Max Planck Research Unit for Neurogenetics

Frankfurt am Main, Germany

Max Planck Research Unit for Neurogenetics

Frankfurt am Main, Germany

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Omura M.,Max Planck Research Unit for Neurogenetics | Grosmaitre X.,University of Pennsylvania | Grosmaitre X.,Center Des Science Du Gout Et Of Lalimentation | Ma M.,University of Pennsylvania | Mombaerts P.,Max Planck Research Unit for Neurogenetics
Molecular and Cellular Neuroscience | Year: 2014

In the mouse, mature olfactory sensory neurons (OSNs) express one allele of one of the ~. 1200 odorant receptor (OR) genes, which encode G-protein coupled receptors (GPCRs). Axons of OSNs that express the same OR coalesce into homogeneous glomeruli at conserved positions in the olfactory bulb. ORs are involved in OR gene choice and OSN axonal wiring, but the mechanisms remain poorly understood. One approach is to substitute an OR genetically with another GPCR, and to determine in which aspects this GPCR can serve as a surrogate OR under experimental conditions. Here, we characterize a novel gene-targeted mouse strain in which the mouse β2-adrenergic receptor (β2AR) is coexpressed with tauGFP in OSNs that choose the OR locus M71 for expression (β2AR→M71-GFP). By crossing these mice with β2AR→M71-lacZ gene-targeted mice, we find that differentially tagged β2AR→M71 alleles are expressed monoallelically. The OR coding sequence is thus not required for monoallelic expression - the expression of one of the two alleles of a given OR gene in an OSN. We detect strong β2AR immunoreactivity in dendritic cilia of β2AR→M71-GFP OSNs. These OSNs respond to the β2AR agonist isoproterenol in a dose-dependent manner. Axons of β2AR→M71-GFP OSNs coalesce into homogeneous glomeruli, and β2AR immunoreactivity is detectable within these glomeruli. We do not find evidence for expression of endogenous β2AR in OSNs of wild-type mice, also not in M71-expressing OSNs, and we do not observe overt differences in the olfactory system of β2AR and β1AR knockout mice. Our findings corroborate the experimental value of the β2AR as a surrogate OR, including for the study of the mechanisms of monoallelic expression. © 2013 Elsevier Inc.


Omura M.,Max Planck Research Unit for Neurogenetics | Mombaerts P.,Max Planck Research Unit for Neurogenetics
Molecular and Cellular Neuroscience | Year: 2015

Chemoreception in the mouse olfactory system occurs primarily at two chemosensory epithelia in the nasal cavity: the main olfactory epithelium (MOE) and the vomeronasal epithelium. The canonical chemosensory neurons in the MOE, the olfactory sensory neurons (OSNs), express the odorant receptor (OR) gene repertoire, and depend on Adcy3 and Cnga2 for chemosensory signal transduction. The canonical chemosensory neurons in the vomeronasal epithelium, the vomeronasal sensory neurons (VSNs), express two unrelated vomeronasal receptor (VR) gene repertoires, and involve Trpc2 for chemosensory signal transduction. Recently we reported the discovery of two types of neurons in the mouse MOE that express Trcp2 in addition to Cnga2. These cell types can be distinguished at the single-cell level by expression of Adcy3: positive, type A and negative, type B. Some type A cells express OR genes. Thus far there is no specific gene or marker for type B cells, hampering further analyses such as physiological recordings. Here, we show that among MOE cells, type B cells are unique in their expression of the soluble guanylate cyclase Gucy1b2. We came across Gucy1b2 in an explorative approach based on Long Serial Analysis of Gene Expression (LongSAGE) that we applied to single red-fluorescent cells isolated from whole olfactory mucosa and vomeronasal organ of mice of a novel Trcp2-IRES-taumCherry gene-targeted strain. The generation of a novel Gucy1b2-IRES-tauGFP gene-targeted strain enabled us to visualize coalescence of axons of type B cells into glomeruli in the main olfactory bulb. Our molecular and anatomical analyses define Gucy1b2 as a marker for type B cells within the MOE. The Gucy1b2-IRES-tauGFP strain will be useful for physiological, molecular, cellular, and anatomical studies of this newly described chemosensory subsystem. © 2015 Published by Elsevier Inc.


