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Cell Growth & Differentiation Vol. 12, 591-601, December 2001
© 2001 American Association for Cancer Research

Cell Cycle Regulation during Mouse Olfactory Neurogenesis

Marie-Emmanuelle Legrier, Angélique Ducray, Alain Propper, Moses Chao and Anne Kastner1

Laboratoire de neurosciences, EA481, Université de Franche-Comté, 25 030 Besançon Cedex, France [A. D., A. P., A. K.]; Cytogénétique moléculaire et oncologie, UMR 147, Institut Curie, Paris 75000, France [M-E. L.]; and Molecular Neurobiology Program, Skirball Institute for Biomolecular Medicine, New York University School of Medicine, New York, New York 10016 [M. C.]


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The development of the nervous system requires a strict control of cell cycle entry and withdrawal. The olfactory epithelium (OE) is noticeable by its ability to yield new neurons not only during development but also continuously during adulthood. The aim of our study was to investigate, by biochemical and immunohistochemical methods, which cell cycle regulators are involved in the control of neuron production during OE development and maturity. At birth, olfactory neural progenitors, the basal cells, exhibited a high mitogenic and neurogenic activity, decreasing in the following weeks together with the drop in expression of several cell cycle regulators. p27Kip1 and p18Ink4c, at birth, were expressed in the whole basal cell layer, whereas p16Ink4a, p19Ink4d, and p21Cip1 were rather located in differentiating or mature neurons. CDK inhibitors may thus act sequentially during this developmental neurogenic process. By comparison, in the adult OE, in which most neural precursors were quiescent, these cells still exhibited p18Ink4c expression but only occasionally p27Kip1 expression. It suggests that p18Ink4c may contribute to maintain basal cells in a quiescent state, whereas p27Kip1 expression in these cells may be rather linked to their neurogenic activity, which declines with age. In keeping with this hypothesis, transgenic mice that lacked p27Kip1 expression displayed a higher rate of cell proliferation versus differentiation in their OE. In these mice, a down-regulation of positive cell cycle regulators was observed that may contribute to compensate for the absence of p27Kip1. Taken together, the present data suggest distinct functions for CDK inhibitors, either in the control of cell cycle exit and differentiation during neurogenesis (respectively, p27Kip1 and p19Ink4d) or in the maintenance of a quiescent state in neural progenitors (p18Ink4c) or neurons (p21Cip1) in adults.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Most neurons are generated only during development and are never renewed during adulthood. However, this is not the case for at least three groups of neurons in mammals: hippocampal granular cells in the dentate gyrus; periglomerular and granular cells in the olfactory bulb that originate from the subventricular region (1) ; and the olfactory receptor neurons located in the OE.2 Cells in the OE are renewed by proliferation and differentiation of local progenitors, the globose basal cells (2) . In hippocampal dentate gyrus, subventricular cell layer, and OE, the proliferation of neural cell precursors is maintained throughout life but decreases with age (3, 4, 5, 6) . The mechanism involved in the control of neural cell proliferation or quiescence remains a central question in developmental neurobiology. It is thought that negative regulators of cell cycle progression in G1 phase may play a decisive role in these processes.

Progression through cell cycle is promoted by holoenzymes composed of regulatory (cyclin) and catalytic CDK subunits. One key substrate of these kinases is the nuclear tumor suppressor Rb, which is phosphorylated on serine and threonine residues during G1 phase, mainly by CDK4/6-cyclin D (7 , 8) . The catalytic activity of CDKs is negatively regulated by two families of molecules: the Ink4 one (including p15Ink4b, p16Ink4a, p18Ink4C, p19Ink4d) and the Cip/Kip gene family (composed of the three members, p21Cip/Waf1, p27Kip1, p57Kip2). The Ink4 molecules inhibit specifically CDK4 and CDK6 by impairing their interaction with cyclin D, whereas Cip/Kip molecules act on CDK-cyclin by forming a ternary complex (9) .

However, the function of each CDK inhibitor may be different as indicated by the distinct phenotypes engendered by their mutations in mouse (10, 11, 12, 13, 14, 15, 16, 17, 18, 19) and their distinct pattern of expression during development (20 , 21) . There is evidence that most of these CDK inhibitors, as well as Rb (22, 23, 24, 25, 26, 27, 28, 29) are implicated in the control of cell differentiation, particularly in the nervous system (30) . Thus, p27Kip1, p21Cip1, p57Kip2 and p19Ink4d are up-regulated during glial and neural differentiation in vitro (8 , 31, 32, 33, 34, 35, 36, 37) or in vivo (20 , 21 , 38, 39, 40, 41) . The involvement of p27Kip1 in neural commitment is suggested by experiments showing that overexpression of this gene in cycling cells in vitro leads to neural cell cycle growth arrest (24 , 40 , 42) , whereas its depletion in vivo causes an increased proliferative capacity during development as reported for glial progenitors (43) and cerebellar granule cells (44) . Alternatively, p27Kip1, but also other cell cycle inhibitors, may operate in postmitotic neurons to support their differentiation or to maintain definitive cell quiescence (19 , 39) . However, the relative contribution of the negative regulators to the control of neural cell renewal or quiescence in adult organism is still poorly understood.

