Apparent apoptotic cell death in the olfactory epithelium of adult rodents: Death occurs at different developmental stages

THE JOURNAL OF COMPARATIVE NEUROLOGY 372457464 (1996)

Apparent Apoptotic Cell Death in the Olfactory Epithelium of Adult Rodents:

Death Occurs at Different Developmental Stages

THOMAS J. MAHALIK Department of Cellular and Structural Biology, University of Colorado Health Sciences

Center, Denver, Colorado 80262

ABSTRACT In the olfactory epithelium of adult rodent, receptor neurons are generated continually.

Despite the ongoing generation of new neurons, no corresponding increase occurs in the thickness of the mature olfactory epithelium. Thus, epithelial cell death must occur to offset the continual generation of new cells. In the present study, a sensitive method to label nicked DNA in dying cells was combined with immunocytochemistry to determine the identity of dying olfactory cells. In addition, the positions of putative apoptotic cells were mapped to provide additional information about the identity of dying cells.

Double labeling experiments revealed that each of the olfactory cell types, i.e., basal cells (keratin-positive), immature neurons (GAP43-positive) and mature receptor neurons (olfactory marker protein (OMPI-positive), were positive for fragmented DNA, suggesting that they undergo apoptotic cell death. The results of the mapping study suggest that apoptotic cell death occurs primarily among GAP43-positive neurons. D 1996 Wiley-rim, Inc.

Indexing terms: immature neurons, mature neurons, programmed cell death

The generation and death of cells in the rodent olfactory system resembles the generation and death of cells during the development of the central nervous system (CNS). In the CNS, germinal cells give rise to undifferentiated stem cells. These undifferentiated cells undergo asymmetric mitosis in which one cell returns to the cell cycle and the other becomes a postmitotic undifferentiated neuron (Jacob- son, 1991). As these primitive neurons move away from the germinal zone, they differentiate into immature neurons and begin to extend axons. As the axons of differentiating neurons reach their targets, up to 50% undergo cell death (Oppenheim, 1991). Neuronal cell death in the CNS may function to control neuronal number and to match the size of a projection to the size of the target (Oppenheim, 1991). The most likely cause of neuronal death is the failure of neurons to successfully compete for limiting amounts of target-derived growth factors (Korsching, 1993).

As in the developing CNS, olfactory neurogenesis occurs in several distinct stages. Horizontal basal cells are in direct contact with the basal lamina; horizontal basal cells are only occasionally labeled after the injection of tritiated thymidine (McKay-Sim and Kittel, 1991). Globose basal cells, which are located above the horizontal basal cells, are labeled at short intervals after the injection of tritiated thymidine (Hinds et al., 1984; McKay-Sim and Kittel, 1991;

Schwob et al., 1992). Moreover, the number of globose basal cells increases after lesions of the olfactory bulb (Schwartz- Levey et al., 1991). Evidence from in vivo (Graziadei and Graziadei, 1979; Mackay-Sim and Kittel, 1991) experiments suggests that the horizontal basal cells undergo asymmetric divisions to produce one horizontal basal cell and one neuronal precursor cell. In vitro experiments suggest that the neuronal precursor divides symmetrically to generate two undifferentiated neurons (Calof and Chi- karaishi, 1989). As the immature neurons differentiate, they migrate away from the basal layer of the epithelium (Graziadei and Graziadei, 1979; Hinds et al., 1984; Mackay- Sim and Kittel, 1991). Thus, newly generated cells are reportedly close to the basal layer, while the oldest neurons are close to the superficial layer of the epithelium.

At least three stages of olfactory cell development can be distinguished with antibody markers. Horizontal basal cells can be identified with a wide spectrum antibody against keratins (Calof and Chikaraishi, 1989). GAP43 is expressed in differentiated immature neurons both in vivo (Verhaa-

Accepted April 22, 1996 Address reprint requests to Thomas J. Mahalik, Department of Cellular

and Structural Biology, U. Colorado Health Sciences Center, 4200 E. 9th Ave., Denver, CO 80262.

