Development Of Submandibular Gland Research Articles

Research progress on Hippo signaling pathway effector molecules in rheumatic immune system diseases.

The core components of the Hippo signaling pathway encompass upstream regulatory molecules, core kinase cascade complexes, and downstream transcriptional regulation complexes. This pathway modulates cellular behaviors by influencing the effector molecules of its core components and plays a pivotal role in immune regulation. Effector molecules,such as Yes-associated protein (YAP), transcriptional coactivator with PDZ-binding motif (TAZ), transcriptional enhanced associate domain transcriptional factor (TEAD), monopolar spindle-one binder (MOB1), large tumor suppressor (LATS), can stimulate fibroblast-like synovial cell migration and invasion in rheumatoid arthritis, regulate osteoarthritis disease progression, promote pathological new bone formation in ankylosing spondylitis, sustain submandibular gland development while delaying Sjogren's syndrome progression, mediate alpha-smooth muscle actin in systemic sclerosis, and refine the regulation of target genes associated with pulmonary fibrosis. This article provides an overview of the regulatory mechanisms involving Hippo signaling pathway-related effector molecules in the pathogenesis and progression of rheumatic immune system diseases, to serve as a reference for exploring novel therapeutic targets of rheumatic immune system diseases.

Expression of glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide in the rat submandibular gland is influenced by pre- and post-natal high-fat diet exposure.

Background: Incretins, i.e., glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) promote insulin secretion to reduce postprandial blood sugar. Previous studies found incretins in the salivary glands. However, the role of GLP-1 and GIP in the submandibular gland (SMG) is unclear. This study investigates the effects of a high-fat diet (HFD) on the expression of GLP-1 and GIP throughout the development of rat SMG. Methods: Pregnant 11-week-old Wistar rats were divided into two groups: those fed on a standard diet (n = 5) and those fed on a HFD (n = 5). From day 7 of pregnancy and throughout the lactation period, all the rats were fed on either a chow diet or HFD. The newborns were divided into four subgroups (n = 6): standard diet males (SM), HFD males (HM), standard diet females (SF), and HFD females (HF). The SMGs of 3- and 10-week-old rats from each subgroup were collected under general anesthesia. Moreover, body weight, food intake, and fasting blood sugar were measured. The mRNA expression of GLP-1 and GIP was quantified, and the localization was observed using immunohistochemistry (p < 0.05). Results: GLP-1 mRNA expression was statistically significantly more upregulated in HM than in HF at 3 weeks. Moreover, GLP-1 mRNA expression was significantly higher in HM than in both SM and HF at 10weeks. Although a decreasing trend was observed in GIP mRNA expression in both 3- and 10-week-old rats fed on a HFD, a significant difference between HM and SM only occurred at 3weeks. Furthermore, the GIP mRNA expression of HM was lower than that of HF at 10weeks. Immunohistochemical staining revealed GLP-1 and GIP expression mainly in the SMG duct system. Moreover, vacuolated cytoplasm in the duct was observed in rats fed on a HFD. Conclusion: Exposure to HFD during pre- and post-natal periods increased GLP-1 mRNA expression in the SMGs of male rats. However, GIP expression decreased following the HFD in male newborns. Furthermore, a decreasing trend of GIP mRNA expression was observed in male newborns after HFD feeding. Sex influenced incretin hormones secretion and obesity-related conditions. HFD during pre- and post-natal periods reprograms the epigenome, contributing to subsequent disease development.

Developmental function of Piezo1 in mouse submandibular gland morphogenesis.

Mechanically activated factors are important in organogenesis, especially in the formation of secretory organs, such as salivary glands. Piezo-type mechanosensitive ion channel component 1 (Piezo1), although previously studied as a physical modulator of the mechanotransduction, was firstly evaluated on its developmental function in this study. The detailed localization and expression pattern of Piezo1 during mouse submandibular gland (SMG) development were analyzed using immunohistochemistry and RT-qPCR, respectively. The specific expression pattern of Piezo1 was examined in acinar-forming epithelial cells at embryonic day 14 (E14) and E16, which are important developmental stages for acinar cell differentiation. To understand the precise function of Piezo1 in SMG development, siRNA against Piezo1 (siPiezo1) was employed as a loss-of-function approach, during invitro organ cultivation of SMG at E14 for the designated period. Alterations in the histomorphology and expression patterns of related signaling molecules, including Bmp2, Fgf4, Fgf10, Gli1, Gli3, Ptch1, Shh, and Tgfβ-3, were examined in acinar-forming cells after 1 and 2days of cultivation. Particularly, altered localization patterns of differentiation-related signaling molecules including Aquaporin5, E-cadherin, Vimentin, and cytokeratins would suggest that Piezo1 modulates the early differentiation of acinar cells in SMGs by modulating the Shh signaling pathway.

