RESUMO
The sense of smell is intricately linked to essential animal behaviors necessary for individual survival and species preservation. During vertebrate evolution, odorant receptors (ORs), responsible for detecting odor molecules, have evolved to adapt to changing environments, transitioning from aquatic to terrestrial habitats and accommodating increasing complex chemical environments. These evolutionary pressures have given rise to the largest gene family in vertebrate genomes. Vertebrate ORs are phylogenetically divided into two major classes; class I and class II. Class I OR genes, initially identified in fish and frog, have persisted across vertebrate species. On the other hand, class II OR genes are unique to terrestrial animals, accounting for ~90% of mammalian OR genes. In mice, each olfactory sensory neuron (OSN) expresses a single functional allele of a single OR gene from either the class I or class II OR repertoire. This one neuron-one receptor rule is established through two sequential steps: specification of OR class and subsequent exclusive OR expression from the corresponding OR class. Consequently, OSNs acquire diverse neuronal identities during the process of OSN differentiation, enabling animals to detect a wide array of odor molecules. This review provides an overview of the OSN differentiation process through which OSN diversity is achieved, primarily using the mouse as a model animal.
Assuntos
Neurônios Receptores Olfatórios , Animais , Camundongos , Neurônios Receptores Olfatórios/metabolismo , Olfato/fisiologia , Odorantes , Células Receptoras Sensoriais , MamíferosRESUMO
Tetrodotoxin (TTX) is a potent neurotoxin that binds to voltage-gated sodium channels and blocks the passage of sodium ions. TTX is widely distributed in both terrestrial and marine organisms, and the toxic puffers are believed to accumulate TTX through the food chain. Although pufferfish was previously thought to be attracted by TTX, recent finding from electroolfactogram (EOG) studies have indicated that the olfactory epithelium of T. alboplumbeus responded to 5, 6, 11-trideoxyTTX (TDT), but not to TTX itself. In this study, we examined behavioral experiments for Takifugu rubripes to distinguish between TTX and TDT under static and flow-through conditions. Our data clearly suggested that T. rubripes juveniles were attracted to TDT, not TTX. Moreover, we determined that the minimum effective dose of TDT to attract the puffer was 1-2 nmol of TDT under static conditions and 50-60 nmol of TDT under flow-through conditions. Following the experiments under static conditions, numerous bite marks by the pufferfish were found solely on the agarose gel infused with TDT. Based on these finding, we hypothesize that the pufferfish are attracted to TDT derived from prey, leading them effectively become toxic.
Assuntos
Neurotoxinas , Takifugu , Animais , Takifugu/metabolismo , Tetrodotoxina/toxicidade , Tetrodotoxina/metabolismo , Neurotoxinas/metabolismo , Cadeia AlimentarRESUMO
Continuous turnover of taste bud cells in the oral cavity underlies the homeostasis of taste tissues. Previous studies have demonstrated that Sox2+ stem cells give rise to all types of epithelial cells including taste bud cells and non-gustatory epithelial cells in the oral epithelium, and Sox2 is required for generating taste bud cells. Here, we show the dynamism of single stem cells through multicolor lineage tracing analyses in Sox2-CreERT2; Rosa26-Confetti mice. In the non-gustatory epithelium, unicolored areas populated by a cluster of cells expressing the same fluorescent protein grew over time, while epithelial cells were randomly labeled with multiple fluorescent proteins by short-term tracing. Similar phenomena were observed in gustatory epithelia. These results suggest that the Sox2+ stem cell population is maintained by balancing the increase of certain stem cells with the reduction of the others. In the gustatory epithelia, many single taste buds contained cells labeled with different fluorescent proteins, indicating that a single taste bud is composed of cells derived from multiple Sox2+ stem cells. Our results reveal the characteristics of Sox2+ stem cells underlying the turnover of taste bud cells and the homeostasis of taste tissues.
