Fungiform taste papillae form a regular array on the dorsal tongue. Taste buds arise from papilla epithelium and, unusually for epithelial derivatives, synapse with neurons, release neurotransmitters and generate receptor and action potentials. Despite the importance of taste as one of our five senses, genetic analyses of taste papilla and bud development are lacking. We demonstrate that Wnt-beta-catenin signaling is activated in developing fungiform placodes and taste bud cells. A dominant stabilizing mutation of epithelial beta-catenin causes massive overproduction of enlarged fungiform papillae and taste buds. Likewise, genetic deletion of epithelial beta-catenin or inhibition of Wnt-beta-catenin signaling by ectopic dickkopf1 (Dkk1) blocks initiation of fungiform papilla morphogenesis. Ectopic papillae are innervated in the stabilizing beta-catenin mutant, whereas ectopic Dkk1 causes absence of lingual epithelial innervation. Thus, Wnt-beta-catenin signaling is critical for fungiform papilla and taste bud development. Altered regulation of this pathway may underlie evolutionary changes in taste papilla patterning.

Mammalian taste buds have properties of both epithelial and neuronal cells, and are thus developmentally intriguing. Taste buds differentiate at birth within epithelial appendages, termed taste papillae, which arise at mid-gestation as epithelial thickenings or placodes. However, the embryonic relationship between placodes, papillae and adult taste buds has not been defined. Here, using an inducible Cre-lox fate mapping approach with the ShhcreER T2 mouse line, we demonstrate that Shh-expressing embryonic taste placodes are taste bud progenitors, which give rise to at least two different adult taste cell types, but do not contribute to taste papillae. Strikingly, placodally descendant taste cells disappear early in adult life. As placodally derived taste cells are lost, we used Wnt1Cre mice to show that the neural crest does not supply cells to taste buds, either embryonically or postnatally, thus ruling out a mesenchymal contribution to taste buds. Finally, using Bdnf null mice, which lose neurons that innervate taste buds, we demonstrate that Shh-expressing taste bud progenitors are specified and produce differentiated taste cells normally, in the absence of gustatory nerve contact. This resolution of a direct relationship between embryonic taste placodes with adult taste buds, which is independent of mesenchymal contribution and nerve contact, allows us to better define the early development of this important sensory system. These studies further suggest that mammalian taste bud development is very distinct from that of other epithelial appendages.

The sense of taste is fundamental to our ability to ingest nutritious substances and to detect and avoid potentially toxic ones. Sensory taste buds are housed in papillae that develop from epithelial placodes. Three distinct types of gustatory papillae reside on the rodent tongue: small fungiform papillae are found in the anterior tongue, whereas the posterior tongue contains the larger foliate papillae and a single midline circumvallate papilla (CVP). Despite the great variation in the number of CVPs in mammals, its importance in taste function, and its status as the largest of the taste papillae, very little is known about the development of this structure. Here, we report that a balance between Sprouty (Spry) genes and Fgf10, which respectively antagonize and activate receptor tyrosine kinase (RTK) signaling, regulates the number of CVPs. Deletion of Spry2 alone resulted in duplication of the CVP as a result of an increase in the size of the placode progenitor field, and Spry1−/−;Spry2−/− embryos had multiple CVPs, demonstrating the redundancy of Sprouty genes in regulating the progenitor field size. By contrast, deletion of Fgf10 led to absence of the CVP, identifying FGF10 as the first inductive, mesenchyme-derived factor for taste papillae. Our results provide the first demonstration of the role of epithelial-mesenchymal FGF signaling in taste papilla development, indicate that regulation of the progenitor field size by FGF signaling is a critical determinant of papilla number, and suggest that the great variation in CVP number among mammalian species may be linked to levels of signaling by the FGF pathway.

Aims/hypothesis Neural tube defects (NTDs) are a common malformation associated with diabetic embryopathy. Maternal hyperglycaemia-induced oxidative stress inhibits the expression of Pax3, a gene that is essential for neural tube closure, and increases the incidence of NTDs. Because oxidative stress can stimulate AMP-activated kinase (AMPK) activity, and AMPK can regulate gene transcription, we hypothesised that increased AMPK activity would mediate the adverse effects of maternal hyperglycaemia-induced oxidative stress on Pax3 expression and NTDs. Methods Pregnant mice were made transiently hyperglycaemic by glucose injection, or hypoxic by housing in a hypoxic chamber, or were treated with antimycin A to induce oxidative stress, and AMPK activity in the embryos was assayed. The effects of stimulating AMPK activity with 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR) on Pax3 expression and NTDs were determined. Vitamin E or glutathione ethyl ester was used to reduce oxidative stress, and compound C was used to inhibit AMPK activation. Murine embryonic stem cells were employed as an in vitro model to study the effects of oxidative stress on AMPK activity and the effects of AMPK stimulation on Pax3 expression. Results Maternal hyperglycaemia stimulated AMPK activity, and stimulation of AMPK with AICAR inhibited Pax3 expression (in vivo and in vitro) and increased NTDs (in vivo). Stimulation of AMPK by hyperglycaemia, hypoxia or antimycin A was inhibited by antioxidants. The AMPK inhibitor compound C blocked the effects of hyperglycaemia or AA on Pax3 expression and NTDs. Conclusions/interpretation Stimulation of AMPK in embryos during a diabetic pregnancy mediates the effects of hyperglycaemia-induced oxidative stress to disturb the expression of the critical Pax3 gene, thereby causing NTDs.

Embryonic stem cell lines are routinely selected and cultured in glucose and oxygen concentrations that are well above those of the intrauterine environment. Supraphysiological glucose and hyperoxia each increase oxidative stress, which could be detrimental to survival in vitro by inhibiting proliferation and/or inducing cell death. The aim of this study was to test whether isolation of new embryonic stem cell lines from murine blastocysts is improved by culture in physiological (5%) oxygen instead of approximately 20%, the concentration of oxygen in room air, or in media containing physiological (100 mg/dL) instead of 450 mg/dL glucose. We found that culturing in either physiological oxygen or physiological glucose improved the success of establishing new murine embryonic stem cell lines, and that culture when concentrations of both oxygen and glucose were physiological improved the success of establishing new lines more than culture in either alone. Physiological oxygen and glucose reduce oxidative stress, as determined by 2',7'-dichloro-dihydrofluorescein fluorescence. BrdU incorporation suggests that physiological oxygen and glucose increase the pool of proliferating cells. Cells isolated in physiological oxygen and glucose are capable of self-renewal and differentiation into all three germ layers in vitro. However, none of the culture conditions prevents cytogenetic instability with prolonged passage. These results suggest that culture of cells derived from murine blastocysts in physiological oxygen and glucose reduces oxidant stress, which increases the success of establishing new embryonic stem cell lines.

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.

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