Chem. Senses 28: 197-205,
2003
© Oxford University Press 2003
Expression of Hes6 and NeuroD in the Olfactory Epithelium, Vomeronasal Organ and Non-sensory Patches
1 Department of Oral Anatomy, School of Dentistry, Health Sciences University of Hokkaido, Ishikari-Tobetsu 061-0293, Japan 2 Department of Orthodontics, School of Dentistry, Health Sciences University of Hokkaido, Ishikari-Tobetsu 061-0293, Japan
Correspondence to be sent to: Yuko Suzuki, Department of Oral Anatomy, School of Dentistry, Health Sciences University of Hokkaido, Ishikari-Tobetsu, 061-0293, Japan. e-mail: suzuki{at}hoku-iryo-u.ac.jp
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Basic helix–loop–helix transcription factors NeuroD and Hes6 promote neuronal differentiation. The expression of their genes in the olfactory epithelium (OE), vomeronasal organ (VNO) and the non-sensory patches of the posterior nasal cavity of mice was examined. As detected by in situ hybridization, Hes6 was expressed in a basal progenitor layer of the embryonic OE. After birth, the expression of Hes6 was detected in a cell layer above the basement membrane, globose basal cells (GBCs). Expression of NeuroD in the embryonic OE was in agreement with that previously described; and in the postnatal OE, it was detected in cells of GBC layer and cells upper to GBCs. In the VNO, Hes6 was expressed throughout the sensory epithelium (S-VNO) at embryonic day 12, and later became restricted to a single layer of cells in the basal region of the S-VNO, where Hes5-expressing undifferentiated cells were present. NeuroD was expressed throughout the S-VNO during the embryonic stage. After birth, Hes6 and NeuroD expressions were observed in the border between the S-VNO and non-sensory VNO. Immunohistochemistry using anti-NeuroD antibody revealed that NeuroD-positive cells were still present not only at the edges but also in the center of the S-VNO until P3. These findings suggest that Hes6 and NeuroD are expressed in progenitors of chemoreceptor neurons and that the expression of Hes6 precedes that of NeuroD. Moreover, in the regenerating VNO of bulbectomized mice, NeuroD-positive cells were observed both at the edges and in the center of the S-VNO, suggesting that neuronal turnover occurred in both regions. Moreover, in the dorsal fossa of the posterior nasal cavity, several non-sensory patches are formed between postnatal (P) days 10 and 21 because of programmed death of ORNs and GBCs. During embryonic stages, the expression of Hes6 and NeuroD in the OE showed no regional differences. At P3–P7, expression of NeuroD and Hes6 disappeared in the region corresponding to the presumptive non-sensory patches. The loss of these genes may stop the differentiation and may cause apoptosis of GBCs and ORNs.
Key words: Hes6, NeuroD, olfactory epithelium, vomeronasal organ, in situ hybridization
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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In the mammalian olfactory epithelium (OE), the olfactory receptor neurons (ORNs) continually die and are replaced by their progenitor cells. Basic helix–loop–helix (bHLH) transcription factors, i.e. Mash1, Neurogenin1, NeuroD, Hes1, Hes5 and Hes6, are known to control the generation of progenitor cells and their differentiation of them to ORNs (Cau et al., 1997
,
2002;
Guillemot, 1999;
Bae et al., 2000).
Mash1 is a determination gene for ORNs since Mash1 null
mutant mice fail to produce progenitor cells
(Cau et al., 1997).
Neurogenin1 is also determination gene, and is required for
activation of genetic programs functioning downstream of Mash1. In
Neurogenin1 null mutant mice, progenitors of ORNs are generated but
their differentiation is blocked (Cau
et al., 2002). Hes1 and Hes5 maintain
progenitors in their undifferentiated form
(Cau et al., 2000). In
contrast, NeuroD acts downstream of the above-mentioned genes and was
demonstrated to have neuronal differentiation activity
(Lee et al., 1995).
In the embryonic OE, as detected by in situ hybridization,
NeuroD was expressed in the basal region
(Cau et al., 1997).
Morphologically, the cell types of the embryonic OE are supporting cells, ORNs
and basally located columnarshaped cells
(Cuschieri and Bannister,
1975). After birth, horizontal basal cells (HBCs) and globose
basal cells (GBCs) are differentiated in the basal region. HBCs are
keratin-positive cells and are in direct contact with the basement membrane.
