The olfactory critical period is determined by activity-dependent Sema7A/PlxnC1 signaling within glomeruli.

In mice, early exposure to environmental odors affects social behaviors later in life. A signaling molecule, Semaphorin 7A (Sema7A), is induced in the odor-responding olfactory sensory neurons. Plexin C1 (PlxnC1), a receptor for Sema7A, is expressed in mitral/tufted cells, whose dendrite-localization is restricted to the first week after birth. Sema7A/PlxnC1 signaling promotes post-synaptic events and dendrite selection in mitral/tufted cells, resulting in glomerular enlargement that causes an increase in sensitivity to the experienced odor. Neonatal odor experience also induces positive responses to the imprinted odor. Knockout and rescue experiments indicate that oxytocin in neonates is responsible for imposing positive quality on imprinted memory. In the oxytocin knockout mice, the sensitivity to the imprinted odor increases, but positive responses cannot be promoted, indicating that Sema7A/PlxnC1 signaling and oxytocin separately function. These results give new insights into our understanding of olfactory imprinting during the neonatal critical period.

Author Correction: Sema7A/PlxnCl signaling triggers activity-dependent olfactory synapse formation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Primary dendrites of mitral cells synapse unto neighboring glomeruli independent of their odorant receptor identity.

In the mouse olfactory bulb, neural map topography is largely established by axon-axon interactions of olfactory sensory neurons (OSNs). However, to make the map functional, the OSNs must make proper connections to second-order neurons, the mitral cells. How do the mitral-cell dendrites find their partner glomeruli for synapse formation with OSN axons? Here, we analyze dendrite connections of mitral cells in various mutant mice in which glomerular formation is perturbed. Our present results support the proximity model, whereby mitral cells tend to connect primary dendrites to the nearest neighboring glomeruli regardless of their odorant receptor identities. The physical location of glomeruli rather than the odorant-receptor specificity appears to play a key role in matching mitral cells with their partner OSN axons.

Sema7A/PlxnCl signaling triggers activity-dependent olfactory synapse formation.

In mammals, neural circuits are formed based on a genetic program and further refined by neuronal activity during the neonatal period. We report that in the mouse olfactory system, the glomerular map is not merely refined but newly connected to second-order neurons by odorant-receptor-derived neuronal activity. Here, we analyzed a pair of molecules, Sema7A, expressed in olfactory sensory neurons (OSNs) in an activity-dependent manner, and PlxnC1, localized to dendrites of mitral/tufted (M/T) cells in the first week after birth. In Sema7A or PlxnC1 knockout (KO) mice, initiation of synapse formation and dendrite selection of M/T cells were perturbed. Reconstitution and rescue experiments demonstrated that Sema7A-PlxnC1 interaction is essential to form the post-synaptic assembly. Pharmacological blocking experiments indicated that synaptic transmission triggers primary dendrite selection by synaptic competition. We conclude that Sema7A signaling is key to inducing activity-dependent post-synapse events and dendrite selection in M/T-cells during the neonatal period.

Nrp2 is sufficient to instruct circuit formation of mitral-cells to mediate odour-induced attractive social responses.

Odour information induces various innate responses that are critical to the survival of the individual and for the species. An axon guidance molecule, Neuropilin 2 (Nrp2), is known to mediate targeting of olfactory sensory neurons (primary neurons), to the posteroventral main olfactory bulb (PV MOB) in mice. Here we report that Nrp2-positive (Nrp2+) mitral cells (MCs, second-order neurons) play crucial roles in transmitting attractive social signals from the PV MOB to the anterior part of medial amygdala (MeA). Semaphorin 3F, a repulsive ligand to Nrp2, regulates both migration of Nrp2+ MCs to the PV MOB and their axonal projection to the anterior MeA. In the MC-specific Nrp2 knockout mice, circuit formation of Nrp2+ MCs and odour-induced attractive social responses are impaired. In utero, electroporation demonstrates that activation of the Nrp2 gene in MCs is sufficient to instruct their circuit formation from the PV MOB to the anterior MeA.

Immobility responses are induced by photoactivation of single glomerular species responsive to fox odour TMT.

