1 Department of Anatomy and Neurobiology, and Developmental Biology Center,
University of California, Irvine, CA 92697-1275, USA
2 Department of Neuroscience and Molecular Genetics, Albert Einstein College of
Medicine of Yeshiva University, Bronx, NY 10461, USA
3 Department of Biological Sciences, Stanford University, Stanford, CA 94305,
USA
4 MRC Centre for Developmental Neurobiology, New Hunt's House, King's College
London, Guy's Campus, London SE1 1UL, UK
Author for correspondence (e-mail:
alcalof{at}uci.edu)
Accepted 23 September 2005
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SUMMARY |
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Key words: FGF, Vomeronasal organ, Neurogenesis, Olfactory epithelium, Nasal cavity, Stem cell, Apoptosis, Cre recombinase, Fgf8, Foxg1, Sox2, Pax6, Mash1, Neurogenin 1, Ncam1, Pyst1, Shh, Dlx5, Neuronal progenitor, Mouse mutant
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Introduction |
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Mouse olfactory epithelium (OE) provides a useful model system with which
to understand how neurogenesis is regulated at the cellular and molecular
levels. Studies in vitro and in vivo have demonstrated four distinct
developmental stages in the neuronal lineage of established OE: neural stem
cells, which express the transcription factor Sox2; Mash1
(Ascl1 - Mouse Genome Informatics)-expressing committed neuronal
progenitors, the progeny of the stem cells; Ngn1-expressing immediate
neuronal precursors (INPs), the progeny of Mash1+ progenitors; and
olfactory receptor neurons (ORNs), which differentiate from daughter cells of
INP divisions and can be identified by expression of the neural cell adhesion
molecule, NCAM1 (Beites et al.,
2005; Calof et al.,
2002
; Kawauchi et al.,
2004
). Interestingly, the OE is one of the few regions of the
nervous system in which neurogenesis and nerve cell renewal take place
throughout life (Murray and Calof,
1999
). This capacity for continual neurogenesis suggests that
cells within the OE produce signals that stimulate this process.
In a previous study, we have shown that several FGFs, particularly FGF2,
are potent stimulators of neurogenesis in cultured OE, where they promote
divisions of OE neuronal transit amplifying progenitors and maintain the stem
cells that give rise to these progenitors
(DeHamer et al., 1994).
Moreover, a number of FGFs, including FGF2, are expressed in and around OE at
various stages of development (DeHamer et
al., 1994
; Hsu et al.,
2001
; Kawauchi et al.,
2004
; Key et al.,
1996
). However, two observations argue that FGF2 is unlikely to be
a crucial regulator of developmental neurogenesis in the OE. First, mice with
targeted inactivation of the Fgf2 gene show few if any defects in
developmental neurogenesis (Dono et al.,
1998
; Ortega et al.,
1998
). Second, Fgf2 is not highly expressed in mouse OE
until postnatal ages (Hsu et al.,
2001
; Kawauchi et al.,
2004
) (S.K. and A.L.C., unpublished). Because Fgf8 has
been reported to be expressed in the frontonasal region near the olfactory
placodes (Bachler and Neubuser,
2001
; Crossley and Martin,
1995
; Mahmood et al.,
1995
), we hypothesized that it may serve as a signal promoting
neurogenesis during early OE development.
To test this hypothesis, we analyzed expression of Fgf8 and its
actions on OE neurogenesis in vivo, using a conditional genetic approach in
which the Fgf8 gene was inactivated in mouse OE from the earliest
stages of OE development. Tissue culture assays were also used to investigate
effects of recombinant FGF8 on OE neurogenesis. Expression analysis indicated
that Fgf8 is initially transcribed in a small domain - which we have
termed the morphogenetic center - at the rim of the invaginating neural pit.
These Fgf8-expressing cells give rise to new cells that both contain
Fgf8 mRNA and express markers of OE neural stem cells. Analysis of
FGF8 signaling and cell death demonstrate that, as a consequence of
Fgf8 inactivation, cells within the morphogenetic center and in
adjacent developing neuroepithelium undergo apoptosis. As a consequence,
although initial invagination of the nasal pit (NP) and initiation of the OE
neuronal lineage take place, both NP morphogenesis and OE neurogenesis halt
shortly thereafter. This in turn results in a failure of development of
definitive OE, vomeronasal organ (VNO) - the pheromone-sensing component of
the primary olfactory system (also derived from the olfactory placode)
(Farbman, 1992) - and the
nasal cavity as a whole. Thus, Fgf8 is required for the survival of
cells in a crucial anterior morphogenetic center, which is responsible not
only for nasal cavity and OE development, but also for the generation and
survival of the stem cells that ultimately generate all cell types in the OE
neuronal lineage and endow this neuroepithelium with its capacity for neuronal
regeneration.