Assuntos
Células-Tronco Adultas , Papilas Gustativas , Animais , Células Epiteliais/metabolismo , Epitélio/metabolismo , Camundongos , Células-Tronco , Papilas Gustativas/metabolismoRESUMO
Individual olfactory sensory neurons (OSNs) in the mouse main olfactory epithelium express a single odorant receptor (OR) gene from the repertoire of either class I or class II ORs. The transcription factor Bcl11b determines the OR class to be expressed in OSNs. The septal organ (SO), a small neuroepithelium located at the ventral base of the nasal septum, is considered as an olfactory subsystem because it expresses a specific subset of ORs. However, the mechanisms underlying the generation and differentiation of SO-OSN remain unknown. In the present study, we show that the generation and differentiation of SO-OSN employ the same genetic pathway as in the OSN lineage, which is initiated by the neuronal fate determinant factor Ascl1. Additionally, the key role of Bcl11b in the SO is demonstrated by the abnormal phenotypes of Bcl11b-deficient mice: significant reduction in the expression of OR genes and in the number of mature SO-OSNs. Although SO-OSNs are specified to express a subset of class II OR genes in wild-type mice, the Bcl11b deletion led to the expression of class I OR genes, while the expression of class II OR genes was significantly decreased, with one exception of Olfr15. These results indicate that Bcl11b is necessary for proper OR expression in SO-OSNs.
Assuntos
Perfuração do Septo Nasal/metabolismo , Mucosa Olfatória/metabolismo , Neurônios Receptores Olfatórios/metabolismo , Receptores Odorantes/metabolismo , Proteínas Repressoras/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Animais , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mucosa Olfatória/citologia , Neurônios Receptores Olfatórios/citologia , OlfatoRESUMO
In the mouse, 129 functional class I odorant receptor (OR) genes reside in a ~ 3 megabase huge gene cluster on chromosome 7. The J element, a long-range cis-regulatory element governs the singular expression of class I OR genes by exerting its effect over the whole cluster. To elucidate the molecular mechanisms underlying class I-specific enhancer activity of the J element, we analyzed the J element sequence to determine the functional region and essential motif. The 430-bp core J element, that is highly conserved in mammalian species from the platypus to humans, contains a class I-specific conserved motif of AAACTTTTC, multiple homeodomain sites, and a neighboring O/E-like site, as in class II OR-enhancers. A series of transgenic reporter assays demonstrated that the class I-specific motif is not essential, but the 330-bp core J-H/O containing the homeodomain and O/E-like sites is necessary and sufficient for class I-specific enhancer activity. Further motif analysis revealed that one of homeodomain sequence is the Greek Islands composite motif of the adjacent homeodomain and O/E-like sequences, and mutations in the composite motif abolished or severely reduced class I-enhancer activity. Our results demonstrate that class I and class II enhancers share a functional motif for their enhancer activity.
