1 Department of Biological Sciences, Stanford University, Stanford, CA 94305,
USA
2 Institute of Biotechnology, University of Helsinki, FIN-00014, Finland
3 S. Lunenfeld Research Institute, University of Toronto, Toronto M5G 1X5, ON,
Canada
* Present address: Department of Neuroscience, Albert Einstein College of
Medicine, Bronx, NY 10461, USA
Author for correspondence (e-mail:
suemcc{at}leland.stanford.edu)
Accepted 12 December 2002
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SUMMARY |
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Key words: Telencephalon, Cre/loxP, Cell fate, Neurogenesis, Forebrain, Patterning
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INTRODUCTION |
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Several experiments suggest that Fgf8 may play a key role in
patterning the telencephalon along the AP axis. Fgf8 is expressed
just anterior to the neural plate in the anterior neural ridge as early as
E8.5, and in the anterior forebrain from E9 to at least E12.5
(Crossley and Martin, 1995).
Mice that are completely deficient for Fgf8 die around the time of
gastrulation (Meyers et al.,
1998
). However, partial loss of function of Fgf8 in E18.5
embryos carrying an Fgf8 null allele over a hypomorphic allele can
result in a small telencephalon that lacks olfactory bulbs and a normal
midline (Meyers et al., 1998
),
demonstrating a requirement for Fgf8 in telencephalic development and
a sensitivity of telencephalic precursors to even a partial loss of
Fgf8 expression. Results from experiments in which FGF8-coated beads
were ectopically applied to forebrain tissue
(Shimamura and Rubenstein,
1997
; Crossley et al.,
2001
) and in which Fgf8 was overexpressed in the
telencephalon (Fukuchi-Shimogori and
Grove, 2001
) also suggest that FGF8 induces the formation of
anterior telencephalic structures.
Fgf genes other than Fgf8 may also be expressed and play a role in
the developing telencephalon. For example, Fgf3, Fgf15, Fgf17 and
Fgf18 are all expressed at the anterior end of the developing
telencephalon (McWhirter et al.,
1997; Maruoka et al.,
1998
; Shinya et al.,
2001
). Fgf2 has also been implicated in telencephalic
development. In particular, analysis of Fgf2-deficient mice
demonstrates a role for this gene in regulating cortical neurogenesis and
promoting neural progenitor cell proliferation
(Dono et al., 1998
;
Ortega et al., 1998
;
Raballo et al., 2000
),
although no patterning defects were observed in Fgf2-deficient
mice.
Although over 20 genes encode FGF ligands (reviewed by
Ornitz and Itoh, 2001;
Ford-Perriss et al., 2001
),
there are only four known genes encoding FGF receptors Fgfr1 to
Fgfr4. Fgfr1, Fgfr2 and Fgfr3, but not Fgfr4, are
expressed in the progenitor cells lining the telencephalic ventricles
throughout development (Orr-Utreger et al., 1991;
Peters et al., 1992
;
Peters et al., 1993
;
Yamaguchi et al., 1992
) (see
Fig. 7). The genetic analysis
of the Fgfr genes has thus far been uninformative as to the role of FGF
signaling in telencephalic development. Embryos deficient for either
Fgfr1 or Fgfr2 die at developmental stages that are too
early for telencephalic development to be assessed
(Deng et al., 1994
;
Yamaguchi et al., 1994
;
Arman et al., 1998
;
Xu et al., 1998
), whereas
Fgfr3-deficient mice survive and show no obvious telencephalic
defects (Deng et al.,
1996
).
|
The olfactory bulb (OB), a telencephalic derivative located at the anterior
most end of the rodent brain, may be particularly susceptible to a loss of FGF
signaling, as observed in embryos carrying a hypomorphic allele of
Fgf8 (Meyers et al.,
1998). However, it is likely that other signaling events are also
involved in initial OB formation. Compared with the rest of the telencephalon,
the OB undergoes unique morphological changes. In mice, OB morphogenesis is
first apparent at E12.5 as an evagination at the anterior end of the
telencephalon. Pre- and perinatally, olfactory axons from the olfactory
epithelium in the nose enter the anterior telencephalon and, together with the
dendrites from mitral cells, the projection neurons in the OB, form structures
called glomeruli. It has been proposed that initial OB morphogenesis is
induced by the arrival of axons from the olfactory epithelium, because these
axons reach the anterior telencephalon prior to any sign of OB formation and
contact ventricular zone cells just before changes in the proliferation rate
of these cells is observed (Gong and
Shipley, 1995
). Another proposed mode of OB induction involves
retinoic acid signaling from the frontonasal mesenchyme to the telencephalon
and induction of Pax6 expression
(LaMantia et al., 1993
;
Anchan et al., 1997
). The OB is
also a site that continues to acquire new neurons throughout life as
neuroblasts born in the anterior subventricular zone migrate anteriorly along
the rostral migratory stream to reach the OB and regenerate the interneuron
population (Luskin, 1993
;
Lois and Alvarez-Buylla,
1994
).