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Materials and methods |
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Detection of gene expression
Whole-mount X-gal staining of mouse embryos was performed as described
(Murray et al., 2003). For
RT-PCR, E10.5 frontonasal tissue (including forebrain and olfactory pit) RNA
was purified using Trizol (Invitrogen). PCR primers were set to detect cDNA
coding for Fgf8 exon 2 and exon 3 (forward,
5'-GTGGAGACCGATACTTTTGG-3'; reverse,
5'-GCCCAAGTCCTCTGGCTGCC-3'). Cycling parameters were denaturation
at 96°C for 20 seconds, annealing at 55°C for 30 seconds and
elongation at 72°C for 1 minute, for 35 cycles.
Section in situ hybridization for E8.5-E17.5 embryos was performed as
described (Murray et al.,
2003). For two-color in situ, one probe was synthesized using
fluorescein-12-UTP and detected using AP-conjugated anti-fluorescein Fab
fragments from sheep with INT/BCIP as the chromagen/substrate mix (Roche). For
whole-mount in situ hybridization, E9.5-10.5 embryos were fixed overnight in
4% paraformaldehyde in PBS with 2.5 mM EGTA at 4°C and processed as
described (Kawauchi et al.,
1999
). Probes used in this study were: ORF of mouse Fgf8
(GenBank Accession Number MMU18673); 463 bp of mouse Fgf18 (520-983
bp of GenBank #AF075291); 748 bp of mouse Sox2 (1281bp-2029 bp of
GenBank Accession Number X94127); and mouse Pyst1 (Dusp6 -
Mouse Genome Informatics) (Dickinson et
al., 2002
). Unless otherwise indicated, Fgf8 expression
was detected using a probe generated from the full-length Fgf8 ORF
cDNA (GenBank Accession Number MMU18673)
(Mahmood et al., 1995
).
Fgf8 ex1, ex2,3 and int probes were generated in our
laboratory by PCR amplification of genomic DNA or cDNA. The int probe
consists of bp 2464-3142 of the 3274 bp intron sequence between exons 3 and 4
(Ensemble #ENSMUST00000026241). Mash1, NgnI, Gdf11 and Ncam1
probes were described previously (Murray
et al., 2003
; Wu et al.,
2003
).
Immunostaining and TUNEL assays
Cells in M-phase were detected by immunostaining using polyclonal rabbit
anti-phospho-histone H3 (Upstate Biotechnology, Cat. No. 06-570) at 1:200
dilution, visualized with Alexa Red-conjugated goat anti-rabbit-IgG (1:1000
dilution; Invitrogen). Cells in S-phase were detected as follows: 1 hour
before sacrifice, timed-pregnant dams were given a single intraperitoneal
injection of 5-bromo-2'-deoxyuridine (BrdU; 50 µg/gm body weight).
Tissue was fixed and sectioned as for in situ hybridization, and
immunostaining for BrdU was performed as described
(Murray et al., 2003).
TdT-mediated dUTP nick end-labeling (TUNEL) staining to detect DNA
fragmentation in apoptotic cells was performed as described
(Murray et al., 2003
), except
that 20 µm cryosections of paraformaldehyde-fixed tissue were used and
incubated with four changes of 10 mM citric acid (70°C; 15 minutes per
wash) following permeabilization and prior to the TdT reaction. Texas
Red-conjugated NeutrAvidin (1:200 dilution; Invitrogen) was used to detect
incorporated biotin-16-dUTP (Roche).
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Results |
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Because the data in Fig. 1A
suggest an overlap in the expression domains of Fgf8 and
Sox2, which marks many early neuroepithelial stem cells as well as OE
neural stem cells once the definitive OE structure has been established
(Beites et al., 2005;
Ellis et al., 2004
;
Graham et al., 2003
;
Kawauchi et al., 2004
;
Wood and Episkopou, 1999
), we
performed two sets of experiments to try to determine if a subpopulation of
the Fgf8-expressing cells in the NP are early OE neural stem cells.