GBCs are devoid of keratin, lie above the HBCs and are generally accepted to
be the progenitors of ORNs (Schwartz-Levey
et al., 1991; Suzuki
and Takeda, 1991; Holbrook
et al., 1995; Huard
et al., 1998). In adult mice, NeuroD was
detected by immunohistochemical means, in the layer superior to the GBCs, a
postmitotic cell layer (Nibu et
al., 1999). However, the same authors described that
NeuroD expression, detected with the same antibody, was observed both
in the basal and middle regions of the OE of mice during postnatal days (P)
1–28 (Nibu et al.,
2001). Therefore, it is not clear what cell type expresses
NeuroD in postnatal and adult stages. Moreover, the recently cloned
bHLH gene Hes6 was shown to promote neuronal differentiation by
inhibiting Hes1 activity
(Koyano-Nakagawa et al.,
2000). Hes6 is known to be expressed in the embryonic OE
(Bae et al., 2000),
but its detailed localization has not been investigated.
The chemoreceptor cells in the vomeronasal organ (VNO) also undergo
continuous neurogenesis during development and after injury. The VNO consists
of two epithelia: a thick sensory epithelium (S-VNO) that is located in the
medial portion of the VNO and contains supporting cells and chemoreceptor
cells at various stages of differentiation, and a thinner non-sensory
epithelium (NS-VNO) that is located in its lateral portion. Unlike the OE,
keratin-containing basal cells are not observed in the basal region of S-VNO
(Witt et al., 2002).
The progenitor cells of chemoreceptor cells are localized at the boundary
region between S-VNO and NS-VNO of adult mice
(Barber and Raisman, 1978).
Studies using the immunohistochemical detection of BrdU in rat and hamster
VNOs have found progenitor cells not only in the margins, but also in the
cells along the basement membrane, of the S-VNO
(Ichikawa et al.,
1998; Weiler et al.,
1999; Martinez-Marcos et
al., 2000). Although Mash1 expression in embryonic
day 14.5 mice has been reported (Cohen
et al., 2000), little is yet known about the expression
of other bHLH transcription factors in the VNO.
In the posterior nasal cavity of rodents, epithelial patches exclusively
consisting of olfactory supporting cells and HBCs are found. Several such
non-sensory patches were located among the OE as small patches, in the dorsal
fossa of the first, second, third and fourth turbinates and corresponding
septa (Suzuki et al.,
2000,
2001). The presence of
Bowman's glands in the patches indicates the origin of the patches to be OE.
In fact, in newborn mice, it was shown that normal OE occupied these regions
and that the patches were generated by programmed cell death of ORNs and the
progenitors, and by the subsequent disappearance of these cells during
postnatal development (Suzuki et
al., 2000). Selective death during development has also been
reported to occur in the VNO of mice: the NS-VNO contains neurons that
disappear after birth for the formation of the respiratory epithelium
(Tarozzo et al.,
1998; Cappello et al.,
1999). Therefore, it is not known whether the expression of bHLH
transcription factors changes in the region where programmed cell death
occurs. In the present study, we examined the expression of two
differentiation factors, NeuroD and Hes6, in the OE, VNO and
non-sensory patches of mice.
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Materials and methods |
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Animals
Timed pregnant and adult ddY mice were obtained from Sankyo Laboratories. All animals were maintained in a heat-and humidity-controlled vivarium on food and water provided ad libitum.
Unilateral bulbectomy
Mice were anesthetized with Nembutal (Abbot Laboratories, North Chicago,
IL), and unilateral bulbectomy was performed as described previously
(Suzuki et al.,
1995). The bulbectomized mice were used at 12 days after
surgery.
Tissue preparation
To obtain embryos, pregnant females were killed by cervical dislocation and their uteri with fetuses embryonic day (E) 10–18 carefully dissected out. Neonatal and adult mice were killed by an overdose of Nembutal given by intraperitoneal injection. The heads were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) overnight or with periodate–lysine–paraformaldehyde (PLP) for 4–6 h at 4°C. The specimen from adult mice were decalcified in 10% EDTA in Tris buffer (pH 7.6), and cryoprotected with 25% sucrose, embedded in OCT compound (TissueTek, Miles, Elkhart, IN), and frozen in a spray freezer (Oken, Japan). The tissues were sectioned coronally at a thickness of 8–10 µm. Sections were collected and placed on silane-coated slides.
