FGF17b and FGF18 have different midbrain regulatory properties from FGF8b or activated FGF receptors
Aimin Liu1,2,
James Y. H. Li2,
Carrie Bromleigh2,
Zhimin Lao2,
Lee A. Niswander1 and
Alexandra L. Joyner2,*
1 Howard Hughes Medical Institute, Developmental Biology Program, Memorial Sloan
Kettering Cancer Center, New York, NY 10021, USA
2 Howard Hughes Medical Institute and Skirball Institute of Biomolecular
Medicine, Departments of Cell Biology, and Physiology and Neuroscience, NYU
School of Medicine, New York, NY 10016, USA
*
Author for correspondence (e-mail:
joyner{at}saturn.med.nyu.edu)
Accepted 28 August 2003
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SUMMARY
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Early patterning of the vertebrate midbrain and cerebellum is regulated by
a mid/hindbrain organizer that produces three fibroblast growth factors (FGF8,
FGF17 and FGF18). The mechanism by which each FGF contributes to patterning
the midbrain, and induces a cerebellum in rhombomere 1 (r1) is not clear. We
and others have found that FGF8b can transform the midbrain into a cerebellum
fate, whereas FGF8a can promote midbrain development. In this study we used a
chick electroporation assay and in vitro mouse brain explant experiments to
compare the activity of FGF17b and FGF18 to FGF8a and FGF8b. First, FGF8b is
the only protein that can induce the r1 gene Gbx2 and strongly
activate the pathway inhibitors Spry1/2, as well as repress the
midbrain gene Otx2. Consistent with previous studies that indicated
high level FGF signaling is required to induce these gene expression changes,
electroporation of activated FGFRs produce similar gene expression changes to
FGF8b. Second, FGF8b extends the organizer along the junction between the
induced Gbx2 domain and the remaining Otx2 region in the
midbrain, correlating with cerebellum development. By contrast, FGF17b and
FGF18 mimic FGF8a by causing expansion of the midbrain and upregulating
midbrain gene expression. This result is consistent with Fgf17 and
Fgf18 being expressed in the midbrain and not just in r1 as
Fgf8 is. Third, analysis of gene expression in mouse brain explants
with beads soaked in FGF8b or FGF17b showed that the distinct activities of
FGF17b and FGF8b are not due to differences in the amount of FGF17b protein
produced in vivo. Finally, brain explants were used to define a positive
feedback loop involving FGF8b mediated upregulation of Fgf18, and two
negative feedback loops that include repression of Fgfr2/3 and direct
induction of Spry1/2. As Fgf17 and Fgf18 are
co-expressed with Fgf8 in many tissues, our studies have broad
implications for how these FGFs differentially control development.
Key words: Fgf8, Fgf17, Fgf18, Mid/hindbrain organizer, FGF receptors, Sprouty, Mouse, Chick
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Introduction
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Early patterning of the vertebrate presumptive midbrain and rhombomere 1
(r1), which dorsally gives rise to the cerebellum, is regulated by a local
organizer situated at the mid/hindbrain junction (reviewed by
Joyner et al., 2000
;
Liu and Joyner, 2001a
;
Wurst and Balley-Cuif, 2001
).
FGF8, a member of the fibroblast growth factor (FGF) family, is expressed in
r1 adjacent to the mid/hindbrain junction and has organizer activity. FGF8 can
induce the patterned expression of many midbrain/r1 genes and the formation of
ectopic midbrain or cerebellar structures depending on the cellular
environment and isoform (a or b) of FGF8 protein used. Furthermore,
loss-of-function studies in mouse and zebrafish have shown that Fgf8
is required for normal development of the midbrain and cerebellum
(Meyers et al., 1998
;
Reifers et al., 1998
;
Chi et al., 2003
).
Fgf17 and Fgf18, which encode proteins more closely related
to FGF8 than the other FGF family members, are also expressed in the
mid/hindbrain junction region in broader domains than Fgf8, including
the posterior midbrain (Maruoka et al.,
1998
; Xu et al.,
1999
). In biochemical and cell culture assays FGF17b and FGF18
have similar receptor binding properties and ability to induce proliferation
when compared with FGF8b (Xu et al.,
1999
; Xu et al.,
2000
). In zebrafish, mRNA injection experiments indicate that
Fgf8 and Fgf17 have similar effects on gastrulation
(Reifers et al., 2000
). Loss
of Fgf17 in mouse results in truncation of the posterior midbrain
(inferior colliculus) and reduced proliferation of the anterior cerebellum
(Xu et al., 2000
), whereas
Fgf18 does not appear to be required for midbrain or cerebellum
development (Liu et al., 2002
;
Ohbayashi et al., 2002
). There
is clearly overlap in function between at least Fgf8 and 17,
as removal of one copy of Fgf8 on an Fgf17 mutant background
leads to an exaggerated cerebellum phenotype
(Xu et al., 2000
). The exact
functions of each FGF protein therefore are not clear.
Fgf8 mRNA is differentially spliced to generate multiple protein
isoforms. FGF8a and FGF8b are the primary isoforms expressed in r1
(Sato et al., 2001
) and they
differ by only 11 amino acids that are included in FGF8b. Surprisingly, we
have shown that these two FGF8 isoforms produce very different phenotypes when
mis-expressed in transgenic mouse embryos
(Liu et al., 1999
). Ectopic
expression of the a isoform of Fgf8 in the midbrain and
caudal forebrain results in both expansion of the midbrain and ectopic
expression of En2, but not other genes expressed in the midbrain and
r1 (Lee et al., 1997
;
Liu et al., 1999
). The EN
transcription factors alone cannot mediate the midbrain expansion, as similar
ectopic expression of En1 does not induce the same phenotype
(Lee et al., 1997
), and
Fgf8a produces midbrain expansion even in En2 mutants (D.
Song and A.L.J., unpublished data). In contrast to FGF8a, the b isoform
produces exencephaly and a rapid transformation of the midbrain and
diencephalon into an anterior r1 fate (Liu
et al., 1999
) that includes repression of the midbrain gene
Otx2, expansion of the hindbrain gene Gbx2 and an anterior
shift in organizer genes (Fgf8/Wnt1). A further study showed that
GBX2 and EN1/2 are both required for FGF8b to regulate some midbrain/r1 genes
(Liu and Joyner, 2001b
).
