Journal of Neurocytology

, Volume 33, Issue 6, pp 657–669 | Cite as

Protocols for two- and three-color fluorescent RNA in situ hybridization of the main and accessory olfactory epithelia in mouse

Article

Abstract

The main and accessory olfactory epithelia of the mouse are composed of many cell populations. Each sensory neuron is thought to express one allele of one of the ∼1000 odorant or ∼300 vomeronasal receptor genes. Sensory neurons die and are replaced by new neurons that differentiate from precursor cells throughout the lifetime of the individual. Neuronal replacement is asynchronous, resulting in the co-existence of cells at various stages of differentiation. Receptor gene diversity and ongoing neuronal differentiation produce complex mosaics of gene expression within these epithelia. Accurate description of gene expression patterns will facilitate the understanding of mechanisms of gene choice and differentiation. Here we report a detailed protocol for two- and three-color fluorescent RNA in situ hybridization (ISH) and its combination with immunohistochemistry, or detection of bromodeoxyuridine (BrdU)-incorporated DNA after labeling. The protocol is applied to cryosections of the main and accessory olfactory epithelia in mouse.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. ALENIUS, M. & BOHM, S. (1997) Identification of a novel neural cell adhesion molecule-related gene with a potential role in selective axonal projection. Journal of Biological Chemistry 272, 26083–26086.CrossRefPubMedGoogle Scholar
  2. BERGHARD, A. & BUCK, L. B. (1996) Sensory transduction in vomeronasal neurons: Evidence for Gαo, Gαi2, and adenylyl cyclase II as major components of a pheromone signaling cascade. The Journal of Neuroscience 16, 909– 918.PubMedGoogle Scholar
  3. BUCK, L. & AXEL, R. (1991) A novel multigene family may encode odorant receptors: A molecular basis for odor recognition. Cell 65, 175–187.CrossRefPubMedGoogle Scholar
  4. CARR, V. MCM. & FARBMAN, A. I. (1992) Ablation of the olfactory bulb up-regulates the rate of neurogenesis and induces precocious cell death in olfactory epithelium. Experimental Neurology 115, 55–59.CrossRefPubMedGoogle Scholar
  5. CAU, E., CASAROSA, S. & GUILLEMOT, F. (2002) Mash1 and Ngn1 control distinct steps of determination and differentiation in the olfactory sensory neuron lineage. Development 129, 1871–1880.PubMedGoogle Scholar
  6. CHERRY, J. A. & PHO, V. (2002) Characterization of cAMP degradation by phosphodiesterases in the accessory olfactory system. Chemical Senses 27, 643–652.CrossRefPubMedGoogle Scholar
  7. CHESS, A., SIMON, I., CEDAR, H. & AXEL, R. (1994) Allelic inactivation regulates olfactory receptor gene expression. Cell 78, 823–834.CrossRefPubMedGoogle Scholar
  8. CLOUTIER, J. F., GIGER, R. J., KOENTGES, G., DULAC, C., KOLODKIN, A. L. & GINTY, D. D. (2002) Neuropilin-2 mediates axonal fasciculation, zonal segregation, but not axonal convergence, of primary accessory olfactory neurons. Neuron 33, 877–892.CrossRefPubMedGoogle Scholar
  9. CLOUTIER, J. F., SAHAY, A., CHANG, E. C., TESSIER-LAVIGNE, M., DULAC, C., KOLODKIN, A. L. & GINTY, D. D. (2004) Differential requirements for semaphorin 3F and Slit-1 in axonal targeting, fasciculation, and segregation of olfactory sensory neuron projections. The Journal of Neuroscience 24, 9087–9096.PubMedGoogle Scholar
