RESUMO
Our goal in this article is to provide a perspective on how to understand the nature of responses to chemical mixtures. In studying responses to mixtures, researchers often identify "mixture interactions"-responses to mixtures that are not accurately predicted from the responses to the mixture's individual components. Critical in these studies is how to predict responses to mixtures and thus to identify a mixture interaction. We explore this issue with a focus on olfaction and on the first level of neural processing-olfactory sensory neurons-although we use examples from taste systems as well and we consider responses beyond sensory neurons, including behavior and psychophysics. We provide a broadly comparative perspective that includes examples from vertebrates and invertebrates, from genetic and nongenetic animal models, and from literature old and new. In the end, we attempt to recommend how to approach these problems, including possible future research directions.
Assuntos
Neurônios Receptores Olfatórios , Olfato , Animais , Células Receptoras Sensoriais , Olfato/fisiologiaRESUMO
The encoding of odors is believed to begin as a combinatorial code consisting of distinct patterns of responses from odorant receptors (ORs), trace-amine associated receptors (TAARs), or both. To determine how specific response patterns arise requires detecting patterns in vivo and understanding how the components of an odor, which are nearly always mixtures of odorants, give rise to parts of the pattern. Cigarette smoke, a common and clinically relevant odor consisting of >400 odorants, evokes responses from 144 ORs and 3 TAARs in freely behaving male and female mice, the first example of in vivo responses of both ORs and TAARs to an odor. As expected, a simplified artificial mimic of cigarette smoke odor tested at low concentration to identify highly sensitive receptors evokes responses from four ORs, all also responsive to cigarette smoke. Human subjects of either sex identify 1-pentanethiol as the odorant most critical for perception of the artificial mimic; and in mice the OR response patterns to these two odors are significantly similar. Fifty-eight ORs respond to the headspace above 25% 1-pentanethiol, including 9 ORs responsive to cigarette smoke. The response patterns to both cigarette smoke and 1-pentanethiol have strongly responsive ORs spread widely across OR sequence diversity, consistent with most other combinatorial codes previously measured in vivo The encoding of cigarette smoke is accomplished by a broad receptor response pattern, and 1-pentanethiol is responsible for a small subset of the responsive ORs in this combinatorial code.SIGNIFICANCE STATEMENT Complex odors are usually perceived as distinct odor objects. Cigarette smoke is the first complex odor whose in vivo receptor response pattern has been measured. It is also the first pattern shown to include responses from both odorant receptors and trace-amine associated receptors, confirming that the encoding of complex odors can be enriched by signals coming through both families of receptors. Measures of human perception and mouse receptor physiology agree that 1-pentanethiol is a critical component of a simplified odorant mixture designed to mimic cigarette smoke odor. Its receptor response pattern helps to link those of the artificial mimic and real cigarette smoke, consistent with expectations about perceptual similarity arising from shared elements in receptor response patterns.
Assuntos
Odorantes , Percepção Olfatória , Olfato , Compostos de Sulfidrila/química , Poluição por Fumaça de Tabaco , Adolescente , Adulto , Animais , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Receptores Odorantes/genética , Receptores Odorantes/metabolismo , Compostos de Sulfidrila/farmacologia , Produtos do TabacoRESUMO
The perception of odors relies on combinatorial codes consisting of odorant receptor (OR) response patterns to encode odor identity. Modulation of these patterns by odorant interactions at ORs potentially explains several olfactory phenomena: mixture suppression, unpredictable sensory outcomes, and the perception of odorant mixtures as unique objects. We determined OR response patterns to 4 odorants and 3 binary mixtures in vivo in mice, identifying 30 responsive ORs. These patterns typically had a few strongly responsive ORs and a greater number of weakly responsive ORs. ORs responsive to an odorant were often unrelated sequences distributed across several OR subfamilies. Mixture responses predicted pharmacological interactions between odorants, which were tested in vitro by heterologous expression of ORs in cultured cells, providing independent evidence confirming odorant agonists for 13 ORs and identifying both suppressive and additive effects. This included 11 instances of antagonism of ORs by an odorant, 1 instance of additive responses to a binary mixture, 1 instance of suppression of a strong agonist by a weak agonist, and the discovery of an inverse agonist for an OR. Interactions between odorants at ORs are common even when the odorants are not known to interact perceptually in humans, and in some cases interactions at mouse ORs correlate with the ability of humans to perceive an odorant in a mixture.