In the first experiment, we used double-label in situ hybridization for
Fgf8 (ORF probe) and Sox2 on the same sections. The results,
shown in Fig. 1B, indicate that
many Fgf8-expressing cells that lie within the neuroepithelium of the
invaginating NP also express Sox2. These cells also express
Pax6 and Dlx5, other markers of definitive OE at this stage
(see Fig. S1 in the supplementary material). Thus, by the criterion of
Sox2 expression, a subpopulation of Fgf8-expressing cells
can be considered to be early OE neural stem cells. The presence of cells that
co-express both Fgf8 and Sox2 suggested to us that
Fgf8 expression defines a region from which neural stem cells emerge
and enter the invaginating olfactory neuroepithelium. Moreover, the sequential
appearance of cells expressing markers of successively more differentiated
stages in the OE neuronal lineage, in NP regions that are further and further
from the Fgf8-expressing domain, is consistent with the idea that OE
morphogenesis and initiation of the OE neuronal lineage proceed in an
outside-in fashion, with early stem cells at the periphery and terminally
differentiated ORNs in the center of the NP (cf.
Cau et al., 1997
).
In a second set of experiments, we investigated the origin of
Fgf8-expressing cells in the OE neuroepithelium, using a technique
similar to that of Dubrulle and colleagues
(Dubrulle and Pourquie, 2004).
In situ hybridization was performed on adjacent sections of invaginating NP,
in one case using a probe to intron sequences to determine the location of
cells that initially transcribe unprocessed Fgf8 RNA (`Fgf8
int' probe), and in the second case a probe for exons 2 and 3 of the
processed mRNA (`Fgf8 ex2,3' probe). The results, shown in
Fig. 1C, demonstrate that the
cells which initially transcribe Fgf8 form a subset of all cells that
actually contain Fgf8 RNA in this region. These
Fgf8-transcribing cells appear to be located preferentially at the
rim of the NP and in the basal region of the invaginating neuroepithelium. By
contrast, cells that contain processed mRNA for Fgf8 (positive for
the Fgf8 ex2,3 probe) are found more extensively both within
invaginating neuroepithelium and in the ectoderm surrounding the NP. As cells
that are Fgf8 ex2,3 positive must be the progeny of the Fgf8
int-positive cells (Dubrulle and
Pourquie, 2004
), these findings suggest a lineal relationship
between the Fgf8-expressing ectodermal cells that define the rim of
the invaginating NP and the neuroepithelial cells that express both
Fgf8 mRNA and Sox2. Thus, at least some of the
Sox2-expressing neural stem cells in the developing OE must be
derived from Fgf8-expressing ectodermal cells. Altogether, these
observations suggest that Fgf8 expression defines a morphogenetic
center that gives rise to at least some of the Sox2-expressing neural
stem cells of the OE, and that the earliest of these neural stem cells
themselves transcribe Fgf8. These ideas are depicted in the cartoon
shown in Fig. 1D.
At later stages (beyond E12-13), OE stem and progenitor cells take on a
more restricted location, and come to lie in a compartment adjacent to the
basal lamina of the epithelium (Kawauchi
et al., 2004). Analyses by in situ hybridization and RT-PCR
indicate that Fgf8 continues to be expressed in scattered cells
located in the basal compartment of OE at E14.5 (see Fig. S2 in the
supplementary material). These data suggest that Fgf8 continues to be
expressed in or near stem and progenitor cells of the OE. Moreover, in vitro
assays show that recombinant FGF8 is capable of stimulating development of
neural stem cells and proliferation of neuronal progenitors in cultures taken
from E14.5 OE (see Fig. S3 in the supplementary material), demonstrating that
OE progenitors are responsive to FGF8 at this stage of development.
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The strategy for generating conditional mutants is outlined in
Fig. 2B.
Fgf8flox/flox;Foxg1+/+ females were crossed
with Fgf8d2,3/+;Foxg1Cre/+ males, and 25%
of embryos generated were
Fgf8flox/d2,3;Foxg1Cre/+ animals (hereafter
referred to as `mutant' animals). Mutant animals were detected at all
embryonic stages and in newborn litters, but died shortly after birth owing to
multiple defects (see below).
