European Journal of Neuroscience, Vol. 12, pp. 1372±1384, 2000
Ó European Neuroscience Association
NFI in the development of the olfactory neuroepithelium
and the regulation of olfactory marker protein gene
expression.
M. Behrens,1 G. Venkatraman,1* R. M. Gronostajski,2 R. R. Reed3 and F. L. Margolis1
1
Department of Anatomy and Neurobiology, University of Maryland at Baltimore, School of Medicine, Baltimore, MD 21201, USA
Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195, USA
3
Department of Molecular Biology and Genetics, and Department of Neuroscience, The Johns Hopkins University, School of
Medicine, Baltimore, MD 21205, USA
2
Keywords: in situ hybridization, mouse, olfactory system, transcription factors
Abstract
Nuclear factor I (NFI) proteins are DNA-binding transcription factors that participate in the tissue speci®c expression of various genes.
They are encoded by four different genes (NFI-A, B, C, and X) each of which generates multiple isoforms by alternative RNA splicing.
NFI-like binding sites have been identi®ed in several genes preferentially expressed in olfactory receptor neurons. Our prior
demonstration that NFI binds to these elements led to the hypothesis that NFI is involved in the regulation of these genes. To analyse
the role of NFI in the regulation of olfactory neuron gene expression we have performed transient transfection experiments in HEK
293 cells using constructs that place luciferase expression under the control of an olfactory marker protein (OMP)-promoter fragment
containing the NFI binding site. In vitro mutagenesis of this site revealed a negative modulation of luciferase expression by
endogenous NFI proteins in HEK 293 cells. In addition, we have used in situ hybridization to analyse the tissue and cellular
distribution of the four NFI gene transcripts during pre- and postnatal mouse development. We have simultaneously characterized the
expression of Pax-6, and O/E-1, transcription factors known to regulate the phenotype of olfactory receptor neurons. We demonstrate
that all of these transcription factors vary in speci®c spatio±temporal patterns during the development of the olfactory system. These
data on NFI activity, and on transcription factor expression, provide a basis to understand the role of NFI in regulating gene
expression in olfactory receptor neurons.
Introduction
The mammalian olfactory epithelium develops from competent
ectoderm that gives rise to olfactory turbinates (Farbman, 1994).
This event and subsequent olfactory receptor neuron (ORN)
development are accompanied by changes in transcription factor
gene expression. The Pax-6 gene (Walther & Gruss, 1991) is
expressed early in this process and Pax-6 null mutants are
characterized by anomalies in nasal, eye and forebrain structures
(Hogan et al., 1986; Hill et al., 1991). Deletion of Mash-1 that is
expressed in olfactory neuronal precursors led to a severe reduction in
the number of ORNs (Guillemot et al., 1993) indicating its critical
role in this process. O/E-1 is mainly expressed in postmitotic neurons
indicating a role in differentiation and maintenance of the ORN
phenotype (Davis & Reed, 1996). Our prior report of nuclear factor 1
(NFI) binding to promoters of several genes preferentially expressed
in ORNs and the detection of NFI mRNAs by in situ hybridization
(Chaudhry et al., 1997; Baumeister et al., 1999) imply that NFI
proteins participate in establishing the ORN phenotype.
Correspondence: Dr Frank L. Margolis, as above.
E-mail: [email protected]
*Present address: Department of Cell Biology, Emory University, School of
Medicine, Atlanta, GA 30329, USA.
Received 28 June 1999, revised 14 January 2000, accepted 18 January 2000
Odour detection is mediated by a multigene family of olfactory
receptors (Buck & Axel, 1991) linked to members of the signal
transduction cascade that are selectively expressed in ORNs (Buck,
1996). Golf (Jones & Reed, 1989), type III adenylate cyclase (AC III)
(Bakalyar & Reed, 1990), and the olfactory cyclic nucleotide-gated
channel-I (oCNGC-I; Dhallan et al., 1990) are components of the
cAMP-mediated pathway. Electrophysiological (Buiakova et al.,
1996) and behavioural (Youngentob & Margolis, 1999) data
implicate another olfactory speci®c component, the olfactory marker
protein (OMP;Margolis, 1980), as part of this signalling pathway.
The 5¢-¯anking regions of the OMP, Golf, ACIII, and oCNGC-I
genes contain several similar sequence motifs. The Olf-1 site
(Kudrycki et al., 1993) binds to Olf-1/EBF-like transcription factors.
O/E-1 was identi®ed in both olfactory mucosa (Olf-1) (Wang &
Reed, 1993) and developing B-cells (EBF) (Hagman et al., 1993).
The isolation of O/E-2 and O/E-3 (Wang et al., 1997) indicate that
Olf-1/EBF-like proteins are predominantly expressed in neurons in
distinct brain regions, retina, and olfactory epithelium. The
importance of this element is demonstrated by in vivo (Danciger
et al., 1989; Walters et al., 1996; Kudrycki et al., 1998) and in vitro
(Tsai & Reed, 1997; Wang et al., 1997) analyses and by its presence
in the promoters of additional genes preferentially expressed in ORNs
(Kudrycki et al., 1993; Wang et al., 1993).
Another sequence motif common to OMP, AC III and oCNGC-I
(Fig. 1a) is similar to binding sites for NFI (Buiakova et al., 1994;
NFI expression in olfactory epithelium of mouse 1373
further puri®ed by extractions with acidic phenol/chloroform. The
mRNA was reverse transcribed using M-MLV reverse transcriptase
(Gibco BRL, Grand Island, NY, USA) and random hexamers. This
step was omitted for a fraction of the puri®ed RNA as a negative
control. The produced cDNA was used as a template for the subsequent
PCR. The 5¢-primer NFI-A (5¢-ACITGGTTCAACCTGCAGGCICG3¢) and the two 3¢-primers NFI-AC (5¢-GGCGCTCGCCRTCRGTRCTYTCC-3¢) and NFI-BX (5-¢GCCGCTCYCCATCRGTACTTTCC-3¢) were chosen from highly conserved regions within the DNAbinding domains of all four human NFI subtypes (Fig. 2a). Primer NFIA was used in combination with primer NFI-AC and primer NFI-BX to
amplify NFI subtypes A and C and NFI subtypes B and X, respectively.
