Pinpoint the Sox10+ taste bud progenitor cells in von Ebner's glands and Sox10 regulation in cell differentiation - Project Summary/Abstract (Abbreviations: CvP – circumvallate papilla; vEG – von Ebner’s gland) In mammals, taste bud 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 taste bud cells for homeostasis. Thorough information of taste bud progenitors and molecular regulation in the cell differentiation is essential to understanding taste disorders caused by taste bud progenitor deficiencies. Taste bud progenitor cells have been thought to reside in the basal layer of the stratified lingual epithelium and express Krt14, Krt5, Gli1, p63, Sox2 or Lgr5. Our recent findings indicate the existence of an underappreciated source of progenitors that express Sox10 (Sox10+) -- cells under, but not within, the stratified tongue epithelium. These Sox10+ taste bud progenitors contribute to a significant proportion of taste bud cells including most of type-III taste cells that transduce sour and salt taste. Our further studies and early work using circumvallate papilla (CvP) transplantation provide compelling data to demonstrate that von Ebner’s minor salivary glands (vEGs) are the niche of these novel Sox10+ progenitors for taste buds. Questions remain regarding the exact locations, specific type(s), molecular signatures of Sox10+ taste bud progenitors in vEGs and regulatory roles of Sox10 transcription factor in cell differentiation. In mice, the single CvP in the midline of posterior tongue houses hundreds of taste buds with abundant vEGs underneath to form the CvP-vEG complex, which enables efficient analyses and will be our focus in this proposal. The goal of this application is to (Aim 1) define the spatial distribution and molecular signature of Sox10+ taste bud progenitors in vEGs, and (Aim 2) test whether Sox10 transcription factor regulates the differentiation of these progenitors to taste bud cells. We will take advantage of the unique strengths of conventional and modern tools (e.g., inducible transgenic mouse lines, proliferating and retaining cell labeling, spatial transcriptomic analysis, and CRISPR/Cas9 technology). In Aim 1, we will use adult Sox10-iCreERT2/tdTTmx mice to trace Sox10+ cell lineages, perform proliferating and retaining cell labeling and MERFISH (Multiplexed Error Robust Fluorescence in situ Hybridization) that combines the power of single-cell transcriptomics with spatial biology by directly visualizing and counting RNA transcripts of ~140 marker genes in tissue slices. In Aim 2, we will analyze phenotypes in Sox10 conditional knockout (cKO) -- adult Sox10-iCreERT2/Sox10 fx mice, and Sox10- iCreERT2/ R26LSL-Cas9-EGFP mice with microinjection of AAV-Sox10 gRNA into CvP-vEG tongue region. The yielded data will allow us to pinpoint the Sox10+ taste bud progenitors in vEGs and to detect the marker gene expression in these progenitors and other cells on the tissue sections, and to collect solid data as to whether Sox10 is essential for the differentiation of Sox10+ taste bud progenitors in vEGs. The studies will enable us to better understand the features (spatial, cellular, molecular) and differentiation of these Sox10+ taste bud progenitors in vEGs and thus better understand the maintenance of taste bud homeostasis.