O 1996 WILEY-LISS, INC.

458 TJ. MAHALIK

gen, 1989; Schwob et al., 1992) and in vitro (Calof and Chikaraishi, 1989), and antibodies against olfactory marker protein (OMP) can be used to identify mature receptors. Single immunochemical markers do not yet exist for the neural progenitor cells, or for undifferentiated neurons. The undifferentiated neurons can be identified in double labeling experiments because they are NCAM-positive, but negative for GAP43 (Schwob et al., 1992). Combined immunochemical and thymidine labeling experiments have shown that epithelial neurons make the transition from basal cells to immature neurons between days 0 and 5 after the last cell division. The transition from GAP43-positive immature neurons to OMP-positive mature neurons occurs between day 5 and day 14 after a cell is generated (Schwob et al., 1992). This pattern of neurogenesis and differentiation takes place throughout the life of the rodent.

In mature rodents, the thickness of the olfactory epithelium does not increase. This means that the continual generation of new olfactory cells must be offset by ongoing death (Farbman, 1990). Indeed, initial thymidine labeling experiments suggested that the average lifespan of mature olfactory neurons was approximately 30 days (Graziadei and Graziadei, 1979). This observation conflicted with what was known about neuronal death in the adult CNS of vertebrates, where the lifespan of neurons is not prepro- grammed (Oppenheim, 1991; Jacobson, 1991).

More recent work, however, has shown that under appropriate conditions, mature rodent olfactory receptors can survive as long as one year in the rodent (Hinds et al., 1984). Nevertheless, most of the cell death in the olfactory epithelium appears to occur in the population of immature receptors within two weeks of their birth (Mackay-Sim and Kittel, 1991; Schwob et al., 1992). As in the CNS, the survival of olfactory neurons depends upon trophic interac- tions with their target tissue (Schwob et al., 1992). In the absence of olfactory bulb, most newly generated olfactory neurons die within 7-10 days (Schwob et al., 1992; Carr and Farbman, 1992); even with an intact bulb, most newly generated cells die within 30 days (Graziadei and Graziadei, 1979; Schwob et al., 1992). In the absence of the olfactory bulb, a small percentage of newly generated neurons differentiate into mature-looking, OMP-positive receptors (Schwob et al., 1992). Thus, the survival and differentiation of olfactory neurons is enhanced by an intact olfactory bulb, but at least some olfactory neurons are not absolutely dependent upon the bulb for trophic support.

In the present study, in situ labeling of fragmented DNA was used to map the distribution of apoptotic cells in the olfactory epithelium of adult rats. The distribution of apoptotic cells was then compared with the distribution of GAP43- and OMP-positive cells. In addition, in situ labeling of apoptotic cells was combined with immunocytochemistry to determine whether apoptosis occurs at each of the different stages of olfactory neuron development.

MATERIALS AND METHODS Twelve adult (250-300 g) Sprague-Dawley rats (Harlan)

were used in the current study. Rats were maintained on a day-night cycle (6 A.M. :on; 6 P.M. off, with standard air (i.e., non-filtered), and received food and water ad libitum. The rats were deeply anesthetized with sodium pentobarbital (100 mgikg) and intracardially perfused with 4% parafor- maldehyde in 0.1 M phosphate buffer (pH 7.4). As in our

previous work (e.g., Mahalik et al., 1995), the rats were not perfused with saline or buffer before the fixative. Olfactory epithelia were dissected from the cranium and postfixed for an additional 6-8 hours. The epithelia were then placed in 0.1 M phosphate buffer containing 15% sucrose for an additional 8-12 hours. The blocks were embedded and frozen in O.C.T. embedding medium and stored at -80°C until use. The blocks were sectioned at 8 pm and mounted onto Superfrost Plus (Fisher) slides and stored at -80°C.