Recently, the functions of FGF8a and b also were elegantly compared in
chick following electroporation of different concentrations of DNA expression
constructs. Similar to what was observed in mouse, Fgf8a causes
expansion of the midbrain and Fgf8b transforms the midbrain into a
cerebellum based on early gene expression changes and later morphology
(Sato et al., 2001
).
Interestingly, the initial effect of FGF8b is to reduce growth of the
midbrain. Thus, FGF8a and b have distinct activities, both on growth and
regulation of gene expression. Of relevance, 100 times lower levels of
Fgf8b induce an expanded midbrain. These results, and other studies
(Martinez et al., 1999
;
Liu et al., 1999
), have led to
the suggestion that a high level of FGF8 signaling induces cerebellum
development and a lower level induces midbrain development. If this is the
case, then strongly inducing the FGF pathway using activating mutations in
FGFRs should mimic the effects of FGF8b. Furthermore, given the dual functions
of FGF8 proteins in midbrain and cerebellum development, it is important to
determine whether FGF17 and 18 are similar to FGF8a or b.
FGF signaling is mediated by fibroblast growth factor receptor (FGFR)
proteins, which belong to a family of tyrosine kinase-containing transmembrane
proteins that bind to FGF molecules and mediate FGF signaling (reviewed by
Powers et al., 2000
).
Loss-of-function studies in mouse have demonstrated that various FGFRs are
essential in processes such as gastrulation, limb outgrowth and lung
morphogenesis (reviewed by Liu and Joyner,
2001a
). In vitro studies have indicated that in the presence of
heparin, all three FGFs present in the mid/hindbrain region bind to the c
isoforms of FGFR2 and FGFR3 with high affinity, but not to FGFR1
(Blunt et al., 1997
;
Xu et al., 1999
).
Interestingly, in mouse and chick embryos Fgfr2 and Fgfr3
are not expressed near the mid/hindbrain organizer and Fgfr1 is
expressed at low levels (Ishibashi and
McMahon, 2002
; Walshe and
Mason, 2000
), raising the question of whether FGFR2/3 mediate FGF
signaling from the organizer. Indeed, a recent study of mice lacking
Fgfr1 specifically in the midbrain and r1 showed that Fgfr1
is the primary FGF receptor required in midbrain and cerebellum development
(Trokovic et al., 2003
).
The Sprouty (Spry) family of proteins are antagonists of multiple tyrosine
kinase-containing receptors including those for epidermal growth factor and
FGF. In Drosophila, spry is expressed in cells receiving Fgf signals,
and loss of spry phenocopies gain-of-function mutations in
fgf (breathless) or fgfr (branchless)
(Hacohen et al., 1998
). There
are multiple Spry members in the vertebrates, two of which (Spry1 and
Spry2) are expressed in the mid/hindbrain region of mouse and chick
embryos and induced by FGF (4 or 8b)-soaked beads in chick embryos
(Chambers et al., 2000
;
de Maximy et al., 1999
;
Minowada et al., 1999
). Thus,
similar to other signaling pathways, FGF induces a negative feedback loop, and
a fine balance between activating and suppressing signaling must be required
for proper midbrain and cerebellum development.
In this study, we compared the activity of FGF17b and FGF18 to FGF8 in
midbrain/cerebellum development using the chick electroporation assay.
Strikingly, mis-expression of Fgf17b or Fgf18 at similar
levels to Fgf8 induced expansion of the midbrain and regulation of
midbrain genes similar to FGF8a. Of significance, among the four FGF proteins
tested, only FGF8b induces Gbx2 and represses Otx2 producing
a broad Gbx2+/Otx2 domain that abuts the Otx2
positive cells in the remainder of the midbrain. Interestingly, FGF8b induces
organizer genes at the new Gbx2/Otx2 border, whereas FGF8a induces
Fgf8 in scattered cells in the midbrain. In addition, only FGF8b
strongly induces the feedback inhibitors Spry1 and Spry2,
and we show that Spry1 is a direct target of FGF8 signaling.
Consistent with the idea that FGF8b induces a higher level of signaling,
mis-expression of activated FGFRs leads to induction of Gbx2 and
Spry1/2 and repression of Otx2 similar to FGF8b, although
the induction is in scattered cells and does not produce a late phenotype of
cerebellum induction.
 |
Materials and methods
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Techniques
Mouse brain explant culture, RNA in situ hybridization of wholemount tissue
and sections was carried out as previously described
(Liu and Joyner, 2001b
;
Liu et al., 1999
). In ovo
electroporation in chicken embryos was performed as described previously
(Timmer et al., 2001
) with
some modifications. Specifically, cDNAs for mouse Fgf8a
(Lee et al., 1997
),
Fgf8b (Liu et al.,
1999
), Fgf17b (Xu et
al., 1999
) and Fgf18
(Maruoka et al., 1998
), and
human mutant FGFR genes were cloned into a chicken expression vector
pMiwIII (Muramatsu et al.,
1997
) such that they are under the control of a chicken
ß-actin promoter. Two mutant forms of human FGFR1, N546K and
K656E (M. Mohammadi, unpublished), as well as one mutant form of human FGFR2,
C342Y (Mansukhani et al.,
2000
), were used in this study. The expression constructs were
injected into the midbrain ventricles of stage 9-12 chicken embryos
(Hamburger and Hamilton, 1992
)
and two electrodes were placed on either side of the rostral brain. Five
rectangular electric pulses of 10 volts, for 50 mseconds were then
delivered.
Reagents
Human FGF17b protein was kindly provided by Shaun K. Olsen and M.
Mohammadi. An in situ probe for chicken Gbx2 was generated by RT-PCR
from stage 18 chicken brain RNA according to chicken sequences published in
GenBank. A probe for chicken Otx2 was made by Dado Boncinelli. The
chick Fgf8 probe was from Brigid Hogan, and Spry1 and
Spry2 probes were from Gail Martin. The chick Wnt1 probe was
from Marion Wassef.
 |
Results
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FGF8b can regulate expression of mouse Fgf18, Spry1 and 2, Fgfr2
and 3
Previous studies in mouse showed that Fgf8, Fgf17 and
Fgf18 are all expressed in the isthmus region of eight- to
nine-somite mouse embryos with Fgf8 in the broadest domain
(Xu et al., 2000
). To
determine the temporal sequence of mid/hindbrain expression of the three Fgf
genes, we examined gene expression in whole mouse embryos. Fgf8
expression was first seen at the four-somite stage
(Fig. 1A,C), whereas
Fgf18 expression was not detected until slightly later at the
five-somite stage (Fig. 1D,E
and data not shown). Fgf17 expression was first detected in the
midbrain/r1 region at the six-somite stage
(Fig. 1I and data not shown).
