The Lim homeobox gene Lhx2 is required for olfactory sensory neuron identity
Åsa Kolterud2,
Mattias Alenius1,
Leif Carlsson2 and
Staffan Bohm1,*
1 Department of Molecular Biology, Umeå University, Umeå, SE901 87,
Sweden
2 Umea Centre for Molecular Medicine, Umeå University, Umeå, SE901
87, Sweden
*
Author for correspondence (e-mail:
staffan.bohm{at}molbiol.umu.se)
Accepted 17 August 2004
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SUMMARY
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Progenitor cells in the mouse olfactory epithelium generate over a thousand
subpopulations of neurons, each expressing a unique odorant receptor (OR)
gene. This event is under the control of spatial cues, since neurons in
different epithelial regions are restricted to express region-specific subsets
of OR genes. We show that progenitors and neurons express the LIM-homeobox
gene Lhx2 and that neurons in Lhx2-null mutant embryos do
not diversify into subpopulations expressing different OR genes and other
region-restricted genes such as Nqo1 and Ncam2.
Lhx2-/- embryos have, however, a normal distribution of
Mash1-positive and neurogenin 1-positive neuronal progenitors that leave the
cell cycle, acquire pan-neuronal traits and form axon bundles. Increased cell
death in combination with increased expression of the early differentiation
marker Neurod1, as well as reduced expression of late differentiation
markers (G
olf and Omp), suggests that neuronal
differentiation in the absence of Lhx2 is primarily inhibited at, or immediate
prior to, onset of OR expression. Aberrant regional expression of early and
late differentiation markers, taken together with unaltered region-restricted
expression of the Msx1 homeobox gene in the progenitor cell layer of
Lhx2-/- embryos, shows that Lhx2 function is not required
for all aspects of regional specification of progenitors and neurons. Thus,
these results indicate that a cell-autonomous function of Lhx2 is required for
differentiation of progenitors into a heterogeneous population of individually
and regionally specified mature olfactory sensory neurons.
Key words: Lhx2, Neurod1, Olfactory, Gene expression, Odorant receptors, Lim, Homeobox, Neuron, Differentiation, Mouse
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Introduction
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A major challenge in our understanding of how axonal projection maps are
generated is to identify gene regulatory pathways that generate neuronal
diversity, both with regard to response properties and axon target selection
of individual neurons. A notable example is the highly heterogeneous
population of olfactory sensory neurons (OSNs) in the olfactory epithelium
(OE) of the nose. Orderly afferent projections of approximately 1000 different
subpopulations of mouse OSNs, each expressing a unique odorant receptor (OR)
gene, form a projection map decoding olfactory sensory information in the
olfactory bulb of the brain (Buck and Axel,
1991
; Ressler et al.,
1994
; Vassar et al.,
1994
). The scattered distribution of cell bodies of a given
OR-specific subpopulation is confined to one of several distinct regions of
OE. OSNs expressing different subsets of OR genes thus partition OE into zones
(Ressler et al., 1993
;
Vassar et al., 1993
). Some
overlap in OR expression between zones divides OE roughly into a dorsomedial,
a middle and a ventrolateral zone (Iwema
et al., 2004
). The zonal organization of OE is also evident from
the differential expression of other types of genes, such as the neural cell
adhesion molecule 2 (Ncam2/Ocam/Rncam) and
NADPH:quinone oxidoreductase 1 (Nqo1/DT-diaphorase)
(Alenius and Bohm, 1997
;
Gussing and Bohm, 2004
;
Yoshihara et al., 1997
). Nqo1
catalyzes reduction quinones and is co-expressed with OR genes of the
dorsomedial zone (Gussing and Bohm,
2004
). Ncam2, which is co-expressed with ORs of the middle and the
ventrolateral zones, has been shown to play a role in axonal guidance of OSNs
(Alenius and Bohm, 2003
).
OE originates from ectodermally derived neurogenic placodes and the
appearance of cell layers and zones becomes evident around embryonic day (E)
12.5-13.5 in mouse (Cau et al.,
1997
; Sullivan et al.,
1995
). The three major cell layers are: a superficial layer of
sustentacular (supporting) cells; a basal cell layer with dividing immediate
neuronal progenitors; and an intermediate cell layer containing OSNs. Also
progenitor and sustentacular cells differentially express certain genes in a
manner that correlates with their zonal position in OE
(Miyawaki et al., 1996
;
Norlin et al., 2001
;
Tietjen et al., 2003
). The
significance of zone-specific gene regulation in the progenitor and
sustentacular cell layers is not known.
Functional analyses of certain basic helix-loop-helix (bHLH) transcription
factors that are expressed in the progenitor cells in all zones have shown
that Mash1 (Ascl1 Mouse Genome Informatics) and neurogenin 1 (Ngn1)
are required for the development of OSNs
(Cau et al., 2002
;
Guillemot et al., 1993
). Mash1
is required for the survival of OSN progenitors at an early stage in the OSN
lineage, whereas the function of Ngn1 appears important in initiating
differentiation, presumably via the bHLH protein Neurod1, which is transiently
expressed at the onset of differentiation
(Cau et al., 2002
). Despite
these advances in knowledge of the essential roles played by bHLH in ensuring
neurogenesis in OE, transcription factors regulating diversification of OSNs,
including the choice of a given OR gene or the formation of OE zones, have not
been identified. A candidate gene that is expressed in a Mash1-dependent
manner in OE is Lhx2 (Cau et al.,
2002
; Tietjen et al.,
2003
). Lhx2 is a LIM-homeodomain transcription factor that is
implied in the specification of several aspects of the neuronal phenotype
(Monuki et al., 2001
;
Porter et al., 1997
;
Xu et al., 1993
). Analyses of
Lhx2-deficient mice have shown that Lhx2 is required for the formation of the
optic cup that gives rise to the multilayered neural retina
(Porter et al., 1997
). In
addition, lack of Lhx2 function results in agenesis of the hippocampus and
profound losses of cortical progenitors and neurons
(Monuki et al., 2001
;
Porter et al., 1997
). Other
LIM homeobox family members are required for the diversification of neuronal
subtypes in the spinal cord (Shirasaki and
Pfaff, 2002
). A specific role for Lhx2 in the generation of
neuronal subtypes has, however, remained elusive. In the present study we have
analyzed the function of Lhx2 during neurogenesis in OE. We provide evidence
that the expression of Lhx2 in the OSN lineage is required for the generation
of OR-and zone-specific subpopulations of OSNs during differentiation.
