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First published online 15 March 2006
doi: 10.1242/dev.02329
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1 Laboratory for Vertebrate Axis Formation, Center for Developmental Biology,
RIKEN, Kobe 650-0047, Japan.
2 Laboratory for Neurobiology of Synapse, Brain Science Institute, RIKEN,
Wako-shi, Saitama 351-0198, Japan.
3 Laboratory for Animal Resources and Genetic Engineering, Center for
Developmental Biology, RIKEN, Kobe 650-0047, Japan.
4 Department of Mental Retardation and Birth Defect Research, National Institute
of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo
187-8502, Japan.
Author for correspondence (e-mail:
hibi{at}cdb.riken.jp)
Accepted 16 February 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Fez, Zinc finger, Olfactory sensory neuron, Olfactory bulb, Axon guidance, Mitral cell, Interneuron, Rostral migratory stream, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Mori and Yoshihara,
1995; Yoshihara and Mori,
1997). Olfactory sensory neurons (OSNs) reside in the olfactory
epithelium (OE) and vomeronasal organs, where they express seven
transmembrane-type odorant or pheromone receptors, respectively. Their axons
extend through the lamina cribrosa and reach the olfactory bulbs (OBs), which
protrude from the anterior telencephalon. The OB is a laminar structure that
contains limited types of neurons and glia. The outermost layer is the
olfactory sensory nerve layer (ONL), which consists of olfactory afferent
axons and ensheathing glia. Beneath the ONL, OSNs converge and synapse onto
the dendrites of glutamatergic projection neurons (mitral and tufted cells) in
structures called glomeruli [the glomerular layer (GL)]. Between the glomeruli
in the GL, local circuit interneurons (periglomerular cells) send their
dendrites into the glomeruli. Below the GL, the external plexiform layer (EPL)
contains the cell bodies of the tufted cells, and the secondary dendrites of
the mitral and tufted cells. Beneath the EPL is the mitral cell layer (MCL),
which contains the mitral cell bodies. The projection neurons send their axons
into the fibrous plexiform layer (IPL). Below the IPL lie the cell bodies of
granule cells, which are interneurons [granule cell layer (GCL)]. Beneath the
GCL, interneuron progenitor cells are localized to the subventricular zone
(SVZ), which continuously generates the interneurons, periglomerular cells and
granule cells from the embryonic stage to adult
(Goldman and Luskin, 1998;
Hinds, 1968a;
Hinds, 1968b;
Lois and Alvarez-Buylla, 1994;
Luskin, 1993).
The OB projection neurons and interneurons are born at distinct time
periods under different kinds of genetic control. The projection neurons are
born earlier than the interneurons (Hinds,
1968a; Hinds,
1968b). They have a pallial origin, whereas the interneurons
originate in the subpallium. The development of the OB can be divided into two
steps. First, evagination of the primordial OB from the telencephalon takes
place on embryonic day (E) 12-E13 in mouse. Second, at E14-E18 in mouse, the
interneuron progenitors arise from the SVZ of the lateral ganglionic eminence
(LGE) (Luskin, 1998;
Wichterle et al., 2001). The
interneurons are continuously generated in the SVZ of the LGE and, thereafter,
travel through the rostral migratory stream (RMS) throughout the life of the
animal (Goldman and Luskin,
1998; Lois and Alvarez-Buylla,
1994; Luskin,
1993).
Analyses of genetically modified mice have revealed that the homeobox Dlx
and Arx genes function cell-autonomously to generate interneurons,
and that these genes are required for layer formation in the OB and the axon
projection of OSNs to the OB (Bulfone et
al., 1998; Levi et al.,
2003; Long et al.,
2003; Qiu et al.,
1995; Yoshihara et al.,
2005). As Arx is not expressed in the OE or in mitral
cells, Arx-dependent instructive signals from the OB are required for
proper innervation of the OB by OSNs, and Arx non-cell-autonomously
controls the MCL formation in the OB. By contrast, there is no direct
molecular or genetic evidence that signals from the OSNs are involved in OB
formation, although primary olfactory axons are reported to alter the cell
cycle of neurons within the OB primordium
(Gong and Shipley, 1995).
