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.

Targeted disruption of semaphorin 3C leads to persistent truncus arteriosus and aortic arch interruption.

Semaphorin 3C is a secreted member of the semaphorin gene family. To investigate its function in vivo, we have disrupted the semaphorin 3C locus in mice by targeted mutagenesis. semaphorin 3C mutant mice die within hours after birth from congenital cardiovascular defects consisting of interruption of the aortic arch and improper septation of the cardiac outflow tract. This phenotype is similar to that reported following ablation of the cardiac neural crest in chick embryos and resembles congenital heart defects seen in humans. Semaphorin 3C is expressed in the cardiac outflow tract as neural crest cells migrate into it. Their entry is disrupted in semaphorin 3C mutant mice. These data suggest that semaphorin 3C promotes crest cell migration into the proximal cardiac outflow tract.

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.

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.

Characterization of a cluster comprising approximately 100 odorant receptor genes in mouse.

With -1000 genes, the odorant receptor (OR) gene repertoire is the largest gene family in the mouse genome. Here we have established a 129/Sv BAC contig for mouse OR gene cluster 7 (Olfr7) on Chromosome (Chr) 9. The assembled approximately 2-Mb contig consists of 75 BACs and may contain as many as 100 OR genes, or approximately 10% of the mouse repertoire. Facilitated by the lack of introns in the coding region, we have determined the nucleotide sequence of 37 full-length, 2 partial, and 3 pseudo coding regions. These 42 OR genes and 3 additional OR genes previously mapped to the mouse Olfr7 cluster can be organized into 13 classes based on OR probe cross-hybridizations with 129/Sv mouse genomic DNA. OR genes belonging to the same class tend to be located next to each other within the cluster. Comparison of published full-length mouse and rat OR coding sequences with those identified here shows that the Olfr7 OR genes are highly related to each other, clustering on two major branches of an unrooted phylogenetic tree. Eight ORs contain an unusual NXC sequon at the amino-terminal extracellular domain that may represent a novel N-linked glycosylation site. The BAC contig presented here provides the substrate for sequencing of the cluster.

Local permutations in the glomerular array of the mouse olfactory bulb.

Olfactory sensory neurons expressing a given odorant receptor gene project their axons with great precision to a few specific glomeruli in the olfactory bulb. It is not clear to which extent the positions of these glomeruli are fixed. We sought to evaluate the constancy of the glomerular array in the mouse by determining the relative positions of glomeruli for various odorant receptors, using a method that affords single-axon resolution, and in a large number of bulbs. We used a genetic strategy to visualize neuronal populations that express one of three members of the mOR37 subfamily. We generated by gene targeting five strains of mice in which expression of a given mOR37 gene is linked to expression of an axonal maker, which is either taulacZ or tauGFP. The patterns of marker expression faithfully mimic those of the cognate receptors. Axons of neurons expressing a given mOR37 gene converge onto one or two glomeruli per bulb. Each mOR37 gene has its own glomeruli, and the mOR37 glomeruli are grouped within a restricted domain of the bulb. Serial sectioning of 214 bulbs reveals that the relative positions of the three types of glomeruli are not fixed but display local permutations. Importantly, this is also the case among the two bulbs from one individual, ruling out the genetic manipulation itself and differences in genetic background or olfactory experience as causes for the observed variability. These local permutations may reflect the developmental history of the glomeruli and are relevant for the construction of spatial odor maps.

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.

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.

Variable patterns of axonal projections of sensory neurons in the mouse vomeronasal system.

The vomeronasal system mediates pheromonal effects in mammals. We have employed gene targeting technology to introduce mutations in a putative pheromone receptor gene, VR2, in the germline of mice. By generating alleles differentially tagged with the histological markers taulacZ and tauGFP, we show that VR2 is monoallelically expressed in a given neuron. Axons of VR2-expressing neurons converge onto numerous glomeruli in the accessory olfactory bulb. The pattern of axonal projections is complex and variable. This wiring diagram is substantially different from that of the main olfactory system. The projection pattern is disrupted by deleting the coding region of VR2, but an unrelated seven-transmembrane protein, the odorant receptor M71, can partially substitute for VR2.

Identification of homeotic target genes in Drosophila melanogaster including nervy, a proto-oncogene homologue.

In Drosophila, the specific morphological characteristics of each segment are determined by the homeotic genes that regulate the expression of downstream target genes. We used a subtractive hybridization procedure to isolate activated target genes of the homeotic gene Ultrabithorax (Ubx). In addition, we constructed a set of mutant genotypes that measures the regulatory contribution of individual homeotic genes to a complex target gene expression pattern. Using these mutants, we demonstrate that homeotic genes can regulate target gene expression at the start of gastrulation, suggesting a previously unknown role for the homeotic genes at this early stage. We also show that, in abdominal segments, the levels of expression for two target genes increase in response to high levels of Ubx, demonstrating that the normal down-regulation of Ubx in these segments is functional. Finally, the DNA sequence of cDNAs for one of these genes predicts a protein that is similar to a human proto-oncogene involved in acute myeloid leukemias. These results illustrate potentially general rules about the homeotic control of target gene expression and suggest that subtractive hybridization can be used to isolate interesting homeotic target genes.

Endoscopic carpal tunnel release in a community-based series.

