Chemical Senses Vol. 30 No. suppl 1 © Oxford University
Press 2005; all rights reserved
Restoring Olfaction: A View from the Olfactory Epithelium
Department of Anatomy and Cellular Biology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111, USA
Correspondence to be sent to: James E. Schwob, e-mail: jim.schwob{at}tufts.edu
Key words: basal cells, bHLH transcription factors, neurogenesis, regeneration, reinnervation, tissue stem cells
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Introduction |
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 Top Introduction AcknowledgementsReferences |
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Damage to the olfactory periphery destroys the population of olfactory sensory neurons and, in the case of direct epithelial lesion, also eliminates other constituents of the epithelium. In marked contrast to other parts of the nervous system, there is substantial anatomical and functional recovery of the olfactory epithelium and its projection into the CNS even in the face of overwhelming injury. For example, in the case of epithelial lesions caused by a single exposure to methyl bromide (MeBr), the olfactory epithelium is restored to a status that is indistinguishable from unlesioned epithelium within 6–8 weeks after damage, despite the severity of the initial damage (>95% of the epithelium is destroyed). The first olfactory neurons reappear in large numbers on day 4 after MeBr, the first mature neurons emerge during week 2, and the accelerated production of neurons falls to normal around week 6 after lesion (Schwob et al., 1995
). The
reconstitution of the epithelium is sufficiently robust and precise that the spatially
restricted distribution of odorant receptor (OR) expression is also restored to normal
(Iwema et al., 2004). In this
case, we assayed a set of eight different ORs, whose expression encompasses the whole of
the tangential extent of the epithelium, by in situ hybridization (ISH). In rats
in which the lesion caused by MeBr had been confined to one side of the epithelium by the
temporary closure of a naris during the exposure to the gas, the boundaries of the
expression territories of each of the eight were indistinguishable on the two sides.
after recovery from the lesion (Iwema et
al., 2004). Remarkably, despite moderate damage to the GBC population,
the spatial patterning of odorant receptor expression is restored, suggesting that
extra-epithelial cues, perhaps emanating from deeper in the mucosa, might direct aspects
of that patterning.
The projection of the epithelium onto the olfactory bulb is also restored quickly. By
2 weeks after lesion, axons fill the olfactory nerve layer and begin to re-enter the
glomeruli. By 3 weeks, the glomeruli are densely innervated, and the projection becomes
largely stabilized by 8 weeks (Schwob et
al., 1999). However, destruction of the neuronal population is not
without consequence. Reinnervation of the olfactory bulb, although robust, does not fully
restore the normally precise receptotopic organization of the projection. The newly
generated axons target roughly the right part of the bulb, but often innervate multiple
glomeruli in that locus as opposed to the usual one or two (C.L. Iwema, G. Ring and J.E.
Schwob, unpublished data). Despite the degradation in the mapping function responsible
for converting odorant stimuli into spatial patterns of neural activity, substantial
function is restored after recovery from lesion (S.L. Youngentob, unpublished data).
In sum, the epithelium accomplishes a remarkable degree of functional as well as anatomical recovery after lesion.
How is that recovery accomplished? A series of investigations over the last few years
has explored the capacity of the basal cell populations and the residual cells of
Bowman’s ducts to generate the various cell types that need reconstituting during
the recovery process. Recall that there are at least two distinct populations of basal
cells in the epithelium: horizontal basal cells (HBCs) and globose basal cells (GBCs)
(Graziadei and Monti Graziadei, 1979;
Holbrook et al., 1995). The
HBCs are highly differentiated cells that attach to the basal lamina by desmosomes,
assemble cytokeratins 5 and 14 into intermediate filaments, enwrap bundles of olfactory
axons as they exit the epithelium, and express a variety of other molecules in common
with the basal cells of the respiratory epithelium. The GBCs are the small, round,
morphologically non-descript cells that sit between the HBCs below and the immature OSNs
above, that proliferate at a high rate in the normal OE, are limited to the OE, and are
poorly characterized at the level of their molecular phenotype.
