1Unite ´MixtedeRecherche5020CentreNationaldelaRechercheScientifique,Universite ´LyonI,69622Villeurbanne,France,2BiologyDivision,California
InstituteofTechnology,Pasadena,California91125,3U407InstitutNationaldelaSante ´etdelaRechercheMe ´dicale,Ho ˆpitalLyon-Sud,69921Pierre-
Be ´nite,France,and4Unite ´MixtedeRecherche5123CentreNationaldelaRechercheScientifique,Universite ´LyonI,69622Villeurbanne,France
The mammalian olfactory epithelium (OE) is composed of primary olfactory sensory neurons (OSNs) that are renewed throughout
vivo involvement of a cytokine in the cellular events leading to the regeneration of the OE. We find that, of many potential mitogenic
signals, only leukemia inhibitory factor (LIF) is induced before the onset of neuronal progenitor proliferation. The rise in LIF mRNA
The mammalian olfactory epithelium (OE) is a pseudostratified
epithelium made up predominantly of odorant-receptive neu-
rons that project to the olfactory bulb (OB). These olfactory sen-
sory neurons (OSNs) are renewed throughout adulthood by the
mitotic activity of local neuronal progenitors, the globose basal
cells (GBCs) (Monti-Graziadei and Graziadei, 1979; Caggiano et
al., 1994; for review, see Schwob, 2002). The GBCs display neu-
ronal phenotypic markers and were shown by lineage analysis to
exclusively generate OSNs and GBCs in the normal, noninjured
adult OE (Caggiano et al., 1994; Hunter et al., 1994).
Olfactory neuron renewal is strongly stimulated by ablating
the OB [olfactory bulbectomy (OBX)], which leads to a wave of
neuronal apoptosis in the OE, peaking at 36–48 hr after lesion
(Michel et al., 1994; Holcomb et al., 1995; Deckner et al., 1997),
and to a subsequent wave of GBC mitosis peaking at 5 d after
lesion in the C57BL/6 adult mouse (Schwartz-Levey et al., 1991).
In this paradigm, mitotic stimulation consists of an increased
number of neuronal progenitors entering the cell cycle
(Schwartz-Levey et al., 1991; Huard and Schwob, 1995), which
leads to the appearance of new neurons in the apical layer of the
OE (Caggiano et al., 1994; Hunter et al., 1994). The delay sepa-
rating apoptosis of mature neurons and the onset of progenitor
proliferation suggests that basal cells are stimulated to divide by
local signals emitted during lesion-induced neuronal apoptosis.
Candidate signals for this stimulation are growth factors and
cytokines (for review, see Mackay-Sim and Chuah, 2000) that
have been shown to stimulate the production of new olfactory
neurons in vitro, including leukemia inhibitory factor (LIF)
(Satoh and Yoshida, 1997a,b), epidermal growth factor (EGF)
(Mahanthappa and Schwarting, 1993; Farbman and Buchholz,
1996), TGF-? (Farbman and Buchholz, 1996), TGF?1 and
TGF?2 (Mahanthappa and Schwarting, 1993; Newman et al.,
2000), FGF 2 (DeHamer et al., 1994; MacDonald et al., 1996;
Goldstein et al., 1997; Satoh and Yoshida, 1997a; Ensoli et al.,
1998; Newman et al., 2000), FGFs 1, 4, 7 (DeHamer et al., 1994),
and the neurotrophin family, NT-3, NT-4/5, and BDNF (Hol-
comb et al., 1995; Roskams et al., 1996). Some of these growth
factors and their receptors have also been localized in the intact
OE of adult rodents in vivo (Deckner et al., 1993; Farbman and
Cunningham, 1999; Ohta and Ichimura, 1999; Hsu et al., 2001),
and systemic injection of EGF or TGF? stimulates receptor-
dependent phosphorylation in basal cells of the OE (Ezeh and
cavity of adult rodent can enhance OSN survival after olfactory
nerve section (Yasuno et al., 2000), whereas subcutaneous injec-
tions of EGF, TGF?, or FGF 2 enhance the regeneration of OSN
after chemical lesion to the OE (Herzog and Otto, 1999).
It remains to be determined, however, whether these or other
paracrine signals are required for the various steps of olfactory
neuron turnover in vivo. To investigate this question we used the
Ge ´rontologie Franc ¸aise and Socie ´te ´ de Biologie du Vieillissement (S.B.), as well as by the National Institute of
1792 • TheJournalofNeuroscience,March1,2003 • 23(5):1792–1803
paradigm of OBX in the adult mouse. Because this operation
(Schwartz-Levey et al., 1991; Michel et al., 1994; Holcomb et al.,
1995), the prediction is that the surge in neurogenesis will be
preceded by an upregulation of the appropriate endogenous mi-
togen(s) (for review, see Mackay-Sim and Chuah, 2000; Schwob,
Animals and surgery. For analysis of cytokine expression, three-month-
old mice of the C57BL/6 strain (Iffa Credo, l’Arbresle, France) were
subjected to bilateral OBX as described previously (Michel et al., 1994).
the skull above the olfactory bulbs, which were then removed by aspira-
tion through a curved glass pipette connected to a vacuum pump. After
the cavities were filled with sterile Gelfoam and the skin was sutured,
mice were allowed to recover and returned to standard cage conditions.
every 8 hr up to 6 d (three to four animals per point). Controls were
sham-operated mice killed 8 hr after skull drilling. In each animal, the
whole bulk of olfactory turbinates (30 mg tissular cube wet weight),
including the septal tissue and cribriform plate but excluding the respi-
sterilized, RNase-free tools, frozen in sterile Eppendorf tubes by immer-
sion in liquid nitrogen, and stored at ?80°C. This tissue sampling is
termed “olfactory organ” throughout this paper; it has been devised and
validated in several previous studies (Michel et al., 1994, 1997b; Kastner
et al., 2000).