We have previously described a CRE/loxP conditional genetic
approach for knocking out or overexpressing genes in the telencephalon using
the Foxg1-Cre mouse line (Hébert
and McConnell, 2000). In mice that carry the Foxg1-Cre allele and
a gene flanked by loxP sites (`floxed'), recombination of the floxed
allele occurs efficiently in the telencephalon from its earliest stages of
development. To investigate the role of FGF signaling further in telencephalic
development and patterning, we have specifically disrupted Fgfr1 in
the telencephalon using Foxg1-Cre mice. We find that Fgfr1 is
essential for the formation of the OB, the most anterior telencephalic
structure, whereas AP patterning in the rest of the telencephalon is largely
normal in the Fgfr1 mutants.
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MATERIALS AND METHODS |
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RNA in situ hybridization analysis
Frozen sections were prepared and hybridized to 35S-labelled
probes as previously described (Frantz et
al., 1994). A minimum of three mutant and three control embryos
were analyzed for each probe at each age. Plasmids used to make probes were
kindly provided by Heike Popperl (Pbx3) and Brian Condie
(Gad67).
DiI tracing
Whole E12.5 embryos, E18.5 heads or E18.5 brains were immersion fixed in 4%
paraformaldehyde for 1 week. A single DiI crystal on the point of a pin was
inserted in the olfactory epithelium (anterograde labeling in whole E12.5
embryos or E18.5 heads) or olfactory cortex (retrograde labeling in E18.5
brains); extraneous crystals were washed away carefully. Diffusion occurred
over 1-3 weeks in the dark at room temperature in 4% formalin/PBS. Samples
were then embedded in 3% low melting temperature agarose in PBS and vibratome
sectioned (125 µm) into phosphate buffer. Sections were mounted onto glass
slides and examined wet using rhodamine optics on an inverted fluorescence
microscope.
BrdU and TUNEL analysis
Females pregnant with E12.5 embryos received an intraperitoneal injection
with BrdU and were euthanized 1 hour later. Embryos were collected, frozen in
OCT and sectioned on a cryostat. Fresh frozen sections were used for either
BrdU staining, as previously described
(O'Rourke et al., 1997), or
for TUNEL analysis according to the manufacturer's specifications (Roche,
Catalog # 2 156 792). Sections were counterstained with Syto11 (Molecular
Probes) to reveal cell nuclei. The fraction of BrdU- or TUNEL-positive cells
was determined by counting the number of labeled cells in a radial segment
spanning from the ventricular surface to the pial surface and dividing by the
total number of Syto11-positive cells in the segment. Segments were taken
either at the anterior telencephalic end, which corresponds to the location of
the OB primordium, or 500 µm caudally in the precursor to the cerebral
cortex. At least two segments from each of three separate embryos were counted
for each area. Expression of Cre in mouse cells has previously been
reported to increase the frequency of chromosomal abnormalities both in mice
(Schmidt et al., 2000
) and in
cultured cells (Loonstra et al.,
2001
). In our studies, a higher rate of apoptosis was observed in
the lateral and ventral telencephalon of embryos carrying the
Foxg1-Cre allele (Foxg1-Cre;Fgfr1flox
embryos), than of controls not carrying the Foxg1-Cre allele
(Fgfr1flox, data not shown). However, the
increased rate of apoptosis due to CRE was not in itself sufficient to cause a
phenotype. Embryos that carried the Foxg1-Cre allele were used as
controls in the TUNEL and BrdU incorporation assays presented here, and all
other experiments presented in this report included
Foxg1-Cre;Fgfr1flox embryos among the
controls.
Fos induction assay
Lateral cortical tissue was dissected from the brains of control and
Fgfr1-deficient embryos at E13.5. Cells from each embryo were
dissociated separately for 10 minutes with trypsin EDTA, split in duplicate 15
mm wells, and allowed to recover 5 hours in DMEM supplemented with N2. FGF2
was added at 50-100 ng/ml to one of each of the duplicate wells 1 hour before
cells were collected for northern blot analysis. The intensity of northern
blot bands from five separate control and mutant samples was quantified by
measuring the integrated density using NIH Image 1.62. The intensity of bands
obtained with Fos was normalized to the intensity of bands obtained
with Gapdh. Fold induction in levels of Fos expression was
calculated by dividing the normalized intensity for Fos bands in
FGF2-treated samples by those in untreated samples.
