1, Fgf2, Fgf3, Pdgfra, Pdgfrb, Mmp2, Bmp4, Lama5, Etv5, and Dusp6 was significantly higher in the SMG/SLG complex of Ap mice. FGF3 and BMP4 exhibited altered detection patterns. The numbers of macrophages were significantly greater in SMGs of Ap mice than in controls. Regarding functional evaluations of the salivary glands, no significant differences were observed., Conclusions: These results suggest that the gain-of-function mutation in FGFR2 in the SMGs of Ap mice enhances branching morphogenesis. Developmental Dynamics 247:1175-1185, 2018. © 2018 Wiley Periodicals, Inc., (© 2018 Wiley Periodicals, Inc.)

Stem and progenitor cells of the submandibular salivary gland (SMG) give rise to, maintain, and regenerate the multiple lineages of mature epithelial cells including those belonging to the ductal, acinar, basal and myoepithelial subtypes. Here we have exploited single cell RNA-sequencing and in vivo genetic lineage tracing technologies to generate a detailed map of the cell fate trajectories and branch points of the basal and myoepithelial cell populations of the mouse SMG during embryonic development and in adults. Our studies show that the transcription factor p63 and alpha-smooth muscle actin (SMA) serve as faithful markers of the basal and myoepithelial cell lineages, respectively and that both cell types are endowed with progenitor cell properties. However, p63 + basal and SMA + myoepithelial cells exhibit distinct cell fates by virtue of maintaining different cellular lineages during morphogenesis and in adults. Collectively, our results reveal the dynamic and complex nature of the diverse SMG cell populations and highlight the distinct differentiation potential of the p63 and SMA expressing subtypes in the stem and progenitor cell hierarchy. Long term these findings have profound implications towards a better understanding of the molecular mechanisms that dictate lineage commitment and differentiation programs during development and adult gland maintenance.

Epithelial progenitor cells are dependent upon a complex 3D niche to promote their proliferation and differentiation during development, which can be recapitulated in organoids. The specific requirements of the niche remain unclear for many cell types, including the proacinar cells that give rise to secretory acinar epithelial cells that produce saliva. Here, using ex vivo cultures of E16 primary mouse submandibular salivary gland epithelial cell clusters, we investigated the requirement for mesenchymal cells and other factors in producing salivary organoids in culture. Native E16 salivary mesenchyme, but not NIH3T3 cells or mesenchymal cell conditioned medium, supported robust protein expression of the progenitor marker Kit and the acinar/proacinar marker AQP5, with a requirement for FGF2 expression by the mesenchyme. Enriched salivary epithelial clusters that were grown in laminin-enriched basement membrane extract or laminin-111 together with exogenous FGF2, but not with EGF, underwent morphogenesis to form organoids that displayed robust expression of AQP5 in terminal buds. Knockdown of FGF2 in the mesenchyme or depletion of mesenchyme cells from the organoids significantly reduced AQP5 levels even in the presence of FGF2, suggesting a requirement for autocrine FGF2 signaling in the mesenchyme cells for AQP5 expression. We conclude that basement membrane proteins and mesenchyme cells function as niche factors in salivary organoids., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

The mouse granular convoluted tubules (GCTs), which are only located in the submandibular gland (SMG) are known to develop and maintain their structure in an androgen-dependent manner. We previously demonstrated that the GCTs are involuted by the epithelial deletion of core binding factor β (CBFβ), a transcription factor that physically interacts with any of the Runt-related transcription factor (RUNX) proteins (RUNX1, 2 and 3). This result clearly demonstrates that the Runx /Cbfb signaling pathway is indispensable in the development of the GCTs. However, it is not clear which of the RUNX proteins plays useful role in the development of the GCTs by activating the Runx /Cbfb signaling pathway. Past studies have revealed that the Runx /Cbfb signaling pathway plays important roles in various aspects of development and homeostatic events. Moreover, the Runx genes have different temporospatial requirements depending on the biological situation. In the present study, the GCTs of the SMG showed a remarkable phenotype of, which phenocopied the epithelial deletion of Cbfb, in epithelial-specific Runx1 conditional knock-out (cKO) mice. The results indicate that Runx1 works as a partner of Cbfb during the development of the GCTs. We also discovered that the depletion of Runx1 resulted in the reduced secretion of saliva in male mice. Consistent with this finding, one of the water channels, Aquaporin-5 (AQP5) was mislocalized in the cytoplasm of the Runx1 mutants, suggesting a novel role of Runx1 in the membrane trafficking of AQP5. In summary, the present findings demonstrated that RUNX1 is essential for the development of the GCTs. Furthermore, RUNX1 could also be involved in the membrane trafficking of the AQP5 protein of the acinar cells in the SMG in order to allow for the proper secretion of saliva.

The importance of macrophages in tissue development and regeneration has been strongly emphasized. However, the specific roles of macrophage colony-stimulating factor (MCSF), the key regulator of macrophage differentiation, in glandular tissue development have been unexplored. Here, we disclose new macrophage-independent roles of MCSF in tissue development. We initially found that MCSF is markedly upregulated at embryonic day (E)13.5, at a stage preceding the colonization of macrophages (at E15.5), in mouse submandibular gland (SMG) tissue. Surprisingly, MCSF-induced branching morphogenesis was based on a direct effect on epithelial cells, as well as indirectly, by modulating the expression of major growth factors of SMG growth, FGF7 and FGF10, via the phosphoinositide 3-kinase (PI3K) pathway. Additionally, given the importance of neurons in SMG organogenesis, we found that MCSF-induced SMG growth was associated with regulation of neurturin expression and neuronal network development during early SMG development in an in vitro organogenesis model as well as in vivo These results indicate that MCSF plays pleiotropic roles and is an important regulator of early SMG morphogenesis., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

Dysfunction of salivary glands leads to several oral health problems, including dental caries, mastication and swallowing dysfunctions and multiple oral infections. Conventional treatments for such condition fell short of providing satisfying therapeutic results. Recent advances in organ regeneration therapy which utilize tissue stem cells to fabricate bioengineered 3D organ buds, have introduced a promising therapeutic tool for full functional organ regeneration. However, finding a sustainable and easily accessible cell source for such approaches is still challenging, especially in case of severely atrophied tissues such as irradiated salivary glands. In response to this, we hypothesized that bone marrow derived mesenchymal stem cells (MSCs) could be used as feeder cells to induce salivary epithelial tissues/cells branching. Indeed, in 2D cultures, MSCs supported branching of embryonic submandibular salivary gland (SMG) epithelium. Interestingly, this enhancing effect was dependent on the initial number of MSC feeder cells. In addition, MSCs supported the self-assembly of SMG epithelial progenitor cells into well-patterned and branched 3D salivary organoids. Therefore, these findings propose MSCs as a valuable candidate cell source for induced SMG epithelial branching, which can potentially be applied in future methods for SMG regeneration approaches.

