Bud Cells Research Articles

TNFα has differential effects on the transcriptome profile of selected populations in murine cartilage

ObjectiveTo further our understanding of the role of tumor necrosis factor (TNF)α on the inflammatory response in chondrocytes. DesignWe explored the effects of TNFα on the transcriptome of epiphyseal chondrocytes from newborn C57BL/6 mice at the total and single cell (sc) resolution. ResultsGene set enrichment analysis of total RNA-Seq from TNFα-treated chondrocytes revealed enhanced response to biotic stimulus, defense and immune response and cytokine signaling and suppressed cartilage and skeletal morphogenesis and development. scRNA-Seq analyzed 14,239 ​cells and 24,320 genes and distinguished 16 ​cell clusters. The more prevalent ones were constituted by limb bud and chondrogenic cells and fibroblasts comprising ∼73 ​% of the cell population. Genes expressed by joint fibroblasts were detected in 5 clusters comprising ∼45 ​% of the cells isolated. Pseudotime trajectory finding revealed an association between fibroblast and chondrogenic clusters which was not modified by TNFα. TNFα decreased the total cells recovered by 18.5 ​% and the chondrogenic, limb bud and mesenchymal clusters by 32 ​%, 27 ​% and 7 ​%, respectively. TNFα had profound effects on the insulin-like growth factor (IGF) axis decreasing Igf1, Igf2 and Igfbp4 and inducing Igfbp3 and Igfbp5, explaining an inhibition of collagen biosynthesis, cartilage and skeletal morphogenesis. Ingenuity Pathway Analysis of scRNA-Seq data revealed that TNFα enhanced the osteoarthritis, rheumatoid arthritis, pathogen induced cytokine storm and interleukin 6 signaling pathways and suppressed fibroblast growth factor signaling. ConclusionsEpiphyseal chondrocytes are constituted by diverse cell populations distinctly regulated by TNFα to promote inflammation and suppression of matrix biosynthesis and growth.

The Buds of Oscarella lobularis (Porifera, Homoscleromorpha): A New Convenient Model for Sponge Cell and Evolutionary Developmental Biology.

The comparative study of the four non-bilaterian phyla (Cnidaria, Placozoa, Ctenophora, and Porifera) provides insights into the origin of bilaterian traits. To complete our knowledge of the cell biology and development of these animals, additional non-bilaterian models are needed. Given the developmental, histological, ecological, and genomic differences between the four sponge classes (Demospongiae, Calcarea, Homoscleromorpha, and Hexactinellida), we have been developing the Oscarella lobularis (Porifera, class Homoscleromorpha) model over the past 15 years. Here, we report a new step forward by inducing, producing, and maintaining in vitro thousands of clonal buds that now make possible various downstream applications. This study provides a full description of bud morphology, physiology, cells and tissues, from their formation to their development into juveniles, using adapted cell staining protocols. In addition, we show that buds have outstanding capabilities of regeneration after being injured and of re-epithelization after complete cell dissociation. Altogether, Oscarella buds constitute a relevant all-in-one sponge model to access a large set of biological processes, including somatic morphogenesis, epithelial morphogenesis, cell fate, body axes formation, nutrition, contraction, ciliary beating, and respiration.

Generation of human expandable limb-bud-like progenitors via chemically induced dedifferentiation.

In certain highly regenerative animals, cellular dedifferentiation occurs after injury, allowing specialized cells to become progenitor cells for regeneration. However, this capacity is restricted in human cells due to reduced plasticity. Here, we introduce a chemical-induced dedifferentiation approach that reverts the differentiated cells to a progenitor-like state, conferring the features of human limb bud cells from human adult somatic cells. These chemically induced human limb-bud-like progenitors (hCiLBP cells) show a high degree of transcriptomic similarity to human embryonic limb bud progenitors. Importantly, we established culture conditions that allow hCiLBP cells to undergo extensive expansion while maintaining population homogeneity and long-term self-renewal capacity. Moreover, hCiLBP cells exhibit increased osteochondrogenic differentiation ability, providing an innovative platform for generation of skeletal lineage cell types. These results highlight a potential therapeutic approach for repairing damaged human tissues through reversal of developmental pathways from mature cells to expandable progenitor cells.