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RESULTS |
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A striking defect caused by loss of Fgfr1 in the telencephalon is a failure of normal olfactory bulb (OB) formation. The OB appears absent in Foxg1-Cre;Fgfr1flox/flox embryos from E12.5, the age at which it is first morphologically distinguishable, to E16.5 (Fig. 2A-B',D,D'). The phenotype is completely penetrant: 27/27 E12.5, 22/22 E13.5, 3/3 E15.5 and 8/8 E16.5 brains from mutant embryos lacked any morphologically apparent OB compared with embryos of control genotypes (Fgfr1flox, Fgfr1flox/flox or Foxg1-Cre;Fgfr1flox) in which the OBs are evident at these ages. From E18.5 to birth, a protrusion resembling a small bulb becomes progressively apparent at the anterior end of the mutant telencephalon (Fig. 2C',E'). Sagittal sections through the telencephalon at E16.5 reveals that the cellular organization of the anterior telencephalon of mutants is disrupted and is no longer arranged in layers as it is in control embryos (Fig. 2D,D'). Therefore, Fgfr1 is required for OB formation. All the other major areas of the telencephalon, the cerebral cortex, the hippocampus, and the medial and lateral ganglionic eminences are present in mutants and appear histologically normal.
|
Anterior-posterior patterning is largely normal in the
Fgfr1-deficient telencephalon
There are several possible mechanisms by which Fgfr1 may be
necessary for OB formation. One possibility is that the cells that form the OB
are never specified in the absence of Fgfr1 because of an AP
patterning defect. An anterior shift of posterior cell fates leading to a loss
of the most anterior types might be expected given that the source of FGF
signaling is likely to be at the anterior end of the telencephalon, where
Fgf8, Fgf15, Fgf17 and Fgf18 are most strongly expressed
(McWhirter et al., 1997;
Maruoka et al., 1998
). To
address the possibility that an AP patterning defect in mutants results in a
failure of OB cell specification, we examined the expression of genes that are
regionally expressed along the AP axis in the telencephalon.
Two genes that are thought to play roles in telencephalic patterning are
the homeodomain transcription factors Emx2 and Pax6
(Bishop et al., 2000;
Mallamaci et al., 2000
;
Muzio et al., 2002
).
Emx2 is expressed at low levels anteriorly and higher levels
posteriorly in the E12.5 cerebral cortex
(Gulisano et al., 1996
)
(Fig. 3A). Pax6 is
expressed in a counter-gradient to Emx2, with higher levels
anteriorly and lower levels posteriorly
(Walther and Gruss, 1991
)
(Fig. 3B). To examine whether
these patterns are disrupted in the Fgfr1-deficient telencephalon,
expression of Emx2 and Pax6 was assessed using RNA in situ
hybridization. In Foxg1-Cre;Fgfr1flox/flox E12.5
embryos, Emx2 and Pax6 expression remain in gradients in the
developing cerebral cortex (Fig.
3A',B'), indicating that AP patterning has occurred
despite the lack of Fgfr1. It should be noted, however, that it
remains possible that the levels of expression of these genes have been
shifted quantitatively along the AP axis in response to loss of
Fgfr1.
|
At later stages of cortical development, genes other than Emx2 and Pax6 are also expressed in regionally restricted patterns in wild-type embryos. For example at E16.5 and P0, Cdh8 (cadherin 8) is expressed strongly in the anterior cortex and weaker posteriorly (Fig. 3C). In mutants, the higher anterior expression of Cdh8 in the cortex is lost (Fig. 3C'), suggesting a slight perturbation in anterior cortical cell fates. However, other genes with regionally restricted expression patterns, such as ephrin A5 and Fgfr3, show no apparent change in their expression patterns in mutants (Fig. 3D,D'; see Fig. 7B,B').
The anterior telencephalon is specified in Fgfr1-deficient
animals
Although patterning appears for the most part normal in the
Fgfr1-deficient telencephalon, the results described above fail to
ascertain clearly whether the anterior telencephalon, from which the OB forms,
has been specified or not in mutants. To address this question, we examined
the expression of anterior telencephalic markers. Ephrin A5 and
Pou3f1 (Scip/Oct6) are two genes expressed at E12.5
in the anterior tip of the telencephalon, where the OB is just beginning to
form in control embryos (Fig.
4A,C). Ephrin A5 is also expressed in the OB itself at E16.5
(Fig. 4B). Surprisingly, in
mutant E12.5 embryos, cells in the anterior tip of the telencephalon still
express Pou3f1 and ephrin A5, even though no morphological changes
indicative of OB formation are occurring
(Fig. 4A',C'). At
E16.5, ephrin A5 remains expressed in the anterior end of both control and
mutant telencephalons (Fig.