For immunocytochemistry alone, the slides were warmed for 15 minutes and then were rinsed in phosphate-buffered saline (PBS) containing 0.3% Triton X-100 for 15 minutes. The sections were incubated in rabbit anti-keratin (1:400, Dako), mouse anti-GAP43 (1:1,000, Boehringer) or goat- anti-OMP (1:5,000) in PBS containing 2% normal donkey serum for 24 to 48 hours at 4°C. After several rinses in PBS, the sections were placed in the appropriate biotin-labeled secondary antiserum (made in donkey, Jackson Labs) for 1-2 hours at room temperature. The slides were then placed in PBS containing an avidin complexed to biotinylated horseradish peroxidase (HRP; ABC) diluted 1 : l O O for 1-2 hours at room temperature. The slides then were placed in 3,3'-diaminobenzidine (0.0075%) and hydrogen peroxide (0.0025%) in PBS for 10 minutes. After several washes in PBS, the slides were dehydrated in an ethanol series and then defatted in histoclear.

For combined immunocytochemistry and in situ labeling of fragmented DNA (TUNEL; Gavrielli et al., 19921, slides were warmed and placed in 100% methanol for 15 minutes, in 3% hydrogen peroxide for 15 minutes, followed by a 15-minute incubation in PBS containing 0.3% Triton X-100. After several rinses in distilled water the sections were reacted with terminal transferase (40 UnitsilOO p1) and biotinylated deoxyUTP (2 nmolesi 100 p1 reaction; Boeh- ringer) for 2 hours at 37°C. The sections were washed in PBS and reacted with the ABC complex (1:100, Vectastain) for 1 hour, and then reacted in 0.0075% 3,3'-diaminobenzidine and 0.0025% hydrogen peroxide in PBS. The slides were washed several times in PBS and prepared for immunocytochemistry as described above. No blocking agents such as normal serum were used to preincubate the tissue before exposure to the primary antisera. Protease or acid treatment was not necessary before TUNEL labeling because the tissue was pretreated in PBS with 0.3% Triton x-100.

Double-labeled material was viewed and imaged with a BioRad MRC 600 scanning confocal microscope, equipped for fluorescence and brightfield confocal microscopy. Counts and the positions of labeled cells were made manually with an Olympus BH-2 microscope. The positions of labeled cells are expressed as a percentage of epithelial width.

The Adobe Photoshop Program (Adobe Systems, Moun- tain View, CA) was used to construct composite images (Figs. 5 and 61, to place arrows and letters on the confocal images, and to adjust the brightness and contrast. Photo- shop was also used to resize images and low pass filters were used to remove noise. No other image processing was carried out. The original unprocessed images are available upon request.

RESULTS TUNEL-positive cells were present throughout the width

of the olfactory epithelium (Fig. 1, arrows); however, the density of labeled cells was greater in the lower two-thirds

OLFACTORYCELLDEATH 459

Fig. 1. Brightfield micrographs of the rat olfactory epithelium. Several TUNEL-labeled cells are shown (arrows). The TUNEL-labeled cells were distributed across the width of the epithelium. Arrowheads in A and B highlight punctate reaction product which may indicate that cells are in the advanced stages of apoptosis (basal lamina, BL). Asterisks in A and B mark the surface of the epithelium.

of the epithelium than in the upper one-third. There was no apparent difference in the density of labeled cells when the septal mucosa was compared with the mucosa in the olfactory turbinates.

In the positive cells, the peroxidase reaction product was usually present at the periphery of cell nuclei (Fig. 2A and B, arrows). This distribution of label is similar to the distribution of basophilic dyes when they are used to stain apoptotic cells (e.g., Owens et al., 1995). Occasionally, several small globules of reaction product were observed (Fig. 2A and B, arrowheads); these globules presumably represent labeled DNA present in apoptotic bodies. In most cases, single TUNEL-positive cells were observed. How- ever, in several cases, 2 TUNEL-positive cells were adjacent, or were in contact with one another. Clusters containing more than 2 cells were never observed. These doublets were typically located within 1-2 cell diameters of the basal layer of the epithelium.