Mutant embryos have severe defects in frontonasal structures
Intact mutant embryos were examined from E9.5 to birth
(Fig. 3A). Defects are evident
as early as E9.5, primarily as a reduction in the size of the forebrain and
frontonasal structures. Strikingly, all mutant animals have at least a
rudimentary olfactory pit at E10.5; however, by this time, defects in medial
nasal process development are often apparent, with this region being flattened
and smaller than in controls. From E12.5 onwards, the forebrains of mutant
embryos show dramatic reductions in size compared with control littermates;
limbs and other posterior structures appear grossly normal, however. At birth,
mutants have a small short snout, and the lower jaw, pinnae (outer ears) and
eyelids are either reduced in size or absent. The size and gross structure of
the eye itself appear to be unaffected in mutants (data not shown). Littermate
embryos with genotypes other than
Fgf8flox/d2,3;Foxg1Cre/+ showed normal
development and survived to adulthood.
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FGF signaling is downregulated in Fgf8 mutant embryos
Experiments described above demonstrate that Fgf8 gene expression
is effectively eliminated in the OE of mutant animals. However, it might still
be the case that FGF8-mediated signaling could somehow be compensated for in
mutants, thus complicating analysis of phenotypes. To test this hypothesis, we
examined expression of Pyst1/Mkp3 in the frontonasal region of E10.5
mutant and control littermate embryos. Pyst1 encodes a tyrosine
phosphatase that is an FGF-inducible antagonist of FGF signaling
(Eblaghie et al., 2003), and
is one of a number of genes in the FGF `synexpression' group, i.e. genes
expressed in the same temporal and spatial pattern when FGFs initiate
signaling (Niehrs and Meinhardt,
2002
). Pyst1 is expressed in many known sites of FGF
signaling in mouse embryos, including the olfactory system
(Dickinson et al., 2002
),
making its expression useful as a read-out for active FGF signaling. As shown
in Fig. 3D, Pyst1 is
normally expressed in the rim of the invaginating olfactory pit (where
Fgf8 itself is expressed) and in the mesenchyme throughout the medial
nasal process. In mutant embryos, expression of Pyst1 is strongly
downregulated in both domains (insets), confirming that loss of Fgf8
leads to severe decrements in FGF signaling in the developing olfactory
region.
We also examined expression of Shh, which, like Fgf8, is
a key signaling molecule in limb and telencephalon
(Ohkubo et al., 2002;
Panman and Zeller, 2003
), and
which evidence suggests may be positively regulated by FGFs
(Niswander et al., 1994
;
Zuniga et al., 1999
). As shown
in Fig. 3D, Shh is not
expressed in OE at E10.5, but instead is expressed in the ventral wall of the
telencephalon next to the medial nasal process and forebrain commissural
plate. In mutants, the basic pattern of Shh expression appears
unaffected. Expression of the SHH receptor patched, which is autoregulated
through SHH signaling in overlapping and complementary patterns, also appeared
unaffected in mutants (data not shown)
(Drossopoulou et al., 2000
;
Marigo and Tabin, 1996
;
Platt et al., 1997
). These
observations indicate that effects on neurogenesis observed in mutants are
unlikely to be mediated indirectly via Shh signaling
(LaMantia et al., 2000
).
Severe reduction or absence of olfactory structures in mutant embryos
To evaluate OE formation and growth in the absence of Fgf8, we
analyzed sections of mutant and control animals from E10.5 to E17.5 (a minimum
of four mutant animals were examined at each age, with a total of 31
analyzed). Normal OE development is shown in
Fig. 4A. At E10.5, some
olfactory placode/nasal pit structure was observed in all animals, even
mutants; however, the OE and nasal cavity were severely reduced in size or
absent in all mutant embryos by E11.5 (Fig.
4B). On the basis of histology, mutants were placed into one of
two categories: Type A (aplastic) mutants had essentially no nasal cavity or
OE detectable from E11.5 onward; Type B (hypoplastic) mutants retained a
vestige of nasal cavity, usually present as an S-shaped tubular structure
lined with an epithelium, at E11.5 and after. The numbers of each type of
mutant found at each age are given in Table
1. Interestingly, at E10.5 some mutant embryos already displayed a
phenotype, in that they had extremely small placodes and obvious reductions in
the sizes of the lateral and medial nasal processes
(Fig. 4B). We surmise that such
embryos would exhibit the more severe, aplastic OE phenotype at later stages.