For in situ studies, we used LIF knock-out (KO) mice and wild-type
(WT) littermates from a colony that was maintained by mating within
the original colony of the mutant strain, and by back crossing with the
C57BL/6 parental stain (Bugga et al., 1998). Genotyping was done ac-
cording to Bugga et al. (1998). Mice were subjected to unilateral OBX
(removal of only one OB) and allowed to survive for either 2 or 5 d after
mg/kg bromodeoxyuridine (BrdU) (Sigma, St. Louis, MO) 12 hr before
animals were killed. They were then anesthetized with pentobarbital (40
mg/kg, i.p.) (Abbott Labs, Chicago, IL) and perfused transcardially with
4°C PBS followed by freshly prepared 4% paraformaldehyde in PBS at
with incomplete surgery were not included in the analysis. Olfactory
organs were removed and postfixed in the same fixative for 12 hr and
cryoprotected in 30% sucrose in PBS at 4°C for 24–48 hr, with a 5–10
min step under vacuum. Each olfactory organ was then immersed in
Tissue-Tek OCT (Miles, Elkhart, IN) under vacuum for 5–10 min and
for 6–12 hr, and frozen at ?20°C until use. Pairs of adjacent sections
were collected for subsequent BrdU immunohistochemistry and termi-
nal deoxynucleotidyl transferase-mediated biotinylated dUTP nick end
as above, olfactory organs were dehydrated and embedded in paraffin;
12-?m-thick sections were made with a microtome and mounted onto
Perkin-Elmer slides (Applied Biosystems, Courtaboeuf, France).
RNA extraction and RT-PCR. Total cellular RNA was extracted from
(Life Technologies, Cergy Pontoise, France), following the manufactur-
er’s instructions. Briefly, each sample of frozen tissue was dry-crushed
and homogenized in 1 ml of TRIzol. Chloroform (0.5 ml) was added to
resuspended in RNase-free water, and RNA concentrations were deter-
al. (1994) was used to measure growth factor and cytokine mRNAs.
Leukemia Virus reverse transcriptase (Life Technologies), 5 ?M random
Reaction mixtures were heated for 60 min at 37°C and for 5 min at 95°C
and then diluted in water and stored at ?20°C.
amplified in the same assay for all samples with a thermocycler (M100
MJ-Research, Lyon, France) in a 20 ?l reaction mixture containing 0.01
U/?l Taq polymerase (Promega, Charbonnie `res, France), manufactur-
er’s Taq buffer, 50 ?M dNTP (Life Technologies), 0.0375 ?Ci of [33P]-
dATP (Amersham, Les Ulis, France), 1 ?M specific primers, and 1.5 mM
MgCl2. Amplification programs included a first step of DNA denatur-
ation (5 min at 94°C), followed by 18–35 cycles each comprising DNA
denaturation (30 sec at 94°C), primer hybridization (30 sec at primer-
respective-temperatures) (Table 1) and elongation (30 sec at 72°C), and
a final elongation step (5 min at 72°C). For some primers (BDNF), am-
the thermocycler program. All oligonucleotides were synthesized by
Genset (Paris, France), and their sequences are presented in Table 1.
PCR products were separated by electrophoresis (Protean-2, Biorad,
Issy-les-Moulineaux, France) on 8% polyacrylamide gels, which were
then vacuum dried and processed for autoradiography on Biomax films
(Kodak, Sevran, France). Exposure times were adjusted to avoid film
ization temperature and minimum number of PCR cycles necessary to
detect radiolabeled reaction products at a unique, expected molecular
size, so that each PCR analysis would be performed in the exponential
phase of amplification. Forward and reverse primers were designed in
separate exons to avoid any bias caused by residual genomic contamina-
tion. PCR-amplified products were checked with restriction enzymes.
For all primer pairs, no amplification was observed when PCR was per-
formed on either RNase-free water or non-reverse-transcribed RNA
samples. At least three independent PCRs were performed on the same
samples for each primer pair.
were measured by densitometric scanning using the Bio-Image scanner
(Bio-Image, Cheshire, UK). The data were normalized to OD values
from ?-actin amplification for the corresponding samples. We then cal-
culated means ? SEM of the values obtained from animals killed at the
same time point, and these ratios were plotted as a function of time. The
occurrence of statistically significant differences from controls among
the normalized set of data for each mRNA sequence was first addressed
by one-way ANOVA using the Instat computer program (Graph Pad
software); this first test was taken as positive if the F value yielded a
tabulated equivalent for p ? 0.05. Any ANOVA-positive set was further
made with the Bonferroni test for multiple comparisons. Differences
were accepted as significant when p ? 0.05. Consistent results were ob-
tained from three separate sets of experiments.
