In chickens, the sensory organs for taste are the taste buds in the oral cavity, of which there are ~240–360 in total number as estimated by scanning electron microscopy (SEM). There is not an easy way to visualize all taste buds in chickens. Here, we report a highly efficient method for labeling chicken taste buds in oral epithelial sheets using the molecular markers Vimentin and α-Gustducin. Immediate tissue fixation following incubation with sub-epithelially injected proteases enabled us to peel off whole epithelial sheets, leaving the shape and integrity of the tissue intact. In the peeled epithelial sheets, taste buds labeled with antibodies against Vimentin and α-Gustducin were easily identified and counted under a light microscope and many more taste buds, patterned in rosette-like clusters, were found than previously reported with SEM. Broiler-type, female-line males have more taste buds than other groups and continue to increase the number of taste buds over stages after hatch. In addition to ovoid-shaped taste buds, big tube-shaped taste buds were observed in the chicken using 2-photon microscopy. Our protocol for labeling taste buds with molecular markers will factilitate future mechanistic studies on the development of chicken taste buds in association with their feeding behaviors.

Taste buds, the sensory organs for taste, have been described as arising solely from the surrounding epithelium, which is in distinction from other sensory receptors that are known to originate from neural precursors, i.e., neural ectoderm that includes neural crest (NC). Our previous study suggested a potential contribution of NC derived cells to early immature fungiform taste buds in late embryonic (E18.5) and young postnatal (P1-10) mice. In the present study we demonstrated the contribution of the underlying connective tissue (CT) to mature taste buds in mouse tongue and soft palate. Three independent mouse models were used for fate mapping of NC and NC derived connective tissue cells: (1) P0-Cre/R26-tdTomato (RFP) to label NC, NC derived Schwann cells and derivatives; (2) Dermo1-Cre/RFP to label mesenchymal cells and derivatives; and (3) Vimentin-CreER/mGFP to label Vimentin-expressing CT cells and derivatives upon tamoxifen treatment. Both P0-Cre/RFP and Dermo1-Cre/RFP labeled cells were abundant in mature taste buds in lingual taste papillae and soft palate, but not in the surrounding epithelial cells. Concurrently, labeled cells were extensively distributed in the underlying CT. RFP signals were seen in the majority of taste buds and all three types (I, II, III) of differentiated taste bud cells, with the neuronal-like type III cells labeled at a greater proportion. Further, Vimentin-CreER labeled cells were found in the taste buds of 3-month-old mice whereas Vimentin immunoreactivity was only seen in the CT. Taken together, our data demonstrate a previously unrecognized origin of taste bud cells from the underlying CT, a conceptually new finding in our knowledge of taste bud cell derivation, i.e., from both the surrounding epithelium and the underlying CT that is primarily derived from NC.

Taste bud cells are specialized epithelial cells that undergo continuous turnover, and thus require active progenitors for their renewal and an intact taste function. Our previous studies suggested that a population of taste bud cells originates from outside of the surrounding tongue epithelium—previously regarded sole source of taste bud progenitors. In this study, we demonstrated that SOX10 (SRY-related HMG-box gene 10)-expressing cells, known to be in the migrating neural crest, were also distributed in taste bud-surrounding tissue compartments under the tongue epithelium, that is, the connective tissue core of taste papillae and von Ebner's glands. By lineage tracing of SOX10 -expressing cells using SOX10-Cre , a Cre model driven by the endogenous SOX10 promoter, crossing with a Cre reporter line R26-tdTomato (tdT), we found SOX10-Cre -labeled tdT ⁺ cells within taste buds in all three types of taste papillae (fungiform, circumvallate, and foliate) as well as in the soft palate in postnatal mice. The tdT ⁺ taste bud cells were progressively more abundant along the developmental stages, from virtually zero at birth to over 35% in adults. Most of tdT ⁺ taste bud cells had a low intensity of immunosignals of Keratin 8 (a widely used taste bud cell marker). In circumvallate taste buds, tdT signals were co-localized principally with a type III taste bud cell marker, less so with type I and II cell makers. Together, our data demonstrate a novel progenitor source for taste buds of postnatal mice— SOX10 - Cre- labeled cells in the connective tissue core and/or von Ebner's glands.