Fez (forebrain embryonic zinc-finger) is a zinc-finger gene that
was originally isolated as a forebrain- and OSN-specific gene in
Xenopus (Matsuo-Takasaki et al.,
2000). Identification of a homolog (Fez-like), and of
orthologs in zebrafish, mouse and human, revealed that there are two highly
related genes, Fez and Fez-like (Fezl), in all of
these animal genomes (Hashimoto et al.,
2000) (see Fig. S1 in the supplementary material). Fezl,
which is more similar to zebrafish fezl than to Xenopus
Fez, is expressed in the prospective forebrain region at E8.5 in
mouse; later, it is expressed in the vomeronasal organ, hypothalamus, pallium
and prethalamus (Hirata et al.,
2004). Recent reports show that Fezl is involved in the
development of neural tissues: the zebrafish fezl mutant too
few displays lost or reduced monoaminergic neurons in the hypothalamus
(Levkowitz et al., 2003);
Fezl-deficient mice show abnormalities in the formation of subplate
neurons and thalamocortical axons, and loss of the fornix/fimbria system
(Hirata et al., 2004). It has
also been recently reported that Fezl is required for the development
of subcerebral projection neurons in the neocortex
(Chen et al., 2005a;
Chen et al., 2005b;
Molyneaux et al., 2005).
However, nothing is known about the function of Fez in the
development of the forebrain and olfactory system in any animal species.
In this study, we examined the expression of Fez during mouse
development and generated Fez-deficient mice. Fez was
expressed in the OE and in forebrain regions, where it partially overlapped
with Fezl expression (Hirata et
al., 2004), but it was not expressed in the OB projection neurons,
interneurons or interneuron progenitors. Fez-deficient mice showed
impaired projection of the OSN axons, abnormal OB layer formation and aberrant
rostral migration of the interneuron progenitors. We found that Fez
expressed in OSNs cell-autonomously controlled the axon targeting of the OSNs,
and non-cell-autonomously regulated layer formation in the OB and the
migration of interneurons. Our data provide genetic evidence that signals from
the OSNs contribute to the formation of the OB.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and
deposited in the Mouse Genome Informatics Database under the accession number
AK014242. Mouse Fez cDNA was isolated by polymerase chain reaction
(PCR) from a mouse E12.5 cDNA library and subcloned into pBluescript II SK+
(pBLSK Fez). To make a riboprobe of Fez, the 5' region
of the cDNA was inserted into the EcoRI and BamHI sites of
pBluescript II SK+ (pBLSK FezISH). The full-length cDNA fragment of
zebrafish fez was isolated by 5' and 3' rapid
amplification of cDNA ends (RACE) using the SMART RACE cDNA Amplification Kit
(BD Biosciences, Clontech), based on the sequence information deposited in the
Database (AI722599) (Matsuo-Takasaki et
al., 2000). The sequence of zebrafish fez was deposited
in the DDBJ databank under the accession number AB207804. The mouse gene
(AI325906) originally reported as the mouse ortholog of Xenopus
Fez was discovered to be more similar to zebrafish fezl
(Hashimoto et al., 2000) than
to zebrafish fez (see Fig. S1 in the supplementary material), and is
referred to as mouse Fezl (full-length sequence, AB042399) in this
report and in our previous publications
(Hashimoto et al., 2000;
Hirata et al., 2004). We
previously reported the expression of Fezl in mouse and the
phenotypes of Fezl-deficient mice
(Hirata et al., 2004).