Khan M.,Max Planck Research Unit for Neurogenetics | Vaes E.,Max Planck Research Unit for Neurogenetics | Mombaerts P.,Max Planck Research Unit for Neurogenetics
Molecular and Cellular Neuroscience | Year: 2013

In the mouse, the sense of smell relies predominantly on the expression of ~. 1200 odorant receptor (OR) genes in the main olfactory epithelium (MOE). Each mature olfactory sensory neuron (OSN) in the MOE is thought to express just one of these OR genes; conversely, an OR gene is expressed in thousands to tens of thousands of OSNs per mouse. Here, we have characterized temporal patterns of OR gene expression in a cohort of inbred C57BL6/N mice from the Aged Rodent Colonies of the National Institute on Aging. We applied the NanoString multiplex platform to quantify RNA abundance for 531 OR genes in whole olfactory mucosa (WOM) tissue samples. The five study groups were females aged 2, 6, 12, 18, and 31. months (mo). We classified the 531 temporal patterns using a step-down quadratic regression method for time course analysis. The majority of OR genes (58.4%) are classified as flat: there is no significant difference from a horizontal line within this time window. There are 32.8% of OR genes with a downward profile, 7.2% with an upward profile, and 1.7% with a convex or concave profile. But the magnitude of these decreases and increases tends to be small: only 4.3% of OR genes are differentially expressed (DE) at 31. mo compared to 2. mo. Interestingly, the variances of NanoString counts for individual OR genes are homogeneous among the age groups. Our analyses of these 15,930 OR gene expression data of C57BL6/N mice that were raised and housed under well-controlled conditions indicate that OR gene expression at the MOE level is intrinsically stable. © 2013 Elsevier Inc.


McClintock T.S.,University of Kentucky | Adipietro K.,Duke University | Adipietro K.,University of Maryland Baltimore County | Titlow W.B.,University of Kentucky | And 5 more authors.
Journal of Neuroscience | Year: 2014

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 ∼1100 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. © 2014 the authors.


Vaes E.,Max Planck Research Unit for Neurogenetics | Khan M.,Max Planck Research Unit for Neurogenetics | Mombaerts P.,Max Planck Research Unit for Neurogenetics
BMC Bioinformatics | Year: 2014

Background: A challenge in gene expression studies is the reliable identification of differentially expressed genes. In many high-throughput studies, genes are accepted as differentially expressed only if they satisfy simultaneously a p value criterion and a fold change criterion. A statistical method, TREAT, has been developed for microarray data to assess formally if fold changes are significantly higher than a predefined threshold. We have recently applied the NanoString digital platform to study expression of mouse odorant receptor genes, which form with 1,200 members the largest gene family in the mouse genome. Our objectives are, on these data, to decrease false discoveries when formally assessing the genes relative to a fold change threshold, and to provide a guided selection in the choice of this threshold. Results: Statistical tests have been developed for microarray data to identify genes that are differentially expressed relative to a fold change threshold. Here we report that another approach, which we refer to as tTREAT, is more appropriate for our NanoString data, where false discoveries lead to costly and time-consuming follow-up experiments. Methods that we refer to as tTREAT2 and the running fold change model improve the performance of the statistical tests by protecting or selecting the fold change threshold more objectively. We show the benefits on simulated and real data. Conclusions: Gene-wise statistical analyses of gene expression data, for which the significance relative to a fold change threshold is important, give reproducible and reliable results on NanoString data of mouse odorant receptor genes. Because it can be difficult to set in advance a fold change threshold that is meaningful for the available data, we developed methods that enable a better choice (thus reducing false discoveries and/or missed genes) or avoid this choice altogether. This set of tools may be useful for the analysis of other types of gene expression data. © 2014 Vaes et al.; licensee BioMed Central Ltd.