Protein extraction and Western blotting. For Western blotting, frozen
idet P40 in 50 mM Tris, pH 8.0, containing (in mM): 120 NaCl, 0.1 NaF,
by centrifugation at 10,000 ? g for 15 min and frozen. After protein
quantitation by Bradford assay, lysates were diluted in NP-40 buffer to a
concentration of 8 mg/ml, mixed with equal volumes of 2? protein
sample buffer, boiled for 5 min, and kept frozen until use. Electrophore-
sis was performed on SDS-9% polyacrylamide mini-gels (Mini-Protean
in each lane (Laemmli, 1970). After electrotransfer to nitrocellulose
membrane (Optitran BAS 85; Schleicher & Schuell, Ecquevilly, France),
blots were blocked for 2 hr in a 5% skim milk solution and incubated
overnight at 4°C with primary antibodies against LIF (polyclonal, 1:100;
Santa-Cruz, Tebu, Le Perray, France) and ?-actin (monoclonal, 1:6000;
Roche Diagnostics) in the same buffer. After 1 hr incubation in appro-
Baueretal.•LIFRegulatesInjury-InducedOlfactoryNeurogenesis J.Neurosci.,March1,2003 • 23(5):1792–1803 • 1793
activity was detected by chemoluminescence (ECL, Amersham).
BrdU immunohistochemistry. Frozen, prefixed sections were brought
pH 5.5, and incubated in the same buffer for 20 min at 98–100°C to
denature endogenous DNA. Sections were then dried at room tempera-
ture, rinsed in PBS, and treated for 2 min with pepsin (Sigma), 0.25
mg/ml in 0.1N HCl to unmask DNA. After three 5 min rinses in PBS,
sections were incubated in 3% H2O2-containing PBS for 20 min, rinsed
in PBS, and incubated for 30 min in PBS containing 0.125% BSA, 0.05%
Triton X-100, and 2.5% normal goat serum. Sections were then incu-
bated with the anti-BrdU antibody (rat monoclonal, Harlan Sera–Lab,
Loughborough, UK) diluted 1:100 in PBS containing 0.125% BSA and
rinses, sections were further incubated in biotinylated anti-rat immuno-
globulin antibody (Vector Laboratories, Burlingame, CA) diluted 1:200
in PBS for 2 hr at room temperature. After three 10 min PBS rinses,
sections were incubated with avidin–peroxidase mixture (Elite-ABC kit,
Vector Laboratories) for 30 min at room temperature. After rinsing two
times for 10 min each in PBS and once in 0.05 M Tris-HCl, pH 7.6,
tissue-bound peroxidase was visualized by 10 min incubation in 50 mM
Tris, pH 7.6, containing 0.05% DAB, 0.03% NiCl2, and 0.6% H2O2.
Sections were then treated with ethanols (70, 95, 100%; two times for 5
min each) and xylene (10 min) and coverslipped with DEPEX (Merck,
Isle d’Abeau, France).
TUNEL staining. Apoptotic nuclei were labeled in situ by the TUNEL
method (Gavrieli et al., 1992). Frozen sections of fixed tissue were
brought to room temperature, rinsed 15 min in PBS, and treated over-
night with 0.1% Triton X-100 (Sigma) in PBS to unmask DNA; this step
replaces the usual 2–5 min treatment with proteinase K, because it was
found to yield the same sensitivity with improved tissue preservation
(Martin-Villalba et al., 1999). After a 10 min rinse in PBS, sections were
treated for 10 min with 3% H2O2in PBS to block endogenous peroxi-
dases. After PBS rinsing, sections were preincubated in TdT buffer (30
chloride). Sections were then drop-incubated in TdT buffer containing
Diagnostics) in a humid chamber for 90 min at 37°C; the reaction was
stopped by a 15 min rinse at room temperature in TB buffer (300 mM
NaCl, 30 mM sodium citrate). Sections were rinsed again in PBS before a
dUTP-labeled sections were incubated with avidin–peroxidase mixture
(Elite-ABC kit, Vector Laboratories) for 30 min at room temperature.
After rinsing twice in PBS and once in 50 mM Tris, pH 7.5, tissue-bound
peroxidase was visualized by 5 min incubation in 50 mM Tris, pH 7.6,
containing 0.05% DAB, 0.03% NiCl2, and 0.6% H2O2. Sections were
then dehydrated in graded ethanols (70, 95, 100°; two times for 5 min
each) and xylene (10 min) and coverslipped with DEPEX.
quantified on adjacent sections, using computer-assisted densitometry
survival time) at two different rostrocaudal levels of the olfactory organ
the septal OE. For inter-animal comparison, nuclei numbers were nor-
malized over the linear length of epithelium used for quantification, as
measured with the Biocom system (arbitrary units). A total of 120 slides
was analyzed. For each staining, at least five sections per animal were
analyzed, corresponding to ?4 mm length of septal OE per animal.
Results were expressed as mean ? SEM of the total number of positive
by Student’s t test and considered significant when p ? 0.05.
Olfactory marker protein in situ hybridization. In situ hybridization
(ISH) was performed under RNase-free conditions according to the
146 1.56218 Micheletal.(1994)/X03765
184 1.55925 Micheletal.(1997a)/M63419
370 1.559 23D26177
2461.5 62 25U65016
442 1.559 22D28526
3321.5 5925 AF039601
389 1.559 25 X55573
1794 • J.Neurosci.,March1,2003 • 23(5):1792–1803Baueretal.•LIFRegulatesInjury-InducedOlfactoryNeurogenesis
protocol of Jankowsky and Patterson (1999) with a few modifications.