At E9.5, both Fgf17 and Fgf18 were strongly expressed in
domains encompassing the posterior midbrain and anterior r1
(Fig. 1F,J), with the
Fgf17 expression domain being broadest. Fgf8 expression, by
contrast, was restricted to a small domain at the anterior border of r1
(Fig. 1B).

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Fig. 1. FGF8 begins to be expressed in the mouse mid/hindbrain region prior to
Fgf18 and can induce Fgf18 expression in mouse midbrain
explants. (A-C) Fgf8 is first expressed (arrowheads) in the
mid/hindbrain region at the four-somite stage (C). (D-F) Fgf18 is
first expressed in the mid/hindbrain region at the five-somite stage
(arrowhead in E) and becomes restricted to a narrow transverse band straddling
the isthmus by E9.5 (arrowhead in F; asterisk in D indicates the presumptive
mid/hindbrain junction region). (G,H) Fgf18 is induced by
FGF8b-soaked beads (arrowhead in H) by 48 hours, but not by BSA-soaked beads
(G). Inset in H shows that Fgf18 is induced by FGF8b by 16 hours.
(I,J) Fgf17 expression is first detectable in the mid/hindbrain
region at the six-somite stage and at E9.5 it is in a broad domain on both
sides of the mid/hindbrain junction (arrowheads). (K,L) Fgf17 is not
induced after 48 hours by either the BSA-soaked or FGF8-soaked beads in
rostral midbrain explants. Arrowhead in L indicates the endogenous
Fgf17 expression sustained in the explant. Broken lines in F and J
indicate the tissues used for the explant assays.
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As Fgf8 expression precedes that of Fgf17 and
Fgf18, we investigated whether FGF8 can regulate the expression of
Fgf17 and Fgf18 in the mouse brain. Explants were taken from
the anterior midbrain at E9.5 and cultured with FGF8b-soaked beads as
described previously (Liu et al.,
1999
). By 40 hours, Fgf18 was induced by FGF8b-soaked
beads (n=4/4, Fig. 1H)
but not by BSA-soaked beads (n=0/4,
Fig. 1G). By contrast,
Fgf17 was not induced by either FGF8b-soaked beads (n=0/4,
Fig. 1L) or BSA-soaked beads
(n=0/4, Fig. 1K). A
time course of Fgf18 induction was then performed. Fgf18
mRNA was not detected in midbrain explants after 8 hours of exposure to FGF8b
(n=4), but was present by 16 hours (n=3) (inset in
Fig. 1H and data not shown). We
have previously shown that Fgf8 is not induced by FGF8 in the same
assay (Liu et al., 1999
).
Taken together, these studies demonstrate that Fgf8 is the first Fgf
expressed in the mid/hindbrain region and suggest that it, in turn, induces
Fgf18 (directly or indirectly) in surrounding cells.
Induction of Spry1 and Spry2 by FGF-soaked beads has been
shown to occur more rapidly in chick embryonic brains than other midbrain/r1
genes such as En and Wnt1
(Chambers et al., 2000
;
Minowada et al., 1999
). We
therefore sought to determine whether the Spry genes are direct
targets of FGF8b using our mouse brain explant assay, as protein synthesis
inhibitors can be added to the medium. First we examined whether expression of
the mouse Spry genes are similarly controlled by FGF signaling, using explants
from prosomere 1 (p1), where neither Spry is expressed
(Fig. 2A,B). Similar to in the
chick, we found that Spry1 and Spry2 were rapidly induced
within 4 hours by FGF8b-soaked beads (n=4/4 for each gene,
Fig. 2D,H and data not shown),
but not BSA-soaked beads (n=0/4 for each gene,
Fig. 2C), in p1 explants. Next
we added 50 µg/ml cyclohexamide or ethanol to the medium, and found that
Spry1 is not induced by this treatment
(Fig. 2E,G), whereas
Spry2 is induced by cyclohexamide alone (data not shown). Regulation
of Spry1 by FGF8b in the presence of the protein synthesis inhibitor
was then tested, and indeed Spry1 was found to be induced
(n=6/6; Fig. 2F). By
contrast, the induction of En1, En2 and Gbx2 by FGF8b-soaked
beads was efficiently blocked by cyclohexamide (data not shown), consistent
with our observation that it takes at least 8 hours for these genes to be
induced by FGF8b-soaked beads (Liu and
Joyner, 2001b
). These results show that induction of
Spry1, but not of En1, En2 and Gbx2 by FGF8b is
direct.

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Fig. 2. Spry1 is induced by FGF8b-soaked beads in prosomere 1 explants in
the absence of protein synthesis. Spry1 (A) and Spry2 (B)
are expressed in broad domains of the mid/hindbrain region at E9.5
Spry1 is induced by FGF8b-soaked beads within 4 hours of culture (D),
but not by BSA-soaked beads (C). (E-H) Spry1 is induced within 6
hours by FGF8 in the presence or absence of 50 µg/ml cyclohexamide (CHX) in
explants grown in medium containing 0.1% ethanol, but not by BSA-soaked beads.
Red arrows in D,F,H indicate induced gene expression. Green circles in C and H
indicate beads that were lost during processing of the tissues. Broken lines
in A and B indicate the tissues used for the explant assays.
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As Fgfr2 and Fgfr3 are not expressed in the cells
surrounding the Fgf8 domain in r1
(Walshe and Mason, 2000
;
Ishibashi and McMahon, 2002
)
(Fig. 3A-F), this raises the
question of whether FGF8b regulates these receptors as well as Fgf18.
We therefore examined the effects of FGF8b on the expression of Fgfr genes in
p1 brain explants. Fgfr1 was expressed at low levels in these
explants and the expression was not altered by FGF8b-soaked beads after 40
hours (Fig. 3G,H;
n=4/4). Fgfr2 expression was maintained in p1 brain explants
in the presence of BSA-soaked beads (n=4/4), with the highest level
being along the dorsal midline (Fig.
3I). Significantly, FGF8b-soaked beads downregulated
Fgfr2 expression in the surrounding cells
(Fig. 3J; n=4/4).
Fgfr3 expression was also maintained in control p1 brain explants
(Fig. 3K; n=4/4), and
was repressed by FGF8b-soaked beads (Fig.
3L; n=4/4). The repression of Fgfr3 by FGF8b
seemed to be more efficient than repression of Fgfr2, consistent with
Fgfr3 expression being more restricted to the rostral midbrain at
E9.5 than Fgfr2.

