{beta}3GnT2 Maintains Adenylyl Cyclase-3 Signaling and Axon Guidance Molecule Expression in the Olfactory Epithelium

In the olfactory epithelium (OE), odorant receptor stimulation generates cAMP signals that function in both odor detection and the regulation of axon guidance molecule expression. The enzyme that synthesizes cAMP, adenylyl cyclase 3 (AC3), is coexpressed in olfactory sensory neurons (OSNs) with poly-N-acetyllactosamine (PLN) oligosaccharides determined by the glycosyltransferase beta3GnT2. The loss of either enzyme results in similar defects in olfactory bulb (OB) innervation and OSN survival, suggesting that glycosylation may be important for AC3 function. We show here that AC3 is extensively modified with N-linked PLN, which is essential for AC3 activity and localization. On Western blots, AC3 from the wild-type OE migrates diffusely as a heavily glycosylated 200 kDa band that interacts with the PLN-binding lectin LEA. AC3 from the beta3GnT2(-/-) OE loses these PLN modifications, migrating instead as a 140 kDa glycoprotein. Furthermore, basal and forskolin-stimulated cAMP production is reduced 80-90% in the beta3GnT2(-/-) OE. Although AC3 traffics normally to null OSN cilia, it is absent from axon projections that aberrantly target the OB. The cAMP-dependent guidance receptor neuropilin-1 is also lost from beta3GnT2(-/-) OSNs and axons, while semaphorin-3A ligand expression is upregulated. In addition, kirrel2, a mosaically expressed adhesion molecule that functions in axon sorting, is absent from beta3GnT2(-/-) OB projections. These results demonstrate that PLN glycans are essential in OSNs for proper AC3 localization and function. We propose that the loss of cAMP-dependent guidance cues is also a critical factor in the severe axon guidance defects observed in beta3GnT2(-/-) mice

Stromal cell-derived factor-1 (chemokine C-X-C motif ligand 12) and chemokine C-X-C motif receptor 4 are required for migration of gonadotropin-releasing hormone neurons to the forebrain

Gonadotropin-releasing hormone (GnRH) neurons migrate from the vomeronasal organ (VNO) in the nasal compartment to the basal forebrain in mice, beginning on embryonic day 11 (E11). These neurons use vomeronasal axons as guides to migrate through the nasal mesenchyme. Most GnRH neurons then migrate along the caudal branch of the vomeronasal nerve to reach the hypothalamus. We show here that stromal cell-derived factor-1 [SDF-1, also known as chemokine C-X-C motif ligand 12 (CXCL12)] is expressed in the embryonic nasal mesenchyme from as early as E10 in an increasing rostral to caudal gradient that is most intense at the border of the nasal mesenchyme and the telencephalon. Chemokine C-X-C motif receptor 4 (CXCR4), the receptor for SDF-1, is expressed by neurons in the olfactory epithelium and VNO. Cells derived from these sensory epithelia, including migrating GnRH neurons and ensheathing glial precursors of the migrating mass (MM), also express CXCR4, suggesting that they may use SDF-1 as a chemokine. In support of this, most GnRH neurons of CXCR4-/- mice fail to exit the VNO at E13, and comparatively few GnRH neurons reach the forebrain. There is also a significant decrease in the total number of GnRH neurons in CXCR4-/- mice and an increase in cell death within the VNO relative to controls. The MM is smaller in CXCR4-/- mice, suggesting that some MM cells also require SDF-1/CXCR4 function for migration and survival

Beta1,3-N-acetylglucosaminyltransferase 1 glycosylation is required for axon pathfinding by olfactory sensory neurons

During embryonic development, axons from sensory neurons in the olfactory epithelium (OE) extend into the olfactory bulb (OB) where they synapse with projection neurons and form glomerular structures. To determine whether glycans play a role in these processes, we analyzed mice deficient for the glycosyltransferase beta1,3-N-acetylglucosaminyltransferase 1 (beta3GnT1), a key enzyme in lactosamine glycan synthesis. Terminal lactosamine expression, as shown by immunoreactivity with the monoclonal antibody 1B2, is dramatically reduced in the neonatal null OE. Postnatal beta3GnT1-/- mice exhibit severely disorganized OB innervation and defective glomerular formation. Beginning in embryonic development, specific subsets of odorant receptor-expressing neurons are progressively lost from the OE of null mice, which exhibit a postnatal smell perception deficit. Axon guidance errors and increased neuronal cell death result in an absence of P2, I7, and M72 glomeruli, indicating a reduction in the repertoire of odorant receptor-specific glomeruli. By approximately 2 weeks of age, lactosamine is unexpectedly reexpressed in sensory neurons of null mice through a secondary pathway, which is accompanied by the regrowth of axons into the OB glomerular layer and the return of smell perception. Thus, both neonatal OE degeneration and the postnatal regeneration are lactosamine dependent. Lactosamine expression in beta3GnT1-/- mice is also reduced in pheromone-receptive vomeronasal neurons and dorsal root ganglion cells, suggesting that beta3GnT1 may perform a conserved function in multiple sensory systems. These results reveal an essential role for lactosamine in sensory axon pathfinding and in the formation of OB synaptic connections