4B,B'). These results suggest that the anterior-most end of
the telencephalon is specified in the absence of Fgfr1.
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In addition, expression of another gene, Tbr1, which marks all
early-born telencephalic neurons including the projection neurons of the OB
(Bulfone et al., 1995),
suggests that neurogenesis occurs normally at the anterior end of the
Fgfr1-deficient telencephalon. At E12.5, Tbr1 expression
reveals a relatively large number of neurons at the anterior end of the
telencephalon compared with most other areas
(Fig. 4E). This pattern of
Tbr1 expression at the anterior tip is consistent with a previous
report showing an increase in postmitotic cells in this region, an early step
in the initial formation of the OB (Gong
and Shipley, 1995
). The Fgfr1-deficient telencephalon
also shows an increase in neuronal differentiation at the anterior end, as
evidenced by Tbr1 expression (Fig.
4E'). This result is confirmed by staining E12.5 with a TUJ1
antibody, which marks an accumulation of newborn neurons in both the control
and mutant anterior telencephalon (data not shown). However, in mutant animals
the accumulation of neurons is displaced ventrally and inwards towards the
ventricle when compared with control embryos, in which the differentiating
cells begin to protrude anteriorly (Fig.
4E,E'; arrowhead). This displacement of anterior neurons in
mutants is even more evident at E16.5 (Fig.
4F,F'; arrowhead). These results suggest that the anterior
telencephalon in mutants has been specified to produce the normal, relatively
large number of neurons compared with the rest of the telencephalon, despite a
lack of normal OB morphogenesis. Tbr1 expression outside of the OB
also suggests that Fgfr1 is not required for normal neurogenesis
throughout the rest of the telencephalon
(Fig. 4E-F').
Another cell class present in the anterior telencephalon at the time of
initial OB evagination is the radial glial cell. It is likely that radial glia
are required to guide the migration of early-born projection neurons into the
OB, as is the case in other CNS areas, and are thus required for OB
morphogenesis. To address the possibility that defects in radial glial cells
are the root of OB malformations in the Fgfr1-deficient
telencephalon, we used antibodies against the radial glial marker RC-2
(Misson et al., 1988) in E13.5
and E16.5 control and Fgfr1-deficient telencephalon. Radial glia are
present in similar numbers and with normal morphologies at the anterior end of
both the control and mutant telencephalon at E13.5 and E16.5
(Fig. 4G-H'), suggesting
that radial glia specification and differentiation are unaffected by loss of
Fgfr1.
Anterior telencephalic neurons in mutants share characteristics with
OB neurons
Although neurons still accumulate at the anterior end of the telencephalon
in mutant embryos, the results above do not establish whether these neurons
are in fact normal OB neurons. The primary projection neuron found within the
OB is the mitral cell. Mitral cells receive inputs through their dendrites
directly from olfactory receptor neurons in the olfactory epithelium of the
nose. The mitral cells then project axons to the olfactory cortex. To
ascertain whether the neurons that accumulate at the anterior end of the
Fgfr1-deficient telencephalon develop connections typical of mitral
cells, we used the tracer DiI to track the afferent and efferent projections
of these cells.
To ascertain whether cells in the anterior telencephalon of mutants receive their normal inputs from olfactory sensory neurons, a DiI crystal was placed in the olfactory epithelium in the posterior part of the nasal cavity in each of three control and mutant embryos for each age. Sections (125 µm) were collected using a vibratome and examined by fluorescence microscopy. Olfactory sensory axons contacted the anterior telencephalon by E12.5 in both control and mutant embryos (Fig. 5A,A'). At E18.5 in control embryos, large numbers of olfactory neurons have penetrated the bulb where they presumably make connections with OB neurons (Fig. 5B). In mutant embryos, these projections are indistinguishable from those in controls (Fig. 5B'). Thus, the pattern of sensory projections to the anterior telencephalon from the olfactory epithelium is indistinguishable in control and mutant brains.
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We next ascertained whether cells in the anterior-most region of the
Fgfr1-deficient telencephalon project normally to olfactory cortex.
DiI crystals were placed in the olfactory (or piriform) cortex of three
control and three mutant E18.5 embryos to retrogradely label neurons
projecting to the olfactory cortex. Neuronal cell bodies in both the OB of
control embryos and the bulb-like region of mutants are labeled with DiI
(Fig. 5C,C'). In both
cases, the retrogradely labeled cells were small and multipolar with short
dendrites, as is typical of young mitral neurons before their dendritic
innervation of glomeruli which have yet to form
(Ramón y Cajal, 1995).