To further assess the involvement of p27Kip1 and other cell cycle regulators during developmental and adult neurogenesis, we analyzed here the expression of various cell cycle regulators in relation to neural cell proliferation in the mouse OE. We, moreover, examined the regulation of neurogenesis in the OE of p27Kip1-deficient mice to evaluate the contribution of this gene to this process. The decrease of olfactory neuron production with age (3 , 6) as well as its sudden increase in response to injury suggest that neurogenesis in the intact adult OE may be highly regulated and normally inhibited (45) . Negative signals may involve bone morphogenetic protein (46) , but the intracellular mechanisms responsible for this repression of neuronal production are still poorly understood. Recent data suggest a contribution of the transcriptional repressor HES1 which may control negatively the level of olfactory neurogenesis during development (47) . Stimulation of neural progenitors proliferation by removal of the synaptic target, the olfactory bulb, was linked to the production of leukemia inhibitory factor (48) and to an increase of CDK activities (49) , which may, thus, stimulate olfactory cell proliferation in response to extracellular signals. In the present report, the comparison of various CDK inhibitors expression in the OE of mice of various ages shows that they may act either in neural precursors (p27Kip1 and p18Ink4c) or in differentiating olfactory neurons, and contribute not only to the control of neurogenesis but also to the maintenance of the quiescence state of these cells during adulthood.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Basal Cell Proliferation and Neurogenesis Decreased in the Aging OE.
The OE lines the deepest part of the nasal cavity at the level of the olfactory turbinates. It is essentially composed of only three cellular types arranged in successive layers, which are, from the nasal cavity to the basal lamina, respectively: supporting cells, olfactory neurons, and their precursors, the basal cells (50) . To investigate the effect of age on postnatal proliferation of the basal cells in the mouse OE, we analyzed by Western blotting techniques, the kinetics of PCNA protein, a marker of S phase. The presence of proteins in the extracts was appreciated by the Coomassie Blue coloration after electrophoretic migration, and the equal loading of samples was checked by Western blots of ß-actin (Fig. 1A)Citation . Western blots showed that PCNA was abundant in newborn mice but dropped progressively to a lower level (about 10-fold) during the first 3 months (Fig. 1B)Citation . We also analyzed using BrdUrd and PCNA immunohistochemistry, the location and number of dividing cells in the OE of young mice (3-week-old) and adults (3-month-old). In neonates and young mice, PCNA and BrdUrd antibody labeled a continuous layer of cells corresponding to the basal ones, but also numerous cells in other parts of the OE (Figs. 2Citation and 4Citation ). In accordance with Western blot results, PCNA and BrdUrd immunoreactive cells were markedly less dense in mature OE, both in the basal cells layer, in which they exhibited a distribution in cluster, and elsewhere in the OE (Fig. 2)Citation . The number of BrdUrd- and PCNA-positive basal cells was about 3-fold higher in young mice than in adults, in good correlation with our biochemical results (Fig. 2Citation and Table 1Citation ) and also with data previously obtained in the rat OE (6) . Thus, PCNA protein levels may serve as an index to quantify cell proliferation in the OE.



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Fig. 1. Neural cell proliferation declines in relation to age in the mouse olfactory mucosa. A, Coomassie Blue staining of proteins on SDS PAGE and Western blotting of ß-actin. Olfactory mucosa extracts were obtained from mice ranging in age from 1 day (d) to 8.5 months (m). B, kinetics of basal cell proliferation and olfactory neuron production by Western blotting experiments. PCNA serves as a marker for the proliferative state of basal cells; GAP 43, ß3 tubulin, and OMP serve as markers of differentiating and mature olfactory neurons. Western blotting was realized with the same lysates as that in A. The densitometric profiles are given for PCNA (•) and OMP ({square}). Neural cell proliferation and mature neuron production have opposite kinetics.

 


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Fig. 2. Immunohistochemical analysis of neural cell proliferation in the olfactory mucosa indicates that it is higher in young mice than in adults. Proliferating cells in the OE were detected using an antibody either against BrdUrd or against PCNA, a marker of proliferating cells. Arrowhead, the basal lamina lining the OE in the olfactory mucosa; arrow, some labeled cells; nc, nasal cavity; ch, chorion; bc, basal cell(s); on, olfactory neuron(s); scale bar, 100 µm. The number of dividing cells are reduced in the OE of 3-month-old mice by comparison with that of 3-week-old mice (see also Table 1Citation ).