Continuously feeding a liquid diet to growing rodents strongly inhibits parotid gland growth, due to suppressed growth of acinar cells. This study investigated whether a liquid diet had a similar effect on submandibular and sublingual glands of growing rats. Rats were weaned on day 21 after birth and then fed a pellet diet in the control group and a liquid diet in the experimental group for 0, 1, 2, 4, and 8 weeks. Their submandibular and sublingual glands were excised, weighed, and examined histologically, immunohistochemically (using antibodies to 5'-bromo-2-deoxyuridine and cleaved caspase 3), and ultrastructurally. The submandibular glands did not significantly differ between the control and experimental groups at all tested points. Only at Week 8, acinar cell area and 5'-bromo-2-deoxyuridine-labeling index of acinar cells in sublingual glands were significantly lower in the experimental group than in the control group. These results show that a liquid diet during rats' growth period had no effect on acinar cells in their submandibular glands, and only a slight effect on acinar cells in their sublingual glands of growing rats, in contrast to the marked effect of a liquid diet on parotid glands., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

In many parts of the nervous system, signals pass across multiple synaptic relays on their way to a destination, but little is known about how these relays form and the function they serve. To get some insight into this question we ask how the connectivity patterns are organized at two successive synaptic relays in a simple, cholinergic efferent pathway. We found that the organization at successive relays in the parasympathetic nervous system strongly resemble each other despite the different embryological origin and physiological properties of the pre- and postsynaptic cells. Additionally, we found a similar developmental synaptic pruning and elaboration strategy is used at both sites to generate their adult organizations. The striking parallels in adult innervation and developmental mechanisms at the relays argue that a general strategy is in operation. We discuss why from a functional standpoint this structural organization may amplify central signals while at the same time maintaining positional targeting.

Objective: Expression of Transient receptor potential (TRP) channels: TRP canonical (TRPC)1, TRP vanilloid (TRPV)3, TRPV4 and TRP melastatin (TRPM)8 in adult rat salivary gland has recently been reported. The authors investigated expression of these TRP channels in the submandibular gland during early developmental stage in which the cell constitution is different, and discussed the function of TRP in the submandibular gland in early development., Design: Using rat submandibular gland at embryonic days (E)18 and E20 and postnatal days (PN)0 and PN5 and PN28, expression of TRPV3, TRPV4, TRPC1 and TRPM8 was investigated using real-time polymerase chain reaction (RT-PCR) and immunohistochemistry., Results: All TRP channels were expressed in cells constituting the submandibular gland in early developmental stage, but an increase in the expression level at PN5 on RT-PCR was significant compared with those at E18, PN0 and PN28 in TRPC1 and TRPV4 channels, whereas an increase was observed but not significant in the others. On immunohistochemical staining at PN5, whereas strong reactions of anti-TRPM8 antibody, anti-TRPV3 and anti-TRPV4 antibodies were observed in cells which proliferated from a terminal portion of cells arranged tubular structure which previously constituted mostly the submandibular gland., Conclusion: It was clarified that TRP channels are expressed in the rat submandibular gland in early developmental stage although cells constituting the submandibular gland are different from those in adult animals, suggesting that these TRP channels are involved in cell differentiation in at PN5 into the adult submandibular gland during early development., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The mandibular glands (MGs) of honeybee workers are vital for the secretion of lipids, for both larval nutrition and pheromones. However, knowledge of how the proteome controls MG development and functionality at the different physiological stages of worker bees is still lacking. We characterized and compared the proteome across different ages of MGs in Italian bees (ITBs) and Royal Jelly (RJ) bees (RJBs), the latter being a line bred for increasing RJ yield, originating from the ITB. All 2000 proteins that were shared by differently aged MGs in both bee lines (>4000 proteins identified in all) were strongly enriched in metabolizing protein, nucleic acid, small molecule, and lipid functional groups. The fact that these shared proteins are enriched in similar groups in both lines suggests that they are essential for basic cellular maintenance and MG functions. However, great differences were found when comparing the proteome across different MG phases in each line. In newly emerged bees (NEBs), the unique and highly abundant proteins were enriched in protein synthesis, cytoskeleton, and development related functional groups, suggesting their importance to initialize young MG development. In nurse bees (NBs), specific and highly abundant proteins were mainly enriched in substance transport and lipid synthesis, indicating their priority may be in priming high secretory activity in lipid synthesis as larval nutrition. The unique and highly abundant proteins in forager bees (FBs) were enriched in lipid metabolism, small molecule, and carbohydrate metabolism. This indicates their emphasis on 2-heptanone synthesis as an alarm pheromone to enhance colony defense or scent marker for foraging efficiency. Furthermore, a wide range of different biological processes was observed between ITBs and RJBs at different MG ages. Both bee stocks may adapt different proteome programs to drive gland development and functionality. The RJB nurse bee has reshaped its proteome by enhancing the rate of lipid synthesis and minimizing degradation to increase 10-hydroxy-2-decenoic acid synthesis, a major component of RJ, to maintain the desired proportion of lipids in increased RJ production. This study contributes a novel understanding of MG development and lipid metabolism, and a potential starting point for lipid or pheromone biochemists as well as developmental geneticists.

Keratin 5 (K5) is a marker of basal progenitor cells in the epithelia of a number of organs. During prenatal development of the submandibular gland (SMG) in mice, K5(+) progenitor cells in the developing epithelia play important roles in its organogenesis. Although K5(+) cells are also present in the adult mouse SMG and may function in tissue regeneration, their histological localization has not yet investigated in detail. In the present study, we examined the immunohistochemical localization of K5 in the SMG in adult and postnatal developing mice. At birth, K5 immunoreactivity was detected in the entire duct system, in which it was localized in the basal cells of a double-layered epithelium, but was not detected in the terminal tubule or myoepithelial cells. At postnatal weeks 1-3, with the development of intercalated ducts (ID), striated ducts (SD), and excretory ducts (ED), K5-immunoreactive basal cells were gradually restricted to the ED and the proximal double-layered portions of the ID connecting to the SD. At the same time, K5 immunoreactivity appeared in myoepithelial cells, in which its positive ratio gradually increased. In adults, K5 immunoreactivity was localized to most myoepithelial cells, most basal cells in the ED, and a small number of ID cells at the boundary between the ID and SD in the female SMG or between the ID and granular convoluted tubules in the male SMG. These results suggest that K5 is a marker of differentiated myoepithelial cells and duct progenitor cells in the mouse SMG.