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Fig. 3. Fgfr2 and Fgfr3 expression is excluded from the
mid/hindbrain junction and repressed by FGF8b. (A,B) Fgfr1 is weakly
expressed throughout the embryo at the five-somite stage and E9.5, with the
exception of the heart (h in B) at E9.5. (C,D) Fgfr2 is expressed in
the brain at E8.5 and E9.5 in the forebrain, rostral midbrain and part of the
posterior hindbrain (arrowheads), but excluded from the mid/hindbrain region
(asterisks). (E,F) In the brain, Fgfr3 is expressed weakly in the
caudal forebrain and part of the posterior hindbrain (arrowheads), but not in
the mid/hindbrain region (asterisks) at the five-somite stage and E9.5. Note
the strong expression in the extra-embryonic tissues at E8.5. (G,H) Weak and
patchy Fgfr1 expression is seen in p1 explants after 48 hours and
this expression is not altered by FGF8b-soaked beads. (I) In control p1
explants, strong Fgfr2 expression is limited to the dorsal midline,
whereas weak expression is maintained in the rest of the explants. (J)
Fgfr2 expression is downregulated by FGF8b-soaked beads. (K)
Fgfr3 is maintained in BSA-treated p1 explants, whereas
Fgfr3 is repressed by FGF8b-soaked beads (L). Broken lines in B,D,F
indicate the tissues used for the explant assays.
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FGF17b and FGF18 have a similar activity to FGF8a
Given that Fgf17 and Fgf18 are expressed in the midbrain,
as well as in r1, it is important to determine the activity of these FGFs
compared with FGF8a and FGF8b. A previous study using beads soaked in FGF18
and placed in the caudal diencephalon showed that it can induce Fgf8
and apparently an ectopic midbrain after 3 days
(Ohuchi et al., 2000
), but did
not address whether it can induce a cerebellum or regulate other genes. The
function of FGF17 in the midbrain/r1 has not been explored in such a
gain-of-function assay. Only one isoform of FGF18 has been described and it
contains a 12 amino acid insert in the same position that FGF8b has an 11
amino acid insert compared to FGF8a (Xu et
al., 1999
). Three isoforms of FGF17 have been described and one
(referred to as FGF17b) has an 11 amino acid insert in a similar position to
FGF8b, whereas FGF17a lacks this insert. FGF17c has a stop codon that
truncates the protein before the conserved FGF domain. To compare the activity
of FGF17b and FGF18 with FGF8a/b in midbrain and cerebellum induction we used
the electroporation assay described by Sato et al.
(Sato et al., 2001
) in which
FGF8a induces midbrain development and FGF8b represses midbrain development
and later induces a cerebellum. Expression constructs containing mouse cDNAs
for Fgf17b or Fgf18 (see Materials and methods) were
electroporated into the midbrain and caudal forebrain region of chick embryos
(Fig. 4A) at stages 9-12
(Hamburger and Hamilton, 1992
)
at a concentration of 1 µg/µl, and examined for changes in midbrain
morphology and midbrain/r1 gene expression.

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Fig. 4. FGF8b represses midbrain development whereas FGF8a, FGF17b and FGF18
promote midbrain development. (A) Schematic diagram showing the in ovo
electroporation experiments. DNA (green) is injected into the midbrain by a
glass needle and five electric pulses are applied. DNA is driven toward the
anode and transfected only on the right side of the brain, whereas the left
side serves as an internal control. (B) Co-transfection of a GFP expression
vector with the experimental vector serves to show that most cells on the
right side of the brain, including the mid/hindbrain region and caudal
forebrain, are transfected. (C) Dorsal view of a wild-type E10.5 chicken
brain. (D) Dorsal view of an E10.5 chicken brain electroporated with
1µg/µl pMiw-Fgf8b; the asterisk indicates lack of midbrain
(mes) on the transfected right side. (E) Dorsal view of an E6.5 chicken brain
electroporated with 1 µg/µl pMiw-Fgf8a; the midbrain on the
transfected side is larger than the one on the control side. (F) Dorsal view
of an E8.5 chicken brain electroporated with 1 µg/µl
pMiw-Fgf17b; the midbrain on the transfected side is larger than the
one on the control side. (G) Dorsal view of an E8.5 chicken brain
electroporated with 1 µg/µl pMiw-Fgf18; the midbrain on the
transfected side is larger than the one on the control side. tel,
telencephalon; di, diencephlon; mes, mesencephalon; ce, cerebellum.
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As a control for our experiments, we repeated the experiments of Sato et
al. (Sato et al., 2001
) by
electroporating mouse Fgf8a and Fgf8b expression vectors at
different concentrations (Table
1). The transfection efficiency was monitored by visualizing the
expression of co-electroporated GFP (Fig.
4B) and by examining the transgene RNA using section in situ
hybridization (see Fig. 5). Similar to recent studies (Sato et al.,
2001
), when Fgf8b was electroporated into the midbrain
and caudal forebrain at a concentration of 0.1 µg/µl to 3 µg/µl,
midbrain development was repressed and the region was probably transformed
into an ectopic cerebellum (n=22/22;
Fig. 4D, compare with the control in
4C, and data not shown). By contrast, electroporation of 0.01
µg/µl Fgf8b (n=6/6, data not shown), or 1-2
µg/µl Fgf8a (n=5/5;
Fig. 4E) led to expansion of
the midbrain and transformation of the diencephalon into a midbrain. Analysis
of expression of the Fgf8a and Fgf8b mRNA produced by the
expression vectors showed that similar levels and patterns of expression were
obtained when 1-2 µg/µl of the DNA was used (inset in
Fig. 5A,B). As expected, no
Fgf8 mRNA was detected when 0.01 µg/µl Fgf8b was used
(data not shown).

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Fig. 5. FGF8b activates more molecular pathways than FGF8a, FGF17b and FGF18. (A-D)
1 µg/µl pMiw-Fgf8a induces Fgf8 (C), but does not
induce Gbx2 (B) or Spry1 (D), or repress Otx2 (A).
(E-H) 1 µg/µl pMiw-Fgf8b induces Gbx2 (F),
Fgf8 (G) and Spry1 (H) and represses Otx2 (E).
(I-L) 1 µg/µl pMiw-Fgf17b fails to induce Gbx2 (J),
Fgf8 (K) or Spry1 (L), or to repress Otx2 (I).