The studies have taken advantage of a series of cell type-specific antibodies, the
use of replication-incompetent, retrovirally derived vectors (RRVV) for lineage tracing,
and a colony forming unit assay which entails FACS isolation of potential progenitor
populations followed by transplantation into a new host. The multiple, converging lines
of evidence indicate that some among the residual GBCs function as broadly multipotent
progenitors capable of giving rise to neurons and all of the cell types of the
epithelium, and hence may be totipotent stem cells of the epithelium. First, marker
studies using antibodies that are selective for GBCs in normal epithelium label cells
that express both GBC and HBC markers, or both GBC and Sus cell markers, during the acute
phase in the recovery after MeBr, suggesting that the GBCs are differentiating into these
non-neuronal cell types (Goldstein and Schwob,
1996). Second, intranasal infusions of RRVV label GBC progenitors that give
rise to neurons and multiple types of non-neuronal cells within the same clone, while
other progenitors give rise to clones containing duct/gland cells and Sus cells
(Huard et al., 1998).
Third, and most directly, GBCs that are harvested from the normal epithelium,
isolated by FACS using a GBC-selective antibody and free from HBC, duct/gland and Sus
cell contaminants (to a level of <0.1% of total) engraft into the MeBr-lesioned
epithelium after infusion into the nasal cavity and give rise to an impressive range of
epithelial cell types (Goldstein et al.,
1998;
Chen et al., 2004). In
aggregate, engrafted GBCs in the mouse can give rise to OSNs, Sus cells, BG/D cells,
ciliated respiratory epithelial cells, and GBCs themselves (a form of self-renewal).
Individual clones, derived from single engrafted GBCs, were composed of OSNs or Sus cells
or OSNs, GBCs and Sus cells, or OSNs and BG/D cells or OSNs and respiratory epithelial
cells (Chen et al., 2004). In
addition, clones that contain multiple cell types arise from GBCs that are infectable
with a RRVV and hence mitotically active in the neurogenic epithelium. Moreover, the
neurons that derive from the engrafted GBCs mature to the extent of making OMP,
projecting axons to the olfactory bulb (specifically to the area of the bulb to which the
surrounding host epithelium would project, and expressing an OR. Transplanted Sus cells
give rise only to themselves. In contrast, HBCs either do not engraft i.e. in the mouse,
or engraft but remain as HBCs, i.e. in rat.
In their totipotency and in their apparent self-renewal, the GBCs satisfy two of the
several criteria that denote a tissue stem cell. Another indicant that at least some GBCs
are bona fide tissue stem cells is that some among them are slowly cycling/quiescent and
retain thymidine label for a prolonged period (X. Chen and J.E. Schwob, unpublished
data). Finally, destruction of the GBCs eliminates the capacity of the epithelium to
recover as olfactory after MeBr injury, forcing the epithelium to undergo respiratory
metaplasia instead (Jang et al.,
2003).
The foregoing data indicate that GBCs are multipotent and probably stem cells, and
that multipotent GBCs are making an active choice between making neurons and making
non-neuronal cells in the normal, neurogenic epithelium. It appears that aspects of that
choice are dictated by expression of members of the bHLH transcription factor family. Of
the seven classes of bHLH markers, we have used ISH to study two of them: (i)
transcriptional activators that drive neuronal differentiation, including Mash1,
Ngn1 and NeuroD, which are normally limited to GBCs; and (ii)
repressors of neuronal differentiation that belong to the Hes group (Manglapus et al., 2004). After MeBr
lesion, the proneuronal factors reappear in a sequence that mimics their expression
during the embryonic development of the olfactory epithelium (Cau et al., 2002;
Manglapus et al., 2004):
Mash1 is eliminated from the epithelium the day after MeBr, but reappears at 2
days post-lesion in increased numbers. Ngn1 and NeuroD also disappear
and then re-emerge at 3 days post-lesion, in advance of the reappearance of large numbers
of differentiating neurons 4 days after MeBr. In contrast, the neuronal repressor
Hes1 is expressed by Sus cells in the normal epithelium, but then materializes
in GBCs 1 day after MeBr. Subsequently the Hes1 (+) GBCs lose their
association with the basal zone, shift apically at the surface of the thickening
epithelium, and differentiate into Sus cells (Manglapus et al., 2004). These data provide a
molecular correlate for the differentiation of engrafted GBCs into Sus cells either alone
or in combination with neurons (Goldstein et
al., 1998;
Chen et al., 2004). Given
that Hes1 protein is known suppress transcription of Mash1 (Davis and Turner, 2001), it appears that the
residual GBCs are first driven to differentiate into Sus cells after lesion and only
later do they begin to make neurons.