In vitro fabricated biological tissue would be a valuable tool to screen newly synthesized drugs or understand the tissue development process. Several studies have attempted to fabricate biological tissue in vitro. However, controlling the growth and morphology of the fabricated tissue remains a challenge. Therefore, new techniques are required to modulate tissue growth. RGD (arginine-glycine-aspartic acid), which is an integrin-binding domain of fibronectin, has been found to enhance cell adhesion and survival; it has been used to modify substrates for in vitro cell culture studies or used as tissue engineering scaffolds. In addition, this study shows novel functions of the RGD peptide, which enhances tissue growth and modulates tissue morphology in vitro. When an isolated submandibular gland (SMG) was cultured on an RGD-modified alginate hydrogel sheet, SMG growth including bud expansion and cleft formation was dramatically enhanced. Furthermore, we prepared small RGD-modified alginate beads and placed them on the growing SMG tissue. These RGD-modified beads successfully induced cleft formation at the bead position, guiding the desired SMG morphology. Thus, this RGD-modified material might be a promising tool to modulate tissue growth and morphology in vitro for biological tissue fabrication.

Objective: To determine the role of Bmi-1 in the submandibular gland (SMG) of mice., Methods: SMG of 4-week wild-type (WT) and Bmi-1 null (Bmi-1(-/-)) mice was analyzed on the weight, salivary flow rate, hematoxylin-eosin staining morphological differences and the changes in proliferation and aging by histology, immunohistochemistry and Western blotting., Results: Compared with WT mice, the average static salivary flow rate [WT:(0.21 ± 0.02) µg/min,Bmi-1(-/-): (0.10 ± 0.02) µg/min] (P = 0.001) and the submandibular gland weight [WT: (1.89 ± 0.15) µg], Bmi-1(-/-): [(1.34 ± 0.07)µg] (P = 0.003) of the male Bmi-1(-/-) mice were significantly decreased, the number of gland duct increased, and the granular convoluted duct showed reduced diameter and branches. More senescence-associated β-galactosidase positive cells existed in SMG of Bmi-1(-/-)mice (WT:0.00, Bmi-1(-/-): 0.18 ± 0.02), and Ki-67 immunopositive cells decreased in SMG of Bmi-1(-/-) mice (WT:0.40 ∼ 0.47, Bmi-1(-/-): 0.18 ∼ 0.20) (P = 0.000). The expression of p16 (WT:1.00 ± 0.12, Bmi-1(-/-): 0.00 ± 0.00) (P = 0.003) and p19 (WT:0.97 ± 0.09, Bmi-1(-/-): 5.09 ± 0.21) (P = 0.004) were up-regulated dramatically in SMG of the Bmi-1(-/-) mice., Conclusions: Bmi-1 gene deficiency causes abnormal function of SMG by inducing senescence phenotype of SMG.

Objective: To determine the effect of perinatal exposure to low doses of genistein and/or vinclozolin on submandibular salivary gland (SSG) development in juvenile and adult male rats and to establish a link with sweet preference., Material and Methods: Female rats received orally (1 mg kg(-1) body weight/day) genistein and vinclozolin, alone or in combination, from the first gestational day up to weaning. Sweet preference was assessed at weaning and in adulthood in male offspring; submandibular glands were then collected to study the morphogenesis and mRNA expression of steroid receptors, growth factors and taste related proteins., Results: Exposure to genistein and/or vinclozolin resulted in a higher saccharin intake on postnatal day 25 (P < 0.05) linked to a higher number of pro-acinar cells (P < 0.01) and mRNA expression of progesterone receptor, growth factors and gustine (P < 0.01). These increases disappeared in adulthood, but mRNA expressions of sex hormone receptors and growth factors were strongly repressed in all treated groups (P < 0.01)., Conclusion: Our findings confirm that the SSG are target for xenohormones and provide evidence that perinatal exposure to low doses of genistein and/or vinclozolin could simultaneously disrupt not only the salivary gland prepubertal development and sweet intake but also endocrine gene mRNA expression later in life., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The mechanical properties of the cellular microenvironment dramatically alter during tissue development and growth. Growing evidence suggests that physical microenvironments and mechanical stresses direct cell fate in developing tissue. However, how these physical cues affect the tissue morphogenesis remains a major unknown. We explain here that the physical properties of the cell and tissue microenvironment, biomimetically reproduced by using hydrogel, guide the tissue morphogenesis in the developmental submandibular gland (SMG). In particular, the softer gel enhances the bud expansion and cleft formation of SMG, whereas the stiffer gel attenuates them. These morphological changes in SMG tissue are led by soluble factors (FGF7/10) induction regulated by cell traction force derived from the tissue deformation. Our findings suggest that cells sense the mechanics of their surrounding environment and alter their properties for self-organization and the following tissue morphogenesis. Also, physically designed hydrogel material is a valuable tool for producing the biomimetic microenvironment to explore how physical cues affect tissue morphogenesis and to modulate tissue morphogenesis for in vitro tissue synthesis., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Amniotic membrane (AM) has been widely used in the reconstruction of oral epithelial defects. However, whether it is also effective in facilitating tissue formation of salivary gland, an appendix of oral epithelia, has never been explored. To investigate the effects and the underlying mechanism of AM on salivary gland morphogenesis, murine fetal submandibular gland (SMG) explants were cultured on different preparations of AM scaffolds. It was found that, on AM stromal scaffold, SMG demonstrated well-developed branching morphogenesis. Nonetheless, on AM epithelial scaffold, SMG epithelial cell converted to a spindle-shape, lost intercellular connection, changed cytoskeletal organization, and exhibited scattering behaviors. Meanwhile, the integrity of SMG basement membrane was dismantled as well. However, when acellular AM epithelial scaffold was used, cultured SMG demonstrated organized morphology, indicating that AM epithelial component provided specific surface features for SMG morphogenesis. To further investigate AM scaffold morphogenetic effect, it was found hepatocyte growth factor (HGF), an epithelial scattering factor, was expressed abundantly in cultivated AM epithelia. After blocking HGF function of AM, cultured SMG regained branching activity, reorganized cell adhesion and subcellular organization, and reproduced basement membranes. Therefore, AM-derived bioactive factor profoundly influences cell behaviors and structure formation of SMG. Together, this study showed that compositional topography of AM scaffold is important in affecting SMG morphogenesis. By understanding the effects of AM scaffold on SMG morphogenesis, it provides important information for rationally designing and fabricating AM scaffold for salivary gland regeneration., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

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.

On the basis of light and electron microscopic observations of the post natal development of the hamster submandibular gland, granules in the acinar cells showed considerably variations in size and shape, as well as electron density of the peroxidase-positive reaction. The present study shows that secretory granules of the hamster submandibular gland undergo changes of area and of intensity for peroxidase activity 6 months after birth.