 


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Fig. 4. Expression patterns in adult OE suggest that p18Ink4c rather than p27Kip1 maintains basal cells in a quiescent state. A, immunohistochemical labeling of PCNA (a marker of cell proliferation), p27Kip1, and p18Ink4c on adjacent sections in the olfactory mucosa from newborn mice (1 week old). Arrowhead, the basal lamina (bl) lining the OE. Some labeled cells are indicated by an arrow; nc, nasal cavity; ch, chorion; bc, basal cells; on, olfactory neurons; sc, supporting cells; scale bar, 100 µm. p27Kip1 and p18Ink4a (arrows) are restricted in areas close to the basal lamina as PCNA. B, the same material as in A but at a higher magnification. Scale bar, 40 µm. Expression of both p27Kip1 and p18Ink4c are seen in the basal cell layer. C and D, immunohistochemical labeling of PCNA, p27Kip1, and p18Ink4c on sections from adult mice without any treatment (C) or 4 days after destruction of the olfactory neurons by ZnSO4 intranasal injections (D); scale bar, 40 µm.

 

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Table 1 Quantitative assessment of basal cells proliferation level in the OE of young (3-week-old) mice and adult (3-month-old) mice, and of adult mice with lesioned OE

 
To investigate the relationship between olfactory cell proliferation and the appearance of olfactory neurons, we also analyzed, by Western blotting, the variations in relation to age of GAP 43, a marker of neuronal differentiation (51) , of OMP, a marker of mature olfactory neurons (52) , and of ß3 tubulin (53) , present in both differentiating and mature neurons (Fig. 1B)Citation . Whereas ß3 tubulin was approximately constant with age, OMP was considerably increased during the first 2 months, a fact corresponding, therefore, to the initial production of olfactory neurons. Thus, at birth, olfactory neurons are mainly in an immature state (OMP negative, GAP 43 positive), whereas in adults, most of them are fully mature.

Immunohistochemical observations showed that in newborn mice, GAP 43 protein was present in the major part of the OE (except in the basal and luminal cell layers; Fig. 5Citation ), whereas in adults it was restricted to patches of cells lining up the basal layer of the OE, which showed that neurogenesis was decreased in the mature OE.



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Fig. 5. The CDK inhibitors p16Ink4a, p19Ink4d, and p21 Cip1 are rather localized in differentiated neurons in the developing and mature OE. A, lectin histochemistry with UEA and DBA on comparative sections were used to localize differentiating and mature neurons, respectively, in the OE of newborn mice. Arrowhead, the basal lamina (bl) lining the OE. Some labeled cells are indicated by an arrow; nc, nasal cavity; ch, chorion; bc, basal cells; on, olfactory neurons; sc, supporting cells; scale bar, 100 µm. Immunohistochemical localization at a higher magnification of GAP 43, a marker of differentiating neurons. Scale bar, 40 µm; nc, nasal cavity; sc, supporting cells; on, olfactory neurons; bc, basal cells; ch, chorion; arrowhead, basal lamina. B, immunohistochemical localization of the CDK inhibitors p16Inka, p19Ink4d, and p21Cip1 in the olfactory mucosa from newborn mice. C, immunohistochemical localization of p16Ink4a, p21Cip1, and, for comparison, Gap43 in the olfactory mucosa of adult mouse.

 
The Expression of Most Cell Cycle Regulators Declined in the Aging OE.
We test whether the age-related changes in olfactory cell proliferation were linked to a modified expression of various CDKs, cyclins, and CDK inhibitors, by Western blotting experiments on olfactory mucosa extracts from 1-day to 8.5-month-old mice. Fig. 3ACitation shows a rapid decrease of CDK1 with age and to a lesser extent of CDK2 and CDK4. Like PCNA, CDK4 immunoreactivity on OE sections was, however, mostly restricted to a subpopulation of basal cells, more numerous in neonates than in adults (not shown). The level of CDK5 protein did not detectably vary with increasing age. All cyclins D were also markedly reduced in relation to age (Fig. 3B)Citation , cyclins D2 and D3 being only detected in newborn mice, and cyclin D1 being detected during the first 2 months after birth. The decline of olfactory cell proliferation in young mice appears, therefore, linked to the decrease in CDK1 and cyclin D.



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Fig. 3. Age-related kinetics of cell cycle regulators in the mouse olfactory mucosa reveal that most of them are down-regulated in the mature OE. Various cell cycle regulators including CDKs (A), cyclins D (B), and CDK inhibitors (C) were assessed by Western blotting experiments on olfactory mucosa protein extracts (the same as in Fig. 1Citation ) from mice of increasing age (d, day; m, month). It shows that the expression of most cell cycle regulators declined with age. A nonspecific band at Mr 27,000 was detected with anti-CDK1 (PSTAIR) antibody, which did not vary with age. The densitometric profiles are given for CDK1, CDK2, and cyclins D1 and D2. There is a distinct temporal pattern for p27Kip1 and p18Ink4c. D, Western blots of CDK4, p19Ink4d, p18Ink4c, and p27Kip1 after immunoprecipitation with anti-CDK4 antibodies. OD, absorbance.