FAHFA promotes intracellular calcium signaling via activating the fat taste receptor, CD36 and Src protein kinases in mice taste bud cells

Two lipid sensors, CD36 and GPR120, are crucial for the orosensory detection of fat taste and for mediating fat preference. However, the mechanism by which endogenous lipid (FAHFA) binds to CD36 to initiate intracellular signaling remains unexplained. Hence, the primary objective of this study is to investigate the binding mechanism of FAHFA to CD36 and its role in isolated mouse taste bud cells (mTBCs). The Schrodinger platform was used to assess the molecular dynamics of protein and ligand interactions, and an in vitro experiment was used to validate the findings. Based on the docking score of the ligand, the molecular mechanistic activities of the targeted complexes, CD36–5-POHSA (−8.2 kcal/mol), were investigated using the dynamic simulation. In comparison to linoleic acid (LA), POHSA rapidly increased [Ca2+]i via acting on CD36, and 5-POHSA treatment in mTBCs activated src-kinase at 20 μM. CD36 siRNA transfection in TBCs downregulate the CD36 protein expression as well as [Ca2+]i flux. This study suggests that 5-POHSA may help combat taste abnormalities and the adverse effects of obesity by binding to the lingual CD36 receptor and activating the tongue-brain axis.

Different taste map for amiloride sensitivity, response frequency, and threshold to NaCl in the rostral nucleus of the solitary tract in rats.

Studies on taste bud cells and brain stem relay nuclei suggest that alternative pathways convey information regarding different taste qualities. Building on the hypothesis that amiloride (epithelial Na channel antagonist)-sensitive neurons respond to palatable salt (low-concentration) and amiloride-insensitive neurons respond to aversive salt (high-concentration), we investigated the histological distribution of taste-sensitive neurons in the rostral nucleus of the solitary tract in rats and their NaCl and amiloride sensitivities. We recorded neuronal activity in extracellular single units using multi-barrel glass micropipettes and reconstructed their locations on the rostrocaudal and mediolateral axes. Seventy-three taste-sensitive neurons were categorized into the best-taste category. The amiloride sensitivities of the 31 neurons were examined for 0.1, 0.2, 0.4, and 0.8 M NaCl. The neuronal distribution of amiloride-sensitive neurons was located in the lateral region, while amiloride-insensitive neurons were located in the medial region. The amiloride-sensitive neurons responded to low salt concentrations, signaling the NaCl levels required by body fluids. Amiloride-insensitive neurons were silent at low salt concentrations but may function as warning signals for high salt concentrations. Low-threshold and/or high-response neurons were located in the rostrolateral region. In contrast, high-threshold and/or low-response neurons were located in the caudal-medial region.

The Expression of Cannabinoid and Cannabinoid-Related Receptors on the Gustatory Cells of the Piglet Tongue.

The gustatory system is responsible for detecting and evaluating the palatability of the various chemicals present in food and beverages. Taste bud cells, located primarily on the tongue, communicate with the gustatory sensory neurons by means of neurochemical signals, transmitting taste information to the brain. It has also been found that the endocannabinoid system (ECS) may modulate food intake and palatability, and that taste bud cells express cannabinoid receptors. The purpose of this study was to investigate the expression of cannabinoid and cannabinoid-related receptors in the gustatory cells of the papillae vallatae and foliatae of ten piglets. Specific antibodies against the cannabinoid receptors (CB1R and CB2R), G protein-coupled receptor 55 (GPR55), transient receptor potential vanilloid 1 (TRPV1) and ankyrin 1 (TRPA1) were applied on cryosections of lingual tissue; the lingual tissue was also processed using Western blot analysis. Cannabinoid and cannabinoid-related receptors were found to be expressed in the taste bud cells and the surrounding epithelial cells. The extra-papillary epithelium also showed strong immunolabeling for these receptors. The results showed that these receptors were present in both the taste bud cells and the extra-gustatory epithelial cells, indicating their potential role in taste perception and chemesthesis. These findings contributed to understanding the complex interactions between cannabinoids and the gustatory system, highlighting the role of the ECS within taste perception and its potential use in animal production in order to enhance food intake.

