Taste papilla cell differentiation requires the regulation of secretory protein production by ALK3-BMP signaling in the tongue mesenchyme.

Taste papillae are specialized organs, each of which comprises an epithelial wall hosting taste buds and a core of mesenchymal tissue. In the present study, we report that during early taste papilla development in mouse embryos, bone morphogenetic protein (BMP) signaling mediated by type 1 receptor ALK3 in the tongue mesenchyme is required for epithelial Wnt/ß-catenin activity and taste papilla differentiation. Mesenchyme-specific knockout (cKO) of Alk3 using Wnt1-Cre and Sox10-Cre resulted in an absence of taste papillae at E12.0. Biochemical and cell differentiation analyses demonstrated that mesenchymal ALK3-BMP signaling governed the production of previously unappreciated secretory proteins, i.e. it suppressed those that inhibit and facilitated those that promote taste papilla differentiation. Bulk RNA-sequencing analysis revealed many more differentially expressed genes (DEGs) in the tongue epithelium than in the mesenchyme in Alk3 cKO versus control. Moreover, we detected downregulated epithelial Wnt/ß-catenin signaling and found that taste papilla development in the Alk3 cKO was rescued by the GSK3ß inhibitor LiCl, but not by Wnt3a. Our findings demonstrate for the first time the requirement of tongue mesenchyme in taste papilla cell differentiation.

NFE2L1 restrains ferroptosis by transcriptionally regulating HJURP and participates in the progress of oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is a common head and neck malignancy with increasing mortality and high recurrence. In this work, we aim to explore the functional role of NFE2 like bZIP transcription factor 1 (NFE2L1) in OSCC progression. Based on databases analysis, we found that NFE2L1 was overexpressed in OSCC tumor tissues, and elevated NFE2L1 level induced poor prognosis of OSCC patients. Our results showed that NFE2L1 is upregulated in OSCC cells and overexpression of NFE2L1 promotes cell proliferation, and reduces the sensitivity of OSCC cells to erastin-induced ferroptosis. NFE2L1 upregulation decreased the levels of Fe2+, lipid reactive oxygen species and content of malondialdehyde, and increased the level of the key negative regulator of ferroptosis, GPX4 and SLC7A11. In NFE2L1 suppressed cells, these trends were reversed. Further results of dual luciferase reporter and chromatin immunoprecipitation assays confirmed that NFE2L1 could bind to the promoter of Holliday junction recognition protein (HJURP) to increase the transcriptional activity of HJURP, thus upregulating its expression. Inhibition of HJURP attenuated the proliferation and ferroptosis inhibition in NFE2L1 upregulated cells. In vivo tumorigenicity assay further proved that NFE2L1 promotes OSCC tumor growth. In summary, NFE2L1 restrains ferroptosis by transcriptionally regulating HJURP and participates in the progress of OSCC. Thus, NFE2L1 plays a key role in OSCC development and may be a promising therapeutic target for OSCC.

Taste buds are not derived from neural crest in mouse, chicken, and zebrafish.

Our lineage tracing studies using multiple Cre mouse lines showed a concurrent labeling of abundant taste bud cells and the underlying connective tissue with a neural crest (NC) origin, warranting a further examination on the issue of whether there is an NC derivation of taste bud cells. In this study, we mapped NC cell lineages in three different models, Sox10-iCreERT2/tdT mouse, GFP+ neural fold transplantation to GFP- chickens, and Sox10-Cre/GFP-RFP zebrafish model. We found that in mice, Sox10-iCreERT2 specifically labels NC cell lineages with a single dose of tamoxifen at E7.5 and that the labeled cells were widely distributed in the connective tissue of the tongue. No labeled cells were found in taste buds or the surrounding epithelium in the postnatal mice. In the GFP+/GFP- chicken chimera model, GFP+ cells migrated extensively to the cranial region of chicken embryos ipsilateral to the surgery side but were absent in taste buds in the base of oral cavity and palate. In zebrafish, Sox10-Cre/GFP-RFP faithfully labeled known NC-derived tissues but did not label taste buds in lower jaw or the barbel. Our data, together with previous findings in axolotl, indicate that taste buds are not derived from NC cells in rodents, birds, amphibians or teleost fish.

