Hong Xiang Liu

Wnt signaling interacts with Shh to regulate taste papilla development

Wnt and Shh signaling pathways are critical for the development and maturation of many epithelial tissues. Both pathways have roles in stem cell maintenance, tissue development, and tumorigenesis. However, linkage between these pathways in mammalian systems had not been well established. Here, we report that Shh expression in fungiform papillae and formation of normal mature fungiform papillae depend on signaling through Wnt and β-catenin. We observed that during fungiform papilla formation in mice, Shh and components of the Wnt/β-catenin signaling pathway are expressed together in the developing placode. The elimination of Wnt/β-catenin signaling in either Lef1 or Wnt10b knockout mice resulted in down-regulation of Shh expression. In addition, the size and number of fungiform papillae were greatly reduced in Lef1 knockout mice. By examining embryonic mouse tongues in culture we determined that activation of Wnt/β-catenin signaling up-regulates Shh expression. We observed that blocking Shh signaling in cultured tongue explants enhanced papillae formation and was accompanied by an up-regulation of Wnt/β-catenin signaling, indicating that Shh inhibits the Wnt/β-catenin pathway. Exogenously added Shh suppressed expression of endogenous Shh and inhibited Wnt/β-catenin signaling (assessed in TOPGAL mice), further implicating Shh as an inhibitor of the Wnt/β-catenin pathway. Our observations indicate that Wnt/β-catenin signaling and interactions between the Wnt and Shh pathways play essential roles in the development of fungiform papillae.

Cyclopamine and jervine in embryonic rat tongue cultures demonstrate a role for Shh signaling in taste papilla development and patterning : fungiform papillae double in number and form in novel locations in dorsal lingual epithelium

From time of embryonic emergence, the gustatory papilla types on the mammalian tongue have stereotypic anterior and posterior tongue locations. Furthermore, on anterior tongue, the fungiform papillae are patterned in rows. Among the many molecules that have potential roles in regulating papilla location and pattern, Sonic hedgehog (Shh) has been localized within early tongue and developing papillae. We used an embryonic, tongue organ culture system that retains temporal, spatial, and molecular characteristics of in vivo taste papilla morphogenesis and patterning to study the role of Shh in taste papilla development. Tongues from gestational day 14 rat embryos, when papillae are just beginning to emerge on dorsal tongue, were maintained in organ culture for 2 days. The steroidal alkaloids, cyclopamine and jervine, that specifically disrupt the Shh signaling pathway, or a Shh-blocking antibody were added to the standard culture medium. Controls included tongues cultured in the standard medium alone, and with addition of solanidine, an alkaloid that resembles cyclopamine structurally but that does not disrupt Shh signaling. In cultures with cyclopamine, jervine, or blocking antibody, fungiform papilla numbers doubled on the dorsal tongue with a distribution that essentially eliminated inter-papilla regions, compared with tongues in standard medium or solanidine. In addition, fungiform papillae developed on posterior oral tongue, just in front of and beside the single circumvallate papilla, regions where fungiform papillae do not typically develop. The Shh protein was in all fungiform papillae in embryonic tongues, and tongue cultures with standard medium or cyclopamine, and was conspicuously localized in the basement membrane region of the papillae. Ptc protein had a similar distribution to Shh, although the immunoproduct was more diffuse. Fungiform papillae did not develop on pharyngeal or ventral tongue in cyclopamine and jervine cultures, or in the tongue midline furrow, nor was development of the single circumvallate papilla altered. The results demonstrate a prominent role for Shh in fungiform papilla induction and patterning and indicate differences in morphogenetic control of fungiform and circumvallate papilla development and numbers. Furthermore, a previously unknown, broad competence of dorsal lingual epithelium to form fungiform papillae on both anterior and posterior oral tongue is revealed.

