A G protein/cAMP signal cascade is required for axonal convergence into olfactory glomeruli

Department of Biological Sciences, Columbia University, New York, New York, United States
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 02/2007; 104(3):1039-44. DOI: 10.1073/pnas.0609215104
Source: PubMed


The mammalian odorant receptors (ORs) comprise a large family of G protein-coupled receptors that are critical determinants of both the odorant response profile and the axonal identity of the olfactory sensory neurons in which they are expressed. Although the pathway by which ORs activate odor transduction is well established, the mechanism by which they direct axons into proper glomerular relationships remains unknown. We have developed a gain-of-function approach by using injection of retroviral vectors into the embryonic olfactory epithelium to study the ORs' contribution to axon guidance. By ectopically expressing ORs, we demonstrate that functional OR proteins induce axonal coalescence. Furthermore, ectopic expression of Galpha mutants reveals that activation of the signal transduction cascade is sufficient to cause axonal convergence into glomeruli. Analysis of Galpha subunit expression indicates that development and odorant transduction use separate transduction pathways. Last, we establish that the generation of cAMP through adenylyl cyclase 3 is necessary to establish proper axonal identity. Our data point to a model in which axonal sorting is accomplished by OR stimulation of cAMP production by coupling to Galphas.

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    • "pCAGGS-FLAGRhoMOR256-17-iresGFP and pCAGGS-FLAGRhoM71-iresGFP were obtained by subcloning FLAGRho from pLNCX2-FLAGRhoβ2AR-iresTauGFP (provided by S. Firestein, Columbia University, NY, USA) [8] into pCAGGS-iresGFP (provided by S. Garel, ENS, Paris, France) [17], and insertion of the MOR256-17 or M71 coding sequences PCR-amplified from genomic DNA. The presence of an Internal Ribosome Entry Site (IRES) sequence enables the expression of the OR and GFP from a single mRNA. "
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    ABSTRACT: In the mouse olfactory system regulated expression of a large family of G Protein-Coupled Receptors (GPCRs), the Odorant Receptors (ORs), provides each sensory neuron with a single OR identity. In the wiring of the olfactory sensory neuron projections, a complex axon sorting process ensures the segregation of >1,000 subpopulations of axons of the same OR identity into homogeneously innervated glomeruli. ORs are critical determinants in axon sorting, and their presence on olfactory axons raises the intriguing possibility that they may participate in axonal wiring through direct or indirect trans-interactions mediating adhesion or repulsion between axons. In the present work, we used a biophysical assay to test the capacity of ORs to induce adhesion of cell doublets overexpressing these receptors. We also tested the β2 Adrenergic Receptor, a non-OR GPCR known to recapitulate the functions of ORs in olfactory axon sorting. We report here the first evidence for homo- and heterotypic adhesion between cells overexpressing the ORs MOR256-17 or M71, supporting the hypothesis that ORs may contribute to olfactory axon sorting by mediating differential adhesion between axons.
    PLoS ONE 12/2013; 8(12):e80100. DOI:10.1371/journal.pone.0080100 · 3.23 Impact Factor
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    • "Differentiation via LSD1 Downregulation The intriguing observation that Adcy3 expression is mutually exclusive with LSD1 expression and depends upon OR expression prompted us to test whether this protein plays a role in the downregulation of LSD1 and the stabilization of OR choice. Adcy3 is the main adenylyl cyclase in OSNs, and previous reports have shown that Adcy3 KO OSNs have severe targeting defects (Chesler et al., 2007; Col et al., 2007; Zou et al., 2007). A role of Adcy3 in stabilization of OR expression could account for these targeting deficits, together with activity-dependent processes that regulate axon guidance. "
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    ABSTRACT: The molecular mechanisms regulating olfactory receptor (OR) expression in the mammalian nose are not yet understood. Here, we identify the transient expression of histone demethylase LSD1 and the OR-dependent expression of adenylyl cyclase 3 (Adcy3) as requirements for initiation and stabilization of OR expression. As a transcriptional coactivator, LSD1 is necessary for desilencing and initiating OR transcription, but as a transcriptional corepressor, it is incompatible with maintenance of OR expression, and its downregulation is imperative for stable OR choice. Adcy3, a sensor of OR expression and a transmitter of an OR-elicited feedback, mediates the downregulation of LSD1 and promotes the differentiation of olfactory sensory neurons (OSNs). This novel, three-node signaling cascade locks the epigenetic state of the chosen OR, stabilizes its singular expression, and prevents the transcriptional activation of additional OR alleles for the life of the neuron.
    Cell 07/2013; 154(2):325-36. DOI:10.1016/j.cell.2013.06.039 · 32.24 Impact Factor
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    • "Knowledge about developmental regulation of βγ subunits in mammals is limited and in the olfactory system, this knowledge is missing (Norgren et al., 1995; Chesler et al., 2007). Yet, studies from other systems in invertebrate species, such as Drosophila and C.elegans have demonstrated important roles for Gβ and Gγ in development (Zwaal et al., 1996; Gotta and Ahringer, 2001; Schaefer et al., 2001; Yu et al., 2003; Izumi et al., 2004). "
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    ABSTRACT: Heterotrimeric G-proteins mediate a variety of cellular functions, including signal transduction in sensory neurons of the olfactory system. Whereas the Gα subunits in these neurons are well characterized, the gene transcript expression profile of Gβγ subunits is largely missing. Here we report our comprehensive expression analysis to identify Gβ and Gγ subunit gene transcripts in the mouse main olfactory epithelium (MOE) and the vomeronasal organ (VNO). Our reverse transcriptase PCR (RT-PCR) and realtime qPCR analyses of all known Gβ (β1,2,3,4,5) and Gγ (γ1,2,2t,3,4,5,7,8,10,11,12,13) subunits indicate presence of multiple Gβ and Gγ subunit gene transcripts in the MOE and the VNO at various expression levels. These results are supported by our RNA in situ hybridization (RISH) experiments, which reveal the expression patterns of two Gβ subunits and four Gγ subunits in the MOE as well as one Gβ and four Gγ subunits in the VNO. Using double-probe fluorescence RISH and line intensity scan analysis of the RISH signals of two dominant Gβγ subunits, we show that Gγ13 is expressed in mature olfactory sensory neurons (OSNs), while Gβ1 is present in both mature and immature OSNs. Interestingly, we also found Gβ1 to be the dominant Gβ subunit in the VNO and present throughout the sensory epithelium. In contrast, we found diverse expression of Gγ subunit gene transcripts with Gγ2, Gγ3, and Gγ13 in the Gαi2-expressing neuronal population, while Gγ8 is expressed in both layers. Further, we determined the expression of these Gβγ gene transcripts in three post-natal developmental stages (p0, 7, and 14) and found their cell-type specific expression remains largely unchanged, except the transient expression of Gγ2 in a single basal layer of cells in the MOE during P7 and P14. Taken together, our comprehensive expression analyses reveal cell-type specific gene expression of multiple Gβ and Gγ in sensory neurons of the olfactory system.
    Frontiers in Cellular Neuroscience 06/2013; 7:84. DOI:10.3389/fncel.2013.00084 · 4.29 Impact Factor
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