Long non-coding RNA SNHG20 promotes the tumorigenesis of oral squamous cell carcinoma via targeting miR-197/LIN28 axis.

Long non-coding RNA (lncRNA) has been verified to participate in the tumour regulation, including oral squamous cell carcinoma (OSCC). Nevertheless, the role of lncRNA SNHG20 on OSCC still remains elusive. Here, we investigate the physiopathologic functions of lncRNA SNHG20 in OSCC tumorigenesis and explore its potential mechanism. LncRNA SNHG20 was up-regulated in OSCC tissue compared with adjacent non-tumour tissue. Meanwhile, SNHG20 was overexpressed in cancer stem-like cells. In vitro and in vivo, loss-of-function experiments showed that lncRNA SNHG20 knockdown inhibited proliferative ability, mammosphere-forming ability, ALDH1 expression, stem factors (LIN28, Nanog, Oct4, SOX2) and tumour growth. Bioinformatics and luciferase reporter assay revealed that miR-197 targeted the 3'-untranslated regions of SNHG20 and LIN28 by complementary binding. Validation experiments confirmed the associated functions of SNHG20/miR-197/LIN28 axis on OSCC proliferation and stemness. In summary, our results reveal the important function of SNHG20/miR-197/LIN28 axis in the oncogenesis and stemness of OSCC, suggesting the vital role of SNHG20 in OSCC tumorigenesis.

SNHG20: A vital lncRNA in multiple human cancers.

Long noncoding RNAs (lncRNAs) act as an initial factor and promoter in different tumors as a kind of ncRNAs. The length of them is >200 nucleotides opposite small ncRNAs. Increasing researches have proved that dysregulation lncRNA has been implicated in tumorigenesis. Small nucleolar RNA host gene 20 (SNHG20), a member of lncRNAs, expresses frequently in cancer types, such as hepatocellular carcinoma, ovarian cancer, colorectal cancer, and bladder cancer, contributing to cancer development and progression by transcriptional or posttranscriptional modifications. Not only does this review show the recent published literature concerning the biological functions but also demonstrates molecular mechanisms of SNHG20 among above multiple malignancies and others.

Bitter taste receptor T2R7 and umami taste receptor subunit T1R1 are expressed highly in Vimentin-negative taste bud cells in chickens.

In the mammalian taste system, the taste receptor type 2 (T2R) family mediates bitter taste, and the taste receptor type 1 (T1R) family mediates sweet and umami tastes (the heterodimer of T1R2/T1R3 forms the sweet taste receptor, and the heterodimer of T1R1/T1R3 forms the umami taste receptor). In the chicken genome, bitter (T2R1, T2R2, and T2R7) and umami (T1R1 and T1R3) taste receptor genes have been found. However, the localization of these taste receptors in the taste buds of chickens has not been elucidated. In the present study, we demonstrated that the bitter taste receptor T2R7 and the umami taste receptor subunit T1R1 were expressed specifically in the taste buds of chickens labeled by Vimentin, a molecular marker for chicken taste buds. We analyzed the distributions of T2R7 and T1R1 on the oral epithelial sheets of chickens and among 3 different oral tissues of chickens: the palate, the base of the oral cavity, and the posterior tongue. We found that the distribution patterns and numbers were similar between taste bud clusters expressing these receptors and those expressing Vimentin. These results indicated broad distributions of T2R7 and T1R1 in the gustatory tissues of the chicken oral cavity. In addition, 3D-reconstructed images clearly revealed that high levels of T2R7 and T1R1 were expressed in Vimentin-negative taste bud cells. Taken together, the present results indicated the presence of bitter and umami sensing systems in the taste buds of chickens, and broad distribution of T2R7 and T1R1 in the chicken oral cavity.

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.

Effect of stainless-steel wire internal fixation on intracapsular condylar fracture.