After 40 cycles (1 min at 62 °C, 1 min at 72 °C, 30 s at 95 °C) the pool
of 364 bp-long amplicons was subjected to analytical digestions with
restriction endonucleases in order to determine which subtypes had
been ampli®ed (Fig. 2b).
Construction of vectors and in vitro mutagenesis
FIG. 1. (a) Schematic of the promoter regions of rat OMP, AC III, and oCNGCI genes. Olf-1/EBF-like binding sites (rectangular) and NFI-like binding sites
(ellipsoid) as well as the starts of transcription are marked. (b) Schematic of
the OMP promoter constructs driving the expression of luciferase. Olf-1 sites
and the NFI element are indicated. The position and sequences of the wildtype (wt) and mutated (mut) NFI elements are shown.
Baumeister et al., 1999). The four NFI genes (NFI-A, -B, -C, and -X)
are each transcribed and processed to several splice variants (Liu
et al., 1997; Nebl & Cato, 1995). NFI proteins have positive (i.e.
Inoue et al., 1990) or negative regulatory roles (Liu et al., 1997) on
different tissue-speci®c expressed genes.
We demonstrate in vitro that NFI proteins are negative modulators
of OMP gene expression. Our in situ data demonstrate spatial and
temporal changes in NFI gene expression during ontogeny and
demonstrate that NFI-C is the only detectable NFI subtype in adult
neuroepithelium.
Materials and methods
RT-PCR
A combination of RT-PCR and restriction endonuclease digestion was
used to identify the NFI isoforms expressed in HEK 293 and JEG-3
cells. To determine which forms of NFI proteins are endogenously
expressed poly(A)+ RNA from these cells was isolated with the Micro
Poly(A)Pure mRNA Isolation Kit (Ambion, Austin, TX, USA)
according to the manufacturer's protocol. To remove contaminating
genomic DNA, the RNA was subjected to DNAse I digestion and
The 2.7 kbp promoter fragment of the rat OMP promoter containing
the proximal and distal Olf-1 elements and the UBE (upstream binding
element, NFI site) was cloned into the polylinker region preceeding the
ORF for the ®re¯y luciferase of the pGL2 basic vector (Promega,
Madison, WI, USA). The 800 bp fragment including both Olf-1
elements and the UBE was subcloned as a Bgl II fragment from the 2.7
kbp reporter construct and subsequently recloned into the pGL2 basic
vector. For in vitro mutagenesis the same 800 bp Bgl II fragment was
recircularized with T4 DNA-ligase (Gibco BRL, Grand Island, NY,
USA). Primers A (5¢-GCTGCCATCTGTCTGGCAGATG-3¢) and B
(5¢-CTCGGCTCCGAGAGGCTGTG-3¢) were used to introduce point
mutations (underlined in the above sequences) into a linear PCR
product generated by Pfu-DNA-Polymerase (Stratagene, La Jolla, CA,
USA) (Fig. 1b). The PCR product was recircularized and cut with BglII
and PstI to obtain a cassette with a defective NFI binding site
(Meisterernst et al., 1988; Baumeister et al., 1999) that was subsequently used to exchange the corresponding wild-type BglII/PstI
fragments within the 800 bp and 2.7 kbp constructs. A defective
oligonucleotide synthesis led to the generation and subsequent cloning
of another cassette containing a deletion-mutated NFI binding site. The
high af®nity binding site CTGGCTTT-GGGCCAA (consensus
sequence shown in bold, highest conserved nucleotides bold and
underlined) was rendered to CTGGCTTT-GGGC-del-GAG by the
deletion of a tri-nucleotide. This exchange affects a 100% conserved
nucleotide (Gronostajski, 1987; Osada et al., 1996; Baumeister et al.,
1999) within one NFI half binding site and should therefore abolish
NFI binding. We chose to include constructs containing this deletionmutated NFI binding site into the reporter gene assays. The region
coding for the b-galactosidase of the pCMVb vector (Clontech, Palo
Alto, CA, USA) was removed by restriction with NotI and the
remainder of this vector was recircularized to generate a noncoding
expression vector for the adjustment of equimolar amounts of this
vector type in transient transfection assays.
Transient transfection assays
HEK 293 cells were cultivated in DMEM (Gibco BRL, Grand Island,
NY, USA); 10% foetal bovine serum (FBS; HyClone, Logan, UT,
USA) at 37 °C in 7% CO2. Twenty-four hours before transfection the
cells were seeded on 6 cm diameter culture plates at a constant 50±60%
con¯uency. The transfection mixture consisted of the following
components: 1 pmol reporter construct, 0.05 pmol expression vector (if
indicated), 0.01 pmol pCMVb to monitor the transfection ef®ciency,
pCMVb vector with the removed b-galactosidase coding region to
adjust the total amount of expression constructs to 0.22 pmole. The
Ó 2000 European Neuroscience Association, European Journal of Neuroscience, 12, 1372±1384