Assuntos
Odorantes , Neurônios Receptores Olfatórios/metabolismo , Receptores Odorantes/metabolismo , Olfato , Aldeídos/farmacologia , Animais , Células Cultivadas , Feminino , Lactonas/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios Receptores Olfatórios/efeitos dos fármacos , Pentanóis/farmacologia , Receptores Odorantes/agonistas , Receptores Odorantes/antagonistas & inibidoresRESUMO
Natural odors are mixtures of volatile chemicals (odorants). Odors are encoded as responses of distinct subsets of the hundreds of odorant receptors and trace amine-associated receptors expressed monogenically by olfactory sensory neurons. This is an elegantly simple mechanism for differentially encoding odors but it is susceptible to complex dose-response relationships and interactions between odorants at receptors, which may help explain olfactory phenomena such as mixture suppression, synthetic versus elemental odor processing, and poorly predictable perceptual outcomes of new odor mixtures. In this study in vivo tests in freely behaving mice confirm evidence of a characteristic receptor response pattern consisting of a few receptors with strong responses and a greater number of weakly responding receptors. Odorant receptors responsive to an odor are often unrelated and widely divergent in sequence, even when the odor consists of a single species of odorant. Odorant receptor response patterns to a citrus odor broaden with concentration. Some highly sensitive receptors respond only to a low concentration but others respond in proportion to concentration, a feature that may be critical for concentration-invariant perception. Other tests find evidence of interactions between odorants in vivo. All of the odorant receptor responses to a moderate concentration of the fecal malodor indole are suppressed by a high concentration of the floral odorant, α-ionone. Such suppressive effects are consistent with prior evidence that odorant interactions at individual odorant receptors are common.
RESUMO
Olfactory sensory neurons (OSNs) are bipolar neurons, unusual because they turn over continuously and have a multiciliated dendrite. The extensive changes in gene expression accompanying OSN differentiation in mice are largely known, especially the transcriptional regulators responsible for altering gene expression, revealing much about how differentiation proceeds. Basal progenitor cells of the olfactory epithelium transition into nascent OSNs marked by Cxcr4 expression and the initial extension of basal and apical neurites. Nascent OSNs become immature OSNs within 24-48 h. Immature OSN differentiation requires about a week and at least 2 stages. Early-stage immature OSNs initiate expression of genes encoding key transcriptional regulators and structural proteins necessary for further neuritogenesis. Late-stage immature OSNs begin expressing genes encoding proteins important for energy production and neuronal homeostasis that carry over into mature OSNs. The transition to maturity depends on massive expression of one allele of one odorant receptor gene, and this results in expression of the last 8% of genes expressed by mature OSNs. Many of these genes encode proteins necessary for mature function of axons and synapses or for completing the elaboration of non-motile cilia, which began extending from the newly formed dendritic knobs of immature OSNs. The cilia from adjoining OSNs form a meshwork in the olfactory mucus and are the site of olfactory transduction. Immature OSNs also have a primary cilium, but its role is unknown, unlike the critical role in proliferation and differentiation played by the primary cilium of the olfactory epithelium's horizontal basal cell.
