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First published online 23 January 2008
doi: 10.1242/dev.011569
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1 Department of Anatomy and Program in Developmental Biology, University of
California, San Francisco, CA 94158, USA.
2 MRC Centre for Developmental Neurobiology, King's College London, London SE1
1UL, UK.
3 Department of Craniofacial Development, King's College London, London SE1 9RT,
UK.
4 Instituto de Neurociencias de Alicante, UMH-CSIC, 03550-San Juan de Alicante,
Spain.
5 Developmental Biology Program, Memorial Sloan-Kettering Cancer Center, New
York, NY 10021, USA.
Author for correspondence (e-mail:
gail.r.martin{at}ucsf.edu)
Accepted 30 November 2007
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: FGF, Midbrain, Cerebellum, Sprouty, Apoptosis, Vermis, Roof plate
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Zervas et al.,
2005). The midbrain develops from the mesencephalon (mes) (see
Fig. 1D). The dorsal mes gives
rise to the tectum, which comprises the superior colliculus (SC) anteriorly
and inferior colliculus (IC) posteriorly, where visual and auditory stimuli
are processed, respectively. The cerebellum develops from dorsal rhombomere 1
(r1): the anterior-most segment of the hindbrain (see
Fig. 1D). In mouse, the
cerebellum consists of a medial vermis, flanked laterally by a pair of
hemispheres (see Fig. 1E). The
cerebellum, where motor activity is coordinated, undergoes dramatic changes
after birth that transform it into a highly foliated structure
(Sillitoe and Joyner, 2007).
The molecular mechanisms that subdivide the tectum and cerebellum into
subregions are poorly understood.
At early stages, signals that pattern both mes and r1 are produced by a
signaling center, the isthmic organizer (IsO), at the mes/r1 boundary
(Echevarria et al., 2003;
Nakamura et al., 2005;
Partanen, 2007), thereby
ensuring coordination of midbrain and cerebellum development. One key
component of the IsO signal is FGF8
(Crossley et al., 1996), a
member of the fibroblast growth factor (FGF) family
(Itoh and Ornitz, 2004). In
mouse, Fgf8 is expressed in r1 from early neural plate [embryonic day
(E) 8.25] through mid-gestation (E12.5) stages. By E10.0, Fgf8
expression is localized in r1, just posterior to the mes/r1 boundary, in a
circular domain that is interrupted by Fgf8-negative regions at the
dorsal and ventral midlines (roof and floor plates, respectively)
(Crossley and Martin, 1995).
When Fgf8 is inactivated in the early neural plate, all mes and r1
cells die between E8.5 and E10. However, when Fgf8 expression is only
moderately reduced, the anterior midbrain appears normal, but posterior
midbrain, isthmus and vermis are lost (Chi
et al., 2003). The reason for such tissue loss in Fgf8
hypomorphs is unknown.
FGF8 regulates the expression of Fgf17
(Chi et al., 2003), which is
detected in a broad domain encompassing both prospective posterior midbrain
and cerebellum (Xu et al.,
2000). In Fgf17-null mice, part of the IC and anterior
vermis are absent, but the remaining midbrain and cerebellum appear normal.
The extent of vermis loss is increased in these mutants by removing one copy
of Fgf8, suggesting that the two FGF family members cooperate to
control cerebellum development (Xu et al.,
2000).
The level of FGF signaling can affect cell fate during mes/r1 development.
Ectopic expression of an Fgf8 splice variant, Fgf8b, which
encodes an FGF8 isoform with high affinity for FGF receptors
(Olsen et al., 2006),
transforms mouse mes cells to a cerebellar fate
(Liu et al., 1999). By
contrast, ectopic expression of Fgf8a, which encodes an FGF8 isoform
with much lower affinity for FGF receptors
(Olsen et al., 2006;
Zhang et al., 2006), expands
the mes and transforms posterior forebrain (diencephalon) progenitors to a
midbrain fate (Lee et al.,
1997; Liu et al.,
1999). Importantly, when Fgf8b is ectopically expressed
at low rather than high level in chicken embryos, the results are similar to
those obtained with Fgf8a or Fgf17. Together, these data
indicate that specification of midbrain and cerebellum require different
levels of FGF signaling (Sato et al.,
2001; Liu et al.,
2003), and highlight the importance of controlling FGF signaling
during mes/r1 development.
