Odorant receptor gene choice
By nuclear transfer of OSNs that express a particular OR, we have demonstrated that OR gene choice does not involve irreversible DNA rearrangements in the genome. We have provided evidence that is inconsistent with the hypothesis of the function of a sequence called the H element as a trans-acting enhancer for OR promoters throughout the genome. We found that the promoters for the OR genes M71 and MOR23 are proximal and short, and that they contain binding sites for homeodomain proteins and O/E transcription factors. We have shown that OR genes of the so-called class II (the majority of OR genes) are dependent on the LIM-homeodomain protein Lhx2 for their expression, either directly or indirectly via maturation of the OSNs. By contrast, class I OR gene expression is independent of Lhx2.
We have developed genetic approaches in mice to visualize or manipulate a population of OSNs that express the same OR, using gene targeting in embryonic stem cells. A typical design is the co-expression of an axonal marker such as taulacZ or tauGFP with an OR gene, which is made possible at the translational level by virtue of an internal ribosome entry site. Gene-targeted mutations in the OR coding sequence, such as its replacement by the coding sequence of another OR, have collectively demonstrated that the OR is instrumental in axonal coalescence itself, and in determining the approximate site in the glomerular layer of the olfactory bulb where axonal coalescence occurs. An extensive series of gene-targeted mice led us to propose a model of homophilic interactions between ORs or OR-containing membrane complexes as a fundamental mechanism of OR-dependent coalescence of axons into glomeruli. We have recently found that there are two OSN types, which express either class I or class II ORs, and that these OSN types are determinants of axonal wiring, operating at a level higher than the ORs.
We have shown that OSNs that express the same OR respond to the same odorous ligands, and that genetic replacement of the OR coding sequence shifts the odorant response pattern accordingly. We have identified odorous ligands for several mouse ORs. We observe, however, a substantial heterogeneity in the electrophysiological responses to a given odorant among OSNs that express the same OR. We have engineered mice in which all mature OSNs express an indicator of synaptic activity, permitting us to record in vivo responses to odorants in the olfactory bulbs of anesthetized mice.
Vomeronasal receptors and pheromones
The vomeronasal organ (VNO) in the nasal cavity of the mouse is specialized in the detection of pheromones, but it is responsive to other chemosensory stimuli as well, and conversely, the main olfactory epithelium can also detect compounds with pheromonal effects. The vomeronasal sensory neurons (VSNs) of the VNO express G-protein coupled receptors of two other large gene families, which are unrelated to ORs: the V1Rs and V2Rs. With our genetic approach we have demonstrated that axons of VSNs that express a given V1R or V2R coalesce into multiple, smaller glomeruli in the accessory olfactory bulb. We have shown that a given VR gene is expressed from one allele in an individual VSN. By generating mice that lack a cluster of 16 V1R genes via chromosome engineering, we have provided in vivo evidence for a role of V1Rs in pheromone behaviors. We have discovered a family of nine non-classical class I Major histocompatibility genes, H2-Mv genes. These genes are expressed only in the VNO, specifically in VSNs that express V2R genes, and in particular combinatorial patterns. Their function and regulation remain elusive.
Cloning mice by nuclear transfer
We have reported the cloning of mice by nuclear transfer from embryonic stem cells. Conversely, we have generated embryonic stem cell lines from blastocysts produced by nuclear transfer. We have shown that OR gene choice is reset upon nuclear transfer with OSNs. We have cloned mice from adult skin cells. We have demonstrated the feasibility of nuclear transfer with somatic cells that were frozen for prolonged periods of time without cryoprotectant.
Peter Mombaerts, Direktor
Abteilung für Molekulare Neurogenetik
Tel.: +49 (0) 69 6303-4000
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