Assuntos
Cílios/metabolismo , Neurônios Receptores Olfatórios/metabolismo , Animais , Axônios/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Regulação da Expressão Gênica , Humanos , Neurogênese/genética , Mucosa Olfatória/citologia , Mucosa Olfatória/metabolismo , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Receptores Odorantes/genética , Receptores Odorantes/metabolismo , Olfato , Sinapses/metabolismoRESUMO
Activity-dependent processes are important to olfactory sensory neurons (OSNs) in several ways, such as cell survival and the specificity of axonal convergence. The identification of activity-dependent mRNAs has contributed to our understanding of OSN axon convergence, but has revealed surprisingly little about other processes. Published studies of activity-dependent mRNAs in olfactory mucosae overlap poorly, but by combining these agreements with meta-analysis of existing data we identify 443 mRNAs that respond to methods that alter OSN activity. Three hundred and fifty of them are expressed in mature OSNs, consistent with the expectation that activity-dependent responses are cell autonomous and driven by odor stimulation. Many of these mRNAs encode proteins that function at presynaptic terminals or support electrical activity, consistent with hypotheses linking activity dependence to synaptic plasticity and energy conservation. The lack of agreement between studies is due largely to underpowered experiments. In addition, methods used to alter OSN activity are susceptible to indirect or off-target effects. These effects deserve greater attention, not only to rigorously identify OSN mRNAs that respond to altered OSN activity, but also because these effects are of significant interest in their own right. For example, the mRNAs of some sustentacular cell enzymes believed to function in odorant clearance (Cyp2a4 and Cyp2g1) are sensitive to unilateral naris occlusion used to reduce odorant stimulation of the ipsilateral olfactory epithelium. Also problematic are odorant receptor mRNAs, which show little agreement across studies and are susceptible to differences in frequency of expression that masquerade as activity-dependent changes in mRNA abundance.
Assuntos
Regulação da Expressão Gênica , Neurônios Receptores Olfatórios/metabolismo , Receptores Odorantes/genética , Animais , Humanos , RNA Mensageiro/análise , RNA Mensageiro/genéticaRESUMO
Our understanding of mammalian olfactory coding has been impeded by the paucity of information about the odorant receptors (ORs) that respond to a given odorant ligand in awake, freely behaving animals. Identifying the ORs that respond in vivo to a given odorant ligand from among the â¼1100 ORs in mice is intrinsically challenging but critical for our understanding of olfactory coding at the periphery. Here, we report an in vivo assay that is based on a novel gene-targeted mouse strain, S100a5-tauGFP, in which a fluorescent reporter selectively marks olfactory sensory neurons that have been activated recently in vivo. Because each olfactory sensory neuron expresses a single OR gene, multiple ORs responding to a given odorant ligand can be identified simultaneously by capturing the population of activated olfactory sensory neurons and using expression profiling methods to screen the repertoire of mouse OR genes. We used this in vivo assay to re-identify known eugenol- and muscone-responsive mouse ORs. We identified additional ORs responsive to eugenol or muscone. Heterologous expression assays confirmed nine eugenol-responsive ORs (Olfr73, Olfr178, Olfr432, Olfr610, Olfr958, Olfr960, Olfr961, Olfr913, and Olfr1234) and four muscone-responsive ORs (Olfr74, Olfr235, Olfr816, and Olfr1440). We found that the human ortholog of Olfr235 and Olfr1440 responds to macrocyclic ketone and lactone musk odorants but not to polycyclic musk odorants or a macrocyclic diester musk odorant. This novel assay, called the Kentucky in vivo odorant ligand-receptor assay, should facilitate the in vivo identification of mouse ORs for a given odorant ligand of interest.
Assuntos
Cicloparafinas/farmacologia , Eugenol/farmacologia , Receptores Odorantes/efeitos dos fármacos , Animais , Humanos , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Odorantes , Receptores Acoplados a Proteínas G/fisiologiaRESUMO
Activity-dependent survival of olfactory sensory neurons (OSNs) may allow animals to tune their olfactory systems to match their odor environment. Activity-dependent genes should play important roles in this process, motivating experiments to identify them. Both unilateral naris occlusion of mice for 6 days and genetic silencing of OSNs decreased S100A5, Lrrc3b, Kirrel2, Slc17a6, Rasgrp4, Pcp4l1, Plcxd3, and Kcnn2 while increasing Kirrel3. Naris occlusion also decreased Eml5, Ptprn, and Nphs1. OSN number was unchanged and stress-response mRNAs were unaffected after 6 days of naris occlusion. This leaves odor stimulation as the most likely cause of differential abundance of these mRNAs, but through a mechanism that is slow or indirect for most because 30-40 min of odor stimulation increased only 3 of 11 mRNAs decreased by naris occlusion: S100A5, Lrrc3b, and Kirrel2. Odorant receptor (OR) mRNAs were significantly more variable than the average mRNA, consistent with difficulty in reliably detecting changes in these mRNAs after 6 days of naris occlusion. One OR mRNA, Olfr855, was consistently decreased, however. These results suggest that the latency from the cessation of odor stimulation to effects on activity-dependent OSN survival must be a week or more in juvenile mice.