Chang I.,Max Planck Research Unit for Neurogenetics | Parrilla M.,Max Planck Research Unit for Neurogenetics
Gene Expression Patterns | Year: 2016

Homeodomain proteins are encoded by homeobox genes and regulate development and differentiation in many neuronal systems. The mouse vomeronasal organ (VNO) generates in situ mature chemosensory neurons from stem cells. The roles of homeodomain proteins in neuronal differentiation in the VNO are poorly understood. Here we have characterized the expression patterns of 28 homeobox genes in the VNO of C57BL/6 mice at postnatal stages using multicolor fluorescent in situ hybridization. We identified 11 homeobox genes (Dlx3, Dlx4, Emx2, Lhx2, Meis1, Pbx3, Pknox2, Pou6f1, Tshz2, Zhx1, Zhx3) that were expressed exclusively in neurons; 4 homeobox genes (Pax6, Six1, Tgif1, Zfhx3) that were expressed in all non-neuronal cell populations, with Pax6, Six1 and Tgif1 also expressed in some neuronal progenitors and precursors; 12 homeobox genes (Adnp, Cux1, Dlx5, Dlx6, Meis2, Pbx2, Pknox1, Pou2f1, Satb1, Tshz1, Tshz3, Zhx2) with expression in both neuronal and non-neuronal cell populations; and one homeobox gene (Hopx) that was exclusively expressed in the non-sensory epithelium. We studied further in detail the expression of Emx2, Lhx2, Meis1, and Meis2. We found that expression of Emx2 and Lhx2 initiated between neuronal progenitor and neuronal precursor stages. As far as the sensory neurons of the VNO are concerned, Meis1 and Meis2 were only expressed in the apical layer, together with Gnai2, but not in the basal layer. © 2016 The Authors


PubMed | Max Planck Research Unit for Neurogenetics
Type: | Journal: Neuroscience | Year: 2017

In the mouse, odorant receptor proteins (ORs) are G-protein coupled receptors expressed in mature olfactory sensory neurons (OSNs) of the main olfactory epithelium (MOE). ORs mediate odorant reception at the level of the OSN cilia. Most the 1100 OR genes in the mouse genome are expressed, at the RNA level, in mature OSNs. The literature on antibodies against ORs is limited, and most reports are with antibodies that are not commercially available. Here we have screened 40 commercial antibodies against human and mouse ORs by immunofluorescence staining of coronal cryosections of the MOE of 21-day old C57BL/6J mice. Various methods of antigen retrieval were tested. Of the 19 antibodies raised against human ORs, three yielded a consistent immunoreactive signal in the mouse MOE; of these three, two appeared to crossreact against one or more, unknown, mouse ORs. Of the 21 antibodies raised against mouse ORs, six yielded a consistent immunoreactive signal in the mouse MOE; of these six, two also stained specific glomeruli in the olfactory bulb. Antibody specificity could be validated with gene-targeted mouse strains in the case of three ORs. The number of OSNs immunoreactive for the MOR28/Olfr1507 antibody is greater in C57BL/6J than in 129S6/SvEvTac wild-type mice. Taken together, our results are encouraging: 20-30% of these commercially available antibodies are informative in immunohistochemical analyses of the mouse MOE. The commercial availability of these antibodies should facilitate the study of OR proteins in the MOE and the olfactory bulb, and the replicability of results in the literature.


