MOLECULAR AND DEVELOPMENTAL NEUROSCIENCE Stem and progenitor cell compartments within adult mouse taste buds Jeremy M. Sullivan, 1,2 Alexander A. Borecki 1 and Sharon Oleskevich 1,2 1 Neuroscience Program, Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, Sydney, NSW 2010, Australia 2 Faculty of Medicine, University of New South Wales, Sydney, NSW 2052, Australia Keywords: adult stem cell, circadian rhythm, gustation, label-retaining cell, sensory nervous system Abstract Adult taste buds are maintained by the lifelong proliferation of epithelial stem and progenitor cells, the identities of which have remained elusive. It has been proposed that these cells reside either within the taste bud (intragemmal) or in the surrounding epithelium (perigemmal). Here, we apply three different in vivo approaches enabling single-cell resolution of proliferative history to identify putative stem and progenitor cells associated with adult mouse taste buds. Experiments were performed across the circadian peak in oral epithelial proliferation (04:00 h), a time period in which mitotic activity in taste buds has not yet been detailed. Using double label pulse-chase experiments, we show that defined intragemmal cells (taste and basal) and perigemmal cells undergo rapid, sequential cell divisions and thus represent potential progenitor cells. Strikingly, mitotic activity was observed in taste cells previously thought to be postmitotic (labelled cells occur in 30% of palatal taste buds after 1 h of BrdU exposure). Basal cells showed expression of the transcription factor p63, required for maintaining the self-renewal potential of various epithelial stem cell types. Candidate taste stem cells were identified almost exclusively as basal cells using the label-retaining cell approach to localize slow-cycling cells (0.06 ± 0.01 cells per taste bud; n = 436 taste buds). Together, these results indicate that both stem- and progenitor-like cells reside within the mammalian taste bud. Introduction Taste buds, the sensory endorgans for taste, are maintained by the lifelong addition and incorporation of new cells (reviewed by Miura et al., 2006). Although taste buds are known to arise from local epithelium (Stone et al., 1995; Okubo et al., 2009; Thirumangalathu et al., 2009), little is known about the locations and phenotypes of the stem and progenitor cell populations maintaining the continuous genesis of taste cells. Proliferation and lineage tracing experiments have led to two differing hypotheses, that the stem and progenitor cells maintaining adult taste buds are located either inside or outside the taste bud (Miura et al., 2006; Okubo et al., 2009). Individual taste buds in mice consist of a tight cluster of 30–150 intragemmal cells of four subtypes (types I–IV) (Murray, 1973; Ma et al., 2007), and are surrounded by perigemmal cells (Sakai et al., 1999). Taste cell types I–III are postmitotic (Hirota et al., 2001) and function as supporting cells (type I), tastant receptors (type II) and presynaptic cells (type III) (Roper, 2007). In contrast, basal cells (type IV), which occur in contact with the basement membrane (Olivieri- Sangiacomo, 1972; Delay et al., 1986), appear to exhibit mitotic activity. The extent of proliferation observed in basal cells has differed markedly between studies, with some authors describing high levels of proliferation (Delay et al., 1986; Hendricks et al., 2004) whereas others have found no evidence of mitotic activity (Beidler & Smallman, 1965; Asano-Miyoshi et al., 2008). In these latter studies, mitotically active cells were observed immediately adjacent to the base of the taste bud. Mitotic activity within the oral epithelia of rodents exhibits one of the strongest circadian rhythms in cell proliferation seen in any tissue (Potten et al., 2002b; Luo et al., 2009). The peak in proliferation (as measured by S phase) occurs around 04:00 h with the nadir at 16:00 h (Potten et al., 2002b; Luo et al., 2009; see also Supporting informa- tion, Fig. S1). Despite this marked circadian rhythm, few studies examining proliferation associated with taste buds have incorporated this feature into their experimental design (but see Hamamichi et al., 2006). Furthermore, a systematic examination of proliferation across the peak in proliferative activity has not yet been undertaken. As identification of taste bud stem and progenitor cell populations has proven elusive, here we used three different in vivo approaches to detect rapid and slow dividing cells associated with taste buds of the tongue (fungiform, circumvallate) and soft palate (palatal). All experiments were performed across the peak of the circadian rhythm in proliferation. To detect rapid re-entry into the cell cycle, we undertook two double labelling pulse-chase experiments using nucleotide analogues. The label-retaining cell approach (Kalabis et al., 2008) was used to localize slow cycling candidate stem cells. The resolution afforded by these techniques provides the opportunity to examine the temporal history of proliferation at the single cell level. Correspondence: Dr J. M. Sullivan, as above. E-mail: j.sullivan@garvan.org.au Received 25 November 2009, revised 29 January 2010, accepted 18 February 2010 European Journal of Neuroscience, Vol. 31, pp. 1549–1560, 2010 doi:10.1111/j.1460-9568.2010.07184.x ª The Authors (2010). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd European Journal of Neuroscience