Analyses of lncRNAs, circRNAs, and the Interactions between ncRNAs and mRNAs in Goat Submandibular Glands Reveal Their Potential Function in Immune Regulation.

As part of one of the main ruminants, goat salivary glands hardly secrete digestive enzymes, but play an important role in immunity. The immune function of goat salivary glands significantly changes with age, while the expression profile and specific function of non-coding RNA during this process are unknown. In this study, transcriptome sequencing was performed on submandibular gland (SMG) tissues of 1-month-old, 12-month-old, and 24-month-old goats, revealing the expression patterns of lncRNA and circRNA at different ages. A total of 369 lncRNAs and 1699 circRNAs were found to be differentially expressed. Functional enrichment analyses showed that the lncRNA regulated target mRNAs and circRNA host genes were significantly enriched in immune-related GO terms and pathways. CeRNA network analysis showed that the key differentially expressed circRNAs and lncRNAs mainly regulate the key immune-related genes ITGB2, LCP2, PTPRC, SYK, and ZAP70 through competitive binding with miR-141-x, miR-29-y, and chi-miR-29b-3p, thereby affecting the natural killer cell-mediated cytotoxicity pathway, the T cell receptor signaling pathway, and other immune-related pathways. It should be noted that the expression of key circRNAs, lncRNAs, and key immune-related genes in goat SMGs decreased significantly with the growth of the goat. This is the first reporting of lncRNAs, circRNAs, and ceRNA network regulation in goat SMGs. Our study contributes to the knowledge of changes in the expression of non-coding RNAs during SMG development in goats and provides new insights into the relationship between non-coding RNAs and salivary gland immune function in goats.

The Semaphorin 3A-AKT axis-mediated cell proliferation in salivary gland morphogenesis and adenoid cystic carcinoma pathogenesis

We recently demonstrated that Semaphorin 3 A (Sema3A), the expression of which is negatively regulated by Wnt/β-catenin signaling, promotes odontogenic epithelial cell proliferation, suggesting the involvement of Sema3A in tooth germ development. Salivary glands have a similar developmental process to tooth germ development, in which reciprocal interactions between the oral epithelium and adjacent mesenchyme proceeds via stimulation with several growth factors; however, the role of Sema3A in the development of salivary glands is unknown. There may thus be a common mechanism between epithelial morphogenesis and pathogenesis; however, the role of Sema3A in salivary gland tumors is also unclear. The current study investigated the involvement of Sema3A in submandibular gland (SMG) development and its expression in adenoid cystic carcinoma (ACC) specimens. Quantitative RT-PCR and immunohistochemical analyses revealed that Sema3A was expressed both in epithelium and in mesenchyme in the initial developmental stages of SMG and their expressions were decreased during the developmental processes. Loss-of-function experiments using an inhibitor revealed that Sema3A was required for AKT activation-mediated cellular growth and formation of cleft and bud in SMG rudiment culture. In addition, Wnt/β-catenin signaling decreased the Sema3A expression in the rudiment culture. ACC arising from salivary glands frequently exhibits malignant potential. Immunohistochemical analyses of tissue specimens obtained from 10 ACC patients showed that Sema3A was hardly observed in non-tumor regions but was strongly expressed in tumor lesions, especially in myoepithelial neoplastic cells, at high frequencies where phosphorylated AKT expression was frequently detected. These results suggest that the Sema3A-AKT axis promotes cell growth, thereby contributing to morphogenesis and pathogenesis, at least in ACC, of salivary glands.

