1 Department of Anatomy and Program in Developmental Biology, School of
Medicine, University of California at San Francisco, San Francisco, CA
94143-0452, USA
2 Institute of Neurosciences, University Miguel Hernandez, San Juan, Alicante,
Spain 03550
3 GSF-Research Center, Institute for Developmental Genetics, 85764
Munich/Neuherberg, Max Planck Institute of Psychiatry, 80804 Munich,
Germany
* Author for correspondence (e-mail: gmartin{at}itsa.ucsf.edu)
Accepted 6 March 2003
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SUMMARY |
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Key words: Brain patterning, Cerebellum, Fgf8, Isthmic organizer, mes/met, Mesencephalon, Metencephalon, Midbrain, Midbrain/hindbrain organizer, Mouse, Rhombomere 1
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INTRODUCTION |
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Mes/met specification and AP patterning begin during gastrulation, with the
induction of gene expression that distinguishes the mes/met from the rest of
the neuroepithelium [paired-related (Pax) and
engrailed-related (En) transcription factor genes] and the
prospective mes from met (Otx2 and Gbx2 transcription factor
genes, respectively). Genetic analysis in mice has demonstrated that
Pax2 and Pax5 (Bouchard
et al., 2000) and En1 and En2
(Hanks et al., 1995
),
respectively, are functionally equivalent in the mes/met, and that in the
absence of either pair of genes, the entire midbrain to cerebellum region is
deleted (Schwarz et al., 1997
;
Wurst et al., 1994
) (W. W. and
A. Joyner, unpublished observations). Within the mes/met, Otx2 and
Gbx2 act antagonistically and are required for positioning and
function of the IsO (reviewed by Simeone,
2000
; Liu and Joyner,
2001a
; Wurst and Bally-Cuif,
2001
; see also Li and Joyner,
2001
; Martinez-Barbera et al.,
2001
).
The patterning activity of the IsO was initially demonstrated in avian
embryos by studies showing that grafts of tissue that include the mes/met
boundary region could induce cells in the caudal forebrain to develop into an
ectopic midbrain and cells in the posterior hindbrain to develop into ectopic
cerebellar structures (reviewed by Puelles
et al., 1996; Wassef and
Joyner, 1997
). Gene expression analysis identified members of the
WNT and FGF families of secreted signaling molecules as candidate mediators of
IsO activity. In the mouse mes/met, Wnt1 expression is first detected
at the 1 somite stage [embryonic day (E) 8.0] throughout the prospective
midbrain, but is soon restricted to a transverse band at its caudal end, and
is also expressed along the lateral edges of the neural plate
(McMahon et al., 1992
;
Parr et al., 1993
).
Fgf8 expression is first detected at approx. the 3-5 somite stage,
and initially extends throughout the prospective cerebellum (rhombomere 1;
r1), but is soon restricted to a narrow band at the anterior end of the
hindbrain. Thus Wnt1 and Fgf8 are expressed in adjacent
bands on either side of the midbrain/hindbrain boundary
(Crossley and Martin,
1995
).
As yet, there is no evidence that WNT1 can mediate IsO patterning activity.
However, Wnt1 is essential for mes/met development, since first
prospective midbrain and then cerebellum are deleted at early stages in
Wnt1-/- embryos
(McMahon and Bradley, 1990;
McMahon et al., 1992
;
Thomas and Capecchi, 1990
).
Further studies have suggested that in the mes/met, Wnt1 is required
to promote cell proliferation (Dickinson
et al., 1994
; Wurst and
Bally-Cuif, 2001
), to maintain En1 expression
(Danielian and McMahon, 1996
),
and to maintain a stable midbrain/hindbrain boundary
(Bally-Cuif et al., 1995
).
Unlike WNT1, FGF8 can mimic the effects of grafts of the mes/met boundary
region in the chick, in some cases inducing diencephalic tissue to form
complete ectopic midbrains as well as cerebellar tissue
(Crossley et al., 1996;
Martinez et al., 1999
;
Shamim et al., 1999
). The
results of experiments aimed at determining how FGF8 affects cells that are
competent to form mes/met tissue have not been entirely consistent, but
generally support the hypothesis that it normally stimulates cell
proliferation in the mes/met and maintains gene expression required for IsO
activity (reviewed by Liu and Joyner,
2001a
; Wurst and Bally-Cuif,
2001
).
