Project description:We performed single-cell RNA-seq (10x Chromium 3' v.3.1) on dissociated single cell suspensions from mouse (C57BL/6) osmosensory brain nuclei - subfornical organ (SFO) to determine transcriptomic cell types and neuronal activation patterns. By performing single cell RNA-seq based stimulus to cell-type mapping, we captured the endogenous immediate early gene expression triggered by distinct physiological state stimuli in SFO cells. We identified a unique excitatory neuron type in SFO that is uniquely tuned to sodium deficiency.
Project description:Taste stem/progenitor cells from the mouse posterior tongue have been recently used to generate taste bud organoids. However, the inaccessible location of the taste receptor cells is observed in conventional organoids. Here, we established a suspension culture method for fine tuning of taste bud organoid by apicobasal polarity alteration to form the accessible localization of taste receptor cells in organoid. Compared to conventional Matrigel-embedded organoids, suspension-cultured organoids showed comparable differentiation and renewal rates to those of taste buds in vivo and exhibited functional taste receptor cells and cycling progenitor cells. Accessible taste receptor cells on the outer region of taste bud organoids enabled the direct application of calcium imaging for evaluating the taste response. Moreover, suspension-cultured organoids could be genetically altered using gene editing methods. Suspension-cultured taste bud organoid harmoniously integrated with the recipient lingual epithelium; maintained the taste receptor cells and gustatory innervation capacity. Thus, we propose that suspension-cultured organoids may provide efficient model for taste research including taste bud development, regeneration and transplantation
Project description:To uncover novel molecules involved in taste detection, we performed a microarray-based screen for genes enriched in taste neurons. Proboscis RNA from flies homozygous for a recessive poxn null mutation was compared to RNA from heterozygous controls. Poxn mutants have a transformation of labellar gustatory chemosensory bristles into mechanosensory bristles and therefore lack most or all taste neurons. Experiment Overall Design: Proboscises of poxn70 homozygous mutant and poxn70 heterozygous mutant males (8-18 days post eclosure) were dissected, and total RNA was harvested in Trizol according to standard trizol protocol. Samples for each microarray were prepared from 164-280 proboscises. We performed 3 biological replicates for each genotype.
Project description:Low-calorie sweetener (LCS) consumption in children has increased dramatically due to widespread presence in the food environment and efforts to mitigate obesity through sugar replacement. However, mechanistic studies on the long-term impact of early-life LCS consumption on cognitive function and physiological processes are lacking. Here, we developed a rodent model to evaluate the effects of daily LCS consumption (acesulfame potassium, saccharin, or stevia) during adolescence on adult metabolic, behavioral, gut microbiome, and brain transcriptomic outcomes. Results reveal that habitual early-life LCS consumption impacts normal post-oral glucose handling and impairs hippocampal-dependent memory in the absence of weight gain. Furthermore, adolescent LCS consumption yielded long-term reductions in lingual sweet taste receptor expression and alterations in sugar-motivated appetitive and consummatory responses. While early life LCS consumption did not produce robust changes in the gut microbiome, brain region-specific RNA sequencing analyses reveal LCS-induced changes in collagen- and synaptic signaling-related gene pathways in the hippocampus and nucleus accumbens, respectively, in a sex-dependent manner. Collectively, these results reveal that habitual early-life LCS consumption has long lasting implications for glucoregulation, sugar-motivated behavior, and hippocampal-dependent memory in rats, which may be based in part on changes in nutrient transporter, sweet taste receptor, and central gene pathway expression.
Project description:Taste substances are received by taste receptors expressed in taste cells. “Salty taste” sensation is evoked when sodium and chloride ions are present together in the oral cavity. The presence of an epithelial cation channel that receives Na+ has previously been reported. However, no molecular entity involving Cl- receptors has been elucidated. We report the strong expression of transmembrane channel-like 4 (TMC4) in the circumvallate and foliate papillae projected to the glossopharyngeal nerve, mediating a high-concentration of NaCl. Electrophysiological analysis using HEK293T cells revealed that TMC4 was a voltage-dependent Cl- channel and the consequent currents were completely inhibited by NPPB, an anion channel blocker. This channel could be activated without an increase in intracellular calcium ion. TMC4 allowed permeation of organic anions including gluconate, but their current amplitudes at positive potentials were less than that of Cl-. Tmc4-deficient mice showed significantly weaker glossopharyngeal nerve response to high-concentration of NaCl than the wild-type littermates. These results indicated that TMC4 is a novel chloride channel that responds to high-concentration of NaCl.
Project description:Previously we showed that taste receptor cells in situ in taste buds synthesize insulin. Here we describe a model of pig taste organoid culture in which we have promoted insulin expression by induction of quiescence. The cellular heterogeneity of the lingual epithelium is maintained in the organoids, and stem cell type and organoid architecture can be controlled through changes in media composition and/or use of static versus dynamic culture. Pig taste organoids were maintained long term and organoids cultured in low sheer stress dynamic exhibited an architecture and expression profile akin to the native tissue. Porcine taste organoids also contained insulin, and the insulin critical transcription factors MAFA and PAX4. These results provide a pig model of taste organoid culture that can be used universally and bring us closer to the use of the taste tissue as a new renewable source of beta cells
Project description:To understand the mechanisms that regulate the renewal and maintenance of taste cells we performed RNA-sequencing analysis on isolated taste cells from 2 and 6 month old mice to determine how alterations in the taste cell-transcriptome regulate taste cell maintenance and function in adults. We found that the Activator Protein-1 (AP1) transcription factors (c-Fos, Fosb and c-Jun) and genes associated with this pathway were significantly downregulated in taste cells from 6 month old mice and further declined at 12 months. We generated conditional c-Fos- knockout mice to target K14-expressing cells, including differentiating taste cells. c-Fos deletion caused a severe perturbation in taste bud structure and resulted in a significant reduction in the taste bud size. c-Fos deletion also affected taste cell turnover as evident by, a decrease in proliferative markers, and upregulation of the apoptotic marker cleaved-PARP. Thus, AP1 factors are important regulators of adult taste cell renewal and their downregulation negatively impacts taste maintenance. Comparison of genes responsible for peripheral Taste system maintenance at age, 3 and 6 months. processed_data.txt: List of B vs S gene expression data gene_exp.diff: cuffdiff data for 2 and 6 month taste cells
Project description:Novel taste memories, critical for animal survival, are consolidated to form long term memories which are dependent on translation regulation in the gustatory cortex We used microarray for identification of genes involved in novel taste learning at two time points- 1 and 3 hours following memory formation for novel taste Adult rats were separated into two groups- novel tatse (0.1% sacchain) and water, two time points were used 1, 3 hours following learnin. Rna was extracted and hybridized on Affymetrix microarray