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.

Little is known about the presence of the various membrane-located water channels, aquaporins (AQP), during the prenatal and postnatal development of the mouse submandibular salivary gland (SMG). To learn more about AQPs in the developing aspect of salivary glands, we investigated trends in the expression patterns of several AQPs using the embryonic, early postnatal, and young adult mouse SMGs as models. We have chosen AQPs previously found in salivary glands in other animals. Transcripts of AQPs 1, 3, 4, 5, and 8 were detected by reverse transcription-polymerase chain reaction (RT-PCR) and quantified. Aquaporin proteins 1, 3, 4, and 5, but not AQP protein 8, were detected and quantified using western blotting. The various AQPs showed distinct transcript and protein-expression patterns. The change in trends may indicate that the importance of the various AQPs varies throughout the developmental stages in the mouse SMG. Their presence might be related to cell adhesion, migration, proliferation, apoptosis, transepithelial transport, osmosensing, or cell volume regulation; all roles that in the literature are linked to the various AQPs. Overall, this study demonstrates that AQP presentation varies and has a specific expression pattern during the development of mouse SMG. This feature may be important for glandular anatomical and physiological development.

Proteolysis is essential during branching morphogenesis, but the roles of MT-MMPs and their proteolytic products are not clearly understood. Here, we discover that decreasing MT-MMP activity during submandibular gland branching morphogenesis decreases proliferation and increases collagen IV and MT-MMP expression. Specifically, reducing epithelial MT2-MMP profoundly decreases proliferation and morphogenesis, increases Col4a2 and intracellular accumulation of collagen IV, and decreases the proteolytic release of collagen IV NC1 domains. Importantly, we demonstrate the presence of collagen IV NC1 domains in developing tissue. Furthermore, recombinant collagen IV NC1 domains rescue branching morphogenesis after MT2-siRNA treatment, increasing MT-MMP and proproliferative gene expression via beta1 integrin and PI3K-AKT signaling. Additionally, HBEGF also rescues MT2-siRNA treatment, increasing NC1 domain release, proliferation, and MT2-MMP and Hbegf expression. Our studies provide mechanistic insight into how MT2-MMP-dependent release of bioactive NC1 domains from collagen IV is critical for integrating collagen IV synthesis and proteolysis with epithelial proliferation during branching morphogenesis.

Background: Members of the Six family of homeoproteins are expressed in numerous tissues during vertebrate embryogenesis, and are critical regulators of both cell proliferation and survival. Here we report the temporal and spatial expression of Six1 during maturation of the mouse submandibular salivary gland (SSG) from embryonic day 18.5 (E18.5) to postnatal day 28. Additionally, we examine the role of Six1 during SSG development using Six1-deficient mice., Methods: Six1 expression was assessed by reverse transcription-polymerase chain reaction, Western blot, and immunofluorescence. Proliferation was measured by bromodeoxyuridine (BrdU) incorporation index, and apoptosis was evaluated by TUNEL assay., Results: Six1 mRNA and protein levels are high in the epithelial SSG cells at E18.5 and decrease progressively in the postnatal maturing SSG. Although SSGs from Six1(-/-) embryos are significantly smaller than wild type SSGs, the histological structures of the SSG acini and ducts are similar. Six1(-/-) salivary epithelial cells exhibit an intrinsic defect in cell proliferation accompanied by a significant reduction in the Six1 target gene cyclin A1, previously shown to be a critical mediator of Six1-induced proliferation., Conclusion: Our results suggest that the reduction in size of Six1(-/-) SSGs is result of a decrease in cell proliferation during development/maturation.

Scurfy (Foxp3(Sf)/Y), Il2(-/-), and Il2ralpha(-/-) mice are deficient in CD4(+)Foxp3(+) regulatory T cells (Treg), but only the latter two develop inflammation in the submandibular gland (SMG), a critical target of Sjögren's syndrome. In this study, we investigated the reason that SMG of Scurfy (Sf), Sf.Il2(-/-), Sf.Il2ralpha(-/-), and the long-lived Sf.Fas(lpr/lpr) mice remained free of inflammation, even though their lymph node cells induced SMG inflammation in Rag1(-/-) recipients. A strong correlation was observed between the development of the granular convoluted tubules (GCT) of the SMG in these mice and SMG resistance to inflammation. Moreover, GCT development in Sf.Rag1(-/-) mice was not impeded, indicating a role of adaptive immunity. In the Sf.Fas(lpr/lpr) mice, this block was linked to atrophy and inflammation in the accessory reproductive organs. Testosterone treatment restored GCT expression, but did not induce SMG inflammation, indicating GCT is not required for inflammation and additional mechanisms were controlling SMG inflammation. Conversely, oral application of LPS induced SMG inflammation, but not GCT expression. LPS treatment induced up-regulation of several chemokines in SMG with little effect on the chemokine receptors on CD4(+) T cells in Sf mice. Our study demonstrates that Sf mutation affects SMG development through adaptive immunity against accessory reproductive organs, and the manifestation of SMG inflammation in Sf mice is critically controlled through innate immunity.

Branching morphogenesis (BrM) is a basic developmental process for the formation of the lung, kidney, and all exocrine glands, including the salivary glands. This process proceeds as follows. An epithelial downgrowth invaginates into underlying mesenchyme, and forms a cleft at its distal end, which is the site of dichotomous branching and elongation; this process of clefting and elongation is repeated many times at the distal ends of the invading epithelium until the desired final extent of branching is reached. The distal ends of the epithelium differentiate into the secretory endpieces, and the elongated segments become the ducts. This presentation is a brief historical review of studies on BrM during the development of the submandibular gland (SMG).

Renin is responsible for initiating the enzymatic cascade that results in the production of angiotensin II, the major effector molecule of the renin-angiotensin system (RAS). Extensive information on the regulatory region of the renin gene has been derived by transient transfection studies in vitro, particularly using the As4.1 cell line. To verify key factors within the regulatory region of renin in vivo, homologous recombination was used to introduce a green fluorescent protein (GFP) cassette into exon one of the renin gene contained within a 240 kb bacterial artificial chromosome (BAC) to create a construct that has GFP expression controlled by the renin regulatory region (RenGFP BAC). Within the regulatory region of the RenGFP BAC construct we independently deleted the enhancer, as well as mutated the HOX-PBX site within the proximal promoter element. Transgenic lines were generated for each of these BAC constructs and GFP expression was analyzed throughout a spectrum of tissues positive for renin expression including the kidney, adrenal gland, gonadal artery, and submandibular gland. The results described within this manuscript support the interpretation that the renin enhancer is critical for regulating baseline expression where as the Hox/Pbx site is important for the tissue specificity of renin expression.