These results indicate that not only do neurons accumulate at the anterior end
of the Fgfr1-deficient telencephalon in the absence of a normal OB,
but that these neurons also establish olfactory pathway projections
characteristic of mitral cells.
Consistent with the finding that mitral cells are likely to be present in mutants, but displaced so that they now reside within the wall surrounding the lateral ventricle as opposed to in a morphologically distinguishable bulb, the size of the telencephalon in mutants appears enlarged compared with the telencephalon in controls (not including the OB) (Figs 2, 3). Although no significant difference was found in the dorsoventral thickness of the telencephalon, the length of the telencephalon from the most anterior point to the most posterior point were measured in whole-mount mutant and control brains (excluding the OB in controls) at E16.5 and P0. The mutant telencephalon was found to be 11% longer (P<0.01).
The rostral migratory stream in Fgfr1 mutant embryos
Neuroblasts from the ventral telencephalon migrate to the OB before birth
and continue to do so after birth from the anterior subventricular zone as
part of the rostral migratory stream. Upon reaching the OB, the cells
differentiate into GABAergic interneurons. It is possible that the failure of
the OB to form in mutants might arise if these neurons no longer migrated to
the anterior telencephalon in the Fgfr1-deficient telencephalon. To
address this possibility, we examined the expression of several genes that
mark these migrating cells by in situ RNA hybridization analysis. In E16.5 and
P0 control animals, Dlx2, Gad67 and Pbx3 are all expressed
in the ganglionic eminence and in cells migrating anteriorly and dorsally
(Fig. 4D and data not shown).
In mutant embryos, this pattern of expression is maintained for all three
genes, although the labeled cells appear to accumulate within the anterior
telencephalon at E16.5 and in the bulb-like protrusion at P0
(Fig. 4D' and data not
shown). This result is consistent with previous findings demonstrating that
cells from the ventral telencephalon still migrate towards the anterior tip of
the telencephalon even when the OB itself has been surgically removed
(Jankovski et al., 1998;
Kirschenbaum et al.,
1999
).
Proliferation and morphogenesis are abnormal in mutant OB
primordia
The experiments described above suggest that OB neurons are born at the
right time and accumulate normally at the anterior end of the telencephalon in
Fgfr1 mutants and that many of these neurons display connections
characteristic of mitral cells. Despite the normal fate specification of
anterior telencephalic cells, OB development is highly abnormal in the mutant
mice. This raises the possibility that the OB phenotype is due to a defect in
the process of morphogenesis itself, whereby anterior telencephalic cells
normally evaginate to form a bulb. It has been suggested that projections from
the olfactory epithelium, which contact the anterior telencephalon, induce
bulb morphogenesis by reducing the number of proliferating telencephalic cells
in this region (Gong and Shipley,
1995). The reduction in proliferation and the accompanying
increase in neuronal differentiation at the anterior end of the telencephalon
are thought to trigger the evagination of the OB relative to the rest of the
telencephalon, where proliferation continues at a high rate. In this model, if
cells in the anterior telencephalon were proliferating at the same rate as
those in the rest of the telencephalon, expansion would occur uniformly in the
lateral plane of the telencephalon and the OB would thus be morphologically
indistinguishable from neighboring areas. Decreased proliferation at the
anterior tip of the telencephalon thus enables the formation of a
morphologically apparent OB.
As illustrated in Fig. 5, projections from the olfactory epithelium to the anterior telencephalon appear normal in the Fgfr1-deficient telencephalon, both at an early age when these projections have just reached their target, and at a later age when functional connections are presumably being established. Therefore, contact between olfactory epithelial projections and the anterior telencephalon is not in itself sufficient to induce OB formation.
To address the second part of the model, that a decrease in the
proliferation of anterior telencephalic cells is required for evagination, we
asked whether proliferation in the mutant telencephalon at the time of initial
bulb formation is affected by loss of Fgfr1. E12.5 embryos were
exposed to BrdU in utero for 1 hour and were then analyzed in situ for BrdU
incorporation. The percentage of BrdU-positive cells was counted in two
sections from each of three separate experiments for both control and mutant
embryos. In control embryos, markedly fewer cells incorporated BrdU in the OB
region (23.0%) compared with neighboring cerebral cortex (45.0%,
Fig. 6A-C,
P<0.0001), consistent with previous findings in rat at a
comparable developmental age (25.4% versus 50.2%)
(Gong and Shipley, 1995). In
mutant embryos, proliferation in the OB region remains nearly as high as in
neighboring cortex (41.6% compared with 44.0%,
Fig. 6A',B',C).
