A putative pheromone receptor gene expressed in human olfactory mucosa.

Pheromones elicit specific behavioural responses and physiological alterations in recipients of the same species. In mammals, these chemical signals are recognized within the nasal cavity by sensory neurons that express pheromone receptors. In rodents, these receptors are thought to be represented by two large multigene families, comprising the V1r and V2r genes, which encode seven-transmembrane proteins. Although pheromonal effects have been demonstrated in humans, V1R or V2R counterparts of the rodent genes have yet to be characterized.

Transgenic mice demonstrate that epithelial homing of gamma/delta T cells is determined by cell lineages independent of T cell receptor specificity.

gamma/delta T cells with different TCR repertoires are compartmentalized in different epithelia. This raises the possibility that the TCR-gamma/delta directs homing of T cells to these epithelia. Alternatively, the signals that induce TCR-gamma/delta expression in developing T cells may also induce homing properties in such cells, presumably in the form of cell surface receptors. We have examined this issue by studying the homing of gamma/delta T cells in transgenic mice constructed with specific pairs of rearranged gamma and delta genes. In such mice, most gamma/delta T cells express the transgene-encoded TCR. We find that homing to both skin and gut epithelia is a property of T cells and is not determined by the type of gamma and delta genes used to encode their TCR. We also studied the effect of TCR replacement on the expression of Thy-1 and CD8 proteins on the gamma/delta T cells associated with gut epithelia. Our results show that the expression of the appropriate type of TCR-gamma/delta is not required for the Thy-1 expression by these T cells, suggesting that Thy-1 is not an activation marker. In contrast, CD8 expression by gut gamma/delta T cells seems to depend on the expression of the appropriate type of TCR.

Seven-transmembrane proteins as odorant and chemosensory receptors.

The olfactory systems of various species solve the challenging problem of general molecular recognition in widely differing ways. Despite this variety, the molecular receptors are invariably G protein-coupled seven-transmembrane proteins, and are encoded by the largest gene families known to exist in a given animal genome. Receptor gene families have been identified in vertebrates and two invertebrate species, the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster. The complexity of the odorant receptor repertoire is estimated in mouse and rat at 1000 genes, or 1 percent of the genome, surpassing that of the immunoglobulin and T cell receptor genes combined. Two distinct seven-transmembrane gene families may encode in rodents the chemosensory receptors of the vomeronasal organ, which is specialized in the detection of pheromones. Remarkably, these five receptor families have practically no sequence homology among them. Genetic manipulation experiments in mice imply that vertebrate odorant receptors may fulfill a dual role, also serving as address molecules that guide axons of olfactory sensory neurons to their precise target in the brain.

Differentiation of embryonic stem cell lines generated from adult somatic cells by nuclear transfer.

Embryonic stem (ES) cells are fully pluripotent in that they can differentiate into all cell types, including gametes. We have derived 35 ES cell lines via nuclear transfer (ntES cell lines) from adult mouse somatic cells of inbred, hybrid, and mutant strains. ntES cells contributed to an extensive variety of cell types, including dopaminergic and serotonergic neurons in vitro and germ cells in vivo. Cloning by transfer of ntES cell nuclei could result in normal development of fertile adults. These studies demonstrate the full pluripotency of ntES cells.

Peripheral olfactory projections are differentially affected in mice deficient in a cyclic nucleotide-gated channel subunit.

Axons of olfactory sensory neurons expressing a given odorant receptor converge to a few glomeruli in the olfactory bulb. We have generated mice with unresponsive olfactory sensory neurons by targeted mutagenesis of a cyclic nucleotide-gated channel subunit gene, OCNC1. When these anosmic mice were crossed with mice in which neurons expressing a given odorant receptor can be visualized by coexpression of an axonal marker, the pattern of convergence was affected for one but not another receptor. In a novel paradigm, termed monoallelic deprivation, axons from channel positive or negative neurons that express the same odorant receptor segregate into distinct glomeruli within the same bulb. Thus, the peripheral olfactory projections are in part influenced by mechanisms that depend on neuronal activity.

Odorant receptor genes in humans.

The sense of smell is highly sophisticated in vertebrates but Homo sapiens ranks low in olfactory performance when compared to other species - why? Olfaction initiates with the interaction of odorants with specific receptors on the surface of olfactory sensory neurons in the nose. The genes encoding odorant receptors form the largest family in the vertebrate genome, numbering as many as 1000 in rodents. It has recently come to light that the repertoire of human odorant receptor genes, unlike in other vertebrates, is riddled with pseudogenes.

How smell develops.

The mouse's sense of smell is built of approximately 1000 input channels. Each of these consists of a population of olfactory sensory neurons that express the same odorant receptor gene and project their axons to the same targets (glomeruli) in the olfactory bulb. A neuron must choose to express a singular receptor gene from a repertoire of approximately 1000 genes, and its axon must be wired to the corresponding glomerulus, from an array of approximately 1800 glomeruli. Genetic experiments have shown that the expressed odorant receptor specifies axonal choice of the innervated glomerulus, but it is not the only determinant. The mechanisms of odorant receptor gene choice and axonal wiring are central to the functional organization of the mammalian olfactory system. Although principles have emerged, our understanding of these processes is still limited.