  10. DEL PUNTA, K., PUCHE, A., ADAMS, N.C., RODRIGUEZ, I. & MOMBAERTS, P. (2002) A divergent pattern of sensory axonal projections is rendered convergent by second-order neurons in the accessory olfactory bulb. Neuron 35, 1057–1066.PubMedGoogle Scholar
  11. DULAC, C. & AXEL, R. (1995) A novel family of genes encoding putative pheromone receptors in mammals. Cell 83, 195–206.CrossRefPubMedGoogle Scholar
  12. ESUMI, S., KAKAZU, N., TAGUCHI, Y., HIRAYAMA, T., SASAKI, A., HIRABAYASHI, T., KOIDE, T., KITSUKAWA, T., HAMADA, S. & YAGI, T. (2005) Monoallelic yet combinatorial expression of variable exons of the protocadherin-α gene cluster in single neurons. Nature Genetics 37, 171–176.CrossRefPubMedGoogle Scholar
  13. FARBMAN, A. I. (1990) Olfactory neurogenesis: Genetic or environmental controls? Trends in Neurosciences 13, 362–365.CrossRefPubMedGoogle Scholar
  14. FARBMAN, A. I. (1992) Cell Biology of Olfaction (Developmental and Cell Biology Series) Cambridge: Cambridge University Press.Google Scholar
  15. GIACOBINI, P., BENEDETTO, A., TIRINDELLI, R. & FASOLO, A. (2000) Proliferation and migration of receptor neurons in the vomeronasal organ of the adult mouse. Developmental Brain Research 123, 33– 40.CrossRefPubMedGoogle Scholar
  16. GUILLEMOT, F., LO, L. C., JOHNSON, J. E., AUERBACH, A., ANDERSON, D. J. & JOYNER, A. L. (1993) Mammalian achaete-scute homolog 1 is required for the early development of olfactory and autonomic neurons. Cell 75, 463–476.CrossRefPubMedGoogle Scholar
  17. GUSSING, F. & BOHM, S. (2004) NQO1 activity in the main and the accessory olfactory systems correlates with the zonal topography of projection maps. European Journal of Neuroscience 19, 2511–2518.CrossRefPubMedGoogle Scholar
  18. HALPERN, M. & MARTINEZ-MARCOS, A. (2003) Structure and function of the vomeronasal system: An update. Progress in Neurobiology 70, 245–318.CrossRefPubMedGoogle Scholar
  19. HAUPTMANN, G. (2001) One-, two-, and three-color whole-mount in situ hybridization to Drosophila embryos. Methods 23, 359–372.CrossRefPubMedGoogle Scholar
  20. HERRADA, G. & DULAC, C. (1997) A novel family of putative pheromone receptors in mammals with a topographically organized and sexually dimorphic distribution. Cell 90, 763–773.CrossRefPubMedGoogle Scholar
  21. HIROTA, J. & MOMBAERTS, P. (2004) The LIM-homeodomain protein Lhx2 is required for complete development of mouse olfactory sensory neurons. Proceedings of the National Academy of Sciences USA 101, 8751–8755.Google Scholar
  22. HIROTA, S., ITO, A., MORII, E., WANAKA, A., TOHYAMA, M., KITAMURA, Y. & NOMURA, S. (1992) Localization of mRNA for c-kit receptor and its ligand in the brain of adult rats: An analysis using in situ hybridization histochemistry. Molecular Brain Research 15, 47–54.CrossRefPubMedGoogle Scholar
  23. HUARD, J. M., YOUNGENTOB, S. L., GOLDSTEIN, B. J., LUSKIN, M. B. & SCHWOB, J. E. (1998) Adult olfactory epithelium contains multipotent progenitors that give rise to neurons and non-neural cells. The Journal of Comparative Neurology 400, 469–486.CrossRefPubMedGoogle Scholar
  24. ISHII, T., HIROTA, J. & MOMBAERTS, P. (2003) Combinatorial coexpression of neural and immune multigene families in mouse vomeronasal sensory neurons. Current Biology 13, 394–400.CrossRefPubMedGoogle Scholar