Immunocytochemistry revealed that there was a difference in the distribution of GAP43-, OMP- and keratin- positive cells. As expected from previous work (Schwob et al., 1992), keratin-positive cells were present in a single layer immediately in contact with the basal lamina (Fig. 3A). The keratin-positive cells were ovoid with their major axis oriented in the horizontal plane. GAP43-positive cells were located superficial to the layer of keratin-positive cells (Fig. 3B). GAP43-positive cells possessed unlabeled nuclei surrounded by thin rims of immunopositive cytoplasm. Occasionally, a GAP43-positive apical process made its way to the surface of the epithelium (Fig. 3B).

OMP-positive cells were typically located more superfi- cially than the GAP43-labeled neurons, although there was substantial overlap between the 2 cell types. OMP-positive cells were round, had a thin immunopositive cytoplasm and

prominent unlabeled nuclei (Fig. 3C). OMP-positive processes often coursed to the surface of the epithelium.

To compare the distributions of GAP43- and OMP- positive cells with the distribution of apoptotic, TUNEL- positive cells, the distance that a labeled cell was from the basal lamina was measured and expressed as percent of epithelial width. More than half of the TUNEL-positive cells were distributed in the lower one-third of the epithelium (Fig. 4A). Nearly 15% of the TUNEL-positive cells were located in the deepest part of the epithelium (Fig. 4A). In this part of the epithelium, no OMP-positive cells (Fig. 4 0 , and only 5% of the GAP43-positive cells (Fig. 4B) were present. At 1 1 4 0 % of the epithelial width, there was a considerable overlap between the TUNEL-positive and GAP43-positive cells (compare Fig. 4A and 4B).

The distribution of the OMP-positive cells (Fig. 4C) was bimodal: most of the OMP cells were located in the middle part of the epithelium, while 20% of the cells were closer to the epithelial surface (Fig. 4). The positions of keratin- positive cells were not plotted because it has been previ- ously shown (Schwob et al., 1992) that these cells reside in a single layer, immediately adjacent to the basal lamina.

The data presented in Figure 4 suggested that with respect to their distribution in the epithelium, there was an overlap among apoptotic cells, GAP43-positive cells and basal cells. There was much less overlap between OMP- positive cells and TUNEL-positive cells, however. Thus, cell death appears more likely in basal cells and in GAP43- positive, immature neurons than in mature receptor cells. These correlative data do not address the question of whether cell death occurs in each of these cell types. To address this issue, we combined TUNEL staining with immunocytochemistry for keratin, GAP43 or OMP.

460 TJ. MAHALIK

Fig. 2. High power micrographs of TUNEL-laheled cells. A A labeled cell adjacent to the basal lamina (arrow). Note the dark reaction product at the periphery of the nucleus. The arrowhead highlights a cell that contains a condensed globule of reaction product. The cell body of this cell, which is faintly visible, surrounds the labeled nucleus. B: A

labeled cell with a thin band of reaction product at the periphery of its nucleus (arrow). Cells with this labeling pattern may bc in the early stages of death. The arrowheads in A & B highlight cells containing globules of reaction product. Asterisks in A and B mark the surface of the epithelium.

Fig. 3. A comparison of the distributions of keratin- (A) GAP43- (B) and olfactory marker protein (OMP) (C) immunoreactivity in the epithelium. A: Keratin-positive basal cells form a single layer above the epithelium. B: GAF’43-positive cells send apical processes to the surface of the epithelium. C: Typical OMP staining of the epithelium. Note the absence of labeling in the deep epithelial layer.

In the double labeling experiments, brightfield and fluo- rescent confocal microscopy was used to image epithelial sections. Figures 5A depicts keratin immunofluorescence in the basal layer of the epithelium; Figure 5B is the corresponding brightfield images depicting TUNEL-positive cells. The composite image in Figure 5C shows the relationship between TUNEL labeling and keratin immunofluorescence. In Figure 5C, TUNEL reaction product is present in the nucleus of a keratin-positive cell (Fig. 5C, arrow) and in keratin negative cell (Fig. 5C, arrowhead). It was more likely to find TUNEL-positive cells above the basal layer, than in the basal layer itself.