Nasal bone structures are present in hypoplastic (Type B) mutants, and closely
surround any remnant of OE, suggesting that bone formation in this region is
at least partially dependent on OE development, possibly through an inductive
signal derived from OE tissue. Of significance also is the fact that no
vomeronasal organ (VNO) structure was observed in any mutant animals, whether
these exhibited aplastic or hypoplastic phenotypes. As the VNO is derived from
the olfactory placode, and starts to develop during the period when olfactory
pits are invaginating (around E11.5)
(Farbman, 1992), this
observation indicates an absolute requirement for Fgf8 in VNO
formation.
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Fig. 5C,D show Type B
(hypoplastic) mutants, which retain small vestiges of OE. Even in mutants with
this less severe phenotype, almost no OE neuronal cells remain, and the OE
itself is very small compared with that of controls. Interestingly, we
detected expression of Fgf18, which is closely related to
Fgf8 in structure and function
(Maruoka et al., 1998;
Ornitz and Itoh, 2001
), in the
OE remnant present in an E14.5 Type B mutant
(Fig. 5C, arrows). This
observation suggests that, in Type B mutants, there may be some compensation
for loss of Fgf8 function by Fgf18, provided that the OE is
able to develop to the stage when Fgf18 normally starts to be
expressed in this region (about E12.5) (S. Kawauchi, data not shown) (see
Bachler and Neubuser, 2001
;
Xu et al., 2000
).
Fig. 5D illustrates that, in
older Type B mutants (E17.5), even when a relatively large remnant of nasal
cavity persists (arrowheads), the epithelium that lines it is essentially
devoid of cells expressing genes specific to OE neuronal progenitors and ORNs
(Mash1, Gdf11, Ncam1) (see Wu et
al., 2003
). These observations indicate that, whatever process
goes awry in Fgf8 mutant OE, deficits appear very early in
development, and the effects of these deficits are long lasting, such that few
if any neuronal cells are able to form.
Loss of Fgf8 results in increased cell death, not decreased cell proliferation
By what mechanism does loss of Fgf8 cause loss of neuronal stem
and progenitor cells in the OE? Potentially, FGF8 could stimulate neurogenesis
by promoting proliferation and/or survival of neuronal stem and progenitor
cells. Indeed, as described above, recombinant FGF8 - like other FGFs - is
able to promote development of OE stem cells and proliferation of INPs in
tissue culture assays (see Fig. S3 in the supplementary material)
(DeHamer et al., 1994;
Shou et al., 2000
). To
determine the mechanism(s) by which Fgf8 acts in vivo, we performed
in situ assays for both apoptotic cells and proliferating cells in mutant and
control animals at early stages of NP invagination and OE development.
To identify apoptotic cells, we performed TUNEL assays on cryosections of OE of mutants and control littermates at E10.5, E12.5 and E14.5. The results are shown in Fig. 6A-C. Large numbers of apoptotic cells were observed throughout the Fgf8-expressing domain in mutants (detected in adjacent sections using a probe to Fgf8 exon 1, which is expressed, but does not generate functional protein, in Fgf8 mutants). By contrast, very few apoptotic cells were observed in the Fgf8-expressing domain (detected using the Fgf8 exon2,3 probe) in control littermates. The increase in the number of TUNEL-positive cells was greatest at E10.5, when mutant animals showed a 37-fold increase compared with controls [mutant: 1220±485 (s.e.m.); control: 37±18 (s.e.m.)]. At E12.5, the difference between mutant and control mice was smaller but still significant (Fig. 6B-C), whereas by E14.5 cell death was low, and had decreased to approximately the same level in mutants as in controls (Fig. 6C). Interestingly, Sox2 expression also appears to be attenuated in the OE regions, showing high levels of apoptosis in mutants, particularly the OE lining the NP in the medial nasal process (Fig. 6A, Sox2 panel, arrowhead), where FGF8 signaling is strongly reduced in mutants (Fig. 3D).