In this study, we investigated the mouse Fez gene (AK014242),
which displays a stronger sequence similarity to zebrafish fez than
to zebrafish fezl. The mouse genomic DNA fragment of Fez was
obtained from the mouse 129sv strain genomic BAC library. The exon-intron
structure was determined from the published mouse genome sequence and by
restriction enzyme site mapping and sequencing. A targeting vector was
constructed by replacing the region encoding amino acids 1-350 with a cassette
composed of a phosphoglycerate kinase I promoter-linked neomycin-resistance
gene (PGK-NEO) and the herpes simplex virus thymidine kinase gene ligated to
the 3' end. Transfected R1 and EB3 embryonic stem cell (ES) colonies
that survived selection with G418 and ganciclovir were subcloned, and the
homologous recombination events determined by PCR and Southern blot
hybridization. Targeted ES cells were injected into C57BL/6 blastulas to
create chimeric male founders. Chimeric offspring were mated to C57BL/6 mice
to generate F1 heterozygous progeny, and the heterozygotes were maintained by
crossing with C57BL/6. Homozygous embryos were obtained by natural mating or
by in vitro fertilization, using sperm and oocytes from the heterozygotes. The
genotypes of embryos were identified by routine PCR. The primers used for the
genotyping were: 5'-CTAATTCAGGGGCAAATGCCAGGCTATA-3' and
5'-CTTGATTCAGGGAATGAGCACCACCTCC-3' (the common primer) for the
wild-type allele, and 5'-CAGCAGCCTCTGTTCCACATACACTTCAT-3' and the
common primer for the targeted allele. Arx-deficient mice have been
described previously (Yoshihara et al.,
2005). Mice were housed in an environmentally controlled room in
the Animal Facility of the Center for Developmental Biology (CDB), RIKEN,
under the guidelines of the CDB for animal experiments.
Nissl and Hematoxylin-Eosin (HE) staining
Brains or embryos were fixed with Carnoy's fixative solution at room
temperature overnight. Specimens were dehydrated and embedded in paraffin wax.
Serial sections (12 µm) were prepared and stained with 0.1% Cresyl Violet.
For HE staining, embryos were fixed overnight with 4% paraformaldehyde,
dehydrated, and embedded in paraffin wax. Serial sections (12 µm) were
prepared and stained with Mayer's Hematoxylin and 1% Eosin (Muto Pure
Chemicals). Pictures of Nissl and HE staining, in situ hybridization and
immunohistochemistry were taken using an AxioPlan2 microscope and AxioCam CCD
camera (Zeiss). The figures were assembled using Adobe PhotoShop version
7.
RNA probes and in situ hybridization
In situ hybridization was performed as described previously
(Hirata et al., 2004).
Single-stranded digoxigenin-UTP-labeled (Roche Applied Science) riboprobes
were used and signals were detected with an anti-digoxigenin antibody and BM
purple substrate (Roche Applied Science) for most of the figures, except
Fig. 8I-L. For
Fig. 8I-L, NBT and BCIP (Roche
Applied Science) were used for staining. The probes used in this study were as
follows: reelin (D'Arcangelo et al.,
1995), Dlx1 (Bulfone
et al., 1993), Gad67
(Condie et al., 1997), tyrosine
hydroxylase (Th, a PCR product), Slit1
(Yuan et al., 1999) and
Arx (Miura et al.,
1997). A Fez riboprobe was generated from
BamHI-digested pBLSK FezISH using T7 RNA polymerase
(Promega).
Immunohistochemistry
Immunohistochemistry was performed as described previously
(Hirata et al., 2004). Immune
complexes were visualized using an ABC kit (Vector) and FAST
3,3'-diaminobenzidine tablet sets (Sigma). For immunofluorescence
detection (Fig. 5M,N,
Fig. 8A-D), Alex488- and
Alex568-conjugated antibodies (Invitrogen/Molecular Probes) were used. The
primary antibodies used in this study were anti-NCAM (Chemicon, AB5032),
anti-GAP43 (Chemicon, AB5220), anti-OMP
(Keller and Margolis, 1975) (a
gift from F. L. Margolis), anti-GFP (MBL), anti-reelin (Chemicon, MAB5364),
and anti-TBX21 and anti-ARX antibodies
(Yoshihara et al., 2005).