1374 M. Behrens et al.
FIG. 2 (a) Schematic of the region within NFI cDNAs ampli®ed for subtype analysis of the human cell lines HEK 293 and JEG-3. (b) The restriction sites used for
the analysis of the RT-PCR amplicons obtained with oligonucleotides speci®c for NFI subtypes A and C and the subtypes B and X, respectively, are shown
together with the anticipated fragment lengths. (c) NFI subtype analysis of HEK 293 cells. Lane 1: DNA-length standard (FX 174/Hae III). Lanes 2 and 3: 364 bp
PCR products speci®c for NFI subtypes A & C and B and X, respectively. Lanes 4 and 5: A and C and B and X amplicons subjected to digestion with EcoRV. The
202 and 162 bp fragments obtained in lane 4 are speci®c for NFI-A. Lanes 6 and 7: A/C and B/X amplicons digested with BamHI. The 334 bp fragment observed in
lane 7 is speci®c for NFI-X. Lanes 8 and 9: A/C and B/X amplicons digested with HphI. The 185 and the 142 bp fragments in lane 9 are speci®c for NFI-B. Notice
that the 327 bp fragment in lane 8 and the 191 and 173 bp fragments are speci®c for subtypes NFI-A and NFI-C, respectively. The 234 and the 93 bp fragments
found additionally in lane 9 indicate the presence of NFI-X. Lanes 10 and 11: A/C and B/X amplicons digested with NarI. The 215 and 149 bp fragments in lane 10
are speci®c for NFI-C. (d) NFI subtype analysis of JEG-3 cells: Sample application to the gel is identical to FIG. 3c. Lanes 8 and 9: A/C and B/X amplicons
digested with HphI. The 185 and the 142 bp fragments in lane 9 are speci®c for NFI-B. Notice that the 191 and 173 bp fragments are speci®c for subtype NFI-C.
Lanes 10 and 11: A/C and B/X amplicons digested with NarI. The 215 and 149 bp fragments in lane 10 are speci®c for NFI-C. The digestion of the A/C amplicons
with NarI appears to be only partially as the EcoRV and HphI digestions (Lanes 4 and 8) did not detect NFI subtype A.
DNA concentration was adjusted to 8.8 mg in 220 mL of 0.1 x TE
buffer; pH 8.0 by the addition of pBluescript KS II(+) (Stratagene, La
Jolla, CA, USA). Calcium phosphate coprecipitation was carried out
after the addition of 250 mL 2 x HBS buffer (in mM: NaCl, 280; KCl,
10; Na2HPO4, 1.5; dextrose, 12; HEPES, 50, pH 7.05) to the plasmid
mixture by slowly adding 31 mL of 2 M CaCl2. The precipitate was
allowed to form for 30 min at room temperature (RT). Following the
addition of the precipitate to the culture plates and incubation for 5 h at
37 °C in 7% CO2 a brief glycerol shock was applied to enhance the
uptake of constructs (1.5 min with 37 °C warm DMEM, 10% FBS, 8%
glycerol). After the exchange of the glycerol-containing medium for
standard culture medium the cells were allowed to express the reporter
constructs for 48 h before collecting the cell extracts in 250 mL lysis
buffer (Tropix, Bedford, MA, USA) containing 0.5 mM DTT. Cell
extracts were stored at ±80 °C for subsequent luminometric measurements. Luminometric determination of ®re¯y luciferase and bgalactosidase levels expressed by the transient transfected cells was
carried out with 10 mL of cell extracts following the manufacturer's
protocol for the Dual-Light reporter gene assay system (Tropix,
Bedford, MA, USA). Authentic ®re¯y luciferase and bacterial bgalactosidase were included as standards. To correct for differences in
transfection effeciency and cell density variations the luciferase
activity was standardized to the b-galactosidase activity obtained for
each measurement. Unless indicated otherwise, each experiment was
performed at least in quadruplicate.
Determination of embryonal ages
Mating was allowed overnight. The next morning the mice were
separated and the female was monitored for pregnancy by daily
weight gain. In cases of successful mating the morning of separation
was considered as E 0.5.
In situ hybridization
Mice were anaesthetized with ketamine/xylazine (3 : 1, 100 mg/kg
body weight and 33 mg/kg body weight, respectively) and
perfused transcardially with ice-cold phosphate-buffered saline
Ó 2000 European Neuroscience Association, European Journal of Neuroscience, 12, 1372±1384
NFI expression in olfactory epithelium of mouse 1375
FIG. 3. Reporter gene assays for the activation of 2.7 kbp and 0.8 kbp OMP
promoter fragments with wild-type (wt) or mutated (mut, del) NFI binding
sites by cotransfection with the O/E-1 expression vector pCIS O/E-1. The
promoter driven luciferase production was measured by luminometry. The
relative increase of luciferase production by cotransfection with O/E-1 is
illustrated. The relative differences between otherwise identical reporter
constructs containing the wild-type and mutated NFI binding site, respectively,
are: 0.8 kbp mut/0.8 kbp wt = + 51%; 0.8 kbp del/0.8 kbp wt = + 12%; 2.7 kbp
mut/2.7 kbp wt = + 55%; 2.7 kbp del/2.7 kbp wt = + 45%. Relative light units
of the induced luciferase per 1 million relative light units of the b-galatosidase
control were: 0.8 kbp wt/0.8 kbp wt + O/E-1 = 1453/9859. 0.8 kbp mut/0.8 kbp
mut + O/E-1 = 1160/11976. 0.8 kbp del/0.8 kbp del + O/E-1 = 1811/13675. 2.7
kbp wt/2.7 kbp wt + O/E-1 = 3099/10232. 2.7 kbp mut/2.7 kbp mut + O/E1 = 1846/9491. 2.7 kbp del/2.7 kbp del + O/E-1 = 3303/15762.