Digoxigenin-labeled antisense and sense RNA probes were generated
from a 517 bp olfactory marker protein (OMP) cDNA plasmid, using an
in vitro transcription kit (Roche Diagnostics). DNA from the reaction
was removed before probe purification. Purified probes were subjected
tions were determined by spectrophotometry.
Frozen sections were fixed by rapid immersion in ice-cold 4% para-
formaldehyde for 20 min, rinsed in PBS, and treated with 2 ?g/ml pro-
min at room temperature. Sections were rinsed and refixed in 4% para-
formaldehyde for 10 min to inactivate the proteinase K. Sections were
then rinsed and acetylated in acetic anhydride for 10 min, rinsed again,
and dehydrated in graded ethanols (70, 95, 100%). Air-dried sections
were incubated in hybridization buffer containing 10% dextran sulfate,
hardt’s solution, with 1 ?g/ml probe overnight at 60°C. Sections were
1% normal goat serum in PBS for 30 min, followed by incubation in
anti-digoxigenin antibody conjugated with alkaline phosphatase (Roche
Diagnostics) diluted 1:1500 in the same buffer for 2 hr at room temper-
tetrazolium and 5-bromo 4-chloro 3-indolyl phosphate (Roche Diag-
nostics) in 100 mM Tris, 100 mM NaCl, 50 mM MgCl2, pH 9.6.
In situ RT-PCR. Indirect in situ RT-PCR was performed as described
previously (Recher et al., 2001). Briefly, dewaxed sections treated with
proteinase K were dehydrated and air dried. In situ RT reaction buffer
contained 50 mM Tris, pH 7.4, 75 mM KCl, 10 mM dithiothreitol, 3 mM
MgCl2, 0.5 mM dNTP, 1 ?M antisense primer, 1 U/?l RNase inhibitor,
and 10 U/?l SuperScript II (Life Technologies). The sections were cov-
ered with 40 ?l of the reaction buffer, sealed with amplicover clips, and
incubated at 42°C for 1 hr. Coverslips were removed and sections were
dNTP, 1 ?M each primer, and 0.2 U/?l TaqDNA polymerase (Eurobio,
Les Ulis, France). The sections were covered with 40 ?l of this reaction
buffer, sealed, and placed on a Perkin-Elmer thermal cycler (Applied
Biosystems). A total of 25 PCR cycles were performed under the same
conditions as for liquid PCR. Sections were then fixed with 4% parafor-
maldehyde (15 min) and washed as described in the ISH procedure. The
mers sense (CTTACTGCTGCTGGTTCTGCACTGGAAACA) and anti-
sense (TTCTTGATCTGGTTCATGAGGTTGCCGTGG) oligonucleo-
tides (Genset) labeled with digoxigenin-11-dUTP by 3? extension using
terminal transferase (Roche Diagnostics) and then ethanol purified.
Washing steps were done in 2? SSC (two times for 30 min), 0.5? SSC
(30 min) at room temperature, and hybrid detection as described above.
Several controls were performed: (1) ISH only, (2) omission of the ISH,
(3) omission of the reverse transcriptase, and (4) omission of the
LIF immunohistochemistry. LIF immunohistochemistry was per-
Minneapolis, MN) as primary antibody, fol-
lowed by biotin-conjugated anti-goat raised in
horse (Vector Laboratories) as secondary anti-
body. Frozen sections were hydrated in PBS,
perature and then in 0.1% Triton X-100 for 5
min at room temperature, and blocked with
at room temperature. Primary antibody di-
luted 1:100 was incubated overnight at 4°C in
the same buffer. After several washes in PBS,
secondary antibody diluted 1:200 was incu-
bated in PBS containing 2% normal horse se-
rum for 90 min at room temperature. After
several washes in PBS and blocking in 2% BSA
in PBS for 10 min at room temperature, sec-
tions were incubated in Elite-ABC kit (Vector
Laboratories), and staining was visualized with DAB/NiCl2as described
Adenoviral delivery of LIF. Recombinant LacZ and LIF adenoviruses
were prepared and assayed as described previously (Zhu et al., 2001).
a Hamilton syringe inserted 1 cm into the right nostril, while the animal
lay on its left side, at a rate of 0.6 ?l/min. Control mice received saline
and frozen sections were prepared. Sections were stained either with a
goat anti-mouse LIF IgG (R & D Systems) and a fluorescent-labeled,
swine anti-goat secondary antibody (Roche Diagnostics, Indianapolis,
IN) or for LacZ (Roche Diagnostics).
sections were stained for TUNEL, as described above. TUNEL-positive
cells were counted in the entire OE of the injected side from 10 sections
for two mice of each type (control, LacZ and LIF adenovirus). To assess
neurogenesis, mice were injected with 50 mg/kg BrdU 5 d after infection
and killed 6 or 12 hr later. BrdU was visualized as described above, and
labeled cells were quantified as for TUNEL. Separate counts were made
for the apical and basal layers of the OE, and statistical analysis was
significant when p ? 0.05.
