RESEARCH ARTICLE 2457
Development 136, 2457-2465 (2009) doi:10.1242/dev.032656
FGF signaling is strictly required to maintain early
telencephalic precursor cell survival
Hunki Paek, Grigoriy Gutin and Jean M. Hébert*
The FGF family of extracellular signaling factors has been proposed to play multiple roles in patterning the telencephalon, the
precursor to the cerebrum. In this study, unlike previous ones, we effectively abolish FGF signaling in the anterior neural plate via
deletion of three FGF receptor (FGFR) genes. Triple FGFR mutant mice exhibit a complete loss of the telencephalon, except the
dorsal midline. Disruption of FGF signaling prior to and coincident with telencephalic induction reveals that FGFs promote
telencephalic character and are strictly required to keep telencephalic cells alive. Moreover, progressively more severe truncations
of the telencephalon are observed in FGFR single, double and triple mutants. Together with previous gain-of-function studies
showing induction of Foxg1 expression and mirror-image duplications of the cortex by exogenous FGF8, our loss-of-function results
suggest that, rather than independently patterning different areas, FGF ligands and receptors act in concert to mediate organizer
activity for the whole telencephalon.
INTRODUCTION
How a morphologically uniform sheet of cells becomes patterned
into molecularly, functionally and structurally distinct areas remains
one of the principal puzzles facing developmental biologists. In
particular, the mechanisms involved in patterning the central
nervous system, which begins as a sheet of neuroepithelial cells
during embryogenesis, remain poorly understood. The anterior
region of the neuroepithelium develops into the telencephalon, the
precursor to the cerebral hemispheres in which our highest cognitive
processes reside. This study addresses how the anterior
neuroepithelium acquires and maintains a telencephalic fate.
The earliest and most specific marker for telencephalic fate is the
expression of Foxg1, a gene encoding a winged helix transcription
factor (Shimamura and Rubenstein, 1997; Tao and Lai, 1992).
Foxg1 expression initiates shortly after the anterior neuroepithelium
(or neural plate) bends to form the head folds. Other genes (Pax6,
Nkx2.1, Otx1, Six3, Dlx2 and Emx genes) are also expressed in the
telencephalic anlage at early stages, although their expression either
extends beyond telencephalic precursors to diencephalic ones or
turns on later than Foxg1 (Puelles and Rubenstein, 2003;
Shimamura et al., 1995; Wilson and Houart, 2004).
The anterior neural ridge (ANR, or anterior neural border, ANB,
in zebrafish), which is the junction between the anterior neural and
non-neural ectoderm, is essential for induction of the telencephalon.
In mice and zebrafish, removal of the ANR/ANB prior to the
specification of telencephalic character causes a failure to induce
Foxg1 expression (Houart et al., 1998; Shimamura and Rubenstein,
1997). Moreover, in zebrafish, transplanting the ANB to the
diencephalon or midbrain at the mid-gastrula stage induces ectopic
expression of markers for the dorsal and ventral telencephalon
(Houart et al., 1998). This indicates that the ANR acts to induce the
telencephalon, perhaps acting as a classic organizer. However, the
molecular mediators of this activity remain unknown.
Departments of Neuroscience and Genetics, Albert Einstein College of Medicine,
Bronx, NY 10461, USA.
*Author for correspondence (e-mail: [email protected])
Accepted 11 May 2009
There are two leading candidates for ANR-expressed
telencephalic inducers. The first are secreted frizzled-related WNT
antagonists (sFRPs). Tlc, an sFRP in zebrafish, is expressed in the
ANB and is necessary and sufficient to induce telencephalic gene
expression (Houart et al., 2002). In mice, however, evidence
supporting the role of sFRPs in telencephalon induction is lacking
(Satoh et al., 2006). The second candidate for ANR-derived
telencephalic inducers are fibroblast growth factors (FGFs). FGFs
are developmentally important intercellular signaling molecules that
can activate the Ras/MAPK, PLCγ, PI3 kinase and STAT1 pathways
upon binding to FGF receptors (Mason, 2007). During early
telencephalic development in mice, at least 5 of the 22 genes
encoding FGF ligands, Fgf3, Fgf8, Fgf15, Fgf17 and Fgf18, are
expressed in the ANR or its early derivatives, and three of the four
receptor genes, Fgfr1, Fgfr2 and Fgfr3, are expressed broadly in the
anterior neuroectoderm (Maruoka et al., 1998; McWhirter et al.,
1997; Orr-Urtreger et al., 1991; Peters et al., 1993; Peters et al.,
1992; Rash and Grove, 2007; Shinya et al., 2001; Yamaguchi et al.,
1992).
FGF signaling acts at the end of gastrulation to posteriorize the
neural plate and later to pattern the telencephalon (Mason, 2007;
Wilson and Houart, 2004; Wilson and Rubenstein, 2000). However,
as no FGF or FGFR mutant in either mouse or zebrafish has yet to
result in a failure to form a telencephalon, FGF signaling has not
been considered a telencephalic inducer or a mediator of organizing
activity for this tissue. Nevertheless, implanting FGF8-soaked beads
in anterior neural explants lacking an ANR induces Foxg1
expression (Shimamura and Rubenstein, 1997), consistent with a
role in inducing the telencephalon. One reason why a telencephalon
is still observed in the mutants examined to date is that functional
compensation may occur between family members with overlapping
expression domains. Alternatively, studies suggest distinct roles for
FGFs in the developing telencephalon (Borello et al., 2008; Cholfin
and Rubenstein, 2007; Cholfin and Rubenstein, 2008).
In this study, the in vivo requirement for FGF signaling in
initiating telencephalon formation is investigated. In order to
effectively abolish FGF signaling in the anterior neural plate, floxed
alleles of Fgfr1 and Fgfr2 are deleted using two different Cre driver
lines in an Fgfr3–/– background. Our results indicate that except for
the dorsal midline, formation of the telencephalon is entirely
DEVELOPMENT
KEY WORDS: Forebrain, FGF receptors, Neural plate, Neural patterning, Mouse
2458 RESEARCH ARTICLE
MATERIALS AND METHODS
Generation of mutant embryos
To study the role of FGF signaling in initiating telencephalon formation, two
crosses were used to generate mice mutant for all three FGFR genes
expressed in the neural plate (Fig. 1A, Fig. 6A). A loss of Fgfr1 or Fgfr2 in
the whole embryo leads to lethality at gastrulation (Arman et al., 1998; Deng
et al., 1994; Yamaguchi et al., 1994), therefore floxed alleles of these genes
(Trokovic et al., 2003; Yu et al., 2003) were used to generate knockouts
when crossed to Foxg1Cre or CAGG-CreER mice (Hayashi and McMahon,
2002; Hébert et al., 2003a) in an Fgfr3–/– background (Deng et al., 1996).
Control mice used were heterozygous for Fgfr1 and Fgfr2 and carried the
Cre allele, and these controls did not exhibit any of the phenotypes described
in this study. Triple FGFR mutant embryos were obtained at the ages
indicated in the expected ratios with no signs of necrosis (1:32 using the
Foxg1Cre driver and 1:8 using CAGG-CreER). A total of 25 triple
homozygous mutant embryos from the Foxg1Cre cross and 28 from the
CGGA-CreER cross were used.
RNA in situ hybridization
In situ hybridizations were performed according to two protocols; one using
radioactive RNA probes on sections and the other using digoxigenin (DIG)labeled probes in whole mount. For the radioactive in situs, frozen sections
were prepared and hybridized to 35S-labeled probes as previously described
(Frantz et al., 1994). For in situ hybridizations on whole-mount mouse
embryos using DIG-labeled probes, the procedure was performed as
described (Henrique et al., 1995), with the exception that BM Purple
(Roche) was used instead of NBT/BCIP to reveal expression patterns.