Assuntos
Motivos de Aminoácidos/genética , Elementos Facilitadores Genéticos/genética , Receptores Odorantes/genética , Animais , Cromossomos Humanos Par 7/genética , Expressão Gênica/genética , Proteínas de Homeodomínio/genética , Humanos , Camundongos , Mutação , Mucosa Olfatória/metabolismo , Mucosa Olfatória/fisiologia , Receptores Odorantes/classificaçãoRESUMO
Taste substances are detected by taste receptor cells in the taste buds in the oral epithelium. Individual taste receptor cells contribute to evoking one of the five taste qualities: sweet, umami, bitter, sour, and salty (sodium). They are continuously replaced every few weeks by new ones generated from local epithelial stem cells. A POU transcription factor, Pou2f3 (also known as Skn-1a), regulates the generation and differentiation of sweet, umami, and bitter cells. However, the molecular mechanisms underlying terminal differentiation into these Pou2f3-dependent taste receptor cells remain unknown. To identify the candidate molecules that regulate the differentiation of these taste receptor cells, we searched for taste receptor type-specific transcription factors using RNA-sequence data of sweet and bitter cells. No transcription factor gene showing higher expression in sweet cells than in bitter cells was found. Eyes absent 1 (Eya1) was identified as the only transcription factor gene showing higher expression in bitter cells than in sweet cells. In situ hybridization revealed that Eya1 was predominantly expressed in bitter cells and also in the putative immature/differentiating taste bud cells in circumvallate and fungiform papillae and soft palate. Eya1 is a candidate molecule that regulates the generation and differentiation of bitter cells.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/biossíntese , Proteínas Nucleares/biossíntese , Proteínas Tirosina Fosfatases/biossíntese , Papilas Gustativas , Animais , Diferenciação Celular , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Paladar , Papilas Gustativas/citologia , Papilas Gustativas/metabolismoRESUMO
Taste bud cells arise from local epithelial stem cells in the oral cavity and are continuously replaced by newborn cells throughout an animal's life. However, little is known about the molecular and cellular mechanisms of taste cell turnover. Recently, it has been demonstrated that SOX2, a transcription factor expressed in epithelial stem/progenitor cells of the oral cavity, regulates turnover of anterior tongue epithelium including gustatory and non-gustatory papillae. Yet, the role of SOX2 in regulating taste cell turnover in the posterior tongue is unclear. Prompted by the fact that there are regional differences in the cellular and molecular composition of taste buds and stem/progenitor cells in the anterior and posterior portions of tongue, which are derived from distinct embryonic origins, we set out to determine the role of SOX2 in epithelial tissue homeostasis in the posterior tongue. Here we report the differential requirement of SOX2 in the stem/progenitor cells for the normal turnover of lingual epithelial cells in the posterior tongue. Sox2 deletion in the stem/progenitor cells neither induced active caspase 3-mediated apoptotic cell death nor altered stem/progenitor cell population in the posterior tongue. Nevertheless, morphology and molecular feature of non-gustatory epithelial cells were impaired in the circumvallate papilla but not in the filiform papillae. Remarkably, taste buds became thinner, collapsed, and undetectable over time. Lineage tracing of Sox2-deleted stem/progenitor cells demonstrated an almost complete lack of newly generated basal precursor cells in the taste buds, suggesting mechanistically that Sox2 is involved in determining stem/progenitor cells to differentiate to gustatory lineage cells. Together, these results demonstrate that SOX2 plays key roles in regulating epithelial tissue homeostasis in the posterior tongue, similar but not identical to its function in the anterior tongue.
Assuntos
Epitélio/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Papilas Gustativas/metabolismo , Língua , Animais , Diferenciação Celular , Homeostase , Hibridização In Situ , Camundongos , Células-Tronco/metabolismo , Língua/citologia , Língua/metabolismoRESUMO
[This corrects the article DOI: 10.1038/s42003-019-0536-x.].
RESUMO
Each olfactory sensory neuron (OSN) expresses a single odorant receptor (OR) gene from the class I or class II repertoire in mice. The mechanisms that regulate OR class choice in OSNs remain unknown. Here, we show that the transcription factor Bcl11b determines the OR class to be expressed in OSNs. Both loss- and gain-of-function analyses demonstrate that class I is a default fate of OSNs and that Bcl11b dictates a class II OR choice by suppressing the effect of the J-element, a class I-OR enhancer. We further demonstrate that OSN-specific genetic manipulations of Bcl11b bias the OR class choice, generating mice with "class I-dominant" and "class II-dominant" noses, which display contrasting innate olfactory behaviors to two distinct aversive odorants. Overall, these findings reveal a unique transcriptional mechanism mediating a binary switch for OR class choice that is crucial to both the anatomical and functional organization of the olfactory system.