Olfactory coding: revealing intrinsic representations of odors.

Recent studies have applied optical imaging of intrinsic signals to the rodent olfactory system, providing a unique view of how odorous molecules are represented in the central nervous system.

Selective deletion of leptin receptor in neurons leads to obesity.

Animals with mutations in the leptin receptor (ObR) exhibit an obese phenotype that is indistinguishable from that of leptin deficient ob/ob mice. ObR is expressed in many tissues, including brain, and the relative importance of leptin's effects on central versus peripheral sites has not been resolved. To address this, we generated mice with neuron-specific (ObR(SynI)KO) and hepatocyte-specific (ObR(Alb)KO) disruption of ObR. Among the ObR(SynI)KO mice, the extent of obesity was negatively correlated with the level of ObR in hypothalamus and those animals with the lowest levels of ObR exhibited an obese phenotype. The obese mice with low levels of hypothalamic ObR also show elevated plasma levels of leptin, glucose, insulin, and corticosterone. The hypothalamic levels of agouti-related protein and neuropeptide Y RNA are increased in these mice. These data indicate that leptin has direct effects on neurons and that a significant proportion, or perhaps the majority, of its weight-reducing effects are the result of its actions on brain. To explore possible direct effects of leptin on a peripheral tissue, we also characterized ObR(Alb)KO mice. These mice weigh the same as controls and have no alterations in body composition. Moreover, while db/db mice and ObR(SynI)KO mice have enlarged fatty livers, ObR(Alb)KO mice do not. In summary, these data suggest that the brain is a direct target for the weight-reducing and neuroendocrine effects of leptin and that the liver abnormalities of db/db mice are secondary to defective leptin signaling in the brain.

Efficient metaphase II transgenesis with different transgene archetypes.

Mammalian genome characterization and biotechnology each require the mobilization of large DNA segments to produce transgenic animals. We recently showed that mouse metaphase II (mII) oocytes could efficiently promote transgenesis (mII transgenesis) when coinjected with sperm and small (<5 kilobases) ubiquitously expressed transgenes (tgs). We have extended this work and now report that mII transgenesis can readily be applied to a range of larger tgs (11.9-170 kilobases), including bacterial and mammalian artificial chromosome (BAC and MAC) constructs. The efficiency of large-construct mII transgenesis was at least as high as that with small constructs; 11-47% of offspring carried the large tgs. More than 95% of these transgenic founders transmitted the tg to offspring. These data demonstrate the ability of mII transgenesis to deliver large tgs efficiently.

Targeting olfaction.

An olfactory sensory neuron most probably expresses a single olfactory receptor gene, out of 1000 choices. Olfactory neurons expressing the same olfactory receptor are distributed over a wide area of the olfactory epithelium, but they project their axons to a few discrete positions in the olfactory bulb, out of 2500 choices. How?

Structure and emergence of specific olfactory glomeruli in the mouse.

Olfactory sensory neurons (OSNs) expressing a given odorant receptor (OR) gene project their axons to a few specific glomeruli that reside at recognizable locations in the olfactory bulb. Connecting approximately 1000 populations of OSNs to the approximately 1800 glomeruli of the mouse bulb poses a formidable wiring problem. Additional progress in understanding the mechanisms of neuronal connectivity is dependent on knowing how these axonal pathways are organized and how they form during development. Here we have applied a genetic approach to this problem. We have constructed by gene targeting novel strains of mice in which either all OSNs or those that express a specific OR gene, M72 or M71, also produce green fluorescent protein (GFP) or a fusion of tau with GFP. We visualized OSNs and their axons in whole mounts with two-photon laser scanning microscopy. The main conclusion we draw from the three-dimensional reconstructions is the high degree of morphological variability of mature glomeruli receiving axonal input from OR-expressing OSNs and of the pathways taken by the axons to those glomeruli. We also observe that axons of OR-expressing OSNs do not innervate nearby glomeruli in mature mice. Postnatally, a tangle of axons from M72-expressing OSNs occupies a large surface area of the bulb and coalesces abruptly into a protoglomerulus at a reproducible stage of development. These results differ in several aspects from those reported for the development of glomeruli receiving input from OSNs expressing the P2 OR, suggesting the need for a more systematic examination of OR-specific glomeruli.

Dismantling the immune system.

During the past year, we have witnessed a veritable explosion in the number of mutant mouse strains produced by gene targeting in embryonic stem cells. Many of the informative targeted mutants have relevance to the field of immunology. At least one mutant mouse strain now exists for most of the important genes in immunology, and this collection of mutant mice has greatly expanded the experimental repertoire of immunologists. New targeting techniques have been developed that have often found their first application in immunology.