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Materials and methods
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Mice
All mice were maintained at the animal facility at Umeå University
under pathogen-free conditions. Generation of mice with a targeted
Lhx2 gene has been described elsewhere
(Porter et al., 1997
), and
mice and embryos were genotyped as previously described
(Monuki et al., 2001
).
Lhx2+/ mice were maintained on a mixed
129/SvxC57BL/6 background and were mated to generate embryos for
analyses. The morning of the vaginal plug was considered as E0.5. No obvious
difference in gene expression patterns could be observed between wild-type or
heterozygous E14.5-15.5 embryos and differences in gene expression between
E14.5 and E15.5 Lhx2-/- embryos were not observed. All
animal experiments were approved by the ethical committee at Umeå
University.
In-situ hybridization
Embryos were postfixed in 4% paraformaldehyde (PFA), cryoprotected (30%
sucrose in PBS), embedded in Tissue-Tek OCT compound and cryosectioned.
Cryosections (10 µm) for in-situ hybridization using 35S-labeled
probes were pretreated according to Breitschopf et al.
(Breitschopf et al., 1992
) and
hybridization and washing conditions were as described
(Sassoon and Rosenthal, 1993
).
Slides were dehydrated and processed for autoradiography using NTB-emulsion
(Kodak), and exposure was for 5-14 days at 4°C. In-situ
hybridization using digoxygenin (Dig)-labeled probes was performed as
described (Schaeren-Wiemers and
Gerfin-Moser, 1993
), with some modifications. Briefly sections
were treated with 5 µg/ml proteinase K (Roche) in PBS for 15 minutes in
Fast Red (Roche). Probes specific for G
olf, Omp and OR genes (M40, K20,
L45, A16, and M50) have been described previously
(Berghard et al., 1996
;
Ressler et al., 1993
;
Sullivan et al., 1995
). The
Ncam2-specific probe corresponded to the extracellular Ncam2
domain (Alenius and Bohm,
1997
). The Lhx2 probe (460-1750 bp, accession no. NM_010710)
hybridized to both wild-type and mutated Lhx2 transcripts
(Monuki et al., 2001
). Probes
corresponded to published sequences of Nqo1 (94-1626 bp, accession
no. NM_008706), Msx1 (894-1600 bp, accession no. BC016426), and
Alk6 (Bmpr1b Mouse Genome Informatics) (1-1935 bp,
accession no. MMALK6A). The Mash1-, Ngn1- and
Neurod1-specific probes corresponded to the published sequences of
Mash1 (256-900 bp, accession no. MUSHASH1X), Ngn1 (75-840
bp, accession no. YO9166), and Neurod1 (1-1207 bp, accession no.
BC018241). Hoechst 33258 (Sigma) was used as nuclear counterstain.
Immunohistochemistry
Embryos were fixed in 4% PFA for 1-2 hours at room temperature and
cryoprotected at 4°C for 24 hours. Retrieval of Nquo1, Rncam and Gap43
antigens was achieved by boiling in 10 mM citrate buffer (pH 6.0) for 10
minutes. Sections were then incubated for 1 hour in blocking solution (3%
normal donkey serum, 0.3% Triton X-100 in PBS) followed by overnight
incubation at 4°C in blocking solution containing affinity-purified
anti-Ncam2 (1:100 dilution (Alenius and
Bohm, 2003
), anti-Gap43 (1:1000 dilution, AB5220, Lot#22010885,
Chemicon), anti-caspase-3 antibody (1:1000 dilution, 557035, Lot#M051497,
PharMingen), anti-Omp (1:2000 dilution, gift from Dr Margolis), anti-Ncam
(1:100 dilution, 556323, clone 12F11, PharMingen), phospho-H3 (1:500 dilution,
06-570, Lot#24019, Upstate biotech), anti-Stathmin/SCG10 antibody (1:1000
dilution (Holmfeldt et al.,
2003
), and neuronal class III ß-tubulin (1:2000 dilution,
PRB-435P, Nordic BioSite AB), anti-LH2A [1:500
(Liem et al., 1997
)]. Positive
immunoreaction was visualized after a 1 hour incubation with Alexa Fluor 546
donkey anti-goat IgG (A-11056) or Alexa Fluor 488 donkey anti-rabbit IgG
(A-21206, Molecular Probes) diluted 1:250 in blocking solution. Sections were
counterstained with Hoechst. Microphotographs of immunohistochemistry and
in-situ hybridization analyses were taken using light, fluorescent or
dark-field optics on a Zeiss Axioskop microscope with a Hamamatsu digital CCD
camera. Confocal microscopy was performed on a Nikon confocal microscope.