Downregulation of ten-eleven translocation-2 triggers epithelial differentiation during organogenesis

DNA methylation of cytosine bases is a major epigenetic modification that regulates gene expression and vertebrate development. The ten-eleven translocation (TET) enzymes oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), and active DNA demethylation influences gene expression specific to each developmental stage, although recent reports have shown that TET also has a non-catalytic function. In fetal mice, the epithelium in the submandibular gland (SMG) buds as a derivative of the oral cavity at embryonic day 11 (E11) and, by E15, it begins to differentiate into the salivary epithelium, which expresses water-channel aquaporin 5 (AQP5). The functional differentiation of the SMG epithelium can be regulated epigenetically, but how TET enzymes contribute is largely unknown. Here, we used several techniques, including hydroxymethylated DNA immunoprecipitation qPCR and histological analysis, to examine the changes in 5hmC levels and AQP5 and TET expression during SMG development. We found that 5hmC levels and AQP5 expression increased in the E15 SMG epithelium, while TET2 expression in the terminal buds decreased at E15. In agreement with the in vivo observations, Tet2 inhibition ex vivo led to the upregulation of AQP5 expression in terminal buds of the SMG epithelium. These results suggest that the downregulation of TET2 expression at E15 is a critical epigenetic event that establishes the epithelial fate for functional SMGs during development.

Single-cell RNA sequencing reveals PDFGRα+ stromal cell subpopulations that promote proacinar cell differentiation in embryonic salivary gland organoids.

Stromal cells can direct the differentiation of epithelial progenitor cells during organ development. Fibroblast growth factor (FGF) signaling is essential for submandibular salivary gland development. Through stromal fibroblast cells, FGF2 can indirectly regulate proacinar cell differentiation in organoids, but the mechanisms are not understood. We performed single-cell RNA-sequencing and identified multiple stromal cell subsets, including Pdgfra+ stromal subsets expressing both Fgf2 and Fgf10. When combined with epithelial progenitor cells in organoids, magnetic-activated cell-sorted PDGFRα+ cells promoted proacinar cell differentiation similarly to total stroma. Gene expression analysis revealed that FGF2 increased the expression of multiple stromal genes, including Bmp2 and Bmp7. Both BMP2 and BMP7 synergized with FGF2, stimulating proacinar cell differentiation but not branching. However, stromal cells grown without FGF2 did not support proacinar organoid differentiation and instead differentiated into myofibroblasts. In organoids, TGFβ1 treatment stimulated myofibroblast differentiation and inhibited the proacinar cell differentiation of epithelial progenitor cells. Conversely, FGF2 reversed the effects of TGFβ1. We also demonstrated that adult salivary stromal cells were FGF2 responsive and could promote proacinar cell differentiation. These FGF2 signaling pathways may have applications in future regenerative therapies.

Differential regulation of transposable elements (TEs) during the murine submandibular gland development

The submandibular gland (SG) is a relatively simple organ formed by three cell types: acinar, myoepithelial, and an intricate network of duct-forming epithelial cells, that together fulfills several physiological functions from assisting food digestion to acting as an immune barrier against pathogens. Successful SG organogenesis is the product of highly controlled and orchestrated genetic and transcriptional programs. Mounting evidence links Transposable Elements (TEs), originally thought to be selfish genetic elements, to different aspects of gene regulation in mammalian development and disease. To our knowledge, the role of TEs during murine SG organogenesis has not been studied. Using novel bioinformatic tools and publicly available RNA-Seq datasets, our results indicate that a significant number of genic and intergenic TEs are differentially expressed during the SG development. Furthermore, changes in expression of specific TEs correlated with that of genes involved in cellular division and differentiation, critical aspects for SG maturation. Altogether, we propose that TEs modulate gene networks that operate during SG development.