(M-P) 1 µg/µl pMiw-Fgf18 fails to induce Gbx2 (N),
Fgf8 (O) or Spry1 (P), or to repress Otx2 (M).
(Q-T) 1 µg/µl pMiw-caFGFR2 induces Gbx2 (R),
Fgf8 (S) and Spry1 (T) in scattered cells. Otx2 is
repressed on the electroporated side but scattered Otx2-expressing
cells still exist (Q). In all panels, coronal or near coronal sections are
shown with the anterior end towards the right. The broken lines indicate the
midline with the electroporated side above the line and the control side
below. In all panels, the red arrowheads indicate ectopic gene expression on
the electroporated side and the green arrowheads indicate endogenous
expression on the control side except for E and Q where the red arrowheads
indicate the electroporated side where Otx2 expression is repressed
(completely in E and incompletely in Q). Insets in A,C,E,G,I,M,Q show
expression of the mouse or human genes electroporated into the right side of
the brain.
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Strikingly, electroporation of 1 µg/µl Fgf17b
(n=15/15; Fig. 4F and
Table 1) or Fgf18
(n=17/17; Fig. 4G and
Table 1) led to expansion of
the midbrain, similar to the phenotype seen with Fgf8a. This
phenotype was not due to reduced levels of expression of the constructs
compared to the Fgf8b (or Fgf8a), as similar levels of mRNA
were produced by the Fgf17b and Fgf18 vectors (inset in
Fig. 5I,M). To analyze the
phenotype in more detail, chick embryos were processed for section RNA in situ
analysis 24 hours after electroporation and analyzed for midbrain/r1 gene
expression. Sato et al. (Sato et al.,
2001
) found that FGF8b induces Gbx2, Pax2/5, En1/2 and
represses Otx2 and Pax6, whereas FGF8a only induces
En1/2. Thus, a key distinction between the activity of FGF8a and
FGF8b is that only FGF8b induces Gbx2 and represses Otx2
(compare Fig. 5A,B with 5E,F).
Consistent with the similar phenotype produced by FGF17b, FGF18 and FGF8a,
neither FGF17b nor FGF18 induced Gbx2 or repressed Otx2
(Fig. 5I,J,M,N). We found that
electroporation of a low level of Fgf8b vector (0.01 µg/µl)
also did not alter Gbx2 and Otx2 expression (data not
shown). The response of the endogenous Fgf8 gene to the four FGFs was
very interesting. FGF8b (1 µg/µl) was found to induce Fgf8 in a
sharp band of cells at the new Gbx2/Otx2 boundary in the dorsal and
lateral midbrain (Fig. 5G; see
Fig. 7A,B). By contrast, FGF8a
induced Fgf8 in scattered cells in the midbrain
(Fig. 5C), whereas FGF17b and
FGF18 or low FGF8a did not induce detectable levels of Fgf8
(Fig. 5K,O and data not shown).
With all expression vectors, En2 was upregulated broadly (data not
shown). Finally, we examined the induction of Spry1 and
Spry2, as we found that Spry1 is a direct target of FGF8b
signaling. Only FGF8b strongly induced the Spry genes in the
midbrain, and FGF17b or FGF18 only weakly induced Spry1 in some
experiments (Fig. 5H,L,P and
data not shown). Taken together, the gene expression studies and morphological
analysis demonstrate that when Fgf17 and Fgf18 are expressed
at similar mRNA levels, they enhance midbrain development similar to
Fgf8a.

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Fig. 7. Activated FGFR1 and FGFR2 produce over-proliferation of the midbrain. (A)
Dorsal view of an E10.5 chicken brain electroporated with 1 µg/µl
pMiw-Fgfr1K656E, the midbrain on the transfected side is
larger than the one on the control side. (B) Dorsal view of an E7.5 chicken
brain electroporated with 3 µg/µl pMiw-Fgfr1N546K,
the midbrain on the transfected side is larger than the one on the control
side. (C) Dorsal view of an E7.5 chicken brain electroporated with 1
µg/µl pMiw-Fgfr2C342Y, the midbrain on the
transfected side is larger than the one on the control side. In all panels,
the right sides are the experimental sides and the left sides serve as
controls. Broken outline indicates the expanded midbrain.
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Given that FGF17b and FGF18 have similar FGF receptor-binding affinities to
FGF8b, at least for FGFR2 and FGFR3 in a tissue culture assay
(Xu et al., 2000
), it might be
expected that the three proteins activate FGF signaling to the same level. One
possible reason why electroporation of Fgf17b and 18
expression constructs does not produce similar changes in gene expression and
proliferation to Fgf8b is that production or secretion of FGF17b and
FGF18 protein is less efficient than FGF8b. To determine whether the same
concentrations of FGF17b or FGF8b have identical activities, we used our mouse
brain explant assay to compare the changes in gene expression induced by beads
soaked in FGF17b compared with FGF8b. Similar to the in vivo results, FGF17b
was found to induce En1 (n=7/7) and only weakly induce
Spry1 (n=6/6) in midbrain explants
(Fig. 6I,L; data not shown).
Furthermore, Gbx2 was induced in only three out of 12 midbrain
explants and FGF17b did not repress Otx2 (n=8/8) in midbrain
explants (Fig. 6C,F; data not
shown). In the same experiments, FGF8 strongly induce En1
(n=5/5), Spry1 (n=3/3) and Gbx2
(n=9/9), and repressed Otx2 (n=3/7)
(Fig. 6B,E,H,K; data not
shown). In all experiments, beads soaked in BSA had no effect on gene
expression (Fig. 6A,D,G,H;
n
2). Thus, the difference in activity of FGF17b compared with
FGF8b is most likely to be due to intrinsic differences in their ability to
induce the FGF signaling pathway.

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Fig. 6. FGF8b and FGF17b proteins differentially regulate genes in mouse brain
explants. Beads soaked in BSA, mouse FGF8b or human FGF17b, as indicated, were
placed in midbrain explants and cultured for 48 hours. Wholemount RNA in situ
analysis was then performed with the indicated probes. FGF8b strongly induces
Gbx2 (E), Spry1 (H) and En1 (K), and represses
Otx2 (B); FGF17b weakly induces En1 (L) and Spry1
(I).