The role of FGF8 in mes/met development cannot be investigated in
Fgf8-/- mouse embryos because they fail to gastrulate
(Sun et al., 1999), but it can
be studied in Fgf8 hypomorphs, which survive to birth. A preliminary
analysis has shown that when Fgf8 expression is reduced, a
substantial portion of the midbrain as well as isthmus and cerebellum are
deleted (Meyers et al., 1998
).
However, it is possible that the brain phenotypes in such hypomorphic embryos
are secondary to mild defects caused by reduced Fgf8 expression
during gastrulation. To address this concern, we have used a conditional gene
inactivation approach to determine the effects of inactivating Fgf8
in the mes/met without perturbing its expression during gastrulation. We show
that this causes extensive cell death in the mes/met before E10, and confirm
that Fgf8 is part of a gene regulatory network that is essential for
mes/met development. Together with the results of previous studies, our data
suggest that loss of Fgf8 function may be the cause of the deletions
that are also observed in embryos that lack Wnt1, En1/2 or
Pax2/5 gene function.
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MATERIALS AND METHODS |
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Fgf8neo/neo and Fgf8neo/null
embryos were obtained from appropriate crosses of
Fgf8neo/+ and Fgf8null/+ mice, and
genotyped as previously described (Meyers
et al., 1998; Sun et al.,
2002
). Their Fgf8neo/+,
Fgf8null/+ and Fgf8+/+ littermates
were used as controls. Mice carrying a Wnt1 null allele on a Swiss
Webster background were kindly provided by A. McMahon.
Wnt1+/- mice were crossed inter se to obtain null mutant
homozygotes. Genotyping by PCR was performed as previously described
(McMahon and Bradley, 1990
).
Their heterozygous and wild-type littermates were used as control embryos. To
obtain En1 null homozygotes, En1Cre/+ mice were
crossed inter se. En1Cre/+ and En1+/+
littermates were used as controls. Mice carrying the R26R
(Soriano, 1999
) Cre reporter
gene were kindly provided by P. Soriano.
Embryos were collected at various stages of gestation, and dissected free
of maternal tissues in phosphate-buffered saline (PBS) before use in specific
assays. Noon of the day of vaginal plug detection was designated embryonic day
(E) 0.5. Prior to E10, embryos were staged by determining somite number
(Jacobson and Tam, 1982). In
the mouse, a new pair of somites is generated every 1.5 to 2 hours
(Tam, 1986
). DNA for genotype
analysis was prepared from embryonic or yolk sac tissue.
Histological analysis, immunolocalization and in situ hybridization
assays
E17.5 embryonic brains were left in situ, but the overlying epithelium was
dissected open to allow penetration of Clarke's fixative (75% ethanol, 25%
glacial acetic acid). Heads were fixed for 14-24 hours at room temperature,
stored in 70% ethanol, then embedded in paraffin wax and sectioned at 12 µm
in the sagittal and horizontal planes. Serial sections were stained with
Cressyl Violet or processed to detect tyrosine hydroxylase (TH). Sections were
incubated overnight in anti-TH antibody (Inst. Jacques BOY S.A., Reims,
France), following the manufacturer's recommendations. Antibody staining was
detected using biotinylated anti-rabbit IgG and streptavidin-conjugated with
peroxidase (Vector Labs, Burlingame, CA). Parallel sections were processed
without primary antibody as controls for the specificity of the
immunolabelling.
Whole-mount immunohistochemistry was performed essentially as described by
O'Connor et al. (O'Connor et al.,
1999) using 2H3 antibody (Dodd
et al., 1988
) supernatant (Developmental Studies Hybridoma Bank,
U. Iowa) and an HRP-linked sheep anti-mouse secondary antibody (Amersham
Pharmacia NA931). Samples were post-fixed, dehydrated and cleared in 1:2
benzyl benzoate:benzyl alcohol.
Whole-mount RNA in situ hybridization was performed on embryos fixed in 4%
PFA (in PBS), and processed essentially as described by Neubuser et al.
(Neubuser et al., 1997).