Tuft cells in the intestine, immunity and beyond.

Tuft cells have gained substantial attention over the past 10 years due to numerous reports linking them with type 2 immunity and microorganism-sensing capacity in many mucosal tissues. This heightened interest is fuelled by their unique ability to produce an array of biological effector molecules, including IL-25, allergy-related eicosanoids, and the neurotransmitter acetylcholine, enabling downstream responses in diverse cell types. Operating through G protein-coupled receptor-mediated signalling pathways reminiscent of type II taste cells in oral taste buds, tuft cells emerge as chemosensory sentinels that integrate luminal conditions, eliciting appropriate responses in immune, epithelial and neuronal populations. How tuft cells promote tissue alterations and adaptation to the variety of stimuli at mucosal surfaces has been explored in multiple studies in the past few years. Since the initial recognition of the role of tuft cells, the discovery of diverse tuft cell effector functions and associated feedback loops have also revealed the complexity of tuft cell biology. Although earlier work largely focused on extraintestinal tissues, novel genetic tools and recent mechanistic studies on intestinal tuft cells established fundamental concepts of tuft cell activation and functions. This Review is an overview of intestinal tuft cells, providing insights into their development, signalling and interaction modules in immunity and other states.

The role of GABA in modulation of taste signaling within the taste bud.

Taste buds contain 2 types of GABA-producing cells: sour-responsive Type III cells and glial-like Type I cells. The physiological role of GABA, released by Type III cells is not fully understood. Here, we investigated the role of GABA released from Type III cells using transgenic mice lacking the expression of GAD67 in taste bud cells (Gad67-cKO mice). Immunohistochemical experiments confirmed the absence of GAD67 in Type III cells of Gad67-cKO mice. Furthermore, no difference was observed in the expression and localization of cell type markers, ectonucleoside triphosphate diphosphohydrolase 2 (ENTPD2), gustducin, and carbonic anhydrase 4 (CA4) in taste buds between wild-type (WT) and Gad67-cKO mice. Short-term lick tests demonstrated that both WT and Gad67-cKO mice exhibited normal licking behaviors to each of the five basic tastants. Gustatory nerve recordings from the chorda tympani nerve demonstrated that both WT and Gad67-cKO mice similarly responded to five basic tastants when they were applied individually. However, gustatory nerve responses to sweet-sour mixtures were significantly smaller than the sum of responses to each tastant in WT mice but not in Gad67-cKO mice. In summary, elimination of GABA signalling by sour-responsive Type III taste cells eliminates the inhibitory cell-cell interactions seen with application of sour-sweet mixtures.

FBLN2 is associated with basal cell markers Krt14 and ITGB1 in mouse mammary epithelial cells and has a preferential expression in molecular subtypes of human breast cancer.

Fibulin-2 (FBLN2) is a secreted extracellular matrix (ECM) glycoprotein and has been identified in the mouse mammary gland, in cap cells of terminal end buds (TEBs) during puberty, and around myoepithelial cells during early pregnancy. It is required for basement membrane (BM) integrity in mammary epithelium, and its loss has been associated with human breast cancer invasion. Herein, we attempted to confirm the relevance of FBLN2 to myoepithelial phenotype in mammary epithelium and to assess its expression in molecular subtypes of human breast cancer. The relationship between FBLN2 expression and epithelial markers was investigated in pubertal mouse mammary glands and the EpH4 mouse mammary epithelial cell line using immunohistochemistry, immunocytochemistry, and immunoblotting. Human breast cancer mRNA data from the METABRIC and TCGA datasets from Bioportal were analyzed to assess the association of Fbln2 expression with epithelial markers, and with molecular subtypes. Survival curves were generated using data from the METABRIC dataset and the KM databases. FBLN2 knockdown in mouse mammary epithelial cells was associated with a reduction in KRT14 and an increase in KRT18. Further, TGFβ3 treatment resulted in the upregulation of FBLN2 in vitro. Meta-analyses of human breast cancer datasets from Bioportal showed a higher expression of Fbln2 mRNA in claudin-low, LumA, and normal-like breast cancers compared to LumB, Her2 +, and Basal-like subgroups. Fbln2 mRNA levels were positively associated with mesenchymal markers, myoepithelial markers, and markers of epithelial-mesenchymal transition. Higher expression of Fbln2 mRNA was associated with better prognosis in less advanced breast cancer and this pattern was reversed in more advanced lesions. With further validation, these observations may offer a molecular prognostic tool for human breast cancer for more personalized therapeutic approaches.