These results indicate that Fgfr1 is required to lower the number of
proliferating cells at the anterior end of the telencephalon and suggest that
OB morphogenesis depends on a reduced rate of proliferation in this area. In
addition, our data show that the cerebral cortex and other telencephalic areas
do not require Fgfr1 to maintain normal rates of proliferation
(Fig. 6B' and data not
shown).
|
To address the possibility that FGF signaling might affect the rate of apoptosis in the telencephalon, cell death was also examined in control and mutant embryos at E12.5 using TUNEL staining. The percentage of TUNEL-positive cells was counted in two sections from each of three separate experiments for both control and mutant embryos. No significant differences between controls and mutants were observed within the OB region (2.6±2.1% in controls versus 2.0±1.4% in mutants), the cerebral cortex (1.8±1.6% in controls versus 1.2±1.1% in mutants) or in any other telencephalic areas (data not shown).
Fgfr2 and Fgfr3 may partially compensate for loss
of Fgfr1 in the telencephalon
Although Fgfr1 has been effectively deleted in the telencephalon
of Foxg1-Cre; Fgfr1flox/flox mice
(Fig. 1), most areas outside of
the OB region remain largely normal. One reason for this might be that
Fgfr2 and Fgfr3 are still being expressed in telencephalic
progenitor cells (Orr-Utreger et al., 1991;
Peters et al., 1992;
Peters et al., 1993
;
Yamaguchi et al., 1992
). In
fact, expression of Fgfr2 and 3 is unaffected by loss of
Fgfr1 (Fig.
7A-B'); consequently, these receptors may partially
compensate for the absence of Fgfr1. To test the possibility that
Fgfr1-deficient telencephalic cells are still responsive to FGF
signaling, cells from the dorsal telencephalon of E13.5 control and mutant
embryos were dissociated and cultured in the presence or absence of FGF2.
Responsiveness to FGF2 was assessed by examining induction of Fos, a
gene whose expression is induced by FGFs in neural cells
(Ghosh and Greenberg, 1995
).
Telencephalic cells from both control and mutant animals expressed
Fos upon FGF2 treatment (Fig.
7C), confirming that Fgfr1-deficient dorsal telencephalic
cells are still responsive to FGF. However, cells from mutant brains showed a
diminished response to FGF2 compared with control cells, as control cells
showed a 41-fold induction of expression (±15), whereas mutant cells
showed a 24-fold induction of expression (±4, P=0.03). It is
unlikely that this responsiveness in mutants is due to residual
Fgfr1, because recombination of the
Fgfr1flox allele is complete by E12.5
(Fig. 1). Based on the findings
that dorsal telencephalic cells express Fgfr2 and 3 and are
still responsive to FGF2, it is likely that FGF signaling takes place in vivo
in the absence of Fgfr1, but that signaling levels are reduced
relative to levels encountered in the wild-type brain.
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DISCUSSION |
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FGF signaling and anterior-posterior patterning of the
telencephalon
FGF signaling has been hypothesized to pattern the AP axis of the
telencephalon (Shimamura and Rubenstein,
1997; Meyers et al.,
1998
; Crossley et al.,
2001
; Fukuchi-Shimogori and
Grove, 2001
). More specifically, FGF8 expressed from its source at
the anterior end of the telencephalon might instruct cells along the AP axis
to express different levels of regulatory genes that in turn could result in
the adoption of different fates. Telencephalic patterning is grossly normal in
Fgfr1-deficient mutants. The expression of Emx2 and
Pax6 in counter gradients indicates that AP patterning has occurred
and, except for the OB, all the major structures cerebral cortex,
hippocampus, choroid plexus, and medial and lateral ganglionic eminences
are present and morphologically normal
(Fig. 2 and data not
shown).
The role of different FGF receptors in brain patterning remains unclear. One possibility for the lack of a major patterning defect in the Fgfr1-deficient telencephalon is that Fgfr1, Fgfr2 and Fgfr3, all of which are expressed in the ventricular zone of the telencephalon throughout development, have overlapping functions. Alternatively, each receptor may support distinct developmental outcomes. The results presented here suggest that levels of FGF signaling are dampened by deleting Fgfr1, rather than completely ablated, and that this does not result in a global disruption or shift in patterning. This is most consistent with the possibility that the different FGF receptors are working in concert to pattern the telencephalon.