 
We analyzed also the temporal pattern of CDK inhibitor expression in the aging olfactory tissue (Fig. 3C)Citation , by Western blotting experiments. All selected antibodies exhibited a single band at the expected molecular weight. Both p27Kip1 and p19Ink4d were expressed at a transient and high level in young mice but became almost undetectable in older mice. Thus, the expression of p27Kip1 and p19Ink4d in the OE coincided with the level of cell cycle withdrawal and neuronal differentiation that is high in newborn mice and low in adults (see previous section in "Results"). By contrast, the levels of p18Ink4c only slightly declined with age. The presence of these different CDK inhibitors in olfactory cells was also found in immortalized olfactory cells in vitro (not shown). To check whether these cell cycle inhibitors were associated with CDK4, the kinase was immunoprecipitated from OE extracts of new-born and adult mice, and the coprecipitated inhibitors were analyzed by Western blotting (Fig. 3D)Citation . p27Kip1, p19Ink4d, and p18Ink4c were detected by Western blotting in association with CDK4 in newborn mice, but they fell to almost nondetectable levels in adult mice.

p18Ink4c and p27Kip1 Are Expressed Mostly in Neural Progenitors in Contrast to Other Inhibitors.
To find out whether distinct CDK inhibitors were differently expressed during developmental neurogenesis, we analyzed their cellular distribution by immunohistochemistry on OE sections from newborn mice in relation to the state of proliferation or differentiation. In neonates, neural progenitors were restricted chiefly to the most basal cell layer as indicated by the position of PCNA-positive cells (Fig. 4)Citation , whereas differentiating and fully differentiated neurons occupied, respectively, the medial and the apical part of the OE as revealed by the complementary patterns of, respectively, UEA I and DBA histochemistry (54) , and also by the pattern of GAP 43 (Fig. 5)Citation . Thus, the location of a cell across the OE in newborn mice may inform us about its differentiation stage during neurogenesis. CDK inhibitors exhibited a specific expression pattern in the developing OE. Thus, p27Kip1 immunoreactivity was detected in a heterogeneous fashion, mainly in the basal cell layer corresponding to neural progenitors and weakly in young differentiating neurons in the basal part of the OE, but was completely absent from the medial and apical OE. In adjacent sections, p18Ink4c was also restricted to the basal border, presumably in neuroblasts and young growing neurons (Fig. 4Citation ; Table 2Citation ). The pattern of p16Ink4a immunolabeling was consistent with a presence of this protein in differentiating neurons, whereas that of p21Cip1 and p19Ink4d, present in the medial and apical part of the OE, corresponded rather to more mature neurons (Fig. 5Citation ; Table 2Citation ). Such various spatial patterns of different cell cycle inhibitors reveal, thus, a sequential temporal expression of, respectively, p27Kip1, p18Ink4c, p16Ink4a, p19Ink4d, and p21Cip1 from neural cell cycle exit to olfactory neuron maturation.


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Table 2 Expression pattern of various CDK inhibitors in the developing and mature OE

 
P18Ink4c and p27Kip1 Are Expressed Differentially in Adult Neural Precursors.
The distribution of p27Kip1 and p18Ink4c in basal cells in newborn mice suggested that these cell cycle inhibitors may be involved in the control of neural cell cycle withdrawal. Alternatively, these cell cycle inhibitors may also maintain the pool of basal cells in a quiescent state, especially in adults. To discriminate between these two functions we analyzed p27Kip1 and p18Ink4c immunolabeling in the OE of adult mice, in which the majority of neural precursors stay in a quiescent state (Fig. 4)Citation . It shows that p18Ink4c was almost ubiquitously expressed in the basal cell layer, in accordance with the latter hypothesis. By contrast, p27Kip1 was present only in occasional basal cells (Fig. 4Citation ; Table 2Citation ). This distinctive pattern between p27Kip1 and p18Ink4c was consistent with our data of Western blotting studies, which showed in the adult olfactory mucosa, a substantial expression of p18Ink4c but not of p27Kip1 (Fig. 3C)Citation . The reduced p27Kip1 expression in the mature OE may be related to its low neurogenic activity. For comparison, we also analyzed the immunohistochemical pattern of p16Inka, p19Ink4d, and p21Cip1 in the mature OE (Fig. 5Citation ; Table 2Citation ). The location of p21Cip1 corresponds to that of mature olfactory neurons. By contrast, only occasional cells were labeled with p16Ink4a antibody, mostly in the basal layer, whereas p19Ink4d was hardly detectable (not shown) in the mature olfactory organ.

The production of olfactory neurons in adults may be stimulated by experimental lesion of the olfactory organ. Indeed, neuronal degeneration in the OE leads after a few days to an induction of neural proliferation and differentiation. In the regenerating OE, PCNA (Table 1)Citation but also p27Kip1 and p18Ink4c positive cells were abundant (Fig. 4)Citation . Such induction of p27Kip1 during OE regeneration was also observed by western blotting experiments (not shown) and attested the putative relation between p27Kip1 expression and the neurogenic activity of neural precursors.

