Identifying Sox9 as a marker of multipotent progenitors in Circumvallate papilla/Von Ebner's gland homeostasis

Project Summary The circumvallate papilla (CVP) and von Ebner's minor salivary glands (VEG) form a complex at the posterior midline of the tongue. The epithelial trenches of the CVP contain hundreds of taste buds (TBs) which each contain taste receptor cells (TRCs) that transduce taste information that is conveyed to the brain. Taste

perception is affected by salivation through tastant dissolution and subsequent clearing from the oral epithelium. Serous saliva produced by VEG acinar cells is transported through salivary ducts that connect to the ventral region of the CVP trenches. Cancer patients undergoing therapy often experience dysgeusia or taste

dysfunction, and can experience xerostomia due to reduced salivary gland function. Further, clinical data show that dysgeusia is detrimental to patient quality of life, potentially leading to poor survival outcomes. Therefore, understanding the maintenance of the CVP/VEG epithelium will allow the development of therapies to mitigate

the effects of cancer treatments on taste and salivary gland function. CVP homeostasis occurs through the proliferation and differentiation of LGR5+ progenitor cells that generate TRCs in TBs as well as cells of the surrounding keratinized non-taste epithelium that supports TB structure. Though CVP epithelial renewal has been well characterized, progenitor cells involved in VEG

homeostasis have yet to be identified. However, our lab performed single-cell RNA sequencing (scRNA-seq) on murine CVP/VEG epithelium, revealing that CVP and VEG lineages may arise from a common progenitor population. Differential gene expression analysis reveals SOX9 marks this common progenitor, and

pseudotime analysis suggests SOX9+ progenitors give rise to LGR5+ progenitor cells. Preliminary lineage tracing and immunostaining of Sox9CreERT2;TdTomato mice shows that SOX9+ cells contribute to CVP TBs and non-taste epithelium, although this technique did not allow me to assess contributions to VEG. However,

organoids generated from single SOX9+ cells express markers of CVP and VEG, supporting a model where SOX9+ cells are multipotent CVP/VEG progenitors. Together, these findings lead to my hypothesis that SOX9 marks a basal multipotent stem population that gives rise to CVP and VEG epithelia. To test this, in Aim 1 I will use genetic barcoding and scRNAseq of SOX9+ progenitors and their progeny to determine

lineage relationships across the CVP/VEG complex. In Aim 2, I will generate organoids from LGR5+-only, SOX9+-only, and LGR5+/SOX9+ double positive cells using CVP or VEG organoid protocols to assess if LGR5 vs SOX9 expression restricts CVP and VEG lineage production. Further, I will ablate LGR5+ cells from

heterogenous organoid cultures to determine if single SOX9+ cells are truly multipotent or if they coordinate with LGR5+ cells to maintain the CVP/VEG complex. Together, these findings will contribute to our understanding of CVP/VEG homeostasis.