Analysis of neurogenesis in a mammalian neuroepithelium: Proliferation and differentiation of an olfactory neuron precursor in vitro

: Development of a culture system for mammalian olfactory epithelium has permitted the process of neurogenesis to be examined in vitro. Antibody markers allowing the unambiguous identification of putative neuroepithelial stem cells (keratin + basal cells) and differentiated neurons (N-CAM + olfactory receptor neurons) are described. In combination with [ 3 H]thymidine uptake analysis, these antibodies have been used to characterize the existence, proliferation, and differentiation of the immediate neuronal precursor in this system. This cell is distinct from basal cells and rapidly sorts out from them, dividing as it migrates. Data are presented which suggest that the precursor follows a simple lineage program, dividing to give rise to two N-CAM + daughter neurons. Although this precursor efficiently generates neurons in defined medium, neurogenesis Summary Development of a culture system for mammalian olfactory epithelium has permitted the process of neuro genesis to be examined in vitro. Antibody markers allowing the unambiguous identification of putative neuroepitheiiil stem cells (keratin+ basal cells) and differentiated neurons (N-CAM+ olfactory receptor neurons) are described. In combination with t3H]thy-midine uptake analysis, these antibodies have been used to characterize the existence, proliferation, and differentiation of the immediate neuronal precursor in this system. This cell is distinct from basal cells and rapidly sorts out from them, dividing as it migrates. Data are presented which suggest that the precursor follows a simple lineage program, dividing to give rise to two N-CAM+ daughter neurons. Although this precursor efficiently generates neurons in defined medium, neurogenesis subsequently ceases because new precursors are not produced, suggesting that epigenetic factors may regulate continual nemogenesis in this system.