Olfactory discrimination largely persists in mice with defects in odorant receptor expression and axon guidance

<p>Abstract</p> <p>Background</p> <p>The defining feature of the main olfactory system in mice is that each olfactory sensory neuron expresses only one of more than a thousand different odorant receptor genes. Axons expressing the same odorant receptor converge onto a small number of targets in the olfactory bulb such that each glomerulus is made up of axon terminals expressing just one odorant receptor. It is thought that this precision in axon targeting is required to maintain highly refined odor discrimination. We previously showed that β3GnT2^−/− mice have severe developmental and axon guidance defects. The phenotype of these mice is similar to adenylyl cyclase 3 (AC3) knockout mice largely due to the significant down-regulation of AC3 activity in β3GnT2^−/− neurons.</p> <p>Results</p> <p>Microarray analysis reveals that nearly one quarter of all odorant receptor genes are down regulated in β3GnT2^−/− mice compared to controls. Analysis of OR expression by quantitative PCR and <it>in situ</it> hybridization demonstrates that the number of neurons expressing some odorant receptors, such as mOR256-17, is increased by nearly 60% whereas for others such as mOR28 the number of neurons is decreased by more than 75% in β3GnT2^−/− olfactory epithelia. Analysis of axon trajectories confirms that many axons track to inappropriate targets in β3GnT2^−/− mice, and some glomeruli are populated by axons expressing more than one odorant receptor. Results show that mutant mice perform nearly as well as control mice in an odor discrimination task. In addition, <it>in situ</it> hybridization studies indicate that the expression of several activity dependent genes is unaffected in β3GnT2^−/− olfactory neurons.</p> <p>Conclusions</p> <p>Results presented here show that many odorant receptors are under-expressed in β3GnT2^−/− mice and further demonstrate that additional axon subsets grow into inappropriate targets or minimally innervate glomeruli in the olfactory bulb. Odor evoked gene expression is unchanged and β3GnT2^−/− mice exhibit a relatively small deficit in their ability to discriminate divergent odors. Results suggest that despite the fact that β3GnT2^−/− mice have decreased AC3 activity, decreased expression of many ORs, and display many axon growth and guidance errors, odor-evoked activity in cilia of mutant olfactory neurons remains largely intact.</p

Developmental profile and sexually dimorphic expression of kiss1 and kiss1r in the fetal mouse brain

The hypothalamic-pituitary-gonadal axis (HPG) is a complex neuroendocrine circuit involving multiple levels of regulation. Kisspeptin neurons play essential roles in controlling the HPG axis from the perspectives of puberty onset, oscillations of gonadotropin releasing hormone (GnRH) neuron activity and the pre-ovulatory LH surge. The current studies focus on the expression of kisspeptin during murine fetal development using in situ hybridization (ISH), quantitative reverse transcription real-time PCR (QPCR) and immunocytochemistry. Expression of mRNA coding for kisspeptin (KISS1) and its receptor KISS1R was observed at embryonic (E) day 13 by ISH. At E13 and other later ages examined, Kiss1 signal in individual cells within the arcuate nucleus (ARC) appeared stronger in females than males. ISH examination of agonadal steroidogenic factor-1 (Sf1) knockout mice revealed that E17 XY knockouts resembled wild-type XX females. These findings raise the possibility that gonadal hormones modulate the expression of Kiss1 in the ARC prior to birth. The sex and genotype differences were tested quantitatively by QPCR experiments in dissected hypothalami from mice at E17 and adulthood. Females had significantly more Kiss1 than males at both ages, even though the number of cells detected by ISH was similar. In addition, QPCR revealed a significant difference in the amount of Kiss1 mRNA in Sf1 mice with wild-type (WT) XY mice expressing less than XY knockouts (KO) and XX mice of both genotypes. The detection of immunoreactive KISS1 in perikarya of the ARC at E17 indicates that early mRNA is translated to peptide. The functional significance of this early expression of Kiss1 awaits elucidation

Olfactory axon guidance: the modified rules

The olfactory system represents a complex model for the investigation of factors that influence the guidance of sensory axon populations to specific targets in the CNS. In the mouse, the projections of approximately 1,000 neuronal subsets, each defined by expression of a distinct odorant receptor (OR), converge at unique glomerular loci in the olfactory bulb (OB). Unlike the case in other sensory systems, proper guidance is achieved without benefit of any known cues in the target itself that are capable of attracting or repelling specific axons. It has long been argued that OR proteins are the critical molecules orchestrating guidance. However, recent studies suggest that axon identity may be dependent on the graded expression of a variety of unique olfactory axon guidance cues. This review focuses attention on these non-OR factors and their roles in olfactory axon guidance