BrdU labeling
BrdU labeling was performed as described previously
(Hirata et al., 2004).
Pregnant female mice were injected intraperitoneally with a single pulse (50
mg/kg body weight) of 5-bromo-2'-deoxyuridine (BrdU) and sacrificed on
the indicated day (sacrificed at 2 hours after injection at E14.5 or E16.5 for
Fig. 6; injected at E14.5 and
sacrificed at E18.5 for Fig.
7A,B). For proliferation analysis, 12-µm serial coronal
sections were incubated with monoclonal anti-BrdU antibodies (Pharmingen). The
signals were detected using the ABC kit (Vector) and FAST
3,3'-diaminobenzidine tablet sets. In the experiments shown in
Fig. 6, sections were
counter-stained with Hematoxylin.
Generation and genotyping of transgenic mice
To rescue Fez expression in Fez-mutant mice, a transgene
plasmid was constructed by inserting Fez cDNA into the OSN-specific
transgene vector between the #123 promoter and the
IRES-GapVenus-polyadenylation signal (#123p-Fez-IRES-GapVenus). The
#123 promoter drives transgene expression specifically in OSNs from E11 (Y.Y.,
unpublished). Transgenic mice were generated by injecting the purified
#123p-Fez-IRES-GapVenus DNA cassette into wild-type C57BL/6 oocytes,
which were fertilized with Fez+/– sperm in vitro.
Transgene-derived Fez expression was monitored by the expression of
membrane-tagged Venus (GAP43-Venus), observed under a dissection microscope
(Leica FLIII), and detected by immunohistochemistry with anti-GFP antibodies.
Genotyping of transgenic mouse lines was carried out by PCR using the primers:
5'-ACTTTACACACGAAGGGTCTGG-3' and
5'-AACGTATTTAGCCGAAAGGAAT-3' (200-bp PCR products were generated).
Three independent #123p-Fez-IRES-GapVenus transgenic lines were
established and used in this study.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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)
(M.H., unpublished, for zebrafish fez), than to mouse Fezl
cDNA (Hashimoto et al., 2000)
(see Fig. S1 in the supplementary material). We also found orthologs of the
fez and fezl genes in the pufferfish and human genomes (data
not shown). These data indicate that the teleost and mammalian genomes contain
only two genes belonging to the fez/fezl family, and that the mouse
cDNA AK014242 is the mouse ortholog of fez.
Fez expression was first detected in the prospective forebrain
region at E8.0 (Fig. 1A). The
expression domain of Fez was slightly wider than that of
Fezl at E8.5 (Hirata et al.,
2004). Fez was continuously expressed in the forebrain
region during early embryogenesis (Fig.
1B-D). At E12.5, Fez transcripts were detected in the OE,
septum, roof of the telencephalon, amygdala, prethalamus (also called ventral
thalamus) and hypothalamus (Fig.
1E-I). Fez expression was barely detected in the
vomeronasal organs at E12.5 (Fig.
1I), and was detected weakly in the OE, amygdala and hypothalamus
at E15.5 (Fig. 1J; data not
shown). Fez expression was not detected in the OB or in the
ganglionic eminences (Fig.
1F,K), where the interneuron progenitors of the OB are
generated.
To reveal the role of Fez in mouse development, we disrupted the Fez gene in embryonic stem cells by replacing the region corresponding to amino acids 1-350 of FEZ with a neomycin-resistance gene transcriptional unit (PGK-NEO) (Fig. 2). The heterozygous (Fez+/–) mice appeared normal; the homozygous (Fez–/–) mice were born alive but most of them died within one day for unknown reasons. The OBs of Fez–/– mice were smaller than those of their wild-type littermates at E18.5 and at postnatal day 0 (P0; Fig. 3). Nissl staining showed disorganization of the Nissl-positive layer, which corresponds to the MCL at E18.5, in the Fez–/– mice. However, the Fez–/– mice did not show abnormalities in the morphology of the telencephalon or diencephalon at E14.5 or E18.5 (Fig. 3).
