A mesenchymal to epithelial switch in Fgf10 expression specifies an evolutionary-conserved population of ionocytes in salivary gland
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ABSTRACT: Salivary glands are essential structures that secrete saliva to the oral cavity and maintain oral health. Development of salivary glands in mice and humans is controlled by mesenchymally expressed fibroblast growth factor-10 (FGF10). Using single cell RNA-seq atlas of the salivary gland and a tamoxifen inducible Fgf10CreERT2:R26-tdTomato mouse we show that FGF10pos cells are exclusively mesenchymal until postnatal day 5 (P5), but after P7, there is a switch in expression and only epithelial FGF10pos cells are observed after P15. Further RNAseq analysis of sorted mesenchymal and epithelial FGF10pos cells shows that the epithelial FGF10pos populations express the hallmark of ancient ionocyte signature Foxi1, Foxi2, Ascl3 and the cystic fibrosis transmembrane conductance regulator (Cftr). We propose that epithelial FGF10pos cells are specialized salivary gland ionocytes that are important for the ionic modification of saliva. In addition, they maintain FGF10-dependent glands homeostasis via communication with FGFR2b-expressing epithelial progenitor and myoepithelial cells
Project description:Salivary glands produce saliva and play essential roles in digestion and oral health. Pluripotent stem cell-derived (PSC) organoids provide a powerful platform for studying salivary gland development and developing new regenerative therapy. The previous protocol of PSC-derived salivary gland organoids required complicated manufacturing processes, which hampered the organoids for basic research and clinical application.Here, by mimicking the regulatory mechanism of developing salivary glands, we reported the differentiation of induced embryonic salivary glands (iE-SGs) from mouse embryonic stem cells by step-wise treatment of retinoic acid and FGF10. We showed that the iE-SGs recapitulated early morphogenetic events, including the thickening and invagination of the salivary gland placode, and then formed initial buds. The iE-SGs also differentiated into developing ducts structures and could develop to striated and excretory ducts when transplanted in vivo. RNA- seq revealed that iE-SGs had gene expression profiles similar to mouse embryonic SMGs. Thus, our study provided an easy and safe method to generate iE-SGs and offered possibilities for studying events during salivary gland morphogenesis in vitro
Project description:Chronic graft-versus-host disease (cGVHD) targets include the oral mucosa and salivary glands after allogeneic hematopoietic stem cell transplant (HSCT). Without incisional biopsy, no diagnostic test exists to confirm oral cGVHD. Consequently, therapy is often withheld until severe manifestations develop. This proteomic study examined saliva and human salivary gland for a biomarker profile at first onset of oral cGVHD prior to initiation of topical steroid therapy. Whole saliva collected at onset of biopsy-proven oral GVHD was assessed using liquid chromatography coupled tandem mass spectrometry with identification of 569 proteins, of which 77 significantly changed in abundance. ZG16B, a secretory lectin protein, was reduced 2-fold in oral cGVHD saliva (p < 0.05), and significantly decreased in salivary gland secretory cells affected by cGVHD. Single-cell RNAseq analysis of healthy MSG localized ZG16B expression to two discreet acinar cell populations. Reduced expression of ZG16B may indicate specific cGVHD activity, general salivary gland dysfunction.
Project description:Salivary glands that produce and secret saliva, which is essential for lubrication, digestion, immunity, and oral homeostasis, consist of diverse cells. The long-term maintenance of diverse salivary gland cells in organoids remains problematic. Here, we established long-term murine salivary gland organoids from 3 major salivary glands, including parotid gland (PG), submandibular gland (SMG), and sublingual gland (SLG). Murine salivary gland organoids expressed gland-specific genes and proteins of acinar, myoepithelial, and duct cells. Organoids were maintained in growth media (named GEM) and further underwent differentiation in differentiation media (named DAM). Our study will provide an experimental platform for the exploration of mechanisms involvled in tissue regeneration, development, or several salivary gland diseases.
Project description:Salivary glands that produce and secret saliva, which is essential for lubrication, digestion, immunity, and oral homeostasis, consist of diverse cells. Maintenance of diverse salivary gland cells in organoids remains problematic. Here, we established human salivary gland organoids, which is composed of multiple cellular subsets, from 3 major salivary glands, including parotid gland (PG), submandibular gland (SMG), and sublingual gland (SLG). Human salivary gland organoids expressed gland-specific genes and proteins of acinar, myoepithelial, and duct cells. Organoids were maintained in growth media (named GEM) and further underwent differentiation in differentiation media (named DAM). Our study will provide an experimental platform for the exploration of mechanisms involvled in tissue regeneration, development, or several salivary gland diseases.