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.

Many glandular organs are developed by branching morphogenesis, an efficient and ubiquitous process for creating a larger cellular area for metabolic requirement. To regenerate a glandular organ, such as salivary glands, recapitulation of branching processes may be requisite. At present, the roles of biomaterials in regenerative branching have never been thoroughly explored. By culturing the embryonal submandibular gland (SMG) on different substrata, including polyvinyl alcohol (PVA), chitosan, and polycarbonate (PC), this study demonstrated for the first time that chitosan was capable of providing a more preferential environment for salivary gland branch formation. After culturing SMG explants on chitosan membranes, secreted extracellular matrices distributed in a reticular manner and formed thicker fibers beyond the extents of cell attachment, which were not found in PVA and PC. In the subsequent culture of explants just on the used chitosan substrates, these conditioned membranes were able to further enhance SMG branching. The fact that the promoting effects were eliminated with collagenase treatment and type I and type III collagen were identified within the adherent fibrillar extracellular matrix raised the possibility that the stimulating factors were collagen-originated. This indicates that, for SMG, chitosan is a bioactive substratum which enables cells to synthesize and deposit essential extracellular matrix, paving an important way for ensuing branch formation. Accordingly, the current study provides a larger scope to use chitosan as a biomaterial for recapitulating branching, which might be useful for the scaffold design of salivary gland regeneration.

Submandibular glands (SMGs) of 11-week-old mice from four strains, ICR, C57BL/6J, BALB/c, and C3H/HeN were examined by immunohistochemistry for epidermal growth factor (EGF). In addition to sex-related differences in granular convoluted tubules (GCTs), the GCT cells were significantly larger in ICR mice than in other three strains. In males from each of the strains, almost all the GCT cells were strongly positive for EGF. The EGF-positive cells in the females, however, were markedly fewer in number, and were stained weaker in C57BL/6J, BALB/c, and C3H/HeN mice than in ICR mice. The GCT cells and their EGF expression in the F1 progeny from ICR and C3H/HeN strains were approximately intermediate between those of the parent strains of the same sex. T(3) and/or dihydrotestosterone (DHT) enhanced the GCT phenotype in the C3H/HeN mice, and remarkably increased the EGF-positive cells in females. Electron microscopy revealed that gold-labeling of EGF was confined to the secretory granules, and that the GCT cells in females, given T(3) + DHT, had a well-developed Golgi apparatus and net-like RER but few basal infoldings, whereas the equivalent cells in the untreated females had poor RER and prominent basal infoldings. These results suggest that the EGF concentration in SMGs is genetically high in ICR mice and low in other strain mice and that, considering the same response of GCT cells to T(3) and/or DHT between the high and low EGF strains, the low EGF concentrations might be partly caused by a lesser sensitivity of the GCT cells to thyroid hormones.

Glucocorticoids (CORT) are known to promote branching of the epithelial cords during the development of the rat submandibular gland. The aim of this study was to examine the effect of CORT (triamcinolone) on the differentiation of cells forming the terminal tubules in the developing fetal rat submandibular gland and the properties of the secretory granules. Light and electron microscopy showed that the terminal tubules of the glands in the experimental group contained more type III cells, which have been identified as proacinar cells, than those in the control group, whereas the relative number of type I cells, which have been identified as terminal tubule cells, was reduced. Immunoelectron microscopy using an antibody against neonatal submandibular gland secretory protein B (SMGB) revealed the presence of more gold particles over type III cell granules in the experimental group than in the control group. Lectin histochemistry demonstrated more wheat germ agglutinin (WGA)-labeled gold particles over type III cell granules in the experimental group than in the control group. These findings suggest that CORT promote the differentiation of type III cells, and moreover stimulate the production of secretory granules reactive for SMGB and WGA by acting on the terminal tubules of the developing rat submandibular gland., (Copyright 2007 S. Karger AG, Basel.)

To identify stem cells in salivary glands, label-retaining cells (LRCs) were established in rat submandibular glands. Developing and regenerating glands were labeled with bromodeoxyuridine (BrdU). To cause gland regeneration, the glands were injured by duct obstruction. BrdU LRCs were observed in all the parenchymal structures except for the acinus of the glands labeled during regeneration. Among these LRCs, a few, but not many, expressed neither keratin18 (K18; an acinar/duct cell marker) nor alpha-smooth muscle actin (alphaSMA; a myoepithelial cell marker), and thus were putative stem cells. These (K18 and alphaSMA)(neg) LRCs were invariably observed in the intercalated duct and the excretory duct. In the intercalated duct, they were at the proximal end bordering the acinus (the neck of the intercalated duct). Next, to test the above identification, gland extirpation experiments were performed. LRCs were established by labeling developing glands with iododeoxyuridine (IdU) in place of BrdU. Removal of one submandibular gland forced the IdU-LRCs in the remaining gland to divide. They were labeled with chlorodeoxyuridine (CldU). The (K18 and alphaSMA)(neg) LRCs in the neck of the intercalated duct and in the excretory duct did not change in number or in IdU label. The CldU label appeared in these cells and then disappeared. These results indicate that the (K18 and alphaSMA)(neg) LRCs have divided asymmetrically and are thus considered salivary gland stem cells.

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.

The characteristics of the submandibular glands of ageing Wistar rats were studied using light and high-resolution scanning electron microscopy (HRSEM) methods. For light microscopy, the samples were fixed in Bouin solution and embedded in paraffin. Sections were stained with hematoxilin-eosin and Azo-Carmin. For conventional scanning electron microscopy, the tissues were fixed in modified Karnovsky solution, and treated with NaOH solution for 3-4 days. The O-D-O method was used for HRSEM. The samples were fixed in 2% osmium tetroxide, macerated in diluted osmium and dehydrated in an increasing series of ethanol. The samples were dried in a critical point dryer, coated with gold-palladium and examined in a Hitachi high-resolution scanning electron microscope, S-900. The results showed that submandibular glands with lobules are separated by connective tissue septum. The acinar formations and the ducts, revealing the serous and mucous cells were observed. After fracture in liquid nitrogen and treatment with NaOH solution to remove the cellular components, the original disposition of the collagen bundles fibers were revealed corresponding to the round, oval or irregular acinar and ductal structures. In the cytoplasm, organelles such as mitochondria, rough endoplasmic reticulum, Golgi apparatus and serous and mucous secretory granules were observed localized in the apical portion in three-dimensional HRSEM images. The serous secretory granules presented different sizes and shapes showing the modifications which occurred in the ageing rats. The striated duct cells revealed the presence of the secretory cells and mitochondria in parallel disposition. The mitochondrial cristae were noted in three-dimensional aspects. The lumen presented numerous cytoplasmic microprojections. The lumen of excretor ducts are covered by polygonal epithelial cells containing numerous microplicae.

Toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to mouse embryonic teeth, sharing features of early development with salivary glands in common, involves enhanced apoptosis and depends on the expression of epidermal growth factor (EGF) receptor. EGF receptor signaling, on the other hand, is essential for salivary gland branching morphogenesis. To see if TCDD impairs salivary gland morphogenesis and if the impairment is associated with EGF receptor signaling, we cultured mouse (NMRI) E13 submandibular glands with TCDD or TCDD in combination with EGF or fibronectin (FN), both previously found to enhance branching morphogenesis. Explants were examined stereo-microscopically and processed to paraffin sections. TCDD exposure impaired epithelial branching and cleft formation, resulting in enlarged buds. The glands were smaller than normal. EGF and FN alone concentration-dependently stimulated or inhibited branching morphogenesis but when co-administered with TCDD, failed to compensate for its effect. TCDD induced cytochrome P4501A1 expression in the glandular epithelium, indicating activation of the aryl hydrocarbon receptor. TCDD somewhat increased epithelial apoptosis as observed by terminal deoxynucleotidyl transferase (TdT)-mediated nick end-labeling method but the increase could not be correlated with morphological changes. The frequency of proliferating cells was not altered. Corresponding to the reduced cleft sites in TCDD-exposed explants, FN immunoreactivity in the epithelium was reduced. The results show that TCDD, comparably with EGF and FN at morphogenesis-inhibiting concentrations, impaired salivary gland branching morphogenesis in vitro. Together with the failure of EGF and FN at morphogenesis-stimulating concentrations to compensate for the effect of TCDD this implies that TCDD toxicity to developing salivary gland involves reduced EGF receptor signaling.

Salivary gland stem/progenitor cells are thought to be present in intercalated ductal cells, but the fact is unclear. In this study, we sought to clarify if stem/progenitor cells are present in submandibular glands using colony assay, which is one of the stem cell assay methods. Using a low-density culture of submandibular gland cells of neonatal rats, we developed a novel culture system that promotes single cell colony formation. Average doubling time for the colony-forming cells was 24.7 (SD=+/-7.02)h, indicating high proliferative potency. When epidermal growth factor (EGF) and hepatocyte growth factor (HGF) were added to the medium, the number of clonal colonies increased greater than those cultured without growth factors (13.2+/-4.18 vs. 4.5+/-1.73). The RT-PCR and immunostaining demonstrated expressing acinar, ductal, and myoepithelial cell lineage markers. This study demonstrated the presence of the salivary gland stem/progenitor cells that are highly proliferative and multipotent in salivary glands.

Previous studies have shown that the blood vessels supplying the endocrine organs and the mucosa of the intestinal canals change in terms of not only their distribution but also their structure with the development and growth of each organ. We examined changes in the distribution and structure of intralobular blood vessels, including capillaries, throughout the postnatal development of the submandibular gland, an exocrine organ. The mouse submandibular gland from days 0 (birth) to 49 was investigated chronologically and ultrastructurally. The capillaries changed from continuous to fenestrated on day 10, coincident with an increase in the number of acini to more than the number of terminal tubules. The number of sections of intralobular blood vessels per unit area gradually decreased with increasing acinar size and was lowest on day 21 when pups were weaned; the same number was maintained from then on. In contrast with the reduction in the number of intralobular blood vessels, the number of capillary pores appeared to increase gradually. Acinar size increased further till day 28. Capillary pore number also increased further, till day 35, apparently in relation to the increasing acinar size. These findings suggest that the changes in distribution and structure of the intralobular blood vessels in the submandibular gland of the postnatally developing mouse are closely related to the development of the parenchymal cells in preparation for weaning and sexual maturity., (Copyright (c) 2005 Wiley-Liss, Inc.)

SCA-9 cell line was developed from an induced tumor of mouse submandibular gland. We have studied some of the phenotypic characteristics of SCA cells cultured on different matrices. On plastic surface, the cells grow as a monolayer; on matrigel, they form branching structures and tubes, a phenomenon termed branching morphogenesis. EGF and HGF promoted cellular growth and branching morphogenesis which was inhibited by anti-EGF antibodies. We have performed RT-PCR and real-time quantitative RT-PCR of cells grown on plastic surface or on matrigel. Grown on plastic, the cells express EGF and renin 2, but no or only trace amounts of NGF. Growth on matrigel for 24 h resulted in a transient 21-fold increase in EGF mRNA and a 3371-fold increase in renin 2 mRNA. There was no change in NGF mRNA level. SCA-9 cells express mRNAs for receptors for the EGF family of ligands. On plastic, mainly ErbB1 and ErbB2 are expressed. Culture on matrigel resulted in 11-fold increase in mRNA levels for ErbB1 and ErbB2, and a 221-fold and 85-fold increase in the mRNA levels for ErbB3 and ErbB4, respectively. Small interfering RNAs siErbB3 and siErbB4 inhibited the growth of the cells grown on plastic or matrigel. Significant growth inhibition was seen also with siErbB1+siErbB3 and siErbB2+siErbB3. siErbB1 and siErbB2 also inhibited branching morphogenesis. Since SCA cells express EGF and receptors for EGF, EGF acts an autocrine regulator in promoting growth and branching morphogenesis. We conclude that SCA cells provide a useful model to analyze the mechanism of branching morphogenesis and the role of matrix in regulating expression of phenotypic characteristics of cultured cells.

Background: Analyses of Fgf10 and Fgfr2b mutant mice, as well as human studies, suggest that FGF10/FGFR2b signaling may play an essential, nonredundant role during embryonic SMG development. To address this question, we have analyzed the SMG phenotype in Fgf10 and Fgfr2b heterozygous and null mutant mice. In addition, although previous studies suggest that the FGF10/FGFR2b and FGF8/FGFR2c signaling pathways are functionally interrelated, little is known about the functional relationship between these two pathways during SMG development. We have designed in vivo and in vitro experiments to address this question., Results: We analyzed Fgf10 and Fgfr2b heterozygous mutant and null mice and demonstrate dose-dependent SMG phenotypic differences. Hypoplastic SMGs are seen in Fgf10 and Fgfr2b heterozygotes whereas SMG aplasia is seen in Fgf10 and Fgfr2b null embryos. Complementary in vitro studies further indicate that FGF10/FGFR2b signaling regulates SMG epithelial branching and cell proliferation. To delineate the functional relationship between the FGF10/FGFR2b and FGF8/FGFR2c pathways, we compared the SMG phenotype in Fgfr2c+/Delta/Fgf10+/- double heterozygous mice to that seen in wildtype, Fgf10+/- (Fgfr2c+/+/Fgf10+/-) and Fgfr2c+/Delta (Fgfr2c+/Delta/Fgf10+/+) single heterozygous mutant littermates and demonstrate genotype-specific SMG phenotypes. In addition, exogenous FGF8 was able to rescue the abnormal SMG phenotype associated with abrogated FGFR2b signaling in vitro and restore branching to normal levels., Conclusion: Our data indicates that FGF10/FGFR2b signaling is essential for the SMG epithelial branching and histodifferentiation, but not earliest initial bud formation. The functional presence of other endogenous signaling pathways could not prevent complete death of embryonic SMG cells in Fgf10 and Fgfr2b null mice. Though we were able to rescue the abnormal phenotype associated with reduced in vitro FGF10/FGFR2b signaling with exogenous FGF8 supplementation, our results indicate that the FGF10/FGFR2b and FGF8/FGFR2c are nonredundant signaling pathways essential for in vivo embryonic SMG development. What remains to be determined is the in vivo functional relationship between the FGF10/FGFR2b signal transduction pathway and other key signaling pathways, and how these pathways are integrated during embryonic SMG development to compose the functional epigenome.