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; Verhaagen et al.,
1989), through the lamina cribrosa, and innervated the OB at E12.5
(Fig. 4A,C,E). In the
Fez–/– mice, the NCAM- and GAP43-positive
olfactory axons passed through the lamina cribrosa, but they stopped before
the OB at E12.5 and never reached it (Fig.
4B,D,F). These data indicate that Fez is required for the
axons of the OSNs to target the OB. Fez–/–
mice showed GAP43, NCAM and OMP expression in OSNs that was comparable to that
of their wild-type littermates (Fig.
5A-H), indicating that the differentiation of the OSNs was not
significantly affected in the Fez–/– mice.
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;
Faedo et al., 2002)
(Fig. 5I,K,M). The
reelin-positive MCL of Fez–/– mice was thicker
than that of wild-type mice and was located in the outermost region of the OB
(Fig. 5J). In wild-type mice,
the expression patterns of the reelin transcripts and proteins indicated the
polarity of the mitral cells, whose dendrites were oriented in the apical
direction (relative to TBX21, which labels the nuclei;
Fig. 5K,M). By contrast, the
polarity of the mitral cells was disorganized in
Fez–/– mice
(Fig. 5L,N). In wild-type mice,
Dlx1 is expressed in interneurons (periglomerular cells and granule
cells) in the GL and GCL, and in interneuron progenitors in the SVZ
(Bulfone et al., 1998)
(Fig. 5O), and Gad67
and Th mark GABAergic and dopaminergic neurons in the OB, and are
expressed in interneurons in the GL and GCL
(Gall et al., 1987;
Kosaka et al., 1995)
(Fig. 5Q,S). In
Fez–/– mice, the expression domains of
Dlx1-, Gad67- and Th-expressing cells were
disorganized, and the GL did not form (Fig.
5P,R,T), indicating that the
Fez–/– mice had defects in the development of
interneurons. The lateral olfactory tracts (LOT) of the OB projection neurons,
a part of which stain with anti-neuropilin1 and lot1 antibodies
(Kawakami et al., 1996;
Sato et al., 1998;
Sugisaki et al., 1996), were
not significantly affected in Fez–/– mice
(data not shown). These data indicate that Fez is required for the
layer formation of the OB and for the development of interneurons.
Fez is required for the rostral stream migration of interneuron progenitors
Because the development of the OB interneurons was impaired in
Fez–/– mice, we next examined the
proliferation and apoptosis of cells in the SVZ of the OB. We labeled
proliferating cells with BrdU (by peritoneal injection into the mother mice)
at E14.5 and E16.5 for 2 hours, and detected them with anti-BrdU antibodies.
At E14.5, the number of BrdU-positive cells in the SVZ, which correspond to
the interneuron progenitor cells, was not significantly different between the
wild-type and Fez–/– mice
(Fig. 6A,B). However, at E16.5,
the number of BrdU-positive cells was greatly reduced in the
Fez–/– mice when compared with the wild-type
mice (55.5±7.6%; Fig.
6C,D). We did not detect a significant difference in the apoptosis
of cells in the OB between wild-type and
Fez–/– mice by TUNEL assay (data not shown).
When we labeled proliferating cells with BrdU at E14.5 and examined the
position of the BrdU-positive cells at 18.5
(Fig. 7A,B), the number of
BrdU-positive interneurons and their progenitors in
Fez–/– mice was comparable to that in their
wild-type littermates. However, the Fez–/–
mice had no BrdU-positive cells in the GL
(Fig. 7B), suggesting that the
migration of OB interneurons to the GL is impaired.