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Activated FGFRs regulate gene expression similar to FGF8b
If the differential effects of FGF8a, 17b and 18 versus FGF8b
mis-expression on midbrain development are because FGF8b can activate the FGF
pathway more efficiently than the other FGFs, then ectopic expression of
activated Fgfr genes should have the same effect as Fgf8b. To test
this, we electroporated human FGFR constructs containing activating mutations
into the midbrain and caudal forebrain and examined gene expression after
24-36 hours and brain morphology at later stages. We chose three mutant forms
of FGFRs to test: one containing a mutation in the extracellular domain
(C342
Y) of FGFR2, which leads to receptor activation possibly by
inducing spontaneous dimerization of the receptors
(Mangasarian et al., 1997
);
and two containing mutations in the tyrosine kinase domains of FGFR3
[N540
K (Bellus et al.,
1995
)] or [K650
E
(Tavormina et al., 1995
)]. As
the kinase domains are very well conserved among different FGFRs, and
Fgfr1 but not Fgfr3 is expressed in the midbrain/r1 region,
activated forms of FGFR1 containing the N546K and K656E mutations were
used.
We examined gene expression changes in embryos electroporated with 1
µg/µl of the FGFR2C342Y or the
FGFR1N546K vector. Similar to FGF8b, Gbx2, Fgf8
and Spry1 and 2 were induced and Otx2 repressed in
the midbrain by FGFR2C342Y (Fig.
5Q,R,T and data not shown). Different from the homogeneous
alterations in gene expression produced by electroporation of Fgf8b,
the expression of the activated FGFR induced Gbx2, Fgf8 and
Spry and repressed Otx2 in patches of cells mainly in the
ventricular zone. This is probably due to the cell autonomous function of the
activated FGFR compared with the secreted FGF8b protein, as the level and
pattern of expression of the FGFR2C342Y mRNA was similar
to the mouse Fgf8 cDNA (inset in
Fig. 5Q). The
FGFR1N546K vector produced similar results, when assayed
for Fgf8 (n=2/3) and Gbx2 (n=3/3)
expression by whole-mount RNA in situ analysis (data not shown).
We next determined the long-term phenotype of transiently expressing
activated FGFRs in the midbrain. Unlike FGF8b, the three activated FGFRs
(FGFR1N546K, FGFR1K656E and
FGFR2C342Y) produced enlargement of the midbrain and
diencephalon (Fig. 7A-C;
Table 1 and data not shown). At
a DNA concentration of as high as 3 µg/µl, ectopic expression of
FGFR2C342Y or FGFR1N546K caused a
similar phenotype to that obtained with 1 µg/µl DNA
(Fig. 7B and data not shown).
The FGFR1K656E mutant at 3 µg/µl led to a
non-specific loss of the entire brain region including midbrain and
cerebellum, preventing a morphological or marker gene analysis
(Table 1 and data not shown).
Histological analysis of sections through E8-10 chicken embryos (n=2)
electroporated with FGFR2C342Y confirmed that the
long-term phenotype of activated FGFRs is an enlarged midbrain, as the
electroporated side showed the same histological features of the midbrain as
on the control side (data not shown).
One possible reason why transient mis-expression of the FGFRs leads to
expansion of the midbrain is that the upregulation of Gbx2 and
Spry1/2, and repression of Otx2 does not happen in a
sufficient number of cells to transform the midbrain into a cerebellum.
Indeed, Sato et al. (Sato et al.,
2001
) showed that co-electroporation of an Otx2
expression vector (1 µg/µl DNA) with an Fgf8b expression vector
(0.1 µg/µl DNA) results in expansion of the midbrain, indicating that
Otx2 positive cells that receive an FGF8b signal respond by expanding
the midbrain, whereas Gbx2 positive cells form a cerebellum.
Alternatively, or in addition, FGF8b may eventually lead to transformation of
the midbrain into a cerebellum because the organizer is extended along the new
Gbx2/Otx2 border and it maintains the transformation. In order to
explore these ideas further, we analyzed whole-mount embryos mis-expressing
Fgf8b or FGFR2C342Y for expression of genes
normally expressed near the organizer region. In embryos electroporated with
the Fgf8b vector, the normal rings of Fgf8 (n=4),
Wnt1 (n=4) and En1 (n=3) at the
mid/hindbrain junction were repressed on the electroporated side and both
genes were induced along the dorsal midline and in a transverse band in the
caudal diencephalon, probably adjacent to the induced Gbx2 domain
(Fig. 8A-D; n=4 for
Fgf8, n=3 for En1). By contrast, expression of the activated
FGFR vector induced Fgf8, En1 and Wnt1 only in
patches of cells in the midbrain and caudal diencephalon
(Fig. 8E,F and data not shown).
The resolution of the experiment is not such that we can determine whether
these genes are induced at new Gbx2/Otx2 borders. Taken together,
these studies are consistent with the idea that a high level of FGF signaling
is required to induce Gbx2 and repress Otx2 and that
transformation of a broad region into Otx2-/Gbx2+ cells is required
for late cerebellum formation.

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Fig. 8. FGF8b transforms the midbrain into a cerebellum and shifts the
mid/hindbrain organizer rostrally. (A) Experiments shown in B, C and D. 24
hours after 1 µg/µl pMiw-Fgf8b was electroporated into the
midbrain, Fgf8 expression is shifted into the caudal forebrain region
on the experimental side, as well as in a thin band along the dorsal midline
(red lines in A and arrowheads in B) that connects the ectopic Fgf8
domain to the endogenous Fgf8 domain on the control side. Green arrow
shows the down regulation of Fgf8 expression on the transfected side
in the isthmus. Inset shows the rear view of the same embryo. (C) 24 hours
after 1 µg/µl pMiw-Fgf8b is electroporated into the chicken
midbrain En1 expression is shifted rostrally on the electroporated
side, and seen in the most dorsal cells in the midbrain and anterior hindbrain
(red arrowheads), whereas the endogenous expression surrounding the isthmus
(green arrow) is downregulated. Inset shows a rear view of the same embryo,
note the normal expression on the control (left) side. (D) 24 hours after 1
µg/µl pMiw-Fgf8b is electroporated into the chicken midbrain,
the endogenous Wnt1 expression in the isthmus (green arrow) is
downregulated, whereas ectopic expression is induced near the dorsal midline
and in a transverse band in the rostral midbrain. Inset shows a rear view of
the same embryo, note the normal expression on the control (left) side. (E)
Scattered expression of Fgf8 is induced in the midbrain and caudal
forebrain (arrowheads) by ectopic expression of activated FGFR2. Note that the
endogenous Fgf8 expression is not repressed. (F) Scattered expression
of Wnt1 is induced in the midbrain and caudal forebrain (arrowheads)
by ectopic expression of activated FGFR2. Inset shows a rear view of the same
embryo.