Omura M.,Max Planck Research Unit for Neurogenetics | Mombaerts P.,Max Planck Research Unit for Neurogenetics
Cell Reports | Year: 2014

The mouse olfactory system contains two distinct chemosensory epithelia, the main olfactory epithelium (MOE) and the vomeronasal epithelium (VNE). Their sensory neurons express odorant receptor genes and vomeronasal receptor genes, respectively, and differ fundamentally in their signal transduction pathways. Genes required for chemosensory transduction are the cyclic nucleotide-gated channel subunit Cnga2 and the transient receptor potential cation channel Trpc2, respectively. Here, we document two previously unrecognized types of Trpc2+ neurons in the MOE of mice of various ages, including adults. These cell types express Cnga2 and can be distinguished by expression of adenylate cyclase Adcy3 (positive: type A; negative: type B). A third of MOE neurons that express the odorant receptor genes Olfr68/Olfr69 coexpress Trpc2 and are type A cells. In Trpc2-IRES-taulacZ gene-targeted mice, some labeled axons coalesce into glomeruli in the main olfactory bulb. Our findings have implications for the conventional VNE-centric interpretation of the behavioral phenotypes of Trpc2 knockout mice. © 2014 The Authors.


News Article | December 2, 2016
Site: phys.org

The green objects represent the type B cells - thus the neurons that are activated by low environmental oxygen. The red objects represent  “canonical” or “conventional” olfactory sensory neurons: these cells each express one of the 1,100 odorant receptor genes in the genome, and respond to conventional odorous ligands. Credit: Max Planck Research Unit for Neurogenetics, Frankfurt am Main The genome of mice harbours more than 1000 odorant receptor genes, which enable them to smell myriad odours in their surroundings. Researchers at the Max Planck Research Unit for Neurogenetics in Frankfurt, the University of Saarland in Homburg, the University of Cambridge and the Karolinska Institute in Stockholm have discovered that mice can also sense the oxygen level of the inhaled air using neurons in their nose. For this newly discovered sensory property, mice rely on two genes termed Gucy1b2 and Trpc2, but apparently not on odorant receptor genes. The research team discovered that a specific type of chemosensory neuron in the mouse olfactory mucosa responds to oxygen decreases in the environment. Chemosensory cells typically detect an increase in the concentration of a substance. In mammals, a lack of oxygen was thought to be detected primarily by the carotid body, a sensory organ situated at the carotid arteries in the neck. Activation of the carotid body results in activation of the respiratory centre in the brain. As mice live in burrows, it appears that during evolution an additional mechanism has developed in order to protect the individuals and their offspring from a shortage of oxygen. The function of these so-called type B cells was enigmatic until this paper. "We activated these cells with low-oxygen air, and discovered a possibly vital function of these cells", says Peter Mombaerts, director of the Max Planck Research Unit for Neurogenetics. The research team investigated how type B cells behave when exposed to various oxygen levels. Using a calcium-sensitive dye, they observed that type B cells in the olfactory mucosa are activated following a modest decrease of the oxygen level in the external environment. The scientists found that the Gucyb12 and Trpc2 genes are essential for signal transduction in type B cells upon exposure to low oxygen. They used genetically altered mice in which either gene is inactivated genetically. These genes encode, respectively, an enzyme that produces the second messenger cGMP and an ion channel through which calcium enters the cells. (Calcium is also another important intracellular messenger.) Without functional Gucy1b2 and Trpc2 genes, calcium-dependent signalling pathways are not activated in type B cells, and mice cannot distinguish or respond properly to decreased oxygen levels. Thus far the molecular sensor that detects the low oxygen level remains unknown. The researchers are investigating further the signalling mechanisms that result in activation of these neurons. Moreover, the scientists found that mice can learn very quickly where locations with low oxygen levels are, and then avoid these areas. By contrast, mice with inactivated Gucy1b2 or Trpc2 genes cannot distinguish between normal and modestly decreased oxygen levels in the external environment, and do not show avoidance behaviour of these areas with a low oxygen level. These genes thus enable mice early on to select locations with an optimal oxygen level. The researchers speculate that type B cells have also a social influence in mice. For instance, mice build their nests preferably in locations with a higher oxygen level in order to protect their offspring. "The offspring needs sufficient oxygen, otherwise they would be underserved", says Frank Zufall, director at the Center for Integrative Physiology and Molecular Medicine in Homburg. Mice may be more sensitive to a lack of oxygen than humans. The human Gucy1b2 and Trpc2 genes are pseudogenes, which means that these genes may not encode proteins. It remains to be determined if type B cells occur in humans at all and if so, whether they detect low oxygen levels. Explore further: Low-oxygen environment leads to heart regeneration in mice, research shows More information: Katherin Bleymehl et al. A Sensor for Low Environmental Oxygen in the Mouse Main Olfactory Epithelium, Neuron (2016). DOI: 10.1016/j.neuron.2016.11.001