Next, we investigated the RMS, the route through which the OB interneuron
progenitors migrate from the lateral ganglionic eminence to the OB
(Lois and Alvarez-Buylla,
1994; Luskin,
1993), by examining Dlx1 expression. In
Fez–/– mice, the RMS was wider and shorter at
E15.5 and E18.5 than in wild type, and the anterior tip of the RMS was
disorganized at E18.5 (Fig.
7C-F). SLIT1 and SLIT2 are repulsive factors that are reported to
be involved in formation of the RMS; they are expressed in the choroid plexus
and septum (Nguyen-Ba-Charvet et al.,
2004; Wu et al.,
1999), where Fez is expressed. However, the expression of
Slit1 and Slit2 in these regions in
Fez–/– mice appeared to be normal
(Fig. 7G,H, data not shown for
Slit2). The data indicate that Fez is required for proper
rostral migration of the interneuron progenitors, but that SLIT1 and SLIT2 may
not be responsible for the abnormal RMS in the
Fez–/– mice.
The phenotypes of Fez–/– mice are similar
to those observed in Arx-deficient mice
(Yoshihara et al., 2005),
including the abnormal projection of the OSNs and the abnormal layer formation
of the OBs. Although Fez and Arx are expressed in different
regions of the olfactory system, the data suggest that Fez and
Arx function in the same genetic cascade. In an attempt to address
this issue, we examined ARX/Arx expression in
Fez–/– mice and Fez expression in
Arx-deficient mice (Fig.
8). We could not, however, detect any significant difference in
ARX/Arx expression between control and
Fez–/– mice
(Fig. 8A-H), or any difference
in Fez expression between control and Arx-deficient mice
(Fig. 8I-L). The results
indicate that Fez and Arx do not regulate the expression of
one another in the formation of the OEs and OBs.
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), and
confirmed transgene-mediated expression in the OE but not the OB at E14.5
(Fig. 9J). We generated three
different Tg(#123p-Fez) transgenic lines that were all heterozygous
for the Fez-mutant allele
[Fez+/–,Tg(#123p-Fez)1-3], and intercrossed them or
crossed them with Fez+/– mice to generate
Fez–/– mice that expressed Fez only
in the OE (Table 1,
Fig. 9). We then examined the
layer formation of the OB at P0 by immunohistochemistry with anti-OMP (for the
ONL), and by in situ hybridization with reelin (MCL) and Gad67 (GL
and GCL) (Fig. 9A,D,G). As
described above, most of the Fez–/– mice
showed abnormal layer formation of the OB bilaterally (with the exception of a
small percentage that showed unilateral abnormalities;
Table 1,
Fig. 9B,E,H). The
transgene-mediated expression of Fez in the OSNs of
Fez–/– mice partly or completely rescued the
layer formation of the OB at a relatively high penetrance
(Table 1). In some of the
rescued mutant embryos, the axons of the OSNs innervated the OB, and layer
formation was indistinguishable from that seen in wild-type mice
(Fig. 9C,F,I), indicating that
the expression of Fez in the OE was able to suppress the
Fez-mutant phenotypes associated with OSN and OB development. The
data suggest that Fez expression is required in OSNs to
cell-autonomously regulate OSN axon projections and non-cell-autonomously
control the layer formation of the OB.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Several molecules have been shown to be involved in the axon sorting and
targeting of OSNs. They include odorant receptors, semaphorin 3A/neuropilin 1,
ephrin As and Slit/Robo. Odorant receptors, neuropilin 1, ephrin As and Robos
are expressed in OSNs (Cutforth et al.,
2003; Feinstein et al.,
2004; Feinstein and Mombaerts,
2004; Miyasaka et al.,
2005; Mombaerts et al.,
1996; Schwarting et al.,
2000; Taniguchi et al.,
2003; Wang et al.,
1998; Yuan et al.,
1999), and the expression of the genes encoding these molecules
may be controlled by Fez. However, FEZ contains an engrailed homology
1 (EH1) repressor motif (see Fig. S1 in the supplementary material), which
interacts with the Groucho/TLE-family transcriptional co-repressors
(Muhr et al., 2001), and FEZL,
the homolog of FEZ, has been shown to function as a transcriptional repressor
(Levkowitz et al., 2003).