The panels in Figure 6 show that GAP43 cells also underwent apoptotic cell death. Figure 6A shows a GAP- immunoreactive cell, one of which (Fig. 6A, arrow) is TUNEL-positive (Fig. 6B, arrow). The composite image in Figure 6C shows that the TUNEL reaction product (shown in red) was restricted to the cell’s nucleus.

Apoptotic OMP-immunoreactive cells are presented in Figure 6D-F. In Figure 6D there is an OMP-positive cell (Fig. 6D, arrow) that is TUNEL-positive (Fig. 6E, arrow). The composite image in Figure 6F shows the outline of the cell and the central location of the TUNEL reaction product (shown in red).


A 100 i2 m E ,g

B

C

0 10 20 30

Proportion of TUNEL Population

0 10 20 30

Proportion of GAY Population

E 0 10 20 1 30

Proportion of OMP Population

Fig. 4. A histogram summarizing the distribution of TUNEL, GAP43 and OMP cells in the epithelium. The proportion of each cell type is plotted as a function of percent epithelial width.

DISCUSSION Summary

In the present study the TUNEL method of Gavrielli et al. (1992) was combined with immunocytochemistry to identify the developmental phenotype of apoptotic cells in the rat olfactory epithelium. Comparison of the distribution of TUNEL-labeled nuclei with the distribution of immunoreactive cells revealed that the TUNEL-positive and GAP43- positive populations of cells overlapped significantly. In comparison, there was a slight overlap between the TUNEL- labeled nuclei and OMP-positive cells. Direct double labeling experiments demonstrated that keratin-positive (basal cells), GAP43-positive (immature neurons) and OMP- positive cells underwent DNA fragmentation indicative of apoptotic cell death.

Methodological issues In the experiments combining immunocytochemistry

and TUNEL labeling, it was evident that numerous TUNEL- positive cells were negative for either keratin, GAP43 or OMP. For example, there were a significant number of TUNEL-positive cells in the basal layer that did not label

Fig. 5. Keratin and TUNEL labeling. A: A single layer of keratin- positive basal cells. The arrow depicts a double-labeled basal cell. B: A brightfield image illustrating TUNEL labeling. The large “blob” of labeling (arrowheads) is located above the basal layer. C: A composite image showing the relationship between TUNEL labeling (grey patches) and keratin labeling. A TUNEL-positive basal cell is highlighted by an arrow.

with the keratin antibody. In addition, when GAP43 was probed, there were many TUNEL-positive cells in the middle and lower portions of the epithelium that were immunochemically negative. A likely explanation is that proteolysis has destroyed many antigens in cells that have reached the late stages of apoptotic death. As a result, it was not possible to determine the actual proportion of cells undergoing apoptosis in three populations of neurons (i.e., keratin, GAP43 and OMP).

462 T.J. MAHALIK

Fig. 6. GAP43-positive neurons undergo apoptosis. A: A GAP43- positive cell (arrow) which is TUNEL-positive. The arrowhead shows the location of GAP43-positive cell that is TUNEL-negative. B: A brightfield confocal image of TUNEL labeling. C: A composite image illustrating the relationship between TUNEL (red patches) and GAP43

labeling. D: An OMP neuron (arrow) that is also TUNEL-positive. E: Brightfield image of TUNEL labeling. F: A composite image showing the relationship between TUNEL labeling and OMP immunoreactivity.

Apoptosis is most likely in immature neurons There was significant overlap between the GAP43-

immunoreactive and TUNEL-labeled cells in the middle and lower parts of the olfactory epithelium. Although this result is correlative, it suggests that immature olfactory neurons are the most likely cells to undergo apoptosis. This finding is consistent with previous studies of neuronal death in the CNS and the peripheral nervous system (PNS)

where cell death is most likely to occur in immature neurons after they have extended their s o n s (Chu-Wang and Oppenheim, 1978).