Post-ganglionic sympathetic neurons express the p75 neurotrophin receptor (p75NTR) and brain-derived neurotrophic factor (BDNF), which together have been implicated in controlling the degree of efferent innervation of peripheral organs [Kohn, J., Aloyz, R.S., Toma, J.G., Haak-Frendscho, M., Miller, F.D. 1999. Functionally Antagonistic Interactions between the TrkA and p75 Neurotrophin Receptors Regulate Sympathetic Neuron Growth and Target Innervation. J. Neurosci. 19, 5393-5408]. To examine this concept further, we developed null mutant mice lacking both p75NTR and BDNF, and assessed whether the loss of this receptor-ligand interaction negatively impacts the degree of sympathetic innervation to various target tissues. Between postnatal days 10 and 14, hearts, urinary bladders, kidneys, and submandibular salivary glands were isolated from p75(-/-)/BDNF-/-, p75-/-, BDNF-/-, and wild type siblings. Sympathetic axons were visualized using tyrosine hydroxylase (TH) immunohistochemistry, and TH protein levels were quantified by immunoblotting. Concerning the sympathetic innervation of the heart, urinary bladder and kidneys, no differences were seen in single and double null mutant mice, as compared with their wild type siblings. Sympathetic innervation of the submandibular salivary gland was, however, increased in both p75-/- and p75(-/-)/BDNF-/- mice over control mice. These results reveal that an absence of p75NTR and/or BDNF expression does not perturb the degree of sympathetic innervation of many peripheral tissues during postnatal development, and that a lack of p75NTR expression may actually enhance the density of these efferent fibers in other target tissues, such as the salivary glands.

Insect mandibular glands are exocrine organs that produce chemical substances known as pheromones that play an important role in intra-specific communication of insects. The mandibular glands of Apis mellifera, which are more highly developed in queens than in workers, present caste-specific polymorphism which seems to be regulated by the juvenile hormone (JH). These glands develop at the pupation stage, during which the titer of JH is higher in queens. In spite of this observation, application recounted here of juvenile hormone on 5th-instar workers' larvae of Apis mellifera did not produce a significant effect on the size of the mandibular glands. Therefore, we may conclude that the response of insect organs to the exogenous application of JH varies according to the type of organ, its developmental program, and its developmental stage, as well as to the amount of hormone applied.

Lectin cytochemistry was performed in vitro on primary cultures from the rat submandibular gland. For this purpose, prepubertal rats (17, 27, 33 days old) of both sexes were used. Several types of medium supplements were tested and it was found that cells survived until 15 days in presence of all medium supplements and extracellular matrix gel. The binding patterns of all FITC/TRITC-labeled lectins, with and without prior sialidase digestion and deacetylation, were analyzed in a confocal laser scanning microscope. In particular, the occurrence of C4 acetylated sialic acid linked to beta-galactose at day 27 and the presence of fucose residues at day 33 indicated that lectin probes applied to cultured cells give results similar to those obtained in intact tissues and can be used as markers of growth and differentiation.

We report the molecular cloning and characterization of SMGC, a major secretory product and a marker of the type I (terminal tubule) cells of the neonatal rat and mouse submandibular gland. SMGC is expressed in the submandibular gland at high levels through postnatal day 20, but in the adult is present only in some intercalated duct cells. Rat and mouse SMGC have deduced molecular weights of 67.8 and 74.4 kDa, respectively, are 37% Ser+Gly+Thr, and contain tandem repeats of between 8 and 60 amino acids. Secreted SMGC visualized by SDS-PAGE and silver staining is 89 kDa in rat and 105 kDa in mouse, although Western blot analyses with anti-SMGC antisera demonstrate multiple additional lower molecular weight forms. Contributions to the heterogeneity of SMGC include alternate splicing, proteolysis and N-glycosylation. Smgc is localized on rat chromosome 7q34-35 and on mouse chromosome 15E3, both immediately upstream of the high molecular weight salivary mucin, Muc19. Amino acid sequence identity between the signal peptides of SMGC, human MUC19 and pig submaxillary mucin suggest that rat and mouse Smgc and Muc19 arose from a single ancestral mucin gene.

We studied the expression and localization of JunD, a component of the transcription factor activator protein-1 (AP-1), in the mouse submandibular gland with immunoblotting and immunohistochemistry. In adult mice, all seven Jun and Fos family members constituting the AP-1 complex were expressed more abundantly in the female gland than in the male gland, and JunD was the most abundant of the members. Immunoreactivity for JunD was localized exclusively in the duct system of the gland, in which it was localized to the nuclei of intercalated duct (ID) cells and a subpopulation of striated duct (SD) cells located adjacent to ID. In contrast, granular convoluted tubule (GCT) cells, which are much more abundant in the male gland, were devoid of JunD. During postnatal development of the male gland, JunD was lost from the duct cells as they differentiated to GCT cells at 3-5 weeks postpartum. When GCT differentiation was induced in adult female gland by testosterone administration, many JunD-negative SD cells were temporarily induced to express JunD after 6-24 hr, but those cells lost JunD as they completely converted to GCT cells by 48 hr. These results suggested that JunD is involved in the differentiation of the duct system of mouse submandibular gland, in which there is crosstalk between the androgen/androgen receptor system and the AP-1 complex.