Inhibition of Aurora Kinase B activity disrupts development and differentiation of salivary glands

Little is known about the key molecules that regulate cell division during organogenesis. Here we determine the role of the cell cycle promoter aurora kinase B (AURKB) during development, using embryonic salivary glands (E-SGs) as a model. AURKB is a serine/threonine kinase that regulates key events in mitosis, which makes it an attractive target for tailored anticancer therapy. Many reports have elaborated on the role of AURKB in neoplasia and cancer; however, no previous study has shown its role during organ development. Our previous experiments have highlighted the essential requirement for AURKB during adult exocrine regeneration. To investigate if AURKB is similarly required for progression during embryonic development, we pharmacologically inhibited AURKB in developing submandibular glands (SMGs) at embryonic day (E)13.5 and E16.5, using the highly potent and selective drug Barasertib. Inhibition of AURKB interfered with the expansion of the embryonic buds. Interestingly, this effect on SMG development was also seen when the mature explants (E16.5) were incubated for 24 h with another cell cycle inhibitor Aphidicolin. Barasertib prompted apoptosis, DNA damage and senescence, the markers of which (cleaved caspase 3, γH2AX, SA-βgal and p21, respectively), were predominantly seen in the developing buds. In addition to a reduction in cell cycling and proliferation of the epithelial cells in response to AURKB inhibition, Barasertib treatment led to an excessive generation of reactive oxygen species (ROS) that resulted in downregulation of the acinar differentiation marker Mist1. Importantly, inhibition of ROS was able to rescue this loss of identity, with Mist1 expression maintained despite loss of AURKB. Together, these data identify AURKB as a key molecule in supporting embryonic development and differentiation, while inhibiting senescence-inducing signals during organogenesis.

Cell signaling regulation in salivary gland development.

The mammalian salivary gland develops as a highly branched structure designed to produce and secrete saliva. This review focuses on research conducted on mammalian salivary gland development, particularly on the differentiation of acinar, ductal, and myoepithelial cells. We discuss recent studies that provide conceptual advances in the understanding of the molecular mechanisms of salivary gland development. In addition, we describe the organogenesis of submandibular glands (SMGs), model systems used for the study of SMG development, and the key signaling pathways as well as cellular processes involved in salivary gland development. The findings from the recent studies elucidating the identity of stem/progenitor cells in the SMGs, and the process by which they are directed along a series of cell fate decisions to form functional glands, are also discussed. Advances in genetic tools and tissue engineering strategies will significantly increase our knowledge about the mechanisms by which signaling pathways and cells establish tissue architecture and function during salivary gland development, which may also be conserved in the growth and development of other organ systems. An increased knowledge of organ development mechanisms will have profound implications in the design of therapies for the regrowth or repair of injured tissues. In addition, understanding how the processes of cell survival, expansion, specification, movement, and communication with neighboring cells are regulated under physiological and pathological conditions is critical to the development of future treatments.

Canonical Wnt signaling regulates branching morphogenesis of submandibular gland by modulating levels of lama5.

Branching morphogenesis is a crucial developmental mechanism for the formation of the typical bush-like structure of the submandibular gland (SMG). However, the detailed mechanism underlying this process remains to be fully understood. Here, we have investigated whether cross-talk may exist between the Wnt/beta-catenin signaling pathway and lama5 during the branching process in SMG development. An embryonic mouse SMG organ culture model was established, and the validity of this model was confirmed. The roles and possible interactions of the Wnt/beta-catenin signaling pathway, FGF signaling, and lama5 in the branching process were investigated by morphogenesis assays and gene expression patterns. Here, we show that the E12 or E13 SMG organ culture model can be used as an ideal approach to study the process of branching morphogenesis. Our branching morphogenesis assay revealed that the epithelial branching process can be promoted when the canonical Wnt pathway is inhibited and significantly suppressed when the wnt pathway is over activated. Further experiments indicated that FGF signaling most likely acts upstream as a negative regulator of the canonical Wnt pathway during the branching process, whose effect could be partially reversed by Wnt3a. Finally, we show that Wnt/beta-catenin signaling regulates branching morphogenesis through Lama5. We conclude that the Wnt/beta-catenin signaling pathway acting downstream of FGF signaling can serve as a negative regulatory mechanism in the process of SMG branching morphogenesis through Lama5.

Morphological differences between regenerating salivary glands after salivary gland duct ligation and embryonic salivary glands

BackgroundMost animals and organs have regenerative capabilities. Whether regeneration is a developmental process or a distinct phenomenon that is independent of development is debatable. MethodWe examined the differences between developing and regenerating salivary glands using duct-ligation models. We performed morphological analyses comparing submandibular gland regeneration and development. To reveal the proliferation processes that occur during salivary gland regeneration and development, we counted the number of Ki67-positive cells over time. In addition, we examined the expression of the following markers: aquaporin 5, smooth muscle actin, cytokeratin 7, and tubulin beta 3. ResultThe proliferation patterns seen during regeneration differed from those observed during development. Different salivary gland marker expression patterns were seen during development and regeneration. ConclusionThis study showed that regenerating salivary glands do not follow the same growth process as developing salivary glands.