Assuntos
Regulação da Expressão Gênica , Neurônios Receptores Olfatórios/fisiologia , Animais , Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Feminino , Inativação Gênica , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Obstrução Nasal/genética , Odorantes , Estimulação Física , RNA Mensageiro/genética , Receptores Odorantes/genéticaRESUMO
An increasing diversity of techniques investigating the biology of specific cell types and individual cells have elevated the importance of dissociation of viable cells from living tissues. Here we describe a method for the dissociation of single cells from samples of adult mouse olfactory mucosae, with an emphasis on maximizing yield of viable single cells from fluorescence-activated cell sorting. Yields are typically in the range of 80,000-150,000 viable cells per adult mouse.
Assuntos
Neurônios Receptores Olfatórios , Animais , Camundongos , Citometria de FluxoRESUMO
Crustacean crustacyanin proteins are linked to the production and modification of carapace colour, with direct implications for fitness and survival. Here, the structural and functional properties of the two recombinant crustacyanin subunits H(1) and H(2) from the American lobster Homarus americanus are reported. The two subunits are structurally highly similar to the corresponding natural apo crustacyanin CRTC and CRTA subunits from the European lobster H. gammarus. Reconstitution studies of the recombinant crustacyanin proteins H(1) and H(2) with astaxanthin reproduced the bathochromic shift of 85-95â nm typical of the natural crustacyanin subunits from H. gammarus in complex with astaxanthin. Moreover, correlations between the presence of crustacyanin genes in crustacean species and the resulting carapace colours with the spectral properties of the subunits in complex with astaxanthin confirmed this genotype-phenotype linkage.
Assuntos
Proteínas de Transporte/química , Nephropidae/química , Sequência de Aminoácidos , Animais , Sequência Conservada , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Proteínas Recombinantes/química , Alinhamento de SequênciaRESUMO
BACKGROUND: Alzheimer's disease (AD) is a progressive age-dependent disorder whose risk is affected by genetic factors. Better models for investigating early effects of risk factors such as apolipoprotein E (APOE) genotype are needed. OBJECTIVE: To determine whether APOE genotype produces neuropathologies in an AD-susceptible neural system, we compared effects of human APOE É3 (E3) and APOE É4 (E4) alleles on the mouse olfactory epithelium. METHODS: RNA-Seq using the STAR aligner and DESeq2, immunohistochemistry for activated caspase-3 and phosphorylated histone H3, glucose uptake after oral gavage of 2-[1,2-3H (N)]-deoxy-D-glucose, and Seahorse Mito Stress tests on dissociated olfactory mucosal cells. RESULTS: E3 and E4 olfactory mucosae show 121 differentially abundant mRNAs at age 6 months. These do not indicate differences in cell type proportions, but effects on 17 odorant receptor mRNAs suggest small differences in tissue development. Ten oxidoreductases mRNAs important for cellular metabolism and mitochondria are less abundant in E4 olfactory mucosae but this does not translate into differences in cellular respiration. E4 olfactory mucosae show lower glucose uptake, characteristic of AD susceptibility and consistent with greater expression of the glucose-sensitive gene, Asns. Olfactory sensory neuron apoptosis is unaffected at age 6 months but is greater in E4 mice at 10 months. CONCLUSION: Effects of human APOE alleles on mouse olfactory epithelium phenotype are apparent in early adulthood, and neuronal loss begins to increase by middle age (10 months). The olfactory epithelium is an appropriate model for the ability of human APOE alleles to modulate age-dependent effects associated with the progression of AD.
