Makoto Ohmoto

CALHM1 ion channel mediates purinergic neurotransmission of sweet, bitter and umami tastes

Recognition of sweet, bitter and umami tastes requires the non-vesicular release from taste bud cells of ATP, which acts as a neurotransmitter to activate afferent neural gustatory pathways. However, how ATP is released to fulfil this function is not fully understood. Here we show that calcium homeostasis modulator 1 (CALHM1), a voltage-gated ion channel, is indispensable for taste-stimuli-evoked ATP release from sweet-, bitter- and umami-sensing taste bud cells. Calhm1 knockout mice have severely impaired perceptions of sweet, bitter and umami compounds, whereas their recognition of sour and salty tastes remains mostly normal. Calhm1 deficiency affects taste perception without interfering with taste cell development or integrity. CALHM1 is expressed specifically in sweet/bitter/umami-sensing type II taste bud cells. Its heterologous expression induces a novel ATP permeability that releases ATP from cells in response to manipulations that activate the CALHM1 ion channel. Knockout of Calhm1 strongly reduces voltage-gated currents in type II cells and taste-evoked ATP release from taste buds without affecting the excitability of taste cells by taste stimuli. Thus, CALHM1 is a voltage-gated ATP-release channel required for sweet, bitter and umami taste perception.

Intestinal epithelial tuft cells initiate type 2 mucosal immunity to helminth parasites.

Helminth parasitic infections are a major global health and social burden. The host defence against helminths such as Nippostrongylus brasiliensis is orchestrated by type 2 cell-mediated immunity. Induction of type 2 cytokines, including interleukins (IL) IL-4 and IL-13, induce goblet cell hyperplasia with mucus production, ultimately resulting in worm expulsion. However, the mechanisms underlying the initiation of type 2 responses remain incompletely understood. Here we show that tuft cells, a rare epithelial cell type in the steady-state intestinal epithelium, are responsible for initiating type 2 responses to parasites by a cytokine-mediated cellular relay. Tuft cells have a Th2-related gene expression signature and we demonstrate that they undergo a rapid and extensive IL-4Rα-dependent amplification following infection with helminth parasites, owing to direct differentiation of epithelial crypt progenitor cells. We find that the Pou2f3 gene is essential for tuft cell specification. Pou2f3−/− mice lack intestinal tuft cells and have defective mucosal type 2 responses to helminth infection; goblet cell hyperplasia is abrogated and worm expulsion is compromised. Notably, IL-4Rα signalling is sufficient to induce expansion of the tuft cell lineage, and ectopic stimulation of this signalling cascade obviates the need for tuft cells in the epithelial cell remodelling of the intestine. Moreover, tuft cells secrete IL-25, thereby regulating type 2 immune responses. Our data reveal a novel function of intestinal epithelial tuft cells and demonstrate a cellular relay required for initiating mucosal type 2 immunity to helminth infection.

Genetic tracing of the gustatory and trigeminal neural pathways originating from T1R3-expressing taste receptor cells and solitary chemoreceptor cells

We established transgenic mouse lines expressing a transneuronal tracer, wheat germ agglutinin (WGA), under the control of mouse T1R3 gene promoter/enhancer. In the taste buds, WGA transgene was faithfully expressed in T1R3-positive sweet/umami taste receptor cells. WGA protein was transferred not laterally to the synapse-bearing, sour-responsive type III cells in the taste buds but directly to a subset of neurons in the geniculate and nodose/petrosal ganglia, and further conveyed to a rostro-central region of the nucleus of solitary tract. In addition, WGA was expressed in solitary chemoreceptor cells in the nasal epithelium and transferred along the trigeminal sensory pathway to the brainstem neurons. The solitary chemoreceptor cells endogenously expressed T1R3 together with bitter taste receptors T2Rs. This result shows an exceptional signature of receptor expression. Thus, the t1r3-WGA transgenic mice revealed the sweet/umami gustatory pathways from taste receptor cells and the trigeminal neural pathway from solitary chemoreceptor cells.