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|
 |
Discussion
|
---|
FGF signaling regulates positive and negative feedback loops
We show here that FGF8b can positively regulate FGF signaling by inducing
Fgf18 expression in brain explants, and that Fgf18
expression is initiated slightly later than Fgf8 in the mouse
mid/hindbrain junction region. Fgf17 is not expressed until later and
is not induced by FGF8 in mouse brain explants. Thus, FGF8 could normally be
required to induce expression of Fgf18. Furthermore, although the
three FGFs have overlapping spatial distributions after the six somite stage,
Fgf17 and Fgf18 should not be able to compensate for a loss
of FGF8 protein because they are expressed too late and Fgf18
expression is dependent on FGF8 function. A recent study of mice lacking
Fgf8 function specifically in the midbrain/r1 region after the five
somite stage showed that both Fgf17 and Fgf18 are actually
dependent on Fgf8, as their expression is greatly reduced at the
seven- to nine-somite stage and gone by the 12- to 15-somite stage in such
mutants (Chi et al., 2003
).
Thus, Fgf8 mutants are equivalent to Fgf8/17/18 triple
mutants, and determination of the normal requirement for Fgf17/18 in
the midbrain and cerebellum will await analysis of Fgf17/18 double
mutants.
Consistent with the expression patterns of Fgfr1, Fgfr2 and
Fgfr3, we found that FGF8b is sufficient to repress expression of
Fgfr2 and Fgfr3 in caudal forebrain explants. A study of
zebrafish ace mutants that have a mutation in fgf8 showed
that FGF8 is also required to restrict fgfr3 from the mid/hindbrain
junction, because in ace mutants fgfr3 is mis-expressed in
the midbrain and r1 (Sleptsova-Friedricha
et al., 2001
). Thus, Fgf8 negatively regulates FGF
signaling by repressing two FGF receptors. Although Fgfr1 is the key
receptor that mediates FGF signaling in r1 and the caudal midbrain
(Trokovic et al., 2003
), the
other two FGF receptors might play a role in mediating a low level of FGF
signaling in anterior regions of the midbrain.
Expression of two negative regulators of FGF signaling, Spry1 and
Spry2, in a broad domain surrounding the mouse mid/hindbrain junction
region has indicated that FGF signaling is attenuated by SPRY proteins in this
region. Interestingly, we found that only FGF8b strongly induces expression of
Spry1 and Spry2 in the chick anterior midbrain or mouse
brain explants. Furthermore, using a brain explant assay we demonstrated that
Spry1 (and probably Spry2) is a direct downstream target of
FGF8b signaling. This indicates that Spry1 expression can be used as
a read-out for FGF signaling. Consistent with this, in mouse embryos lacking
Fgf8 in the midbrain/r1 after the six-somite stage, Spry2 is
maintained at the 7- to 9-somite stage, but greatly reduced by the 13-16
somite stage (Chi et al.,
2003
).
Taken together, our studies and others show that in mouse FGF8b regulates
at least three components of the FGF signaling pathway. First, FGF8b induces
expression of another FGF protein, FGF18. FGF8b also directly induces two
negative modulators of the pathway (SPRY 1/2), and thus produces a
negative-feedback loop. Furthermore, our finding that FGF8b also represses
Fgfr2 and Fgfr3 demonstrates that a second negative feedback
loop contributes to fine regulation of the level of FGF signaling in r1 and
the midbrain to ensure appropriate patterning and growth.
FGF8b has a distinct activity from FGF8a, FGF17b and FGF18 in the
midbrain
To gain insight into how three Fgf genes orchestrate midbrain and
cerebellum development, we explored the activity of FGF17b and FGF18 in
comparison to FGF8a and FGF8b in their ability to regulate cell proliferation
and gene expression when mis-expressed in the midbrain. Of the four proteins,
only FGF8b has the ability to transform the midbrain into a cerebellum.
Associated with this unique activity, only FGF8b can induce Gbx2 and
repress Otx2 when expressed in the midbrain. Furthermore, and likely
of crucial importance for maintaining the transformation, only FGF8b induces
an ectopic organizer region at the new Gbx2/Otx2 border in the
midbrain. By contrast, FGF8a, FGF17b and FGF18 induce expansion of the
midbrain, and do not alter Gbx2 or Otx2 expression.
Spry1/2 is strongly induced by FGF8b and only weakly by FGF17b and
FGF18, whereas endogenous Fgf8 is only induced locally by FGF8a. This
different activity of FGF8b protein can not be due to a higher level of
expression of the Fgf8b construct, as it is only at a 100-fold lower
DNA concentration at which the mouse Fgf8b mRNA can not even be
detected that FGF8b induces a midbrain. As Fgf17and 18 are
expressed in the midbrain, although Fgf8 is restricted to r1,
Fgf17 and Fgf18 could be the main FGFs that normally
directly regulate growth and patterning of the midbrain.
Mouse mutant analyses have shown that Fgf17 is more important in
the midbrain than Fgf18, because only Fgf17 mutants have a
truncation of the posterior midbrain (Xu
et al., 2000
; Liu et al.,
2002
; Ohbayashi et al.,
2002
). Our comparison of the activities of FGF17 and FGF18 show
that Fgf18 could also function with Fgf17 in regulating
midbrain development. Loss-of-function studies have also shown that
Fgf17 plays a role, along with Fgf8, in regulating late
proliferation of the anterior cerebellum
(Xu et al., 2000
). Our finding
that FGF17b and FGF18 have such distinct activities from FGF8b in the midbrain
are in contrast to previous tissue culture studies that indicated the proteins
have similar binding affinities to FGFR2c and FGFR3c and similar functions in
regulating proliferation (Xu et al.,
1999
; Xu et al.,
2000
). One possibility was that FGF17b and FGF18 proteins are not
produced or secreted as efficiently as FGF8b in the chick midbrain. We have
ruled out this possibility by showing that when similar concentrations of
FGF17b and FGF8b protein are compared in mouse brain explant assays, they
differentially regulate gene expression similar to the electroporation
experiments. Thus, the intrinsic activity of FGF8b is different from that of
FGF17b, possibly because the 11 amino acid inserts in the two proteins are
distinct. Our study demonstrates the importance of testing the activity of
proteins in vivo where they normally function. Finally, although Fgf8
alone encodes two proteins sufficient for directing development of both the
midbrain and cerebellum, Fgf17 and Fgf18 probably augment
the proliferative and midbrain inducing ability of Fgf8a or a low
level of Fgf8b.