Because mitogens are often regulated at the transcriptional level,
we assayed changes in mRNA levels of candidate mitogens after
of whole olfactory organs from OBX mice have been used suc-
cessfully to analyze molecular events in apoptosis (Michel et al.,
1994, 1997b; Farbman et al., 1999; Suzuki and Farbman, 2000),
cell cycle control (Kastner et al., 2000), and signal transduction
(Ezeh and Farbman, 1998). In the present study, artifacts were
products of the expected molecular size, (2) restriction enzyme
analysis of these products, and (3) systematic assessment of neg-
ative controls in each PCR assay (data not shown). The signals
tory organs sampled at various times after lesion were fairly con-
stant (Fig. 1) and therefore were used as a reference for the semi-
observations, including (1) kinetics of the decline in OMP after
OBX (Figs. 1, 2) being consistent with previously documented
timing of OBX-induced OSN degeneration (Schwartz-Levey et
al., 1991; Michel et al., 1994), (2) the duplication of kinetics of
c-fos mRNA expression (data not shown) found in a previous
LIF mRNA is induced and OMP mRNA disappears after OBX. Autoradiograms are shown of typical RT-PCR data
Baueretal.•LIFRegulatesInjury-InducedOlfactoryNeurogenesis J.Neurosci.,March1,2003 • 23(5):1792–1803 • 1795
RT-PCR study in the same murine strain
1994), and (3) the diversity of expression
time courses among the 13 genes assayed
Most of the cytokines and growth fac-
tors assessed (EGF, TGF?, TGF ?1, ?2,
?3, FGF2) are already expressed in the ol-
factory organ before surgery (Fig. 2). In
contrast, LIF (Fig. 1) and BDNF (Fig. 2)
are barely detectable in the controls. Al-
though expression levels of all of these
growth factors and cytokines show slight
variations after OBX, we find that LIF is
the only factor with levels that are signifi-
cantly increased after OBX and before the
onset of progenitor cell proliferation
(Schwartz-Levey et al., 1991; Bauer et al.,
pearing to peak 8 hr after the lesion ( p ?
to nonsignificant values by 1 d after OBX,
although LIF signal shows apparently
nonsignificant fluctuations at later time
Among the four receptor mRNAs that
were assessed, the LIF receptor (LIFR) is
also the only one to display upregulation
then decreases by 3–6 d (Fig. 2) ( p ?
0.05), a time corresponding to the maxi-
mum progenitor cell proliferation in the
OE after OBX (Schwartz-Levey et al.,
is not detectably expressed. After OBX, LIF protein accumulates
quickly, reaching maximal levels 4–8 hr after lesion and then
gradually decaying by 24 hr. Levels of ?-actin remain constant
over time on the same blots (Fig. 3).
Thus, OBX-induced LIF mRNA induction is paralleled by an
accumulation of LIF protein in the olfactory organ. The lack of a
delay between mRNA and protein upregulation as well as the
absence of internal stores of mature signal are characteristic fea-
tures of growth factor/neurotrophin production (cf. pulse-chase
analysis of NGF secretion) (Mowla et al., 1999).
situ RT-PCR for LIF mRNA and immunocytochemistry for LIF
protein. In the OE, as seen 8 hr after unilateral OBX, LIF mRNA
OE (Fig. 4A,B). These correspond to OMP-expressing perikarya
of OSNs, as visualized by conventional in situ hybridization on
adjacent sections (Fig. 4D). However, LIF mRNA signal is also
detected in the sustentacular cell layer and in the basal compart-
ment of the OE, and some sparse labeled cells are found in the
on the unoperated, contralateral side of olfactory organ sections
or on sections from intact mouse olfactory organ (data not
shown), consistent with the very low level of mRNA expression
RT-PCR approach (Fig. 1).
Consistent with the localization of LIF mRNA signal in the
found in olfactory axon bundles in the lamina propria that un-
derlies the OE (Fig. 4E). No staining is observed on the unoper-
ated, contralateral side of olfactory organ sections (Fig. 4F).
of the OSNs are committed to apoptosis by OBX (see Introduc-
be assessed directly because the earliest apoptosis markers in
OSNs become detectable 16 hr after OBX (Michel et al., 1994,
1997b), when LIF mRNA levels are returning to baseline (see
above). In addition, other cell types, such as sustentacular, basal,
or ensheathing glial cells, could also be involved in LIF synthesis
To investigate the biological significance of LIF signaling in vivo
in the OE, we analyzed the effects of OBX on apoptosis (TUNEL
of LIF KO mice compared with WT littermate controls. Labeling
conditions and consisted of manually scoring positive cell pro-
labeling indices for TUNEL and BrdU staining: the ratio of the
1796 • J.Neurosci.,March1,2003 • 23(5):1792–1803Baueretal.•LIFRegulatesInjury-InducedOlfactoryNeurogenesis
total number of positive cells to the linear length of septal OE
in this system because of the variations in OE thickness induced
by OBX (Schwartz-Levey et al., 1991; Holcomb et al., 1995).
Five days after OBX, the number of BrdU-labeled cells is
significantly increased over sham controls by 2.6-fold in the
OE of WT mice (Fig. 5A) ( p ? 0.001), consistent with previ-
ously reported effects of OBX (Schwartz-Levey et al., 1991). In
contrast, the number of BrdU-labeled cells in the OE of LIF
0.43). Therefore, the induction of proliferation 5 d after OBX
does not occur in the absence of LIF. Moreover, the difference
in the number of BrdU-labeled cells between WT and LIF KO
animals 5 d after OBX is highly significant ( p ? 0.003) and is
similar to that in WT mice when comparing the 5 d OBX and
the sham animals (Fig. 5A).
Interestingly, 2 d after OBX the number of BrdU-labeled cells
in the WT compared with the sham significantly decreases (Fig.
