Chemical Senses Vol. 30 No. suppl 1 © Oxford University
Press 2005; all rights reserved
Expression of bHLH Transcription Factors and IGFs in the Non-sensory Patches, Olfactory Epithelium and Vomeronasal Organ
Department of Oral Anatomy, School of Dentistry, Health Sciences University of Hokkaido, Ishikari-Tobetsu 061-0293, Japan
Correspondence to be sent to: Yuko Suzuki, e-mail: suzuki{at}hoku-iryo-u.ac.jp
Key words: IGFBP-5, Mash1, Neurogenin 1, non-sensory patch, olfactory epithelium
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Introduction |
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 Top Introduction Materials and methodsResultsDiscussionSummaryReferences |
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Non-sensory patches in the posterior dorsal fossa of the rodent nasal cavity are formed between postnatal (P) days 10 and 21 due to programmed death of ORNs and GBCs (Suzuki et al., 2000
). In a
previous study, we found bHLH transcription factors Hes6 and NeuroD to
be expressed in the GBC layer in the olfactory epithelium (OE) of mice. During embryonic
days, there were no regional differences in the expression of these two genes, whereas at
postnatal days 3–7, expression disappeared in the region corresponding to the
presumptive non-sensory patches (Suzuki et
al., 2003). Hes 6 and NeuroD are known to promote
neuronal differentiation, NeuroD acting downstream of Mash1 and
Neurogenin 1, which are determination genes for ORNs. Mash 1 null
mutant mice failed to produce progenitor cells and Neurogenin 1 null mutant
mice, progenitors of ORNs are generated but their differentiation is blocked (Cau et al., 2002). It is not yet known
whether Mash1 and Neurogenin1 are related to the formation of
non-sensory patches. On the other hand, insulin-like growth factor (IGF) family and
related molecules were reported to be expressed in the rat OE where IGFs may have
functions of differentiation and survival of ORNs (Suzuki and Takeda, 2002). Six high-affinity IGF binding
proteins (IGFBP-1 to -6) modulate IGF effects. In the present study, we examined whether
the expression of bHLH transcription factors and IGFBPs changes in the region where the
programmed death of ORNs and GBCs occurs. |
Materials and methods |
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TopIntroductionMaterials and methodsResultsDiscussionSummaryReferences |
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Timed pregnant and postnatal ddY mice were obtained from Sankyo Labs. All animals were maintained in a heat- and humidity-controlled vivarium on food and water provided ad libitum. Tissue preparation and in situ hybridization methods are described elsewhere (Suzuki et al., 2003
). For the synthesis of cRNA probes, cDNA fragments of Mash1,
Neurogenin 1, IGFBP-2 and IGFBP-5 were cloned by RT-PCR using
the total RNA extracted from the olfactory mucosa of adult mice. Each resulting fragment
was cloned into HindIII/EcoRI sites of pT7/T3 
18 and sequenced.
DIG-UTP-labeled RNA probes were synthesized by use of RNA transcription kit (Roche
Diagnostics, Mannheim, Germany). For double labeling of NeuroD and BrdU,
sections were immersed with a mixture of goat anti-NeuroD antibody (Santa Cruz)
and monoclonal anti-BrdU antibody (Becton-Dickinson) for overnight at 4°C. The
immunoreacted sections were reacted with Alexa fluor 488-conjugated donkey anti-goat IgG
(Molecular Probes, Eugene, OR) and rhodamine-conjugated donkey anti-mouse IgG (Santa
Cruz) for 2 h at room temperature, and then examined with a Leica confocal laser scanning
electron microscope. |
Results |
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TopIntroductionMaterials and methodsResultsDiscussionSummaryReferences |
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At E12–18, Mash1 was expressed in both apical and basal regions of the OE and VNO as determined by in situ hybridization. Neurogenin 1 was expressed in a single layer of the cells just above the basement membrane, termed basal progenitors. At P1, GBCs differentiated in the basal region of the OE, and Mash1 and Neurogenin 1 were expressed in these cells. At P 3, the expression of Mash1 in GBCs disappeared from the dorsal fossa of the posterior nasal cavity (Figure 1). At the same time, Neurogenin1 expression in that region also disappeared. Since BrdU is a marker for proliferating GBCs, immunohistochemistry using anti-NeuroD and BrdU antibodies was performed. In the basal region of the OE, some NeuroD-immunoreactive cells overlapped with BrdU-immunoreactive cells, however, most of the NeuroD-immunoreactive cells were located above the BrdU-positive cell layer. From embryonic days to P1, the expression of NeuroD and BrdU labeling showed no regional differences. At P3, NeuroD-immunoreactive cells decreased in number in the dorsal fossa. At that time, BrdU-immunoreactive cells were present in that region. At P7, NeuroD-immunoreactive cells were not observed. BrdU-immunoreactive cells were decreased in number as development proceeded further.