Cell Cycle Regulation in p27Kip1-deficient Mice.
Our analysis of p27Kip1 expression was consistent with an involvement of this protein in the process of cell cycle withdrawal and the onset of neuronal differentiation of olfactory cells. To directly test the contribution of this protein to OE cell proliferation and differentiation, we analyzed the OE of young and adult mice lacking the p27Kip1 gene (Fig. 6)Citation . Examination of BrdUrd staining on sections of olfactory mucosa showed that in the young, p27Kip1-null OE (1-month-old), BrdUrd-positive cells were abundant and present all along the OE chiefly at a basal location (Fig. 6Citation ; among 43 +/- 7 cells/mm versus 24 +/- 4 cells/mm in control OE). In older animals (2-month-old), BrdUrd and PCNA were restricted only to clusters of basal cells, similarly in p27Kip1 deficient and wild-type mice (respectively 13 +/- 4 cells per mm and 12 +/- 3 cells per mm). Thus, basal cells proliferation slows down even in the absence of p27Kip1. To quantitatively assess the influence of p27Kip1 on the level of olfactory neuron production in the OE, we compared, using Western blotting methods, various markers of olfactory cell proliferation and differentiation. p27Kip1-deficient mice displayed increased protein amounts in the OE (Fig. 6)Citation . No difference in the electrophoretic pattern was seen after Coomassie Blue staining of the proteins (not shown). On Western blots, the levels of various markers of differentiation (ß3 tubulin, CDK5) and of mature neurons (OMP) were not noticeably changed (Fig. 6)Citation . Olfactory cell proliferation was evaluated quantitatively by measurement of PCNA protein amounts as described earlier. Quantification of PCNA signals (on Western blots obtained from 10 independent experiments) showed that the amount of PCNA protein was significantly increased in young p27Kip1-deficient mice (n = 3; 3-week-old; ~80%; P < 0.01) but not in adults (2- and 4-month-old; Fig. 6DCitation ). Measurement of the level of PCNA and GAP 43 performed on distinct membranes showed that GAP 43 was not significantly different in young p27Kip1 mice (22 +/- 6 versus 27 +/- 7 in controls), so that PCNA/GAP 43 level was ~100% higher in these mice. Both in wild-type and p27Kip1-deficient animals, PCNA levels were significantly lower in adults in comparison with young mice, in accordance with our previous observations (Figs. 1CCitation and 2Citation ). These results suggested that regulatory mechanisms may occur to compensate for the lack of p27Kip1 in adult mice. p18Ink4c was detected in the whole layer of basal cells in p27Kip1-deficient animals as in wild-type animals. Analysis of the positive cell cycle regulators showed that cyclin D1 and, more surprisingly, CDK1 were markedly lower in p27Kip1 null mice, whereas CDK2 and CDK4 were not markedly modified (Fig. 6D)Citation . Interestingly, in p27Kip1 null mice, Western blots with CDK4 antibody showed an additional band at Mr 20,000.



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Fig. 6. Neural cell proliferation and cell cycle regulation is modified in p27Kip1-null mice. A, Western blot assessment of different markers of proliferation and differentiation in the olfactory mucosa of wild-type and p27Kip1-null mice that were 3 weeks (3w), 2 months (2m), or 4 months (4m) old. B, comparison of the protein amounts in the olfactory mucosa from wild-type and p27Kip1 mice that were 3 weeks (3w), 2 months (2m), or 4 months (4m) old. C, densitometric values of PCNA signal. PCNA serves as a marker for the proliferative state of basal cells. The values are the mean of 10 independent Western blotting experiments. The differences were significant between 3 weeks and 2–4 months, and between 3 weeks +/+ and -/- (P < 0.01). D, Western blot assessment of cell cycle regulators in the olfactory mucosa of wild-type and p27Kip1-null mice of various ages: 3 weeks (3w); 2 months (2m), and 4 months (4m). E, immunohistochemical location of BrdUrd- positive cells, PCNA, and GAP 43 on OE sections from p27Kip1-null (right) and wild-type (left) mice. Scale bar, 100 µm. BrdUrd-positive cells are more numerous in 1-month-old p27Kip1-null mice than in wild-type mice.

 

    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Neural Cell Proliferation and Differentiation in the Postnatal OE.
The effect of age on olfactory cells proliferation and neurogenesis was principally analyzed by Western blotting and immunohistochemical experiments. We show that olfactory cell proliferation decreases ~10 to 15 times after birth principally during the 3 first months of life (about three times between the age of 3 weeks and 3 months). Our results confirm those previously obtained by counting BrdUrd- or PCNA-positive cells in the rat and mouse OE (6 , 55) and show that olfactory cells proliferation can be appreciated semiquantitatively by Western blotting of PCNA. Because of the heterogeneous distribution of dividing cells in the tissue and also of technical considerations, this biochemical method appeared more suitable for the present study.