Digoxigenin-labeled ribroprobes were used to detect expression of En1
(Danielian and McMahon, 1996
),
Fgf8-exon3 (Lewandoski et al.,
2000
), full-length Fgf8
(Crossley and Martin, 1995
),
Fgf17 and Fgf18 (Maruoka
et al., 1998
), Gbx2
(Wassarman et al., 1997
),
Hoxa2 (Wilkinson et al.,
1989
), Otx2 (Simeone
et al., 1993
), Pax2
(Dressler et al., 1990
),
Spry2 (Minowada et al.,
1999
) and Wnt1
(McMahon et al., 1992
). For
simultaneous detection of Otx2 and Hoxa2 RNA, the probes
were mixed in a 1:2 ratio. ß-Galactosidase was assayed essentially as
described previously (Logan et al.,
1993
).
Assays for cell death
For whole-mount Nile Blue Sulfate (NBS) staining, embryos were washed in
PBS and incubated at 37°C in filtered NBS solution [10 µg/ml NBS (Sigma
N-5632) in PBS containing 0.1% Tween 20 (PBT)]. Incubation times varied with
embryo stage: 0-10 somite stages, 15 minutes; 10-30 somite stages, 20 minutes;
>30 somite stage, 40 minutes. Embryos were then washed in PBS at room
temperature and photographed immediately.
Whole-mount TUNEL was performed with the `In Situ Cell Death Detection, POD' kit (Roche) following a modified protocol (A. Strickler, personal communication). Embryos were fixed in 4% PFA and stored in methanol, then rehydrated into PBT and treated with proteinase K (20 µg/ml in PBT) for 2 to 10 minutes. They were then post-fixed in 4% PFA/0.2% glutaraldehyde for 20 minutes, incubated for 1 hour in 3% H2O2 in methanol to inactivate endogenous peroxidases, then permeabilized in 0.1% sodium citrate/0.1% Triton X-100 for 5 minutes on ice. The treated embryos were then incubated in Roche kit TUNEL Reaction Mix for 1 hour at 37°C, and washed in PBT. To block nonspecific antibody binding, embryos were next incubated in KTBT buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 mM KCl, 1% Triton X-100) containing 2% blocking reagent (Roche 1096176) and 20% sheep serum. Embryos were then incubated in Roche kit converter POD for 30 minutes at 37°C, and washed in PBT. Finally, specimens were reacted in diaminobenzidine for 30 to 90 minutes.
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RESULTS |
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Deletion of mes/met derivatives increases in severity with
progressive loss of Fgf8 function
Previously, we reported that homozygosity for an Fgf8 hypomorphic
allele, Fgf8neo, caused deletion of posterior midbrain and
cerebellar tissue. In Fgf8neo/null compound heterozygotes,
larger deletions were observed. Reduced Fgf8 function in these mutant
embryos also caused other defects, including absence of the olfactory bulbs in
both genotypes, and severe forebrain, craniofacial, heart and kidney
abnormalities in Fgf8neo/null embryos
(Meyers et al., 1998). In
contrast, in E17.5 MHB KO mutants, we found that the midbrain, isthmus and
cerebellum was deleted, but olfactory bulb, forebrain and craniofacial
development appeared normal (Fig.
2A-B'), and we detected no obvious abnormalities in other
organs (M. Lewandoski and G.R.M., unpublished observations). These data
demonstrate that inactivating Fgf8 using En1Cre
causes defects specifically in structures that develop from the mes/met.
|
In Fgf8neo/neo embryos, in which the level of
functional Fgf8 RNA has been roughly estimated to be 40% of the
amount in wild-type embryos (Meyers et
al., 1998
), the rostral midbrain appeared normal, but caudal
regions of the midbrain, including a substantial portion of the posterior
superior colliculus and the entire inferior colliculus were missing, as was
the isthmus. Dorsal anterior hindbrain tissue (cerebellum) was largely
deleted, except in the most lateral regions. The locus ceruleus, a marker of
ventrolateral anterior hindbrain tissue (the pons), was present but reduced in
size (Fig. 2C-D', and
data not shown). This suggests that when Fgf8 expression is
moderately reduced, at least part of the midbrain and intermediate and basal
anterior hindbrain still develops.
In Fgf8neo/null embryos, in which functional
Fgf8 RNA is presumably reduced to approximately half the level in
Fgf8neo/neo embryos, the extent of the deletion was
greater. Rostrally, all midbrain tissue except the posterior pretectal nucleus
(PPT) was absent. Although its name implies that the PPT is of forebrain
origin, comparative anatomical studies indicate that it is a midbrain
structure derived from the rostral mesencephalon
(Lagares et al., 1994).