Fox odour 2,4,5-trimethyl thiazoline (TMT) is known to activate multiple glomeruli in the mouse olfactory bulb (OB) and elicits strong fear responses. In this study, we screened TMT-reactive odourant receptors and identified Olfr1019 with high ligand reactivity and selectivity, whose glomeruli are located in the posterodorsal OB. In the channelrhodopsin knock-in mice for Olfr1019, TMT-responsive olfactory-cortical regions were activated by photostimulation, leading to the induction of immobility, but not aversive behaviour. Distribution of photoactivation signals was overlapped with that of TMT-induced signals, but restricted to the narrower regions. In the knockout mice, immobility responses were reduced, but not entirely abolished likely due to the compensatory function of other TMT-responsive glomeruli. Our results demonstrate that the activation of a single glomerular species in the posterodorsal OB is sufficient to elicit immobility responses and that TMT-induced fear may be separated into at least two different components of immobility and aversion.

Developmental regulation of neural map formation in the mouse olfactory system.

In the mouse olfactory system, various odorants are detected by approximately 1000 different odorant receptors (ORs) expressed in the olfactory sensory neurons (OSNs). It is well established that each OSN expresses only one functional OR gene in a monoallelic manner. Furthermore, OSN axons expressing the same OR converge to a set of glomeruli in the olfactory bulb (OB). During embryonic development, a coarse map is formed by the combination of two genetically programmed processes. One is OR-independent axonal projection along the dorsal-ventral (D-V) axis, and the other is OR-dependent projection along the anterior-posterior (A-P) axis. D-V projection is regulated by the anatomical location of OSNs within the olfactory epithelium (OE), whereas A-P projection is instructed by expressed OR molecules using cyclic adenosine monophosphate (cAMP) signals. After birth, the map is further refined in an activity-dependent manner by its conversion from a continuous to a discrete map through segregation of glomerular structures. Here, we summarize recent progress from our laboratory in understanding neural map formation in the mouse olfactory system.

Agonist-independent GPCR activity regulates anterior-posterior targeting of olfactory sensory neurons.

G-protein-coupled receptors (GPCRs) are known to possess two different conformations, active and inactive, and they spontaneously alternate between the two in the absence of ligands. Here, we analyzed the agonist-independent GPCR activity for its possible role in receptor-instructed axonal projection. We generated transgenic mice expressing activity mutants of the ß2-adrenergic receptor, a well-characterized GPCR with the highest homology to odorant receptors (ORs). We found that mutants with altered agonist-independent activity changed the transcription levels of axon-targeting molecules--e.g., Neuropilin-1 and Plexin-A1--but not of glomerular segregation molecules--e.g., Kirrel2 and Kirrel3--thus causing shifts in glomerular locations along the anterior-posterior (A-P) axis. Knockout and in vitro experiments demonstrated that Gs, but not Golf, is responsible for mediating the agonist-independent GPCR activity. We conclude that the equilibrium of conformational transitions set by each OR is the major determinant of expression levels of A-P-targeting molecules.

Possible roles of Robo1+ ensheathing cells in guiding dorsal-zone olfactory sensory neurons in mouse.

In the mouse olfactory system, the anatomical locations of olfactory sensory neurons (OSNs) correlate with their axonal projection sites along the dorsoventral axis of the olfactory bulb (OB). We have previously reported that Neuropilin-2 expressed by ventral-zone OSNs contributes to the segregation of dorsal and ventral OSN axons, and that Slit is acting as a negative land mark to restrict the projection of Robo2+, early-arriving OSN axons to the embryonic OB. Here, we report that another guidance receptor, Robo1, also plays an important role in guiding OSN axons. Knockout mice for Robo1 demonstrated defects in targeting of OSN axons to the OB. Although Robo1 is colocalized with dorsal-zone OSN axons, it is not produced by OSNs, but instead by olfactory ensheathing cells. These findings indicate a novel strategy of axon guidance in the mouse olfactory system during development.

A major allogenic leukocyte antigen in the agnathan hagfish.

All vertebrates, from jawless fish to mammals, possess adaptive immune systems that can detect and inactivate non-self-antigens through a vast repertoire of antigen receptors. Unlike jawed vertebrates, the hagfish utilizes variable lymphocyte receptors (VLRs) that are unrelated to immunoglobulin molecules but are diversified by copy-choice gene conversion mechanism. Here, we report that hagfish VLRs react with allogenic leukocyte antigens but not with self-antigens. We found that a highly polymorphic membrane protein, NICIR3, is recognized by VLRs as an allogenic leukocyte antigen (ALA). In a serological cross-reactivity test, a close correlation was observed between the amino acid differences in the protein sequences and the VLR cross-reactivities. This leukocyte antigen was predominantly expressed in phagocytic leukocytes, where it was associated with phagocytosed protein antigens. These findings suggest that a polymorphic leukocyte antigen, NICIR3/ALA, plays a pivotal role in jawless vertebrate adaptive immunity.