Structural comparisons of human and mouse fungiform taste buds.

Taste buds are commonly studied in rodent models, but some differences exist between mice and humans in terms of gustatory mechanisms and sensitivities. Whether these functional differences are reflected in structural differences between species is unclear. Using immunofluorescent image stacks, we compared morphological and molecular characteristics of mouse and human fungiform taste buds. The results suggest that while the general features of fungiform taste buds are similar between mice and humans, several characteristics differ significantly. Human taste buds are larger and taller than those of mice, yet they contain similar numbers of taste cells. Taste buds in humans are more heavily innervated by gustatory nerve fibers expressing the purinergic receptor P2X3 showing a 40% higher innervation density than in mice. Like Type II cells of mice, a subset (about 30%) of cells in human taste buds is immunoreactive for PLCβ2. These PLCβ2-immunoreactive cells display CALHM1-immunoreactive puncta closely apposed to gustatory nerve fibers suggestive of channel-type synapses described in mice. These puncta, used as a measure of synaptic contact, are however significantly larger in humans compared to mice. Altogether these findings suggest that while many similarities exist in the structural organization of murine and human fungiform taste buds, significant differences do exist in taste bud size, innervation density, and size of synaptic contacts that may impact gustatory signal transmission.

Piezo 1 and Piezo 2 in the Chemosensory Organs of Zebrafish (Danio rerio).

The ion channels Piezo 1 and Piezo 2 have been identified as membrane mechano-proteins. Studying mechanosensitive channels in chemosensory organs could help in understanding the mechanisms by which these channels operate, offering new therapeutic targets for various disorders. This study investigates the expression patterns of Piezo proteins in zebrafish chemosensory organs. For the first time, Piezo protein expression in adult zebrafish chemosensory organs is reported. In the olfactory epithelium, Piezo 1 immunolabels kappe neurons, microvillous cells, and crypt neurons, while Calretinin is expressed in ciliated sensory cells. The lack of overlap between Piezo 1 and Calretinin confirms Piezo 1's specificity for kappe neurons, microvillous cells, and crypt neurons. Piezo 2 shows intense immunoreactivity in kappe neurons, one-ciliated sensory cells, and multi-ciliated sensory cells, with overlapping Calretinin expression, indicating its olfactory neuron nature. In taste buds, Piezo 1 immunolabels Merkel-like cells at the bases of cutaneous and pharyngeal taste buds and the light and dark cells of cutaneous and oral taste buds. It also marks the dark cells of pharyngeal taste buds and support cells in oral taste buds. Piezo 2 is found in the light and dark cells of cutaneous and oral taste buds and isolated chemosensory cells. These findings provide new insights into the distribution of Piezo channels in zebrafish chemosensory organs, enhancing our understanding of their sensory processing and potential therapeutic applications.

Macroscopic, microscopic, and immunofluorescent characterization of the Greek tortoise (Testudo graeca graeca) oropharyngeal floor with concern to its feed adaptation as a herbivorous land reptile.