Activated FGFRs regulate midbrain/r1 genes similar to FGF8b
It is possible that the difference in the phenotypes produced by
mis-expression of Fgf8a versus Fgf8b is quantitative,
because in vitro studies have shown that FGF8b has a much higher affinity for
FGFRs than FGF8a. Consistent with this, electroporation of a low concentration
of Fgf8b expression vector has similar effects to high concentrations
of Fgf8a (Sato et al.,
2001
), and some Wnt1-Fgf8a transgenics have phenotypes
similar to Wnt1-Fgf8b transgenics
(Liu et al., 1999
). By
contrast, FGF17b and FGF18 have similar binding affinities and proliferation
activities to FGF8b in vitro (Xu et al.,
1999
; Xu et al.,
2000
), but do not behave like FGF8b when mis-expressed in the
midbrain or applied to brain explants. However, the biochemical studies were
carried out using FGFR2 and FGFR3, which are not the major receptors that
mediate midbrain/r1 patterning (Trokovic
et al., 2003
). It is possible that there are qualitative
differences in the way FGF8b interacts with FGFR1, that allow FGF8b to
activate the downstream pathway more efficiently. We addressed this
possibility by asking whether high level FGF signaling is sufficient to induce
Gbx2 and repress Otx2 using activating mutations in FGFR1
and FGFR2. Indeed, the activated FGFRs regulate key target genes similar to
FGF8b. Of significance, the activated FGFRs strongly induce Spry1/2
and Gbx2 and repress Otx2.
Given the changes in gene expression induced by activated FGFRs, it was
perhaps surprising that the long-term phenotype of transient expression of
activated FGFRs is expansion of the midbrain. We suggest that in transient
mis-expression studies such as electroporations, Gbx2 must be induced
in a homogeneous domain so that a new organizer can form along the extended
Gbx2/Otx2 border, and the organizer can then maintain the long-term
transformation of the midbrain into a cerebellum. In support of this idea,
when Gbx2 is electroporated into the midbrain, Otx2 is only
transiently repressed in scattered cells in the anterior midbrain, and
although the isthmus is expanded anteriorly, no ectopic cerebellum forms
(Katahira et al., 2000
). As
electroporation produces mosaic gene expression, the secreted protein FGF8b,
but not the activated FGF receptor, can induce Gbx2 throughout the
electroporated region. In addition, although the activated FGFRs can induce
Fgf8 as well as Wnt1 and En1 in the midbrain, it is
in patches of cells because of the cell-autonomous nature of the receptors. As
the response of Otx2-expressing midbrain cells to FGF8b is
proliferation of the midbrain (Sato et
al., 2001
), and there are Otx2-positive cells present on
the side of the midbrain electroporated with the activated FGFRs, this could
account for the later expansion of the midbrain.
 |
Conclusions
|
---|
Based on our studies and those of others, we suggest the following steps in
midbrain and cerebellum development in mouse
(Fig. 9). At the four-somite
stage, Fgf8 is induced in the presumptive r1 territory by an unknown
factor. Pax2 is required for this induction
(Ye et al., 2001
) and OTX2
inhibits Fgf8 from being induced in the midbrain
(Li and Joyner, 2001
;
Martinez-Barbera et al.,
2001
). FGF8b then induces Fgf18 in the surrounding cells,
producing a larger domain and gradient of Fgf mRNA that extends into
the midbrain. FGF8b also maintains two negative feedback loops by inducing
Spry1 and Spry2 expression and inhibiting Fgfr2 and
Fgfr3. Fgf17 is then induced by an unknown mechanism that is
dependent on Fgf8 (Chi et al.,
2003
) in a broader domain than Fgf18, further extending
the gradient of Fgf mRNA expression. FGF17 and FGF18 protein, and possibly
FGF8a and a low level of FGF8b, then regulate proliferation of the midbrain
and cerebellum and En expression. The narrow domain where
Fgf8 is expressed becomes the isthmus because of the activity of
FGF8b (Li et al., 2002
), and
the adjacent Otx2-negative r1 cells become the cerebellum. We have
recently shown that by the 15-somite stage Gbx2 is not required in r1
for cerebellum development, but is required earlier to specify r1
(Li et al., 2002
). Thus, once
Fgf8 expression in r1 is stabilized, perhaps by a secreted factor
from the midbrain (Irving and Mason,
1999
), a key function of high level signaling by FGF8b is to
maintain a cascade of gene expression in the midbrain/r1 that maintains an
Otx2-negative domain in r1 in which the cerebellum develops.

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Fig. 9. FGF signaling is autoregulated at multiple levels and multiple FGF proteins
regulate midbrain and cerebellum development. In the mouse, FGF8 expression in
the isthmus at the four-somite stage represses the expression of
Fgfr2 and Fgfr3 and activates the expression of
Fgf18 at the five-somite stage. Fgf17 expression is
initiated in a broader domain slightly later, and by E9 the three Fgfs are
expressed in overlapping gradients radiating from the isthmus, whereas
Fgfr2 and Fgfr3 are absent in this region. Spry1/2
genes are upregulated by FGFs. FGF8b is required to maintain a cascade of gene
expression that includes absence of Otx2 in r1, allowing cerebellum
development to occur. FGF17 and FGF18, and possibly FGF8a and a low level of
FGF8b regulate growth and En expression in the midbrain.
|
|
 |
ACKNOWLEDGMENTS
|
---|
We are grateful to Shaun Olsen and M. Mohammadi for providing the FGF17b
protein. We thank Drs MacArthur, Mohammadi, and Mansukhani and Basilico for
providing us with the cDNAs for Fgf8b, mutant FGFR1, and
mutant FGFR2, respectively. We also thank Drs Martin, Ornitz, Hogan
and Wassef for providing us with in situ probes for mouse Spry1 and
Spry2, mouse Fgfr1, Fgfr2, Fgfr3, Fgf17, Fgf18, chicken
Fgf8, Wnt1 and chicken Otx2. We are grateful to Mark Zervas
and Sema Sgaier for critically reading the manuscript. L.A.N. and A.L.J. are
investigators of the Howard Hughes Medical Institute, and A.L. is a Howard
Hughes Medical Institute research fellow. Some of this work was supported by a
grant from NINDS (R01-NS35876) to A.L.J. and an MSKCC support grant to L.A.N.
J.Y.H.L. is supported by an NIH fellowship.
 |
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