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 -轮廓乳头; vEG - von Ebner腺） 在哺乳动物中，味觉的感觉末端器官是味蕾（TB），其主要存在于胃粘膜的上皮中。 舌头上的味觉乳头菌状、叶状和轮廓状。在啮齿类动物中， (CvP)容纳数百个TB，这使得TB细胞的有效分析成为可能，这将是我们在这方面的重点。 提议TB细胞是有丝分裂后的细胞，它们的寿命很短，并经历不断的更新，因此， 直接周围组织中的祖细胞必须可用于更新TB细胞以实现体内平衡。 TB细胞功能、起源和分化的全面信息对于理解味觉障碍至关重要 是由结核病前体缺陷引起的 TB祖细胞被认为存在于复层舌上皮的基底层中， 表达Krt 14、Krt 5、p63、SOX 2和Lgr 5。然而，我们最近公布的数据表明，某些结核病 细胞（CvP中约35%的TB细胞）来源于以前未被重视的祖细胞来源，但 而不是在复层的舌上皮--表达SOX 10的（SOX 10+）细胞中。标记的TB细胞是 主要是III型，具有酸味和咸味，并且具有显著较低水平的泛味细胞标记物 Krt8.对SOX 10+细胞的组织定位和谱系作图的进一步研究导致了这样的想法，即von Ebner腺（vEG）是CvP中TB细胞的SOX 10+祖细胞的组织巢。都支持这一看法 报道了在移植的CvP组织中vEG导管所在的位置形成含有TB的“环形结构”。 本申请的目的是收集关于S 0X 10+导管细胞是否为乳腺癌的直接和可靠的证据。 vEGs迁移和分化为TB细胞（Aim 1），并建立CvP/vEG的转录组学谱 上皮细胞，包括SOX 10+祖细胞及其衍生的TB细胞（Aim 2）。在目标1中，我们将采用 先前证明的用于检查味觉细胞分化的方法-解离细胞的3D培养物， CvP/vEG移植。除了利用这些体外和离体系统，我们将利用 基因标记的（RFP+）vEG的SOX 10+导管细胞和来自SOX 10-iCreERT 2/RFP的CvP/vEG复合物 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，使一个高效， 信息分析，以建立所有类型的CvP/vEG上皮细胞的转录组学谱，包括 vEG和衍生的TB细胞中的S 0X 10+祖细胞。 所产生的数据将提供关于SOX 10 + vEG细胞分化为TB细胞的明确信息 并带来所有CvP TB细胞和周围上皮细胞基因表达的全面信息 包括那些TB祖细胞如SOX 10 + vEG导管细胞。这些数据将在很长一段时间内具有重要价值。 这一术语有助于研究结核细胞功能和机制， CvP/vEGs中TB祖细胞的区室和细胞类型。 关键词：味蕾; vonEbner腺;轮廓乳头; SOX 10;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.