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;
Casci et al., 1999), the
expression of which is induced by signaling through FGF receptors (FGFRs) and
other receptor tyrosine kinases (RTKs)
(Mason et al., 2006). There
are four sprouty genes in mouse and human. Spry1 and Spry2
are strongly expressed in mes/r1 from early neural plate stages, in cells near
the IsO (Minowada et al.,
1999). In zebrafish (Furthauer
et al., 2001) and chicken
(Suzuki-Hirano et al., 2005)
embryos, increasing sprouty gene expression above normal levels can antagonize
FGF activity and interfere with mes/r1 development.
Recent studies have demonstrated that the roof plate in r1 is another
important source of signals, including BMP ligands, for cerebellar development
(Chizhikov et al., 2006;
Machold et al., 2007). Roof
plate development itself is controlled by Lmx1a
(Millonig et al., 2000), a
gene positively regulated by BMP signaling
(Chizhikov and Millen, 2004a;
Chizhikov et al., 2006). It is
not known whether signals emanating from the IsO affect the development or
function of the cerebellar roof plate or vice versa.
Here, we have employed a mouse line carrying a conditional Spry2 gain-of-function transgene in conjunction with an Fgf8null allele to produce the equivalent of an FGF loss-of-function allelic series specifically in mes/r1. By studying the phenotypic effects of these genetic manipulations, we have uncovered potential mechanism(s) by which FGF signaling differentially regulates the development of specific regions within the midbrain and cerebellum.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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), which
was produced essentially as described by Lu et al.
(Lu et al., 2006), was
detected using primers that amplify human PLAP (ALPP).
Spry2 gain-of-function homozygotes were identified using a
quantitative PCR assay for PLAP sequences (primers and conditions
available upon request). Other alleles were detected as previously described:
En1Cre and Fgf8null
(Chi et al., 2003);
Fgf8neo (Sun et al.,
1999); and Fgf17 (Xu
et al., 2000).
Histological analysis and assays for cell death and gene expression
Noon of the day when a vaginal plug was detected was considered E0.5.
Embryos were staged more precisely by counting somites posterior to the
forelimb bud and scoring the first one counted as somite 13. Histological
analysis of late gestation and postnatal brains, and immunolocalization in
brain tissue were performed as previously described
(Chi et al., 2003).
Assays for cell death were performed in whole mount by staining with
LysoTrackerT (Molecular Probes L-7528), as previously described by Grieshammer
et al. (Grieshammer et al.,
2005). We confirmed that LysoTrackerT staining gave results on
En1Cre/+;Fgf8flox/- (referred to here
as mes/r1-Fgf8-KO) embryos similar to those we previously obtained
using Nile Blue Sulfate staining and TUNEL assays to detect cell death
(Chi et al., 2003).
For gene expression analysis, embryos were collected in cold PBS, fixed in 4% paraformaldehyde and stored in 70% ethanol at -20°C. Some embryos were embedded in paraffin and sectioned at 7 µm. Standard protocols were employed for RNA in situ hybridization on sections and in whole mount, and for assaying β-GAL and PLAP activity in whole mount.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). In
these mice, transgene-expressing cells produce β-GEO, a fusion protein
with both neomycin-resistance and β-galactosidase (β-gal) activities
(Friedrich and Soriano, 1991)
(Fig. 1A). β-Gal assays
demonstrated that Spry2-GOF is expressed in most cells of the embryo
from E7.5 to at least E11.5 (Fig.
1B, and data not shown). Cells in which the transgene has
undergone Cre-mediated recombination express a bicistronic mRNA containing
both Spry2- and human placental alkaline phosphatase
(PLAP)-coding sequences (Fig.
1A). PLAP protein thus functions as a convenient reporter for
expression of the recombined transgene.