Neuropilin-2 is required for the proper targeting of ventral glomeruli in the mouse olfactory bulb.

Recent evidence shows that olfactory sensory neurons expressing a given odorant receptor (OR) are not necessarily confined to one of four zones, rather arranged in an overlapping manner in the olfactory epithelium (OE). In this study, in situ hybridization of OE sections with the OR probes indicated that the OR genes, the mRNAs of which were detected in an array of glomeruli on olfactory bulb (OB) along the anterodorsal/posteroventral (AD/PV) axis, are expressed in subareal zones within the most ventral zone, zone 4, along the dorsomedial/ventrolateral (DM/VL) axis. We also found that Neuropilin-2 (Nrp2) is expressed in a DM-low to VL-high gradient within zone 4 of OE. Furthermore, in Nrp2 mutant mice, we observed multiple glomeruli for zone 4 ORs in OB. These results suggest that the graded expression of Nrp2 in OE is required for the proper targeting of ventral glomeruli along the AD/PV axis in OB.

Regulation of antigen-receptor gene assembly in hagfish.

Variable lymphocyte receptors (VLRs) are antigen receptors in the jawless vertebrates lamprey and hagfish. VLR genes are classified into VLRA and VLRB, and lymphocytes expressing VLRA are T-cell-like, whereas those expressing VLRB are B-cell-like in the sea lamprey. Diverse VLR genes are assembled somatically in lymphocytes; however, how the assembly is regulated is still largely unknown. Here, we analyse VLR gene assembly at the single-cell level in the inshore hagfish (Eptatretus burgeri). Each lymphocyte assembles and transcribes only one type of VLR gene, either VLRA or VLRB. In general, monoallelic assembly of VLR was observed, but diallelic assembly was found in some cases--in many of which, one allele was functional and the other was defective. In fact, all VLR-assembled lymphocytes contained at least one functional VLR gene. Together, these results indicate a feedback inhibition of VLR assembly and selection of VLR-positive lymphocytes.

Deletion of the core-H region in mice abolishes the expression of three proximal odorant receptor genes in cis.

We have previously reported that a 2.1-kb homology (H) sequence, conserved between mouse and human, regulates the odorant receptor (OR) gene MOR28 in transgenic mice. Here, we narrowed down the essential sequences of the H to a core of 124 bp by using a transient expression system in zebrafish embryos. Transgenic experiments in mice demonstrated that the core-H sequence is sufficient to endow expression of the MOR28 minigene. Deletion and mutation analyses of the core-H region revealed two homeodomain sequences to be essential for the H enhancer activity. Targeted deletion of the core-H abolished expression of three proximal OR genes, MOR28, MOR10, and MOR83, in cis, indicating the presence of another locus control region/enhancer in the downstream region, that regulates four distal OR genes in the same MOR28 cluster. In the heterozygous mice, the H(-) phenotype of the mutant allele was not rescued by the wild-type H(+) allele in trans.

Antigen-receptor genes of the agnathan lamprey are assembled by a process involving copy choice.

Jawless vertebrates have acquired immunity but do not have immunoglobulin-type antigen receptors. Variable lymphocyte receptors (VLRs) have been identified in lamprey that consist of multiple leucine-rich repeat (LRR) modules. An active VLR gene is generated by the assembly of a series of variable gene segments, including many that encode LRRs. Stepwise assembly of the gene segments seems to occur by replacement of the intervening DNA between the 5' and 3' constant-region genes. Here we report that lamprey (Lethenteron japonicum) assemble their VLR genes by a process involving 'copy choice'. Regions of short homology seemed to prime copying of donor LRR-encoding sequences into the recipient gene. Those LRR-encoding germline sequences were abundant and shared extensive sequence homologies. Such genomic organization permits initiation of copying anywhere in an LRR-encoding module for the generation of various hybrid LRRs. Thus, a vast repertoire of recombinant VLR genes could be generated not only by copying of various LRR segments in diverse combinations but also by the use of multiple sites in an LRR gene segment for priming.