Images were processed using Adobe Photoshop 7.0.1. Brightness and contrast
adjustments of images were done linearly.
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Results
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Lack of OR gene expression in Lhx2-null embryos
Double Lhx2 and neuronal class III ß-tubulin (TubIII)
immunohistochemistry analysis of E15.5 mouse embryos showed nuclear Lhx2 in
TubIII-positive OSNs (Fig.
1A-C). A fraction of TubIII-negative cells in the basal progenitor
cell layer stained positive with the Lhx2 antibody (arrow in 1C). The
sustentacular cell layer or in cells located in lamina propria under the OE
were negative. OR gene expression was first observed at E11.5 in the
developing OE. The number of OR-positive cells increases dramatically
thereafter and zonal OR expression is apparent by E13
(Sullivan et al., 1995
).
Homozygous Lhx2 knockout mouse (Lhx2-/-) die in
utero after E15.5 (Porter et al.,
1997
). Lhx2-/- and littermate control embryos
(Lhx2+/ and Lhx2+/+) at two
ages (E14.5 and E15.5) were analyzed in this study. Lhx2 and OR gene
expression was examined by in-situ hybridization
(Fig. 1D-J). Lhx2 mRNA
was, in concordance with the result of the immunohistochemical analysis,
expressed in both OSNs and a fraction of cells in the basal progenitor cell
layer in control embryos (Fig.
1D). The targeted Lhx2 allele produces a fusion
transcript of Lhx2 exon 1 and the neomycin resistance gene that
allows for the identification of cells that have an active Lhx2
promoter also in Lhx2-/- embryos
(Monuki et al., 2001
).
Importantly, we detected a similar cell layer organization and distribution of
Lhx2-promoter active cells in control and homozygous mutant mice
(Fig. 1D,E). The result
suggested that Lhx2 is not required for formation of the progenitor and
neuronal cell layers of OE. Next we analyzed for the expression of different
OR genes that are expressed in different zones, i.e. K21
(Olfr145 Mouse Genome Informatics), L45, M50
(Olfr6 Mouse Genome Informatics) and A16 (Olfr140
Mouse Genome Informatics). As expected, all four OR probes hybridized
to scattered OSNs in sections from control mice
(Fig. 1F-I). Interestingly,
parallel analyses of Lhx2-/- littermates revealed no
hybridizing cells (Fig. 1F-I),
even though sections throughout the anteriorposterior extent of the
nasal cavity were examined. Neither did in-situ hybridization analyses using
more sensitive radioactively labeled cRNA probes reveal any OR positive OSNs
in mutant embryos (Fig. 1J). These results indicated that the function of Lhx2 was required for OR
expression.

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Fig. 1. Unaltered distribution of Lhx2 expression and lack of OR
expression in OE of Lhx2 -/- embryos. (A-C)
Double-immunohistochemical analysis of Lhx2 and TubIII expression in OE of
E15.5 mouse embryo is shown. (A) Nuclear Lhx2 immunoreactivity in the neuronal
cell layer (N) and in a fraction of cells in the basal progenitor cell layer
(P). No Lhx2-positive cells are present in the sustentacular cell layer (S)
and the lamina propria (l.p.). (B) TubIII immunoreactivity in neuronal soma
and neuronal processes. (C) Images in A and B combined, showing that Lhx2 and
TubIII are co-expressed in OSNs. A limited number of TubIII-negative
Lhx2-positive cells are present in the progenitor cell layer (arrows in C).
(D-I) In-situ hybridization analyses of serial OE sections from control (left
panel) and Lhx2-/- embryos (right panel) with Dig-labeled
cRNA probes that hybridize to Lhx2 and mutated Lhx2
transcripts (Lhx2m) (D,E), and OR genes (K21, L45,
M50 and A16) (F-I). (J) Autoradiographs of in-situ hybridization
analyses with a 35S-labeled cRNA probe specific to the OR gene
K20. Scattered cells expressing the different OR genes are present in
control mice. The zones in which each the OR gene is expressed is indicated,
i.e. dorsomedial (dm), the middle (m) or the ventrolateral (vl) zone. In
Lhx2 -/- mice, there are no cells expressing ORs in any
epithelial region (representative results are shown).
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Lack of zone-specific Nqo1 and Ncam2 in Lhx2-null embryos
Lack of OR expression in Lhx2-/- embryos suggested that
the function of Lhx2 was important specifically for OR expression.
However, since mutant mice lacked expression of several ORs that are expressed
in different zones, an alternative interpretation was that Lhx2 is
required for zone-specific gene expression in general and not OR gene
expression per se. To address this possibility, we analyzed for the expression
of other zone-specific markers. Nqo1 is co-expressed with ORs of the
dorsomedial zone, whereas Ncam2 is co-expressed with ORs of both the
middle and the ventrolateral zones (Alenius
and Bohm, 2003
; Gussing and
Bohm, 2004
). In-situ hybridization analyses revealed zonal
Nqo1 and Ncam2 expression in control mice
(Fig. 2A, parts B,C and
Fig. 2B, parts A,B), whereas no
hybridizing cells were observed in the OE of Lhx2-/-
embryos (Fig. 2A, parts E,F and
Fig. 2B, parts C,D). Other cell
types that hybridized to Nqo1 and Ncam2 probes were, however, present in both
control and Lhx2-/- embryos (arrowheads in
Fig. 2A, parts B,E,F). Thus,
the function of Lhx2 was required for the development of OSNs that
express both ORs and zone-specific genes.

