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

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 ß-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 ß-catenin signaling was not explored. Here, we define discrete spatiotemporal functions of ß-catenin in fungiform taste bud development. Specifically, we show that early epithelial activation of ß-catenin, before taste placodes form, diverts lingual epithelial cells from a taste bud fate. By contrast, ß-catenin activation a day later within Shh(+) placodes, expands taste bud precursors directly, but enlarges papillae indirectly. Further, placodal activation of ß-catenin drives precocious differentiation of Type I glial-like taste cells, but not other taste cell types. Later activation of ß-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 ß-catenin.

Wnt-beta-catenin signaling initiates taste papilla development.

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.

FGF signaling regulates the number of posterior taste papillae by controlling progenitor field size.

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.

Establishment of new mouse embryonic stem cell lines is improved by physiological glucose and oxygen.

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.

Trocarin, a blood coagulation factor Xa homologue from snake venom, causes inflammation and mitogenesis.

Trocarin, a Group D prothrombin activator from Tropidechis carinatus snake venom, has high sequence similarity to blood coagulation factor Xa (FXa). Both trocarin and FXa activate prothrombin to mature thrombin and have similar requirements for cofactors, such as factor Va, Ca2+ ions and phospholipids. In addition to its hemostatic functions, human FXa causes inflammation and induces mitogenesis in several cell types due to its interaction with effector protease receptor-1 (EPR-1). The inter-EGF domain region (L83FTKRL88) of FXa implicated in EPR-1-binding is distinctly different in trocarin (K83VLYQS88). Here we show that, interestingly, trocarin also causes edema in the mouse footpad; the inflammation, accompanied by a large purplish clot, is more persistent than the transient edema caused by FXa. Histological examination indicates significant differences between edema induced by FXa and trocarin. Moreover, trocarin-induced edema is not inhibited by a synthetic peptide based on the FXa-binding region of EPR-1, indicating that the inflammation is probably mediated by a mechanism independent of EPR-1-binding. Trocarin, like FXa, also has a mitogenic effect on bronchial smooth muscle cells mediated by an EPR-1-independent mechanism. Hence trocarin, being closely related to FXa, has similar non-hemostatic functions in mediating inflammation and mitogenesis, yet appears to act by distinctly different mechanisms.

In vivo fate tracing studies of mammalian taste cell progenitors.

In mammals, the homogeneous lingual epithelium in the process of development forms specialized placodal cells that undergo a series of morphogenetic changes to form a papilla. Taste buds appear in the papillary epithelium around birth and thus papillae serve to house the taste buds in the adult. However, evidence for a precise lineage relationship between a putative embryonic taste progenitor population and functional adult taste buds has so far been elusive and is primarily indirect. Also, mammalian taste papillae are reminiscent of epithelial appendages suggesting that the mesenchymal tissue of the papillae could be involved in the formation of these lingual structures. These major questions in the field of mammalian taste development have remained unanswered due to lack of fate mapping studies that would label embryonic cell populations and remain indelibly marked in the adult. Taking advantage of a genetic fate mapping approach to label cell populations both in the lingual epithelium and mesenchyme and following their fate during development would be an ideal way to assess each of these tissues contribution in taste bud formation. Fate mapping studies using tissue specific cre strains crossed with reporter alleles would uncover unique features in the formation of these specialized sensory cells and also provide us with an in vivo model system for taste organ specific experimental manipulations during development.

Fate mapping of mammalian embryonic taste bud progenitors.

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.