A retrospective analysis of 61 endoscopic carpal tunnel releases was performed. The surgical technique adhered strictly to the Inside Job protocol, as recommended between August and October of 1990. The objective was to provide a baseline for expected results and complications in a community practice setting. Results were rated excellent in 37 cases, good in 11, and poor in 4. Nine patients were not available for follow-up. Decreased postoperative morbidity and a more rapid return to work were found with endoscopic carpal tunnel release. The contribution of pillar pain, scar tenderness, pinch and grip weakness, and persistent numbness to good and poor outcomes were analyzed. One patient required repair of the median nerve. Another patient underwent conventional carpal tunnel release 1 year after the endoscopic procedure. Endoscopic carpal tunnel release promises to reduce morbidity. The results do not justify continued use of the Inside Job device described when conventional release is used as the standard for comparison.

The Drosophila learning and memory gene rutabaga encodes a Ca2+/Calmodulin-responsive adenylyl cyclase.

Four putative adenylyl cyclase genes from Drosophila melanogaster were identified by virtue of their extensive sequence homology with mammalian cyclases. One corresponds to the learning and memory gene rutabaga and is most similar to the mammalian brain Ca2+/calmodulin (CaM)-responsive cyclase. In a mammalian expression system, rutabaga cyclase activity was stimulated approximately 5-fold by the presence of Ca2+/CaM. A point mutation, identified at this locus in rut1 mutant flies, resulted in loss of detectable adenylyl cyclase activity. New P element insertion-induced rutabaga mutations mapped to within 200 nucleotides of the 5' end of the rutabaga cDNA. These data confirm the identity of the rutabaga locus as the structural gene for the Ca2+/CaM-responsive adenylyl cyclase and show that the inactivation of this cyclase leads to a learning and memory defect.

Molecular cloning and characterization of a Ca2+/calmodulin-insensitive adenylyl cyclase from rat brain.

Biochemical, immunological, and molecular cloning studies have suggested the existence of multiple forms of adenylyl cyclase (EC 4.6.1.1). An adenylyl cyclase cDNA clone (type II) was isolated from a rat brain library and found to encode a protein of 1090 amino acids that was homologous to but distinct from the previously described Ca2+/calmodulin-stimulated adenylyl cyclase from bovine brain. Expression of the type II cDNA in an insect cell line resulted in an increased level of adenylyl cyclase activity that was insensitive to Ca2+/calmodulin. Addition of activated Gs alpha protein to type II-containing membranes increased enzyme activity. The mRNA encoding the type II protein was expressed at high levels in brain tissue and at low levels in olfactory epithelium and lung. The existence of multiple adenylyl cyclase enzymes may provide for complex and distinct modes of biochemical regulation of cAMP levels in the brain.

Adenylyl cyclase amino acid sequence: possible channel- or transporter-like structure.

Complementary DNA's that encode an adenylyl cyclase were isolated from a bovine brain library. Most of the deduced amino acid sequence of 1134 residues is divisible into two alternating sets of hydrophobic and hydrophilic domains. Each of the two large hydrophobic domains appears to contain six transmembrane spans. Each of the two large hydrophilic domains contains a sequence that is homologous to a single cytoplasmic domain of several guanylyl cyclases; these sequences may represent nucleotide binding sites. An unexpected topographical resemblance between adenylyl cyclase and various plasma membrane channels and transporters was observed. This structural complexity suggests possible, unappreciated functions for this important enzyme.

Molecular cloning of odorant-binding protein: member of a ligand carrier family.

Odorant-binding protein (OBP) is found in nasal epithelium, and it selectively binds odorants. Three complementary DNAs encoding rat odorant-binding protein have now been cloned and sequenced. One clone contains an open reading frame predicted to encode an 18,091-dalton protein. RNA blot analysis confirms the localization of OBP messenger RNA in the nasal epithelium. This OBP has 33 percent amino acid identity to alpha 2-microglobulin, a secreted plasma protein. Other members of an alpha 2-microglobulin superfamily bind and transport hydrophobic ligands. Thus, OBP probably binds and carries odorants within the nasal epithelium to putative olfactory receptors.

Evaluating and improving the measurement of hospital case mix.

The foundation of case-based prospective payment is the case classification system. The purpose of classification systems is to group together patients with similar treatment requirements. The systems described in this issue take a variety of theoretical and practical approaches to classification. The critical issue in comparing these systems is whether the variation in treatment requirements which is not explained by the classification system is associated with particular groups of patients, particular hospitals, or particular groups of hospitals in such a way as to result in unfair reimbursement. We suggest criteria for comparing classification systems and a research agenda for clarifying the fairness of different approaches.

External fixator management of unstable colles' fractures: an alternative method.

Eighty-seven comminuted Colles' fractures of the Frykman III-VIII variety have been treated with the Medium-C-Hoffmann® external fixator, as an alternative to pins in plaster management, over a three-year period, with an average length of followup of 19 months. A standard technique had been used. The results were assessed on the basis of subjective, objective, and roentgenographic findings. The overall goal of a painless wrist with excellent motion, good strength, and satisfactory cosmesis appears to have been achieved by this method of management.

The child abuse program in a child guidance center.

This paper is a case example of how one private child guidance clinic responded to problems of child abuse seen in the clinic and how the center moved from the "micro-level" of treating individual victims and their families to the "macro-level" of education and community involvement. The authors show how case-by-case treatment of abused families can be used as a guide for program development and community education.