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Fig. 2. No expression of the zone-specific genes Nqo1 and Ncam2
in OE of Lhx2-/- embryos. (A) Autoradiographs of in-situ
hybridization analyses of consecutive coronal sections (dorsal up and medial
left) of one nasal cavity of control embryos (A-C) and
Lhx2-/- embryos (D-F). Sections were hybridized with
35S-labeled cRNA probes specific for Lhx2 and mutated
Lhx2 transcripts (Lhx2m) (A,D), Nqo1
(B,E) and Ncam2 (C,F). Lhx2 expression is distributed
throughout the OE in both control (A) and Lhx2-/- mice
(D). Nqo1 is expressed in OSNs located in the dorsomedial zone in
control embryos (arrow in B), whereas no signal is present in the neuronal
cell layer of Lhx2-/- embryos (E). Ncam2 is
expressed in OSNs located in the middle and the ventrolateral zones in control
embryos (arrow in C), whereas no hybridizing signal is present in the neuronal
cell layer of Lhx2-/- embryos (F). The border between
neurons in the dorsomedial zone and the middle zone is indicated (dotted line
in B,C). Nqo1-specific hybridization to cells in lamina propria
(arrowheads in B,E) and Ncam2-specific hybridization to cells in the
telencephalon (arrowhead in F) are also indicated. (B) High-power
magnifications of OE sections hybridized with Dig-labeled cRNA probes specific
for Nqo1 (A,C) and Ncam2 (B,D) are shown. Both probes
hybridize to OSN in control embryos (A,B), whereas no signal over background
is evident in sections of Lhx2-/- embryos (C,D).
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Inhibited OSN differentiation and increased apoptosis in Lhx2-/- embryos
Formally, the phenotype observed might stem from a complete lack of
neuronal progenitors, altered commitment of multipotent progenitors or that
neuronal differentiation was inhibited at a stage prior to onset of OR and
zone-specific gene expression. To address the question of whether
Lhx2 is required for the generation of OSN progenitors, we analyzed
for the expression of Mash1 and Ngn1. It has been suggested
that the developing OE contains two populations of neuronal progenitors: a
Mash1-positive population that divides in the apical part of OE and a
population of Mash1- and Ngn1-positive secondary progenitor cells that settle
on the basal side of OE, where they continue to divide before differentiating
into OSNs (Cau et al., 1997
).
Expression levels and distribution of Mash1- and Ngn1-positive cells in OE of
control and Lhx2 mutant mice were similar
(Fig. 3A,B). Thus, Lhx2
function did not appear to be required for Mash1/Ngn1-dependent neurogenesis.
Ngn1 regulates the differentiation-promoting gene Neurod1, which is
transiently expressed during the onset of OSN differentiation
(Cau et al., 2002
).
Interestingly, Lhx2 mutant embryos had an increased number of
Neurod1-positive cells (Fig.
3C). Neurod1 was expressed in the one- to two-cell thick
layer of progenitor cells in control mice, whereas Neurod1-positive cells had
expanded into the presumptive neuronal cell layer in Lhx2 mutants
(Fig. 3C). Thus, it appeared
that lack of Lhx2 resulted in accumulation of Neurod1-positive
progenitor cells and/or immature OSNs. To characterize this phenotype further,
we analyzed for expression of OSN-specific genes G
olf and
Omp, which are expressed at terminal stages of OSN differentiation.
The gene for the G-protein alpha subunit G
olf is turned on at a stage
between E13.5-16.5, which is at least 2 days subsequent to the onset of OR
expression (Sullivan et al.,
1995
). G
olf was expressed in neurons of both
control and Lhx2-/- embryos at E14.5-15.5
(Fig. 3D). This result
suggested that the observed phenotype was not a consequence of a developmental
delay in the absence of Lhx2. The expression level of
G
olf was, however, reduced compared with littermate control
embryos (Fig. 3D). We next
analyzed for the expression of Omp, which is a hallmark of mature OSN
(Youngentob et al., 2003
).
Studies of neuronal differentiation in the OE show that ORs are expressed
prior to onset of Omp expression
(Iwema and Schwob, 2003
).
In-situ hybridization analysis showed that a limited number of neurons in
Lhx2-/- mice expressed Omp to markedly reduced levels
(Fig. 3E). The total number of
Omp-positive cells was 133±61 and 13±5 (P<0.001,
Student's t-test) per section and nasal cavity in control and
Lhx2-/- embryos, respectively. The presence of neurons
expressing Omp in Lhx2-/- mice was confirmed by
immunohistochemical analyses (Fig.
3F). Unexpectedly, analyses of sections throughout the nasal
cavity revealed that Omp-positive and G
olf-positive
cells were located selectively in the dorsomedial part of OE (for result and
discussion see below). The reduced expression of Omp and
G
olf suggested that neuronal maturation and/or survival was
reduced. To address if reduced expression of G
olf and
Omp coincided with increased apoptosis, we utilized activated
(cleaved) caspase 3 immunohistochemistry analysis. Caspase 3-positive cells
were evenly distributed throughout OE (data not shown) and the number of cells
with activated caspase 3 in the progenitor and neuronal layers was
5.4±2.1 and 19.4±8.9 (P<0.001, Student's
t-test) per OE section in control and Lhx2-/-
embryos, respectively. The aberrant differentiation of OSNs in
Lhx2-/- embryos was thus associated with an overall
3.5-fold increased rate of apoptosis. Collectively, these results indicated
that a fraction of neurons in the dorsomedial OE acquires an OSN identity in
the absence of Lhx2 but fails to become and/or survive as OR- and
zonally specified bona fide OSNs.