rinses in 0.4 x SSC (15 min at RT, 2 x at 55 °C) were followed by
the colourimetric detection of hybridization signals. The slides
were incubated for 5 min at RT in buffer 1 (150 mM NaCl,
100 mM maleic acid, pH 7.5) and blocked for 1 h at RT in buffer
1 containing 1% blocking reagent (Boehringer Mannheim,
Indianapolis, IN, USA). After draining the blocking solution from
the slides fresh blocking solution with 1 : 750 diluted Anti-Dig-AP
was applied and left for 1 h at RT on the slides. Following two
30 min washes in buffer 1 (at RT, gentle shaking), the slides were
equilibrated in buffer 3 (in mM: NaCl, 100; MgCl2, 50; Tris-HCl,
100, pH 9.5) for 5 min at RT. The chromogenic substrate (NBT
and BCIP in buffer 3) was applied to the slides and the
production of the coloured product by the alkaline phosphatase
moiety of the antibody at RT in the dark was monitored and
stopped in TE buffer when the signal to background ratio
appeared optimal (usually after overnight to 24 h incubations). The
sections were mounted with a water-based mounting medium
(¯uorescent mounting medium; Dako, Carpinteria, CA, USA) and
examined microscopically. Although the length of riboprobes used
in this study vary, the detection sensitivity and speci®city appears
to be similar. The high signal in hippocampus and the absence of
signal in olfactory epithelium obtained with the smallest riboprobe
for NFI-X (220 bp) in P9 mice is complementary to the signals
detected with the longest riboprobe for Pax-6 (2040 bp). This indicates
that the detection of signals in this study primarily depended on the
availability of target mRNA in the examined tissues.
Results
NFI gene expression in human cell lines
(PBS), pH 7.4 and subsequently with 4% paraformaldehyde in
PBS. Noses were removed, post®xed for a few hours at 4 °C, and
allowed to sink in cold 30% sucrose in PBS overnight at 4 °C.
The tissue was transferred into OCT medium (Tissue-Tek, Miles
Scienti®c, Elkhart, IN, USA) and kept for 30 min at 4 °C to allow
complete penetration. After quickly freezing by submersion in
±80 °C cold isopentane, the specimens were stored at ±80 °C.
Cryostat sections were cut in the coronal plane at 10 mm thickness
and thaw-mounted onto siliconized slides (Schmale & Behrens,
1995). Pretreatment, prehybridization and hybridization of the
specimen were mainly performed as described previously (Behrens
et al., 1997). Digoxigenin-labelled homologous antisense riboprobes of NFI-A (430 bp KpnI/EcoRI fragment of the 3¢
untranslated region), NFI-B (320 bp XbaI/EcoRI fragment of the
3¢ untranslated region), NFI-C (760 bp XcmI/EcoRI fragment
containing part of the coding region and the 3¢ untranslated
region), NFI-X (220 bp SmaI/BamHI fragment covering the 3¢ part
of the coding region), Olf-1 (O/E-1) (780 bp fragment corresponding to part of the coding region and the 3¢ untranslated region),
Pax-6 (2040 bp EcoRI fragment covering the entire clone
Pax6sc35 (Walther & Gruss, 1991) alkali digested to an average
fragment length of 200 bp (Angerer et al., 1987), and OMP
(760 bp BamHI/XbaI fragment of the entire coding region) were
prepared and hybridized at a concentration of 250±500 ng/mL
(100 ng/mL for the OMP antisense probe) overnight at 55 °C in a
humid chamber. In addition to these antisense probes an OMP
sense probe was generated to monitor unspeci®c binding of
riboprobes. After hybridization, the slides were washed three
times for 5 min in 2 x SSC (RT with gentle shaking) and
incubated with RNAse A (0.5 M NaCl, 10 mM Tris-HCl, 1 mM
EDTA, 10 mg/mL RNAse A; pH 7.5) for 30 min at 37 °C. Three
RT-PCR revealed the presence of mRNA for all four NFI subtypes in
HEK 293 cells (Fig. 2c). The relative levels of ampli®cation suggest
that subtypes NFI-B, -C, and -X are of similar abundance and in excess
of subtype NFI-A. Transfection of HEK 293 cells with the pCMVb
vector by the calcium phosphate coprecipitation method and
subsequent colourimetric detection of b-galactosidase in successfully
transfected cells revealed a high transfection ef®ciency (» 65%). In the
human choriocarcinoma cell line JEG-3 only the NFI subtypes B and X
were detectable at about equal levels (Fig. 2d). Transfection experiments showed a lower number of positive cells (» 15%) than obtained
with HEK 293 cells. A very attractive system for the regulation of
olfactory speci®c expressed genes would be the use of immortalized
olfactory neurons. The mouse cell line 4.4.2. has been immortalized by
the retroviral insertion of the n-myc oncogene into mitotically active
basal cells of adult mouse olfactory epithelium. Differentiation and
process outgrowth is reported to be induced by the removal of foetal
bovine serum (Puche et al., 1996). However, in our hands although
serum removal led to neurite outgrowth little or no OMP was
expressed. This cell line has been reported to express Golf and OMP as
olfactory speci®c markers of maturation (MacDonald et al., 1996). The
report that overexpression of myc suppresses NFI dependent promoters
by alteration of NFI proteins (Yang et al., 1993) coupled with the poor
transfection ef®ciency (< 5%) we obtained, with both calcium
phosphate coprecipitation and lipofectamine (Gibco BRL, Grand
Island, NY, USA) mediated transfection, encouraged us not to use this
cell line. Instead, we performed all reporter gene assays in HEK 293
cells because of the reliably high transfection ef®ciency and because
this cell line provides a background in which all four NFI genes are
expressed endogeneously permitting the investigation of complex
promoters in which NFI binding elements are present as one of several
components.