Assuntos
Doença de Alzheimer/genética , Apolipoproteína E3/genética , Apolipoproteína E4/genética , Mucosa Olfatória/patologia , Olfato/genética , Adulto , Alelos , Animais , Apolipoproteínas E , Encéfalo/patologia , Feminino , Genótipo , Humanos , Masculino , CamundongosRESUMO
We have shown that deficiency of neutral sphingomyelinase 2 (nSMase2), an enzyme generating the sphingolipid ceramide, improves memory in adult mice. Here, we performed sphingolipid and RNA-seq analyses on the cortex from 10-month-old nSMase2-deficient (fro/fro) and heterozygous (+ /fro) mice. fro/fro cortex showed reduced levels of ceramide, particularly in astrocytes. Differentially abundant transcripts included several functionally related groups, with decreases in mitochondrial oxidative phosphorylation and astrocyte activation transcripts, while axon guidance and synaptic transmission and plasticity transcripts were increased, indicating a role of nSMase2 in oxidative stress, astrocyte activation, and cognition. Experimentally induced oxidative stress decreased the level of glutathione (GSH), an endogenous inhibitor of nSMase2, and increased immunolabeling for ceramide in primary + /fro astrocytes, but not in fro/fro astrocytes. ß-galactosidase activity was lower in 5-week-old fro/fro astrocytes, indicating delayed senescence due to nSMase2 deficiency. In fro/fro cortex, levels of the senescence markers C3b and p27 and the proinflammatory cytokines interleukin 1ß, interleukin 6, and tumor necrosis factor α were reduced, concurrent with twofold decreased phosphorylation of their downstream target, protein kinase Stat3. RNA and protein levels of the ionotropic glutamate receptor subunit 2B (Grin2b/NR2B) were increased by twofold, which was previously shown to enhance cognition. This was consistent with threefold reduced levels of exosomes carrying miR-223-3p, a micro-RNA downregulating NR2B. In summary, our data show that nSMase2 deficiency prevents oxidative stress-induced elevation of ceramide and secretion of exosomes by astrocytes that suppress neuronal function, indicating a role of nSMase2 in the regulation of neuroinflammation and cognition.
Assuntos
Astrócitos , Esfingomielina Fosfodiesterase , Animais , Astrócitos/metabolismo , Ceramidas/metabolismo , Camundongos , Plasticidade Neuronal , Estresse Oxidativo , RNA/metabolismo , Esfingolipídeos/metabolismo , Esfingomielina Fosfodiesterase/genética , Esfingomielina Fosfodiesterase/metabolismoRESUMO
Uncx (Phd1, Chx4) is a paired homeobox transcription factor gene. It and its probable functional partners, Tle co-repressors, were expressed by neurally-fated basal progenitor cells and olfactory sensory neurons of the olfactory epithelium. Uncx expression was rare in olfactory epithelia of Ascl1(-/-) mice, but common in Neurog1(-/-) mice. In Uncx(-/-) mice olfactory progenitor cell proliferation, progenitor cell number, olfactory sensory neuron survival, and Umodl1 and Kcnc4 mRNAs were reduced. Evidence of sensory neuron activity and functional connections to the olfactory bulb argue that decreased neuronal survival was not due to loss of trophic support or activity-dependent mechanisms. These data suggest that UNCX acts downstream of neural determination factors to broadly control transcriptional mechanisms used by neural progenitor cells to specify neural phenotypes.