Differentiation is inhibited at, or just prior to, onset of OR expression
Lhx2 deficiency appeared to influence differentiation primarily at
a stage that was subsequent to onset of Neurod1 expression and prior
to onset of G
olf and Omp expression. To determine if
there was an increased number of Neurod1-positive progenitors and/or
Neurod1-positive immature neurons in Lhx2-/- embryos we
analyzed for Neurod1 expression in relation to dividing cells and
onset of early pan-neuronal differentiation markers. Immunohistochemical
analyses with antibodies specific for Ncam1, Gap43, Stmn/SCG10 (Stmn2
Mouse Genome Informatics) and TubIII showed that immature neurons were present
throughout OE in both control and mutant embryos
(Fig. 4A-F,I,J). Moreover, the
staining patterns showed immunoreactivity in axon bundles in the lamina
propria and in the OE of Lhx2-/- mice (arrows in
4A-F,I,J). Thus, progenitor cells appeared to leave the cell cycle and
differentiate to a stage associated with the onset of pan-neuronal markers and
axonal outgrowth. Analyses of serial sections revealed that regions in OE that
showed high Neurod1 and TubIII expression correlated
(Fig. 4I-L). Confocal
microscopy indicated an increased number of cells that co-expressed Neurod1
and TubIII in Lhx2-/- embryos
(Fig. 4M,N) which is compatible
with the suggestion that Lhx2-/- embryos have a larger
fraction of newly differentiated Neurod1-positive neurons and not
proliferating progenitors. To substantiate this suggestion, we analyzed for
the mitosis marker phosphorylated histone 3 (phospho-H3). The increased number
of Neurod1-positive cells in Lhx2-/- embryos did not
correlate to an increase in dividing progenitors
(Fig. 4G,H,O,P). Thus, these
results indicated that the transient differentiation stage, at which Neurod1
and pan-neuronal markers are co-expressed, is prolonged in
Lhx2-/- embryos. Since there is a lag between onset of
pan-neuronal gene expression and onset of OR expression
(Iwema and Schwob, 2003
), this
suggests that neuronal differentiation in Lhx2-/- embryos
is primarily inhibited at a stage that is normally associated with the onset
of OR gene expression.

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Fig. 4. The neuronal cell layer in OE of Lhx2-/- embryos
largely contains newly differentiated neurons that have acquired pan-neuronal
traits and form axon bundles. Coronal sections (dorsal up and medial left) of
one nasal cavity from control embryos (A,C,E,G,I,K,M,O) and
Lhx2-/- embryos (B,D,F,H,J,L,N,P). (A-F)
Immunohistochemical analyses for expression of immature neuronal markers is
shown. Ncam1 (A,B), Gap43 (c-d), and
Stathmin/SCG10 (E,F) are expressed in OE and axon bundles of
the lamina propria (arrows) in both control and Lhx2-/-
embryos. (G,H) Double phospho-H3 (PH3; in green) immunohistochemistry and
Neurod1 (ND; in red) in-situ hybridization analyses showing that the
increased expression of Neurod1, primarily apparent in the ventral OE
(arrowhead in H), does not correlate with the number and distribution of
mitotic cells. (I-L) Analyses of consecutive OE sections, showing that cells
immunoreactive using an anti-TubIII antibody (I,J) and cells that express
Neurod1 transcripts (K,L) co-localize predominantly in ventrolateral
regions of OE (arrowheads in J,L). Expression of TubIII in axon bundles of the
lamina propria is indicated (arrows in I,J). (M,N) High magnification confocal
images of double TubIII immunohistochemical (in green) and Neurod1
in-situ hybridization (in red) analyses showing an increased number of cells
in Lhx2-/- embryos that co-express Neurod1 and
TubIII (yellow signal; insert in N). (O,P) High magnification confocal images
of double phospho-H3 immunohistochemical (in green) and Neurod1
in-situ hybridization (in red) analyses showing that OE in
Lhx2-/- embryos does not contain an increased number of
progenitors that co-express phospho-H3 and Neurod1 (in yellow;
arrowheads).
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Lack of Lhx2 function reveals zone-specific differences in neuronal maturation
Although neurons in Lhx2-/- embryos were not zonally
specified with regard to OR, Nqo1 or Ncam2 expression, we
found evidence that Lhx2 deficiency did not abolish all zonal
characteristics of the OE. The ventrolateral region contained a higher
fraction of Neurod1-positive cells than did the dorsomedial region (arrowheads
in Fig. 4H,L). This regional
difference in distribution of Neurod1-positive cells was more apparent in
Lhx2-/- embryos than controls. Analyses of consecutive
sections indicated that the increased expression of Neurod1 in the
ventrolateral part coincided with a high expression of TubIII (arrowheads in
Fig. 4J,L). Since phospho-H3
immunoreactive cells were distributed equally throughout the OE
(Fig. 4G,H), this result
suggested that the ventrolateral region contained a larger fraction of newly
differentiated postmitotic neurons compared with the dorsomedial OE region in
Lhx2-/- embryos. Interestingly, neurons expressing the
late differentiation markers (G
olf and Omp) were
selectively located in the dorsomedial OE region in
Lhx2-/- embryos (Fig.
5A-D). The G
olf- and Omp-positive region
appeared to correspond to the dorsomedial zone, namely the dorsal meatus, part
of the medial septum, and the tips of the ethmoturbinate projecting into the
nasal cavity (Fig. 5B,D and
compare with expression of Nqo1 in
Fig. 5E). To confirm the
unequal distribution of Omp expression, we quantified the number of
Omp-positive cells that were located on either side of a tentative
half circle dividing the OE into two roughly equal parts, one dorsomedial and
one ventrolateral part. This analysis revealed that 59.7±14.5% and
95.2±10.9% (P<0.001, Student's t-test) of the
Omp-positive cells were located in the dorsomedial part in control
and Lhx2-/- embryos, respectively. Thus, the absence of
Lhx2 expression revealed zone-specific differences in the regulated
control of neuronal differentiation.