News Article | December 2, 2016
Site: www.chromatographytechniques.com

The genome of mice harbors more than 1,000 odorant receptor genes, which enable them to smell myriad odors in their surroundings. Researchers at the Max Planck Research Unit for Neurogenetics in Frankfurt, the University of Saarland in Homburg, the University of Cambridge and the Karolinska Institute in Stockholm have discovered that mice can also sense the oxygen level of the inhaled air using neurons in their nose. For this newly discovered sensory property, mice rely on two genes termed Gucy1b2 and Trpc2, but apparently not on odorant receptor genes. The research team discovered that a specific type of chemosensory neuron in the mouse olfactory mucosa responds to oxygen decreases in the environment. Chemosensory cells typically detect an increase in the concentration of a substance. In mammals, a lack of oxygen was thought to be detected primarily by the carotid body, a sensory organ situated at the carotid arteries in the neck. Activation of the carotid body results in activation of the respiratory centre in the brain. As mice live in burrows, it appears that during evolution an additional mechanism has developed in order to protect the individuals and their offspring from a shortage of oxygen. The function of these so-called type B cells was enigmatic until this paper. "We activated these cells with low-oxygen air, and discovered a possibly vital function of these cells", says Peter Mombaerts, director of the Max Planck Research Unit for Neurogenetics. The research team investigated how type B cells behave when exposed to various oxygen levels. Using a calcium-sensitive dye, they observed that type B cells in the olfactory mucosa are activated following a modest decrease of the oxygen level in the external environment. The scientists found that the Gucyb12 and Trpc2 genes are essential for signal transduction in type B cells upon exposure to low oxygen. They used genetically altered mice in which either gene is inactivated genetically. These genes encode, respectively, an enzyme that produces the second messenger cGMP and an ion channel through which calcium enters the cells. (Calcium is also another important intracellular messenger.) Without functional Gucy1b2 and Trpc2 genes, calcium-dependent signalling pathways are not activated in type B cells, and mice cannot distinguish or respond properly to decreased oxygen levels. Thus far the molecular sensor that detects the low oxygen level remains unknown. The researchers are investigating further the signalling mechanisms that result in activation of these neurons. Moreover, the scientists found that mice can learn very quickly where locations with low oxygen levels are, and then avoid these areas. By contrast, mice with inactivated Gucy1b2 or Trpc2 genes cannot distinguish between normal and modestly decreased oxygen levels in the external environment, and do not show avoidance behavior of these areas with a low oxygen level. These genes thus enable mice early on to select locations with an optimal oxygen level. The researchers speculate that type B cells have also a social influence in mice. For instance, mice build their nests preferably in locations with a higher oxygen level in order to protect their offspring. "The offspring needs sufficient oxygen, otherwise they would be underserved," says Frank Zufall, director at the Center for Integrative Physiology and Molecular Medicine in Homburg. Mice may be more sensitive to a lack of oxygen than humans. The human Gucy1b2 and Trpc2 genes are pseudogenes, which means that these genes may not encode proteins. It remains to be determined if type B cells occur in humans at all and if so, whether they detect low oxygen levels.

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