To obtain mice in which the transgene was recombined in mes/r1, we crossed
animals carrying Spry2-GOF and En1Cre, an
En1 allele with cre inserted in the first exon
(Kimmel et al., 2000). In
their En1Cre/+;Spry2-GOF offspring, we detected
PLAP activity only in the domains in which En1Cre is known
to function (Li et al., 2002;
Chi et al., 2003). Thus, in the
developing brain, no PLAP activity was detected prior to E8.25 (not shown),
but strong PLAP activity was observed throughout mes/r1 from E8.75
(Fig. 1C). Subsequently,
because the CAGG promoter remains active in most embryo and adult cells, PLAP
activity was detected throughout the mes and r1 at E10.75
(Fig. 1D), and the midbrain and
cerebellum on postnatal day (P) 22 (Fig.
1E). In addition, by E10.75, PLAP activity was detected in the
first branchial arch (Fig. 1D),
presumably in mes/r1-derived neural crest cells that continue to express the
recombined Spry2-GOF transgene under the control of the CAGG
promoter. PLAP activity was also detected in other domains in which
En1 is normally expressed (Kimmel
et al., 2000) (Fig.
1D). No PLAP activity was detected in control littermates (wild
type or mutants carrying only En1Cre/+ or only
Spry2-GOF; not shown).
En1Cre/+;Spry2-GOF mutants will hereafter be
referred to as mes/r1-S2GOF embryos, to indicate that the recombined transgene
was expressed in mes/r1.
To assess Spry2 expression directly from the recombined transgene,
we performed a whole-mount RNA in situ hybridization analysis on mes/r1-S2GOF
mutants and control littermates. Consistent with previous studies
(Minowada et al., 1999), in
control embryos we detected Spry2 RNA at E8.75 in a domain
encompassing most of mes/r1 (Fig.
1F), which subsequently became progressively more restricted, such
that by E9.25 and at E10.75 it encompassed the posterior mes and r1 but not
the anterior mes (Fig. 1H,J).
In mes/r1-S2GOF mutants at these stages
(Fig. 1C,D), the level of
Spry2 RNA appeared to be only slightly increased within the normal
domains of Spry2 expression (Fig.
1G,I,K). However, because transgene expression persisted in
regions where Spry2 expression is normally downregulated, we detected
ectopic Spry2 expression in anterior mes and posterior r1 at later
stages (E9.25 and E10.75; Fig.
1I,K). In situ hybridization analysis on sections of mes/r1-S2GOF
mutants at 42 somites demonstrated that Spry2 RNA was distributed
uniformly within the ectopic expression domain (not shown). Together, our data
suggest that in mes/r1-S2GOF embryos, the level of Spry2 RNA is
slightly elevated within its normal expression domain and is ectopically
expressed throughout the remainder of the developing midbrain and cerebellum
from at least E8.75.
Similar effects on midbrain and cerebellum are obtained by expressing one copy of recombined Spry2-GOF in mes/r1 or by reducing Fgf17 and Fgf8 gene dose
Histological analysis of mes/r1-S2GOF mutants shortly before birth revealed
the effects of expressing the recombined transgene on midbrain and cerebellum
development. In midsagittal sections of E17.5 mes/r1-S2GOF mutants
(n=4), the dorsal midbrain appeared normal at its anterior end, some
tissue loss was observed at the posterior end of the SC, and the IC was absent
(Fig. 2A,B; data not shown).
Staining for calretinin confirmed the absence of the IC, which is normally
calretinin-negative (Fig.
2C,D). Posterior to the midbrain, the dorsal isthmus was also
absent (Fig. 2A-D). The medial
cerebellum (vermis) also appeared to have lost tissue, but only at its
anterior end (Fig. 2A,B, and
data not shown). By contrast, basal plate derivatives in the midbrain
[oculomotor nucleus (nIII), substantia nigra and ventral tegmental area
(SN-VTA)], and the isthmus [trochlear nucleus (nIV)], were present, as was the
r1-derived locus coeruleus, and all appeared normal (not shown).
Mes/r1-S2GOF mutants were viable, and lacked the IC and isthmus postnatally
(Fig. 2G,H), demonstrating that
the apparent tissue loss observed at E17.5 was not due to a developmental
delay. In wild-type mice, there are strain-specific variations in vermis
foliation pattern, such that some strains have three distinct lobules (I, II
and III) anterior to the lobule of culmen (IV-V), and other strains have only
two (indicated as I-II and III in Fig.