The current investigation focuses on gross anatomy, light, and scanning electron microscopy (SEM) of the Testudo graeca oropharyngeal floor, with particular reference to the immunofluorescence technique to examine its tongue. The T. graeca oropharyngeal floor showed many anatomical structures: the lower rhamphotheca, paralingual ridge, lower alveolar ridge, tongue, laryngeal mound, and glottis. The lower rhamphotheca appeared as a V-shaped jaw line with a highly serrated edge and a median tomium (beak). SEM observations of the lingual apex and the lingual body showed rectangular and conical filiform papillae with porous surfaces and taste pores. Meanwhile, the lingual root had two wings that carried papillae with different shapes: dagger-shaped, conical, bifurcated, and leaf-like papillae, and these papillae lacked taste pores. The laryngeal mound had openings for the laryngeal mucus gland and its secretions. Light microscopy findings showed mucous glands in the propria submucosa and near the mucosal surface of the lingual apex. The lingual root had lingual papillae and two hyaline cartilaginous skeletons between skeletal muscles, and the lingual papillae were elongated filiform, rectangular filiform papillae, and fungiform papillae. The lamina propria constituted the core of the lingual papillae and the mucous gland, they had a positive reaction with the periodic acid schiff (PAS) reagent. The apical surface of the fungiform papillae had taste pores. Under immunofluorescence, the vimentin was detected in taste bud cells, and synaptophysin reacted to the taste buds and nerve bundles. The current study of the Greek tortoise oropharyngeal floor investigated its herbivorous eating habits using its serrated lower rhamphotheca, a large tongue with differently shaped papillae, and numerous mucous glands. RESEARCH HIGHLIGHTS: The Greek tortoise (T. graeca graeca) oropharyngeal floor showed many anatomical structures: lower rhamphotheca, paralingual ridge, lower alveolar ridge, tongue, laryngeal mound, and glottis. SEM and light microscopy observations of the tongue revealed varied types and shapes of lingual papillae with a porous surface on the tongue apex (rectangular or conical filiform papillae), on the tongue body (filiform and fungiform papillae), and on the tongue root (dagger-shaped, conical, bifurcated, and leaf-like papillae). Light microscopy findings: the lamina propria constituted the core of the lingual papillae and had numerous mucous glands that had a slightly magenta-red color with PAS reagent. The apical surface of the fungiform papillae had taste pores. Vimentin and synaptophysin gave a reaction to the taste buds.

The existence of cells exhibiting characteristics of both Type II and Type III cells in rat taste buds. An immunohistochemical and electron-microscopic study.

Taste bud cells are classified into four types by their ultrastructural features. Immunohistochemical detection of taste-signaling molecules is used to distinguish cell types of taste bud cells; however, the characteristics of taste cell types such as the immunoreactivity for taste-signaling molecules have long remained unclear. We investigated the detailed characteristics of taste cells in rat vallate taste buds by electron microscopy and immunohistochemistry for gustducin, neural cell adhesion molecule (NCAM) and vesicle-associated membrane protein 2 (VAMP2), which are known as markers of Type II cells, Type III cells and both cell types, respectively. Triple immunostaining for these molecules discriminated seven kinds of cell, including the totally immunopositive cell. Electron microscopy revealed Type III cells with a typical synaptic structure and subsurface cisterna as a specialized contact between a nerve and a Type II cell. The present study clarified the existence of cells with features of both Type II and TypeIII cells as a subtype of taste bud cells in the rat taste bud.

The basement membrane regulates the cellular localization and the cytoplasmic interactome of Yes-Associated Protein (YAP) in mammary epithelial cells.