Skn-1a (Pou2f3) specifies taste receptor cell lineage

Functional diversification of taste cells is crucial for proper discrimination of taste qualities. We found the homeodomain protein Skn-1a (Pou2f3) to be expressed in sweet, umami and bitter taste cells. Skn-1a–deficient mice lacked electrophysiological and behavioral responses to sweet, umami and bitter tastes, as a result of a complete absence of sweet, umami and bitter cells and the concomitant expansion of sour cells. Skn-1a is critical for generating and balancing the diverse composition of taste cells.

Bcl11b/Ctip2 Controls the Differentiation of Vomeronasal Sensory Neurons in Mice

The transcription factor Bcl11b/Ctip2 plays critical roles in the development of several systems and organs, including the immune system, CNS, skin, and teeth. Here, we show that Bcl11b/Ctip2 is highly expressed in the developing vomeronasal system in mice and is required for its proper development. Bcl11b/Ctip2 is expressed in postmitotic vomeronasal sensory neurons (VSNs) in the vomeronasal epithelium (VNE) as well as projection neurons and GABAergic interneurons in the accessory olfactory bulb (AOB). In the absence of Bcl11b, these neurons are born in the correct number, but VSNs selectively die by apoptosis. The critical role of Bcl11b in vomeronasal system development is demonstrated by the abnormal phenotypes of Bcl11b-deficient mice: disorganization of layer formation of the AOB, impaired axonal projections of VSNs, a significant reduction in the expression of vomeronasal receptor genes, and defective mature differentiation of VSNs. VSNs can be classified into two major types of neurons, vomeronasal 1 receptor (V1r)/Gαi2-positive and vomeronasal 2 receptor (V2r)/Gαo-positive VSNs. We found that all Gαi2-positive cells coexpressed Gαo during embryogenesis. This coexpression is also observed in newly differentiated neurons in the adult VNE. Interestingly, loss of Bcl11b function resulted in an increased number of V1r/Gαi2-type VSNs and a decreased number of V2r/Gαo-type VSNs, suggesting that Bcl11b regulates the fate choice between these two VSN types. These results indicate that Bcl11b/Ctip2 is an essential regulator of the differentiation and dichotomy of VSNs.

Functional organization of the rodent parabrachial nucleus.

The rodent parabrachial nucleus (PBN) is not merely a sensory relay station but also plays an important role in integrating various ascending and descending inputs together with plastic changes of neuronal responses after learning and experience. The limbic and reward systems receive ingestion‐related information via the cortical areas in primates, whereas in rodents the information is sent to these systems mostly via the PBN. To explore how the rat PBN is functionally organized, we detected activation patterns of neurons mainly by means of c‐fos immunohistochemistry to show neuronal activation in different situations of ingestive behavior. The expression pattern was different under nutritionally replete and deficient conditions, perceptually new and familiar conditions, and learned and unlearned conditions. As for the possible functions, the rostral part of the external lateral subnucleus is related to general visceral inputs; the caudal part of the external lateral subnucleus, aversive behavior; the dorsal lateral subnucleus, ingestive behavior; and the central medial subnucleus, taste of NaCl. Because several genes were localized in specific subnuclei, we are trying to correlate the gene expressions with possible functional significance.

Skn-1a/Pou2f3 is required for the generation of Trpm5-expressing microvillous cells in the mouse main olfactory epithelium