2G) (Inouye and Oda,
1980). In postnatal mes/r1-S2GOF mutants (n=5), lobules
I-III were either reduced or absent, whereas the remaining lobules appeared
essentially normal (Fig. 2H;
data not shown). Thus, expressing a single copy of the recombined
Spry2-GOF transgene in mes/r1 from an early developmental stage
resulted in absence of the posterior dorsal midbrain (IC), isthmus and
anterior vermis. There were no obvious abnormalities in these animals in other
tissues that developed from cells in which the Spry2-GOF transgene
underwent Cre-mediated recombination.
The phenotype of mes/r1-S2GOF mutants described above is remarkably similar
to, although slightly more severe than, that observed in Fgf17-null
homozygotes carrying one copy of an Fgf8 null allele
(Fgf17-/-; Fgf8+/- animals)
(Xu et al., 2000). At birth,
the IC, isthmus and anterior tissue in the developing cerebellum appeared to
be absent in such compound FGF mutant animals
(Fig. 2E,F), and at 3-4 weeks
after birth, only one major lobule was present anterior to lobules IV-V
(Fig. 2I). These results
suggest that expressing a single copy of the recombined Spry2-GOF
allele results in a reduction in FGF signaling during midbrain and cerebellar
development to approximately the same degree as in
Fgf17-/-; Fgf8+/- mutants.
|
),
appeared normal; tissue was missing at the posterior end of the SC; and the
IC, dorsal isthmus and vermis were absent
(Fig. 3C-E). Mes/r1-S2GOF;F8
mutants could be classified in two groups: group I (n=3/5) had some
tissue loss at the posterior end of the SC, and basal structures including
nIII, nIV and the SN-VTA were present although somewhat reduced; group II
(n=2/5) had more tissue loss at the posterior end of the SC and the
basal structures were absent (Fig.
3C-K). Lateral r1-derived tissue was present in both groups, as
evidenced by the presence of the locus coeruleus, but it was clearly reduced
in group II (Fig. 3I-K).
We next examined animals carrying En1Cre/+ and two
copies of Spry2-GOF (mes/r1-S2GOF;S2GOF; n=3), which
appeared similar to mes/r1-S2GOF;F8 mutants in group I (not shown). Thus,
expressing a second copy of the recombined Spry2-GOF allele appeared
to inhibit FGF signaling to approximately the same extent as removing one copy
of the Fgf8 gene in mes/r1-S2GOF animals. The phenotype of the group
I mes/r1-S2GOF;F8 and mes/r1-S2GOF;S2GOF mutants is similar to that observed
in embryos homozygous for Fgf8neo
(Chi et al., 2003), a
hypomorphic allele of Fgf8, that has been estimated to express
Fgf8 RNA at 
40% of the level in wild-type embryos
(Meyers et al., 1998). Unlike
Fgf8neo/neo animals, which have numerous developmental
abnormalities caused by reduced FGF8 signaling in all tissues and die at
birth, several mes/r1-S2GOF;F8 and mes/r1-S2GOF;S2GOF mutants survived to
adulthood. Thus, we were able to determine the postnatal consequences of the
midbrain and cerebellum defects that were detected just before birth. In P21
mes/r1-S2GOF;F8 animals, a complete loss of vermis was readily observed in
intact brains, whereas the cerebellar hemispheres were present, but appeared
somewhat reduced (Fig. 3L,M).
Section analysis at P28 revealed a hemisphere-like foliation pattern in
lateral sections and further showed that the granule cell layer was of normal
thickness; staining with calbindin demonstrated that the density of Purkinje
cells was apparently normal, as were their axon and dendrite projections (see
Fig. S1 in the supplementary material). Consistent with the absence of the
vermis, the Fastigial nucleus was absent, whereas the Dentate nucleus appeared
relatively unaffected (not shown). The lack of vermis, which controls posture
and locomotion, presumably explains our finding that mes/r1-S2GOF;F8 mutants
exhibited a widened gait and pronounced ataxia that increased in severity with
age (not shown).