Cell proliferation and TUNEL assays
Embryos were collected at E9.0 (14- to 18-somite stages) and frozen in
O.C.T. (Tissue-Tek). Fresh frozen sections were used for either phosphohistone H3 (P-HH3) staining, as previously described (Storm et al., 2006),
or for TUNEL analysis, according to the manufacturer’s specifications
(Roche, 2156792). Horizontal sections throughout the anterior tissue of
control and mutant embryos were compared. Sections were counterstained
with Hoechst 33342. The fraction of P-HH3+ or TUNEL+ cells was
determined by counting the number of these cells in a segment of Foxg1-
expressing neuroepithelium divided by the total number of cells
(Hoechst+) in that segment. An example of a segment of Foxg1+ cells used
for counting is illustrated by the neuroepithelial area between the
arrowheads in Fig. 5 and contains ~200 cells. At least three segments were
counted in each case.
Tamoxifen treatment
For the experiments using the CGGA-CreER mice, 5, 9 or 10 mg of
tamoxifen per 40 g body weight was administered via intraperitoneal
injection to pregnant females at E6.75 and, in cases where a second injection
was performed, again at E7.75. Tamoxifen-treated embryos were collected,
fixed and subjected to whole-mount RNA in situ hybridization as described
above.
Explant cultures
Telencephalic neuroepithelium was dissected from E8.5 (5- to 8-somite
stage) embryos and when indicated the ANR was removed with a ‘Bonn’
Microprobe (Fine Science Tools, 10030-13) as described previously
(Shimamura and Rubenstein, 1997). Isolated tissue was electroporated with
20 μg of a plasmid vector expressing a human Foxg1 cDNA (OriGene,
SC116871). The pEGFP-N1 vector (Clontech, 6085-1) was used to monitor
the efficiency of electroporation. For electroporation, five pulses of 100 V,
each for 50 mseconds, were administered. Electroporated telencephalic
tissue was cultured on a Micropore floating filter (Falcon, 353090) on
Dulbecco’s Modified Eagle Medium (DMEM; GIBCO, 11960)
supplemented with 10% fetal bovine serum and 1⫻ streptomycin/penicillin
(GIBCO) in a 5% CO2 incubator at 37°C for 24 hours.
RESULTS
Deletion of FGFR genes in the anterior neural
plate leads to a loss of FGF signaling
The role of FGF signaling in the induction and initial formation of
the telencephalon remains obscure. All FGF and FGFR mutants
examined to date in either zebrafish or mice have a telencephalon,
albeit in some cases mispatterned. Therefore, either expression of
FGF and FGFR genes is not required for initial telencephalon
formation or compensation by related genes has masked the full role
of FGF signaling in the mutants examined. To distinguish between
these two possibilities, FGF signaling was abolished in this study by
conditionally deleting floxed alleles of Fgfr1 and Fgfr2 in an Fgfr3null background (Fig. 1A). Cre-mediated recombination was first
Fig. 1. Knocking out FGF signaling leads to a loss of telencephalic structures. (A) Genetic cross required to generate embryos that lack FGFR
expression in the anterior neural plate. The Foxg1Cre allele is used to recombine floxed alleles of Fgfr1 and Fgfr2 in an Fgfr3-null background.
(B) RNA in situ hybridization on horizontal sections (anterior up, posterior down) using a radioactive probe that recognizes the sequences between
the lox sites in the floxed Fgfr1 allele. By the 10- to 12-somite stage (E8.75), Fgfr1 expression is not detectable above background levels in a
Fgfr1;Fgfr2 double mutant (right). Bottom panels are enlargements of the boxed regions in the top panels. (C) Whole-mount RNA in situ
hybridization for Spry1, a reporter for FGF signaling. Expression is absent in the 13-somite stage triple FGFR mutant (right) in the anterior
neuroepithelium that normally gives rise to the telencephalon (arrowheads). (D) By E10.5, the bilaterally symmetrical telencephalic hemispheres are
apparent in the control (left), but not the mutant (right; arrowheads).
DEVELOPMENT
dependent on FGF signaling. Moreover, the phenotype observed in
the FGFR triple mutant together with those observed in less severe
single and double FGFR mutants suggest that FGF ligands and
receptors, rather than having distinct functions, act in concert to
pattern the early telencephalon.
Development 136 (14)
carried out using the Foxg1Cre mice in which recombination can start
as early as embryonic day (E) 7.5 in the anterior neuroectoderm
(Fuccillo et al., 2004), but is consistent and uniform in telencephalic
precursors by E8.75-9 (Fuccillo et al., 2004; Hébert and McConnell,
2000; Storm et al., 2003).
In order to examine whether Foxg1Cre-mediated recombination is
efficient in the anterior neural plate, RNA in situ hybridization
analysis was performed on sections of E8.75 controls and
Foxg1Cre/+;Fgfr1fx/fx;Fgfr2fx/fx double mutants using an antisense
probe complementary to the transcribed Fgfr1 sequences found
between the loxP sites. In E8.75 controls (10- to 12-somite stage),
Fgfr1 was expressed ubiquitously throughout the embryo, whereas
in mutants of the same age, the expression of Fgfr1 was greatly
reduced or absent specifically in the anterior neural plate (Fig. 1B),
indicating that recombination began earlier and was essentially
complete by E8.75. Similarly, Fgfr2 is presumably also deleted from
the anterior neural plate in the Foxg1Cre/+;Fgfr1fx/fx;Fgfr2fx/fx;
Fgfr3–/– mutants, as Fgfr1;Fgfr3 double mutant littermates did not
display any of the phenotypes described here.
To confirm loss of FGF signaling in the Foxg1Cre/+;Fgfr1fx/fx;
Fgfr2fx/fx;Fgfr3–/– triple mutants (from now on referred to as
Foxg1Cre;FGFR mutants), we examined the expression of the
downstream gene sprouty 1 (Spry1), which, as in other species is
induced by FGF signaling (Kim and Bar-Sagi, 2004; Liu et al., 2003;
Minowada et al., 1999). At E8.75 (12-13 somites), Spry1 expression
normally occurs in the anterior neural plate and mid-hindbrain
junction, whereas in the Foxg1Cre;FGFR triple mutant, it was
specifically absent from the anterior neural plate (Fig. 1C,
arrowheads), consistent with an absence of FGF signaling in this
region by E8.75.
FGF signaling is required for telencephalic tissue
to develop
Examination of whole Foxg1Cre;FGFR triple mutant embryos at
E10.5 revealed an apparent truncation of anterior structures,
including the telencephalon and facial tissues (Fig. 1D, arrowheads).
At E12.5, a stage at which the major structures of the telencephalon
are morphologically distinguishable, mutants lacked all anterior
head structures, with no visible telencephalon, nasal process, upper
or lower jaw (Fig. 1D, Fig. 7D). The facial phenotype is not
analyzed in this study and is not altogether unexpected, as FGF
signaling in the branchial arch ectoderm is important for facial
development, and Foxg1Cre is also expressed in these cells (Hébert
and McConnell, 2000; Trumpp et al., 1999). The morphology of the
diencephalon, midbrain, hindbrain and rest of the body remained
unaffected. No variability in this morphological phenotype could be
detected and it was 100% penetrant (5/5 mutants at E10.5, 2/2 at
E11.5 and 9/9 at E12.5).
