Staffan Bohm

Evidence for gradients of gene expression correlating with zonal topography of the olfactory sensory map

Signals regulating diversification of olfactory sensory neurons to express odorant receptors and other genes necessary for correct assembly of the olfactory sensory map persist in the olfactory epithelium of adult mouse. We have screened for genes with an expression pattern correlating with the topography odorant receptor-expression zones. The Msx1 homeobox gene and a semaphorin receptor (Neuropilin-2) showed graded expression patterns in the olfactory epithelium. The gradients of Msx1 and Neuropilin-2 expression in basal cells and neurons, respectively, correlated with expression of a retinoic acid-synthesizing enzyme (RALDH2) in lamina propria. A BMP-type I receptor (Alk6) showed a reverse gradient of expression in the supporting cells of the epithelium. Considering known functions of identified genes in cell specification and axon guidance this suggests that zonal division of the olfactory sensory map is maintained, during continuous neurogenesis, as a consequence of topographic counter gradients of positional information.

Identification of a Novel Neural Cell Adhesion Molecule-related Gene with a Potential Role in Selective Axonal Projection

We describe here the cloning of mouse complementary DNAs encoding a novel protein, Rb-8 neural cell adhesion molecule (RNCAM), with a predicted extracellular region of five immunoglobulin C2-type domains followed by two fibronectin type III domains. Alternative splicing is likely to generate two RNCAM isoforms, which are differently attached to the cell membrane. These structural features and overall sequence identity identify this protein as a novel member of a cell adhesion molecule subgroup together with vertebrate neural cell adhesion molecule, Aplysia cell adhesion molecule, and Drosophila fasciclin II. In insects, fasciclin II is present on a restricted subset of embryonic central nervous system axons where it controls selective axon fasciculation. Intriguingly, RNCAM likewise is expressed in subsets of olfactory and vomeronasal neurons with topographically defined axonal projections. The spatial expression RNCAM corresponds precisely to that of certain odorant receptor expression zones of the olfactory epithelium. These expression patterns thus render RNCAM the first described cell adhesion molecule with a potential regulatory role in formation of selective axonal projections important for olfactory sensory information coding.

The Lim homeobox gene Lhx2 is required for olfactory sensory neuron identity.

Progenitor cells in the mouse olfactory epithelium generate over a thousand subpopulations of neurons, each expressing a unique odorant receptor (OR) gene. This event is under the control of spatial cues, since neurons in different epithelial regions are restricted to express region-specific subsets of OR genes. We show that progenitors and neurons express the LIM-homeobox gene Lhx2 and that neurons in Lhx2-null mutant embryos do not diversify into subpopulations expressing different OR genes and other region-restricted genes such as Nqo1 and Ncam2. Lhx2-/- embryos have, however, a normal distribution of Mash1-positive and neurogenin 1-positive neuronal progenitors that leave the cell cycle, acquire pan-neuronal traits and form axon bundles. Increased cell death in combination with increased expression of the early differentiation marker Neurod1, as well as reduced expression of late differentiation markers (Gαolf and Omp), suggests that neuronal differentiation in the absence of Lhx2 is primarily inhibited at, or immediate prior to, onset of OR expression. Aberrant regional expression of early and late differentiation markers, taken together with unaltered region-restricted expression of the Msx1 homeobox gene in the progenitor cell layer of Lhx2-/- embryos, shows that Lhx2 function is not required for all aspects of regional specification of progenitors and neurons. Thus, these results indicate that a cell-autonomous function of Lhx2 is required for differentiation of progenitors into a heterogeneous population of individually and regionally specified mature olfactory sensory neurons.

NQO1 activity in the main and the accessory olfactory systems correlates with the zonal topography of projection maps