Cell death occurs in the anterior mesencephalon in Spry2-GOF mutants and Fgf8 hypomorphs
We have previously shown that eliminating Fgf8 function in mes/r1
causes extensive cell death between the 11 and 28 somite stages, resulting in
complete absence of the midbrain, isthmus and cerebellum
(Chi et al., 2003). However,
the effects of only moderately reducing the level of FGF gene expression on
cell survival in mes/r1 were not examined. We therefore sought to determine
whether abnormal cell death could account for the tissue loss in embryos that
were homozygous for Fgf8neo or that expressed the
recombined Spry2-GOF transgene, by staining with LysotrackerT (see
Materials and methods) at 4- to 6-hour intervals between E8.75 and E10.75 (12
to 38 somites).
|
), the domain
of abnormal cell death was localized at the anterior end of the dorsal mes
(Fig. 4A-E; data not shown). To
confirm the location of this abnormal cell death, we performed an in situ
hybridization assay on the LysotrackerT-labeled embryos using a probe for
Pax6. This gene is expressed throughout the prospective forebrain,
with the posterior limit of its expression domain defining the boundary
between the developing diencephalon (di) and mes. The domain of abnormal cell
death, as detected by LysotrackerT staining, was indeed localized immediately
posterior to the di/mes boundary (Fig.
4C,C', and data not shown).
One explanation for the abnormal cell death in the mutants is that a
certain level of FGF signaling from the IsO is required for cell survival, and
that it falls below that level in the anterior mes when either Spry2
is ectopically expressed or Fgf8 expression is reduced (as in
Fgf8neo/neo embryos). To investigate this hypothesis, we
determined the range of FGF signaling in mes/r1 by assaying for Spry1
expression, which is induced by and thus serves as a reporter for a high level
of FGF signaling (Liu et al.,
2003; Olsen et al.,
2006). We found that at the 18- to 20-somite stage, the distance
from the isthmic constriction to the anterior limit of the Spry1
expression domain was reduced in mes/r1-S2GOF and mes/r1-S2GOF;F8 mutants
compared with that in control embryos (Fig.
4F-H). These data provide evidence that the range of FGF signaling
from the IsO is decreased in the mes of these mutants, supporting the
hypothesis that the observed anterior cell death is a consequence of reduced
FGF signaling.
Similar results were obtained in assays for En1, En2 and
Efna2 expression (Fig.
4 and data not shown). At 33-35 somites, the Efna2
expression domain was smaller than normal in mes/r1-Spry2-GOF and even smaller
in mes/r1-Spry2-GOF;F8 embryos (Fig.
4I-K). These data are consistent with studies showing that
En1 and En2 expression is regulated by FGF signaling
(Trokovic et al., 2003), and
that ephrin gene expression is controlled by engrailed genes
(Nakamura, 2001). In addition,
as expected for mutants with reduced FGF signaling from the IsO
(Zervas et al., 2005;
Partanen, 2007), we found that
the posterior limit of Otx2 expression was slightly less sharp in
mes/r1-S2GOF;F8 mutants than in controls, and a few scattered
Otx2-positive cells were detected in r1. Furthermore, Gbx2
and Wnt1 expression were significantly reduced in anterior r1 and the
posterior midbrain, respectively (see Fig. S2 in the supplementary
material).
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30 cell diameters wide
(Fig. 5C-D').
We next examined gene expression patterns at earlier stages. In control
embryos at E10.5, as well as in mes/r1-S2GOF embryos, the near circular
Fgf8 expression domain in anterior r1 is interrupted at the dorsal
midline by a small group of Fgf8-negative roof plate cells
(Fig. 5E,F; data not shown). In
mes/r1-S2GOF;F8 embryos, the Fgf8 expression pattern appeared almost
normal up to E9.5 (28 somites; Fig.
5H,I), but by E10.5 (34 somites) the Fgf8-negative domain
in dorsal r1 was much wider than normal
(Fig. 5G, compare with
5E,F). In contrast to Fgf8,
Bmp7 expression is a positive marker of the roof plate. In control and
mes/r1-S2GOF embryos at E10.5, it was detected in a narrow domain in anterior
r1 that progressively widened towards posterior r1
(Alder et al., 1999)
(Fig. 5J,K). However, in
mes/r1-S2GOF;F8 embryos, a wider Bmp7-positive roof plate domain was
observed in anterior r1 (Fig.