To ascertain whether telencephalic tissue is completely missing
in the E12.5 mutant, we examined the expression of several
telencephalic markers, starting with Foxg1, which is expressed in all
undifferentiated and differentiated telencephalic cells, except dorsal
midline cells after E9.5, as well as in part of the optic stalk (Hatini
et al., 1994; Pratt et al., 2004; Tao and Lai, 1992). In the mutant
embryos, expression of Foxg1 was completely lacking except for a
small region underlying the diencephalon, in the region of the
anterior hypothalamus (Fig. 2A, arrowhead). To test whether the
remaining small region of Foxg1 expression corresponds to the optic
stalk, expression of Foxd1 [a marker for the ventral optic stalk and
hypothalamus (Hatini et al., 1994)] and Tcf4b [Tcf7l2 – Mouse
Genome Informatics; a marker for the dorsal diencephalon (Cho and
Dressler, 1998)] was examined. In the triple mutant, the Foxg1
RESEARCH ARTICLE 2459
Fig. 2. Expression of telencephalic markers is absent in the FGFR
triple mutant. (A-D) RNA in situ hybridization of coronal brain sections
from E12.5 control (left) and mutant (right) embryos. Expression of the
telencephalic markers Foxg1 (A), Dlx2 (C) and Emx1 (D) are not
detected in the mutant, whereas expression of the diencephalic marker
Tcf4b (B) is apparent. Asterisks in the mutant in A and D mark an
outpocketing of cells (see text). Arrowhead in A indicates residual
Foxg1 expression in the mutant. (E) Whole-mount RNA in situ
hybridization analysis of Shh expression at E10.5 reveals diminished
expression specifically in the most anterior areas in the mutant (right;
arrows).
expression domain was immediately adjacent to the Foxd1 and
Tcf4b domains underlying the diencephalon (Fig. 2B and data not
shown), suggesting that the remaining Foxg1 expression in the
mutant is primarily in the optic stalk rather than in the telencephalon,
and that the mutant completely lacks telencephalic areas that express
Foxg1 at E12.5. Consistent with this interpretation, expression of the
dorsal telencephalic marker Emx1 and ventral markers Dlx2, Nkx2.1
and Gli1 was also missing at E12.5 (Fig. 2C,D and data not shown),
and at least Emx1 and Dlx2 were already missing by E10-10.5 (see
Fig. S1 in the supplementary material). The only area expressing the
ventral markers was diencephalic, and little or no neuroectodermal
tissue was present in a position anterior to these expression domains.
Together, loss of expression of both dorsal and ventral markers
demonstrates a requirement for FGF signaling in forming not only
the ventral telencephalon, as previously reported (Gutin et al., 2006;
Kuschel et al., 2003; Shanmugalingam et al., 2000; Shinya et al.,
2001; Storm et al., 2006; Walshe and Mason, 2003), but also the
dorsal telencephalon.
Sonic hedgehog (Shh) expression in the prechordal plate
maintains the expression of FGF ligands in the ANR and depends
on FGF signaling to form the ventral telencephalon (Hébert and
Fishell, 2008). This does not exclude the possibility that FGF
signaling also promotes the expression of Shh in a positive-feedback
DEVELOPMENT
FGFs mediate telencephalic cell survival
2460 RESEARCH ARTICLE
Development 136 (14)
loop. To test this possibility, expression of Shh was examined in the
prechordal plate. At E8.75 (at the 12- and 18-somite stages), no
detectable difference in the level of Shh expression was observed in
the triple mutant (see Fig. S2 in the supplementary material). At
E10.5, however, Shh expression appeared significantly reduced in
the mutant (Fig. 2E), suggesting that in addition to Shh maintaining
FGF expression, FGFs also play some role in maintaining Shh
expression, although this might be indirectly due to loss of anterior
tissue. Since this reduction in Shh expression does not precede the
truncation of tissue, however, loss of Shh is unlikely to contribute
significantly to the observed phenotype.
Fig. 3. The dorsal midline remains in the FGFR triple mutant.
(A-D⬘) RNA in situ hybridization of coronal brain sections from E12.5
control (left) and mutant (middle and right) embryos. Expression of the
midline markers Ttr (choroid plexus; A-A⬙), Lhx5 (eminentia thalami;
B-B⬙), Wnt3a (cortical hem; C,C⬘) and Wnt8b (whole midline; D,D⬘)
remains in the mutant. In the most anterior regions of the mutant
(A⬙,B⬙), only the choroid plexus (Ttr expression) is detected.
(E) Comparative schematic of the tissues in control and mutant brains.
FGF signaling is essential to keep telencephalic
cells alive
At least two possible mechanisms, which are not mutually exclusive,
can underlie the loss of Foxg1-expressing tissue observed at E12.5
(Figs 2 and 3). First, FGF signaling might be required to induce
anterior neural plate cells to adopt a telencephalic fate and express
telencephalic markers. Second, anterior neural plate cells might
adopt a telencephalic fate, but then fail to proliferate or survive. To
distinguish between these possibilities, expression of Foxg1 was
examined shortly after anterior neural tissue first exhibits
telencephalic character (E8.75-9.25), times at which no FGFR or
Spry1 transcripts are detected in the mutant (Fig. 1B,C).
Surprisingly, at E8.75 (11-somite stage), Foxg1 expression was
clearly detected in the telencephalic anlage of mutants (3/3 mutant
embryos), whereas at E9.25 (20-somite stage) it was undetectable
(4/4 mutant embryos) (Fig. 4). This suggests that FGF signaling is
required to maintain the survival of telencephalic cells. Alternative
explanations that involve a loss of Foxg1 expression without a loss
of cells are not likely because they would not account for the
truncation of anterior neural tissue that is apparent from E9.75
onwards (Fig. 1D, Fig. 7D), as well as the loss of expression of other
telencephalic markers (Fig. 2).
To test the hypothesis that telencephalic cells die between E8.75
and E9.25 in the triple mutant, TUNEL staining was performed on
E9.0 embryos (16-somite stage) to determine the amount of cell
death in the anterior neuroectoderm. High levels of cell death were
detected specifically in the anterior Foxg1-expressing domain of
Foxg1Cre;FGFR mutants but not in controls that also carry Foxg1Cre
and are heterozygous for the FGF receptor genes (Fig. 5A,B,D).
DEVELOPMENT
FGF signaling is dispensable for dorsal midline
formation
Although Foxg1 is not expressed at detectable levels in the dorsal
midline area from E9.5 onwards, the use of Foxg1Cre mice results in
efficient recombination of floxed alleles in most or all dorsal midline
cells of the telencephalon (Hébert and McConnell, 2000; Hébert et al.,
2002; Hébert et al., 2003b; Fernandes et al., 2007). A particularly good
example of this was observed with the Bmp4lox-lacZ allele, in which the
coding region for Bmp4 is replaced by lacZ upon recombination. In
the Foxg1Cre;Bmp4lox-lacZ embryos, all cells of the dorsal midline,
including those of the cortical hem and choroid plexus, are
recombined and express lacZ (Hébert et al., 2003b). Moreover, at a
functional level, using Foxg1Cre mice to recombine the BMP receptor
gene Bmpr1afx leads to a loss of midline characteristics in most or all
dorsal midline cells (Fernandes et al., 2007).