Project description:Saliva aids in the predigestion of food and perception of taste. It helps to maintain the integrity of the mineralized tooth and epithelial surfaces in the mouth, and shields the oro-digestive tract from environmental hazards and invading pathogens. Although salivary glands and saliva fluid are biologically and functionally inseparable, they have thus far been investigated as separate entities. To bridge this gap, we performed an integrative analysis of the transcriptome of 27 samples collected from the major human adult and fetal major salivary glands - submandibular, sublingual, and parotid - along with mass-spectrometry-based saliva proteome data and immunohistochemical localization in glandular tissue. Our results suggest that functional maturation at the transcriptome level occurs late in gland development, and is driven mainly by the transcription of genes that code for secreted saliva proteins. We further provide evidence that protein dosage of the most abundant salivary proteins secreted by the salivary glands is predominantly regulated at the transcriptome level. Finally, we demonstrate distinct transcriptomic profiles of each major salivary gland type that reveal functional specialization and will aid in future clinical analyses. Our study provides the hitherto most comprehensive RNAseq dataset of healthy salivary glands in humans, thus establishing a robust framework for deeper studies of saliva and salivary gland biology, development, and evolution, ultimately paving the way for better understanding the importance of these craniofacial secretory organs in health and their malfunctions in disease.
Project description:We performed this experiment to identify the primary target of Fgf10 in lung development. We used a double transgenic system to either attenuate FGFR2b ligands Rosa26rtTAtet(O)solubleR2b based on the use of doxycycline we generated the samples after 6 and 9 hours of doxycyline induction, or to increase Fgf10 using Rosa26rtTAtet(O)fgf10 double transgenic system we generated samples after 9 of doxycycline induction, controls lungs are provided
Project description:Saliva (oral fluids) is an emerging biofluid poised for clinical diseases detection. Although the rationale for oral diseases applications (e.g. oral cancer) is clear, the rationale and relationship between systemic diseases and saliva biomarkers are unknown. In this study, we used mouse models of melanoma and non-small cell lung cancer and compared the transcriptome biomarker profiles of tumor-bearing mice to those of control mice. Microarray analysis showed that salivary transcriptomes were significantly altered in tumor-bearing mice vs. controls. Analysis of the transcriptomes in the mouse tumors, serum, salivary glands and saliva revealed that salivary biomarkers have multiple origins. Furthermore, we identified that the expression of two groups of significantly altered transcription factors Runx1, Mlxipl, Trim30 and Egr1, Tbx1, Nr1d1 in melanoma-bearing mice that can potentially be responsible for 82.6% of the up-regulated genes expression and 62.5% of the down-regulated gene expression in the mice saliva, respectively. We also confirmed that the ectopic production of nerve growth factor (NGF) in the melanoma tumor tissue as a tumor-released mediator that can induce expression of the transcription factor Egr-1 in the salivary gland. Taken together, our data support the conclusion that upon systemic disease development, a disease-specific change occurs in the salivary biomarker profile. Although the origins of the disease-specific salivary biomarkers are both systemic and local, stimulation of salivary gland by mediators released from remote tumors play an important role in regulating the salivary surrogate biomarker profiles.
Project description:MyD88 is an important adaptor protein for signal transduction downstream of Toll-like receptors and TACI, receptors for regulation of innate immunity and B cell responses, respectively. The surfaces of oral mucosa are protected from infections by antimicrobial proteins and natural immunoglobulins that are constantly secreted in saliva, serving as principal innate immune defense in the oral cavity. Although MyD88-mediated signaling has a regulatory role in the intestinal mucosal immunity, its specific role in the oral cavity has been remained elusive. To identify the specific roles of MyD88-dependent signaling in gene expression profiles in salivary glands, we performed microarray analysis of the major salivary gland tissues (submandibular gland plus sublingual gland) from control (wild-type C57BL/6) mice and C57BL/6 background Myd88-null mice using Agilent Whole Mouse Genome Oligo Microarrays (8x60K, Design ID 028005).
