A Cre-inducible diphtheria toxin receptor mediates cell lineage ablation after toxin administration

A new system for lineage ablation is based on transgenic expression of a diphtheria toxin receptor (DTR) in mouse cells and application of diphtheria toxin (DT). To streamline this approach, we generated Cre-inducible DTR transgenic mice (iDTR) in which Cre-mediated excision of a STOP cassette renders cells sensitive to DT. We tested the iDTR strain by crossing to the T cell- and B cell-specific CD4-Cre and CD19-Cre strains, respectively, and observed efficient ablation of T and B cells after exposure to DT. In MOGi-Cre/iDTR double transgenic mice expressing Cre recombinase in oligodendrocytes, we observed myelin loss after intraperitoneal DT injections. Thus, DT crosses the blood-brain barrier and promotes cell ablation in the central nervous system. Notably, we show that the developing DT-specific antibody response is weak and not neutralizing, and thus does not impede the efficacy of DT. Our results validate the use of iDTR mice as a tool for cell ablation in vivo.     

Long-term transplantation of mature sensory neurons

The spinal ganglia were transplanted into the mesocolon of adult cats for periods of from 1 day to 9 months. About 30% of differentiated sensory neurons survived during the longterm transplantation. The intensive regeneration of the sensory neurons processes was characteristic of the transplanted neurons. Total myelinization of the regenerating nerves occurred during the 3rd--5th month. Potential regeneration capacity of the differentiated neurons and possibly of their prolonged transplantation were revealed.     

Developmental biology of olfactory sensory neurons

Proliferation, differentiation and death of olfactory neurons occur continually, even in adult animals. New data suggest that growth factors regulate the rate of cell proliferation. Early growth of olfactory axons in embryonic development is accompanied by the migration of epithelial cells from the olfactory placode toward the presumptive olfactory bulb. Maturation and ciliogenesis at the dendritic end of the cell is apparently dependent on a signal(s) from the bulb. The total life span of the neuron depends on maintenance of contact with the bulb. Olfactory life span is normally variable but is curtailed substantially in the absence of the bulb.     

Gal-NCAM is a differentially expressed marker for mature sensory neurons in the rat olfactory system

A new monoclonal antibody, 2E11, was produced by immunizing mice with the microsomal fraction of rat accessory olfactory bulb cells. This IgM recognizes a previously described complex alpha-galactosyl containing glycolipid, as well as N-linked glycoproteins at 170 and 210 kD. These proteins correspond to a new nerve cell adhesion molecule (NCAM) glycoform, Gal-NCAM, which contains a blood group B-like oligosaccharide. During embryonic development, the 2E11 epitope is expressed by a subset of mature olfactory sensory neurons randomly dispersed throughout the olfactory epithelium, whereas in the olfactory bulb, immunostaining is restricted to medial areas of the nerve layer. When compared to PSA-NCAM, another NCAM glycoform, Gal-NCAM has a mutually exclusive distribution pattern both in the olfactory epithelium and in the olfactory bulb. We propose a model for the hierarchy of neuronal maturation in the olfactory epithelium, including a switch from PSA-NCAM expression by immature neurons to the expression of Gal-NCAM by mature neurons.     

Axon guidance of mouse olfactory sensory neurons by odorant receptors and the beta2 adrenergic receptor

Odorant receptors (ORs) provide the core determinant of identity for axons of olfactory sensory neurons (OSNs) to coalesce into glomeruli in the olfactory bulb. Here, using gene targeting in mice, we examine how the OR protein determines axonal identity. An OR::GFP fusion protein is present in axons, consistent with a direct function of ORs in axon guidance. When the OR coding region is deleted, we observe OSNs that coexpress other ORs that function in odorant reception and axonal identity. It remains unclear if such coexpression is normally prevented by negative feedback on OR gene choice. A drastic reduction in OR protein level produces axonal coalescence into novel, remote glomeruli. By contrast, chimeric ORs and ORs with minor mutations perturb axon outgrowth. Strikingly, the beta2 adrenergic receptor can substitute for an OR in glomerular formation when expressed from an OR locus. Thus, ORs have not evolved a unique function in axon guidance.     