In the E12.5 FGFR triple mutant, small bilateral ‘outpocketings’
are observed where the telencephalic hemispheres would normally
be found (asterisks in Fig. 2A,D). Since these outpocketings do not
express Foxg1 (Fig. 2A), this raises the possibility that they are
remnants of the telencephalic dorsal midline. The presence of dorsal
midline cell types was assessed in the E12.5 control and
Foxg1Cre;FGFR triple mutant telencephalon using dorsal midline
markers Wnt3a (a marker for the cortical hem), Ttr (choroid plexus),
Msx1 (choroid plexus and cortical hem) and Lhx5 (eminentia thalami
and septum). In the most anterior region of the mutant, the remaining
tissue expressed only Ttr and Msx1 (Fig. 3A⬙ and data not shown),
indicating that the remnant cells are choroid plexus in nature. In more
posterior regions, Ttr, Wnt3a, Msx1 and Lhx5 were all expressed (Fig.
3), indicating that the telencephalic dorsal midline remains in the
Foxg1Cre;FGFR triple mutant. Consistent with this finding, this tissue
does not express the ventral or dorsal telencephalic markers Dlx2 and
Emx1, or a marker for the diencephalon, Tcf4b (Fig. 2).
Two possibilities can explain why the dorsal midline remains in
the FGFR triple mutant. The first is that FGF signaling is not
required for dorsal midline formation. The second is that Fgfr1 and
Fgfr2 are not recombined early or efficiently enough in dorsal
midline precursor cells so that the remaining signaling is sufficient
to promote dorsal midline formation. Several points argue against
this latter possibility. First, no residual Fgfr1 and Fgfr2 expression
was detectable in presumptive dorsomedial precursors from E8.75
onwards (Fig. 1B and data not shown). Second, the loss of
expression observed at E8.75 is likely to be early and efficient
enough, as recombination using Foxg1Cre mice can cause the loss of
the dorsal midline in another mutant, Bmpr1afx (Fernandes et al.,
2007). Although it remains possible that the cell fate of dorsal
midline precursors is already induced by FGF signaling before Cre
recombination effectively abolishes it, or that some cells still express
Fgfr1 and Fgfr2 in the FGFR triple mutant, the results presented
here suggest that FGF signaling is not required for forming the
dorsal midline.
Fig. 4. Foxg1-expressing tissue disappears between the 11- and
20-somite stages. (A,B) Whole-mount RNA in situ hybridization of
E8.75 (11-somite stage) embryos indicates apparently normal levels of
Foxg1 expression (arrowheads) in the FGFR triple mutant (B). (C,D) At
E9.25 (20-somite stage), Foxg1 expression (arrowheads) is undetectable
in the FGFR triple mutant (D).
This high level of cell death, along with the complete loss of Foxg1expressing telencephalic cells, demonstrates a strict requirement for
FGF signaling in keeping telencephalic precursor cells alive.
To test whether a decrease in cell proliferation also contributes to
the truncation of anterior neural tissue observed in the mutants, we
analyzed the expression at E9.0 (16 somites) of the M-phase cell
cycle marker phospho-histone H3 (P-HH3) in controls and
Foxg1Cre;FGFR mutants. Surprisingly, despite the high levels of cell
death in this region, no significant difference in the percentage of Mphase cells over the total number of neuroepithelial cells could be
detected in controls versus mutants (Fig. 5C,E), suggesting that
proliferation is not a key factor in the loss of telencephalic precursors
observed in the mutants from E8.75 to E9.25.
FGF signaling promotes expression of Foxg1 in
the anterior neuroectoderm
The requirement for FGF signaling in maintaining telencephalic
precursor cell survival between E8.75 and E9.25 does not preclude
an earlier role for FGFs at E8.5 (7-9 somites) in inducing anterior
neuroectoderm to adopt telencephalic character. Such a role could
have been missed, as Foxg1 cis regulatory elements drive Cre
expression, and Foxg1 expression itself is used as the earliest
telencephalic marker (first detectable at E8.5 in the neuroectoderm
(Shimamura and Rubenstein, 1997). Therefore a lag, albeit a short
one, is expected between initial Foxg1 expression and loss of any
FGF-mediated induction of telencephalic character.
To assess the role of FGF signaling in inducing the earliest signs
of telencephalic character to the anterior neural plate, FGFR
expression would need to be disrupted as early as E7.5-8.0. For this
reason, a CAGG-CreER driver was used, in which recombination is
reported to occur ubiquitously in embryos upon tamoxifen
administration (Hayashi and McMahon, 2002). To maximize
recombination of the Fgfr1 and Fgfr2 alleles and increase the
number of cells that lack functional FGF receptor signaling, several
regimens of tamoxifen administration were assessed: tamoxifen was
administered once at E6.75 or twice, with a second administration
at E7.75, with doses of 5, 9 or 10 mg/40 g of body weight. Even with
two injections of tamoxifen at the highest concentrations,
recombination was found to be variable. Nevertheless, levels of
RESEARCH ARTICLE 2461
Fig. 5. Without Fgfr expression, Foxg1-expressing cells die.
(A-C) Serial horizontal sections from 16-somite stage control (left) and
mutant (right) embryos (anterior up). The areas between the
arrowheads are the Foxg1-expressing telencephalic precursors, as
determined by in situ hybridization (B). (D) Few TUNEL+ cells (A, purple)
are detected in the Foxg1 domain of the control, whereas in mutants
~30% of cells are labeled. (E) The number of P-HH3+ (C, purple) mitotic
cells, however, is similar in both controls and mutants. Hoechst-labeled
DNA is blue.
FGFR expression and signaling in mutants were reduced compared
with controls (Fig. 6B,C) and this resulted in a decrease in the level
of Foxg1 expression in the anterior neuroepithelium from the earliest
stages at which it is detected (Fig. 6D,E arrowhead).
One possible cause for decreased Foxg1 expression could be cell
death. TUNEL labeling revealed an increase in the percentage of
dying cells in these mutants compared with controls at E8.5, from
1.2±0.3% (mean±standard error) to 4.1±1.1%; P=0.013. However,
this remains a small percentage of cells that are dying and is unlikely
to be the sole contributor to the reduction in Foxg1 expression.
Together, these results suggest that FGF signaling is a positive
regulator of Foxg1 expression. Moreover, these loss-of-function
studies are consistent with previous gain-of-function studies
suggesting that FGFs induce telencephalic character (Shimamura
and Rubenstein, 1997).
Conversely, Foxg1 expression is essential for the expression of at
least one FGF ligand gene, Fgf8 (Martynoga et al., 2005). It has been
difficult to determine the epistatic relationship between Foxg1 and
FGFs because they reciprocally promote each others expression and
exhibit similar loss-of-function phenotypes. An explant culture
paradigm was used to address this question, at least with regards to
promoting cell survival. Wild-type explants of anterior neural plate
from E8.5 (5- to 8-somite stage) embryos were cultured with and
without an ANR, which is likely to be the main or only source of
FGFs at this stage. In fact, removal of the ANR recapitulates the
FGFR triple mutant phenotype. Whereas most explants displayed
scattered cell death within ~25 μm from their periphery regardless
of the presence or absence of an ANR, only explants devoid of an
ANR displayed extensive cell death throughout the tissue (n=5/5;
see Fig. S3 in the supplementary material), suggesting that the
source of FGFs that conveys cell survival in vivo is the ANR. We
then tested whether electroporation of a Foxg1 expression construct
could rescue the cell death observed in the explants lacking an ANR.
A GFP expression construct was coelectroporated to monitor the
DEVELOPMENT
FGFs mediate telencephalic cell survival
2462 RESEARCH ARTICLE
Development 136 (14)
Fig. 6. Reduced FGF signaling prior to telencephalon
development leads to reduced Foxg1 expression.