Ó 2000 European Neuroscience Association, European Journal of Neuroscience, 12, 1372±1384
1376 M. Behrens et al.
FIG. 4. NFI, O/E-1, Pax-6, and OMP expression in the olfactory epithelium of E15.5 mouse embryos. In situ hybridization using Digoxigenin-11-UTP labelled
riboprobes on 10 mm coronal sections. Positive cells are labelled by a dark coloured precipitate. Note the inhomogeneous distribution of weakly stained cells
positive for NFI subtypes A, B, and X, whereas no signal could be obtained for NFI-C mRNA. The mRNA for O/E-1 is most abundant in cells that reside in the
centre of the embryonic epithelium, whereas Pax-6 seems to be expressed throughout the entire structure. Few OMP positive cells indicate the low number of
mature olfactory receptor neurons at this stage of development. Hybridization with a riboprobe comprising the OMP sense strand gave no signal attesting to the
speci®city of the in situ hybridization. Scale bar, 100 mm.
Ó 2000 European Neuroscience Association, European Journal of Neuroscience, 12, 1372±1384
NFI expression in olfactory epithelium of mouse 1377
Promoter analysis in vitro
To con®rm the feasibility of our reporter gene assay system we tested
our ability to reproduce previous results on the activation of OMP
promoter fragments by cotransfection of O/E-1 (Tsai & Reed, 1997;
Wang et al., 1997). The cotransfection of the 0.8 or 2.7-kbp reporter
constructs with the O/E-1 expression vector into HEK 293 cells
resulted in an increase in the expression of luciferase in both cases.
The activation of both wild-type promoter fragments is similar,
FIG. 5. NFI, O/E-1, Pax-6, and OMP expression in the olfactory epithelium of E17.5 mouse embryos. In situ hybridization using Digoxigenin-11-UTP labelled
riboprobes on 10 mm coronal sections. Whereas the signal obtained with the NFI-A speci®c antisense riboprobe seems to persist at this stage of development no
mRNA for the NFI subtypes B and X could be detected. However, the onset of NFI-C expression is indicated by a low number of labelled cells. The progressive
strati®cation of the olfactory epithelium is indicated by the segregation of O/E-1 and Pax-6 positive cells. Whereas Pax-6 signals are highest at the apical and basal
poles of the epithelium where the sustentacular and basal cell layers are formed, O/E-1 mRNA is present in the entire neuronal cell population. The OMP
expression has increased and is now restricted to cells within the most apical neuronal layer. Scale bar, 20 mm.
Ó 2000 European Neuroscience Association, European Journal of Neuroscience, 12, 1372±1384
1378 M. Behrens et al.
indicating the absence of signi®cant interference by additional
regulatory elements in the 2.7 kbp 5¢-¯anking region, with binding
sites within the 0.8 kbp promoter fragment. O/E-1 cotransfection
stimulates the luciferase expression driven by the 0.8 kbp promoter
fragment 6.8-fold. The same experiment performed with the 2.7 kbp
promoter fragment resulted in a 3.3-fold stimulation (Fig. 3).
To determine if NFI proteins are involved in regulating OMP gene
expression transient transfections were carried out using OMP
promoter constructs containing the point-mutated and the deletionmutated NFI binding sites. Constructs containing these mutated
elements gave higher O/E-1 stimulation of the 0.8 kbp promoter
fragment. The activation of 10.3-fold observed with the point-
FIG. 6. NFI, O/E-1, Pax-6, and OMP expression in the olfactory epithelium of P1 mouse. In situ hybridization using Digoxigenin-11-UTP labelled riboprobes on
10 mm coronal sections. The only detectable NFI subtype is C. Cells expressing high amounts of NFI-C mRNA seem to preferentially reside in apical and basal parts
of the neuroepithelium. The O/E-1 expression pattern did not change compared with E17.5. The Pax-6 signal is clearly restricted to sustentacular cells and cells within
the basal cell layer. OMP positive cells form a con¯uent layer within the apical neuroepithelium indicating the most mature population of neurons. Scale bar, 50 mm.
Ó 2000 European Neuroscience Association, European Journal of Neuroscience, 12, 1372±1384
NFI expression in olfactory epithelium of mouse 1379
Ó 2000 European Neuroscience Association, European Journal of Neuroscience, 12, 1372±1384
FIG. 7. NFI, O/E-1, Pax-6, and OMP expression in the vomeronasal organ of P1 mouse. In situ hybridization using Digoxigenin-11-UTP labelled riboprobes on 10 mm coronal sections. The colocalization of the
NFI-C, O/E-1, and OMP mRNAs as seen in the main olfactory epithelium can also be seen in the vomeronasal organ. All three riboprobes label the entire neuronal part of this structure. Pax-6 is only expressed in
the proliferative zone of the vomeronasal organ. Scale bar, 100 mm.
1380 M. Behrens et al.
mutated construct corresponds to an upregulation by the mutated NFI
binding site of 51%. Similarly O/E-1 induced activation in the wildtype and point-mutated 2.7 kbp fragments is 3.3-fold and 5.1-fold,
respectively. This re¯ects an increase of O/E-1 induced activation by
mutation of the NFI binding site of 55%
mutated NFI element raised the activation
(12%) and 4.8-fold (45%) in the 0.8 kbp
fragments, respectively (Fig. 3). Although
(Fig. 3). The deletionby O/E-1 by 7.6-fold
and 2.7 kbp promoter
fewer analyses were
FIG. 8. NFI, O/E-1, Pax-6, and OMP expression in the olfactory epithelium of P9 mouse. In situ hybridization using Digoxigenin-11-UTP labelled riboprobes on
10 mm coronal sections. Notice the complete overlap between the hybridization signals obtained with the NFI-C and O/E-1 riboprobes and the growing number of
OMP positive neurons localized at the apical pole of the neuroepithelium. Scale bar, 100 mm.