Therefore, regulation of the genes involved in the axonal termination by FEZ
is likely to be indirect and may be mediated by another transcriptional
regulator. Intriguingly, the axon termination phenotype is similar to that
observed in Arx-deficient embryos
(Yoshihara et al., 2005).
Arx is expressed in interneurons and radial glia in the OB, but not
in the OE, and Arx is proposed to regulate the expression of putative
instructive signals for the proper innervation of OSN axons
(Yoshihara et al., 2005). We
have found that the expression of Fez and Arx was mutually
independent (Fig. 8),
indicating that the phenotypes of Fez and Arx-deficient mice
are not due to the reduced expression of Arx and Fez,
respectively. It is tempting to speculate that Arx and Fez
coordinately control the expression of guidance molecules and their receptors.
Future studies isolating the genes that function downstream of FEZ and ARX
will clarify the mechanisms underlying the bidirectional interaction between
the developing OE and OB.
In Fez–/– mice, axonal growth of OSNs was
perturbed after the axons passed through the lamina cribrosa, which is located
on the boundary between the OE and OB. The responsiveness of axonal growth
cones is known to be controlled spatially and temporary by intrinsic factors
(Tessier-Lavigne and Goodman,
1996). In zebrafish, inhibition of PKA in the OSNs affects axonal
growth in the olfactory placodes, whereas the activation of PKA affects it
only in the OB, suggesting that PKA activity in OSNs is differently controlled
in the OE and OB, and that the regulation of PKA may confer changes in the
responsiveness of OSNs to guidance molecules
(Yoshida et al., 2002). Such a
change in the responsiveness to guidance molecules may play a role in the
long-distance axon projection through the intermediate checkpoints. It is an
intriguing possibility that Fez cell-autonomously controls the
expression of molecules that are involved in the responsiveness to guidance
molecules.
Non-cell-autonomous roles for Fez in OB layer formation and interneuron development
In addition to the defects in the axonal contribution to the OB layers
(i.e. lack of the ONL), we also found abnormal layer formation of the OB, and
abnormal development and migration of the interneurons and their progenitors
in Fez–/– mice. There are several reports
showing a role for the olfactory placode and/or OE in the formation of the OB.
Removal of the olfactory placode in Xenopus influences the
development of the OB, for example, by reducing the number of OB projection
neurons (Byrd and Burd, 1993;
Graziadei and Monti-Graziadei,
1992). In rats, the arrival of pioneer olfactory axons to the OB
is linked to cell-cycle control of the OB primordium
(Gong and Shipley, 1995).
Here, we have demonstrated that Fez expressed in the OSNs
non-cell-autonomously controls OB layer formation and the development of
interneurons.
Among cells in the olfactory system, at E12.5 we detected Fez
expression in only the OSNs, and not in the other cell types that contribute
to the formation of the OB. Fez was not expressed in the ganglionic
eminence, where the interneuron progenitors are generated, supporting a
non-cell-autonomous role for Fez in the formation of the OB layers,
and in the migration and development of the interneurons and their
progenitors. However, we cannot completely exclude the possibility that
Fez is transiently expressed in the precursors of the OB projection
neurons and interneuron progenitors, as Fez is globally expressed in
the forebrain at E8.5-E9.5. To address this issue, Cre-loxP-mediated cell-fate
mapping of the Fez-expressing cells
(Zinyk et al., 1998) will be
required. However, when evagination of the OB from the telencephalon and
migration of the interneuron progenitors take place, Fez is not
expressed in the projection neurons, the interneuronsor interneuron
progenitors. More importantly, transgenic Fez expression in the OSNs
of Fez–/– mice rescued the formation of all of
the OB layers. Taken together, these data indicate that Fez expressed
in the OSNs non-cell-autonomously controls the formation of the OB. Our data
provide genetic evidence that signals from the OSNs are required for the
proper formation of the OB.