(A) To disrupt FGF signaling earlier than the start of
telencephalon development, a CAGG-CreER allele was
used to recombine floxed alleles of Fgfr1 and Fgfr2 in an
Fgfr3-null background using tamoxifen administration at
E6.75 and E7.75. (B) Expression of Fgfr1 at the 11- to 12somite stage, which is found ubiquitously in the control
(left), is reduced but not abolished in the mutant (right).
(C) Similarly, at the same stage, Spry1 expression is
reduced, but not abolished, in the isthmus (arrowhead),
anterior neurectoderm (arrow) and facial tissue of the
mutant. (D,E) Slightly earlier (E8.5, 9-somite stage), when
Foxg1 has just started to be expressed in the control
embryo (left), it is already significantly reduced in the
anterior neuroepithelium (arrowhead) and to a lesser
extent in the anterior neural ridge (arrow) of the mutant
(right). D, frontal view; E, side view.
FGF signaling patterns and expands the
telencephalon
FGF8 mediates organizer activity for the midbrain and cerebellum.
In conditional mutants deficient for Fgf8 at the mid-hindbrain
junction, or isthmus, early cell death of precursors leads to the loss
of the midbrain and cerebellum (Chi et al., 2003), whereas ectopic
application of FGF8-soaked beads in the diencephalon leads to a
repatterning of this tissue into an ectopic mirror-imaged midbrain
(Crossley et al., 1996; Martinez et al., 1999). The results of the
present study suggest that FGF signaling might act in a similar
manner to pattern and expand the telencephalon. The extent of
telencephalic truncation was compared in littermates in which an
increasing number of FGF receptor genes are deleted in the anterior
neural plate (Fig. 7). In the mutant in which only Fgfr1 was lost,
there was only a slight reduction in telencephalon size, with loss of
only the anterior olfactory bulbs and differentiated ventromedial
neurons (Fig. 7B) (Gutin et al., 2006; Hébert et al., 2003a). In a
mutant in which both Fgfr1 and Fgfr2 were lost, the telencephalon
was more severely truncated along the anterior-posterior axis and
there was a complete loss of all ventral cell types (Fig. 7C) (Gutin et
al., 2006). Finally, when Fgfr1, Fgfr2 and Fgfr3 were deleted, the
telencephalon was essentially gone (Fig. 7D).
When levels of cell death were compared between controls,
Fgfr1;Fgfr2 double mutants and FGFR triple mutants at the 15somite stage (~E8.75), a progressive increase in cell death was
observed. In controls, an average of 2.3% of cells (±0.8%) were
TUNEL-positive. In Fgfr1;Fgfr2 double mutants, no significant
difference in the level of cell death was observed in the dorsal
neuroepithelium at this age compared with controls, but an average
of 8.8% of cells (±3.5%) were TUNEL positive in the ventral
neuroepithelium, consistent with the loss of this part of the
telencephalon. Finally, in the FGFR triple mutant, 32.5% of cells
(±5.2%) were TUNEL-positive throughout the telencephalic
neuroepithelium (Fig. 5A,D). Coincident with the incidences of cell
death was the loss of expression of Dlx2 in the Fgfr1;Fgfr2 double
mutant (see Fig. S4 in the supplementary material) and of both Emx1
and Dlx2 in the triple mutant (see Fig. S1 in the supplementary
material).
Hence with diminishing levels of FGF signaling there is a gradual
truncation of telencephalic areas, starting with ventrorostral areas
and progressing to include dorsocaudal areas. Furthermore, the fact
that all combinations of double mutants show a similar and relatively
intact dorsal telencephalon strongly suggests that there is a
remarkable differential in the need for FGF signaling along the
dorsoventral axis. The data presented here, together with previous
reports demonstrating roles for FGF ligands in patterning both the
ventral and dorsal telencephalon (Hébert and Fishell, 2008), suggest
that FGF signaling acts as an organizer to induce, pattern and expand
the telencephalon, as it does in the mid-hindbrain region.
DISCUSSION
FGFs as candidate mediators of organizer activity
for the telencephalon
Previous studies in which FGF signaling was partially disrupted
demonstrated essential roles for this pathway in specifying,
patterning and expanding anterior and ventral areas of the
telencephalon (Gutin et al., 2006; Kuschel et al., 2003; Marklund et
al., 2004; Shanmugalingam et al., 2000; Shinya et al., 2001; Storm
et al., 2006; Walshe and Mason, 2003). From these studies, however,
it remained unknown whether FGF signaling is required as a
telencephalic inducer and a mediator of organizing activity. The
results presented here are consistent with these functions. First,
reducing FGFR gene expression prior to telencephalon induction
leads to a corresponding decrease in Foxg1 expression. And second,
deletion of the FGFR genes coincident with induction leads to death
of all Foxg1-expressing telencephalic precursors, resulting in a lack
of not only all basal ganglia but also all cortical areas. This role for
FGFs in the anterior neural plate is reminiscent of FGF functions in
mediating organizer activity in other tissues such as the midbrain
and limb, where FGFs are required to keep cells alive and where
progressive reduction of FGF signaling leads to progressive loss of
tissue (Basson et al., 2008; Mariani et al., 2008; Chi et al., 2003;
Crossley et al., 1996; Martinez et al., 1999).
In addition to being necessary to form tissues such as the limbs,
mid-hindbrain and telencephalon, FGFs are also sufficient to induce
the formation of normally organized ectopic midbrains and limbs.
Although this has not been demonstrated for the telencephalon,
DEVELOPMENT
efficiency of electroporation (all controls were electroporated with
GFP alone). Expression of Foxg1 was found to significantly reduce
the amount of cell death observed in the explants lacking ANRs
(n=3/3; see Fig. S3 in the supplementary material), suggesting that
Foxg1 can act downstream of FGFs in mediating survival.
Importantly, exogenous Foxg1 does not induce Fgf8 expression in
the explants (data not shown), consistent with Foxg1 acting
downstream of FGFs.
FGFs mediate telencephalic cell survival
RESEARCH ARTICLE 2463
and Fgfr1;Fgfr2 double mutants exhibit similar phenotypes in that
the ventral areas fail to develop and the overall size of the
telencephalon is reduced (Gutin et al., 2006; Hanashima et al., 2002;
Martynoga et al., 2005; Xuan et al., 1995). From the data presented
here, it appears that at least for maintaining cell survival, Foxg1 can
act independently of FGFs (see Fig. S3 in the supplementary
material).
Despite these overlapping and cooperative functions, the finding
that the FGFR triple mutant phenotype is more severe than the
Foxg1 mutant phenotype indicates that genes other than Foxg1 must
regulate FGF gene expression, and conversely FGFs must function
through genes other than Foxg1 in forming the telencephalon, the
cortical regions in particular. The identity of these other genes
remains unclear. It also remains untested whether FGFs act directly
in a dose-dependent manner to assign cell fates as a morphogen
along the anterior-ventral to posterior-dorsal axis.
FGFs are also likely to be sufficient to induce ectopic telencephalic
tissue. An FGF8-soaked bead can rescue induction of Foxg1
expression in cultured explants of anterior neural plate from which
the ANR is removed (Shimamura and Rubenstein, 1997). Moreover,
ectopically expressing Fgf8 in the early posterior telencephalon can
result in a mirror-image duplication of parts of the dorsal
telencephalon (Fukuchi-Shimogori and Grove, 2001; Shimogori and
Grove, 2005). Earlier ectopic expression prior to telencephalon
induction, if technically feasible, would be likely to result in a
complete duplication.