Conditional cell ablation via diphtheria toxin reveals distinct requirements for the basal plate in the regional identity of diencephalic subpopulations

The mammalian diencephalon is the caudal derivative of the embryonic forebrain. Early events in diencephalic regionalization include its subdivision along the dorsoventral and anteroposterior axes. The prosomeric model by Puelles and Rubenstein (1993) suggests that the alar plate of the posterior diencephalon is partitioned into three different prosomeres (designated p1–p3), which develop into the pretectum, thalamus, and prethalamus, respectively. Here, we report the developmental consequences of genetic ablation of cell populations from the diencephalic basal plate. The strategy for conditionally regulated cell ablation is based on the targeted expression of the diphtheria toxin gene (DTA) to the diencephalic basal plate via tamoxifen‐ induced, Cre‐mediated recombination of the ROSA^DTA allele. We show that activation of DTA leads to specific cell loss in the basal plate of the posterior diencephalon, and disrupted early regionalization of distinct alar territories. In the basal plate‐deficient embryos, the p1 alar plate exhibited reduced expression of subtype‐specific markers in the pretectum, whereas p2 alar plate failed to further subdivide into two discrete thalamic subpopulations. We also show that these defects lead to abnormal nuclear organization at later developmental stages. Our data have implications for increased understanding of the interactive roles between discrete diencephalic compartments. genesis 53:356–365, 2015. © 2015 Wiley Periodicals, Inc.     

Purification of diphtheria toxin receptor from Vero cells

Diphtheria toxin receptor has been solubilized from Vero cell membranes with octyl beta-D-glucoside. CRM197, the product of a mutated diphtheria toxin gene, was used for the identification of the receptor. The binding activity of the solubilized receptor was assayed by precipitating the receptor with acetone in the presence of phospholipids and carrier proteins. The solubilized receptor was purified by the combination of several chromatographic steps in the presence of the detergent, resulting in about a 10(6)-fold purification of the receptor. The purified receptor showed essentially a single band of 14.5 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When partially purified receptor fractions were subjected to ligand blotting analysis using 125I-CRM197 as the probe, the 14.5-kDa protein and a few minor protein bands were identified as diphtheria toxin-binding molecules. These results show clearly that the 14.5-kDa protein is the diphtheria toxin receptor, or at least the major diphtheria toxin-binding molecule. When partially purified receptor was applied to a Sephacryl S-300 column in the presence of detergent, the receptor was eluted in the fractions corresponding to the 60-90-kDa size range. This suggests that the protein forms a complex with itself or with another protein.     

Extension of juxtamembrane domain of diphtheria toxin receptor arrests translocation of diphtheria toxin fragment A into cytosol

Diphtheria toxin (DT) binds to the EGF-like domain of the DT receptor (DTR), followed by internalization and translocation of the enzymatically active fragment A into the cytosol. The juxtamembrane domain (JM) of the DTR is the linker domain connecting the transmembrane and EGF-like domains. We constructed mutants of DTRs with altered JMs and studied their abilities for DT intoxication. Although DTR mutants with extended JMs showed normal DT binding activity, the cells expressing the mutants showed both reduced translocation of DT fragment A into the cytosol and reduced sensitivity to DT, when compared with cells expressing wild-type DTR. These results indicate that the JM contributes to DT intoxication by providing a space appropriate for the interaction of DT with the cell membrane. The present study also indicates that consideration of epitopes of an immunotoxins would be an important factor in the design of potent immunotoxins.     

Chemotaxis behavior mediated by single larval olfactory neurons in Drosophila

BACKGROUND: Odorant receptors (ORs) are thought to act in a combinatorial fashion, in which odor identity is encoded by the activation of a subset of ORs and the olfactory sensory neurons (OSNs) that express them. The extent to which a single OR contributes to chemotaxis behavior is not known. We investigated this question in Drosophila larvae, which represent a powerful genetic system to analyze the contribution of individual OSNs to odor coding. RESULTS: We identify 25 larval OR genes expressed in 21 OSNs and generate genetic tools that allow us to engineer larvae missing a single OSN or having only a single or a pair of functional OSNs. Ablation of single OSNs disrupts chemotaxis behavior to a small subset of the odors tested. Larvae with only a single functional OSN are able to chemotax robustly, demonstrating that chemotaxis is possible in the absence of the remaining elements of the combinatorial code. We provide behavioral evidence that an OSN not sufficient to support chemotaxis behavior alone can act in a combinatorial fashion to enhance chemotaxis along with a second OSN. CONCLUSIONS: We conclude that there is extensive functional redundancy in the olfactory system, such that a given OSN is necessary and sufficient for the perception of only a subset of odors. This study is the first behavioral demonstration that formation of olfactory percepts involves the combinatorial integration of information transmitted by multiple ORs.     