To determine if absence of functional Fgf8 results in a change in
cell proliferation, we performed immunostaining for the M-phase specific
marker, phosphorylated histone H3 (Galli
et al., 2004), and for BrdU incorporation (which detects cells in
S phase), at E10.5. M-phase cells were observed primarily in the surface
(apical) layer of OE in both control and mutant animals, but there was no
obvious difference in either the pattern or number of immunopositive cells
(Fig. 6D). To confirm this,
total numbers of phosphohistone H3-immunopositive cells were counted in serial
sections through the entire extent of the nasal pits in mutant and control
animals. The results, shown in Fig.
6D,E confirm that there is no significant change in the mitotic
index of OE cells in the absence of Fgf8. Levels of BrdU
immunostaining were high in OE in both mutants and controls, and BrdU+ cells
were particularly dense in the basal half of the epithelium in both cases
(Fig. 6F). However,
quantification of these sections again demonstrated that there was no
significant decrease in the number of BrdU-immunopositive cells (i.e. cells in
S phase) in the OE of mutant animals, compared with control littermates
(Fig. 6G). Thus, the changes in
OE neurogenesis seen at E10.5 appear to be the result of increased cell death,
not decreased cell proliferation.
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Discussion |
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|
Fgf8 acts by controlling cell survival
A dramatic phenotype observed in Fgf8 mutants was the very high
level of programmed cell death at E10.5
(Fig. 6). In mutants at this
time, cells in the Fgf8-expressing domain, as well as surrounding
neuroepithelium, are unable to survive and the numbers of all neuronal cell
types are subsequently depleted. The few cells already committed to the
neuronal lineage appear to be able to continue through the maturation process,
as some neuronal cells can be observed in Type B mutants as late as E14.5
(Fig. 5). However, as neuronal
stem cells ultimately die in the absence of Fgf8, the mature
characteristics of the OE and neurogenesis within this epithelium are never
established, even in the least severe mutants
(Fig. 5). These findings are
consistent with observations made in studies of various Fgf8 mutants
in other tissues. For example, when Fgf8 expression is eliminated
from the apical ectodermal ridge (AER) of developing limbs, AER cells and the
mesenchyme that underlies them undergo apoptosis
(Sun et al., 2002). In
addition, Storm and colleagues have reported that telencephalic neural
progenitors undergo apoptosis when Fgf8 is eliminated, the likely
cause of the defects in forebrain development observed in our study
(Storm et al., 2003
) (compare
with Fig. 3). Our results, in
addition to these and results from a number of labs studying Fgf8
function, suggest that FGF8 acts as a survival factor for crucial stem cell
populations in a wide variety of tissues in which it is expressed
(Abu-Issa et al., 2002
;
Chi et al., 2003
;
Frank et al., 2002
;
Storm et al., 2003
;
Sun et al., 2002
;
Trumpp et al., 1999
).
Although the molecular details of how Fgf8 prevents cell death are
incompletely understood, our analysis of Pyst1, which encodes a
MAPK-specific phosphatase whose expression is dependent on MAPK signaling
(Eblaghie et al., 2003),
indicate that the RAS/MAPK pathway activity may be involved. This pathway is
known to regulate apoptosis in other systems
(Downward, 1998
). One
possibility, suggested by our data, is that absence of Fgf8 at this
early crucial juncture leads to downregulation of the RAS/MAPK pathway, which
ultimately acts as a trigger for death of the primordial neural stem cells
that are ultimately responsible for establishing the neurogenic pathway in the
OE. Consistent with this notion, in vitro studies have shown a relationship
between maintenance of FGF signaling and prevention of apoptosis in P19 cells
(Miho et al., 1999
).
Are effects of loss of Fgf8 direct or indirect?