It is unknown how FGFs exert both a survival and a patterning
function. In several tissues in which FGFs have a role in patterning or
in mediating organizer activity, including the mid-hindbrain and the
limb, loss of FGFs leads to massive cell death (Chi et al., 2003;
Crossley et al., 1996; Martinez et al., 1999). In particular in the midhindbrain, FGF signaling, at least in part mediated by FGF8, is
required from the 14- to the 20-somite stages for both survival and
patterning (Chi et al., 2003; Basson et al., 2008), suggesting that these
processes continue to occur concurrently throughout early tissue
development and that it is difficult to separate them. The simplest
explanation is that continuous FGF signaling as a single event conveys
simultaneously both survival and patterning functions.
Interaction between FGF signaling and Foxg1
The function of FGFs and Foxg1 appear tightly linked through a
positive-feedback loop in early telencephalon development. Both
Foxg1 and Fgf8 expression are turned on at apparently the same time
in the ANR and anterior neural plate (Shimamura and Rubenstein,
1997). FGF signaling is necessary and sufficient for Foxg1
expression and Foxg1 is necessary for the expression of at least one
FGF ligand gene, Fgf8 (Figs 2, 4 and 6) (Martynoga et al., 2005;
Shimamura and Rubenstein, 1997). In addition, both Foxg1 mutants
FGF ligands and receptors act in part redundantly
to set the level of signaling
The finding that simultaneous deletion of Fgfr1, Fgfr2 and Fgfr3
leads to a dramatically more severe truncation of the telencephalon
than loss of any one or two FGFR genes alone indicates that, rather
than having distinct functions, these receptors can to a large extent
compensate for each other. This compensation is not complete,
however, as knockouts of individual receptor genes and pairs of
genes do result in phenotypes, albeit mild compared with the triple
mutant phenotype described here (Hébert et al., 2003a; Gutin et al.,
2006). This partial non-overlap in function could be due to the
receptors either having somewhat divergent mechanisms of
intracellular signaling or simply functioning with different
efficiencies to more or less affect the overall level of signaling.
This latter possibility appears likely when one compares the
phenotypes of all FGF and FGFR mutants. As progressively more
FGFR genes are deleted, the phenotypes observed increase in
severity, with truncations expanding from the anterior regions of the
ventral-medial telencephalon to the posterior regions of the dorsal
telencephalon. Disruption of Fgfr2 and Fgfr3 on their own or
together does not lead to gross patterning defects (Gutin et al., 2006).
In fact, in mutants in which Fgfr2 and Fgfr3 are deleted, a single
functional allele of Fgfr1 is sufficient to confer grossly normal
DEVELOPMENT
Fig. 7. Deletion of increasing numbers of FGFR genes leads to
progressively more severe telencephalic truncations. The heads of
four E12.5 littermates with deletions of zero (A), one (B), two (C) or
three (D) receptors (genotypes shown on left) suggest a gradual loss of
telencephalic tissue with decreasing FGF signaling. Panels on the right
depict the remaining telencephalic tissue (black) for each genotype.
FGFs and dorsal midline development
The only area that appears unaffected by the loss of FGF signaling
in the Foxg1Cre;FGFR mutants described here is the dorsal midline,
which suggests that FGFs are not required for the formation of this
part of the telencephalon. However, hypomorphic levels of Fgf8 lead
to a lack of dorsal midline characteristics, suggesting that FGFs may
indeed play a role (Storm et al., 2003; Storm et al., 2006). An
explanation that might reconcile these opposing findings is that, in
the Fgf8 hypomorph, FGF signaling is reduced throughout
development, whereas in the conditional FGFR triple mutant it is not
abolished until E8.75. Therefore, if FGF signaling is required earlier,
a dorsal midline phenotype might occur in the Fgf8 hypomorph, but
not the FGFR triple mutant. The likelihood of this possibility is
lessened, however, as if the Fgf8 hypomorphic allele is converted to
a null allele using the same Foxg1Cre mice used in this report, dorsal
midline features develop, indicating that it is not the timing of FGF8
loss that matters, but the level of FGF8 (Storm et al., 2003). Hence
there may be a window within the level of FGF signaling that
disrupts dorsal midline formation, perhaps by repressing dorsal
midline determinants such as Zic2 and/or BMPs (Brown et al., 1998;
Fernandes et al., 2007; Nagai et al., 2000; Storm et al., 2003).
patterning of the telencephalon. Disruption of both copies of Fgfr1
alone, on the other hand, leads to a lack of olfactory bulbs,
differentiated ventral medial neurons and specialized midline glia
(Gutin et al., 2006; Hébert et al., 2003a; Tole et al., 2006). Similar
phenotypes are also observed in the Fgfr1;Fgfr3 double mutants. In
the Fgfr1;Fgfr2 double mutants, the anterior ventral truncations are
more severe, with a lack of most or all ventral precursors and of all
anterior telencephalic regions (Fig. 7) (Gutin et al., 2006). These
phenotypes are recapitulated by progressive loss of Fgf8 expression
(Storm et al., 2006). Finally, if FGF signaling is abolished by
eliminating expression of Fgfr1, Fgfr2 and Fgfr3, as shown in this
study, then all telencephalic regions are lacking except for a small
dorsomedial region from which the choroid plexus and cortical hem
are derived. Since loss of the dorsal telencephalon is not observed in
any of the double mutants and is only observed when all three
receptors are eliminated, dorsal precursor cells are very likely to
require less FGF signaling than ventral ones. Moreover, the FGFR
mutant phenotypes suggest that FGFR1 carries the bulk of the
signaling load, followed by FGFR2, then FGFR3, and that together
they set the overall level of signaling.
Given that the disruption of any single FGF ligand gene does not
result in as severe a phenotype as that observed in the FGFR triple
mutant, FGF ligands must also act in a large part redundantly. In fact,
of the five FGF genes expressed in the most anterior part of the early
mouse CNS, Fgf3, Fgf8, Fgf15, Fgf17 and Fgf18, only mutations in
Fgf8 and Fgf17 on their own lead to patterning defects (Cholfin and
Rubenstein, 2007; Garel et al., 2003; Storm et al., 2006). Comparing
the phenotypes of severely hypomorphic and null alleles of Fgf8
reveals a progressive loss of ventral structures along with a gradual
reduction in the size of the neocortex, with preferential loss of anterior
markers (Storm et al., 2006). In moderately hypomorphic Fgf8
mutants or in embryos in which FGF signaling is reduced after neural
tube closure by ectopic expression of a dominant-negative receptor,
gradients of transcription factors in the cortical neuroepithelium also
shift anteriorly (Fukuchi-Shimogori and Grove, 2001; Garel et al.,
2003). Fgf17 mutants display an even milder phenotype, with reduced
size of the dorsal part of the frontal cortex (Cholfin and Rubenstein,
2007). Together, the phenotypes of both the ligands and receptors
support the idea that it is the overall levels of FGF signaling, rather
than distinct functions for each ligand or receptor, that act to induce,
pattern and maintain the early telencephalon.
Acknowledgements
We are grateful to our laboratory members for insightful discussions and
comments on the manuscript; to Juha Partanen and Janet Rossant (Fgfr1), Kai
Yu and David Ornitz (Fgfr2), and Chu-Xia Deng and Philip Leder (Fgfr3) for
mice; and to Sonia Garel, Alex Joyner, John Rubenstein, Gord Fishell, Stewart
Anderson, Celine Zimmer and Gregory Dressler for plasmids. This work was
supported in part by the James S. McDonnell Foundation for Brain Tumor
Research, the Feinberg Foundation for neurofibromatosis research, NIH NIMH
70596, and the Alexandrine and Alexander L. Sinsheimer Foundation.