Fc{epsilon}RI-mediated mast cell degranulation requires calcium-independent microtubule-dependent translocation of granules to the plasma membrane.

The aggregation of high affinity IgE receptors (Fcepsilon receptor I [FcepsilonRI]) on mast cells is potent stimulus for the release of inflammatory and allergic mediators from cytoplasmic granules. However, the molecular mechanism of degranulation has not yet been established. It is still unclear how FcepsilonRI-mediated signal transduction ultimately regulates the reorganization of the cytoskeleton and how these events lead to degranulation. Here, we show that FcepsilonRI stimulation triggers the formation of microtubules in a manner independent of calcium. Drugs affecting microtubule dynamics effectively suppressed the FcepsilonRI-mediated translocation of granules to the plasma membrane and degranulation. Furthermore, the translocation of granules to the plasma membrane occurred in a calcium-independent manner, but the release of mediators and granule-plasma membrane fusion were completely dependent on calcium. Thus, the degranulation process can be dissected into two events: the calcium-independent microtubule-dependent translocation of granules to the plasma membrane and calcium-dependent membrane fusion and exocytosis. Finally, we show that the Fyn/Gab2/RhoA (but not Lyn/SLP-76) signaling pathway plays a critical role in the calcium-independent microtubule-dependent pathway.

Joining mutants of RAG1 and RAG2 that demonstrate impaired interactions with the coding-end DNA.

In V(D)J joining of antigen receptor genes, two recombination signal sequences (RSSs), 12- and 23-RSSs, form a complex with the protein products of recombination activating genes, RAG1 and RAG2. DNaseI footprinting demonstrates that the interaction of RAG proteins with substrate RSS DNA is not just limited to the signal region but involves the coding sequence as well. Joining mutants of RAG1 and RAG2 demonstrate impaired interactions with the coding region in both pre- and postcleavage type complexes. A possible role of this RAG coding region interaction is discussed in the context of V(D)J recombination.

Abrogation of autoimmune disease in Lyn-deficient mice by the deletion of IL-5 receptor alpha chain gene.

Lyn, the src-family protein tyrosine kinase, plays a crucial role in the regulation of B cell antigen receptor (BCR)- and IL-5-receptor (IL-5R)-mediated signaling. Lyn-deficient mice have been reported to exhibit an increase in B-1 cell numbers, splenomegaly and accumulation of lymphoblast-like cells in the spleen with age, resulting in hyperimmunoglobulinemia and glomerulonephritis caused by the deposition of autoantibody complexes. To elucidate the role of IL-5 in B-1 cell activation, autoantibody production and autoimmune diseases, Lyn-deficient mice were crossed with IL-5Ralpha chain (IL-5Ralpha)-deficient mice and generated Lyn- and IL-5Ralpha-deficient (DKO) mice. In contrast to Lyn-deficient mice, DKO mice showed significantly reduced splenomegaly and lymphoadenopathy and reduced B-1 cell number in the peritoneal cavity. DKO mice also secreted low levels of IgM and IgG autoantibodies. Biochemical and histological analyses revealed that DKO mice showed milder pathogenesis of autoimmune-like disorders than Lyn-deficient mice. These results suggest involvement of IL-5 in enhanced B-1 cell activation, autoantibody production, and development of autoimmune disease in Lyn-deficient mice.

In vitro processing of the 3'-overhanging DNA in the postcleavage complex involved in V(D)J joining.

The postcleavage complex involved in V(D)J joining is known to possess a transpositional strand transfer activity, whose physiological role is yet to be clarified. Here we report that RAG1 and RAG2 proteins in the signal end (SE) complex cleave the 3'-overhanging structure of the synthetic coding-end (CE) DNA in two successive steps in vitro. The 3'-overhanging structure is attacked by the SE complex imprecisely, near the double-stranded/single-stranded (ds/ss) junction, and transferred to the SE. The transferred overhang is then resolved and cleaved precisely at the ds/ss junction, generating either the linear or the circular cleavage products. Thus, the blunt-end structure is restored for the SE and variably processed ends are generated for the synthetic CE. This 3'-processing activity is observed not only with the core RAG2 but also with the full-length protein.