Function and origin of circumvallate taste bud cells from SOX10 expressing von Ebners glands

项目摘要/摘要(缩写：TB-味蕾；CVP-环状乳头；VEG-von Ebner腺体) 在哺乳动物中，味觉的感觉末端器官是味蕾，它主要驻留在 品尝舌头中的乳突--菌状、叶状和环状。啮齿动物的单个环状乳头 (CVP)拥有数百个TB，这使我们能够高效地分析结核病细胞，这将是我们的重点 求婚。TB细胞是有丝分裂后的，它们的寿命很短，可以持续更新，因此， 紧邻周围组织中的祖细胞必须可用于更新结核细胞以实现内稳态。 全面了解结核细胞的功能、来源和分化是理解味觉障碍的关键 由结核病前体细胞缺陷引起。 TB祖细胞一直被认为存在于复层舌上皮的基底层， 表达Krt14、Krt5、P63、SOX2和Lgr5。然而，我们最近公布的数据表明，某些结核病 细胞(~35%的结核细胞在CVP中)来自以前未被认识的祖细胞来源，但 而不是在复层舌上皮内--表达Sox10+的细胞。标记的结核细胞是 主要是转导酸味和盐味的III型，其泛味细胞标志物水平明显较低 Krt8.对Sox10+细胞的组织定位和谱系图谱的进一步研究导致了von Ebner腺(VEG)是CVP中的TB细胞的Sox10+祖细胞的组织巢。这是由 报道在移植的CVP组织中，在蔬菜导管所在的位置形成了含有TBS的“环形结构”。 这项应用的目标是收集直接和确凿的证据，证明Sox10+导管细胞 VEGS向TB细胞迁移和分化(目标1)，并建立CVP/VEG的转录图谱 上皮细胞，包括Sox10+祖细胞及其衍生的结核细胞(目标2)。在目标1中，我们将采用 先前证实的检查味觉细胞分化的方法-分离细胞的3D培养和 CVP/VIG移植。除了利用这些体外和体外系统，我们还将利用 基因标记(RFP+)Sox10+蔬菜导管细胞和Sox10-iCreERT2/RFP的CVP/VEG复合体 小鼠在3D培养和移植组织中分化味觉细胞。在目标2中，我们将利用 现代高通量技术的力量，单细胞(Sc)RNA-Seq，使高效和 信息量分析，以建立所有类型的CVP/VEG上皮细胞的转录图谱，包括 SOX10+祖细胞在蔬菜和来源的TB细胞中的表达。 所获得的数据将为Sox10+VEG细胞向TB细胞的分化提供明确的信息 并提供了所有CVP TB细胞及其周围上皮细胞的基因表达的全面信息 包括那些结核前体细胞，如Sox10+VEG导管细胞。这些数据将在很长一段时间内具有重大价值 用于促进研究以清楚地了解结核细胞的功能和机制、组织 CVP/VEGS中结核前体细胞的区段和细胞类型。 关键词：味蕾(TB)；von Ebner腺(VEG)；环状乳头(CVP)；Sox10；祖细胞。

Project Summary/Abstract (abbreviations: TB - taste bud; CvP – circumvallate papilla; vEG – von Ebner's gland) In mammals, the sensory end organs for taste are taste buds (TBs) that reside primarily in the epithelium of taste papillae in the tongue -- fungiform, foliate, and circumvallate. In rodents the single circumvallate papilla (CvP) houses hundreds of TBs, which enables efficient analyses of TB cells and will be our focus in this proposal. TB cells are post-mitotic and they have a short lifespan and undergo continuous renewal, therefore, progenitors in the immediate surrounding tissue must be available to renew TB cells for homeostasis. Thorough information of TB cell function, origin, and differentiation is essential to understanding taste disorders caused by TB progenitor deficiencies. TB progenitor cells have been thought to reside in the basal layer of the stratified lingual epithelium and express Krt14, Krt5, p63, SOX2 and Lgr5. However, our recent published data have shown that certain TB cells (~35% of TB cells in the CvP) originate from a previously unappreciated source of progenitors under, but not within, the stratified tongue epithelium -- the SOX10-expressing (SOX10+) cells. The labeled TB cells were mainly type III that transduce a sour and a salt taste, and had a significantly lower level of pan-taste cell marker Krt8. Further studies on the tissue locations and lineage mapping of SOX10+ cells have led to the idea that von Ebner's gland (vEG) is the tissue nest of SOX10+ progenitors for TB cells in the CvP. This is supported by the report that TBs-containing “circular structures” formed where vEG ducts locate in transplanted CvP tissue. The goal of this application is to collect direct and solid evidence about whether SOX10+ ductal cells of vEGs migrate and differentiate to TB cells (Aim 1), and establish the transcriptomic profiles of CvP/vEG epithelial cells, including SOX10+ progenitors and their derived TB cells (Aim 2). In Aim 1, we will adopt the previously proven approaches for examining taste cell differentiation - the 3D cultures of dissociated cells and CvP/vEG transplantations. In addition to taking advantage of these in vitro and ex vivo systems, we will utilize genetically labeled (RFP+) SOX10+ ductal cells of vEGs and CvP/vEG complex from SOX10-iCreERT2/RFP mice to differentiate taste cells in 3D cultures and transplanted tissues. In Aim 2, we will take advantage of the power of a modern high-throughput technique, single cell (sc) RNA-Seq that enables a highly efficient and informative analysis, to establish the transcriptomic profiles of all types of CvP/vEG epithelial cells including SOX10+ progenitors in vEGs and derived TB cells. The yielded data will provide definitive information about the differentiation of SOX10+ vEG cells to TB cells and bring comprehensive information of gene expression for all CvP TB cells and surrounding epithelial cells including those TB progenitors such as SOX10+ vEG ductal cells. The data will be of significant value for a long term in facilitating studies for a clear understanding of TB cell functions and the mechanisms, tissue compartments and cell types of TB progenitors in CvP/vEGs. Key words: taste bud (TB); von Ebner's gland (vEG); circumvallate papilla (CvP); SOX10; progenitor.