5B) ( p ? 0.04) and, to a greater extent, in the LIF KO animals
(Fig. 5B) ( p ? 0.003); the number of BrdU-labeled cells is also
WT 2 d after OBX (Fig. 5B) ( p ? 0.01).
Regarding cell death, OBX also induces a ?500-fold increase
0.0009). The number of TUNEL-positive cells then declines rap-
idly by 5 d after lesion (Fig. 6B) ( p ? 0.06 when compared with
sham controls). These results are consistent with earlier reports
demonstrating OBX-induced neuronal cell death in the OE of
adult mice (Michel et al., 1994; Holcomb et al., 1995).
Bulbectomy-induced cell death also occurs in LIF KO mice 2 d
after OBX (Fig. 6A) ( p ? 0.00002), although its extent appears
somewhat less than in the WT animals. The inter-strain differ-
ence does not reach statistical significance in this set of animals,
however (Fig. 6A) ( p ? 0.1).
Our analysis of cell death and proliferation in the OE of un-
operated WT animals reproduces published results, i.e., very low
levels of TUNEL staining (Holcomb et al., 1995; Mahalik 1996)
et al., 1991). No significant differences in these two parameters
are seen between unoperated OE in the WT and LIF KO mice
the normal adult OE being similar in both genotypes (data not
increased proliferation of OSN progenitor cells after OBX, we
asked whether exogenously delivered LIF could stimulate the
generation of new neurons in the uninjured, WT adult mouse
OE. Using our adenoviral vectors that had been shown to be
skin (Zhu et al., 2001), we unilaterally injected the nostrils of
unoperated WT mice with either virus. All analyses were per-
formed 5 d after viral infection. At this time point, LacZ and
exogenous LIF expression are detected in
the OE (Fig. 7B,C). Infection, detection,
and transgene expression are more effec-
tive with the LacZ than the LIF virus (Ta-
ble 2). Nonetheless, both vectors are very
in this system.
Comparing OE from infected versus
thickness ( p ? 0.01), an effect not seen
with LacZ adenovirus or with sham infec-
tion (Table 2). Hypothesizing that thin-
tion could be caused by cell death, we
determined the effects of exogenous LIF
expression on TUNEL staining in the OE
of uninjured, WT mice. The sham-
infected and LacZ-expressing OEs display
very few TUNEL-positive cells (Fig.
7D,E), indicating an absence of toxicity
from adenovirus administration in these
exogenous LIF displays strong TUNEL
staining in both apical and basal cellular
layers (Fig. 7F). Quantification reveals a
90-fold increase in TUNEL-positive cells in
specific labeling localized in olfactory axon bundles (Ax) of ipsilateral septum. F, No significant signal is found in contralateral,
In situ localization of LIF in the olfactory mucosa. A, Using indirect in situ RT-PCR, LIF mRNA expression 8 hr after
OBX. Duplicate samples from two independent mice are shown for each OBX time point. LIF
mobility is known to vary according to carbohydrate composition; in this preparation the LIF
Baueretal.•LIFRegulatesInjury-InducedOlfactoryNeurogenesisJ.Neurosci.,March1,2003 • 23(5):1792–1803 • 1797
To further analyze the effects of exogenous LIF on cell turn-
over, we assessed proliferation by injecting BrdU 5 d after viral
infection (OBX-induced proliferation peaks 5 d after lesion in
sion of specific cellular differentiation markers, the number of
done to help distinguish between OSN progenitors (basal layer),
immature/mature OSN (middle layer), and sustentacular cells
(apical layer), although the disruption of the OE observed after
LIF adenovirus infection hampers a simple analysis. After sham
infection or infection with the LacZ virus, most of the BrdU-
positive cells are found in the basal compartment of the OE (Fig.
7G,H, Table 2). This indicates that under these conditions, the
adenoviral infection itself does not markedly perturb cell prolif-
eration. In striking contrast, LIF overexpression yields many
BrdU-positive cells in the apical OE (Fig. 7I), the quantitation of
which revealed a 24-fold increase of labeled cells (Table 2) ( p ?
0.0001). Interestingly, the increase in BrdU labeling of the apical
OE after LIF overexpression is accompanied by a corresponding
OE (Table 2).
Given their laminar position in the OE after LIF overexpres-
cellular lineages. Indeed, the normal adult OE is known to con-
tain proliferating sustentacular (Weiler and Farbman, 1998) and
graphs), are shown stained for BrdU at the peak of proliferation, 5 d after unilateral OBX. When
indicated by the sham values does not differ between the genotypes. Although BrdU labeling is
significant (**p ? 0.003). B, The same analysis was performed 2 d after OBX. Interestingly, cell
peak of apoptosis, 2 d after OBX. As expected, there is a large increase in staining in the WT
experiment (3–5 animals per point). B, At 5 d after OBX, the number of TUNEL-labeled cells
1798 • J.Neurosci.,March1,2003 • 23(5):1792–1803Baueretal.•LIFRegulatesInjury-InducedOlfactoryNeurogenesis
basal cells, both horizontal and globose (Monti-Graziadei and
Graziadei, 1979; Schwartz-Levey et al., 1991). All of these cell
vivo retroviral lineage analysis in the normal adult OE (Caggiano et
al., 1994; Hunter et al., 1994; Schwob, 2002). Therefore, our results
expression could induce the proliferation of sustentacular cells.