In conclusion, the remarkable capacity for anatomical and functional recovery by the olfactory periphery reflects the interplay between the GBC progenitors and the environment of the lesioned epithelium. Molecular signals control the choice made by the GBCs between making neurons vs. non-neuronal cells. In addition, cues from extra-epithelial tissue are likely responsible for directing which OR to express. The nature of the corresponding molecules and mechanisms remains a subject of ongoing investigation.
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TopIntroductionAcknowledgementsReferences |
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The author thanks the members of the lab, past and present, whose heroic efforts and tireless dedication are responsible for the body of work presented here. Supported by NIH grants R01 DC00467 and R01 DC02167.
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References |
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TopIntroductionAcknowledgementsReferences |
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Cau, E., Casarosa, S. and Guillemot, F. (2002) Mash1 and Ngn1 control distinct steps of determination and differentiation in the olfactory sensory neuron lineage. Development, 129, 1871–1880.
Chen, X., Fang, H. and Schwob, J.E. (2004) Multipotency of purified, transplanted globose basal cells in olfactory epithelium. J. Comp. Neurol., 469, 457–474.[Medline]
Davis, R.L. and Turner, D.L. (2001) Vertebrate hairy and Enhancer of split related proteins: transcriptional repressors regulating cellular differentiation amd embryonic patterning. Oncogene, 20, 8342–8357.[CrossRef][ISI][Medline]
Goldstein, B.J. and Schwob, J.E. (1996) Analysis of the globose basal cell compartment in rat olfactory epithelium using GBC-1, a new monoclonal antibody against globose basal cells. J. Neurosci., 16, 4005–4016.
Goldstein, B.J., Fang, H., Youngentob, S.L. and Schwob, J.E. (1998) Transplantation of multipotent progenitors from the adult olfactory epithelium. Neuroreport, 9, 1611–1617.[Medline]
Graziadei, P.P.C. and Monti Graziadei, G.A. (1979) Neurogenesis and neuron regeneration in the olfactory system of mammals. I. Morphological aspects of differentiation and structural organization of the olfactory sensory neurons. J. Neurocytol., 8, 1–18.[CrossRef][ISI][Medline]
Holbrook, E.H., Szumowski, K.E.M. and Schwob, J.E. (1995) An immunohistochemical, ultrastructural, and developmental characterization of the horizontal basal cells of rat olfactory epithelium. J. Comp. Neurol., 363, 129–146.[CrossRef][ISI][Medline]
Huard, J.M., Youngentob, S.L., Goldstein, B.J., Luskin, M.B. and Schwob, J.E. (1998) Adult olfactory epithelium contains multipotent progenitors that give rise to neurons and non-neural cells. J. Comp. Neurol., 400, 469–486.[CrossRef][ISI][Medline]
Iwema, C.L., Fang, H., Kurtz, D.B., Youngentob, S.L. and Schwob, J.E. (2004) Odorant receptor expression patterns are restored in lesion-recovered rat olfactory epithelium. J. Neurosci., 24, 356–369.
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