The Hippo pathway, a signaling cascade involved in the regulation of organ size and several other processes, acts as a conduit between extracellular matrix (ECM) cues and cellular responses. We asked whether the basement membrane (BM),a specialized ECM component known to induce quiescence and differentiation in mammary epithelial cells, would regulate the localization, activity, and interactome of YAP, a Hippo pathway effector. To address this question, we used a broad range of experimental approaches, including 2D and 3D cultures of both mouse and human mammary epithelial cells, as well as the developing mouse mammary gland. In contrast to malignant cells, nontumoral cells cultured with a reconstituted BM (rBM)displayed higher concentrations of YAP in the cytoplasm. Incidentally, when in the nucleus of rBM-treated cells, YAP resided preferentially at the nuclear periphery. In agreement with our cell culture experiments, YAP exhibited cytoplasmic predominance in ductal cells of developing mammary epithelia, where a denser BM is found. Conversely, terminal end bud (TEB)cells with a thinner BM displayed higher nucleus-to-cytoplasm ratios of YAP. Bioinformatic analysis revealed that genes regulated by YAP were overrepresented in the transcriptomes of microdissected TEBs. Consistently, mouse epithelial cells exposed to the rBM expressed lower levels of YAP-regulated genes, although the protein level of YAP and Hippo components were slightly altered by the treatment. Mass spectrometry analysis identified a differential set of proteins interacting with YAP in cytoplasmic fractions of mouse epithelial cells in the absence or presence of rBM. In untreated cells, YAP interactants were enriched in processes related to ubiquitin-mediated proteolysis, whereas in cells exposed to rBM YAP interactants were mainly key proteins related to amino acid, amino sugar, and carbohydrate metabolism. Collectively, we unraveled that the BM induces YAP translocation or retention in the cytoplasm of nontumoral epithelial cells and that in the cytoplasm YAP seems to undertake novel functions in metabolic pathways.

#1344 Molecular signatures of assembloids generated from stem cell derived nephron progenitors and ureteric bud cells as potential kidney organoid therapy

Abstract Background and Aims Novel therapies are needed to address the unmet clinical need of patients suffering from end stage kidney disease (ESKD) and cell therapy is an innovative strategy to deliver effective, curative treatment options to renal patients. There are several reports that have developed protocols to differentiate human pluripotent stem cells (hPSCs) into kidney-lineage progenitor derived organoid models and we have developed a robust and scalable method to produce kidney organoids. Method hPSCs were used to generate nephron progenitor cells (NPCs) and ureteric bud cells (UB) through previously described stem cell differentiation protocols. NPCs on day 9 of differentiation and UB on day 9 were combined for 2 days at 3:1 ratio respectively, to form ‘assembloids’ in-vitro and implanted in-vivo for 2 weeks. We used kidney capsule implantation in a NOD background mouse model and studied integration and phenotype using micro-CT and immunohistochemistry. Results RNA sequencing, flow cytometry and histology of NPCs, UB and assembloids in vitro shows broad representation of renal cell types by expression of markers representing several cell populations present in the developing nephron. We can currently show in-vitro molecular signatures of NPC derived organoids versus assembloids, with superiority of UB derived structures such as connecting tubules and intercalated cells. In-vivo histological examination of implanted assembloids showed maturation of human renal compartments such as collecting ducts, distal tubules as well as glomeruli-like structures. Micro-CT analysis showed integration with host and host derived endothelial vascularisation. Conclusion These observations demonstrate the use and differentiation potential of hPSCs and organoids in a pre-clinical setting towards more functional like kidney compartments by combining different progenitor populations. More work is needed evaluating these and other renal progenitor populations for future efforts to examine efficacy studies to develop regenerative therapies for ESKD.

Linking Altered Nephrogenesis with Hypertension using Single Nuclei RNA Sequencing of Fetal Kidney at E15.5, E16.5 and E17.5 in a Novel Nephron Deficient Rat Model