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Fig. 5. Abnormal region-specific OSN differentiation and normal region-specific
gene expression in sustentacular and progenitor cells. In-situ hybridization
analyses of serial coronal (dorsal up and medial left) sections of OE from
control (A,C,E,G,I,K,M) and Lhx2-/- embryos
(B,D,F,H,I,L,N) using Dig-labeled cRNA probes specific for
G olf (A,B), Omp (C,D), Nqo1 (E,F),
Alk6 (G-J) and Msx1 (K-N). (A-F) G olf- and
Omp-positive cells are present in all OE zones in control embryos
(A,C) but show a restricted dorsomedial distribution in OE of
Lhx2-/- embryos (arrows in B,D). The distribution of
G olf- and Omp-positive cells in
Lhx2-/- embryos is similar to the distribution of the
dorsomedial-specific marker Nqo1 in control embryos (arrows in E). No
Nqo1 expression over background is detected in
Lhx2-/- embryos (F). (G-N) The gradient of Alk6
expression (G,H; high dorsomedial and low ventrolateral) and the reverse
gradient of Msx1 expression (K,L) are present in both control and
Lhx2-/- embryos. High-power magnifications of
Alk6 expression in the sustentacular cell layer (I,J) and
Msx1 expression in the basal progenitor cell layer (M,N) is shown.
Thus, cells expressing Alk6 and Msx1 show a similar
distribution in OE cell layers in control and Lhx2-/-
mice.
|
|
Intact region-specific expression in sustentacular and progenitor cell layers
One possible scenario was that positional signals determining zone-specific
gene expression in OE were absent or altered in Lhx2-/-
embryos. The type I BMP receptor Alk6 is expressed in a dorsomedial to
ventrolateral gradient by the sustentacular cells, whereas differential
expression of the homeobox gene Msx1 defines a ventrolateral to
dorsomedial counter gradient in the progenitor cell layer
(Norlin et al., 2001
). This
spatially limited expression of Alk6 and Msx1 was unaltered
in Lhx2-/- embryos
(Fig. 5G-N). Since
sustentacular and progenitor cells showed the expected regional differences in
gene expression, the result strongly suggested that positional cues are
present in OE of Lhx2-/- embryos.
 |
Discussion
|
---|
In this study we show that the function of the LIM homeobox transcription
factor Lhx2 is required for the generation of a heterogeneous population of
individually and zonally specified OSNs. Lhx2 is apparently not required for
the development of zonally specified progenitor cells or supporting cells of
OE. Our results thus indicate that Lhx2 regulates a gene program that
is intrinsic to OSN lineage that includes ORs and zone-specific genes. The
neurons generated in the absence of Lhx2 function, and thereby also
the functions of ORs and zone-specific genes, express pan-neuronal markers and
have axons projecting toward the telencephalic vesicle. Progenitors thus
apparently leave the cell cycle and acquire pan-neuronal traits characteristic
of postmitotic neurons. We detect an accumulation of neurons that express the
differentiation-promoting bHLH transcription factor Neurod1. Since OSNs
normally co-express Neurod1 and pan-neuronal genes only transiently
during differentiation (Cau et al.,
1997
), our results suggest that a majority of neurons in
Lhx2-/- embryos correspond to immature neurons that have
just begun to express pan-neuronal marker genes. Analyses of OR expression in
the OE of adult animals indicate that there is a short delay period between
the onset of pan-neuronal genes and the start of OR expression
(Iwema and Schwob, 2003
). Thus
our results are compatible with the suggestion that neuronal differentiation
in the Lhx2-/- embryos does not proceed past this delay
period in an appropriate manner. In this regard it is interesting to note that
accelerated differentiation as a consequence of OR expression has been
suggested to underlie the feedback mechanism in which a given OR protein
ensures high levels of expression of its corresponding gene
(Lewcock and Reed, 2004
). It
is therefore conceivable that an Lhx2-dependent block in OR
expression inhibits neuronal differentiation and/or prolongs the period during
which OSN under normal circumstances are competent to select an OR gene for
high-level expression. Even though the data does not allow us to determine if
Lhx2 exerts its primary effect in progenitor cells or OSNs, it is
interesting to note that Lhx2 has been identified by yeast one-hybrid
screening as a protein that binds to conserved sequence motifs in the promoter
regions of a defined subfamily of OR genes
(Hoppe et al., 2003
). Previous
studies have demonstrated that promoter proximal regions of certain OR genes
are sufficient to fully recapitulate proper expression in transgenic mice
(Lewcock and Reed, 2004
;
Vassalli et al., 2002
).
Moreover, the feedback control mechanism of OR gene choice was shown to
require a defined cis-acting locus control region with capacity to
enhance transcription of OR genes belonging to different zonal sets
(Lewcock and Reed, 2004
).
Similarly, we find that Lhx2 function appears required for expression of OR
genes belonging to different zonal sets, which is compatible with the
possibility that Lhx2 regulates transcription by binding to conserved
DNA motifs in zonally expressed genes.
The OE in Lhx2-/- embryos shows an aberrant zonal
distribution of neurons that express early (Neurod1 and pan-neuronal)
and late (G
olf and Omp) differentiation markers,
respectively. This result suggests that neuronal differentiation and/or
survival is under the influence of zonal factors and thus, that Lhx2
is not required for all aspects of zonal specification of cells in OE.