Assuntos
Proteínas de Homeodomínio/metabolismo , Células-Tronco Neurais/citologia , Neurogênese/fisiologia , Mucosa Olfatória/citologia , Neurônios Receptores Olfatórios/citologia , Animais , Diferenciação Celular/fisiologia , Proliferação de Células , Imunofluorescência , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Camundongos Knockout , Células-Tronco Neurais/metabolismo , Mucosa Olfatória/metabolismo , Neurônios Receptores Olfatórios/metabolismo , Fenótipo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição GênicaRESUMO
Neurogenesis of projection neurons requires that axons be initiated, extended, and connected. Differences in the expression of axon growth and guidance genes must drive these events, but comprehensively characterizing these differences in a single neuronal type has not been accomplished. Guided by a catalog of gene expression in olfactory sensory neurons (OSNs), in situ hybridization and immunohistochemistry revealed that Cxcr4 and Dbn1, two axon initiation genes, marked the developmental transition from basal progenitor cells to immature OSNs in the olfactory epithelium. The CXCR4 immunoreactivity of these nascent OSNs overlapped partially with markers of proliferation of basal progenitor cells and partially with immunoreactivity for GAP43, the canonical marker of immature OSNs. Intracellular guidance cue signaling transcripts Ablim1, Crmp1, Dypsl2, Dpysl3, Dpysl5, Gap43, Marcskl1, and Stmn1-4 were specific to, or much more abundant in, the immature OSN layer. Receptors that mediate axonal inhibition or repulsion tended to be expressed in both immature and mature OSNs (Plxna1, Plxna4, Nrp2, Efna5) or specifically in mature OSNs (Plxna3, Unc5b, Efna3, Epha5, Epha7), although some were specific to immature OSNs (Plxnb1, Plxnb2, Plxdc2, Nrp1). Cell adhesion molecules were expressed either by both immature and mature OSNs (Dscam, Ncam1, Ncam2, Nrxn1) or solely by immature OSNs (Chl1, Nfasc1, Dscaml1). Given the loss of intracellular signaling protein expression, the continued expression of guidance cue receptors in mature OSNs is consistent with a change in the role of these receptors, perhaps to sending signals back to the cell body and nucleus.
Assuntos
Axônios/ultraestrutura , Neurogênese/genética , Neuropeptídeos/genética , Mucosa Olfatória/crescimento & desenvolvimento , Células Receptoras Sensoriais/citologia , Animais , Diferenciação Celular/genética , Imunofluorescência , Perfilação da Expressão Gênica , Cones de Crescimento/metabolismo , Cones de Crescimento/ultraestrutura , Imuno-Histoquímica , Hibridização In Situ , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores CXCR4/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Receptoras Sensoriais/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismoRESUMO
Odor discrimination requires differential expression of odor detectors. In fact, olfactory input to the brain is organized in units (glomeruli) innervated only by olfactory sensory neurons that express the same odorant receptor (OR). Therefore, discriminatory capacity is maximized if each sensory neuron expresses only one allele of a single OR gene, a postulate sometimes canonized as the "one neuron-one receptor rule." OR gene choice appears to result from a hierarchy of processes: differential availability of the alleles of each OR gene, zonal exclusion (or selection), OR gene switching during the initiation of OR gene transcription, and OR-dependent feedback to solidify the choice of one OR gene. The mechanisms underlying these processes are poorly understood, though a few elements are known or suspected. For example, the mechanism of activation of OR gene transcription appears to work in part through a few homeobox transcription factors (Emx2, and perhaps Lhx2) and the Ebf family of transcription factors. Further insights will probably come from several directions, but a promising hypothesis is that epigenetic mechanisms contribute to all levels of the hierarchical control of OR gene expression, especially the repressive events that seem to be necessary to achieve the singularity of OR gene choice.
Assuntos
Receptores Odorantes/genética , Animais , Montagem e Desmontagem da Cromatina , Epigênese Genética , Regulação da Expressão Gênica , Inativação Gênica , Regiões Promotoras GenéticasRESUMO
Recently, cDNAs encoding prepro-orcokinins were cloned from the crayfish Procambarus clarkii; these cDNAs encode multiple copies of four orcokinin isoforms as well as several other peptides. Using the translated open reading frames of the P. clarkii transcripts as queries, five ESTs encoding American lobster Homarus americanus orthologs were identified via BLAST analysis. From these clones, three cDNAs, each encoding one of two distinct prepro-hormones, were characterized. Predicted processing of the deduced prepro-hormones would generate 13 peptides, 12 of which are conserved between the 2 precursors: the orcokinins NFDEIDRSGFGFN (3 copies), NFDEIDRSGFGFH (2 copies) and NFDEIDRSGFGFV (2 copies), FDAFTTGFGHN (an orcomyotropin-related peptide), SSEDMDRLGFGFN, GDY((SO3))DVYPE, VYGPRDIANLY and SAE. Additionally, one of two longer peptides (GPIKVRFLSAIFIPIAAPARSSPQQDAAAGYTDGAPV or APARSSPQQDAAAGYTDGAPV) is predicted from each prepro-hormone. MALDI-FTMS analyses confirmed the presence of all predicted orcokinins, the orcomyotropin-related peptide, and three precursor-related peptides, SSEDMDRLGFGFN, GDYDVYPE (unsulfated) and VYGPRDIANLY, in H. americanus neural tissues. SAE and the longer, unshared peptides were not detected. Similar complements of peptides are predicted from P. clarkii transcripts; the majority of these were detected in its neural tissues with mass spectrometry. Truncated orcokinins not predicted from any precursor were also detected in both species. Consistent with previous studies in the crayfish Orconectes limosus, NFDEIDRSGFGFN increased mid-/hindgut motility in P. clarkii. Surprisingly, the same peptide, although native to H. americanus, did not affect gut motility in this species. Together, our results provide the framework for future investigations of the regulation and physiological function of orcokinins/orcokinin precursor-related peptides in astacideans.