OBJECTIVES: The aim of the study was to investigate the clinical effect of stainless-steel wire fixation on the early mouth-opening movement of an intracapsular fracture involving the condylar process. MATERIALS AND METHODS: In this study, patients who underwent mandibular condylar intracapsular fracture surgery in our hospital from 2012 to 2020 were selected as research subjects. A total of 44 patients received steel wire internal fixation treatment, 32 patients received titanium plate-and-nail rigid internal fixation, and 28 patients underwent conservative non-surgical treatment. RESULTS: For the patients in the stainless-steel wire group, the degree of mouth opening reached normal levels of 3.7 cm approximately 10 days after surgery. The recovery time for the patients in the titanium plate-and-nail rigid internal-fixation group was 21 days, while the patients in the conservative treatment group needed 60 days to recover. CONCLUSION: The treatment of fixation with a stainless-steel wire for intracapsular condylar fracture reduced the time taken to perform mouth-opening exercises and improved the recovery rate of patients.

OBJETIVO: Explorar el efecto clínico de la fijación de alambre de acero inoxidable en el movimiento temprano de apertura de la boca en la fractura interna del cóndilo. MÉTODO: Este estudio seleccionó a pacientes que se sometieron a cirugía de fractura intracapsular de cóndilo en nuestro hospital de 2012 a 2020 como sujetos de investigación. Un total de 44 pacientes recibieron tratamiento de fijación interna de alambre de acero, 32 recibieron placa de titanio y fijación interna con clavos, y 28 recibieron tratamiento conservador no quirúrgico. RESULTADOS: En los pacientes del grupo de alambre de acero inoxidable, alrededor de 10 días después de la cirugía el grado de apertura de la boca alcanzó un valor normal de 3.7 cm. El tiempo de recuperación de los pacientes en el grupo de fijación interna con clavos y placa de titanio fue de 21 días, mientras que los pacientes en el grupo de tratamiento conservador tardaron 60 días en recuperarse. CONCLUSIONES: La fijación con alambre de acero inoxidable para el tratamiento de la fractura intracapsular del cóndilo acorta el tiempo hasta la apertura de la boca y mejora la tasa de recuperación de los pacientes.

The main duct of von Ebner's glands is a source of Sox10 + taste bud progenitors and susceptible to pathogen infections.

We have recently demonstrated that Sox10 -expressing ( Sox10 + ) cells give rise to mainly type-III neuronal taste bud cells that are responsible for sour and salt taste. The two tissue compartments containing Sox10 + cells in the surrounding of taste buds include the connective tissue core of taste papillae and von Ebner's glands (vEGs) that are connected to the trench of circumvallate and foliate papillae. In this study, we used inducible Cre mouse models to map the cell lineages of connective tissue (including stromal and Schwann cells) and vEGs and performed single cell RNA-sequencing of the epithelium of Sox10-Cre/tdT mouse circumvallate/vEG complex. In vivo lineage mapping showed that the distribution of traced cells in circumvallate taste buds was closely linked with that in the vEGs, but not in the connective tissue. Sox10 , but not the known stem cells marker Lgr5 , expression was enriched in the cell clusters of main ducts of vEGs that contained abundant proliferating cells, while Sox10-Cre/tdT expression was enriched in type-III taste bud cells and excretory ductal cells. Moreover, multiple genes encoding pathogen receptors are enriched in the vEG main ducts. Our data indicate that the main duct of vEGs is a source of Sox10 + taste bud progenitors and susceptible to pathogen infections.

Taste papilla cell differentiation requires tongue mesenchyme via ALK3-BMP signaling to regulate the production of secretory proteins.

Taste papillae are specialized organs each of which is comprised of an epithelial wall hosting taste buds and a core of mesenchymal tissue. In the present study, we report that during the early stages of embryonic development, bone morphogenetic protein (BMP) signaling mediated by type 1 receptor ALK3 in the tongue mesenchyme is required for the epithelial Wnt/ß-catenin activity and taste papilla cell differentiation. Mesenchyme-specific knockout ( cKO ) of Alk3 using Wnt1-Cre and Sox10-Cre resulted in an absence of taste papillae at E12.0. Biochemical and cell differentiation analyses demonstrated that mesenchymal ALK3-BMP signaling governs the production of previously unappreciated secretory proteins, i.e., suppresses those that inhibiting and facilitates those promoting taste cell differentiation. Bulk RNA-Sequencing analysis revealed many more differentially expressed genes (DEGs) in the tongue epithelium than in the mesenchyme in Alk3 cKO vs control. Moreover, we detected a down-regulated epithelial Wnt/ß-catenin signaling, and taste papilla development in the Alk3 cKO was rescued by GSK3ß inhibitor LiCl, but not Wnt3a. Our findings demonstrate for the first time the requirement of tongue mesenchyme in taste papilla cell differentiation. Summary statement: This is the first set of data to implicate the requirement of tongue mesenchyme in taste papilla cell differentiation.