Small subfamily of olfactory receptor genes: structural features, expression pattern and genomic organization.

Olfactory receptors of the OR37 subfamily are characterized by distinct sequence features and are expressed in neurons segregated in a restricted area of the olfactory epithelium. In the present study, we have characterized the complement of OR37-like genes in the mouse. Five OR37-like genes were identified. They reside within only 60kb of DNA on chromosome 4. About 70kb distant from this cluster, two additional olfactory receptor genes are located, which are members of distinct receptor subfamilies. Phylogenetic analysis demonstrated that the two physically linked receptors are closely related to the OR37 subfamily. Studies of gene expression showed that both genes are also expressed in clustered neuron populations located in the typical OR37 region of the epithelium. These data suggest the involvement of locus-dependent mechanisms for the spatial control of OR gene expression.

Fibrinolytic response and fibrin fragment D-dimer levels in patients with deep vein thrombosis.

An enzyme-linked immunosorbent assay for fragment D-dimer was developed with the use of two monoclonal antibodies directed against specific non-overlapping antigenic determinants, present in fragment D-dimer of crosslinked fibrin but not in fragment D of non crosslinked fibrin or of fibrinogen. The lower limit of sensitivity of the assay when applied to human plasma, is 25 ng/ml. Concentration of fragment D-dimer in plasma from healthy individuals was 177 +/- 83 ng/ml (mean +/- SD). In plasma of 11 out of 12 patients with phlebographically confirmed acute deep vein thrombosis, fragment D-dimer levels were significantly increased. Fragment D-dimer was not increased in 9 out of 10 patients with recurrent idiopathic deep vein thrombosis during clinically silent episodes. Total t-PA antigen and free t-PA antigen concentrations were measured using previously developed ELISAs. Nine of the 12 patients with acute deep vein thrombosis showed a significant increase of total t-PA antigen (from 8.6 +/- 6.9 ng/ml to 21 +/- 16 ng/ml) after venous occlusion but in 3 of these free t-PA remained undetectable. Five of the 10 patients with recurrent deep vein thrombosis responded to venous occlusion with a significant increase of total t-PA antigen (from 6.7 +/- 3.2 ng/ml to 14 +/- 7.9 ng/ml) but, in all patients, free t-PA antigen remained undetectable. It is concluded that the combined assays of total and free t-PA antigen and of fragment D-dimer may be useful for the evaluation of the dynamics of the fibrinolytic system in physiological and pathological conditions.

Fibrinolytic response to venous occlusion and fibrin fragment D-dimer levels in normal and complicated pregnancy.

The fibrinolytic response to venous occlusion was assessed in 29 women with normal or complicated pregnancy, by measurements of total t-PA and free t-PA with specific ELISAs. The release of t-PA from the vessel wall was 11 +/- 9 ng/ml in non-pregnant women (mean +/- SD, n = 6) but was markedly reduced throughout pregnancy. Following venous occlusion, free t-PA increased by 12 +/- 11 ng/ml in non-pregnant women but remained below the detection limit of 2 ng/ml towards the end of pregnancy. A markedly reduced t-PA release with absence of free t-PA was also observed during late pregnancy in patients with insulin-dependent diabetes mellitus, intra-uterine growth retardation and pre-eclampsia. Plasma levels of fragment D-dimer of cross-linked fibrin were measured with a specific ELISA in 79 pregnant women. D-dimer levels were 129 +/- 36 ng/ml (mean +/- SD, n = 8) in non-pregnant women and increased to 400 +/- 170 ng/ml (n = 25) and 440 +/- 220 ng/ml (n = 22) during the second and third trimester of pregnancy respectively. Significantly higher levels than observed in uncomplicated third trimester pregnancies were found in 3 out of 6 diabetic and in 2 out of 7 pre-eclamptic women. It is concluded that the t-PA release after venous occlusion is significantly reduced during pregnancy. In addition, released t-PA is rapidly inhibited. The levels of fragment D-dimer increase during pregnancy, suggesting that, notwithstanding the marked impairment of the fibrinolytic response to venous occlusion, the fibrinolytic system remains functionally active.

Lymphocyte development and function in T-cell receptor and RAG-1 mutant mice.

We have used gene targeting in embryonic stem cells to generate mice with mutations in T-cell receptor (TCR) gene-alpha, TCR-beta, TCR-delta or the Recombination Activating Gene-1 (RAG-1). TCR-alpha or TCR-beta mutant mice are deficient in alpha beta T cells, but still contain gamma delta T cells and B cells. TCR-delta mutant mice are deficient in gamma delta T cells, but still contain alpha beta T cells and B cells. Mice doubly mutant for TCR-beta and TCR-delta do not have any mature T cells, but still have B cells. RAG-1 mutant mice are totally deficient in both mature T cells and B cells. Here, I describe recent studies of thymocyte development in the mutant mice, and I review experiments addressing the function of the immune system of the mutant mice.