Salivary gland ionocyte organization and function during homeostasis, repair, and disease

唾液腺是产生唾液的外分泌腺。的充足供应 唾液对维持口腔组织至关重要。唾液由多种电解质组成 唾液中还含有免疫球蛋白、蛋白质、酶和粘蛋白，这表明唾液中含有 复杂函数唾液腺导管的一个重要功能是调节离子 稳态[1]。上皮细胞离子稳态功能障碍可导致几种人类 疾病[2]。我们使用他莫昔芬诱导的FGF 10 CreERT 2：R26进行谱系追踪实验， 番茄小鼠在横纹肌中鉴定出Fgf 10表达细胞的新上皮细胞群， (SD)腮腺（PG）和下颌下（SMG）的颗粒管（GCT）和排泄管（艾德） 腺体重要的是，Fgf 10谱系追踪显示，直到出生后第7天（P7）， 存在间充质Fgf 10表达细胞，然而，在P10后，Fgf 10在间充质中表达。 间充质细胞关闭，而导管上皮细胞的子集开始表达Fgf 10。使用 荧光激活细胞分选（FACS），我们分离了间充质和上皮细胞， Fgf 10+细胞群中的细胞，并进行RNA测序（RNA-seq）。分析这些RNA- seq数据与来自P1和成人的单细胞RNA-Seq（scRNAseq）数据的分析相结合 SMG显示上皮Fgf 10+群体与已知的导管群体重叠， Ascl3此外，这些上皮Fgf 10+细胞表达许多基因特征， 古老类型的离子细胞：Bsnd，Foxi 1，Foxi 2，Asgr 1，Stap 1，H+- ATP酶（V-ATP酶）和SLC 1/2A 1/2和Slc 9a 4转运蛋白的标志物。最重要 Fgf 10+细胞表达囊性纤维化跨膜传导调节因子（Cftr），该因子在纤维化中起作用。 在外分泌分泌（包括唾液腺）中发挥关键作用。我们认为这些上皮细胞 Fgf 10 +/Ascl 3 +/Cftr+细胞是专门的导管唾液腺离子细胞。此外，使用两个 干燥综合征模型：NOD. B10 Sn-H2 b/J和血小板反应蛋白-1缺失（TSP 1-/-） 在小鼠中，我们发现Fgf 10+表达和离子细胞数量显著减少， 疾病进展。我们还发现，从SMG分离的上皮Fgf 10+细胞是 能够维持祖细胞和肌上皮细胞的增殖和生长，这表明 离子细胞在导管维持中的额外作用。在此应用程序中，我们将确定角色 Fgf 10信号传导在SMG再生和离子细胞功能中的作用，定义控制 离子细胞分化和确定慢性炎症如何影响离子细胞功能。在 此外，与霍夫曼博士的小组合作，使用批量RNA-seq和scRNA-seq数据， 将研究唾液腺是否有一种类型或几种类型的离子细胞。我们 这项研究将为将来在基础生物学中研究离子细胞提供一个起始平台 和临床研究。

The salivary glands are exocrine glands that produce saliva. An adequate supply of saliva is critical to the maintenance of oral tissue. Saliva is composed of a variety of electrolytes and also contains immunoglobulins, proteins, enzymes and mucins, suggesting that saliva has a complex function. An important function of salivary gland ducts is to regulate ion homeostasis[1]. Dysfunction of epithelial ion homeostasis can lead to several human diseases[2, 3]. Our lineage tracing experiments using a tamoxifen inducible FGF10CreERT2:R26- Tomato mouse identified new epithelial populations of the Fgf10 expressing cells in the striated (SD), granular (GCT) and excretory ducts (ED) of submandibular (SMG) and parotid (PG) glands. Importantly Fgf10 lineage tracing shows that until postnatal day 7 (P7) only mesenchymal Fgf10 expressing cells are present, however after P10, Fgf10 expression in the mesenchyme shuts down, while a subset of ductal epithelial cells starts to express Fgf10. Using Fluorescence Activated Cell Sorting (FACS) we isolated both mesenchymal and epithelial populations of Fgf10+ cells and performed RNA sequencing (RNA-seq). Analysis of this RNA- seq data combined with analysis of single cell RNA-Seq (scRNAseq) data from P1 and adult SMGs showed that epithelial Fgf10+ populations overlap with the known duct population marked by Ascl3. Moreover, these epithelial Fgf10+ cells expressed many gene characteristics for an ancient type of ionocyte cell: Bsnd, Foxi1, Foxi2, Asgr1, Stap1, several subunits of the H+- ATPase (V-ATPase) and markers of SLC12A1/2 and Slc9a4 transporters. Most importantly Fgf10+ cells expressed cystic fibrosis transmembrane conductance regulator (Cftr) that plays a key role in exocrine secretion, including in salivary glands. We propose that these epithelial Fgf10+/Ascl3+/Cftr+ cells are specialized duct salivary gland ionocytes. Moreover, using two models of Sjogrens syndrome: the NOD.B10Sn-H2b/J and the thrombospondin-1 null (TSP1-/-) mice, we showed a significant decrease in Fgf10+ expression and the number of ionocytes with disease progression. We also showed that epithelial Fgf10+ cells isolated from the SMG are able to maintain proliferation and growth of progenitor and myoepithelial cells, suggesting an additional role for ionocytes in ductal maintenance. In this application we will determine the role of Fgf10 signaling in SMG regeneration and ionocyte function, define factors that control ionocyte differentiation and determine how chronic inflammation affects ionocyte function. In addition, in collaboration with Dr. Hoffman's group using bulk RNA-seq and scRNA-seq data we will investigate whether salivary glands have one type or several types of ionocytes. Our proposed study will provide a starting platform for future studies of ionocytes in basic biology and clinical research.