  25. ISHII, T., SERIZAWA, S., KOHDA, A., NAKATANI, H., SHIROISHI, T., OKUMURA, K., IWAKURA, Y., NAGAWA, F., TSUBOI, A. & SAKANO, H. (2001) Monoallelic expression of the odourant receptor gene and axonal projection of olfactory sensory neurons. Genes to Cells 6, 71–78.CrossRefPubMedGoogle Scholar
  26. ISHII, Y., NAKAMURA, S. & OSUMI, N. (2000) Demarcation of early mammalian cortical development by differential expression of fringe genes. Developmental Brain Research 119, 307–320.CrossRefPubMedGoogle Scholar
  27. JIA, C. & HALPERN, M. (1996) Subclasses of vomeronasal receptor neurons: Differential expression of G proteins (Gia2 and Goa) and segregated projections to the accessory olfactory bulb. Brain Research 719, 117–128.CrossRefPubMedGoogle Scholar
  28. KALUZA, J. F., GUSSING, F., BOHM, S., BREER, H. & STROTMANN, J. (2004) Olfactory receptors in the mouse septal organ. Journal of Neuroscience Research 76, 442–452.CrossRefPubMedGoogle Scholar
  29. KEVERNE, E. B. (1999) The vomeronasal organ. Science 286, 716–720.CrossRefPubMedGoogle Scholar
  30. KNOLL, B., SCHMIDT, H., ANDREWS, W., GUTHRIE, S., PINI, A., SUNDARESAN, V. & DRESCHER, U. (2003) On the topographic targeting of basal vomeronasal axons through Slit-mediated chemorepulsion. Development 130, 5073–5082.CrossRefPubMedGoogle Scholar
  31. KNOLL, B., ZARBALIS, K., WURST, W. & DRESCHER, U. (2001) A role for the EphA family in the topographic targeting of vomeronasal axons. Development 128, 895–906.PubMedGoogle Scholar
  32. KOHMURA, N., SENZAKI, K., HAMADA, S., KAI, N., YASUDA, R., WATANABE, M., ISHII, H., YASUDA, M., MISHINA, M. & YAGI, T. (1998) Diversity revealed by a novel family of cadherins expressed in neurons at a synaptic complex. Neuron 20, 1137–1151.CrossRefPubMedGoogle Scholar
  33. KOSMAN, D., MIZUTANI, C. M., LEMONS, D., COX, W. G., MCGINNIS, W. & BIER, E. (2004) Multiplex detection of RNA expression in Drosophila embryos. Science 305, 846.CrossRefPubMedGoogle Scholar
  34. LAU, Y. E. & CHERRY, J. A. (2000) Distribution of PDE4A and Goα immunoreactivity in the accessory olfactory system of the mouse. Neuroreport 11, 27–32.PubMedGoogle Scholar
  35. LEVSKY, J. M., SHENOY, S. M., PEZO, R. C. & SINGER, R. H. (2002) Single-cell gene expression profiling. Science 297, 836–840.CrossRefPubMedGoogle Scholar
  36. LI, J., ISHII, T., FEINSTEIN, P. & MOMBAERTS, P. (2004) Odorant receptor gene choice is reset by nuclear transfer from mouse olfactory sensory neurons. Nature 428, 393–399.CrossRefPubMedGoogle Scholar
  37. LOCONTO, J., PAPES, F., CHANG, E., STOWERS, L., JONES, E. P., TAKADA, T., KUMANOVICS, A., FISCHER LINDAHL, K. & DULAC, C. (2003) Functional expression of murine V2R pheromone receptors involves selective association with the M10 and M1 families of MHC class Ib molecules. Cell 112, 607–618.CrossRefPubMedGoogle Scholar
  38. MA, Q., KINTNER, C. & ANDERSON, D. J. (1996) Identification of neurogenin, a vertebrate neuronal determination gene. Cell 87, 43–52.CrossRefPubMedGoogle Scholar
  39. MARTINEZ-MARCOS, A., UBEDA-BANON, I., DENG, L. & HALPERN, M. (2000) Neurogenesis in the vomeronasal epithelium of adult rats: Evidence for different mechanisms for growth and neuronal turnover. Journal of Neurobiology 44, 423–435.PubMedGoogle Scholar
