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Taste bud cells are specialized epithelial cells that undergo continuous turnover, and thus require active progenitors for their renewal and an intact taste function. Our previous studies suggested that a population of taste bud cells originates from outside of the surrounding tongue epithelium—previously regarded sole source of taste bud progenitors. In this study, we demonstrated that SOX10 (SRY-related HMG-box gene 10)-expressing cells, known to be in the migrating neural crest, were also distributed in taste bud-surrounding tissue compartments under the tongue epithelium, that is, the connective tissue core of taste papillae and von Ebner's glands. By lineage tracing of SOX10-expressing cells using SOX10-Cre, a Cre model driven by the endogenous SOX10 promoter, crossing with a Cre reporter line R26-tdTomato (tdT), we found SOX10-Cre-labeled tdT(+) cells within taste buds in all three types of taste papillae (fungiform, circumvallate, and foliate) as well as in the soft palate in postnatal mice. The tdT(+) taste bud cells were progressively more abundant along the developmental stages, from virtually zero at birth to over 35% in adults. Most of tdT(+) taste bud cells had a low intensity of immunosignals of Keratin 8 (a widely used taste bud cell marker). In circumvallate taste buds, tdT signals were co-localized principally with a type III taste bud cell marker, less so with type I and II cell makers. Together, our data demonstrate a novel progenitor source for taste buds of postnatal mice—SOX10-Cre-labeled cells in the connective tissue core and/or von Ebner's glands.

Objectives The mammalian tongue develops from the branchial arches (1–4) and comprises highly organized tissues compartmentalized by mesenchyme/connective tissue that is largely derived from neural crest (NC). This study aimed to understand the roles of tumour suppressor Neurofibromin 2 (Nf2) in NC‐derived tongue mesenchyme in regulating Hippo signalling and cell proliferation for the proper development of tongue shape and size. Materials and methods Conditional knockout (cKO) of Nf2 in NC cell lineage was generated using Wnt1‐Cre (Wnt1‐Cre/Nf2cKO ). Nf2 expression, Hippo signalling activities, cell proliferation and tongue shape and size were thoroughly analysed in different tongue regions and tissue types of Wnt1‐Cre/Nf2cKO and Cre ‐/Nf2 fx/fx littermates at various stages (E10.5–E18.5). Results In contrast to many other organs in which the Nf2/Hippo pathway activity restrains growth and cell proliferation and as a result, loss of Nf2 decreases Hippo pathway activity and promotes an enlarged organ development, here we report our observations of distinct, tongue region‐ and stage‐specific alterations of Hippo signalling activity and cell proliferation in Nf2cKO in NC‐derived tongue mesenchyme. Compared to Cre −/Nf2fx / fx littermates, Wnt1‐Cre/Nf2cKO depicted a non‐proportionally enlarged tongue (macroglossia) at E12.5–E13.5 and microglossia at later stages (E15.5–E18.5). Specifically, at E12.5 Nf2cKO mutants had a decreased level of Hippo signalling transcription factor Yes‐associated protein (Yap), Yap target genes and cell proliferation anteriorly, while having an increased Yap, Yap target genes and cell proliferation posteriorly, which lead to a tip‐pointed and posteriorly widened tongue. At E15.5, loss of Nf2 in the NC lineage resulted in distinct changes in cell proliferation in different regions, that is, high in epithelium and mesenchyme subjacent to the epithelium, and lower in deeper layers of the mesenchyme. At E18.5, cell proliferation was reduced throughout the Nf2cKO tongue., Schematic diagram to represent the stage‐ and region‐specific roles of Nf2‐mediated Hippo signaling in the mesenchyme during tongue organogenesis. Nf2 deletion in Neural crest‐derived tongue mesenchyme resulted in distinct alterations of Yap level and cell proliferation causing non‐proportionately larger tongue during the early stages of tongue development (i.e., E12.5‐E13.5) and microglossia during the later stages of tongue development (i.e., E15.5‐E18.5).

Cell dissociation has been an essential procedure for studies at the individual-cell level and/or at a cell-population level (e.g., single cell RNA sequencing and primary cell culture). Yielding viable, healthy cells in large quantities is critical, and the optimal conditions to do so are tissue dependent. Cell populations in the tongue epithelium and underlying mesenchyme/connective tissue are heterogeneous and tissue structures vary in different regions and at different developmental stages. We have tested protocols for isolating cells from the mouse tongue epithelium and mesenchyme/connective tissue in the early developmental [embryonic day 12.5 (E12.5)] and young adult (8-week) stages. A clean separation between the epithelium and underlying mesenchyme/connective tissue was easy to accomplish. However, to further process and isolate cells, yielding viable healthy cells in large quantities, and careful selection of enzymatic digestion buffer, incubation time, and centrifugation speed and time are critical. Incubation of separated epithelium or underlying mesenchyme/connective tissue in 0.25% Trypsin-EDTA for 30 min at 37 °C, followed by centrifugation at 200 x g for 8 min resulted in a high yield of cells at a high viability rate (>90%) regardless of the mouse stages and tongue regions. Moreover, we found that both dissociated epithelial and mesenchymal/connective tissue cells from embryonic and adult tongues could survive in the cell culture-based medium for at least 3 h without a significant decrease of cell viability. The protocols will be useful for studies that require the preparation of isolated cells from mouse tongues at early developmental (E12.5) and young adult (8-week) stages requiring cell dissociation from different tissue compartments.