FGF receptor-ligand specificities in the early telencephalon
Both Fgf8 and Fgfr1 are essential for normal
telencephalic development, as both Fgfr1-deficient and
Fgf8-hypomorphic telencephalons lack normal OBs
(Meyers et al., 1998) (this
report). This suggests that FGF8 acts through FGFR1 in the early anterior
telencephalon, an unexpected finding given previous reports in which FGF8 had
little or no significant affinity for FGFR1 in cell mitogenicity and binding
assays (Ornitz et al., 1996
;
Chellaiah et al., 1999
). The
basis for this discrepancy remains unexplored.
Although our data are consistent with the view that FGF8 acts through
FGFR1, FGF8 must also act through at least one other FGF receptor because the
telencephalic phenotype of Fgf8 hypomorphic embryos can be more
severe than that seen in Fgfr1-deficient brains. In addition to loss
of the OBs, some Fgf8 hypomorphs also have smaller telencephalons
with midline defects (Meyers et al.,
1998). FGF8 is reported to have little affinity for FGFR2 and
significant affinity for FGFR3 (Ornitz et
al., 1996
; Chellaiah et al.,
1999
), but it is clear that FGFR3 on its own can not be a
predominant receptor for transmitting FGF8 signals in the telencephalon
because mice deficient for Fgfr3 have normal OBs and the rest of the
telencephalon appears phenotypically normal
(Deng et al., 1996
).
Elucidation of the pertinent interactions between FGF receptors and ligands in
the developing telencephalon will benefit from further analysis of animals
bearing mutations in single Fgf and Fgfr genes, as well as mutants deficient
in several genes at once.
The role of FGF signaling in OB morphogenesis
The most striking phenotype of Fgfr1-deficient brains is the
absence of normal OBs. Indeed, it was initially tempting for several reasons
to speculate that the lack of OBs in Fgfr1 mutants arises from a
failure in the induction or specification of the most anterior telencephalic
cells. First, as summarized above, FGF signaling is hypothesized to specify
cell fates along the AP axis (Meyers et
al., 1998; Fukuchi-Shimogori
and Grove, 2001
), and mice hypomorphic for Fgf8, which is
expressed at the anterior end of the telencephalon
(Crossley and Martin, 1995
;
McWhirter et al., 1997
), also
lack OBs (Meyers et al.,
1998
), although the mechanism leading to this defect remained
unexplored. Second, like the Fgfr1 mutants described here, mice
deficient for Pax6, a gene that is expressed at high levels in the
anterior telencephalon and is known to be required for normal telencephalic
patterning, lack OBs (Hogan et al.,
1986
). These correlations are consistent with a role for FGF
signaling in elevating Pax6 expression to promote OB formation.
However, there are at least two significant differences between the
Pax6 mutants and those described here. While both lack OBs,
Pax6 mutants also lack nasal cavities and olfactory epithelia, which
may play a role in OB induction. In addition, Pax6 mutants completely
lack Pax6 expression, whereas Pax6 expression is at best
reduced in the Fgfr1-deficient telencephalon.
Our data argue that the absence of the OB in Fgfr1-deficient mice
is not due to defective anterior cell fate specification. First, markers for
the early anterior telencephalon, ephrin A5 and Pou3f1, are induced
and expressed normally in mutants. Second, Tbr1 expression in the
anterior telencephalon indicates that the normal increase in neurogenesis at
the anterior end occurs in mutants. And third, these neurons make connections
that are characteristic of mitral cells, receiving afferents from the
olfactory epithelium and projecting axons to the olfactory cortex. Moreover,
it has been shown previously that in Tbr1/
mice, mitral cells are lacking, but morphogenesis of the bulb still occurs
(Bulfone et al., 1998),
indicating that mitral cell differentiation and bulb morphogenesis can occur
independently of one another. Therefore, it seems likely that in the
Fgfr1-deficient telencephalon, the deficiency in OB formation is not
likely to be due to the absence of mitral cells, which appear to be specified
and to differentiate, but instead to abnormal morphogenesis that results in
the misplacement of these cells. In addition, a global delay in OB development
cannot account for the phenotype because anterior telencephalic markers are
expressed normally and anterior neurogenesis occurs on a normal schedule in
the mutants.
The failure of bulb morphogenesis in the Fgfr1-deficient
telencephalon is also not likely to result from defects in radial glia
differentiation, which appears normal, or neuronal migration from the ventral
telencephalon (Fig.
4D,D',G-H'). In E16.5 to P0 mutants, neuroblasts from
the ventral telencephalon migrate as they would normally towards the anterior
telencephalon. Once these cells reach the anterior telencephalon, however,
they accumulate abnormally in this location (perhaps because of the
misplacement of OB neurons such as mitral cells), instead of continuing
anteriorly as they normally would into a protruding OB. Nevertheless, because
cells still migrate from the ventral telencephalon, it is unlikely that
defects in this process are responsible for the initial lack of bulb
evagination in the Fgfr1-deficient telencephalon. Moreover, previous
reports indicate that cell migration to the OB from the ventral telencephalon
is not required for initial OB morphogenesis
(Anderson et al., 1997;
Bulfone et al., 1998
).