BrdU injection (Fig. 7K), our observation of what appear to be
chains of BrdU-labeled cells extending from the basal to the apical
OE suggest that cells of the neuronal lineage could also proli-
ferate and be generated in the presence of exogenous LIF. On the
other hand, given the fact that exogenous LIF overexpression
in the normal OE appears to disrupt the OE, other cell types
could be induced to proliferate in the
OE, such as duct cells of Bowman’s
liferate in the OE after methyl-bromide
injury (Schwob et al., 1994, 1995).
and virally delivered exogenous LIF dem-
onstrates for the first time the in vivo in-
volvement of a specific endogenous cyto-
kine in the olfactory neuron turnover in
necessary for OBX-induced proliferation
of GBCs, (2) LIF is produced in part by
injured OSNs, and (3) exogenous LIF
stimulates cell turnover, increasing cell
layer of the normal adult OE.
Among the factors previously reported to
stimulate olfactory neuron production in
vitro (LIF, EGF, TGF?, TGF?1 and ?2,
BDNF, FGF2), we show that LIF is the
vivo before mitosis rises in the GBC pop-
ulation. Supporting these and our previ-
reports showed that LIF mRNA and pro-
tein are expressed after OBX in the adult
2002), although the peak of expression is
time course of OBX-induced LIF expres-
sion may be explained by the age of mice
used (6 weeks vs 3 months in our study).
In addition, Nan et al. (2001) suggest that
LIF immunoreactivity in the OE 2–3 d af-
ter OBX is associated with cellular pro-
cesses of macrophages; however, in situ
hybridization reveals that LIF mRNA is
also detected in OSNs (Getchell et al.,
the cellular source of LIF in those two
studies may be explained by the difficulty
in detecting LIF at these low levels. In the
present study, a highly sensitive in situ
RT-PCR approach was required to localize the site of early LIF
induction (8 hr after OBX) to mature OSNs. This localization is
lamina propria, although this staining could be in axons of in-
jured OSNs or in the ensheathing glia. A neuronal localization
would be in agreement with previously reported expression of
LIF in neuronal subpopulations of the adult rat brain (Lemke et
al., 1996, 1997) and in peripheral ganglia (Cheng and Patterson,
1997). Thus, although macrophages can indeed be recruited in
the OE after OBX (Nan et al., 2001; F. Jourdan, unpublished
observations), it seems unlikely that these cells will be the source
with the LIF virus yields labeled cells in all layers of the OE (K), and in some cases there appears to be a chain of labeled cells
Baueretal.•LIFRegulatesInjury-InducedOlfactoryNeurogenesis J.Neurosci.,March1,2003 • 23(5):1792–1803 • 1799
time after OBX or by other cell types in the OE and the lamina
Our finding that LIFR mRNA is upregulated after OBX, in
parallel to LIF itself, is reminiscent of induction of interleukin
signaling in the immune system. It is also consistent with the
OE and to presumptive ensheathing glial cells in the lamina pro-
pria of adult mice after OBX (Nan et al., 2001). Thus, LIF action
on these cells is very likely to be increased after injury.
is revealed by our findings in LIF KO mice, in which the OBX-
the lesion-induced proliferation of GBCs, without showing
whether LIF triggers the entry into, or the acceleration of, the
progenitor cell cycle. However, previous studies support the
former interpretation; OBX-induced proliferation in the adult
mouse OE involves an increased number of
incorporating GBCs, i.e., an increased number of progenitors
committed to enter into the cell cycle (Schwartz-Levey et al.,
1991). Conversely, the absence of mitogen results in a simple
blockade of the cell cycle, the length of which is constant in a
to study proliferation in the LIF KO at longer times after OBX.
A role for LIF in stimulating cells to enter the cell cycle is
supported by our results with LIF overexpression in the normal
adult OE. Here LIF enhances cell turnover, increasing both cell
death and cell replacement in the presumptive OSN layer. It
uninjured, normal adult are apparently unchanged in LIF KO
mice (Figs. 5, 6). Nonetheless, a tendency for reduced cell turn-
over in the OE of the LIF KO mouse, i.e., reduced proliferation
and cell death, is already apparent 2 d after OBX (Figs. 5B, 6A).
Thus, our findings suggest that both exogenous and endoge-
nous LIF in vivo can stimulate the OSN production that was
reported previously in vitro (Satoh and Yoshida, 1997a,b). An-
ena such as maintaining the steady-state rate of neurogenesis,
neuronal differentiation, and axon outgrowth.