Ó 2000 European Neuroscience Association, European Journal of Neuroscience, 12, 1372±1384
NFI expression in olfactory epithelium of mouse 1381
performed with the deletion-mutated fragments than with the wildtype and the point-mutated constructs, the increase of O/E-1
stimulation using two differently mutated NFI elements attests to
the negative regulation by the wild-type NFI site in HEK 293 cells
(Fig. 3). The complex mixture of NFI proteins present in this cell line
therefore behave as negative regulators on the wild-type OMP
promoter fragments.
be seen in the apical layers of the epithelium. The relative signal
intensities indicate that NFI-A may have a more important role for the
embryonal development of the olfactory epithelium than NFI-B. The
hybridization signal obtained with the probe against NFI-C mRNA is
similar to background signals of the OMP sense probe. Very low
levels of NFI-X mRNA are visualized.
Developmental expression of NFI mRNAs
The mature mammalian olfactory neuroepithelium is a pseudostrati®ed structure consisting of olfactory receptor neurons (ORNs),
sustentacular cells and their precursor cells (globose and
horizontal basal cells). Dying ORNs and sustentacular cells are
replenished throughout life by basal cells re¯ecting the regenerative capacity of this epithelium (Graziadei & Graziadei, 1979a,
b; Schwartz-Levey et al., 1991; Huard et al., 1998). The development of this structure from a single layer of invaginating
competent ectoderm to the mature state is marked by a transition
from a predominantly proliferative to a predominantly differentiating neuroepithelium and is accompanied by changes in the
expression of key molecules involved in this process.
OMP positive neurons are evident primarily in the apical layers of the
olfactory neuroepithelium, with gaps evident and a few OMP positive
cells in deeper layers (Fig. 5). This indicates that strati®cation is not a
uniform process. O/E-1 mRNA can be seen in all cells within the
epithelium except in the well developed non-neuronal sustentacular
cell layer. NFI-A mRNA can still be detected in isolated cells within
the centre of the neuroepithelium. The weak signals obtained at E15.5
for NFI-B and -X mRNAs (Fig. 4) are no longer detectable. However,
a few NFI-C positive cells have appeared. NFI-C positive cells are
con®ned to more apical areas overlapping with OMP expression
zones but, unlike OMP, also extend into deeper layers. Pax-6 mRNA
is present in sustentacular cells and in cells in the lower third of the
epithelium including the basal cell layer.
E15.5
P1
At this stage only a few olfactory neurons have reached maturity as
indicated by OMP gene expression (Fig. 4). Although most of the
OMP positive cells are found in the upper third of the epithelium
some can be seen in deeper layers. The number of O/E-1 positive
cells which are mainly localized in the centre of the developing
neuroepithelium vastly exeeds the number of OMP expressing cells.
In contrast to O/E-1 mRNA the signal for Pax-6 mRNA is more
intense at the apical and basal regions of the epithelium. The rather
scattered distribution of OMP, O/E-1 and Pax-6 mRNAs indicate that
mature strati®cation has not yet occurred. NFI signals at this stage of
development are weak. Very few NFI-A and NFI-B positive cells can
The layer of OMP-positive cells is a single to a few cell layers deep
indicating that the neuroepithelium is still expanding (Fig. 6). O/E-1
mRNA is evident in both immature and mature olfactory receptor
neurons. In contrast to the olfactory neuroepithelium at E17.5 (Fig. 5)
a basal cell layer with only scattered O/E-1 positive cells has been
established. The O/E-1 containing cells are surrounded by Pax-6
expressing cells in the sustentacular and basal cell layers as
previously reported (Davis & Reed, 1996). The progressive decrease
in the intensity of the NFI-A signal between E15.5 (Fig. 4) and E17.5
(Fig. 5) has continued and at this age NFI-A mRNA, NFI-B and NFIX are all undetectable. The differential distribution of NFI-C positive
E 17.5
FIG. 9. NFI, O/E-1, and Pax-6 expression in the hippocampal area of the brain of P9 mouse. In situ hybridization using Digoxigenin-11-UTP labelled riboprobes
on 10 mm sagittal sections. The most prominent hybridization in both the dentate gyrus and the CA1 region of the hippocampus was obtained with the antisense
probe for NFI-X. NFI-A and NFI-B mRNA is found in the dentate gyrus and weaker in the CA1 region. mRNAs for NFI-C, O/E-1 and Pax-6 were not detectable
within the hippocampus. Scale bar, 400 mm.
Ó 2000 European Neuroscience Association, European Journal of Neuroscience, 12, 1372±1384
1382 M. Behrens et al.
FIG. 10. Alignment of NFI-like binding sites within the promoter regions of
rat olfactory marker protein (OMP), Type III adenylyl cyclase (AC III),
olfactory cyclic-nucleotide gated channel-I (oCNGC-I), and the NFI consensus
element (CONS). The core motifs (TGGN6G/TCCA) are boxed. Important
differences from the consensus sequence in the NFI binding sites of the OMP
gene and the adenylyl cyclase gene are in bold. As the NFI site in the oCNGCI shows high homology to the consensus sequence in all crucial positions it can
be considered a high af®nity binding site. The nucleotide exchange found in
the AC III element creates a lower af®nity binding site closely related to the
NFI element in the a-globin promoter (Meisterernst et al., 1988). The
expanded spacer region with otherwise high conservation of core motif
positions (Gronostajski, 1987) also generates a lower af®nity binding site
within the OMP gene promoter. The different af®nities for NFI of these
binding sites have recently been experimentally demonstrated (Baumeister
et al., 1999).
cells at E17.5 (Fig. 5) is more pronounced because of the increased
thickness of the epithelium. The majority of NFI-C mRNA is detected
in apical cell layers overlapping with OMP-positive cells and in
scattered cells near the basal part of the epithelium with very few
cells showing a hybridization signal in the centre of the epithelium.