This study sought to elucidate molecular mechanisms linking altered nephrogenesis in the HSRA rat, a novel model of nephron deficiency, to the development of hypertension and chronic kidney disease (CKD) later in life. The HSRA model gives rise to offspring born with a single kidney (-S) 50-75% of the time. The single kidneys from HSRA-S animals exhibit reduced nephron endowment compared to either kidney from two-kidney offspring (-C). We hypothesize that early molecular factors during development contribute to the observed reduction in nephron number in -S animals. Timed breeding and embryonic kidney isolation from -S and -C animals were performed at gestational days (GD) 15.5, 16.5, or 17.5. Single nuclei RNA sequencing (snRNA-seq) was conducted using n=3 -S kidneys and n=3 -C kidneys for each timepoint. The library was prepared using a 10x Chromium Single Cell 3’ kit, followed by sequencing on an Illumina NextSeq2000. The average number of cells captured per sample was 16,997, yielding an average of 25,335 reads/cell, with a median of 1,512 genes/cell identified. Bioinformatic analysis using Cell Ranger and Seurat (v4) revealed various renal progenitor cell types including metanephric mesenchymal (MM) and ureteric bud cells, alongside tubular, stromal, and glomerular endothelial progenitors. Podocytes emerged by GD17.5, originating from the MM cell population, as evidenced by their extension from this cluster. Global differential expression analysis revealed minimal transcriptional differences between -S and -C fetal kidneys at GD 16.5 and 17.5. However, a down-regulation of ribosomal genes was observed on GD15.5 in the -S group, suggesting premature cellular differentiation contributing to the diminished nephron formation. Markedly, cell type-specific gene expression differences were observed at GD 16.5 and 17.5, highlighting the superior resolution that snRNAseq provides compared to bulk RNA sequencing methods. We are currently performing trajectory analysis using Monocle3 to investigate cellular fate pathways and uncover genes involved during nephron progenitor cell differentiation. These preliminary findings shed light on the cellular and molecular landscape during critical developmental stages in the kidney of the HSRA rat that could link developmental factors affecting nephron number to hypertension and CKD pathologies later in life. This research was generously supported by the Graduate School of Health Sciences at UMMC. MRG is supported by NIH R01HL137673. The UMMC Molecular and Genomics Facility is supported, in part, by funds from the NIGMS, including Molecular Center of Health and Disease-COBRE (P20GM144041-Garrett), Mississippi INBRE (P20GM103476-Flynt), and Cardiorenal and Metabolic Diseases-COBRE (P20GM104357-Hall). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Fasting duration impacts ribosome protein 6 phosphorylation in zebrafish brain: New insights in aquatic organisms’ welfare

BackgroundShort- or mid-term fasting, full or partial, triggers metabolic response known to have in turn health effects in an organism. At central level, the metabolic stimulus triggered by fasting is known to be perceived firstly by hypothalamic neurons.In the field of neuroscience, ribosomal protein S6 (S6) phosphorylation is commonly used as a readout of the mammalian target of rapamycin complex 1 signalling activation or as a marker for neuronal activity. The aim of this study is addressed to evaluate whether the phosphorylation of S6 occurs in the central neurons of zebrafish exposed to four (short-term) and seven (mid-term) days of complete fasting. MethodsGroup-housed adult zebrafish were exposed to four and seven days of complete food withdrawal. At the end of the experimental period, Western blotting analyses were carried out to measure the expression levels of the phosphorylated S6 (pS6) by comparing the two experimental conditions versus the control group. The same antibody was then used to identify the distribution pattern of pS6 immunoreactive neurons in the whole brain and in the taste buds. ResultsWe did not observe increased pS6 levels expression in the brain of animals exposed to short-term fasting compared to the control, whereas the expression increased in brain homogenates of animals exposed to mid-term fasting. pS6 immunoreactivity was reported in some hypothalamic neurons, as well as in the dorsal area of telencephalon and preoptic area, a neurosecretory region homolog to the mammalian paraventricular nucleus. Remarkably, we observed pS6 immunostaining in the sensory cells of taste buds lining the oral epithelium. ConclusionsTaken together, our data show that in zebrafish, differently from other fish species, seven days of fasting triggers neuronal activity. Furthermore, the immunostaining on sensory cells of taste buds suggests that metabolic changes may modulate also peripheral sensory cells. This event may have valuable implications when using zebrafish to design metabolic studies involving fasting as well as practical consequences on the animal welfare, in particularly stressful conditions, such as transportation.

The physiological roles of anoctamin2/TMEM16B and anoctamin1/TMEM16A in chemical senses