Deposited in PMC for release after 12 months.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/14/2457/DC1
References
Arman, E., Haffner-Krausz, R., Chen, Y., Heath, J. K. and Lonai, P. (1998).
Targeted disruption of fibroblast growth factor (FGF) receptor 2 suggests a role
for FGF signaling in pregastrulation mammalian development. Proc. Natl. Acad.
Sci. USA 95, 5082-5087.
Basson, M. A., Echevarria, D., Ahn, C. P., Sudarov, A., Joyner, A. L., Mason, I.
J., Martinez, S. and Martin, G. R. (2008). Specific regions within the
embryonic midbrain and cerebellum require different levels of FGF signaling
during development. Development 135, 889-898.
Development 136 (14)
Borello, U., Cobos, I., Long, J. E., Murre, C. and Rubenstein, J. L. (2008).
FGF15 promotes neurogenesis and opposes FGF8 function during neocortical
development. Neural Dev. 3, 17.
Brown, S. A., Warburton, D., Brown, L. Y., Yu, C. Y., Roeder, E. R., StengelRutkowski, S., Hennekam, R. C. and Muenke, M. (1998). Holoprosencephaly
due to mutations in ZIC2, a homologue of Drosophila odd-paired. Nat. Genet.
20, 180-183.
Chi, C. L., Martinez, S., Wurst, W. and Martin, G. R. (2003). The isthmic
organizer signal FGF8 is required for cell survival in the prospective midbrain and
cerebellum. Development 130, 2633-2644.
Cho, E. A. and Dressler, G. R. (1998). TCF-4 binds beta-catenin and is expressed
in distinct regions of the embryonic brain and limbs. Mech. Dev. 77, 9-18.
Cholfin, J. A. and Rubenstein, J. L. (2007). Patterning of frontal cortex
subdivisions by Fgf17. Proc. Natl. Acad. Sci. USA 104, 7652-7657.
Cholfin, J. A. and Rubenstein, J. L. (2008). Frontal cortex subdivision patterning
is coordinately regulated by Fgf8, Fgf17, and Emx2. J. Comp. Neurol. 509, 144155.
Crossley, P. H., Martinez, S. and Martin, G. R. (1996). Midbrain development
induced by FGF8 in the chick embryo. Nature 380, 66-68.
Deng, C. X., Wynshaw-Boris, A., Shen, M. M., Daugherty, C., Ornitz, D. M.
and Leder, P. (1994). Murine FGFR-1 is required for early postimplantation
growth and axial organization. Genes Dev. 8, 3045-3057.
Deng, C., Wynshaw-Boris, A., Zhou, F., Kuo, A. and Leder, P. (1996). Fibroblast
growth factor receptor 3 is a negative regulator of bone growth. Cell 84, 911921.
Fernandes, M., Gutin, G., Alcorn, H., McConnell, S. K. and Hébert, J. M.
(2007). Mutations in the BMP pathway in mice support the existence of two
molecular classes of holoprosencephaly. Development 134, 3789-3794.
Frantz, G. D., Weimann, J. M., Levin, M. E. and McConnell, S. K. (1994). Otx1
and Otx2 define layers and regions in developing cerebral cortex and
cerebellum. J. Neurosci. 14, 5725-5740.
Fuccillo, M., Rallu, M., McMahon, A. P. and Fishell, G. (2004). Temporal
requirement for hedgehog signaling in ventral telencephalic patterning.
Development 131, 5031-5040.
Fukuchi-Shimogori, T. and Grove, E. A. (2001). Neocortex patterning by the
secreted signaling molecule FGF8. Science 294, 1071-1074.
Garel, S., Huffman, K. J. and Rubenstein, J. L. (2003). Molecular regionalization
of the neocortex is disrupted in Fgf8 hypomorphic mutants. Development 130,
1903-1914.
Gutin, G., Fernandes, M., Palazzolo, L., Paek, H., Yu, K., Ornitz, D. M.,
McConnell, S. K. and Hébert, J. M. (2006). FGF signalling generates ventral
telencephalic cells independently of SHH. Development 133, 2937-2946.
Hanashima, C., Shen, L., Li, S. C. and Lai, E. (2002). Brain factor-1 controls the
proliferation and differentiation of neocortical progenitor cells through
independent mechanisms. J. Neurosci. 22, 6526-6536.
Hatini, V., Tao, W. and Lai, E. (1994). Expression of winged helix genes, BF-1 and
BF-2, define adjacent domains within the developing forebrain and retina. J.
Neurobiol. 25, 1293-1309.
Hayashi, S. and McMahon, A. P. (2002). Efficient recombination in diverse
tissues by a tamoxifen-inducible form of Cre: a tool for temporally regulated
gene activation/inactivation in the mouse. Dev. Biol. 244, 305-318.
Hébert, J. M. and McConnell, S. K. (2000). Targeting of cre to the Foxg1 (BF-1)
locus mediates loxP recombination in the telencephalon and other developing
head structures. Dev. Biol. 222, 296-306.
Hébert, J. M. and Fishell, G. (2008). The genetics of early telencephalon
patterning: some assembly required. Nat. Rev. Neurosci. 9, 678-685.
Hébert, J. M., Mishina, Y. and McConnell, S. K. (2002). BMP signaling is
required locally to pattern the dorsal telencephalic midline. Neuron 35,10291041.
Hébert, J. M., Lin, M., Partanen, J., Rossant, J. and McConnell, S. K. (2003a).
FGF signaling through FGFR1 is required for olfactory bulb morphogenesis.
Development 130, 1101-1111.
Hébert, J. M., Hayhurst, M., Marks, M. E., Kulessa, H., Hogan, B. and
McConnell, S. K. (2003b). BMP ligands act reduntantly to pattern the dorsal
telencephalic midline. Genesis 35, 214-219.
Henrique, D., Adam, J., Myat, A., Chitnis, A., Lewis, J. and Ish-Horowicz, D.
(1995). Expression of a Delta homologue in prospective neurons in the chick.
Nature 375, 787-790.
Houart, C., Westerfield, M. and Wilson, S. W. (1998). A small population of
anterior cells patterns the forebrain during zebrafish gastrulation. Nature 391,
7887-7892.
Houart, C., Caneparo, L., Heisenberg, C., Barth, K., Take-Uchi, M. and
Wilson, S. (2002). Establishment of the telencephalon during gastrulation by
local antagonism of Wnt signaling. Neuron 35, 255-265.
Kim, H. J. and Bar-Sagi, D. (2004). Modulation of signalling by Sprouty: a
developing story. Nat. Rev. Mol. Cell Biol. 5, 441-450.
Kuschel, S., Ruther, U. and Theil, T. (2003). A disrupted balance between
Bmp/Wnt and Fgf signaling underlies the ventralization of the Gli3 mutant
telencephalon. Dev. Biol. 260, 484-495.
DEVELOPMENT
2464 RESEARCH ARTICLE
Liu, A., Li, J. Y., Bromleigh, C., Lao, Z., Niswander, L. A. and Joyner, A. L.
(2003). FGF17b and FGF18 have different midbrain regulatory properties from
FGF8b or activated FGF receptors. Development 130, 6175-6185.
Mariani, F. V., Ahn, C. P. and Martin, G. R. (2008). Genetic evidence that FGFs
have an instructive role in limb proximal-distal patterning. Nature 435, 402-405.
Marklund M., Sjödal M., Beehler B. C., Jessell T. M., Edlund T. and Gunhaga
L. (2004). Retinoic acid signalling specifies intermediate character in the
developing telencephalon. Development 131, 4323-4332.
Martinez, S., Crossley, P. H., Cobos, I., Rubenstein, J. L. and Martin, G. R.