Assuntos
Astacoidea/metabolismo , Nephropidae/metabolismo , Neuropeptídeos/química , Peptídeos/química , Precursores de Proteínas/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Dados de Sequência Molecular , Peso Molecular , Isoformas de Proteínas/química , Alinhamento de Sequência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
In mammals, cilia are critical for development, sensation, cell signaling, sperm motility, and fluid movement. Defects in cilia are causes of several congenital syndromes, providing additional reasons to identify cilia-related genes. We hypothesized that mRNAs selectively abundant in tissues rich in highly ciliated cells encode cilia proteins. Selective abundance in olfactory epithelium, testes, vomeronasal organ, trachea, and lung proved to be an expression pattern uniquely effective in identifying documented cilia-related genes. Known and suspected cilia-related genes were statistically overrepresented among the 99 genes identified, but the majority encoded proteins of unknown function, thereby predicting new cilia-related proteins. Evidence of expression in a highly ciliated cell, the olfactory sensory neuron, exists for 73 of the genes. In situ hybridization for 17 mRNAs confirmed expression of all 17 in olfactory sensory neurons. Most were also detected in vomeronasal sensory neurons and in neighboring tissues rich in ciliated cells such as respiratory epithelium. Immunoreactivity for one of the proteins identified, Spa17, colocalized with acetylated tubulin in the cilia layer of the olfactory epithelium. In contrast, the ciliary rootlet protein, Crocc, was located in discrete structures whose position was consistent with the dendritic knobs of the olfactory sensory neurons. A compilation of >2,000 mouse genes predicted to encode cilia-related proteins revealed a strong correlation (R = 0.99) between the number of studies predicting a gene's involvement in cilia and documented evidence of such involvement, a fact that simplifies the selection of genes for further study of the physiology of cilia.
Assuntos
Cílios/genética , Perfilação da Expressão Gênica , Animais , Cílios/metabolismo , Biologia Computacional , Feminino , Imuno-Histoquímica , Hibridização In Situ , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios Aferentes/metabolismo , Mucosa Olfatória/metabolismo , Neurônios Receptores Olfatórios/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
The mechanisms selecting a single odorant receptor (OR) gene for expression in each olfactory sensory neuron (OSN) establish an OR expression pattern critical for odor discrimination. These mechanisms are largely unknown, but putative OR promoters contain homeodomain-like sites, implicating homeobox transcription factors such as Emx2. At embryonic day 18.5, expression of 49-76% of ORs was decreased in mice lacking Emx2, depending on the metric used. The decreases were due to fewer OSNs expressing each OR. Affected ORs showed changes that were disproportionately greater than the 42% reduction in mature neurons and similar decreases in unrelated olfactory neuron-enriched messenger RNAs in Emx2(-/-) mice. Both Class I and Class II ORs decreased, as did ORs expressed in both the dorsal and ventral regions of the epithelium. Conversely, 7% of Class II ORs tested were expressed more frequently, suggesting that some ORs are independent of Emx2. Emx2 helps stimulate transcription for many OR genes, which we hypothesize is through direct action at OR promoters, but Emx2 appears to have no significant role in regulating other aspects of OR gene expression, including the zonal patterns, OR gene cluster selection mechanisms, and singularity of OR gene choice.