In other neuronal systems, neuronal death commonly occurs when neurons are innervating their targets. For example, in the chick spinal cord, 40-50% of motor neurons die after axons innervate the limb bud (Chu-Wang and Oppenheim, 1978). Removal of the chick limb bud increases


vitro and TGF-ps increase the differentiation and proliferation of neural progenitors cells (Mahanthappa and Schwart- ing, 1993). Whether these factors have the same action in vivo is unknown; however, the OE contains a TGF-p-like factor (Mahanthappa and Schwarting, 1993). Other factors that control the survival of olfactory basal and neural progenitors have yet to be identified; it is not known whether such factors act via an autocrine or paracrine mechanism as may be the case in other systems (Acheson et al., 1995).

How cell death is regulated in mature olfactory neurons is not known. One explanation of the TUNEL labeling of OMP cells described here, is that fragmented DNA from necrotic cells was labeled, since two recent studies (Portera- Cailiau et al., 1995; Grasl-Kraupp et al., 1995) have shown that in some circumstances, necrotic cells can be labeled with the TUNEL assay. This seems to be an unlikely explanation of the present results because the TUNEL- labeled cells described here were morphologically similar (i.e., possessed condensed chromatin) to apoptotic cells. The present results raise the possibility that, in addition to catastrophic cell death (Wyllie, 1980), the death of mature receptors occurs by other mechanisms. Removal of the olfactory bulb results in the apoptotic cell death of mature receptor cells from 1 to 4 days after the lesion (Costanzo and Graziadei, 1990), suggesting that mature receptor neurons are dependent on bulb-derived trophic factors for their survival. The results presented here do not rule out the possibility that some cell death in the olfactory epithelium occurs without DNA fragmentation (Wood et al., 1993). The apparent apoptotic death of mature receptors in the intact epithelium could result from the failure of some cells to transport sufficient quantities of growth factors. Alternatively, mature receptor cells may become less respon- sive to trophic factors as they age, and thus require greater amounts of these factors for survival. Another possibility is that environmental trauma results in a type of death in mature receptors that resembles apoptosis.

neuronal death to 90% (Oppenheim et al., 1978), while increasing the size of the target decreases cell death (Holly- day and Hamburger, 1976). Similarly, the survival of developing chick retinal neurons depends upon an intact target (Hughes and McCloon, 1979). The relationship between the size of a target and cell death in neurons innervating the target, has led to the idea that target tissues provide limiting amounts of neurotrophic factors to developing neurons (Korsching, 1993).

Immature GAP43-positive neurons innervate the rodent olfactory bulb (Verhaagen et al., 1989) and are dependent on an intact bulb for their survival (Schwob et al., 1992). Removal of the olfactory bulb results in a large increase in olfactory cell death, particularly in the population of immature olfactory receptors. In the intact olfactory system, approximately 41% of newly generated olfactory cells die within 15 days; after bulbectomy, greater than 90% of these cells die (Schwob et al., 1992). These findings, taken together with the results of the current study, suggest that epithelial cells begin to undergo cell death after they have made the transition from GAP43-negative undifferentiated neurons to GAP-positive immature neurons. The results of lesion studies suggest that OE neurons become dependent on the olfactory bulb for survival shortly after they have made this transition. Prior to their expression of GAP43, epithelial cells are much less likely to undergo cell death. Nevertheless, both keratin-positive horizontal basal cells and putative horizontal basal cells undergo cell death. An important issue raised by this finding is the way in which cell death in horizontal basal cells and globose basal cells is controlled.

The horizontal basal cells may be considered slowly dividing stem cells, while the globose basal cells are rapidly dividing neural progenitor cells (Mackay-Sim and Kittel, 1991; Schwob et al., 1992,1994; Caggiano et al., 1994). The present study showed directly that keratin-positive horizontal basal cells underwent apoptotic cell death. Moreover, approximately 12% of all cells with fragmented DNA in the OE were present in deepest layer of the epithelium, where horizontal basal cells are located. It is likely that globose basal cells undergo apoptotic cell death, although it was not possible to demonstrate this directly because no immunochemical markers for globose basal cells exist. Numerous TUNEL-positive cells were located in the cell layer immediately superficial to the basal layer of the epithelium. Cell death in the basal cells populations of neurons may depend on either a program that is cell autonomous (Ellis et al., 1991), or based on the availability of local growth factors.