The laminin alpha1 chain G domain has multiple biological activities. Previously, we identified cell binding sequences in the laminin alpha1 chain G domain by screening 113 synthetic peptide-polystyrene beads for cell attachment activity. Here, we have used a recombinant protein of the laminin alpha1 G domain (rec-alpha1G) and a large set of synthetic peptides to further identify and characterize heparin, cell, and syndecan-4 binding sites in the laminin alpha1 chain G domain. The rec-alpha1G protein promoted both cell attachment and heparin binding (K(D) = 19 nM). Cell attachment to the rec-alpha1G protein was inhibited 60% by heparin and 30% by EDTA. The heparin binding sites were identified by competing heparin binding to the rec-alpha1G protein with 110 synthetic peptides in solution. Only two peptides, AG73 (IC(50) = 147 microM) and AG75 (IC(50) = 206 microM), inhibited heparin binding to rec-alpha1G. When the peptides were compared in a solid-phase heparin binding assay, AG73 showed more heparin binding than AG75. AG73 also inhibited fibroblast attachment to the rec-alpha1G protein, but AG75 did not. Cell attachment to the peptides was studied using peptide-coated plates and peptide-conjugated sepharose beads. AG73 promoted cell attachment in both assays, but AG75 only showed cell attachment activity in the bead assay. Additionally, AG73, but not AG75, inhibited branching morphogenesis of mouse submandibular glands in organ culture. Furthermore, the rec-alpha1G protein bound syndecan-4, and both AG73 and AG75 inhibited this binding. These results suggest that the AG73 and AG75 sites are important for heparin and syndecan-4 binding in the laminin alpha1 chain G domain. These sites may play a critical role in the diverse biological activities involving heparin and syndecan-4 binding.

We investigated the involvement of hepatocyte growth factor (HGF) in salivary gland (SG) branching morphogenesis. The mouse submandibular gland (SMG) starts to develop at embryonic day 11.5-12 (E11.5-E12), and branching morphogenesis occurs in the area between the mandibular bone and tongue between E14 and E16.5. Real-time reverse transcriptase-polymerase chain reaction showed that the expression of the c-met/HGF receptor gene in SMG increased and peaked between E14 and E16.5, concomitant with epithelial branching, and high levels of HGF mRNA were detected in the surrounding mesenchyme at E14-E15.5. Although strong expression of the HGF and c-met transcripts was observed in the tongue muscles, this expression was limited at E13.5-E14.5. Serum-free organ cultures were established, in which SG rudiments that contained SMG and sublingual gland (SLG) primordia (explant 1) and SMG/SLG rudiments with peripheral tissue that included part of the tongue muscle (explant 2) were isolated from E13.5 or E14 embryos. Mesenchyme-free SMG epithelium was obtained by the removal of mesenchymal tissue from explant 1. In the explant 1 and 2 organ cultures, SMG/SLG rudiments showed growth and branching morphogenesis, while mesenchyme-free epithelium failed to grow. When E13.5 or E14 mesenchyme-free epithelium and a recombinant human HGF (rh-HGF) -soaked bead were placed on Matrigel, the epithelium migrated toward the bead and formed branches, while the E13 epithelium failed to branch. The exogenous application of rh-HGF and anti-HGF antibody to the SMG/SLG rudiment cultures resulted in stimulation and inhibition, respectively, of branching morphogenesis. However, the response of E13.5 SMG to rh-HGF was very weak, while the branching of E14 SMG was enhanced strongly by rh-HGF. The branching morphogenesis of SMG was also inhibited by the addition of either antisense HGF or c-met oligodeoxynucleotides to the cultures. The development of SMG in explant 2, which was significantly better than in explant 1, was comparable to that seen in vivo. Moreover, the expression of both HGF and c-Met in the SMG of explant 2 was higher than in the SMG of explant 1. These findings provide the first demonstration that the branching morphogenesis of SMG is regulated by interactions with the surrounding mesenchyme-derived HGF and c-met expression in SMG, which occur concomitant with epithelial branching. The present data also suggest that the HGF that is released transiently from tongue muscles may contribute to the rapid development of SMG at the branching stage., (Copyright 2003 Wiley-Liss, Inc.)

The expression and localization of aquaporins (AQP1-AQP5), members of the water channel family, in the developing rat submandibular gland were analysed using RT-PCR, Northern blotting and immunohistochemistry to explore their relation to the development of this salivary gland. RT-PCR analysis revealed unique expression patterns of each AQP. AQP1 was expressed constitutively during prenatal development, whereas the expression of AQP5 became more intense in the course of development from embryonic day 16.5 (E16) to E20. These expression patterns concurred with the results of Northern blot analysis. AQP3 and AQP4 mRNAs in the prenatal development were not detected in Northern blots, although they were detected by RT-PCR. During postnatal development, AQP5 and AQP1 mRNAs were expressed continuously, but no message for AQP3 or AQP4 was detected. AQP2 mRNA was not detected during either prenatal or postnatal development in this tissue. Immunohistochemical studies revealed that AQP5 was first localized at the apical membrane of proacinar cells at E18, and then became clearly distributed at the apical membrane of acinar cells in accordance with the differentiation and establishment of the mature acini. In addition, some vasculature also showed immunoreactivity for AQP5. AQP1 was immunolocalized in the blood vessels, including capillaries, of the gland throughout development. These observations suggest the existence of transcriptional regulation of rat AQP5, which is one of the most probable regulators of saliva production and secretion, during the establishment of the functional submandibular salivary gland.

The function of the mouse submaxillary gland/prolactin inducible protein (mSMGP/mPIP), the homologue of the human gross cystic disease fluid protein 15 (GCDFP-15)/prolactin inducible protein (hPIP) remains unknown. The human gene, normally expressed in apocrine glands of healthy individuals, is aberrantly expressed in human breast cancers where it is regulated by hormones including androgens, and in prostate cancers. We have previously reported that in the adult mouse and rat, gene expression is tissue-specific for the salivary and lacrimal glands, and is hormonally regulated. In this study, we examine the endogenous pattern of mouse SMGP/PIP (mSMGP/mPIP) gene expression in mid- and late-embryonic, and in early postnatal development. Gene expression was analyzed by RT-PCR followed by Southern blot analysis, and by in situ hybridization. Gene expression was detected in the submandibular gland as early as embryonic day 14 (E14), a period that coincides with the initiation of submandibular gland development in the embryo, suggesting that mSMGP/mPIP may have a functional role in the developing gland. Nearing the end of gestation, E18, mSMGP/mPIP transcripts were localized in the proacinar cells of the gland, and gene expression continued to be maintained following birth. In addition, during early postnatal development, mSMGP/mPIP gene expression was detected in the other two major salivary glands, the sublingual and parotid, as well as in the lacrimal gland and in reproductive tissues. In the prostate, gene expression was turned off by 10 weeks of age. The spatial and temporal pattern of the mSMGP/mPIP gene expression, in addition to our recent demonstration that mSMGP/mPIP is found in mouse saliva and can bind bacteria, suggest that this protein may have a protective role in the mouse.