Olfactory neuron production in postnatal mice was assessed by comparing the levels of various cellular markers. The level of CDK5, a CDK involved in neural differentiation, remains constant with advancing age, a result recently observed in the rat brain (56) . PCNA and OMP (a marker of mature olfactory neurons) varied in a nearly reciprocal fashion. Thus, cell proliferation decrease during the 1st month goes along with the production of olfactory neurons, in agreement with a previous study showing that the number of sensory cells in the rat OE increases by a factor of >10 during the first 25 days of life (57) . Thus, cell proliferation in the postnatal and adult OE is first related to the initial production of olfactory neurons, then to their continuous replacement throughout life. Moreover, at birth, the majority of cells stay in an immature state (as indicated by the expression of GAP 43 and the absence of OMP), whereas the density of mature neurons increases with advancing age, a result in keeping with a previous observations (50) . Thus, at birth, the levels of basal cell proliferation and neurogenesis are high in relation to the establishment and growth of the olfactory organ whereas they are both reduced in adult animals. The number of olfactory neurons seemed to remain constant as suggested by OMP levels.

Age-related changes in neural cell proliferation went along with a decrease of the regulatory subunits cyclins D, especially D2 and D3, in keeping with their general role in cell cycle driving. A similar relationship between neural cell proliferation in vivo and expression of cyclin D protein has also been observed after lesion-induced proliferation in the OE (49) and in the postnatal cerebellum, in which expression of cyclin D1 was restricted to the proliferating external granular cells (38) . However, in some conditions in vitro, D cyclins were implied in cell cycle exit and neuronal differentiation (24 , 36 , 37 , 58 , 59) . In our model, the relationship between proliferation and the presence of positive cell cycle regulators was also evident for CDK1 but less strict for other CDKs. Thus, the expression of CDK4 was maintained to almost constant levels throughout life. Previous studies showed that CDK4 expression was neither affected by lesion-induced proliferation in the OE (49) nor in neuroblastoma cells during in vitro differentiation (24) . Similar results were reported also during growth arrest of Schwann cells (60) . CDK4 activity appears, thus, merely regulated by its association with cyclin D1, whereas CDK2 activity in proliferating neural cells is chiefly modulated by its expression levels.

CDK Inhibitors in the Developing and Mature OE.
Thus, developing and mature OE differ by the rate of neurogenesis and, relatively, by the states of basal cells either quiescent or cycling. We showed that such changes were accompanied by a distinct spatial and temporal pattern of CDK inhibitors. At birth, p27Kip1 and p18Ink4c were present in almost all basal cells and in young, differentiating olfactory neurons, whereas p16Ink4a, p19Ink4d, and p21Cip1 were rather expressed in more differentiated ones. Thus, CDK inhibitors may act at different stages of the neurogenic process. In the mature OE, the reduced neurogenesis was linked to the disappearance of p27Kip1 in most basal cells, in contrast to p18Ink4c which remained present in the whole cell layer. Only p21Cip1 was detected in the mature olfactory neurons in adults. Such differential pattern suggests that some CDK inhibitors are related to neural cell cycle exit (p27Kip1) and differentiation (p16Ink4a and p19Ink4d), whereas other may be more specifically involved in the quiescence of neural precursors (p18Ink4c) or of mature olfactory neurons (p21Cip1) in the adult OE.

Our results are concordant with previous studies reporting that p27Kip1 (20 , 40 , 61) or p18Ink4c (21) were predominantly expressed in neuroblast and young neurons. Furthermore, an induction of p27Kip1 expression has been reported in vitro after induction of glial cells (32 , 33 , 35) or neurons differentiation in vitro (31 , 34 , 44 , 62) , demonstrating a relationship between p27Kip1 expression and cell cycle exit. In accordance with this hypothesis, overexpression of this gene in cycling cells leads to neural cell cycle arrest (24 , 41 , 42) . Neural withdrawal from cell cycle has also been related to a high but transient level of Rb-E2F1 complex (26 , 27) , which suggests that p27Kip1 may contribute to maintain pRb in an underphosphorylated state in neuroblasts as they undergo differentiation. By contrast, the ubiquitous expression of p18Ink4c in the OE basal cell layer both in young and adult animals suggests that this gene may control the state of quiescence in olfactory cell precursors rather than their commitment to neuronal differentiation.