It seems more likely that the survival of the olfactory basal cells depends upon local growth factors because there is little evidence that cell death in vertebrate neurons is under the exclusive control of an autonomous program (Jacobson et al., 1994). Receptors for FGFl and FGF2 are present in the olfactory epithelium (DeHamer et al., 19941, and several members of the FGF family enhance the proliferation of olfactory progenitor cells (Calof and Chikaraishi, 1989) in explants of olfactory epithelium (Dehamer et al., 1994). I t is not known whether members of the FGF family have the same effect in vivo, or whether FGFs are necessary for the survival of olfactory progenitor cells. Similarly, EGF and TGF-Ps do not affect the survival of basal or neuronal cells, but do affect proliferation and differentiation (Mahanthappa and Schwarting, 1993). EGF increases the proliferation of keratin-positive basal cells in

ACKNOWLEDGMENTS I thank Drs. Thomas E. Finger and Gregory P. Owens for

their comments on this manuscript. This work was sup- ported by an NIH Shannon Award (HS DC01916-01A1 R55) to T.J.M.

LITERATURE CITED Acheson, A,, J.C. Conover, J.P. Fandl, T.M. Dichiara, M. Russell, A. Thadani,

S.P. Squinto, G.D. Yancopoulos, and R.M. Lindsay (1995) A BDNF autocrine loop in adult sensory neurons prevents cell death. Nature 374:450453.

Caggiano, M., J.S. Kauer, and D.D. Hunter (1994) Globose basal cells are neuronal progenitors in the olfactory epithelium: A lineage analysis using a replication incompetent retrovirus. Neuron 13r339-352.

Calof, A.L., and D.M. Chikarishi (1989) Analysis of neurogenesis in a mammalian neuroepithelium: Proliferation and differentiation of an olfactory neuron precursor in vitro. Neuron 3:115-127.

Carr, V.M., and A.I. Farbman (1992) Ablation of the olfactory bulb up- regulates the rate of neurogenesis and induces precocious cell death in olfactory epithelium. Exp. Neurology 115:55-59.

Chu-Wang, I-W., and R.W. Oppenheim (1978) Cell death of motoneurons in the chick embryo spinal cord 11. A quantitative and qualitative analysis of degeneration in the ventral dorsal root, including evidence for axon outgrowth and limb innervation prior to cell death. J. Comp. Neurol. 177:59-86.

464 TJ. MAHALIK

Costanzo, R.M., and P.P.C. Graziadei (1990) A quantitative analysis of changes in the olfactory epithelium following hulbectomy in the ham- ster. J. Comp. Neurol. 215370-381.

DeHamer, M.K., J.L. Guevara, K. Hannon, B.B. Olwin, and A.L. Calof (1994) Genesis of olfact~ry receptor neurons in vitro: Regulation of progenitor cell divisions by fibroblast growth factors. Neuron 13: 1083-1097.

Ellis, R.E., J. Yuan, and R. Horvitz (1991) Mechanisms and functions of cell death. Ann. Rev. Cell Biol. 7:663-698.

Farbman, A.I. (1990) Olfactory neurogenesis: Genetic or environmental controls. TINS 13:362-365.

Gavrielli, Y., Y. Sherman, and S.A. Ben-Sasson (1992) Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J. Cell Biol. 119:493-501.

Grasl-Kraupp, B., B. Ruttkay-Nedecky, H. Koudelka, K. Bukowska, W. Bursch, and R. Schulte-Hermann (1995) In situ detection of fragmented DNA (TUNEL Assay) fails to discriminate among apoptosis, necrosis and autolytic cell death: A cautionary note. Hepatology21:1465- 1468.