RNA probes and in situ hybridization
cDNA fragments of NeuroD, Hes6, Hes5 and NCAM were cloned by reverse transcriptase polymerase chain reaction (RT-PCR) using the total RNA extracted from the olfactory mucosa of adult mice and then used for the synthesis of cRNA probes. The sequences of the primers were 5'-ATGACCAAGGCGCGCCTAGA-3' (55–74) and 5'ACAGGACAGTCACTGTACGCAC-3' (920–899; Genbank U28068) for NeuroD, 5'-ATGAGGTGCACACGTTC-GTG-3' (371–390) and 5'-GCGCAACTGTGTTACAAA-CG-3' (1222–1203; Genbank AF260236) for Hes6, 5'-GGATGCTAATGAGGACGAGCG-3' (63–83) and 5'-CAGCTTCATCTGCGTGTCGC-3' (905–886; Genbank D32132) for Hes5, and 5'-CTACCCTCACCATCTACAA-CGC-3' (376–397) and 5'-GACTGGGAGTCCTGGCC-GAT-3' (1354–1335; Genbank X15049) for NCAM.
The PCR was carried out for 35 cycles. Each resulting fragment was cloned
into HindIII/EcoRI sites of pT7/T3 
18 (Ambion, TX)
and sequenced. Digoxigenin (DIG)-UTP-labeled RNA probes were synthesized by
use of an RNA transcription kit (Roche Diagnostics, Mannheim, Germany).
Sections were immersed in absolute ethanol for 5 min and in 0.2 N HCl for 20 min, and then washed twice in PBS for 5 min each time. Next, the sections were treated with 2 µg/ml of proteinase K (Takara, Kyoto) at 37°C for 15–20 min, washed in PBS, and refixed with 4% paraformaldehyde in 0.1 M phosphate buffer for 20 min. After having been washed twice in PBS, the sections were air-dried and hybridized. Hybridization was performed at 47°C for 16 h with a DIG-labeled RNA probe in a hybridization solution containing 50% formamide, 0.3 M NaCl, 0.02 M Tris–HCl (pH 8.0), 1 mM EDTA, 10% dextran sulfate, 1 x Denhardt's solution, 1 mg/ml yeast tRNA and 0.02% SDS. Hybridized sections were washed at 47°C in a solution containing 50% formamide and 2 x SSC for 1 h, and thereafter twice in 2 x SSC for 5 min each time. They were then treated with 20 µg/ml of RNase (Type II-A, Sigma Chemical Co., St Louis, MO) at 37°C for 30 min, and washed at 47°C in 50% formamide/2 x SSC followed by 50% formamide/1 x SSC for 1 h for each. After having been washed three times in PBS, the sections were incubated with 1% blocking reagent (Boeringer Mannheim GmbH, Mannheim, Germany) in maleic acid buffer (pH 7.5) for 1 h at room temperature. Subsequently, they were incubated overnight at 4°C with alkaline phosphatase-conjugated anti-DIG Fab fragments diluted 1:500 in PBS. After three washes in TBS, chromogenic reactions were carried out by using NBT/BCIP (Boeringer Mannheim).
Immunohistochemistry
A goat polyclonal antibody to NeuroD was purchased from Santa Cruz
Biotechnology (Santa Cruz, CA). The sections were incubated with anti-neuroD
antibody for 1 h at 37°C, and then stained by using a labeled
streptoavidin–biotin (LSAB) kit (Dako, Kyoto). The immunoreactive
product was colored by use of diaminobenzidine (DAB). Control reactions
included: (i) PBS used instead of primary antibody, and (ii) primary antibody
adsorbed with NeuroD peptide (Santa Cruz). The specificity of the
antibody has been examined previously
(Suzuki et al.,
2002).
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Results |
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Olfactory epithelium
At E10, the olfactory placodes, which were oval-shaped epithelial patches,
appeared in the anterolateral region of the head. Hes6 was weakly
expressed in the entire olfactory placodes and telencephalon
(Figure 1A). At E12, the
olfactory placode differentiated into the OE and the VNO. Hes6 was
expressed in the basal region of the OE and throughout the S-VNO, a thick
sensory epithelium of the VNO (Figure
1B). Until birth, Hes6 expression was observed in a
single layer of cells just above the basement membrane
(Figure 1C), termed basal
progenitors. At E15, near the base of the septal wall, which corresponds to
the presumptive septal organ of Masera, a group of Hes6-expressing
cells was observed. The epithelium surrounding this patch was devoid of
Hes6 expression (Figure
1D). At the same time, nasal turbinates arose and developed as a
series of elevated folds on the lateral wall. Strong expression of
Hes6 was observed in several layers of these developing turbinates
(Figure 1E). After birth,
Hes6 was expressed in the cell layer above the basement membrane,
GBCs. HBCs, which were directly against the basement membrane, were devoid of
Hes6 mRNA (Figure 1F).