Proper development of taste organs including the tongue and taste papillae requires interactions with the underlying mesenchyme through multiple molecular signaling pathways. The effects of bone morphogenetic proteins (BMPs) and antagonists are profound, however, the tissue-specific roles of distinct receptors are largely unknown. Here, we report that constitutive activation (ca) of ALK2-BMP signaling in the tongue mesenchyme (marked by Wnt1-Cre) caused microglossia-a dramatically smaller and misshapen tongue with a progressively severe reduction in size along the anteroposterior axis and absence of a pharyngeal region. At E10.5, the tongue primordia (branchial arches 1-4) formed in Wnt1-Cre/caAlk2 mutants while each branchial arch responded to elevated BMP signaling distinctly in gene expression of BMP targets (Id1, Snai1, Snai2, and Runx2), proliferation (Cyclin-D1) and apoptosis (p53). Moreover, elevated ALK2-BMP signaling in the mesenchyme resulted in apparent defects of lingual epithelium, muscles, and nerves. In Wnt1-Cre/caAlk2 mutants, a circumvallate papilla was missing and further development of formed fungiform papillae was arrested in late embryos. Our data collectively demonstrate that ALK2-BMP signaling in the mesenchyme plays essential roles in orchestrating various tissues for proper development of the tongue and its appendages in a region-specific manner.

Mammalian taste buds emerge perinatally and most become mature 3–4 weeks after birth. Mature taste bud cells in rodents are known to be renewed by the surrounding K14(+) basal epithelial cells and potentially other progenitor source(s), but the dynamics between initially developed taste buds and surrounding tissue compartments are unclear. Using the K14-Cre and Dermo1-Cre mouse lines to trace epithelial and mesenchymal cell lineages, we found that early taste buds in E18.5 and newborn mouse tongues are not derived from either lineage. At E11.5 when the tongue primordia (i.e., lingual swellings) emerge, the relatively homogeneous sonic hedgehog-expressing (Shh(+)) epithelial cells express Keratin (K) 8, a marker that is widely used to label taste buds. Mapping lineage of E11.0 Shh(+) epithelium of the tongue rudiment with Shh-CreER(T2)/RFP mice demonstrated that both the early taste buds and the surrounding lingual epithelium are from the same population of progenitors – Shh(+) epithelial cells of the tongue primordium. In combination with previous reports, we propose that Shh(+)K8(+) cells in the homogeneous epithelium of tongue primordium at early embryonic stages are programmed to become taste papilla and taste bud cells. Switching off Shh and K8 expression in the Shh(+) epithelial cells of the tongue primordium transforms the cells to non-gustatory cells surrounding papillae, including K14(+) basal epithelial cells which will eventually contribute to the cell renewal of mature taste buds.

Sweet orange (Citrus sinensis), a key fruit species, is considered as a primary ingredient in herbal medical formulations against ailments such as food borne diseases. Sour orange (C. aurantium) is also very famous as a medicinal plant. There are six commonly grown sweet orange cultivars in Sri Lanka (Arogya, Bibila sweet, MKD, Sisila, BAN and MT) but the antibacterial activity present in their fruit juice is not well documented. Therefore, the present study was conducted to characterize the antibacterial activity of the fruit juice of these sweet oranges in comparison to sour orange and also to establish DNA barcodes for the tested cultivars for precise identification. Fruit juice was collected from sweet orange cultivars and sour orange and antibacterial activity was measured against three model pathogenic bacterial species, Escherichia coli, Staphylococcus aureus and methicillin-resistant S. aureus. After employing filter paper disc method, the diameter of zone of bacterial inhibition (DZI) was measured as the parameter of antibacterial activity. The genomic DNA was extracted from all the tested plants and PCR amplified using trnH–psbA primer pair and subjected to DNA sequencing, followed by alignment analysis and dendrogram construction. Arogya and MKD did not show any antibacterial activity (DZI = 0.0 mm), whereas Sisila, BAN and MT showed antibacterial activity only against E. coli and S. aureus (mean DZI of 8.2 mm and 8.4 mm respectively). Bibila sweet and sour orange showed significantly higher antibacterial activity against all E. coli, S. aureus and methicillin-resistant S. aureus (mean DZI of 10.2 mm, 10.5 mm and 7.8 mm respectively). DNA barcoding provided unique sequence identifiers for each cultivar. These antibacterial activity data in combination with DNA barcodes could help to develop new cultivars through breeding to promote the sweet orange industry in Sri Lanka.The Journal of Agricultural Sciences, vol.11, No 1, January 2016, pp.13-23

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.

Chili pepper ( Capsicum spp.) is important as a spice, flavour enhancer, vegetable and component in herbal medicine. The numerous phytochemicals and their medicinally important properties present in diverse germplasm of chili pepper have been characterized and documented. Capsaicinoids, carotenoids, vitamins, flavonoids such as anthocyanins are present as the major phytochemicals in chili pepper fruits. Capsaicinoids, pungent analogues of capsinoids, are the most important group of phytochemicals in which capsaicin and dihydrocapsaic in are prominent in providing the basis for pungency and medicinal properties. The detailed studies conducted on the phytochemicals in chili pepper fruits using mouse models and human cell lines have reported an array of anticancer effects on leukemia, myeloma and various carcinomas associated with the digestive tract. In addition, chili pepper possesses anti-inflammatory, antidiabetic, antimicrobial, anticholesteremic, anticlotting and antioxidant activities. Therefore, this review is compiled to provide a comprehensive assessment of the phytochemical profile and medicinal values of chili pepper. Collectively, numerous studies performed to date demonstrated the potential medicinal significance of chili pepper for development of herbal medicine and to plan further studies to elucidate other hitherto unknown medicinal values.