Caudally, no cerebellar tissue was observed, and the locus ceruleus was
absent. However, the pontine nucleus, which is derived primarily from tissue
caudal to the prospective cerebellum, was present and appeared normal
(Fig.
2E,E', and
data not shown).
MHB KO mutants exhibited the most severe phenotype. The entire midbrain, including the PPT, and the anterior hindbrain were deleted, but the pontine nucleus was normal (Fig. 2F,F', and data not shown). The posterior commissure, which comprises axons of pretectal (forebrain) nuclei, appeared abnormally extended (Fig. 2F), perhaps because of the lack of caudal tissue to confine it. These data show that Fgf8 is essential for development of the mes/met, and that even a moderate decrease in the level of functional Fgf8 RNA results in deletions of posterior midbrain and most cerebellar tissue. A more substantial decrease results in the absence of almost the entire midbrain, except for the PPT, and all anterior hindbrain structures. Finally, elimination of Fgf8 function results in complete deletion of the territory spanning from the rostral end of the midbrain through the cerebellum, whereas tissues derived from adjacent anterior or posterior regions are intact. However, because Fgf8 MHB KO mutants are heterozygous for a null allele of En1, we cannot formally rule out the possibility that reduced En1 dosage played some role in the deletion of the PPT in those mutants.
To study the phenotype at an earlier stage, we performed
immunohistochemistry using an anti-neurofilament antibody that detects the
cranial nerves (n) and their associated ganglia (g) that mark derivatives of
specific portions of the mes/met by E10.5 (reviewed by
Cordes, 2001
;
Fig. 2G). In E10.5
Fgf8neo/neo embryos, nIV (trochlear), which marks the
isthmus, was absent, but other markers appeared normal (2/2;
Fig. 2H, and data not shown).
In contrast, in both Fgf8neo/null (2/2) and MHB KO (3/3)
embryos, not only nIV, but also nIII (oculomotor), which marks the ventral
midbrain, and the r1-derived component of the trigeminal nV were either absent
or severely truncated. Cranial nerves and ganglia originating from r2 and more
caudal territory, including the trigeminal gV, n/gVII (facial), and n/g VIII
(acoustic), appeared normal (Fig.
2I,J,
and data not shown). These data are consistent with the results of our
histological analysis at E17.5, in that they show a correlation between the
amount of functional Fgf8 expression and the amount of
mes/met-derived tissue present. Furthermore, since the trigeminal and more
posterior ganglia appeared to be intact in all three mutants, the results
indicate that the effects of loss of Fgf8 function in the
midbrain/hindbrain boundary region do not extend caudally beyond r1. In
addition, these data demonstrate that the deletions, including loss of ventral
structures, occur by
E10.5.
Inactivation of Fgf8 causes extensive cell death in the
mes/met
Although previous studies have suggested that Fgf8 functions as a
proliferation factor in the mes/met (Lee
et al., 1997; Xu et al.,
2000
), in several other developmental contexts Fgf8 has
been found to be required for cell survival
(Moon et al., 2000
;
Storm et al., 2003
;
Sun et al., 2002
;
Trumpp et al., 1999
). To
determine if cell death might be a cause of the deletions described above, we
assayed for cell death in MHB KO mutants at E8.0-E10.0 by staining with Nile
Blue Sulfate (NBS), which specifically marks dying cells
(Tone et al.,1983
), and by
using the TUNEL assay, which detects apoptotic cells. Because some cell death
is a normal part of brain development
(Weil et al., 1997
), dying
cells were observed in control embryos at all stages assayed
(Fig. 3, and data not shown).