BackgroundThe main olfactory epithelium (MOE) in mammals is a specialized organ to detect odorous molecules in the external environment. The MOE consists of four types of cells: olfactory sensory neurons, supporting cells, basal cells, and microvillous cells. Among these, development and function of microvillous cells remain largely unknown. Recent studies have shown that a population of microvillous cells expresses the monovalent cation channel Trpm5 (transient receptor potential channel M5). To examine functional differentiation of Trpm5-expressing microvillous cells in the MOE, we investigated the expression and function of Skn-1a, a POU (Pit-Oct-Unc) transcription factor required for functional differentiation of Trpm5-expressing sweet, umami, and bitter taste bud cells in oropharyngeal epithelium and solitary chemosensory cells in nasal respiratory epithelium.ResultsSkn-1a is expressed in a subset of basal cells and apical non-neuronal cells in the MOE of embryonic and adult mice. Two-color in situ hybridization revealed that a small population of Skn-1a-expressing cells was co-labeled with Mash1/Ascl1 and that most Skn-1a-expressing cells coexpress Trpm5. To investigate whether Skn-1a has an irreplaceable role in the MOE, we analyzed Skn-1a-deficient mice. In the absence of Skn-1a, olfactory sensory neurons differentiate normally except for a limited defect in terminal differentiation in ectoturbinate 2 of some of MOEs examined. In contrast, the impact of Skn-1a deficiency on Trpm5-expressing microvillous cells is much more striking: Trpm5, villin, and choline acetyltransferase, cell markers previously shown to identify Trpm5-expressing microvillous cells, were no longer detectable in Skn-1a-deficient mice. In addition, quantitative analysis demonstrated that the density of superficial microvillous cells was significantly decreased in Skn-1a-deficient mice.ConclusionSkn-1a is expressed in a minority of Mash1-positive olfactory progenitor cells and a majority of Trpm5-expressing microvillous cells in the main olfactory epithelium. Loss-of-function mutation of Skn-1a resulted in complete loss of Trpm5-expressing microvillous cells, whereas most of olfactory sensory neurons differentiated normally. Thus, Skn-1a is a critical regulator for the generation of Trpm5-expressing microvillous cells in the main olfactory epithelium in mice.

Mutually exclusive expression of Gαia and Gα14 reveals diversification of taste receptor cells in zebrafish

A comprehensive reevaluation of the G protein alpha subunit genes specifically expressed in taste buds in the tongue epithelium of rodents revealed that Gq and G14 of the Gq class and Gi2 and Ggust (Gt3, also known as gustducin) of the Gi class are expressed in mammalian taste buds. Meanwhile, a database search of fish genomes revealed the absence of a gene encoding an ortholog of the mammalian Ggust gene, which mediates sweet, umami, and bitter taste signals in mammalian taste receptor cells (TRCs). Histochemical screening identified two G protein alpha subunit genes, zfGia and zfG14, expressed in subsets of TRCs in zebrafish. The expression patterns of zfGia and zfG14 in taste buds were mutually exclusive, and the expression of known T1R and T2R genes in zebrafish was restricted to a subset of zfGia‐expressing TRCs. These findings highlight the existence of a novel subset of TRCs in zebrafish that is absent in mammals and suggest that unidentified G protein‐coupled receptors are expressed in zfG14‐expressing TRCs and in zfGia‐expressing TRCs where known T1R and T2R genes were not expressed in zebrafish. The existence of not only generalized but also specialized subsets of TRCs may imply a strong connection between the evolution of the peripheral gustatory system and the evolution of particular species. J. Comp. Neurol. 519:1616–1629, 2011.

Genetic tracing of the neural pathway for bitter taste in t2r5-WGA transgenic mice

To visualize the neural pathways originating from bitter taste receptor cells (TRCs), we generated transgenic mice expressing the transneuronal tracer wheat germ agglutinin (WGA) under the control of the mouse T2R5 gene promoter/enhancer (t2r5-WGA mice). WGA mRNA was specifically expressed in bitter TRCs. The WGA protein was detected in bitter TRCs and nerve processes in taste buds, but not in sweet, umami, or sour TRCs. The WGA protein was transferred to a subset of sensory neurons in the geniculate and nodose/petrosal ganglia. These results suggest that bitter TRCs, which are devoid of synaptic structures, are innervated by gustatory neurons and that bitter sensory information is directly transmitted to specific gustatory neurons. The t2r5-WGA mice provide a useful tool for identifying gustatory relay neurons in the peripheral sensory ganglia responsible for aversive sensations.

Normal Taste Acceptance and Preference of PANX1 Knockout Mice.

Taste compounds detected by G protein-coupled receptors on the apical surface of Type 2 taste cells initiate an intracellular molecular cascade culminating in the release of ATP. It has been suggested that this ATP release is accomplished by pannexin 1 (PANX1). However, we report here that PANX1 knockout mice do not differ from wild-type controls in response to representative taste solutions, measured using 5-s brief-access tests or 48-h two-bottle choice tests. This implies that PANX1 is unnecessary for taste detection and consequently that ATP release from Type 2 taste cells does not require PANX1.