Assuntos
Regulação da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Receptores Odorantes/genética , Fatores de Transcrição/metabolismo , Animais , Forma Celular , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Proteínas de Homeodomínio/genética , Camundongos , Camundongos Knockout , Família Multigênica/genética , Bulbo Olfatório/citologia , Bulbo Olfatório/crescimento & desenvolvimento , Bulbo Olfatório/metabolismo , Fatores de Transcrição/deficiência , Fatores de Transcrição/genéticaRESUMO
Olfactory epithelial cells from olfactory marker protein-green fluorescent protein (OMP-GFP) mice were separated by fluorescence-activated cell sorting into a GFP+ sample enriched in mature olfactory sensory neurons (OSNs) and a GFP- sample enriched in all other cells. GeneChip expression profiling of these samples provided a predictive measure of expression in OSNs. Validation tests comparing the ratio of GFP+/GFP- signal intensity against expression patterns from in situ hybridization for 189 mRNAs proved statistically significant and provided probabilities of expression in OSNs scaled according to the signal intensity ratios. These probabilities predict that, among 11,596 mRNAs detected in the GFP+ sample, more than 10,000 are expressed in OSNs. Transcripts and overrepresented categories of mRNAs detected in the GFP+ sample agreed with known properties of OSNs and predict additional properties. For example, ciliogenesis and spermatogenesis were overrepresented, consistent with similarities between OSN cilia and sperm flagella. Chromatin assembly mRNAs were expressed throughout the OSN cell lineage, consistent with the hypothesis that chromatin remodeling plays a role in OSN differentiation. We detected numerous signaling proteins and receptors, such as 30 nonchemosensory G-protein-coupled receptors, including the presynaptic glutamate receptor mGlur4 and the Wnt receptor Fzd3. The largest group of mRNAs, however, was the hundreds of transcriptional regulators that presumably determine the OSN phenotype. The absence of OMP protein in OMP-GFP mice had no detectable effect on mRNA abundance. Within limits prescribed by the nature of microarray data and the in situ hybridization validation, these data should be useful in directing further experiments on OSN function.
Assuntos
Perfilação da Expressão Gênica/métodos , Expressão Gênica/genética , Proteínas do Tecido Nervoso/genética , Mucosa Olfatória/metabolismo , Neurônios Receptores Olfatórios/metabolismo , RNA Mensageiro/genética , Animais , Adesão Celular/genética , Diferenciação Celular/genética , Células Cultivadas , Montagem e Desmontagem da Cromatina/genética , Cílios/genética , Cílios/metabolismo , Proteínas de Ligação a DNA/genética , Feminino , Proteínas de Fluorescência Verde/genética , Hibridização In Situ , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mucosa Olfatória/citologia , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/análise , Receptores Acoplados a Proteínas G/genética , Fatores de Transcrição/genéticaRESUMO
The lobster olfactory organ is an important model for investigating many aspects of the olfactory system. To facilitate study of the molecular basis of olfaction in lobsters, we made a subtracted cDNA library from the mature zone of the olfactory organ of Homarus americanus, the American lobster. Sequencing of the 5'-end of 5,184 cDNA clones produced 2,389 distinct high-quality sequences consisting of 1,944 singlets and 445 contigs. Matches to known sequences corresponded with the types of cells present in the olfactory organ, including specific markers of olfactory sensory neurons, auxiliary cells, secretory cells of the aesthetasc tegumental gland, and epithelial cells. The wealth of neuronal mRNAs represented among the sequences reflected the preponderance of neurons in the tissue. The sequences identified candidate genes responsible for known functions and suggested new functions not previously recognized in the olfactory organ. A cDNA microarray was designed and tested by assessing mRNA abundance differences between two of the lobster's major chemosensory structures: the mature zone of the olfactory organ and the dactyl of the walking legs, a taste organ. The 115 differences detected again emphasized the abundance of neurons in the olfactory organ, especially a cluster of mRNAs encoding cytoskeletal-associated proteins and cell adhesion molecules such as 14-3-3zeta, actins, tubulins, trophinin, Fax, Yel077cp, suppressor of profilin 2, and gelsolin.