At the 2-5 somite stage (
E8.0-E8.25), the number and distribution of
NBS-positive (NBS+) cells appeared similar in MHB KO mutants
(n=5) and control littermates (n=13). However, by the 7-9
somite stage (
E8.5), a small amount of ectopic cell death was detected in
the mes/met of all MHB KO mutants assayed (NBS, n=6 mutants, 21
controls; TUNEL, n=1 mutant, 1 control;
Fig. 3A, and data not shown),
and by the 11-13 somite stage (
E8.75), the amount of ectopic cell death
was dramatically increased throughout the mes/met (NBS, n=9 mutants,
17 controls; TUNEL, n=2 mutants, 3 controls). Interestingly, the
dying cells appeared to be localized primarily in the alar plate (dorsal
portion) (Fig. 3B, and data not
shown). It seems unlikely that this is an artifact of the whole mount staining
procedure because the mes/met section of the neural tube is open at these
early stages, and therefore dorsal and ventral regions should have been
equally accessible to NBS stain. Thus, our data show that cell death in the
prospective mes/met increased as the level of functional Fgf8 RNA
decreased (Fig. 1D).
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Cell death in the mes/met of Wnt1-/- and
En1-/- embryos
Deletions of midbrain and cerebellum have been observed following loss of
function of several other genes, including Wnt1 and En1.
Although the phenotype of Wnt1-/- embryos can vary, tissue
loss similar in extent to what we observed in Fgf8neo/null
or MHB KO mutants has been observed
(McMahon and Bradley, 1990;
Thomas and Capecchi, 1990
).
Interestingly, it was previously reported that ectopic cell death was detected
in the rostral dorsal metencephalon of Wnt1-/- embryos at
the 20-30 somite stage, but not in the prospective midbrain at earlier stages
(Serbedzija et al., 1996
).
However, we detected ectopic cell death throughout the mes/met of
Wnt1-/- embryos at the 7-8 somite stage (n=8
mutants, 5 wild-type controls; Fig.
4A, and data not shown) and the 12-14 somite stage (n=6
mutants, 4 controls; Fig. 4B,
and data not shown). At the 26-28 somite stage, most of the mutant midbrain
was deleted, and ectopic cell death continued to be detected in what remained
of the rostral metencephalon (n=3 mutants, 3
Wnt1+/+ or Wnt1+/- controls;
Fig. 4C, and data not
shown).
Deletions of posterior midbrain and cerebellar tissue were also observed in
embryos that lack Engrailed gene function. On some genetic backgrounds,
En1-/- embryos have an altered midbrain and cerebellum
phenotype (Wurst et al., 1994)
similar to that of Fgf8neo/neo embryos at E17.5, whereas
loss of function of both En1 and En2, which are co-expressed
in the mes/met, results in a more extensive deletion
(Joyner, 1996
) (W.W. and A.
Joyner, unpublished observations), similar to what we observed in
Fgf8 MHB KO mutants. We detected an abnormal number of
NBS+ cells in both the prospective posterior midbrain and
cerebellum of embryos homozygous for En1Cre (hereafter
referred to as En1-) at the 22-28 somite stage
(n=9 mutants, 13 En1+/- or
En1+/+ controls; Fig.
4C, and data not shown). Together our data suggest that cell death
is a likely cause of the midbrain and cerebellum deletions observed in embryos
that lack Wnt1 or En1 function. Moreover, the pattern and
timing of cell death in Wnt1-/- embryos is remarkably
similar to what we observed in Fgf8 MHB KO mutants, with deletion of
the prospective midbrain occurring before deletion of the prospective
cerebellum.
FGF8 is required to maintain gene expression in the mes/met
Gain-of-function studies have demonstrated that FGF8 can induce ectopic
expression of several genes that are normally expressed in the mes/met,
raising the possibility that FGF8 regulates their expression during normal
brain development (reviewed by Liu and
Joyner, 2001a; Wurst and
Bally-Cuif, 2001
). However, to definitively determine whether
Fgf8 is required to induce or maintain the expression of specific
genes in the mes/met, loss-of-function experiments are required. We assayed
gene expression in our MHB KO mutants at the 7-9 somite stage, when the level
of functional Fgf8 RNA was very reduced compared to normal
(Fig. 1D), but before much
ectopic cell death was detected (Fig.
3A, and data not shown). Four genes were found to be negatively
affected by the loss of Fgf8 function. Wnt1 expression is
normally upregulated and detected in a band at the caudal end of the
prospective midbrain at the 7-9 somite stage
(Parr et al., 1993
)
(Fig. 5A). In MHB KO mutants,
no band of Wnt1 expression was observed, although Wnt1 RNA
was detected at normal levels along the lateral edges of the open neural tube
(Fig. 5B). Similarly,
Gbx2 RNA, which is normally detected in a band at the rostral end of
the metencephalon (Bouillet et al.,
1995
; Fig. 5C), was
not detected in this domain in MHB KO mutants
(Fig. 5D). In contrast,
Gbx2 RNA was readily detected in the posterior hindbrain of both MHB
KO mutants and control embryos (Fig.