P1 vomeronasal organ
The distribution of signals in the vomeronasal organ is similar to that
in the main olfactory epithelium (Fig. 7). OMP staining can be seen
throughout the mature sensory neurons. The proliferative zone near
the point of the crescent contains immature, OMP negative neurons.
The O/E-1 signal overlaps with the OMP staining but extends into the
proliferative zones that are also marked by an elevated Pax-6
expression. The zones of strong expression of NFI-C and weak
expression of NFI-A overlap with areas where O/E-1 mRNA is
detectable. As in the main olfactory epithelium the NFI-B and NFI-X
isoforms are not detectable.
P9
The young olfactory epithelium is marked by several layers of OMPpositive cells residing in the apical 50% of the olfactory receptor cells
indicating the border between mature (OMP-positive) and immature
neurons (Fig. 8). In contrast to OMP mRNA O/E-1 mRNA is present
in the entire population of mature and immature neurons. NFI-C
mRNA distribution is identical to that of O/E-1 mRNA, whereas NFIA, -B, and -X mRNAs are absent. The mRNA for the developmental
control gene Pax-6 which plays an important role as an inducer of
early development of the olfactory epithelium is restricted to the nonneuronal sustentacular cells and to the basal cells.
P9 hippocampus
Walters et al. (1996) reported the ectopic expression of the 800 bp
OMP promoter fragment driven expression of b-galactosidase in
dentate gyrus and CA1 region of the hippocampus in transgenic mice.
The in situ hybridization of this area shows high signals of NFI-X
mRNA in these regions (Fig. 9). In addition NFI subtypes A and B are
expressed in the dentate gyrus and to a lesser extend in the CA1 area.
No signals could be obtained with probes for NFI-C, O/E-1, and
Pax-6. The expression pattern observed for the NFI subtypes in
FIG. 11. Model of the regulation of OMP gene transcription. The ®rst step of
OMP gene transcription, the competition of NF-I with nucleosome formation
is hypothesized. Later steps have been demonstrated experimentally.
hippocampus is in agreement with previous reports by Chaudhry and
colleagues (Chaudhry et al., 1997).
Discussion
DNAseI footprinting analysis of the OMP gene promoter identi®ed a
region whose sequence is conserved across species and was named
UBE (Kudrycki et al., 1993; Buiakova et al., 1994). Similar elements
were found in two other genes selectively expressed in ORNs, the AC
III (UCY) and the oCNGC-I (UC). Subsequently, the speci®c
interaction of oligonucleotides comprising these elements with
authentic NFI protein as well as with protein extracts from olfactory
mucosa was reported (Baumeister et al., 1999) identifying the UBE,
UCY and UC as NFI binding sites. However, functional data was
missing. The present study demonstrates that the UBE is functional in
reporter gene assays where it acts as a repressor element. Further, we
demonstrate by in situ hybridization that NFI genes are expressed in a
highly ordered spatio-temporal pattern during the development of
mouse olfactory epithelium and that NFI-C is present in olfactory
receptor neurons of postnatal mice. The presence of NFI-C mRNA in
postnatal olfactory epithelium supports the biological signi®cance of
the reporter gene assays.
The expression of the OMP gene in ORNs is considered a hallmark
of the mature neuronal phenotype (Margolis, 1980). The identi®cation of the Olf-1 motif (Kudrycki et al., 1993) and the subsequent
isolation of Olf-1, a DNA-binding transcription factor preferentially
expressed in olfactory epithelium (Wang & Reed, 1993), was a
crucial step for the understanding of OMP (and ORN) gene
regulation. The demonstration of transcriptional activation by Olf-1
(Tsai & Reed, 1997; Wang et al., 1997) of OMP promoter fragments
linked to reporter genes and the ®nding that a promoter construct
containing only the proximal Olf-1 binding site was suf®cient to drive
cell type speci®c expression of b-galactosidase in transgenic mice
(Walters et al., 1996) provided the ®rst insights into the transcriptional regulatory cascade. As Olf-1 (recently renamed O/E-1) and the
subsequently isolated related factors O/E-2 and O/E-3 (Wang et al.,
1997) are expressed in the neuronal precursor cells (globose basal
cells), immature olfactory receptor cells, as well as in the mature
Ó 2000 European Neuroscience Association, European Journal of Neuroscience, 12, 1372±1384
NFI expression in olfactory epithelium of mouse 1383
neurons these factors alone could not explain the pattern of gene
expression in ORNs. The isolation of Roaz, a negative regulatory
protein binding to all three O/E subtypes, sequestering them, and
preventing their binding to DNA promoter elements, closed this gap,
as Roaz was shown to be present in immature olfactory neurons and
precursor cells that are not expressing OMP (Tsai & Reed, 1997).