  40. MARTINI, S., SILVOTTI, L., SHIRAZI, A., RYBA, N. J. & TIRINDELLI, R. (2001) Co-expression of putative pheromone receptors in the sensory neurons of the vomeronasal organ. The Journal of Neuroscience 21, 843–848.PubMedGoogle Scholar
  41. MATARAZZO, V., COHEN, D., PALMER, A. M., SIMPSON, P. J., KHOKHAR, B., PAN, S. J. & RONNETT, G. V. (2004) The transcriptional repressor Mecp2 regulates terminal neuronal differentiation. Molecular and Cellular Neuroscience 27, 44–58.CrossRefPubMedGoogle Scholar
  42. MATSUNAMI, H. & BUCK, L. B. (1997) A multigene family encoding a diverse array of putative pheromone receptors in mammals. Cell 90, 775–784.CrossRefPubMedGoogle Scholar
  43. MOMBAERTS, P. (2004a) Genes and ligands for odorant, vomeronasal and taste receptors. Nature Reviews Neuroscience 5, 263–278.CrossRefGoogle Scholar
  44. MOMBAERTS, P. (2004b) Odorant receptor gene choice in olfactory sensory neurons: The one receptor-one neuron hypothesis revisited. Current Opinion in Neurobiology 14, 31–36.CrossRefGoogle Scholar
  45. MONTMAYEUR, J. P., LIBERLES, S. D., MATSUNAMI, H. & BUCK, L. B. (2001) A candidate taste receptor gene near a sweet taste locus. Nature Neuroscience 4, 492–498.PubMedGoogle Scholar
  46. NORLIN, E. M. & BERGHARD, A. (2001) Spatially restricted expression of regulators of G-protein signaling in primary olfactory neurons. Molecular and Cellular Neuroscience 17, 872–882.CrossRefPubMedGoogle Scholar
  47. OKA, Y., KOBAYAKAWA, K., NISHIZUMI, H., MIYAMICHI, K., HIROSE, S., TSUBOI, A. & SAKANO, H. (2003) O-MACS, a novel member of the medium-chain acyl-CoA synthetase family, specifically expressed in the olfactory epithelium in a zone-specific manner. European Journal of Biochemistry 270, 1995–2004.CrossRefPubMedGoogle Scholar
  48. PANTAGES, E. & DULAC, C. (2000) A novel family of candidate pheromone receptors in mammals. Neuron 28, 835–845.CrossRefPubMedGoogle Scholar
  49. PARATORE, C., SUTER, U. & SOMMER, L. (1999) Embryonic gene expression resolved at the cellular level by fluorescence in situ hybridization. Histochemistry and Cell Biology 111, 435–443.CrossRefPubMedGoogle Scholar
  50. RAWSON, N. E., EBERWINE, J., DOTSON, R., JACKSON, J., ULRICH, P. & RESTREPO, D. (2000) Expression of mRNAs encoding for two different olfactory receptors in a subset of olfactory receptor neurons. Journal of Neurochemistry 75, 185–195.CrossRefPubMedGoogle Scholar
  51. RODRIGUEZ, I., FEINSTEIN, P. & MOMBAERTS, P. (1999) Variable patterns of axonal projections of sensory neurons in the mouse vomeronasal system. Cell 97, 199–208.CrossRefPubMedGoogle Scholar
  52. ROTHMAN, A., FEINSTEIN, P., HIROTA, J. & MOMBAERTS, P. (2005) The promoter of the mouse odorant receptor gene M71. Molecular and Cellular Neuroscience 28, 535–546.CrossRefPubMedGoogle Scholar
  53. RUNNENBURGER, K., BREER, H. & BOEKHOFF, I. (2002) Selective G protein β-subunit compositions mediate phospholipase C activation in the vomeronasal organ. European Journal of Cell Biology 81, 539–547.CrossRefPubMedGoogle Scholar
  54. RYBA, N. J. & TIRINDELLI, R. (1997) A new multigene family of putative pheromone receptors. Neuron 19, 371–379.CrossRefPubMedGoogle Scholar
  55. SCHWARZENBACHER, K., FLEISCHER, J., BREER, H. & CONZELMANN, S. (2004) Expression of olfactory receptors in the cribriform mesenchyme during prenatal development. Gene Expression Patterns 4, 543–552.CrossRefPubMedGoogle Scholar