Assuntos
Comportamento Animal , Odorantes , Bulbo Olfatório/metabolismo , Percepção Olfatória , Neurônios Receptores Olfatórios/metabolismo , Proteínas Repressoras/metabolismo , Olfato , Proteínas Supressoras de Tumor/metabolismo , Animais , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos Endogâmicos BALB C , Camundongos Knockout , Receptores Odorantes/genética , Receptores Odorantes/metabolismo , Proteínas Repressoras/deficiência , Proteínas Repressoras/genética , Transdução de Sinais , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genética , XenopusRESUMO
Pheromones are crucial for eliciting social and sexual behaviors in diverse animal species. The vomeronasal receptor type-1 (V1R) genes, encoding members of a pheromone receptor family, are highly variable in number and repertoire among mammals due to extensive gene gain and loss. Here, we report a novel pheromone receptor gene belonging to the V1R family, named ancient V1R (ancV1R), which is shared among most Osteichthyes (bony vertebrates) from the basal lineage of ray-finned fishes to mammals. Phylogenetic and syntenic analyses of ancV1R using 115 vertebrate genomes revealed that it represents an orthologous gene conserved for >400 My of vertebrate evolution. Interestingly, the loss of ancV1R in some tetrapods is coincident with the degeneration of the vomeronasal organ in higher primates, cetaceans, and some reptiles including birds and crocodilians. In addition, ancV1R is expressed in most mature vomeronasal sensory neurons in contrast with canonical V1Rs, which are sparsely expressed in a manner that is consistent with the "one neuron-one receptor" rule. Our results imply that a previously undescribed V1R gene inherited from an ancient Silurian ancestor may have played an important functional role in the evolution of vertebrate vomeronasal organ.
Assuntos
Evolução Biológica , Receptores de Feromônios/genética , Células Receptoras Sensoriais/metabolismo , Vertebrados/genética , Órgão Vomeronasal/metabolismo , Animais , Humanos , Receptores de Feromônios/metabolismo , Seleção Genética , Homologia de Sequência , Vertebrados/metabolismoRESUMO
Transient receptor potential channel M5 (Trpm5)-expressing cells, such as sweet, umami, and bitter taste cells in the oropharyngeal epithelium, solitary chemosensory cells in the nasal respiratory epithelium, and tuft cells in the small intestine, that express taste-related genes function as chemosensory cells. Previous studies demonstrated that Skn-1a/Pou2f3, a POU homeodomain transcription factor is expressed in these Trpm5-expressing chemosensory cells, and is necessary for their generation. Trpm5-expressing cells have recently been found in trachea, auditory tube, urethra, thymus, pancreatic duct, stomach, and large intestine. They are considered to be involved in protective responses to potential hazardous compounds as Skn-1a-dependent bitter taste cells, respiratory solitary chemosensory cells, and intestinal tuft cells are. In this study, we examined the expression and function of Skn-1a/Pou2f3 in Trpm5-expressing cells in trachea, auditory tube, urethra, thymus, pancreatic duct, stomach, and large intestine. Skn-1a/Pou2f3 is expressed in a majority of Trpm5-expressing cells in all tissues examined. In Skn-1a/Pou2f3-deficient mice, the expression of Trpm5 as well as marker genes for Trpm5-expressing cells were absent in all tested tissues. Immunohistochemical analyses demonstrated that two types of microvillous cells exist in trachea, urethra, and thymus, Trpm5-positive and Trpm5-negative cells. In Skn-1a/Pou2f3-deficient mice, a considerable proportion of Trpm5-negative and villin-positive microvillous cells remained present in these tissues. Thus, we propose that Skn-1a/Pou2f3 is the master regulator for the generation of the Trpm5-expressing microvillous cells in multiple tissues.