Cell Dissociation from the Tongue Epithelium and Mesenchyme/Connective Tissue of Embryonic-Day 12.5 and 8-Week-Old Mice.

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.

Deletion of Nf2 in neural crest-derived tongue mesenchyme alters tongue shape and size, Hippo signalling and cell proliferation in a region- and stage-specific manner.

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- /Nf2fx/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.

[Stability analysis of on-board calibration system of spatially modulated imaging Fourier transform spectrometer].

Spatially modulated imaging Fourier transform spectrometer (SMIFTS) was an instrument that depended on interference, and after calibration, the reconstruction spectrum can quantificationally reflect the diffuse reflection of target under sunshine. On-board calibration of SMIFTS confirmed the change of SMIFTS according to relative spectrum calibration, inspected long-time attenuation of SMIFTS optical system, and corrected export data of SMIFTS. According to the requirement of remote sensor application, it must stay in vacuum environment for a long time. As a radiant standard, the stability of lamp-house in long time is the most important characteristic of on-board calibration system. By calculation and experimentation, analyses of on-board calibration of SMIFTS, and testing spaceflight environment characteristic of on-board calibration, the difficulty in the key parts of on-board calibration of SMIFTS such as lamp-house, spectrum filter and integrating sphere was solved. According to the radiation-time stability testing for lamp-house and optics, particle-radiation testing, environmental-mechanics testing and hot vacuum examination, good result was obtained. By whole-aperture and part-field comparative wavelength calibration, the spectrum curve and before-launch interferogram were obtained. After comparison with reconstruction spectrum under different condition, the stability and credibility of SMIFTS on-board calibration system was proved.

[Modeling and simulation of effect of optical distortion on the large aperture static imaging spectrometer].

As a new type Fourier transform imaging spectrometry, large aperture static imaging spectrometry (LASIS) has come forth in recent years, which has many advantages such as simple principle, high stability and so on. However, the requirement for the optical system design of LASIS was very harsh. As one of the optical aberrations, optical distortion degrades the data quality acquired by LASIS, consequently limits its applications. According to the analysis of the data acquisition principle of LASIS, the data model with the effect of optical distortion was presented, which could be used for LASIS performance pre-evaluation. Finally, the computer simlulation of the data model was achieved with supposed parameters. The simulation results indicated that the relative error more than 5% was induced in the recovery spectrum, and approximate 8 nm spectral line deviation was occurred at the long wavelength region. The results show that the 4% optical distortion was inapplicable for LASIS although it is acceptant for common optical imaging system.

[Spatially modulated Fourier transform imaging spectrometer data compression research].

Fourier transform imaging spectrometer is a new technic, and has been developed very fast in recent ten years. When it is used in satellite, because of the limit by the data transmission, the authors need to compress the original data obtained by the Fourier transform imaging spectrometer, then, the data can be transmitted, and can be incepted on the earth and decompressed. Then the authors can do data process to get spectrum data which can be used by user. Data compression technic used in Fourier transform imaging spectrometer is a new technic, and few papers introduce it at home and abroad. In this paper the authors will give a data compression method, which has been used in EDIS, and achieved a good result.

SARS-CoV-2 Receptor ACE2 Is Enriched in a Subpopulation of Mouse Tongue Epithelial Cells in Nongustatory Papillae but Not in Taste Buds or Embryonic Oral Epithelium.