5C,D).
Fgf17 and Fgf18 expression, which is normally detected in
the midbrain/hindbrain boundary region
(Maruoka et al., 1998
;
Xu et al., 2000
) (Fig.
5E,G)
was either barely detected (Fig.
5F, and data not shown) or not detected
(Fig. 5H) in MHB KO mutants. At
a later stage (12-15 somite stage), no expression of these genes was detected
(data not shown). Together with studies of the normal expression patterns of
these genes, our data suggest that FGF8 is required to induce or maintain
Fgf18 and to maintain Wnt1, Gbx2 and Fgf17
expression in the mes/met.
|
At early neural plate stages, Otx2 is normally expressed in a
domain that stretches from the rostral end of the prospective forebrain to the
caudal end of the prospective midbrain
(Fig. 2O). Numerous studies
have suggested that Gbx2 expressed in the rostral metencephalon
negatively regulates Otx2 expression and restricts its caudal border
to the midbrain/hindbrain boundary (Li and
Joyner, 2001; Li et al.,
2002
; Martinez-Barbera et al.,
2001
), and that FGF8 may play a role in this process by regulating
Gbx2 expression in r1 (Garda et
al., 2001
; Irving and Mason,
2000
; Liu et al.,
1999
; Martinez et al.,
1999
; Sato et al.,
2001
). In order to determine if the posterior limit of the
Otx2 expression domain was altered in MHB KO mutants, we assayed for
Otx2 RNA in combination with a probe for Hoxa2, which is
strongly expressed in r3. At the 7-9 somite stage, we detected no obvious
difference between the distance from the caudal end of Otx2
expression domain to r3 in control and MHB KO mutants (Fig.
5O,P).
However, by the 12-13 somite stage stage, this distance appeared to be
slightly reduced in MHB KO as compared with control embryos (Fig.
5W,X),
possibly reflecting a posterior shift in the caudal limit of Otx2
expression, or a small amount of tissue loss.
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DISCUSSION |
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Deletions of the midbrain and cerebellum in Fgf8, Wnt1, En
and Pax mutant mice may be due to cell death
In our Fgf8 MHB KO mice, those portions of the brain normally
derived from the mes/met are deleted. Likewise, on certain genetic
backgrounds, similar deletions are found in Wnt1
(McMahon and Bradley, 1990),
En1/2 (Joyner, 1996
)
(W.W. and A. Joyner, unpublished observations) and Pax2
(Bouchard et al., 2000
) mutant
mouse embryos. In zebrafish embryos, loss of function of a Pax2
ortholog likewise causes deletion of the midbrain to cerebellum
(Brand et al., 1996
;
Lun and Brand, 1998
), whereas
homozygosity for ace, a mutant allele of Fgf8, causes
deletion of only the isthmus and cerebellum
(Brand et al., 1996
;
Reifers et al., 1998
). One
possible reason why the midbrain is intact in ace mutants is that
ace is a hypomorphic rather than a null allele
(Draper et al., 2001
).
Alternatively, the midbrain may be rescued by some other FGF family member
that is expressed in the zebrafish midbrain/hindbrain boundary region and is
functionally redundant with ace.
Our data show that cell death is a major cause of the midbrain to
cerebellum deletion in Fgf8 MHB KO mutants, and that the cells die
soon after they are deprived of FGF8. However, since FGF8 has been shown to
stimulate cell proliferation in the mes/met
(Lee et al., 1997;
Xu et al., 2000
), it is
possible that some of the tissue is lost because the cells do not proliferate.