  56. SCHWOB, J. E. (2002) Neural regeneration and the peripheral olfactory system. The Anatomical Record 269, 33–49.CrossRefPubMedGoogle Scholar
  57. SERIZAWA, S., MIYAMICHI, K., NAKATANI, H., SUZUKI, M., SAITO, M., YOSHIHARA, Y. & SAKANO, H. (2003) Negative feedback regulation ensures the one receptor-one olfactory neuron rule in mouse. Science 302, 2088–2094.CrossRefPubMedGoogle Scholar
  58. SHYKIND, B. M., ROHANI, S. C., O'DONNELL, S., NEMES, A., MENDELSOHN, M., SUN, Y., AXEL, R. & BARNEA, G. (2004) Gene switching and the stability of odorant receptor gene choice. Cell 117, 801– 815.CrossRefPubMedGoogle Scholar
  59. STROTMANN, J., CONZELMANN, S., BECK, A., FEINSTEIN, P., BREER, H. & MOMBAERTS, P. (2000) Local permutations in the glomerular array of the mouse olfactory bulb. The Journal of Neuroscience 20, 6927–6938.PubMedGoogle Scholar
  60. TIAN, H. & MA, M. (2004) Molecular organization of the olfactory septal organ. The Journal of Neuroscience 24, 8383–8390.PubMedGoogle Scholar
  61. TSUBOI, A., YOSHIHARA, S., YAMAZAKI, N., KASAI, H., ASAI-TSUBOI, H., KOMATSU, M., SERIZAWA, S., ISHII, T., MATSUDA, Y., NAGAWA, F. & SAKANO, H. (1999) Olfactory neurons expressing closely linked and homologous odorant receptor genes tend to project their axons to neighboring glomeruli on the olfactory bulb. The Journal of Neuroscience 19, 8409–8418.PubMedGoogle Scholar
  62. WALZ, A., RODRIGUEZ, I. & MOMBAERTS, P. (2002) Aberrant sensory innervation of the olfactory bulb in neuropilin-2 mutant mice. The Journal of Neuroscience 22, 4025–4035.PubMedGoogle Scholar
  63. WEILER, E. & FARBMAN, A. I. (1997) Proliferation in the rat olfactory epithelium: Age-dependent changes. The Journal of Neuroscience 17, 3610–3622.PubMedGoogle Scholar
  64. WEILER, E., MCCULLOCH, M. A. & FARBMAN, A. I. (1999) Proliferation in the vomeronasal organ of the rat during postnatal development. European Journal of Neuroscience 11, 700–711.PubMedGoogle Scholar
  65. WILKIE, G. S. & DAVIS, I. (1998) Visualizing mRNA by in situ hybridization using high resolution and sensitive tyramide signal amplification. Elsevier Trends Journals, Technical Tips Online:T01458.Google Scholar
  66. YOSHIHARA, Y., KAWASAKI, M., TAMADA, A., FUJITA, H., HAYASHI, H., KAGAMIYAMA, H. & MORI, K. (1997) OCAM: A new member of the neural cell adhesion molecule family related to zone-to-zone projection of olfactory and vomeronasal axons. The Journal of Neuroscience 17, 5830–5842.PubMedGoogle Scholar
  67. YU, T. T., MCINTYRE, J. C., BOSE, S. C., HARDIN, D., OWEN, M. C. & MCCLINTOCK, T. S. (2005) Differentially expressed transcripts from phenotypically identified olfactory sensory neurons. The Journal of Comparative Neurology 483, 251–262.PubMedGoogle Scholar
  68. ZAIDI, A. U., ENOMOTO, H., MILBRANDT, J. & ROTH, K. A. (2000) Dual fluorescent in situ hybridization and immunohistochemical detection with tyramide signal amplification. Journal of Histochemistry & Cytochemistry 48, 1369–1375.Google Scholar
  69. ZHANG, X., RODRIGUEZ, I., MOMBAERTS, P. & FIRESTEIN, S. (2004) Odorant and vomeronasal receptor genes in two mouse genome assemblies. Genomics 83, 802–811.PubMedGoogle Scholar

Copyright information

© Springer Science + Business Media, Inc. 2005

Authors and Affiliations

  1. 1.The Rockefeller UniversityNew YorkUSA

Personalised recommendations