(1999). FGF8 induces formation of an ectopic isthmic organizer and
isthmocerebellar development via a repressive effect on Otx2 expression.
Development 126, 1189-1200.
Martynoga, B., Morrison, H., Price, D. J. and Mason, J. O. (2005). Foxg1 is
required for specification of ventral telencephalon and region-specific regulation
of dorsal telencephalic precursor proliferation and apoptosis. Dev. Biol. 283,
113-127.
Maruoka, Y., Ohbayashi, N., Hoshikawa, M., Itoh, N., Hogan, B. L. and
Furuta, Y. (1998). Comparison of the expression of three highly related genes,
Fgf8, Fgf17 and Fgf18, in the mouse embryo. Mech. Dev. 74, 175-177.
Mason, I. (2007). Initiation to end point: the multiple roles of fibroblast growth
factors in neural development. Nat. Rev. Neurosci. 8, 583-596.
McWhirter, J. R., Goulding, M., Weiner, J. A., Chun, J. and Murre, C. (1997).
A novel fibroblast growth factor gene expressed in the developing nervous
system is a downstream target of the chimeric homeodomain oncoprotein E2APbx1. Development 124, 3221-3232.
Minowada, G., Jarvis, L. A., Chi, C. L., Neubuser, A., Sun, X., Hacohen, N.,
Krasnow, M. A. and Martin, G. R. (1999). Vertebrate Sprouty genes are
induced by FGF signaling and can cause chondrodysplasia when overexpressed.
Development 126, 4465-4475.
Nagai, T., Aruga, J., Minowa, O., Sugimoto, T., Ohno, Y., Noda, T. and
Mikoshiba, K. (2000). Zic2 regulates the kinetics of neurulation. Proc. Natl.
Acad. Sci. USA 97, 1618-1623.
Orr-Urtreger, A., Givol, D., Yayon, A., Yarden, Y. and Lonai, P. (1991).
Developmental expression of two murine fibroblast growth factor receptors, flg
and bek. Development 113, 1419-1434.
Peters, K. G., Werner, S., Chen, G. and Williams, L. T. (1992). Two FGF receptor
genes are differentially expressed in epithelial and mesenchymal tissues during
limb formation and organogenesis in the mouse. Development 114, 233-243.
Peters, K., Ornitz, D., Werner, S. and Williams, L. (1993). Unique expression
pattern of the FGF receptor 3 gene during mouse organogenesis. Dev. Biol. 155,
423-430.
Pratt, T., Tian, N. M., Simpson, T. I., Mason, J. O. and Price, D. J. (2004). The
winged helix transcription factor Foxg1 facilitates retinal ganglion cell axon
crossing of the ventral midline in the mouse. Development 131, 3773-3784.
Puelles, L. and Rubenstein, J. L. (2003). Forebrain gene expression domains and
the evolving prosomeric model. Trends Neurosci. 26, 469-476.
Rash, B. G. and Grove, E. A. (2007). Patterning the dorsal telencephalon: a role
for sonic hedgehog? J. Neurosci. 27, 11595-11603.
Satoh, W., Gotoh, T., Tsunematsu, Y., Aizawa, S. and Shimono, A. (2006).
Sfrp1 and Sfrp2 regulate anteroposterior axis elongation and somite
segmentation during mouse embryogenesis. Development 133, 989-999.
Shanmugalingam, S., Houart, C., Picker, A., Reifers, F., Macdonald, R., Barth,
A., Griffin, K., Brand, M. and Wilson, S. W. (2000). Ace/Fgf8 is required for
RESEARCH ARTICLE 2465
forebrain commissure formation and patterning of the telencephalon.
Development 127, 2549-2561.
Shimamura, K. and Rubenstein, J. L. (1997). Inductive interactions direct early
regionalization of the mouse forebrain. Development 124, 2709-2718.
Shimamura, K., Hartigan, D. J., Martinez, S., Puelles, L. and Rubenstein, J. L.
(1995). Longitudinal organization of the anterior neural plate and neural tube.
Development 121, 3923-3933.
Shimogori, T. and Grove, E. A. (2005). Fibroblast growth factor 8 regulates
neocortical guidance of area-specific thalamic innervation. J. Neurosci. 25, 65506560.
Shinya, M., Koshida, S., Sawada, A., Kuroiwa, A. and Takeda, H. (2001). Fgf
signalling through MAPK cascade is required for development of the subpallial
telencephalon in zebrafish embryos. Development 128, 4153-4164.
Storm, E. E., Rubenstein, J. L. and Martin, G. R. (2003). Dosage of Fgf8
determines whether cell survival is positively or negatively regulated in the
developing forebrain. Proc. Natl. Acad. Sci. USA 100, 1757-1762.
Storm, E. E., Garel, S., Borello, U., Hébert, J. M., Martinez, S., McConnell, S.
K., Martin, G. R. and Rubenstein, J. L. (2006). Dose-dependent functions of
Fgf8 in regulating telencephalic patterning centers. Development 133, 18311844.
Tao, W. and Lai, E. (1992). Telencephalon-restricted expression of BF-1, a new
member of the HNF-3/fork head gene family, in the developing rat brain.
Neuron 8, 957-966.
Tole, S., Gutin, G., Bhatnagar, L., Remedios, R. and Hébert, J. M. (2006).
Development of midline cell types and commissural axon tracts requires Fgfr1 in
the cerebrum. Dev. Biol. 289, 141-151.
Trokovic, R., Trokovic, N., Hernesniemi, S., Pirvola, U., Vogt Weisenhorn, D.
M., Rossant, J., McMahon, A. P., Wurst, W. and Partanen, J. (2003). FGFR1
is independently required in both developing mid- and hindbrain for sustained
response to isthmic signals. EMBO J. 22, 1811-1823.
Trumpp, A., Depew, M. J., Rubenstein, J. L., Bishop, J. M. and Martin, G. R.
(1999). Cre-mediated gene inactivation demonstrates that FGF8 is required for
cell survival and patterning of the first branchial arch. Genes Dev. 13, 31363148.
Walshe, J. and Mason, I. (2003). Unique and combinatorial functions of Fgf3 and
Fgf8 during zebrafish forebrain development. Development 130, 4337-4349.
Wilson, S. W. and Rubenstein, J. L. (2000). Induction and dorsoventral
patterning of the telencephalon. Neuron 28, 641-651.
Wilson, S. W. and Houart, C. (2004). Early steps in the development of the
forebrain. Dev. Cell 6, 167-181.
Xuan, S., Baptista, C. A., Balas, G., Tao, W., Soares, V. C. and Lai, E. (1995).
Winged helix transcription factor BF-1 is essential for the development of the
cerebral hemispheres. Neuron 14, 1141-1152.
Yamaguchi, T. P., Conlon, R. A. and Rossant, J. (1992). Expression of the
fibroblast growth factor receptor FGFR-1/flg during gastrulation and
segmentation in the mouse embryo. Dev. Biol. 152, 75-88.
Yamaguchi, T. P., Harpal, K., Henkemeyer, M. and Rossant, J. (1994). fgfr-1 is
required for embryonic growth and mesodermal patterning during mouse
gastrulation. Genes Dev. 8, 3032-3044.
Yu, K., Xu, J., Liu, Z., Sosic, D., Shao, J., Olson, E. N., Towler, D. A. and
Ornitz, D. M. (2003). Conditional inactivation of FGF receptor 2 reveals an
essential role for FGF signaling in the regulation of osteoblast function and bone
growth. Development 130, 3063-3074.
DEVELOPMENT
FGFs mediate telencephalic cell survival