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Among IGFBPs, IGFBP-2 and -5 were found in the OE and the VNO by in situ hybridization.
During embryonic stages, specific IGFBP-5 signals were detected in some of the ORNs and receptor neurons of the VNO. Strong expression was observed in ORNs of the developing nasal turbinates and at the boundary of the respiratory epithelium. From P1, the expression was restricted to the apical layer of presumptive non-sensory patches in the dorsal fossa. This expression pattern persisted up to P14. Moreover, strong expression was observed in the mitral cells of the olfactory bulb.
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Discussion |
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TopIntroductionMaterials and methodsResultsDiscussionSummaryReferences |
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Mash1 and Neurogenin 1 are determination genes for ORNs (Cau et al., 2002
). The results of the
present study suggest that loss of expression of Mash 1 and Neurogenin
1 cause the formation of non-sensory patches. This loss may consequently prevent the
activation of the downstream genes NeuroD and Hes6. The lack of these
gene functions may stop differentiation and may cause apoptosis of GBCs and ORNs in the
presumptive non-sensory patches. A similar finding was reported in the case of the
non-sensory epithelium of the VNO (NS-VNO), which contains receptor neurons during
embryonic stages: neither NeuroD nor Hes6 was expressed before
apoptosis of the receptor neurons (Suzuki et
al., 2003).
IGFBPs have been shown to inhibit or potentiate IGF actions. Furthermore, some IGFBPs
have been demonstrated to have IGF-independent actions (Zhou et al., 2003). In the present study, a change
in the expression pattern of IGFBP-5 during development of the nasal cavity was
observed. During embryonic stages, IGFBP-5 may affect growth of the OE and
differentiation or maintenance of ORNs and receptor neurons of the VNO. Postnatally,
IGFBP-5 may influence growth and survival of non-sensory patches.
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Summary |
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TopIntroductionMaterials and methodsResultsDiscussionSummaryReferences |
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Molecules related to the formation of non-sensory patches in the rodent nasal cavity were examined. During embryonic stages, bHLH transcription factors Mash1 and Neurogenin 1 were expressed in the progenitor layers of the olfactory epithelium (OE) and vomeronasal organ (VNO). After birth, in the basal region of the OE, Mash 1 and Neurogenin 1 were expressed in globose basal cells (GBCs). In the posterior nasal cavity, the expression of Mash1 and Neurogenin 1 disappeared from GBCs of the region corresponding to the presumptive non-sensory patches. Downstream genes Hes6 and NeuroD were also absent in that region as described previously (Suzuki et al., 2003
). The loss of these genes may stop the differentiation and may cause
apoptosis of olfactory receptor neurons (ORNs) and GBCs. Moreover, the expression pattern
of insulin-like growth factor binding protein (IGFBP)-5 was unique: it
was expressed in ORNs and vomeronasal receptor neurons during embryonic stages. At
postnatal days 1, 3, 7 and 14, the signals of IGFBP-5 were detected in the
apical layer of the presumptive non-sensory patches. IGFBP-5 may influence the
growth and survival of non-sensory patches.
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References |
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TopIntroductionMaterials and methodsResultsDiscussionSummaryReferences |
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Cau, E., Casarosa, S. and Guillemot, F. (2002) Mash1 and Ngn 1 control distinct steps of determination and differentiation in the olfactory sensory neuron lineage. Development, 129, 1871–1880.
Suzuki, Y. and Takeda, M. (2002) Expression of insulin-like growth factor family in the rat olfactory epithelium. Anat. Embryol., 205, 401–405.[Medline]
Suzuki, Y., Takeda, M., Obara, N., Suzuki, N. and Takeichi, N. (2000) Olfactory epithelium consisting of supporting cells and horizontal basal cells in the posterior nasal cavity of mice. Cell Tissue Res., 299, 313–325.[CrossRef][Web of Science][Medline]
Suzuki, Y., Mizoguchi, I., Nishiyama, H., Takeda, M. and Obara, N. (2003) Expression of Hes6 and NeuroD in the olfactory epithelium, vomeronasal organ and non-sensory patches. Chem. Senses, 28, 197–205.
Zhou, R., Diehl, D., Hoeflich, A., Lahm, H. and Wolf, E. (2003) IGF-binding protein-4: biochemical characteristics and functional consequences. J. Endocrinol., 178, 177–193.[Abstract]
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