Assuntos
Fatores de Transcrição de Octâmero/fisiologia , Canais de Cátion TRPM/fisiologia , Animais , Sistema Digestório/citologia , Sistema Digestório/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Traqueia/citologia , Traqueia/metabolismoRESUMO
Individual olfactory sensory neurons express a single odorant receptor gene from either class I genes residing in a single cluster on a single chromosome or class II genes spread over multiple clusters on multiple chromosomes. Here, we identify an enhancer element for mouse class I genes, the J element, that is conserved through mammalian species from the platypus to humans. The J element regulates most class I genes expression by exerting an effect over ~ 3 megabases within the whole cluster. Deletion of the trans J element increases the expression frequencies of class I genes from the intact J allele, indicating that the allelic exclusion of class I genes depends on the activity of the J element. Our data reveal a long-range cis-regulatory element that governs the singular class I gene expression and has been phylogenetically preserved to retain a single cluster organization of class I genes in mammals."Each olfactory sensory neuron expresses a single odorant receptor gene from either class I or class II genes. Here, the authors identify an enhancer for mouse class I genes, that is highly conserved, and regulates most class I genes expression by acting over ~ 3 megabases within the whole cluster."
Assuntos
Elementos Facilitadores Genéticos , Neurônios Receptores Olfatórios/metabolismo , Receptores Odorantes/genética , Animais , Sequência Conservada , Regulação da Expressão Gênica , Camundongos , Família Multigênica , FilogeniaRESUMO
Taste cells in taste buds are epithelial sensory cells. Old taste bud cells die and are replaced by new ones generated from taste stem cells. Identifying and characterizing adult taste stem cells is therefore important to understand how peripheral taste tissues are maintained. SOX2 is expressed in oral epithelium including gustatory papillae and has been proposed to be a marker of adult taste stem/progenitor cells. Nevertheless, this hypothesis has never been directly tested. Here, by single-color genetic lineage tracing using Sox2-CreERT2 strain, we reveal that all types of taste bud cells distributed throughout the oral epithelium are derived from stem cells that express SOX2. Short-term tracing shows that SOX2-positive taste stem cells actively supply taste bud cells. At the base of epithelium outside taste buds are distributed proliferation marker- and SOX2-positive cells. Consistently, taste stem cells identified by Lgr5 expression in the circumvallate papillae also express SOX2. Together, taste stem cells distributed in oral epithelia express SOX2.
Assuntos
Fatores de Transcrição SOXB1/metabolismo , Papilas Gustativas/metabolismo , Animais , Técnicas de Introdução de Genes , Ligação Genética , Imuno-Histoquímica , Canal de Potássio KCNQ1/genética , Canal de Potássio KCNQ1/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Mucosa Bucal/metabolismo , Mucosa Bucal/patologia , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Fatores de Transcrição SOXB1/genética , Células-Tronco/citologia , Células-Tronco/metabolismo , Papilas Gustativas/citologiaRESUMO
Molecular mechanisms underlying the development and morphogenesis of oral epithelia, comprising the gustatory and nongustatory epithelium, remain unclear. Here, we show that Bcl11b, a zinc finger transcription factor, plays an important role in the development of lingual papillae, especially filiform papillae. In both gustatory and nongustatory epithelium, Bcl11b was expressed in keratin 14-positive epithelial basal cells, which differentiate into keratinocytes and/or taste cells. Loss of Bcl11b function resulted in abnormal morphology of the gustatory papillae: flattened fungiform papillae, shorter trench wall in the foliate and circumvallate papillae, and ectopic invagination in more than half of circumvallate papillae. However, Bcl11b loss caused no effect on differentiation of taste receptor cells. In nongustatory epithelium, the impact of Bcl11b deficiency was much more striking, resulting in a smooth surface on the tongue tip and hypoplastic filiform papillae in the dorsal lingual epithelium. Immunohistochemical analyses revealed that a keratinocyte differentiation marker, Tchh expression was severely decreased in the Bcl11b(-/-) filiform papillae. In addition, expression of Pax9, required for morphogenesis of filiform papillae and its downstream target genes, hard keratins, almost disappeared in the tongue tip and was decreased in the dorsal tongue of Bcl11b(-/-) mice. Gene expression analyses demonstrated a delayed onset of expression of epithelial differentiation complex genes, which disturbed barrier formation in the mutant tongue. These results indicate that Bcl11b regulates the differentiation of keratinocytes in the tongue and identify Bcl11b as an essential factor for the lingual papilla morphogenesis.