The immunocytochemical localization of LIFR complexes to
GBCs and presumptive ensheathing glial cells (Nan et al., 2001)
suggests that LIF can act on these cell types after OBX, both of
which could be involved in the neurogenic response. LIF could
directly trigger entry into the cell cycle, most likely via cyclin D1
induction (Lavoie et al., 1996; Sherr, 2000). This general mito-
of GBC proliferation in adult mouse OE (Kastner et al., 2000;
tion pathways, by JAK/STAT3 (Janus kinase/signal transducer
and activator of transcription 3), which is known to mediate LIF
in the olfactory mucosa after OBX (Getchell et al., 2002). Alter-
other paracrine messenger. For instance, LIF-producing cells
grafted in the spinal cord selectively induce neurotrophin-3 in
vivo (Blesch et al., 1999). However, the 2–3 d delay between the
peak of LIF mRNA/protein expression (8 hr) and the onset of
is in keeping with previously reported delays for the direct
vitro (Ciccolini and Svendsen, 1998) and in vivo (Craig et al.,
1996; for review, see Scheffler et al., 1999). Also, LIF stimulation
of OE progenitor cell proliferation would be reminiscent of the
recently demonstrated LIF mitogenic action in the developing
cerebral cortex (Hatta et al., 2002). That LIF induction is found
transient (?24 hr), whereas OBX-induced increase of GBC pro-
liferation in OE is sustained for several weeks (Schwartz-Levey et
al., 1991), suggests that LIF would be involved specifically in the
level of progenitor proliferation would be maintained by other
It has been suggested recently that mature OSNs produce
of progenitor proliferation (Shou et al., 1999, 2000). Olfactory
neurogenesis could then be regulated by independent factors de-
pending on the physiological context. This leads to an attractive
model for in vivo combinatorial control of olfactory neurogen-
produced by mature OSNs might be primarily involved in the
inhibition of GBC proliferation in basal conditions. Under con-
ditions of increased neuronal loss, OSNs then synthesize LIF,
which could act in synergy with BMP disappearance to stimulate
GBC production of new neurons. Interestingly, LIF and BMPs
can co-regulate and counter-regulate the expression of the same
target genes in sympathetic neurons (Fann and Patterson,
P. H. Patterson, unpublished observations).
composed of colony-forming unit cells, hypothesized to be the
OSN stem cells, and of two consecutive types of neuronal pro-
genitors, i.e., transit amplifying cells that generate immediate
neuronal precursors, which finally produce new neurons (for
with the LIF KO OE demonstrate that LIF is required for injury-
induced neurogenesis, and the LIFR localization points to GBCs
as a likely target for LIF action, the steps for how LIF stimulates
new neuron production are not yet clear. Our results with LIF
ulate the production of putative neuronal progeny, without,
however, enhancing the number of proliferating basal cells.
This could be explained by the observation that LIF also stim-
ulates cell death in this experiment, which makes any conclu-
Interestingly, however, a subpopulation of GBCs has been
thetransgene OEthickness(?m) TUNEL?cells
measured in the dorsal and the mediodorsal (septal) OE at reproducible anatomical levels. Statistical analysis between treatment groups was performed with Student’s t test (*p ? 0.05 and ***p ? 0.001 when comparing LIF
1800 • J.Neurosci.,March1,2003 • 23(5):1792–1803Baueretal.•LIFRegulatesInjury-InducedOlfactoryNeurogenesis
identified as being true olfactory stem cells, i.e., giving rise to
different OE cell types, including neurons, horizontal basal cells,
and sustentacular cells. This occurs after the OE has been com-
pletely destroyed by methyl-bromide exposure (Huard et al.,
1998). Only in that case is the multilineage potential of some
enous LIF experiments, the question is whether LIF could stim-
ulate the multilineage potential of some GBCs.
We here further document LIF upregulation after injury in the
CNS (Minami et al., 1991; Banner et al., 1997; Jankowsky and
al., 1994; Sun et al., 1994; Carlson et al., 1996; Kurek et al., 1996;
Sun and Zigmond, 1996). The cytokine LIF thus appears as a key
messenger in injury-induced reactions of the adult nervous sys-
and phenotype, astrocyte activation, and inflammatory cell infil-
Sun and Zigmond, 1996; Sugiura et al., 2000). In the previously
and Patterson, 1994; Curtis et al., 1994; Banner et al., 1997;
Jankowsky and Patterson, 1999). In the OE in contrast, which is
devoid of classical glia (Crews and Hunter, 1994), LIF appears to
be produced in part by the lesioned neurons themselves and is
involved in OSN turnover. Interestingly, LIF expression is also
detected in presumptive sustentacular cells, which have been as-
signed a glial-like function in the OE, and in presumptive en-
at later times after lesion than LIF induction (Michel et al.,
Interestingly, LIFR signaling has been shown recently to be
involved in the control of neurogenesis in the adult subventricu-
lar zone (SVZ) stem cell system. Using adult heterozygous LIFR
KO mice, Shimazaki et al. (2001) demonstrated a 55% reduction
It is worth noting that this effect was not seen during develop-
period. This leads to the hypothesis, supported by the present
tenance of the self-renewing potency of neural stem cells
neuronal progenitors) that migrate into the OB (Kerr et al.,
2002). Interestingly, as is potentially the case for the OE, it is
possible that there is an interplay between LIF (Shimazaki et al.,
2001) and BMP (Lim et al., 2000) signaling in the SVZ. Such a
relationship has also been characterized recently in embryonic
stem (ES) cells plated at low density, where LIF, through an au-
tocrine FGF-2 signaling pathway, induces the formation of com-
mitted neural stem cells from undifferentiated, multipotent ES
cells, a process that is inhibited by endogenous BMP signaling
(Tropepe et al., 2001).
In vivo demonstration of mitogenic signaling by
The present study provides an example of a phenomenon that
could prove to be of general importance: cells emitting a self-
renewing mitogenic signal while entering apoptosis. Such a
cell dynamics in the adult OE allowed us to observe this for OSN
Concerning adult brain repair, there are reports of neuronal re-
placement after induction of neuronal apoptosis (Magavi et al.,
Thus, understanding the molecular signals that lead to neuronal
replacement after injury is a key step in developing therapeutic
approaches for brain repair. Our study on the adult mammalian
OE, which is well known for its capacity for neuronal replace-
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per therapies relying on induction of cell apoptosis.
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