Other distinct patterns have been observed for other cell cycle inhibitors that were expressed in differentiating (p16Ink4a) or mature neurons (p21Cip1 and p19Ink4d). Previous data concerning the mouse brain have indeed shown that p19Ink4d was present primarily in postmitotic neurons and that its expression continued postnatally during adulthood in different brain regions (21) . Moreover, in the developing cerebral cortex, p19Ink4d immunohistochemical labeling increase as cells undergo differentiation during their ascent toward the cortical plate (41) . The presence of p16Ink4a and p21Cip1 has also been reported in differentiated neurons in some discrete regions of the mouse embryonic or postnatal brain (20) . In addition, induction in PC12 cells of neuronal differentiation leads to a progressive accumulation of p21Cip1 (34 , 35) . Moreover, a recent study establishes that p21Cip1 is required for proper differentiation of oligodendrocyte, by contrast to p27Kip1 (63) . Thus, we suggest that distinct CDK inhibitors operate in a sequential fashion during neurogenesis from neuronal cell cycle exit to mature neurons. Otherwise, a few reports mention that p19Ink4d (21) and p21Cip1 (20) as well as p27Kip1 (39) are detectable in substantial levels in the adult brain, probably in fully mature neurons. Thus, these genes may control not only neuron production but also other functions, as the maintenance of cells in a postmitotic state (19) . In the adult OE, we showed that only p21Cip1 remains expressed in olfactory neurons, which suggests a specific function of this gene in these fully mature neurons.

The function of p27Kip1 in the control of neural cell cycle exit during development is attested by our experiments showing that the loss of this gene in mice leads to an increased proliferation of olfactory precursor cells and to a higher rate of proliferating versus differentiating cells. The higher protein amounts in p27Kip1-null mice OE reveals, moreover, that the size of the organ is larger. It corroborates previously reported data obtained for the oligodendrocyte lineage (32 , 43) and recently reported data for cerebellar neurons in vitro (44) , indicating that p27Kip1 is a major regulatory component for neural cell cycle exit, both in the olfactory organ and in the brain (64) . Moreover, in the retina of newborn mice, it has been recently reported that the period of photoreceptor and Müller cell production was extended for 2 weeks in p27Kip1-null mice, which suggests that p27Kip1 is also required for the timing of cell cycle withdrawal of neural progenitors (40) . However, in these different reports, p27Kip1 did not prevent the slowing down of neural proliferation during development or aging so that neural proliferative activity in adults was similar in p27Kip1-null and wild-type mice, either absent as in retina (40) , or low as in the OE (present data). Compensatory mechanisms in p27Kip1 mice may be achieved by down-regulation of positive regulators as demonstrated here for cyclin D1 and CDK1. An effect of the lack of p27Kip1 has been mentioned previously for the regulation of cyclin D1 activity in fibroblasts (65) and for cyclin E in brain (43) . Moreover, the deletion of p27Kip1 restores almost normal development in cyclin D1-deficient mice (66 , 67) . It suggests that a positive feedback loop exists in normal cells between p27Kip1 and positive regulators that may contribute to protect cells from putative cell cycle disregulations.

A unique feature of the OE is its ability to produce neuron continuously throughout life and to modulate such neurogenesis in relation to age or in response to environmental injuries (68) . The rate of neural cell proliferation may depend on local extracellular signals that may act positively (as fibroblast growth factor 2, leukemia inhibitory factor, and also neuropeptide Y; Refs. 44 , 69 , 70 ) or negatively (as bone morphogenic proteins; Refs. 45 , 46 ). The present report establishes that some CDK inhibitors such as p27Kip1 or p18Ink4C, expressed in olfactory neural precursors, contribute to regulate negatively the proliferation of these cells and thus to maintain the proper number of olfactory basal cells and olfactory neurons throughout life.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Animals.
The study was performed on OF1 mice ranging in age from birth to 1 year. Mice were anesthetized with chloralhydrate and killed by decapitation, the bulk of olfactory turbinates being immediately dissected out of each mouse head and incubated either in NP40 lysis buffer (for biochemical studies, see below) or in 4% paraformaldehyde in PBS buffer (for immunohistochemistry, see below). Two 2–3-month-old mice were subjected to an intranasal injection of 10% ZnSO4 or to bilateral bulbectomies to induce olfactory neuron degeneration and regeneration. Four mice that were 3 weeks old (n = 2) or 3 months old (n = 2) received BrdUrd injection i.p. 1 day before dissection. p27Kip1-null and wild-type mice were obtained from heterozygous p27Kip1 breeding pairs C57 black/SJL (14) . Their genotype was determined by PCR analysis of genomic tail DNA. Six mice of each type were used for Western blotting and four for immunohistochemistry, the latest having received a BrdUrd injection.

Antibodies.
Antibodies used were: anti-PCNA, anti-ß3 tubulin, and anti-ß-actin from Sigma Chemical Co.; anti-cyclin D1 (DCS-6 and Ab-3), anti-p15INK4b (Ab-3), anti-p18INK4c (Ab1 and Ab2), and anti-p27KIP1 (DCS-72F6 and Ab-2) from NeoMarker; anti-cyclin D2 (M20), anti-cyclin D3 (H292), anti-CDK1 (PSTAIR), anti-CDK2 (M2), anti-CDK4 (C22), anti-CDK5 (DC17), anti-p19INK4d (M167; previously used in the studies reported in Refs. 21 , 41 , 42 ), p21CIP1 (C19; previously used in the studies reported in Refs. 38 , 43 ), E2F1 (KH95) and anti-p27KIP1 (C19; previously used in the studies reported in Refs. 20 , 39 ) from Santa Cruz Biotechnology; anti-p16 INK4a (18.070.247) and anti-GAP 43 from Chemicon; anti-BrdUrd from NeoMarker and Becton Dickinson. Anti-OMP antibodies were a gift from Professor F. Margolis.