Assuntos
Proteínas Repressoras/fisiologia , Língua/embriologia , Fatores de Transcrição/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Animais , Diferenciação Celular , Queratinócitos/citologia , Camundongos , Morfogênese/genética , Mucosa Bucal/citologia , Mucosa Bucal/embriologia , Proteínas Repressoras/genética , Papilas Gustativas/embriologia , Língua/ultraestrutura , Proteínas Supressoras de Tumor/genéticaRESUMO
BACKGROUND: The Bacillus subtilis genome (BGM) vector is a novel cloning system based on the natural competence that enables B. subtilis to import extracellular DNA fragments into the cell and incorporate the recombinogenic DNA into the genome vector by homologous recombination. The BGM vector system has several attractive properties, such as a megabase cloning capacity, stable propagation of cloned DNA inserts, and various modification strategies using RecA-mediated homologous recombination. However, the endogenous RecA activity may cause undesirable recombination, as has been observed in yeast artificial chromosome systems. In this study, we developed a novel BGM vector system of an inducible recA expression BGM vector (iREX), in which the expression of recA can be controlled by xylose in the medium. RESULTS: We constructed the iREX system by introducing the xylose-inducible recA expression cassette followed by the targeted deletion of the endogenous recA. Western blot analysis showed that the expression of recA was strictly controlled by xylose in the medium. In the absence of xylose, recA was not expressed in the iREX, and the RecA-mediated recombination reactions were greatly suppressed. By contrast, the addition of xylose successfully induced RecA expression, which enabled the iREX to exploit the same capacities of transformation and gene modifications observed with the conventional BGM vector. In addition, an evaluation of the stability of the cloned DNA insert demonstrated that the DNA fragments containing homologous sequences were more stably maintained in the iREX by suppressing undesirable homologous recombination. CONCLUSIONS: We developed a novel BGM vector with inducible recA expression system, iREX, which enables us to manipulate large DNA fragments more stably than the conventional BGM vector by suppressing undesirable recombination. In addition, we demonstrate that the iREX can be applied to handling the DNA, which has several homologous sequences, such as multiple-reporter expression cassettes. Thus, the iREX expands the utility of the BGM vector as a platform for engineering large DNA fragments.
Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/genética , Vetores Genéticos/metabolismo , Genoma Bacteriano , Recombinases Rec A/genética , Cromossomos Artificiais Bacterianos/genética , Cromossomos Artificiais Bacterianos/metabolismo , Clonagem Molecular , DNA/metabolismo , Expressão Gênica/efeitos dos fármacos , Genes Reporter , Vetores Genéticos/genética , Xilose/farmacologiaRESUMO
BACKGROUND: The 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. RESULTS: Skn-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. CONCLUSION: Skn-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.
Assuntos
Fatores de Transcrição de Octâmero/metabolismo , Bulbo Olfatório/metabolismo , Mucosa Olfatória/metabolismo , Neurônios Receptores Olfatórios/metabolismo , Neurônios Receptores Olfatórios/ultraestrutura , Canais de Cátion TRPM/metabolismo , Animais , Células Cultivadas , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microvilosidades/metabolismoRESUMO
Solitary chemosensory cells in the non-neuronal epithelium of the anterior nasal cavity have bitter taste cell-like molecular characteristics and are involved in the detection of noxious substances. Here, we demonstrate that Pou2f3/Skn-1a, which is necessary for generation of sweet, umami, and bitter taste cells, is also necessary for the generation or differentiation of solitary chemosensory cells.