Indeed, a failure to proliferate might possibly be the cause of the cell death
we observed. Our data are consistent with previous studies showing that loss
of Fgf8 function results in cell death in the first branchial arch
(Trumpp et al., 1999
), the
developing limb bud (Moon et al.,
2000
; Sun et al.,
2002
), and the early forebrain
(Storm et al., 2003
). There
has been no evidence that cell death is the cause of the deletions in Pax or
En mutant mouse embryos, but in Wnt1-/- embryos cell death
was observed in the rostral metencephalon
(Serbedzija et al., 1996
). We
show here that in Wnt1-/- embryos, there is also extensive
cell death in the prospective midbrain, and that overall the pattern of
ectopic cell death and deletion is very similar to the one we observed in
Fgf8 MHB KO mutants, except that in the Wnt1 mutants a small
portion of the prospective midbrain survived. This presumably reflects the
variability that has been observed in the extent of the midbrain and anterior
hindbrain deletion in Wnt1-/- embryos
(McMahon and Bradley, 1990
;
Thomas and Capecchi,
1990
).
We also detected ectopic cell death in both the prospective midbrain and
cerebellum of En1-/- embryos. This is consistent with the
phenotype of En1 mutants described by Wurst et al.
(Wurst et al., 1994), in which
the posterior midbrain and much of the cerebellum was deleted. Although an
analysis of cell death in the mes/met has not yet been reported in mouse Pax
mutants, death of a large block of predominantly dorsal cells has been
observed in the zebrafish Pax2.1 mutant midbrain
(Brand et al., 1996
). Together,
the data indicate that the midbrain to cerebellum deletions caused by loss of
Fgf8, Wnt1, En and Pax gene function can be explained, in large
measure, by a failure of cell survival.
Interestingly, our data indicate that the cell death that occurs as a result of loss of Fgf8 function in the mes/met is localized primarily in the dorsal portion of the neural plate/tube. However, we also found that as early as E10.5, some ventral structures are absent. At present we do not know why dorsal and ventral mes/met cells respond differently to a loss of Fgf8 function, or what causes the loss of ventral structures.
Loss of Fgf8 function may be the cause of midbrain and
cerebellum deletion phenotypes in other mutants
It has been suggested that loss of function of genes involved in specifying
the mes/met very early in neuroectoderm development, such as Pax and En,
causes cells to assume the wrong fate, to fail to develop normally, and
therefore to die (Wurst et al.,
1994). An alternative possibility is that the loss of function of
such genes results in a failure to induce and/or maintain Fgf8
expression, which is required for cell survival in the mes/met. This
hypothesis is supported by the finding that Fgf8 expression is not
induced in Pax2-/- embryos
(Ye et al., 2001
), and is
detected but then rapidly down-regulated in En1/2 double null
homozygotes (Liu and Joyner,
2001b
). Thus loss of Fgf8 function in both Pax and En
mutant embryos occurs before there is any significant loss of mes/met tissue.
In Wnt1-/- embryos, Fgf8 expression appears
normal at the 6 somite stage but is rapidly down-regulated
(Lee et al., 1997
), raising
the possibility that the ectopic cell death that we observed in Wnt1
null homozygotes is also due to loss of Fgf8 function.
However, the tissue deletions observed in all these mutant might be due to
a loss of Wnt1 function, since the midbrain domain of Wnt1
expression is not maintained in the absence of FGF8. There is ample evidence
that both the FGF and WNT signaling pathways can regulate cell survival
(reviewed by Feig and Buchsbaum,
2002; Patapoutian and
Reichardt, 2000
), so either FGF8 or WNT1, or both, could be the
essential factor(s) required for cell survival in the mes/met. One intriguing
possibility, consistent with their different domains of expression, is that
Wnt1 is required for cell survival in the prospective midbrain
whereas Fgf8 is required in the prospective cerebellum. If so, it
should be possible to rescue the cerebellum in Wnt1-/-
embryos by expressing an Fgf8 transgene, and the midbrain in
Fgf8 MHB KO mutants by expressing a Wnt1 transgene in the
midbrain/hindbrain boundary region.