Project description:Primary Sjogren’s syndrome (pSS) is an autoimmune disease characterized by xerostomia (dry mouth), lymphocytic infiltration into salivary glands and the presence of SSA and SSB autoantibodies. Xerostomia is caused by hypofunction of the salivary glands and has been considered a driver in pSS development. Saliva production is regulated by sympathetic input into the gland initiating intracellular Ca2+ signals that activate the store operated Ca2+ entry (SOCE) pathway eliciting sustained Ca2+ influx. SOCE is mediated by the STIM1 and STIM2 proteins and the ORAI1 Ca2+ channel. However, there are no studies on the effects of lack of STIM1/2 function in salivary acini in animal models or its impact on pSS. Here we report that male and female mice lacking Stim1 and Stim2 (Stim1/2K14Cre) in salivary glands hyposalivate upon pilocarpine stimulation and showed reduced intracellular Ca2+ levels via SOCE in parotid acini. Bulk RNASeq of the parotid glands of Stim1/2K14Cre showed a decrease in Stim1/2 genes but not in other Ca2+ associated genes mediating saliva fluid secretion, yet SOCE was functionally required for the activation of the Ca2+ activated chloride channel ANO1. Ageing Stim1/2K14Cre mice showed no evidence of lymphocytic infiltration in the glands or elevated levels of SSA or SSB autoantibodies in the serum which may be linked to the downregulation of the toll-like receptor 8 (Tlr8) in Stim1/2K14Cre mice. This is supported by an increase in TLR8 gene expression in a salivary gland cell line following SOCE stimulation. Moreover, salivary gland biopsies of pSS patients showed increased STIM1 and TLR8 expression. These results implicate SOCE as an important activator of ANO1 and saliva fluid secretion in salivary glands but loss of SOCE does not result in pSS. Importantly, our data suggest a link between SOCE and TLR8 signaling which may have implications in inflammatory responses in salivary glands.
Project description:Saliva (oral fluids) is an emerging biofluid poised for clinical diseases detection. Although the rationale for oral diseases applications (e.g. oral cancer) is clear, the rationale and relationship between systemic diseases and saliva biomarkers are unknown. In this study, we used mouse models of melanoma and non-small cell lung cancer and compared the transcriptome biomarker profiles of tumor-bearing mice to those of control mice. Microarray analysis showed that salivary transcriptomes were significantly altered in tumor-bearing mice vs. controls. Analysis of the transcriptomes in the mouse tumors, serum, salivary glands and saliva revealed that salivary biomarkers have multiple origins. Furthermore, we identified that the expression of two groups of significantly altered transcription factors Runx1, Mlxipl, Trim30 and Egr1, Tbx1, Nr1d1 in melanoma-bearing mice that can potentially be responsible for 82.6% of the up-regulated genes expression and 62.5% of the down-regulated gene expression in the mice saliva, respectively. We also confirmed that the ectopic production of nerve growth factor (NGF) in the melanoma tumor tissue as a tumor-released mediator that can induce expression of the transcription factor Egr-1 in the salivary gland. Taken together, our data support the conclusion that upon systemic disease development, a disease-specific change occurs in the salivary biomarker profile. Although the origins of the disease-specific salivary biomarkers are both systemic and local, stimulation of salivary gland by mediators released from remote tumors play an important role in regulating the salivary surrogate biomarker profiles. Experiment Overall Design: Mice (either C57BL/6 mice or DBA/2 mice) were randomly assigned to control group and tumor group (15 animals per group). Melanoma mice model was induced by subcutaneous (s.c.) injection of B16-F1 cells in 0.1 ml PBS into the lower-right flank of C57BL/6 mice. The lung cancer model was established by s.c. injection of KLN-205 cells in DBA/2 mice. Control animal were injected with PBS alone. Established tumors were observed after 2-3 weeks. Experiment Overall Design: When tumors reached 15 mm in diameter saliva was collected and the mice were sacrificed. Mild anesthesia was induced. Mice saliva was stimulated, obtained and immediately placed in pre-schilled 1.5-ml microcentrifuge tubes. Collection was completed in 20 minutes and samples were stored at -80ºC until analyzed.In addition, Blood was collected in BD Vacutainer tubes containing clot activator (BD Biosciences). Salivary gland and tumor tissue were removed from mice, snap-frozen in liquid nitrogen and stored at -80ºC. Experiment Overall Design: Experiment Overall Design: Salivary,salivary gland, serum or tumor RNA was isolated using the RNeasy Mini Kit (Qiagen) as described previously. There are 15 mice in the control group or tumor group (totally 30 C57BL/6 mice for melanoma mouse model, another 30 DBA/2 mice for lung cancer mouse model). Samples derived from 5 mice in each group were pooled and RNA extracted. The pooling is necessary to ensure sufficient salivary mRNA can be obtained for microarray analyses. Isolated total RNA was treated with recombinant DNase (Ambion, Austin, TX). For microarray analysis, mRNA from mouse saliva, gland or tumor was linearly amplified using the RiboAmp RNA Amplification kit (Molecular Devices, Sunnyvale, CA). After purification, cDNA were in vitro transcribed and biotinylated using GeneChip Expression 3â-Amplification Reagents for in vitro transcription labeling (Affymetrix, Santa Clara, CA). The labeled RNAs was subsequently fragmented, hybridization and scanning.