Multiple Shh signaling centers participate in fungiform papilla and taste bud formation and maintenance

The adult fungiform taste papilla is a complex of specialized cell types residing in the stratified squamous tongue epithelium. This unique sensory organ includes taste buds, papilla epithelium and lateral walls that extend into underlying connective tissue to surround a core of lamina propria cells. Fungiform papillae must contain long-lived, sustaining or stem cells and short-lived, maintaining or transit amplifying cells that support the papilla and specialized taste buds. Shh signaling has established roles in supporting fungiform induction, development and patterning. However, for a full understanding of how Shh transduced signals act in tongue, papilla and taste bud formation and maintenance, it is necessary to know where and when the Shh ligand and pathway components are positioned. We used immunostaining, in situ hybridization and mouse reporter strains for Shh, Ptch1, Gli1 and Gli2-expression and proliferation markers to identify cells that participate in hedgehog signaling. Whereas there is a progressive restriction in location of Shh ligand-expressing cells, from placode and apical papilla cells to taste bud cells only, a surrounding population of Ptch1 and Gli1 responding cells is maintained in signaling centers throughout papilla and taste bud development and differentiation. The Shh signaling targets are in regions of active cell proliferation. Using genetic-inducible lineage tracing for Gli1-expression, we found that Shh-responding cells contribute not only to maintenance of filiform and fungiform papillae, but also to taste buds. A requirement for normal Shh signaling in fungiform papilla, taste bud and filiform papilla maintenance was shown by Gli2 constitutive activation. We identified proliferation niches where Shh signaling is active and suggest that epithelial and mesenchymal compartments harbor potential stem and/or progenitor cell zones. In all, we report a set of hedgehog signaling centers that regulate development and maintenance of taste organs, the fungiform papilla and taste bud, and surrounding lingual cells. Shh signaling has roles in forming and maintaining fungiform papillae and taste buds, most likely via stage-specific autocrine and/or paracrine mechanisms, and by engaging epithelial/mesenchymal interactions.

Fungiform papilla pattern: EGF regulates inter-papilla lingual epithelium and decreases papilla number by means of PI3K/Akt, MEK/ERK, and p38 MAPK signaling.

Fungiform papillae are epithelial taste organs that form on the tongue, requiring differentiation of papillae and inter‐papilla epithelium. We tested roles of epidermal growth factor (EGF) and the receptor EGFR in papilla development. Developmentally, EGF was localized within and between papillae whereas EGFR was progressively restricted to inter‐papilla epithelium. In tongue cultures, EGF decreased papillae and increased cell proliferation in inter‐papilla epithelium in a concentration‐dependent manner, whereas EGFR inhibitor increased and fused papillae. EGF preincubation could over‐ride disruption of Shh signaling that ordinarily would effect a doubling of fungiform papillae. With EGF‐induced activation of EGFR, we demonstrated phosphorylation in PI3K/Akt, MEK/ERK, and p38 MAPK pathways; with pathway inhibitors (LY294002, U0126, SB203580) the EGF‐mediated decrease in papillae was reversed, and synergistic actions were shown. Thus, EGF/EGFR signaling by means of PI3K/Akt, MEK/ERK, and p38 MAPK contributes to epithelial cell proliferation between papillae; this biases against papilla differentiation and reduces numbers of papillae. Developmental Dynamics 237:2378–2393, 2008.

Separate and distinctive roles for Wnt5a in tongue, lingual tissue and taste papilla development

Although canonical Wnt signaling is known to regulate taste papilla induction and numbers, roles for noncanonical Wnt pathways in tongue and taste papilla development have not been explored. With mutant mice and whole tongue organ cultures we demonstrate that Wnt5a protein and message are within anterior tongue mesenchyme across embryo stages from the initiation of tongue formation, through papilla placode appearance and taste papilla development. The Wnt5a mutant tongue is severely shortened, with an ankyloglossia, and lingual mesenchyme is disorganized. However, fungiform papilla morphology, number and innervation are preserved, as is expression of the papilla marker, Shh. These data demonstrate that the genetic regulation for tongue size and shape can be separated from that directing lingual papilla development. Preserved number of papillae in a shortened tongue results in an increased density of fungiform papillae in the mutant tongues. In tongue organ cultures, exogenous Wnt5a profoundly suppresses papilla formation and simultaneously decreases canonical Wnt signaling as measured by the TOPGAL reporter. These findings suggest that Wnt5a antagonizes canonical Wnt signaling to dictate papilla number and spacing. In all, distinctive roles for Wnt5a in tongue size, fungiform papilla patterning and development are shown and a necessary balance between non-canonical and canonical Wnt paths in regulating tongue growth and fungiform papillae is proposed in a model, through the Ror2 receptor.