Introduction
.  Figure  1A). In 15 day mouse embryos, this antiserum stains a large number of cells, many of which appear to span the entire width of the developing OE ( Figure  1C). This is consistent with the findings of Smart (1971), who has shown that the nuclei to keratins stains the layer of basal cells immediately adjacent to the basement membrane (A); short processes of some of these cells can be seen extending into more apical regions of the epithelium. In OE of the same age, monoclonal anti-N-CAM (B) exclusively stains neurons in the extensive receptor neuron layer fn), but does not stain cells in the basal cell layer (b) or the luminal sustentacular cell layer fs). N-CAM is present on the entire surface of olfactory receptor neurons: the apical dendrites (arrow) and the bundles of axons (asterisk) of the receptor neurons are both brightly stained. In El5 OE in situ, anti-keratins antiserum stains about half of the cells (C; see Table 1 also), many of which extend from the basal to the apical surface. This staining pattern is consistent with the observation that, in the OE at this age, the nuclei of proliferating cells are present throughout the basal-apical extent of the epithelium and only later become restricted to the basal layer (see Smart, 1971 Figure  1D). In addition, a large proportion of total cells in the OE are proliferating at this age (Smart, 1971;Cuschieri and Bannister, 1975) -Fraser, 1985). Cultures were then incubated for 30 min at 37"C, rinsed twice in HBSS, and fixed immediately in 3.7% formaldehyde. (A) shows a culture grown in normal calcium medium for 16 hr. In this micrograph the epithelial sheet fe), which is fluorescent, is out of the plane of focus, which was chosen to show the bundles of round cells lying on top of the epithelial sheet. Processes extending between these bundles of cells (arrow) suggest that they contain at least some neurons. At 16 hr in LCM (81,   Cultures were pulsed with f3H]thymidine (5 pCi/ml, 80 Ci/mmol) for 2 hr at 10 hr after plating. Some cultures were fixed immediately, and the rest were re-fed with medium containing an excess of cold thymidine.
At 6 hr intervals, duplicate cultures were fixed, stained with monoclonal anti-N-CAM, and processed for autoradiography as described  (8); it is labeled with [jH]thymidine.
Other clusters of silver grains, also over the nuclei of N-CAM-migrating cells, can be seen as well. Bar, 50 urn.  Figure  6. The two N-CAM+ neurons seen in Figure  6A Figure  3A with Figure  5). in these cultures ( Figure  6), reinforcing the notion that the proposed lineage program is correct. Second, selective cell death does not seem to be a problem.
In Figure  3A,  et al., 1988) and visualized by rabbit anti-sheep biotin (1:2CO) followed by avidin-alkaline phosphatase (as specified in Vectastain ABC-AP kit). Negative controls for this staining were performed using exactly the same methods, except that a 25 &ml solution of sheep IgG (Sigma) was used in place of the primary antibody. Tissue Sections For staining with antiserum to keratins, embryos were fixed by immersion and postnatal mice by perfusion with 4% paraformaldehyde. Embryos or dissected postnatal turbinates were embedded in gelatin (Lallier and Bronner-Fraser, 1988) and cryostat-sectioned at 14 pm. Sections were permeabilized with Triton X-100 (0.2%) prior to staining and were visualized with the secondary antibodies listed above. To avoid cross-reactivity of secondary antisera with endogenous mouse immunoglobulin during staining with monoclonal anti-N-CAM, AC1 IgG was purified from ascites fluid by protein A affinity chromatography (Bio-Rad MAPS II system) and coupled to long-chain biotin (Pierce LC-NHS-biotin) according to the supplier's instructions.
Biotinylated anti-N-CAM was visualized with Texas red avidin (1 :lOOO). For anti-N-CAM staining, embryos and dissected postnatal turbinates were fixed by freezing in isopentane chilled on dry ice, then rehydrated by freeze-substitution, embedded in gelatin, and sectioned as above.
Analysis of Dissociated Cells Cultures were rinsed in CMF-PBS, then incubated for 15 min in 1 ml of CMF-PBS containing 1 mg/ml trypsin (Sigma type III-S) and 1 mM EDTA. Enzymatic digestion was stopped by the addition of 250 pg of soybean trypsin inhibitor.
Cells were pelleted for 10 min at 100 x g, resuspended to a single-cell suspension by extensive trituration in cold CMF-PBS, and plated for 1 hr at 4OC on glass slides that had been treated with 3-aminopropyl-triethoxysilane (Berger, 1986) and activated with 0.25% glutaraldehyde. Cells were then fixed onto slides by incubation in acetone for 5 min at room temperature. lmmunocytochemistry was performed as described above for cultured cells. Cells were counted under phase-contrast and epifluorescence optics using a 40x objective. For each data point, dissociated cells from two cultures were counted and the mean and range were calculated.
For each culture, a minimum of 200 cells in a minimum of 20 fields were scored.

Autoradiographic
Analysis of Cell Proliferation For pulse-fix analysis, cultures were incubated at designated times for 2 hr in 5 pCi/ml [3H]thymidine (80 Ci/mmol; New England Nuclear), then fixed prior to immunocytochemical processing. Follow-ing incubation with secondary antibody, coverslips were dehydrated and reverse-mounted on microscope slides, dipped in NTB2 emulsion diluted 1 :l in water, exposed for 48 hr at -7O"C, and developed in D-19 developer. Nuclei were stained with Hoechst 33258 fbisbenztmide, 1 uglml). For pulse-chase analysis, cultures were treated exactly as above, except that following the 2 hr incubation with [3H]thymidine, cultures were re-fed with complete LCM containing 50 pM unlabeled thymidine and incubated for designated times prior to fixation and subsequent processing. Quantitative analysis was performed by examining processed cultures with a 63x objective.
Cells were scored for morphology, fluorescence, and the presence of silver grains over their nuclei. Each experiment was performed a minimum of 3 times; each figure shows data taken from a single experiment.
For each data point, duplicate cultures were examined; for each culture, a minimum of 1CO labeled cells in a minimum of 15 fields were scored.