Project Summary/Abstract (abbreviations: taste bud (TB); connective tissue (CT); neural crest (NC)) In mammals, the sensory end organs for taste are taste buds (TBs), which reside primarily in the epithelium of taste papillae in the tongue. Although the location and shape of taste papillae (fungiform, foliate, and circumvallate) vary, each papilla is composed of an epithelium containing TBs, and an underlying mesenchyme/connective tissue (CT) core including various cell types derived primarily from the neural crest (NC). Like other epithelial cells, TB cells have a short lifespan and undergo continuous turnover. Therefore, progenitor/stem cells in the surrounding tissue compartments must be available to renew taste cells for homeostasis. Information regarding these TB progenitors is necessary to understand both TB formation and maintenance, and how taste disorders emerge from TB progenitor defects. TBs emerge perinatally, and undergo maturation and maintenance postnatally. Our recent findings indicate that a previously unappreciated source of progenitors, SOX10-expressing (hereafter SOX10+) progenitors, may differentiate into a unique population of taste cells. Using a model of Cre under the control of the SOX10 endogenous promoter, which is NC-specific in early embryos, we found that SOX10-Cre labeled a unique population of TB cells (faint for the widely used pan-taste cell marker K8) concurrently with the underlying CT, which originates primarily from the NC. These data suggest that (1) SOX10+ cells in NC-derived CT are potentially progenitors that differentiate to unique TB cells during TB maturation and maintenance, and (2) TB cells are more diverse than previously thought. Further, we identified tamoxifen-induced SOX10-iCreERT2/RFP cells in the CT core of taste papillae in 2-wk-old mice while labeled cells were seen in TBs at 2 wks after tamoxifen. This new data suggests the existence of SOX10+ progenitors in the CT for TB cell renewal, warranting further studies. In this application, we will test our hypothesis that SOX10+ TB progenitors exist in the underlying CT, undergo a transition from mesenchyme to TB-surrounding epithelium, and differentiate to form unique TB cells. (Aim 1) To demonstrate firmly the contribution of SOX10+ progenitors to specific TB cell types, we will administer tamoxifen to inducible SOX10-iCreERT2/RFP mice at different postnatal stages and trace SOX10+ progenitors and labeled TB cells at various time points after tamoxifen treatment. (Aim 2) To determine whether NC cells give rise to SOX10+ progenitors and the derived unique TB cells, we will provide a single dose of tamoxifen to dams at ~E8.5 pregnancy of SOX10-iCreERT2/RFP embryos, and determine whether SOX10-iCreERT2 labels SOX10+ CT cells. We will also determine whether the labeled cells localize within the TBs of postnatal mice. Finally, (Aim 3) we will demonstrate the differentiation of isolated SOX10+ mesenchymal progenitors to TB cells in 3D cultures in vitro. Demonstration of the contribution, derivation, and differentiation of SOX10+ TB progenitors will significantly advance our knowledge regarding the progenitor source(s) required for TB cell renewal, and thereby taste as a whole. Key words: taste bud (TB); connective tissue (CT); neural crest (NC); SOX10; 3D cell cultures.