Specific and spatial labeling of P0-Cre versus Wnt1-Cre in cranial neural crest in early mouse embryos

P0‐Cre and Wnt1‐Cre mouse lines have been widely used in combination with loxP‐flanked mice to label and genetically modify neural crest (NC) cells and their derivatives. Wnt1‐Cre has been regarded as the gold standard and there have been concerns about the specificity of P0‐Cre because it is not clear about the timing and spatial distribution of the P0‐Cre transgene in labeling NC cells at early embryonic stages. We re‐visited P0‐Cre and Wnt1‐Cre models in the labeling of NC cells in early mouse embryos with a focus on cranial NC. We found that R26‐lacZ Cre reporter responded to Cre activity more reliably than CAAG‐lacZ Cre reporter during early embryogenesis. Cre immunosignals in P0‐Cre and reporter (lacZ and RFP) activity in P0‐Cre/R26‐lacZ and P0‐Cre/R26‐RFP embryos was detected in the cranial NC and notochord regions in E8.0–9.5 (4–19 somites) embryos. P0‐Cre transgene expression was observed in migrating NC cells and was more extensive in the forebrain and hindbrain but not apparent in the midbrain. Differences in the Cre distribution patterns of P0‐Cre and Wnt1‐Cre were profound in the midbrain and hindbrain regions, that is, extensive in the midbrain of Wnt1‐Cre and in the hindbrain of P0‐Cre embryos. The difference between P0‐Cre and Wnt1‐Cre in labeling cranial NC may provide a better explanation of the differential distributions of their NC derivatives and of the phenotypes caused by Cre‐driven genetic modifications.

Labeling and analysis of chicken taste buds using molecular markers in oral epithelial sheets

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.

Distribution of α-Gustducin and Vimentin in premature and mature taste buds in chickens

The sensory organs for taste in chickens (Gallus sp.) are taste buds in the oral epithelium of the palate, base of the oral cavity, and posterior tongue. Although there is not a pan-taste cell marker that labels all chicken taste bud cells, α-Gustducin and Vimentin each label a subpopulation of taste bud cells. In the present study, we used both α-Gustducin and Vimentin to further characterize chicken taste buds at the embryonic and post-hatching stages (E17-P5). We found that both α-Gustducin and Vimentin label distinct and overlapping populations of, but not all, taste bud cells. A-Gustducin immunosignals were observed as early as E18 and were consistently distributed in early and mature taste buds in embryos and hatchlings. Vimentin immunoreactivity was initially sparse at the embryonic stages then became apparent in taste buds after hatch. In hatchlings, α-Gustducin and Vimentin immunosignals largely co-localized in taste buds. A small subset of taste bud cells were labeled by either α-Gustducin or Vimentin or were not labeled. Importantly, each of the markers was observed in all of the examined taste buds. Our data suggest that the early onset of α-Gustducin in taste buds might be important for enabling chickens to respond to taste stimuli immediately after hatch and that distinctive population of taste bud cells that are labeled by different molecular markers might represent different cell types or different phases of taste bud cells. Additionally, α-Gustducin and Vimentin can potentially be used as molecular markers of all chicken taste buds in whole mount tissue.

WNT5a in tongue and fungiform Papilla development.

Fungiform papillae are complex taste organs that develop in a pattern on anterior tongue in rodent embryos. Several intrinsic secreted molecules are important for papilla development and patterning, including sonic hedgehog, bone morphogenetic proteins, Noggin, epidermal growth factor, and WNTs. Recent data about roles of WNTs in regulation of tongue and fungiform papilla development lead to new insights about the importance of tissue and timing contexts when studying the effects of morphogenetic proteins. WNT/β‐catenin signaling is required for formation of fungiform papillae, but not for determining tongue size and shape. In contrast, WNT5a apparently is important for tongue outgrowth, but not papilla development. Preliminary data from WNT5a mutant mice separate genetic programs for papilla number from those for tongue shape and size.

Contribution of Underlying Connective Tissue Cells to Taste Buds in Mouse Tongue and Soft Palate.

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.