Overall, our observations suggest a model in which FGF8 acts in an
autocrine and/or paracrine manner in cells of the developing OE and
surrounding anterior ectoderm. However, the dramatic effects of loss of
Fgf8 function on both craniofacial morphogenesis and forebrain
development, observed by us in the present study and by others in this and
other Fgf8 mutants (Abu-Issa et
al., 2002; Storm et al.,
2003
; Trumpp et al.,
1999
), raise the alternative possibility that effects on OE
neurogenesis observed in mutants could be caused indirectly, owing to effects
on these other tissues. As we observe a reduction is FGF-mediated signaling in
the mesenchyme surrounding the invaginating olfactory neuroepithelium, as well
as in the Fgf8-expressing neuroepithelium itself
(Fig. 3C), the possibility that
loss of Fgf8 disrupts an epithelial-mesenchymal signaling loop that
may feed back to promote OE neurogenesis cannot be discounted totally by our
data. Because by far the most cell death we observe in mutants is in
Fgf8-expressing ectoderm and neuroepithelium, and not in underlying
mesenchyme (Fig. 6B and data
not shown), we do not think that alterations in FGF8-mediated signaling in
mesenchyme are responsible for the defects - especially cell death - that we
observe in OE. It may still be the case that loss of FGF8 signaling in
mesenchyme contributes to the defects in nasal cavity formation we observe,
however, possibly by interrupting BMP-mediated signaling in this tissue; we
are currently exploring this possibility (S.K., C. E. Crocker and A.L.C.,
unpublished).
Because Fgf8 mutants also exhibit strong defects in the developing
telencephalon, it might also be argued that proper OE development is dependent
on proper formation of the olfactory bulbs (OBs), and thus that effects on OE
neurogenesis in mutants might be indirectly mediated via effects on the OB. To
test this idea, we examined OE development and OB structure using in situ
hybridization in Fgf8 hypomorphs (Fgf8neo/neo),
which have been reported to lack most or all OB tissue
(Garel et al., 2003;
Meyers et al., 1998
). Our
observations indicate that the normal complement of Ncam1-expressing
ORNs is present in the OE of Fgf8 hypomorphs at P0, even though these
animals fail to develop any proper OB and have profound reductions in the
number of OB neurons and neuronal progenitors (see Fig. S4 in the
supplementary material). Similar results have been obtained from studies of
other mutant mouse strains in which OBs fail to form during development
because of defects in genes other than Fgf8 [e.g.
extra-toesJ (XtJ) mutant mice
(Sullivan et al., 1995
)].
These observations indicate that Fgf8 regulates neurogenesis in the
OE independently from its regulation of neurogenesis in the OB. Taken
together, the data support the conclusion that defects in OE neurogenesis
observed in Fgf8 mutants in the present study are due to direct
effects of loss of endogenous Fgf8 on developing OE, and not to
indirect effects resulting from failure of OB formation.
What cell type is dying in Fgf8 mutants?
Our data from adjacent serial sections indicate that it is
Fgf8-expressing cells themselves that undergo apoptosis in the
absence of FGF8 signaling (Fig.
6). The results of in situ hybridization analysis using probes to
intronic versus exonic sequences indicate that ectodermal cells that initially
transcribe Fgf8 RNA give rise to a larger population of
neuroepithelial cells, some of which continue to express processed
Fgf8 mRNA (Fig. 1C).
Taken together with double-label in situ hybridization results showing overlap
between the domains of
Fgf8 and Sox2 expression
(Fig. 1B), these findings
strongly suggest that at least some Fgf8-expressing cells become what
we have termed primordial neural stem cells of the OE (Fgf8+/Sox2+
cells) [compare Fig. 7 with
Kawauchi et al. (Kawauchi et al.,
2004)]. Therefore, as apoptosis in mutant OE is most extensive in
the Fgf8+ domain (Fig.
6B), we infer that at least some of the cells that are dying in
mutants are primordial neural stem cells, an idea supported by the finding
that Sox2 expression is attenuated in mutant OE in the region where
Fgf8 expression and Sox2 expression normally overlap
(Fig. 6A). However, as the
regions of both Fgf8 expression and apoptosis in the mutant extend
beyond the neuroepithelial domain defined by Sox2 expression, it is
also likely to be the case that some of the dying cells in mutants are not
committed neural stem cells. Thus, we conclude that apoptotic cells include
both Fgf8-expressing cells committed to the neural lineage of the OE
(i.e. primordial neural stem cells), as well Fgf8-expressing cells
that may not be destined to undergo this commitment step. Ultimately, the
decreased numbers of all neuronal cell types, the failure of the OE and the
VNO to develop, and abortive nasal cavity morphogenesis in mutant animals,
together demonstrate the profound dependence of both OE neurogenesis and
anterior craniofacial development on developmental expression of
Fgf8.
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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/132/23/5211/DC1
* Present address: Lilly Research Labs-Functional Genomics, Eli Lilly and
Company, Indianapolis, IN 46285, USA
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