Coupling between sensory neurons in the olfactory epithelium

Coupling of olfactory sensory neurons (OSNs) in the olfactory epithelium of Necturus maculosus was demonstrated by dye-transfer with Lucifer yellow CH; however, the incidence of dye-transfer was low. Immunocytochemistry and Western blot analysis indicated that connexin 43, a gap junction channel subunit, was widely expressed by cells in the olfactory epithelium. Electrical coupling by presumptive gap junctions was assessed using electrophysiological recordings, heptanol block, tracer-uptake through hemi-junctions, and tracer-injection into tissue whole-mounts. Coupling, which involved pairs of OSNs only, was detected in approximately 3-10% of the OSN population; there was no evidence that OSNs were coupled into extended neural syncitia. These results suggest that coupling of OSNs by gap junctions is unlikely to have a general role in olfactory responses by mature (odor responsive) OSNs. Instead, the incidence of inter-neuronal coupling was small, similar to the fraction of immature OSNs, suggesting a possible role of gap junctions in the continual turnover and development of OSNs or possibly their senescence.     

Molecular characterization of key diphtheria toxin:receptor interactions

The major amino acids necessary for diphtheria toxin (DT) binding to its receptor have been identified previously. Studies by W. H. Shen et al. (J. Biol. Chem. 269, 29077-29084, 1994) and by J. H. Cha et al. (Mol. Microbiol. 29 (5), 1275-1284, 1998) suggested that the positively charged nature of the single amino acid residue, (516)Lys of DT, is crucial for binding to the DT receptor, whereas the negatively charged (141)Glu of the DT receptor is the most important residue for toxin binding. Here, we hypothesize that key interactions occur between these two oppositely charged amino acid residues. Reciprocal substitution of the residues at these positions between the toxin and the receptor was performed, which resulted in a partial reconstitution of the toxin:receptor interaction. This study provides the first biological data that characterizes the specific interaction of these two key residues with each other and also the additional interactions between other positively charged residues of DT and (141)Glu of the DT receptor.     

Gal‐NCAM is a differentially expressed marker for mature sensory neurons in the rat olfactory system

A new monoclonal antibody, 2E11, was produced by immunizing mice with the microsomal fraction of rat accessory olfactory bulb cells. This IgM recognizes a previously described complex α‐galactosyl containing glycolipid, as well as N‐linked glycoproteins at 170 and 210 kD. These proteins correspond to a new nerve cell adhesion molecule (NCAM) glycoform, Gal‐NCAM, which contains a blood group B‐like oligosaccharide. During embryonic development, the 2E11 epitope is expressed by a subset of mature olfactory sensory neurons randomly dispersed throughout the olfactory epithelium, whereas in the olfactory bulb, immunostaining is restricted to medial areas of the nerve layer. When compared to PSA‐NCAM, another NCAM glycoform, Gal‐NCAM has a mutually exclusive distribution pattern both in the olfactory epithelium and in the olfactory bulb. We propose a model for the hierarchy of neuronal maturation in the olfactory epithelium, including a switch from PSA‐NCAM expression by immature neurons to the expression of Gal‐NCAM by mature neurons. © 2000 John Wiley & Sons, Inc. J Neurobiol 43: 173–185, 2000     

Transgenic mice expressing the diphtheria toxin receptor are sensitive to the toxin

Whereas diphtheria and the mechanism of action of diphtheria toxin, the bacterial molecule that induces the disease, have been studied and understood for some time, the receptor that allows animal cells to bind the toxin escaped identification until recently. The receptor was identified by its ability to confer toxin-sensitivity to mouse cells, which are normally toxin-resistant. Although mice are also naturally resistant, we now demonstrate that transgenic mice expressing the diphtheria toxin receptor are as sensitive to the toxin as are humans and other toxin-sensitive animals. These transgenic mice provide a suitable model for studying modern antidotes for diphtheria.     

Odorant receptor gene choice in olfactory sensory neurons: the one receptor-one neuron hypothesis revisited

Designed for general chemical recognition, the mammalian olfactory system shares many similarities with the immune system. Among these is the popular notion that a single olfactory sensory neuron expresses a single odorant receptor gene, while all other approximately 1000 genes of this type remain silent. Here, I examine the evidence supporting the one receptor-one neuron hypothesis. I conclude that, contrary to widespread belief, it is far from being proven. I propose an hypothesis of a developmental phase of oligogenic expression that is followed by positive and negative selection resulting usually in cells with one expressed receptor. Curiously, selective processes are well established and widely accepted for lymphocytes, but these concepts are essentially ignored for olfactory sensory neurons, despite the analogies that are frequently made between these two systems. More attention must be paid to odorant receptor gene choice and expression during development and neuronal differentiation.