Loss of the disease-associated glycosyltransferase Galnt3 alters Muc10 glycosylation and the composition of the oral microbiome

Loss of the disease-associated glycosyltransferase Galnt3 alters Muc10 glycosylation and the composition of the oral microbiome

Initial stages of development of the submandibular gland (human embryos at 5.5-8 weeks of development).

The aim of this study was to determine the main stages of submandibular salivary gland development during the embryonic period in humans. In addition, we studied submandibular salivary gland development in rats on embryonic days 14-16 and expression in the submandibular salivary gland region with the monoclonal antibody HNK-1. Serial sections from 25 human embryos with a greatest length ranging from 10 to 31mm (Carnegie stages 16-23; weeks 5.5-8 of development) and Wistar rats of embryonic days (E) 14-16 were analysed with light microscopy. Five stages of submandibular salivary gland development were identified. The prospective stage (1), between weeks 5.5 and early week 6, is characterized by a thickening of the epithelium of the medial paralingual groove in the floor of the mouth corresponding to the primordium of the submandibular salivary gland parenchyma. At this stage, the primordium of the parasympathetic ganglion lies below the lingual nerve. The primordium of the submandibular salivary gland parenchyma is observed in rats on E14 in the medial paralingual groove with mesenchymal cells, underlying the lingual nerve. These cells are HNK-1-positive, corresponding to the primordium of the parasympathetic ganglion. The bud stage (2), at the end of week 6 in humans and on E15 in rats, is characterized by the proliferation and invagination of the epithelial condensation, surrounded by an important condensation of the mesenchyme. The pseudoglandular stage (3) at week 6.5 is characterized by the beginning of the formation of lobes in the condensed mesenchyme. The canalicular stage (4), between week 7 and 7.5, is characterized by the appearance of a lumen in the proximal part of the submandibular duct. The innervation stage (5) occurs during week 8, with the innervation of the submandibular and interlobular ducts. Nervous branches arriving from the parasympathetic ganglion innervate the glandular parenchyma. Numerous blood vessels are observed nearby. Our results suggest that submandibular salivary gland development requires interactions among epithelium, mesenchyme, parasympathetic ganglion and blood vessels.

Regulation of Transforming Growth Factor-Alpha Expression by Thyroid Hormone during the Post-Natal Development of Rat Submandibular and Sublingual Salivary Glands

Regulation of Transforming Growth Factor-Alpha Expression by Thyroid Hormone during the Post-Natal Development of Rat Submandibular and Sublingual Salivary Glands

Postnatal changes in the development of rat submandibular glands in offspring of diabetic mothers: Biochemical, histological and ultrastructural study.

Development and maturation of submandibular salivary glands are influenced by intrauterine diabetic environment. Several studies investigated the effects of diabetes on the salivary glands. However, the effects of maternal diabetes on the submandibular glands of the offspring was not properly examined. Therefore, the present study was designed to describe the changes in the development of the submandibular glands of the offspring of diabetic mothers. The submandibular glands of the offspring of Streptozotocin (STZ)-induced diabetic female rats were examined at two and four weeks after birth. Detection of mRNA demonstrated that maternal diabetes affects the level of different indicators. The reduction of expression of epidermal growth factor (EGF); a protein mitogen, cytokeratin 5 (CK5); an epithelial cell progenitor, CK7 and aquaporin 5 (AQP5); differentiation markers and B cell lymphoma 2 (Bcl2); an antiapoptotic marker were found. Increase in Bcl2-associated X protein (Bax); an apoptotic marker was detected. These changes indicate their effects on saliva secretion, glands tumorigenesis, growth of normal oral flora and oral microbes, with decreased protein synthesis and production of xerostomia and dental caries. Loss of normal glandular architecture, significant increase in fibrosis, by the detection of collagen fibers, and stagnation of secretory granules were found with atrophic changes in the acinar cells. Marked defect of polysaccharides in the acinar cells, denoting functional changes, was manifested by significant reduction of the intensity of periodic acid-Schiff (PAS) reaction. The positive immunoreactivity of caspase-3, denoting cellular apoptosis, and minimal reaction of alpha-smooth muscle actin (α SMA) and proliferating cell nuclear antigen (PCNA) were evident in the offspring of diabetic mothers. We conclude that maternal diabetes produces degenerative effects in the structure and function of the submandibular salivary glands of the offspring, reflecting possible influences on their secretory activity affecting oral and digestive health.