As a result of the COVID-19 pandemic, evidence revealed that SARS-CoV-2 infection caused taste loss at a rate higher than that of influenza. ACE2, the entry receptor of SARS-CoV-2, has been identified in the oral epithelium; however, it is unclear at what developmental stage ACE2 expression emerges and whether ACE2 is expressed in taste buds. To identify the specific developmental stage, we analyzed RNA-Seq data from embryonic and newborn mouse oral tissue. We found that robust ACE2 expression was observed in the newborn oral epithelium. In contrast, only extremely low levels, if any, of ACE2 transcripts in the embryonic stage oral tissue were found (E12.5 and E14.5). Analyses of three public scRNA-seq data sets of adult mouse tongue epithelial cells showed that receptors for various viruses were enriched in distinct clusters of tongue epithelial cells. ACE2 was enriched in a subpopulation of epithelial cells in the basal region of nongustatory filiform papillae but not in the taste papillae or taste buds. Expression of ACE2 was detected in a small proportion of type III taste cells. Our results indicate that when applied across species, nongustatory papilla epithelial cells are the prime targets for SARS-CoV-2 infection in the tongue; thus, taste loss in COVID-19 patients is likely not caused by a direct infection of SARS-CoV-2 to taste bud cells. Additionally, fetuses at different stages of development may have distinct susceptibility to SARS-CoV-2 infection.

Resistance of Scratched Fused Silica Surface to UV Laser Induced Damage.

Scratches in fused silica are notorious laser damage precursors to UV laser damage initiation. Ductile and brittle scratches were intentionally generated using various polishing slurries. The distribution, profile and the dimension of scratches were characterized. The damage resistance of polished surfaces was evaluated using raster scanning damage testing protocol. The results show that both ductile and brittle scratches greatly increase area proportion of laser damage about one to two orders of magnitude relative to unscratched surface and brittle scratches are more deleterious. Moreover, finite difference time domain (FDTD) simulation was used to numerically calculate the light field distribution around scratches on rear surface (i.e. exit surface for light) which indicates that modulated light intensity is susceptible to the profile and size of scratches. FDTD simulation results also indicate that the light field intensification is elevated with the dimension of scratches and light modulation effects in triangular scratches are usually not as notable as serrated and parabolic scratches.

Abundant proliferating cells within early chicken taste buds indicate a potentially "built-in" progenitor system for taste bud growth during maturation in hatchlings.

Like other epithelial cells, taste bud cells have a short life span and undergo continuous turnover. An active stem or progenitor cell niche is essential for taste bud formation and maintenance. Early taste bud cells have a life span of ~4 days on average in chicken hatchlings when taste buds grow rapidly and undergo maturation. The average life span is shorter than that of mature taste bud cells of rodents (~10-12 days on average). To better understand the mechanism underlying taste bud growth and homeostasis in chickens, we analyzed the distribution of proliferating cells in different tissue compartments, including taste buds, the surrounding epithelium and the underlying connective tissue in P1-3 hatchlings and P45 chickens. Unlike rodents, which lack proliferating cells within both early and mature taste buds, chickens possessed abundant proliferating cells within early taste buds. Further, at post-hatch day 45, when taste buds are mature and undergo continuous cell renewal, taste buds also contained proliferating cells, though to a lesser extent. These proliferating cells in early taste buds, indicated by PCNA⁺ and BrdU⁺ cells, primarily localized to the basal region of taste buds and were largely unlabeled by the two known molecular markers for chicken taste bud cells (Vimentin and α-Gustducin), suggesting their undifferentiated status. Our data indicate that early chicken taste buds have "built-in" progenitors in order to grow to and maintain their large size and rapid cell turnover in hatchlings.

An Update on the Sense of Taste in Chickens: A Better Developed System than Previously Appreciated.

Taste is important in guiding nutritive choices and motivating food intake. The sensory organs for taste are the taste buds, that transduce gustatory stimuli into neural signals. It has been reported that chickens have a low taste bud number and thus low taste acuity. However, more recent studies indicate that chickens have a well-developed taste system and the reported number and distribution of taste buds may have been significantly underestimated. Chickens, as a well-established animal model for research, are also the major species of animals in the poultry industry. Thus, a clear understanding of taste organ formation and the effects of taste sensation on nutrition and feeding practices is important for improving livestock production strategies. In this review, we provide an update on recent findings in chicken taste buds and taste sensation indicating that the chicken taste organ is better developed than previously thought and can serve as an ideal system for multidisciplinary studies including organogenesis, regenerative medicine, feeding and nutritional choices.

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.