The signals of Hes6 became weaker and were detected in scattered GBCs
at P7. The expression pattern of NeuroD during embryonic stages was
in agreement with that previously described
(Cau et al., 1997).
After birth, it was expressed in GBCs and cells upper to GBCs
(Figure 1G). The signals of
NeuroD mRNA also became weak as the mice grew. Sense controls
displayed no reactivity (Figure
1H).
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Vomeronasal organ
At E12, the VNO appeared as a tubular structure. A thick epithelium of
S-VNO and a thinner epithelium of NS-VNO could be distinguished. The
expression of Hes6 was detected throughout the S-VNO
(Figure 1B). The expression of
NeuroD was similar to that of Hes6. At E15, strong
expression of Hes6 was observed in the basal region of the S-VNO
(Figure 2A). To examine whether
cells in the basal region remain undifferentiated or enter a differentiation
pathway, Hes5 probe was used. Hes5 is known to be a negative
regulator to inhibit differentiation in the OE
(Cau et al., 2000). A
few scattered cells were reactive with the Hes5 probe
(Figure 2B). NeuroD
was expressed throughout the S-VNO (Figure
2C). In the NS-VNO, these genes were not expressed (Figure
2A-C). A marker of mature and
immature ORN and VNO receptor cells, NCAM, was expressed in both
S-and NS-VNOs. In the S-VNO, NCAM expression was detected throughout
the epithelium except the basal layer. In the NS-VNO, a few
NCAM-expressing cells were observed
(Figure 2D). At P3, expression
of Hes6 was restricted to the border between the S-and NS-VNO
(Figure 2E). At the same time,
NeuroD was expressed also in this border region
(Figure 2F).
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Non-sensory patches
From E12 to P1, the expression of NeuroD and Hes6 showed no regional differences. However, at P3, NeuroD expression disappeared from the dorsal fossa of the posterior nasal cavity (Figure 3A). At the same time, Hes6 expression in that region was weak as compared with that in other regions (Figure 3B). The expression of Hes6 disappeared from the dorsal fossa by P7. Immunohistochemical detection using anti-NeuroD antibody also failed to detect the immunoreactive cells in the dorsal fossa of P3 mice (Figure 3C). At that time, NCAM expression was present in both dorsal fossa and other regions (Figure 3D).
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NeuroD expression after bulbectomy
Immunohistochemistry using anti-NeuroD antibody revealed that the expression pattern of NeuroD in the embryonic OE and VNO was similar to that of the in situ hybridization data (not shown). In the stages of postnatal development and in the adult, when in situ signals were weak, immunoreactive cells could be detected. In the VNO, NeuroD-immunoreactive cells were observed both in the center and at the edges of the S-VNO at P3 (Figure 4A). During postnatal development, the immunoreactive cells gradually decreased in number and became restricted to the border between the S-and NS-VNO (Figure 4B). At 12 days after unilateral bulbectomy, NeuroD-immunoreactive cells in the margin of the S-VNO of the operated side (Figure 4C) were more abundant than those of the unoperated side (Figure 4B). Furthermore, NeuroD-immunoreactive cells appeared in the central region of the S-VNO (Figure 4C). In the OE, localization of NeuroD-immunoreactive cells was restricted to the basal region from P1 to adult, the number of immunoreactive cells decreased as development proceeds. A subset of NeuroD-immunoreactive cells was observed in the GBC region of adult mice; and after bulbectomy, NeuroD-immunoreacive cells were more abundant on the operated side than on the unoperated side (Figure 4D). In slides for the two control reactions, i.e. PBS in place of primary antibody and preadsorbed primary antibody, the sections were completely unstained.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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In the embryonic OE, dividing progenitors are present in the apical and basal regions (Cuschieri and Bannister, 1975
; Cau et al.,
2002). The present study clarified that Hes6 was
expressed in olfactory placodal cells and in basal progenitor cells of the
embryonic OE and VNO. Hes6 acts as a positive regulator of
differentiation by inhibiting the action of bHLH-negative regulator,
Hes1 (Bae et al.,
2000). However, Hes1 is expressed in apical side of the
embryonic OE and is absent from the basal side. Another negative regulator,
Hes5, in contrast, is expressed in the cells on the basal side of the
OE (Cau et al., 2000).