Chemical senses allow animals to detect and discriminate a vast array of molecules. The olfactory system is responsible of the detection of small volatile molecules, while water dissolved molecules are detected by taste buds in the oral cavity. Moreover, many animals respond to signaling molecules such as pheromones and other semiochemicals through the vomeronasal organ. The peripheral organs dedicated to chemical detection convert chemical signals into perceivable information through the employment of diverse receptor types and the activation of multiple ion channels. Two ion channels, TMEM16B, also known as anoctamin2 (ANO2) and TMEM16A, or anoctamin1 (ANO1), encoding for Ca2+-activated Cl¯ channels, have been recently described playing critical roles in various cell types. This review aims to discuss the main properties of TMEM16A and TMEM16B-mediated currents and their physiological roles in chemical senses. In olfactory sensory neurons, TMEM16B contributes to amplify the odorant response, to modulate firing, response kinetics and adaptation. TMEM16A and TMEM16B shape the pattern of action potentials in vomeronasal sensory neurons increasing the interspike interval. In type I taste bud cells, TMEM16A is activated during paracrine signaling mediated by ATP. This review aims to shed light on the regulation of diverse signaling mechanisms and neuronal excitability mediated by Ca-activated Cl¯ channels, hinting at potential new roles for TMEM16A and TMEM16B in the chemical senses.

The scale of zebrafish pectoral fin buds is determined by intercellular K+ levels and consequent Ca2+-mediated signaling via retinoic acid regulation of Rcan2 and Kcnk5b.

K+ channels regulate morphogens to scale adult fins, but little is known about what regulates the channels and how they control morphogen expression. Using the zebrafish pectoral fin bud as a model for early vertebrate fin/limb development, we found that K+ channels also scale this anatomical structure, and we determined how one K+-leak channel, Kcnk5b, integrates into its developmental program. From FLIM measurements of a Förster Resonance Energy Transfer (FRET)-based K+ sensor, we observed coordinated decreases in intracellular K+ levels during bud growth, and overexpression of K+-leak channels in vivo coordinately increased bud proportions. Retinoic acid, which can enhance fin/limb bud growth, decreased K+ in bud tissues and up-regulated regulator of calcineurin (rcan2). rcan2 overexpression increased bud growth and decreased K+, while CRISPR-Cas9 targeting of rcan2 decreased growth and increased K+. We observed similar results in the adult caudal fins. Moreover, CRISPR targeting of Kcnk5b revealed that Rcan2-mediated growth was dependent on the Kcnk5b. We also found that Kcnk5b enhanced depolarization in fin bud cells via Na+ channels and that this enhanced depolarization was required for Kcnk5b-enhanced growth. Lastly, Kcnk5b-induced shha transcription and bud growth required IP3R-mediated Ca2+ release and CaMKK activity. Thus, we provide a mechanism for how retinoic acid via rcan2 can regulate K+-channel activity to scale a vertebrate appendage via intercellular Ca2+ signaling.

Characteristics of A-type voltage-gated K+ currents expressed on sour-sensing type III taste receptor cells in mice

Sour taste is detected by type III taste receptor cells that generate membrane depolarization with action potentials in response to HCl applied to the apical membranes. The shape of action potentials in type III cells exhibits larger afterhyperpolarization due to activation of transient A-type voltage-gated K+ currents. Although action potentials play an important role in neurotransmitter release, the electrophysiological features of A-type K+ currents in taste buds remain unclear. Here, we examined the electrophysiological properties of A-type K+ currents in mouse fungiform taste bud cells using in-situ whole-cell patch clamping. Type III cells were identified with SNAP-25 immunoreactivity and/or electrophysiological features of voltage-gated currents. Type III cells expressed A-type K+ currents which were completely inhibited by 10 mM TEA, whereas IP3R3-immunoreactive type II cells did not. The half-maximal activation and steady-state inactivation of A-type K+ currents were 17.9 ± 4.5 (n = 17) and − 11.0 ± 5.7 (n = 17) mV, respectively, which are similar to the features of Kv3.3 and Kv3.4 channels (transient and high voltage-activated K+ channels). The recovery from inactivation was well fitted with a double exponential equation; the fast and slow time constants were 6.4 ± 0.6 ms and 0.76 ± 0.26 s (n = 6), respectively. RT-PCR experiments suggest that Kv3.3 and Kv3.4 mRNAs were detected at the taste bud level, but not at single-cell levels. As the phosphorylation of Kv3.3 and Kv3.4 channels generally leads to the modulation of cell excitability, neuromodulator-mediated A-type K+ channel phosphorylation likely affects the signal transduction of taste.