Graziadei, P.P.C., and M. Graziadei (1979) Neurogenesis and neuron regeneration in the olfactory system of mammals. I. Morphological aspects of differentiation and structural organization of the olfactory sensory neurons. J. Neurocytol. 8: 1-18.

Hinds, J.W., P.L. Hinds, and N.A. McNelly (1984) An autoradiographic study of the mouse olfactory epithelium: Evidence for long-lived receptors. Anat. Rec. 210:375-383.

Hollyday, M., and V. Hamburger (1976) Reduction of naturally occurring motoneuron loss by enlargement of the periphery. J. Comp. Neurol. 170:311-320.

Hughes, W.F., and S.C. McCloon (1979) Ganglion cell death during normal retinal development in the chick: Comparisons with cell death induced by early target field destruction. Exp. Neurol. 66:587-601.

Jacobson, M. (1991) Developmental Biology, Third Edition. New York: Plenum.

Jacobson, M.D., J.F. Burne, and M.C. Raff (1994) Programmed cell death and bcl-2 protection in the absence ofa nucleus. EMBO J. 161889-1910.

Korsching, S. (1993) The neurotrophic factor concept: A reexamination. J. Neurosci. 13:2739-2748.

Mackay-Sim, A., and P. Kittel (1991) Cell dynamics in the mouse olfactory epithelium: A quantitative autoradiographic study. J. Neurosci. 11: 979-984.

Mahalik, T.J., W.E. Hahn, G.H. Clayton, and G.P. Owens (1996) Pro- grammed cell death in developing grafts of fetal substantia nigra. Exper. Neurol. 129:27-36.

Mahanthappa, N.K., and G.A. Schwarting (1993) Peptide growth factor control of olfactory neurogenesis and neuron survival in vitro: Roles of EGF and TGF-6’s. Neuron 20:293-305.

Oppenheim, R.W. (1991) Cell death during development of the nervous system. Ann. Rev. Neurosci. 14:453-501.

Oppenheim, R.W., I-W. Chu-Wang, and J.L. Maderdrut (1978) Cell death of motoneurons in the chick embryo spinal cord 111. The differentiation of motoneurons prior to induced degeneration following limb-bud removal. J. Comp. Neurol. 177%-112.

Owens, G.P., T.J. Mahalik, and W.E. Hahn (1995) Expression of the death-associated gene RP8 in granule neurons undergoing postnatal cell death in the cerebellum of weaver mice. Dev. Brain Res. 86:3547.

Portera-Cailiau, C., J.C. Hedreen, D.L. Price, and V.E. Koliastos (1995) Evidence for apoptotic cell death in Huntington Disease and excitotoxic animal models. J. Neurosci. 153775-3787.

Schwartz-Levey, M., D.M. Chikaraishi, and J.S. Kauer (1991) Characteriza- tion of potential precursor populations in the mouse olfactory epithelium using immunocytochemistry and autoradiography. J. Neurosci. 11:355€- 3564.

Schwoh, J.E., K.E. Mielesko, and A.A. Stasky (1992) Olfactory sensory neurons are trophically dependent on the olfactory bulb for their prolonged survival. J. Neurosci. 123896-3919.

Schwob, J.E., J.M. Huard, M.B. Luskin, and S.L. Youngentoh (1994) Retroviral lineage studies of the rat olfactory epithelium. Chem. Senses 19:671-682.

Verhaagen, J., A.B. Oestreicher, and F.L. Margolis (1989) The expression of the growth associated protein B50iGAP43 in the olfactory system of neonatal and adult rats. J. Neurosci. 9t683691.

Wood, K.A., €3. Dipasquale, and R.J. Youle (1993) In situ labeling of granule cells for apoptosis-associated DNA fragmentation reveals different mechanisms of cell loss in the developing cerebellum. Neuron 11:621632.

Wyllie, A.H. (1980) Cell death: The significance of apoptosis. Int. Rev. Cytol. 68:25 1-306.

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Apparent apoptotic cell death in the olfactory epithelium of adult rodents: Death occurs at different developmental stages