It was confirmed also in the S-VNO of E15 that Hes5 was expressed in
scattered cells on the basal region. Therefore, the positive regulator
Hes6 and the negative regulator Hes5 are co-expressed in the
basal progenitor cells. In the embryonic OE, progenitors give rise to not only
the ORNs, but also supporting cells, Bowman's glands and the cells outside the
OE, i.e. LHRH neurons, ensheathing cells of the olfactory bulb and Schwann
cells of olfactory axons (Farbman,
1992). In the postnatal OE, Hes6 was expressed in cells
of the GBC layer, suggesting its action is intrinsic to the ORN lineage. GBCs
are identified as various cell-cycle marker-positive cells,
[3H]thymidine (Schwartz-Levey
et al., 1991), BrdU
(Suzuki and Takeda, 1991),
Ki67 and cyclin D1 (Ohta and Ichimura,
2001); and they differentiate into post-mitotic neurons. The
present in situ hybridization and immunohistochemical study confirmed
that NeuroD is expressed in GBCs, and post-mitotic neurons. In fact,
Lee et al. showed that NeuroD was expressed not only in
mitotic cells but also in post-mitotic cells of the developing central nervous
system (Lee et al.,
2000). Moreover, Nibu et al. reported that
NeuroD-immunoreactive cells were located in the layer superior to GBC
and that after axotomy they were detected in higher layer in the regenerating
OE than in the unlesioned side (Nibu
et al., 1999). However, in our data on regenerating OE,
the number of NeuroD-immunoreactive cells increased, but their
localization within the OE did not change.
In the VNO, the expression of Hes6 and NeuroD in
postnatal periods was similar to that of progenitors of chemoreceptor cells,
i.e. the pool of dividing cells observed at the boundary between NS- and
S-VNO. Also, NeuroD-immunoreactive cells appeared in the central
region of regenerating S-VNO, where progenitor cells are present
(Barber and Raisman, 1978;
Ichikawa et al.,
1998; Weiler et al.,
1999; Martinez-Marcos et
al., 2000). It is believed that dividing cells at the
boundary do not migrate to the central region of the S-VNO, and represent a
pool for growth, whereas cells in the central region would participate in cell
turnover (Jia and Halpern,
1998; Weiler et al.,
1999). However, abundant NeuroD-immunoreactive cells at
the edges of the regenerating VNO revealed that cell turnover occurred in that
region. Moreover, in the regenerating and embryonic S-VNO,
NeuroD-expressing cells showed a vertically diffused pattern of
localization. This may reflect the arrangement of chemoreceptor cells, for the
mature chemoreceptor cells were diffusely distributed within the thick layer
of chemoreceptor neurons, in contrast to the OE, where ORNs appeared more
apically (Witt et al.,
2002).
During neurogenesis, bHLH genes are sequentially expressed as a result of
activation cascade in which the early genes activate the expression of the
late genes. The spatial pattern of expression of Hes6 and
NeuroD in the VNO and the OE suggests that expression of
Hes6 precedes that of NeuroD. NeuroD may activate downstream
of Hes6 in ORN and VNO receptor cell lineage. This expression pattern
is also true in the developing retina: Hes6 is expressed in both
undifferentiated and differentiated cells
(Bae et al., 2000),
whereas NeuroD is expressed in the differentiated population of
retinal cells (Ahmad et al.,
1998).
Our previous study showed that non-sensory patches were generated by
programmed cell death of ORNs and their progenitor GBCs, and by the subsequent
disappearance of these cells from P10 to P21
(Suzuki et al.,
2000). The present study clarified that expression of
Hes6 and NeuroD disappeared from the OE before apoptosis
occurred. Moreover, the embryonic NS-VNO contains receptor cells
(Tarozzo et al.,
1998) (see also this study), which disappear by apoptosis after
birth to produce the respiratory epithelium
(Cappello et al.,
1999). Neither Hes6 nor NeuroD was expressed in
the embryonic NS-VNO. These genes might have been expressed there in earlier
stages for a short time and then disappeared. Studies using bHLH gene-null
mutant mice have shown that these genes regulate not only differentiation but
also apoptosis. In NeuroD2-null mice, brain areas that would normally
express NeuroD2 showed apoptosis
(Olsen et al., 2001).
The endocrine pancreas of BETA2/NeuroD-deficient mice
undergoes massive apoptosis and, consequently, animals die of diabetes shortly
after birth (Naya et al.,
1997). In the present study, upstream genes, such as
Mash1 and neurogenin1, may also disappear from the
presumptive non-sensory patches or NS-VNO before overt apoptosis. The loss of
expression of these genes may stop the differentiation into ORNs or
chemoreceptor cells and lead to apoptosis of these cells and their
progenitors.
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Accepted February 5, 2003
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