Assuntos
Diferenciação Celular , Células Epiteliais/citologia , Cavidade Nasal/citologia , Fatores de Transcrição de Octâmero/metabolismo , Animais , Células Epiteliais/metabolismo , Técnicas de Inativação de Genes , Camundongos , Fatores de Transcrição de Octâmero/deficiência , Fatores de Transcrição de Octâmero/genéticaRESUMO
BACKGROUND: The Bacillus subtilis genome (BGM) vector is a novel cloning system for large DNA fragments, in which the entire 4.2 Mb genome of B. subtilis functions as a vector. The BGM vector system has several attractive properties, such as a large cloning capacity of over 3 Mb, stable propagation of cloned DNA and various modification strategies using RecA-mediated homologous recombination. However, genetic modifications using the BGM vector system have not been fully established, and this system has not been applied to transgenesis. In this study, we developed important additions to the genetic modification methods of the BGM vector system. To explore the potential of the BGM vector, we focused on the fish-like odorant receptor (class I OR) gene family, which consists of 158 genes and forms a single gene cluster. Although a cis-acting locus control region is expected to regulate transcription, this has not yet been determined experimentally. RESULTS: Using two contiguous bacterial artificial chromosome clones containing several class I OR genes, we constructed two transgenes in the BGM vector by inserting a reporter gene cassette into one class I OR gene. Because they were oriented in opposite directions, we performed an inversion modification to align their orientation and then fused them to enlarge the genomic structure. DNA sequencing revealed that no mutations occurred during gene manipulations with the BGM vector. We further demonstrated that the modified, reconstructed genomic DNA fragments could be used to generate transgenic mice. Transgenic mice carrying the enlarged transgene recapitulated the expression and axonal projection patterns of the target class I OR gene in the main olfactory system. CONCLUSION: We offer a complete genetic modification method for the BGM vector system, including insertion, deletion, inversion and fusion, to engineer genomic DNA fragments without any trace of modifications. In addition, we demonstrate that this system can be used for mouse transgenesis. Thus, the BGM vector system can be an alternative platform for engineering large DNA fragments in addition to conventional systems such as bacterial and yeast artificial chromosomes. Using this system, we provide the first experimental evidence of a cis-acting element for a class I OR gene.
Assuntos
Bacillus subtilis/genética , Cromossomos Artificiais Bacterianos/genética , Técnicas de Transferência de Genes , Vetores Genéticos , Animais , Clonagem Molecular , Análise Mutacional de DNA , Feminino , Genes Reporter , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Elementos Reguladores de TranscriçãoRESUMO
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)/Gα(i2)-positive and vomeronasal 2 receptor (V2r)/Gα(o)-positive VSNs. We found that all Gα(i2)-positive cells coexpressed Gα(o) 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/Gα(i2)-type VSNs and a decreased number of V2r/Gα(o)-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.
Assuntos
Diferenciação Celular/fisiologia , Proteínas Repressoras/metabolismo , Células Receptoras Sensoriais/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Órgão Vomeronasal/citologia , Órgão Vomeronasal/metabolismo , Animais , Interneurônios/metabolismo , Camundongos , Camundongos Knockout , Bulbo Olfatório/citologia , Bulbo Olfatório/metabolismo , Ácido gama-Aminobutírico/metabolismoRESUMO
Odorant receptor (OR) genes can be classified into two types: fish-like class I OR genes and mammalian-specific class II OR genes. We have previously shown that Lhx2, a LIM-homeodomain protein, binds to the homeodomain site in the promoter region of mouse M71, a class II OR, and that a knockout mutation in Lhx2 precludes expression of all tested class II OR genes including M71. Here, we report that most class I OR genes, which are expressed in a dorsal region of the olfactory epithelium, are still expressed in Lhx2-deficient embryos. There are two exceptions: two class I OR genes, which are normally expressed in a more ventral region, are no longer expressed in Lhx2 mutant mice. Lhx2 is transcribed in olfactory sensory neurons irrespective of expression of class I or class II OR genes. Thus, a deficiency of Lhx2 has a differential impact on class I and class II OR gene expression.