In addition to the OSNs, Fez was expressed in the ventrolateral
pallium, septum, roof of the telencephalon, hypothalamus and prethalamus. We
did not find significant abnormalities in the development of these regions in
the forebrain, but it is possible that their functions contribute to OB layer
formation and the migration/development of interneurons. Slit1 and
Slit2 are expressed in the septum and are reported to be involved in
migration of interneuron progenitors through the RMS
(Nguyen-Ba-Charvet et al.,
2004). However, the expression of Slit1 and
Slit2 was not affected in Fez–/– mice
(Fig. 7, data not shown),
suggesting that SLIT1 and SLIT2 are not responsible for the abnormal RMS
formation in these mice. We do not rule out the possibility that Fez
expressed in regions other than the OE functions redundantly with
Fezl in the formation of the OB. Fezl is expressed in
overlapping regions of the telencephalon and hypothalamus
(Hirata et al., 2004), and
mice deficient in both the Fez and Fezl genes showed a much
greater size reduction of the OB (T.H., M.N., O. Muraoka, R. Nakayama, Y. Suda
and M.H., unpublished) than do Fez-deficient mice
[Fezl-deficient mice do not show abnormalities in OB development
(Hirata et al., 2004)]. These
data suggest that Fez has functions other than in the OE for the
development of the OB. A study involving region-specific disruption of the
Fez and Fezl genes in the forebrain will clarify this
issue.
Functional interactions between OSNs and the OB
How do signals from OSNs control OB layer formation and interneuron
development? The abnormal OB layer formation and RMS phenotypes in
Fez–/– mice were similar to those observed in
Dlx5- and Arx-deficient mice; these mutants also show
absence of the ONL, a disorganized MCL, a reduction in the number of
interneurons, and aberrant RMS (Levi et
al., 2003; Long et al.,
2003; Yoshihara et al.,
2005). Both Dlx5 and Arx are expressed in
interneurons and their progenitors, and they cell-autonomously control the RMS
and interneuron development, and non-cell-autonomously control MCL formation
(Levi et al., 2003;
Long et al., 2003;
Yoshihara et al., 2005).
Therefore, the abnormal development of the MCL can be explained, at least in
part, by the abnormal migration of the interneuron progenitors from the
lateral ganglionic eminence. Because axons of the OSNs do not directly contact
the interneuron progenitors at the beginning of OB development, the axon
terminals of the OSNs could provide diffusible guidance molecules that control
the RMS and the migration of interneurons from the SVZ to the GL in the OB.
Alternatively, the absence of olfactory sensory input into the OB could
disrupt the formation of glomeruli and lead to the disorganized layer
formation of the OB projection neurons (MCL), which may secondarily affect the
development of the interneurons. Tbr1-mutant mice, which lack
projection neurons, show aberrant layer formation of the GL and GCL, and
reduced size of the OB (Bulfone et al.,
1998), implying a role for projection neurons in the development
of interneurons. The abnormal layer formation of the projection neurons may
contribute to the impaired development of the interneurons. It is less likely,
but also possible, that the tangled structure of the olfactory sensory axons
in Fez–/– mice places a physical pressure on
the OB primordium, which interferes with the formation of the RMS and the OB
layer. We favor a hypothesis combining the first two possibilities: that
signals from OSNs independently control the development of the OB projection
neurons and interneurons/interneuron progenitors, and that these two cell
populations functionally interact with each other later to form the correct
layers in the OB.
In summary, we found that Fez is an essential factor that cell-autonomously controls the axonal projection of the OSNs, and non-cell-autonomously regulates layer formation of the OB and the development of the interneurons. Our data provide evidence that signals from the OSNs control OB development.
|
ACKNOWLEDGMENTS |
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|
Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/133/8/1433/DC1
* Present address: Department of Neuroscience, Georgetown University School
of Medicine, Washington, DC 20057, USA 
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