Issue Information

COVER PHOTOGRAPH: Whole mount co‐immunostaining for anti‐Tubb3 (green) and anti‐Krt8 (red) on e14.5 heads in which primary cilia have been conditionally knocked out in neural crest cells (Kif3af/f;Wnt1‐Cre). From: A novel role for cilia‐dependent Sonic hedgehog signaling during submandibular gland development; Kelsey H. Elliotta, Grethel Millingtona and Samantha A. Brugmann; Developmental Dynamics 247:pp 818–831

A novel role for cilia-dependent sonic hedgehog signaling during submandibular gland development.

Submandibular glands (SMGs) are specialized epithelial structures which generate saliva necessary for mastication and digestion. Loss of SMGs can lead to inflammation, oral lesions, fungal infections, problems with chewing/swallowing, and tooth decay. Understanding the development of the SMG is important for developing therapeutic options for patients with impaired SMG function. Recent studies have suggested Sonic hedgehog (Shh) signaling in the epithelium plays an integral role in SMG development; however, the mechanism by which Shh influences gland development remains nebulous. Using the Kif3af/f ;Wnt1-Cre ciliopathic mouse model to prevent Shh signal transduction by means of the loss of primary cilia in neural crest cells, we report that mesenchymal Shh activity is necessary for gland development. Furthermore, using a variety of murine transgenic lines with aberrant mesenchymal Shh signal transduction, we determine that loss of Shh activity, by means of loss of the Gli activator, rather than gain of Gli repressor, is sufficient to cause the SMG aplasia. Finally, we determine that loss of the SMG correlates with reduced Neuregulin1 (Nrg1) expression and lack of innervation of the SMG epithelium. Together, these data suggest a novel mechanistic role for mesenchymal Shh signaling during SMG development. Developmental Dynamics 247:818-831, 2018. © 2018 Wiley Periodicals, Inc.

Bcl-2 expression is essential for development and normal physiological properties of tooth hard tissue and saliva production

BackgroundApoptosis plays a fundamental role in appropriate tissue development and function. Although expression of Bcl-2 has been reported during tooth and submandibular gland (SMG) development, the physiological role Bcl-2 plays during these processes has not been addressed. This study was performed to evaluate the impact of Bcl-2 expression on the formation and properties of tooth hard tissue, and saliva production. MethodsTwenty-four mice (12 males and 12 females) were divided into three groups of eight (n=8): group A (Bcl-2 +/+), group B (Bcl-2 +/-), and group C (Bcl-2 -/-) and subjected to micro-CT analyses. The mineral content of first molars was analyzed by X-Ray diffraction (XRD) and scanning electron microscopy (SEM) color dot map. The surface microhardness was determined by Vickers test on labial surfaces of incisors. Saliva was collected from different groups of mice after subcutaneous injection of pilocarpine. ResultsSamples from Bcl-2 -/- mice showed significantly smaller micro-CT values, lower and poor crystallinity of hydroxyapatite (HA), and lowest surface micro hardness. SMG from Bcl-2 -/- mice showed remarkable reduction in size, consistent with reduced saliva accumulation. ConclusionsThe absence of Bcl-2 expression in SMG did not affect the expression of other Bcl-2 family members. Thus, Bcl-2 expression influence on the formation and properties of tooth hard tissue, and saliva accumulation. SignificanceBcl-2 expression has a significant impact on the mineralogical content of enamel crystals of tooth structure. Lack of Bcl-2 expression led to impaired production of enamel ACP crystals.

Immunohistochemical localization and expression of TRPs in rat submandibular gland development

Immunohistochemical localization and expression of TRPs in rat submandibular gland development