A model for initial OB evagination has been proposed previously
(Gong and Shipley, 1995).
According to this model, just prior to the earliest signs of OB evagination,
sensory axons from the olfactory epithelium arrive at the anterior
telencephalic end, where they initiate OB morphogenesis
(Fig. 8, step 1). Subsequent to
the arrival of axons from the olfactory epithelium and their contact with
anterior telencephalic progenitor cells, a decrease in the proliferation of
these cells is observed (Fig.
8, step 2), suggesting that olfactory epithelial axons inhibit
progenitor cell proliferation to trigger bulb evagination. In this model, a
decrease in the number of proliferating cells along with an increase in the
number of differentiating cells leads to an initial radial (or outwards)
expansion at the anterior telencephalic tip
(Fig. 8, step 3). If cells at
the anterior telencephalon were proliferating at the same rate as the rest of
the telencephalon, uniform expansion would occur in the lateral plane of the
telencephalon and the OB would be morphologically indistinguishable from
neighboring areas (Gong and Shipley,
1995
).
|
As shown in Fig. 1, the olfactory epithelium normally expresses Fgfr1 and this expression is lost in mutants, raising the possibility that the olfactory epithelium itself is defective and thus cannot induce OB formation. However, the olfactory epithelium is histologically normal in the Fgfr1-deficient telencephalon, and olfactory axons reach the telencephalon on time at E12.5 (Fig. 5A). The normal behavior of olfactory epithelial axons suggests that projections from the olfactory epithelium to the telencephalon are not sufficient to induce OB evagination. However, it remains possible that the arrival of olfactory axons still plays an important role in OB formation, as the olfactory axons could either exert their effect on the anterior telencephalon directly or indirectly through an FGF-dependent mechanism (e.g. if the axons secreted an FGF to which the anterior telencephalon normally responds), or through an FGF-independent mechanism that would be required in addition to FGF signaling to initiate OB evagination. In this latter case, disrupting either the FGF-dependent or -independent pathways would cause a failure of OB evagination.
It is interesting to note that between E16.5 and birth, a bulb-like
structure begins to appear in the Fgfr1 mutants. Previous studies in
mice have shown that olfactory epithelial axons can induce OB-like neurons in
the anterior telencephalon of bulbectomized neonates
(Graziadei and Monti-Graziadei,
1986). It is possible that during later development, the inductive
effect of sensory axons, along with the arrival of migrating neurons from the
ventral telencephalon, account for the late forming bulb-like evagination in
the Fgfr1-deficient telencephalon.
What is clear from the results presented here is that in the
Fgfr1-deficient telencephalon, a decrease in proliferation in the
region of normal bulb evagination fails to occur, indicating that FGF
signaling, either directly or indirectly, is required to inhibit proliferation
at the anterior tip of the forebrain (Fig.
8, step 2) and that the initial evagination of the OB requires a
decrease in the number of proliferating cells at the anterior end of the
telencephalon (Fig. 8, step 3).
The mechanism by which Fgf8 hypomorphic embryos fail to form the OB
remains to be elucidated, but the results presented here suggest that it may
also be due to a failure to decrease anterior telencephalic cell
proliferation. Consistent with this possibility, FGF8-soaked beads placed
ectopically in the chick telencephalon generate extra sulci, which are thought
to be the result of reduced proliferation
(Crossley et al., 2001).
FGF signaling has previously been implicated in promoting proliferation of
neuroepithelial progenitor cells in vitro
(Gensburger et al., 1987;
Ghosh and Greenberg, 1995
) and
in vivo (Dono et al., 1998
;
Ortega et al., 1998
;
Raballo et al., 2000
). Hence,
the result that Fgfr1 is required to inhibit proliferation of
anterior telencephalic cells was unexpected. However, FGF signaling has
previously been implicated in inhibiting the proliferation of cell types other
than those described here. For example, Fgfr3-deficient mice show an
increase in chondrocyte proliferation
(Deng et al., 1996
), whereas
activation of FGFR3 inhibits their proliferation
(Sahni et al., 1999
),
demonstrating a role for FGF signaling in inhibiting proliferation. FGF5
either directly of indirectly inhibits proliferation of hair cells
(Hébert et al., 1994
).
Hence, the roles that FGF signaling plays throughout telencephalic development
may yet be through unexpected mechanisms.
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ACKNOWLEDGMENTS |
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