Western Blotting.
For biochemical studies, olfactory organs were homogenized in NP40 lysis buffer [0.5% NP40 in 50 mM Tris-HCl (pH = 8), containing 120 mM NaCl, 0.1 mM NaF, 0.1 mM Na3VO4, 1 mM EDTA, and a protease inhibitor cocktail]. After 1-h incubation at 4°C under stirring, lysates were cleared by centrifugation at 10,000 x g for 2 min. Lysates originating from mice of the same age (2 or 3 mice per each age) were then pooled, and, after protein titration by Bradford assay on each sample, they were diluted in NP40 buffer to a final concentration of 8mg/ml For Western blotting experiments, the lysates were mixed to equal volumes of 2x protein sample buffer and kept frozen until use. Electrophoresis was performed on SDS polyacrylamide mini-gels (Bio-Rad) by loading 20–40 µg of protein in each lane, according to the standard technique of Laemmli. After electrotransfer onto nitrocellulose membrane (Optitran BAS 85, Schleicher & Schull) and blocking with a 5% milk solution (in PBS-0.1% Tween), blots were incubated overnight at 4°C (or 3 h at room temperature) with the diluted primary antibody (dilution ranging from 1:200 to 1:5000 in PBS-milk-Tween solution). After incubation for 1 h with the appropriate peroxidase-linked secondary antibody, immunoreactivity was detected by chemiluminescence using a commercial kit (Pierce). For quantitative analysis, the intensity of the bands were determined by densitometry and normalized using the Molecular Analyst software.

Immunoprecipitation.
For immunoprecipitation of complexes with CDK4, 250-µg protein aliquots in NP40 lysis buffer were precleaned in the presence of 50 µl of protein A-Sepharose beads (Sigma Chemical Co.) diluted 1 vol:1 vol, then incubated for 1 h at 4°C with 3 µl of anti-CDK4 antibodies. Immunocomplexes were collected on 40-µl diluted protein A-Sepharose beads, gently shacked for 2 h at 4°C, washed four times with NP40 lysis buffer and eluted in 20 µl of 2x protein sample buffer. Western blotting analyses were then performed as described above.

Immunohistochemistry and Histochemistry.
For immunohistochemical experiments, olfactory mucosa were fixed in 4% paraformaldehyde in PBS for 8 h at 4°C. The fixed tissues were washed in PBS, then decalcified (1 night), dehydrated in rising concentrations of alcohol and butanol baths and embedded in paraffin. A similar protocol was used for whole brain inclusion. After paraffin removal and rehydration, sections were incubated for 1 h in PBS containing 10% normal horse serum and 0.3% Tween. Sections were then incubated with diluted primary antibodies (1:50 to 1:500 in PBS, 10% normal horse serum, and 0.3% Tween). After washing, tissue sections were incubated for 3 h at room temperature in 1:20 to 1:50 diluted solution of appropriate peroxidase-linked secondary antibody. The peroxidase activity was revealed in a solution containing diaminobenzidine tetrahydrochloride (0.07 M), nickel chloride 0.001%, and 0.01% hydrogen peroxide. For quantitative analysis, the number of BrdUrd or PCNA-positive cells in comparative OE sections was determined in at least five fields per mouse using a x40 objective. For lectin histochemistry, sections were incubated with biotin-labeled lectins (Sigma Chemical Co.) from Ulex europaeus (UEAI) and DBA and processed as described previously (54) .


    Acknowledgments
 
We thank Dr. F. L. Margolis for the gracious gift of anti-OMP antibody. We express our gratitude to Germaine Michel for technical help, we thank both reviewers for their helpful comments and criticisms.


    Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom requests for reprints should be addressed, at Laboratoire de neurosciences, EA481, Université de Franche-Comté, 25 030 Besançon Cedex, France. Phone: 33-3-81-66-57-84; Fax: 33-3-81-66-57-54; E-mail: anne.kastner{at}univ-fcomte.fr. Back

2 The abbreviations used are: OE, olfactory epithelium; CDK, cyclin-dependent kinase; PCNA, proliferating cell nuclear antigen; GAP, growth-associated protein; OMP, olfactory marker protein; DBA, Dolichos biflorus agglutinin; UEA, Ulex europaeus agglutinin; BrdUrd, bromodeoxyuridine. Back

Received for publication 2/23/01. Revision received 9/25/01. Accepted for publication 10/ 9/01.


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 Materials and Methods
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D. N. Abrous, M. Koehl, and M. Le Moal
Adult Neurogenesis: From Precursors to Network and Physiology
Physiol Rev, April 1, 2005; 85(2): 523 - 569.
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