The mouse olfactory epithelium (OE) is divided into spatial zones, each containing neurons expressing zone‐specific subsets of odorant receptor genes. Likewise, the vomeronasal (VN) organ is organized into apical and basal subpopulations of neurons expressing different VN receptor gene families. Axons projecting from the different OE zones and VN subpopulations form synapses within circumscribed regions in the glomerular layer of the olfactory bulb (OB) and accessory olfactory bulb (AOB), respectively. We here show that mature neurons in one defined zone selectively express NADPH:quinone oxidoreductase (NQO1), an enzyme that catalyses reduction of quinones. Immunohistochemistry and in situ hybridization analyses show non‐overlapping expression of NQO1 and the Rb8 neural cell adhesion molecule (RNCAM/OCAM) in OE and axon terminals within glomeruli of the OB. In addition, NQO1 immunoreactivity reveals selective, zone‐specific axon fasciculation in the olfactory nerve. VN subpopulations do not show complementary patterns of RNCAM and NQO1 immunoreactivity, instead both genes are co‐expressed in apical VN neurons that project to the rostral AOB. These results indicate that one division of both the accessory and the main olfactory projection maps are composed of sensory neurons that are specialized to reduce environmental and/or endogenously produced quinones via an NQO1‐dependent mechanism. The role of NQO1 in bioactivation of quinoidal drugs also points to a connection between zone‐specific NQO1 expression and zone‐specific toxicity of certain olfactory toxins.

Differential function of RNCAM isoforms in precise target selection of olfactory sensory neurons

Olfactory sensory neurons (OSNs) are individually specified to express one odorant receptor (OR) gene among ∼1000 different and project with precision to topographically defined convergence sites, the glomeruli, in the olfactory bulb. Although ORs partially determine the location of convergence sites, the mechanism ensuring that axons with different OR identities do not co-converge is unknown. RNCAM (OCAM, NCAM2) is assumed to regulate a broad zonal segregation of projections by virtue of being a homophilic cell adhesion molecule that is selectively expressed on axons terminating in a defined olfactory bulb region. We have identified NADPH diaphorase activity as being an independent marker for RNCAM-negative axons. Analyses of transgenic mice that ectopically express RNCAM in NADPH diaphorase-positive OSNs show that the postulated function of RNCAM in mediating zone-specific segregation of axons is unlikely. Instead, analyses of one OR-specific OSN subpopulation (P2) reveal that elevated RNCAM levels result in an increased number of P2 axons that incorrectly co-converge with axons of other OR identities. Both Gpi-anchored and transmembrane-bound RNCAM isoforms are localized on axons in the nerve layer, while the transmembrane-bound RNCAM is the predominant isoform on axon terminals within glomeruli. Overexpressing transmembrane-bound RNCAM results in co-convergence events close to the correct target glomeruli. By contrast, overexpression of Gpi-anchored RNCAM results in axons that can bypass the correct target before co-converging on glomeruli located at a distance. The phenotype specific for Gpi-anchored RNCAM is suppressed in mice overexpressing both isoforms, which suggests that two distinct RNCAM isoform-dependent activities influence segregation of OR-defined axon subclasses.

Olfactory receptors in the mouse septal organ

In this study we have identified a repertoire of chemosensory receptors expressed in the septal organ (SO). The results suggest that septal organ neurons are specified to express receptor genes belonging to class II olfactory receptors that are also expressed in the main olfactory epithelium. We found no evidence for the expression of members from the vomeronasal receptor gene families. In the SO, no topography analogous to the receptor expression zones of the main olfactory epithelium was evident. The majority of identified receptors corresponds to genes with restricted expression in the medial and lateral zones of the main olfactory epithelium. This coincides with the expression of olfactory cell adhesion molecule (OCAM) throughout the SO, which is considered as a marker for the medial‐lateral zones. In contrast, NADPH:quinone oxidoreductase 1 expression, a characteristic marker for the dorsal zone, was lacking in the SO. Most of the receptor types were found to be expressed in rather few SO neurons; as an exception, the receptor mOR244‐3 was observed in a very high proportion of cells. Although a very high fraction of SO neurons expressed mOR244‐3, we found no evidence for the coexpression of different receptors in individual cells.

Retinoic Acid Receptor-Dependent Survival of Olfactory Sensory Neurons in Postnatal and Adult Mice

To address the hypothesis that retinoids produced by synthesizing enzymes present in the primary olfactory system influence the mouse olfactory sensory map, we expressed a dominant-negative retinoic acid receptor selectively in olfactory sensory neurons. We show that neurons deficient in nuclear retinoid signaling are responsive to odors and form correct odorant receptor-specific axonal projections to target neurons in the olfactory bulb of the brain. Subsequent to the formation of the map, the neurons die prematurely by retrograde-driven caspase-3 activation, which resembles the previously described mechanism of neural death after olfactory bulb ablation. This neurodegenerative event is initiated the second postnatal week and occurs in the adult animal without a compensatory increase of progenitor cell proliferation. In addition, we find that nuclear retinoid signaling is required for the expression of a retinoic acid-degrading enzyme, Cyp26B1, in a small fraction of mature neurons. Collectively, the results provide evidence for a role of locally regulated retinoid metabolism in neuroprotection and in determining population size of neurons at a late stage of neural circuit formation.