Regulation and function of axon guidance and adhesion molecules during olfactory map formation

The olfactory system presents a practical model for investigating basic mechanisms involved in patterning connections between peripheral sensory neurons and central targets. Our understanding of olfactory map formation was advanced greatly by the discovery of cAMP signaling as an important determinant of glomerular positioning in the olfactory bulb. Additionally, several cell adhesion molecules have been identified recently that are proposed to regulate homotypic interactions among projecting axons. From these studies a model has emerged to partially explain the wiring of axons from widely dispersed neuron populations in the nasal cavity to relatively stereotyped glomerular positions. These advances have revitalized interest in axon guidance molecules in establishing olfactory topography, but also open new questions regarding how these patterns of guidance cues are established and function, and what other pathways, such as glycosylation, might be involved. This review summarizes the current state of this field and the important molecules that impact on cAMP-dependent mechanism in olfactory axon guidance

N-linked polylactosamine glycan synthesis is regulated by co-expression of beta3GnT2 and GCNT2

Poly-N-acetyllactosamine (PLN) is a unique glycan composed of repeating units of the common disaccharide (Galbeta1,4-GlcNAcbeta1,3)n . The expression of PLN on glycoprotein core structures minimally requires enzyme activities for beta1,4-galactosyltransferase (beta4GalT) and beta1,3-N-acetylglucosminyltransferase (beta3GnT). Because beta4GalTs are ubiquitous in most cells, PLN expression is generally ascribed to the tissue-specific transcription of eight known beta3GnT genes in mice. In the olfactory epithelium (OE), beta3GnT2 regulates expression of extended PLN chains that are essential for axon guidance and neuronal survival. N-glycan branching and core composition, however, can also modulate the extent of PLN modification. Here, we show for the first time that the beta1,6-branching glycosyltransferase GCNT2 (formerly known as IGnT) is expressed at high levels specifically in the OE and other sensory ganglia. Postnatally, GCNT2 is maintained in mature olfactory neurons that co-express beta3GnT2 and PLN. This highly specific co-expression suggests that GCNT2 and beta3GnT2 function cooperatively in PLN synthesis. In support of this, beta3GnT2 and GCNT2 co-transfection in HEK293T cells results in high levels of PLN expression on the cell surface and on adenylyl cyclase 3, a major carrier of PLN glycans in the OE. These data clearly suggest that GCNT2 functions in vivo together with beta3GnT2 to determine PLN levels in olfactory neurons by regulating beta1,6-branches that promote PLN extension

Patterning the developing and regenerating olfactory system

The olfactory system is a remarkable model for investigating the factors that influence the guidance of sensory axon populations to specific targets in the CNS. Since the initial discovery of the vast odorant receptor (ORs) gene family in rodents and the subsequent finding that these molecules directly influence targeting, several additional olfactory axon guidance cues have been identified. Two of these, ephrins and semaphorins, have well-established functions in patterning axon connections in other systems. In addition, lactosamine-containing glycans are also required for proper targeting and maintenance of olfactory axons, and may also function in other sensory regions. It is now apparent that these and likely other additional molecules are required along with ORs to orchestrate the complex pattern of convergence and divergence that is unique to the olfactory system

Lactosamine differentially affects olfactory sensory neuron projections to the olfactory bulb

During embryonic development, olfactory sensory neurons extend axons that form synapses with the dendrites of projection neurons in glomeruli of the olfactory bulb (OB). The glycosyltransferase beta3GnT1 regulates the expression of 1B2-reactive lactosamine glycans that are mosaically distributed among glomeruli. In newborn beta3GnT1-/- mice, lactosamine expression is lost, and many glomeruli fail to form. To determine the role of lactosamine in OB targeting, we analyzed the trajectories of specific OR axon populations and their reactivity with 1B2 in beta3GnT1-/- mice. mI7 axons and P2 axons, both of which are weakly 1B2+ in wild-type mice, fail to grow to their normal positions in the glomerular layer during early postnatal development and never recover in adult mutant mice. In contrast, many M72 axons, which are always lactosamine negative in wild-type mice, survive but are misguided to the extreme anterior OB in neonatal mutant mice and persist as heterotypic glomeruli, even in adult null mice. These results show that the loss of lactosamine differentially affects each OR population. Those that lose their normal expression of lactosamine fail to form stable connections with mitral and tufted cells in the OB, disappear during early postnatal development, and do not recover in adults. Neurons that are normally lactosamine negative, survive early postnatal degeneration in beta3GnT1-/- mice but extend axons that converge on inappropriate targets in the mutant OB