Evidence for the existence of zone-specific cues in the absence of
Lhx2 is that the region-specific expression of Alk6 in
sustentacular cells and Msx1 in progenitors is unperturbed in mutant
embryos. Several other genes have been found to be expressed in progenitors in
middle and ventrolateral zones exclusively
(Tietjen et al., 2003
).
Interestingly, the expression of these genes in progenitors is either
dependent or independent of Mash1 function. Since multipotent
progenitors are present in OE it is tempting to speculate that a
Mash1- and Lhx2-independent mechanism specify sustentacular
cells to differentially express genes such as Alk6 in a zone-specific
manner. Rbtn1 (Lmo1 Mouse Genome Informatics), which belongs to the
LIM only family, is among the transcription factors that were identified to be
zonally expressed in a Mash1-dependent manner. LIM only factors interact with
both LIM homeobox and bHLH proteins and can regulate Neurod1
expression (Bao et al., 2000
;
Jurata et al., 1996
). Thus,
the zone-specific differentiation phenotype may be a consequence of lack of a
zone-specific signaling protein in OSNs and/or absence of Lhx2
function in progenitors. In either case it is likely that Lhx2
deficiency accentuates a zone-specific difference in OSN maturation that is
not as apparent in the OE of control animals. Evidence for regional
differences under normal conditions has come from the finding that the
regenerative capacity of OSNs in an adult animal differs between OE zones
(Konzelmann et al., 1998
).
Moreover, the anterior OE, with a high proportion of the dorsomedial zone,
appears to contain more OSNs that co-express early (Gap43) and late
(Omp) differentiation markers compared with middle and ventrolateral
zones (Iwema and Schwob,
2003
).
Collectively, the results presented show a role for Lhx2 in
supplying a fundamental function in the regulated control of neuronal
diversity within the OSN lineage. Further studies of Lhx2 function in
the olfactory system will help clarify the precise role of Lhx2 in
the gene regulatory mechanism, whereby an individual OSN selects a given
response profile and acquires topographic precision in axon targeting.
 |
Note added in proof
|
---|
During the revision of this manuscript, similar results were reported
(Hirota and Mombaerts,
2004
).
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr Frank Margolis for the Omp antibody. This work was supported by
grants from the Swedish Natural Science Research Council (B5101-1250/2001),
the Swedish Cancer Society, the Wallenberg Foundation and the
Vàsterbotten County.
 |
REFERENCES
|
---|
Alenius, M. and Bohm, S. (1997). Identification
of a novel neural cell adhesion molecule-related gene with a potential role in
selective axonal projection. J. Biol. Chem.
272,26083
-26086.[Abstract/Free Full Text]
Alenius, M. and Bohm, S. (2003). Differential
function of RNCAM isoforms in precise target selection of olfactory sensory
neurons. Development
130,917
-927.[Abstract/Free Full Text]
Bao, J., Talmage, D. A., Role, L. W. and Gautier, J.
(2000). Regulation of neurogenesis by interactions between HEN1
and neuronal LMO proteins. Development
127,425
-435.[Abstract/Free Full Text]
Berghard, A., Buck, L. B. and Liman, E. R.
(1996). Evidence for distinct signaling mechanisms in two
mammalian olfactory sense organs. Proc. Natl. Acad. Sci.
USA 93,2365
-2369.[Abstract/Free Full Text]
Breitschopf, H., Suchanek, G., Gould, R. M., Colman, D. R. and
Lassmann, H. (1992). In situ hybridization with
digoxigenin-labeled probes: sensitive and reliable detection method applied to
myelinating rat brain. Acta Neuropathol.
84,581
-587.[Medline]
Buck, L. and Axel, R. (1991). A novel multigene
family may encode odorant receptors: a molecular basis for odor recognition.
Cell 65,175
-187.[Medline]
Cau, E., Gradwohl, G., Fode, C. and Guillemot, F.
(1997). Mash1 activates a cascade of bHLH regulators in olfactory
neuron progenitors. Development
124,1611
-1621.[Abstract/Free Full Text]
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.[Medline]
Guillemot, F., Lo, L. C., Johnson, J. E., Auerbach, A.,
Anderson, D. J. and Joyner, A. L. (1993). Mammalian
achaete-scute homolog 1 is required for the early development of olfactory and
autonomic neurons. Cell
75,463
-476.[Medline]
Gussing, F. and Bohm, S. (2004). NQO1 activity
in the main and the accessory olfactory systems correlates with the zonal
topography of projection maps. Eur. J. Neurosci.
19, 1-8.[CrossRef][Medline]
Hirota, J. and Mombaerts, P. (2004). The LIM
homeodomain protein Lhx2 is required for complete development of mouse
olfactory sensory neurons. Proc. Natl. Acad. Sci. USA
101,8751
-8755.[Abstract/Free Full Text]
Holmfeldt, P., Brannstrom, K., Stenmark, S. and Gullberg, M.
(2003). Deciphering the cellular functions of the Op18/Stathmin
family of microtubule-regulators by plasma membrane-targeted localization.
Mol. Biol. Cell 14,3716
-3729.[Abstract/Free Full Text]
Hoppe, R., Frank, H., Breer, H. and Strotmann, J.
(2003). The clustered olfactory receptor gene family 262: genomic
organization, promotor elements, and interacting transcription factors.
Genome Res. 13,2674
-2685.[Abstract/Free Full Text]
Iwema, C. L. and Schwob, J. E. (2003). Odorant
receptor expression as a function of neuronal maturity in the adult rodent
olfactory system. J. Comp. Neurol.
