ß-Catenin signaling regulates temporally discrete phases of anterior taste bud development

Abstract

The sense of taste is mediated by multicellular taste buds located within taste papillae on the tongue. In mice, individual taste buds reside in fungiform papillae, which develop at mid-gestation as epithelial placodes in the anterior tongue. Taste placodes comprise taste bud precursor cells, which express the secreted factor sonic hedgehog (Shh) and give rise to taste bud cells that differentiate around birth. We showed previously that epithelial activation of β-catenin is the primary inductive signal for taste placode formation, followed by taste papilla morphogenesis and taste bud differentiation, but the degree to which these later elements were direct or indirect consequences of β-catenin signaling was not explored. Here, we define discrete spatiotemporal functions of β-catenin in fungiform taste bud development. Specifically, we show that early epithelial activation of β-catenin, before taste placodes form, diverts lingual epithelial cells from a taste bud fate. By contrast, β-catenin activation a day later within Shh + placodes, expands taste bud precursors directly, but enlarges papillae indirectly. Further, placodal activation of β-catenin drives precocious differentiation of Type I glial-like taste cells, but not other taste cell types. Later activation of β-catenin within Shh + precursors during papilla morphogenesis also expands taste bud precursors and accelerates Type I cell differentiation, but papilla size is no longer enhanced. Finally, although Shh regulates taste placode patterning, we find that it is dispensable for the accelerated Type I cell differentiation induced by β-catenin.

Onset of taste bud cell renewal starts at birth and coincides with a shift in SHH function

“…Once specified, taste placodes/taste bud primordia do not increase in cell number for the remainder of embryogenesis. This stasis is consistent with numerous reports that taste placodes are mitotically quiescent, while the rest of the lingual epithelium actively proliferates (Farbman and Mbiene, 1991;Liu et al, 2008;Mbiene and Roberts, 2003;Thirumangalathu and Barlow, 2015). Rather we find taste cell number first increases in the first postnatal…”

“…However, the effects of this disruption on later aspects of taste bud development, including TRC differentiation, have not been assessed. Previously, we observed differentiated Type I and II TRCs within a subset of taste buds at E18.5 (Thirumangalathu and Barlow, 2015) and further analysis here reveals differentiation of all three TRC types is underway at E17.5 ( Fig S1). To determine if loss of Shh signaling affects TRC differentation, we induced Shh deletion in Shh CreERT2/fl (Shh-ShhcKO) mice (Castillo-Azofeifa et al, 2017;El Shahawy et al, 2017).…”

“…Because taste placode cells are largely KRT14-immunonegative at E13.5 ( Fig 1D-F), we reasoned that tomato-labeling at E16.5 from lineage trace induced at E14.5 ( Fig 1J, K) could reflect continued addition of new KRT14+ progenitor-derived cells post-placode specification. Taste placodes are post-mitotic (Farbman and Mbiene, 1991;Mbiene and Roberts, 2003;Thirumangalathu and Barlow, 2015); thus if embryonic KRT14+ progenitors contribute new cells to placodes, the number of cells per placode should increase during development. To determine if cells were added to taste bud primordia after specification, we tallied the cell number and volume of taste placodes and bud primordia at E13.5 and E16.5, respectively (Fig 1L-O and see Methods), and found developing taste buds were static in both cell number and volume ( Fig 1P, Q), ruling out an embryonic KRT14 progenitor contribution to taste bud primordia following placode specification.…”

“…Additionally, E12.5 Shh-ShhcKO significantly increased the proportion of taste buds containing differentiated type I (NTPdase2+), II (PLCß2+), and III (NCAM+) TRCs compared to Shh wt/fl littermate controls ( Fig S2G-I), indicating that Shh reduction during placode specification accelerated taste bud differentiaton. This outcome is likely due to the loss of Shh's repressive role on Wnt/ß-catenin signaling during placode specification that in turn leads to precocious TRC differentiation (Iwatsuki et al, 2007;Thirumangalathu and Barlow, 2015) Pharmacologic inhibition of the Shh pathway effector, Smoothened, in embryonic rat tongue explants suggests Shh no longer regulates placode development once these structures have formed (Liu et al, 2004). Consistent with this, ShhcKO induced at E16.5 did not alter the number or size of FFP or KRT8+ taste buds ( Fig S2B, J, K).…”

Early taste buds are from Shh+ epithelial cells of tongue primordium in distinction from mature taste bud cells which arise from surrounding tissue compartments

“…Multiple studies have examined the effects of signaling pathways on papilla patterning [13]. For example, inhibition of Shh signaling [18,26,27] or BMP signaling [28][29][30], and activation of Wnt/β-catenin [31][32][33] signaling induces the formation of many more papillae -even outside regions of normal papilla development. These papillae were capable of developing after K14 expression first appears in the dorsal tongue epithelium, indicating that cell fate is reversible, i.e., from Shh + K8 + to K14 + and vice versa.…”