Regional differences in olfactory epithelial homeostasis in the adult mouse

The olfactory sensory neurons in the nasal cavity of the adult mouse are organized into a few regions that differ in their molecular properties, as several classes of genes show regional expression. Most renowned is the fact that expression of each of hundreds of different odorant receptor genes is limited to one such region, or zone, of the olfactory neuroepithelial sheet. Zone differences are in place at birth, as exemplified here by the expression of neuronal progenitor marker Foxg1. We herein describe that an adult pattern showing regional differences in neurogenesis develops during the first few weeks of postnatal life which, e.g., is reflected in the temporal and regional regulation of the neuronal progenitor marker Ascl1. The most dorsomedial zone shows significantly fewer cells in S‐phase in the adult but not in newborn mice by two different measures. Moreover, we show that there are regional differences in the relative differentiation, cell survival, and thickness of the olfactory epithelium. These findings are compatible with the view that zones are inherently distinct and that such differences contribute to generate regional differences in cellular homeostasis that in turn may modulate the capacity of a region to adjust to extrinsic influence. J. Comp. Neurol. 513:375–384, 2009.

Lhx2-dependent specification of olfactory sensory neurons is required for successful integration of olfactory, vomeronasal, and GnRH neurons

Inactivation of the LIM‐homeodomain 2 gene (Lhx2) results in a severe defect in specification of olfactory sensory neurons (OSNs). However, the ramifications of lack of Lhx2‐dependent OSN specification for formation of the primary olfactory pathway have not been addressed, since mutant mice die in utero. We have analyzed prenatal and postnatal consequences of conditionally inactivating Lhx2 selectively in OSNs. A cell‐autonomous effect is that OSN axons cannot innervate their target, the olfactory bulb. Moreover, the lack of Lhx2 in OSNs causes unpredicted, non‐cell‐autonomous phenotypes. First, the olfactory bulb shows pronounced hypoplasia in adults, and the data suggest that innervation by correctly specified OSNs is necessary for adult bulb size and organization. Second, absence of an olfactory nerve in the conditional mutant reveals that the vomeronasal nerve is dependent on olfactory nerve formation. Third, the lack of a proper vomeronasal nerve prevents migration of gonadotropin‐releasing hormone (GnRH) cells the whole distance to their final positions in the hypothalamus during embryo development. As adults, the conditional mutants do not pass puberty, and these findings support the view of an exclusive nasal origin of GnRH neurons in the mouse. Thus, Lhx2 in OSNs is required for functional development of three separate systems.—Berghard, A., Hägglund, A.‐C., Bohm, S., and Carlsson, L. Lhx2‐dependent specification of olfactory sensory neurons is required for successful integration of olfactory, vomeronasal, and GnRH neurons. FASEB J. 26, 3464–3472 (2012). www.fasebj.org

Odorant-dependent, spatially restricted induction of c-fos in the olfactory epithelium of the mouse

Volatile odorous chemicals are detected by around a thousand different G protein‐coupled odorant receptors in the mouse. We demonstrated that exposure of the behaving mouse to odorant for a few minutes led to induction of the immediate early gene c‐fos for several hours in a fraction of the olfactory sensory neurones in the nasal cavity. Associated with this odorant‐specific induction event was activation of extracellular‐regulated kinase (ERK)1/2 that preceded increased c‐fos expression. The distribution of odorant‐activated neurones mimicked the scattered and spatially limited distribution of neurones expressing a single odorant receptor gene. A small change in odorant chemical structure caused a zonal shift in the spatial distribution of activated neurones, suggesting that the gene expression change resulted from specific receptor interaction. Repeated exposure to odorant or use of different concentrations did not change the pattern of c‐fos induction. These results indicate that odorant‐induced c‐fos expression can be used to visualize odorant representations in the olfactory epithelium that reflect late cellular events regulated by adequate odorant receptor stimulation.