Effects of loss of Fgf8 function on gene expression in the
mes/met
The observation that the various mutations that cause midbrain and
cerebellum deletions also cause extensive cell death makes it difficult to
interpret the significance of gene expression data in such mutants, except at
the earliest stages of mes/met development. In our Fgf8 MHB KO
mutants, the stages at which we could perform a useful analysis were further
restricted by the fact that Fgf8 was transiently expressed before it
was inactivated. Nevertheless, we were able to determine that in addition to
maintaining Wnt1 expression, Fgf8 function is also required
to maintain Gbx2 expression in the rostral metencephalon, consistent
with what has been concluded from gain-of-function experiments
(Garda et al., 2001;
Irving and Mason, 2000
;
Liu et al., 1999
;
Martinez et al., 1999
;
Sato et al., 2001
). The
expression of other genes thought to be regulated by Fgf8, such as
Pax2, En1, and Spry2 (reviewed by
Liu and Joyner, 2001a
;
Wurst and Bally-Cuif, 2001
),
was not significantly reduced in Fgf8 MHB KO embryos at early stages,
when Fgf8 expression is very low but before many cells have begun to
die. This was particularly surprising for Spry2, since previous
loss-of-function studies have demonstrated that Spry2 expression is
dependent on FGF signaling (Minowada et
al., 1999
). Presumably the low level of Fgf8 expression
at the 7 somite stage was sufficient to maintain Spry2
expression.
The results of our analysis of Otx2 expression in Fgf8
MHB KO embryos are consistent with the extensive body of evidence that
Gbx2 expression in the rostral metencephalon determines the posterior
limit of Otx2 expression in the mes/met
(Li and Joyner, 2001;
Martinez-Barbera et al., 2001
;
Li et al., 2002
). In the
absence of Gbx2 expression in r1, we detected a small decrease in the
distance between the posterior limit of Otx2 expression and r3, as
marked by Hoxa2 expression, in our mutant embryos at 13 som, which
might reflect a caudal expansion of the Otx2 expression domain.
However, we cannot exclude the possibility that this effect is due to cell
death.
One hypothesis about the role of FGF8 in mes/met development is that it
functions to define the rostral limit of Hoxa2 expression and
restrict it to the r1/r2 boundary (Irving
and Mason, 2000). Support for this hypothesis comes from an
elegant series of studies in the chick embryo, including the demonstration
that blocking FGF8 function in r1 by applying anti-FGF8 antibody resulted in
anterior expansion of the Hoxa2 expression domain into r1. We made an
effort to test this hypothesis in our Fgf8 MHB KO embryos.
Unfortunately, although Hoxa2 expression is relatively strong in
chicken r2, it is extremely weak in mouse r2, and we were unable to determine
whether it was expressed to a more rostral limit than normal in the mutant
embryos we assayed at the 7 and 13 somite stages.
Interestingly, we found that FGF8 is essential for maintaining the
expression of two other FGF family members, Fgf17 and Fgf18,
in the mes/met (Maruoka et al.,
1998). These two FGF genes are closely related to Fgf8
and encode proteins with similar biochemical and inductive properties
(Ohuchi et al., 2000
;
Xu et al., 2000
). It remains
to be determined whether Fgf17 and/or Fgf18 are functionally
redundant with Fgf8 in mes/met development. Analysis of the
phenotypes of Fgf17-/- and
Fgf17-/-,Fgf8+/- mice showed that
Fgf17 contributes to midbrain and cerebellar development, but
abnormalities were not detected until
E11.5, two days after the IsO is
thought to function. This led Xu et al.
(Xu et al., 2000
), to propose
that FGF genes have both an early and a late function in midbrain and
cerebellar development: prior to E10, they contribute to IsO activity, and
after E10, they maintain precursors of the cerebellar vermis in a
proliferative, undifferentiated state. As yet, there is no evidence that
Fgf18 is required for midbrain or cerebellar development, since the
brain appears normal in Fgf18-/- embryos
(Liu et al., 2002
;
Ohbayashi et al., 2002
).
Analysis of Fgf17/18 double null homozygotes should help to clarify
this point. It would also be interesting to know if, in addition to its
function at early stages, Fgf8 also plays a role at a later stage.
This could be determined by varying the timing of cre expression so that
Fgf8 is inactivated after E10.
Concluding remarks
The finding that a bead soaked in recombinant FGF8 can induce one or even
two nearly complete, ectopic midbrains, as well as cerebellar tissue, led to
the hypothesis that FGF8 functions to pattern the mes/met region. However,
another interpretation of this observation is that FGF8 induces these
structures only because it is capable of initiating the genetic cascade that
leads to the formation of an ectopic IsO. Our data show that without FGF8 the
entire mes/met fails to survive. Whether FGF8 directly participates in
patterning this region remains unknown. Methods that enable cells to survive
in the absence of FGF8 will need to be developed, to fully explore the role
that FGF8 plays in mes/met development.
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ACKNOWLEDGMENTS |
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