459,209
-222.[CrossRef][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.[Abstract/Free Full Text]
Jurata, L. W., Kenny, D. A. and Gill, G. N.
(1996). Nuclear LIM interactor, a rhombotin and LIM homeodomain
interacting protein, is expressed early in neuronal development.
Proc. Natl. Acad. Sci. USA
93,11693
-11698.[Abstract/Free Full Text]
Konzelmann, S., Saucier, D., Strotmann, J., Breer, H. and Astic,
L. (1998). Decline and recovery of olfactory receptor
expression following unilateral bulbectomy. Cell Tissue
Res. 294,421
-430.[CrossRef][Medline]
Lewcock, J. W. and Reed, R. R. (2004). A
feedback mechanism regulates monoallelic odorant receptor expression.
Proc. Natl. Acad. Sci. USA
101,1069
-1074.[Abstract/Free Full Text]
Liem, K. F., Jr, Tremml, G. and Jessell, T. M.
(1997). A role for the roof plate and its resident
TGFbeta-related proteins in neuronal patterning in the dorsal spinal cord.
Cell 91,127
-138.[CrossRef][Medline]
Miyawaki, A., Homma, H., Tamura, H., Matsui, M. and Mikoshiba,
K. (1996). Zonal distribution of sulfotransferase for phenol
in olfactory sustentacular cells. EMBO J.
15,2050
-2055.[Abstract]
Monuki, E. S., Porter, F. D. and Walsh, C. A.
(2001). Patterning of the dorsal telencephalon and cerebral
cortex by a roof plate-Lhx2 pathway. Neuron
32,591
-604.[Medline]
Norlin, E. M., Alenius, M., Gussing, F., Hagglund, M., Vedin, V.
and Bohm, S. (2001). Evidence for gradients of gene
expression correlating with zonal topography of the olfactory sensory map.
Mol. Cell. Neurosci. 18,283
-295.[CrossRef][Medline]
Porter, F. D., Drago, J., Xu, Y., Cheema, S. S., Wassif, C.,
Huang, S. P., Lee, E., Grinberg, A., Massalas, J. S., Bodine, D. et al.
(1997). Lhx2, a LIM homeobox gene, is required for eye,
forebrain, and definitive erythrocyte development.
Development 124,2935
-2944.[Abstract/Free Full Text]
Ressler, K. J., Sullivan, S. L. and Buck, L. B.
(1993). A zonal organization of odorant receptor gene expression
in the olfactory epithelium. Cell
73,597
-609.[Medline]
Ressler, K. J., Sullivan, S. L. and Buck, L. B.
(1994). Information coding in the olfactory system: evidence for
a stereotyped and highly organized epitope map in the olfactory bulb.
Cell 79,1245
-1256.[Medline]
Sassoon, D. and Rosenthal, N. (1993). Detection
of messenger RNA by in situ hybridization. Methods
Enzymol. 225,384
-404.[Medline]
Schaeren-Wiemers, N. and Gerfin-Moser, A.
(1993). A single protocol to detect transcripts of various types
and expression levels in neural tissue and cultured cells: in situ
hybridization using digoxigenin-labelled cRNA probes.
Histochemistry 100,431
-440.[Medline]
Shirasaki, R. and Pfaff, S. L. (2002).
Transcriptional codes and the control of neuronal identity. Annu.
Rev. Neurosci. 25,251
-281.[CrossRef][Medline]
Sullivan, S. L., Bohm, S., Ressler, K. J., Horowitz, L. F. and
Buck, L. B. (1995). Target-independet pattern specification
in the olfactory epithelium. Neuron
15,779
-789.[CrossRef][Medline]
Tietjen, I., Rihel, J. M., Cao, Y., Koentges, G., Zakhary, L.
and Dulac, C. (2003). Single-cell transcriptional analysis of
neuronal progenitors. Neuron
38,161
-175.[Medline]
Vassalli, A., Rothman, A., Feinstein, P., Zapotocky, M. and
Mombaerts, P. (2002). Minigenes impart odorant
receptor-specific axon guidance in the olfactory bulb.
Neuron 35,681
-696.[Medline]
Vassar, R., Ngai, J. and Axel, R. (1993).
Spatial segregation of odorant receptor expression in the mammalian olfactory
epithelium. Cell 74,309
-318.[Medline]
Vassar, R., Chao, S. K., Sitcheran, R., Nunez, J. M., Vosshall,
L. B. and Axel, R. (1994). Topographic organization of
sensory projections to the olfactory bulb. Cell
79,981
-992.[Medline]
Xu, Y., Baldassare, M., Fisher, P., Rathbun, G., Oltz, E. M.,
Yancopoulos, G. D., Jessell, T. M. and Alt, F. W. (1993).
LH-2: a LIM/homeodomain gene expressed in developing lymphocytes and neural
cells. Proc. Natl. Acad. Sci. USA
90,227
-231.[Abstract]
Yoshihara, Y., Kawasaki, M., Tamada, A., Fujita, H., Hayashi,
H., Kagamiyama, H. and Mori, K. (1997). OCAM: A new member of
the neural cell adhesion molecule family related to zone-to-zone projection of
olfactory and vomeronasal axons. J. Neurosci.
17,5830
-5842.[Abstract/Free Full Text]
Youngentob, S. L., Kent, P. F. and Margolis, F. L.
(2003). OMP gene deletion results in an alteration in
odorant-induced mucosal activity patterns. J.
Neurophysiol. 90,3864
-3873.[Abstract/Free Full Text]