Although the promoters of OMP, AC III, and oCNGC-I all contain a
combination of Olf-1 and NFI binding sites their onset of expression in
ORNs varies considerably. In the rat OMP (Allen & Akeson, 1985;
Baker & Farbman, 1993) and AC III (Menco et al., 1994) expression
are evident at E14 whereas oCNGC-I expression is ®rst detected at E19
(Margalit & Lancet, 1993). This variability cannot be explained solely
by the expression of members of the O/E-family. Comparison of the
sequences of the NFI binding sites of OMP, AC III, and oCNGC-I in
Fig. 10 shows that only the NFI binding site in the oCNGC-I promoter
can be considered a high af®nity binding site whereas the site within
the OMP and AC III promoters are lower af®nity binding sites. In the
OMP gene, because of an expanded linker region between the core
motifs, and in the AC III promoter, because of a nucleotide exchange
within the core motif. We have con®rmed these predictions
experimentally (Baumeister et al., 1999). Our data presented here
suggest that NFI acts as a negative regulatory factor in a promoter that
combines Olf-1 with NFI elements (Fig. 3). Therefore, we hypothesize
that the relative af®nities of NFI for the NFI binding elements within
these promoters contributes to the differential onset of expression
during development. This is further supported by the previous
observation that transgenic mice generated with a 300-bp fragment
of the OMP promoter that lacks the distal Olf-1 and the NFI site show
in general a higher expression of reporter gene and a higher number of
positive cells than transgenic mice that have been created using the
800 bp OMP promoter fragment that includes the second Olf-1 binding
site and the NFI element (Walters et al., 1996).
It remains to be determined whether the negative regulation of the
OMP gene by the NFI site is due to the presence of inhibitory NFI
variants in ORNs; if the unique promoter context itself favours
inhibitory interactions of NFI proteins; or if the cellular environment is
the main determinant of the observed phenomenon. Examples of all
three possibilities have been demonstrated experimentally. Thus, NFIA is a potent stimulator of the myelin basic promoter in NG108±15
cells (Inoue et al., 1990) but an identical NFI-A variant shows only
weak stimulation of the MMTV promoter in JEG-3 cells (Chaudhry
et al., 1997). Analysis of the differential function of NFI subtypes
utilizing one promoter in one cell line also add to the variety of
observed effects. In HepG2 cells the expression of NFI-A and NFI-B
could stimulate the phosphoenolpyruvate carboxykinase gene promoter while NFI-C and NFI-X were slightly inhibitory. All four subtypes,
however, were potent inhibitors of PKAc (catalytic subunit of the
protein kinase A) induced transcription (Crawford et al., 1998). In
JEG-3 cells the potency of glucocorticoid-dependent stimulation of the
MMTV promoter was NFI-B > NFI-X > NFI-C > NFI-A (Chaudhry
et al., 1997). Even different splice variants of a single NFI subtype can
show positive or negative regulation as demonstrated by the
interaction of fusion proteins containing GAL4/NFI-X C-terminal
variants in different cell lines and promoter constellations (Nebl &
Cato, 1995). Additional complexity derives from the observation that
the regulatory functions of NFI proteins are not solely based on their
direct interaction with components of the basal transcription
machinery but by speci®c interactions with additional cellular
components, such as the ski oncoprotein (Tarapore et al., 1997).
Alternatively, NFI proteins, in addition to their direct role in gene
expression may also be involved in nucleosomal reorganization
within the promoter region of the OMP gene. It is important to note
the growing evidence that NFI proteins, like other transcription
factors, cannot bind to their target sequences in a nucleosomal
context (Blomquist et al., 1996). Interestingly, it has been shown that
histone H1 can bind to the NFI binding site within the P2 promoter of
the alpha1b adrenergic receptor gene and that the sequence speci®c
binding of NFI-X can compete for the same DNA element (Gao et al.,
1998). As all NFI subtypes are reported to have the same af®nity for
given NFI binding elements (Goyal et al., 1990) this competition (that
is most likely to occur during replication) should not be restricted to
NFI-X. This additional activity, together with the observation that
NFI binding causes strong bending of DNA surrounding its binding
site in adenovirus (Zorbas et al., 1989), might create a permissive
state in gene activation. Our in situ hybridization data show that NFI
is already present in immature neurons. It therefore could compete
successfully for its binding site when the last cell division of globose
basal cells occurs and immature neurons are formed. As O/E proteins
are maintained in an inactive state by their interaction with Roaz at
this stage of differentiation (Tsai & Reed, 1997), and NFI acts as a
negative regulator in the context of the OMP promoter, NFI could
transfer the promoter into a permissive state without inducing
premature gene expression. We postulate that the acute transcription
modulated by NFI is initiated when O/E proteins are released from
the complex with Roaz by a yet unknown process during the
formation of mature ORNs (Fig. 11). Curiously this corresponds to
the increase of NFI-C expression suggesting that NFI proteins may
play a complex role in regulating ORN gene expression.
Acknowledgements
We are grateful to Dr P. Gruss for the gift of the cDNA clone of the mouse
Pax-6 gene. We thank Dr D.M. Cummings for providing the embryonal mouse
stages used in this study and the late Dr Robert Wade for providing access to a
luminometer (Tropix, Bedford, MA, USA). Supported in part by NIH grant
DCO3112 (FLM), a grant to MB from the DFG (BE 2091/1±1), NIH grant
T32-DC00055±01 (GV), and NIH grant HD34908 (RMG).
Abbreviations
AC III, type III adenylate cyclase; Anti-Dig-AP, anti-Digoxigenin antibody
coupled to alkaline phosphatase; BCIP, 5-Bromo-4-chloro-3-indolyl phosphate; DMEM, Dulbecco's modi®ed Eagle's medium; DTT, Dithiothreitol;
EBF, early B-cell factor; EMSA, electromobility shift assay; FBS, foetal
bovine serum; Mash-1, mammalian achaete-scute homologue 1; NBT, Nitro
blue tetrazolium; NFI, nuclear factor I; oCNGC-I, olfactory cyclic nucleotidegated channel-I; O/E, Olf-1/EBF; OMP, olfactory marker protein; ORN,
olfactory receptor neuron; RT-PCR, reverse transcription-polymerase chain
reaction, M-MLV, Moloney-murine leukaemia virus, RT, room temperature.
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