By comparison with the rest of the nervous system, the olfactory epithelium has an unparalleled ability to renew and repair itself throughout life. However, the identity and capacity of the various types of progenitor cells that underlie that ability are not well established. We used selective isolation, transplantation, and engraftment of various types of marker-labeled cells into the epithelium of methyl bromide-lesioned, unmarked host mice to dissect progenitor cell capacity. Globose basal cells were purified from other potential progenitors using the monoclonal antibody GBC-2 (GBC, globose basal cell) and fluorescence activated cell sorting. Transplanted globose basal cells engraft and, in aggregate, give rise to globose basal cells, neurons, sustentacular cells, and several other kinds of non-neuronal cells. Individual clones, derived from single engrafted globose basal cells, can consist of a mixture of neurons and non-neuronal cells, only neurons, or only non-neuronal cells. Neurons that arise after transplantation mature to the point of expressing odorant receptors and olfactory marker protein and of projecting axons to the olfactory bulb. In contrast, other kinds of epithelial cells are neither neurogenic nor multipotent. For example, sustentacular and duct cells give rise only to themselves after transplantation. Furthermore, horizontal basal cells do not engraft in mice, in which the endogenous population is spared after lesion. Thus, some subtype(s) of GBC is a multipotent progenitor cell, whose multipotency is activated after destruction of both neurons and non-neuronal cells. The results suggest that progenitor cell transplantation may prove useful as a therapeutic modality as well as an analytical tool.

The technique of in vitro selection was used to generate variants of the human immunodeficiency virus type 1 that are resistant to 2',3'-dideoxycytidine (ddC). Most of the pol regions of such viruses, including the complete reverse transcriptase open reading frame and portions of flanking protease and integrase genes, were cloned and sequenced, using PCR-based procedures. Mutations were variously detected at amino acid site 65 (Lys-*Arg; AAA--AGA) and at a previously reported codon, site 184 (Met->Val; ATG->GTG). We introduced the site 65 mutation into the pol gene of infectious, cloned HxB2-D DNA by site-directed mutagenesis in order to confirm by viral replication assay the importance of this site in conferring resistance to ddC. The recombinant virus possessed greater than 10-fold resistance against this compound in conmparison with parental HxB2-D. Cross-resistance of approximately 20-and 3-fold, respectively, was detectable against the (-) enantiomer of 2',3'-dideoxy-3'-thiacytidine and 2',3'-dideoxyinosine but not against 3'-azido-3'-deoxythymidine. Combinations of the site 65 and 184 mutations did not yield levels of resistance higher than those attained with the site 65 mutation alone. The presence of the site 65 mutation was confirmed by PCR analysis of peripheral blood mononuclear cells from patients on long-term ddC therapy in 4 of 11 cases tested. Viruses that possessed a ddC resistance phenotype were isolated from subjects whose viruses contained the site 65 mutation in each of four instances. Four of these clinical samples were also demonstrated to possess the Met-184-Wal mutation, and one of them possessed both the Lys-65->Arg and Met-184-Wal substitutions.Direct cloning and sequencing revealed the site 65 mutation in viruses isolated from these individuals.

Lesions of the olfactory periphery provide a means for examining the reconstitution of a diverse and highly regulated population of sensory neurons and the growth, en masse, of nascent axons to the bulb. The olfactory epithelium and its projection onto the bulb are reconstituted after ablation by methyl bromide gas, and some measure of olfactory function is restored. The extent to which the system regenerates the full repertoire of odorant receptor-expressing neurons, particularly their spatially restricted distribution across the epithelial sheet, is unknown, however, and altered odorant receptor expression might contribute to the persistent distortion of odorant quality that is observed in the lesioned-recovered animals. To address the question of receptor expression in the recovered epithelium, we performed in situ hybridization with digoxigenin-labeled riboprobes for eight odorant receptors on the olfactory epithelium from unilaterally methyl bromide-lesioned and control rats. The data demonstrate that the distribution of sensory neuron types, as identified and defined by odorant receptor expression, is restored to normal or nearly so by 3 months after lesion. Likewise, the numbers of probe-labeled neurons in the lesioned-recovered epithelium are nearly equivalent to the unlesioned side at this time. Finally, our evidence suggests that odorant receptors are distributed in multiple overlapping bands in the normal, unlesioned, and lesioned-recovered epithelium rather than in the conventionally accepted three or four zones. Thus, the primary sensory elements required for functional recovery of the olfactory system after damage are restored, and altered function implies the persistence of a more central failure in regeneration.

Mammalian olfactory epithelium produces new neurons rapidly throughout adulthood. Here, we demonstrate that precursor cells harvested from the adult olfactory epithelium, when transplanted into the nasal mucosa of host rats exposed previously to an olfactotoxic gas, engraft and participate in neuroepithelial reconstitution. In contrast to their normal neuronal fate in situ, grafted precursors harvested from bulbectomized donors produced non-neuronal cells as well as neurons. These results demonstrate that epithelial precursors activated following olfactory bulbectomy are not irreversibly committed to making neurons. Thus, olfactory progenitors are subject to a form of feedback control in vivo that regulates the types of cells that they produce within a broader-than-neuronal repertoire.

Developing olfactory sensory neurons are guided to the glomeruli of the olfactory bulb by an increasingly stringent process that is influenced by expression of odorant receptors, cell adhesion molecules (CAMs), and other kinds of signaling cascades. A fundamental feature of the projection is the connecting of broad zones in the epithelium to broad zones in the bulb, also termed rhinotopy. One molecule that parallels and may aid neurons in establishing rhinotopy is the mammalian homologue of fasciclin II (OCAM/mamFas II; also known as RNCAM and NCAM-2), an immunoglobulin superfamily CAM that is differentially expressed in the developing and mature olfactory epithelium (OE): Axons elaborated by ventral and lateral epithelium express the protein at high levels, whereas dorsomedial axons express little or no OCAM/mamFas II. Our investigation has demonstrated that OCAM/mamFas II is detectable early in the development of the rat OE. mRNA is evident on RT-PCR and in situ hybridization by E12.5, and protein is apparent by immunohistochemistry by E13.5. By using a tissue culture system that separates ventral septal epithelium (OCAM/mamFas II-positive) from dorsal (OCAM/mamFas II-negative), we find that explants maintain protein expression levels in vitro that are characteristic of the phenotype at the original location in vivo. At least some neurons are born in culture, suggesting that any cues that direct differential expression are also maintained in vitro. Finally, high OCAM/mamFas II expression correlates with increased growth and fasciculation of olfactory axons in vitro. These data and the similarity between OCAM/mamFas II, on the one hand, and fasciclin II and NCAM, on the other, suggest that OCAM/mamFas II might play a role in growth and fasciculation of primary olfactory axons during development of the projection.