5L, compare with
5J,K).
Together, these data provide strong evidence that the roof plate had
expanded laterally in anterior r1 in mes/r1-S2GOF;F8 embryos. Fate-mapping
studies in the mouse embryo have localized vermis progenitors to a small
domain flanking the dorsal midline in the anterior-most region of r1 at E12.5,
and there is evidence that at earlier stages these cells are likewise
localized close to the dorsal midline
(Sgaier et al., 2005). Thus,
it appears that the region containing the vermis progenitors, which is
normally positive for Fgf8 and negative for Bmp7 expression,
has been replaced by Fgf8- negative Bmp7-positive roof plate
cells in mes/r1-S2GOF;F8 embryos. These results indicate that reducing FGF
signaling to the level attained in mes/r1-S2GOF;F8 embryos causes an expansion
of the roof plate between the 28- and 34-somite stages, possibly at the
expense of vermis progenitors.
BMP target gene expression is increased and BMP antagonist gene expression is decreased in mes/r1-S2GOF;F8 mutants
Previous studies have indicated that roof plate cells express several BMP
family members in addition to Bmp7, and that BMP signaling is
necessary and sufficient for roof plate development in the chick neural tube
(Lee and Jessell, 1999;
Chizhikov and Millen, 2004b;
Chizhikov and Millen, 2004c;
Liu et al., 2004). The
transcription factor gene Msx1 is a downstream target of BMP
signaling in the dorsal neural tube, and misexpression of Msx1 in the
chick spinal cord results in expansion of the roof plate
(Liu et al., 2004). Therefore,
to determine whether excess BMP signaling might be responsible for the roof
plate expansion we observed in mes/r1-S2GOF;F8 embryos at E10.5, we assayed
for Msx1 expression as a readout for BMP signaling in dorsal r1. The
domain of Msx1 expression was indeed significantly expanded
mediolaterally in anterior r1 in mes/r1-S2GOF;F8 embryos as compared with
control embryos at 33 somites (Fig.
5M,N). These data support the hypothesis that a decrease in FGF
signaling in r1 results in an increase in BMP signaling and expansion of the
roof plate.
|
), which is
expressed in the roof plate of anterior r1
(Pearce et al., 1999;
Louvi et al., 2003). We found
that in control embryos at E9.25 (22 somites), Grem1 RNA was readily
detected in the roof plate and adjacent neuroepithelium
(Fig. 5O, and data not shown),
whereas in mes/r1-S2GOF;F8 embryos Grem1 expression was barely
detected in anterior r1 (Fig.
5P). By contrast, Grem1 expression was only moderately
reduced in mes/r1-S2GOF embryos (not shown). These results suggest that a
normal function of FGF signaling from the IsO is to inhibit BMP signaling in
dorsal r1 via a positive effect on Grem1 expression. |
DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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E8.5, we obtained mes/r1-S2GOF mutants with a
phenotype similar to that of
Fgf17-/-;Fgf8+/- mutants
(Xu et al., 2000). When we
reduced Fgf8 gene dose in mes/r1-S2GOF mutants, or produced animals
with two copies of the recombined Spry2-GOF allele, we obtained a
more severe phenotype, which resembled that of embryos homozygous for an
Fgf8 hypomorphic allele (Fgf8neo)
(Chi et al., 2003). Together,
these data strongly support the hypothesis that expression of the recombined
Spry2-GOF allele results in an inhibition of signaling via FGF
receptors rather than other RTKs during midbrain and cerebellum development. A
dorsal mes/r1 phenotype similar to that in mes/r1-S2GOF;F8 embryos has also
been observed following conditional inactivation of Fgfr1 in mes/r1
(Trokovic et al., 2003),
suggesting that the FGF signaling that is affected in the Spry2
gain-of-function mutants is primarily relayed by FGFR1.
The results of our analysis provide genetic evidence consistent with the
proposal that the processes that shape the midbrain and cerebellum require
distinct levels of FGF signaling (Liu et
al., 1999; Sato et al.,
2001; Liu et al.,
2003), and further reveal that anatomically and functionally
distinct regions within the midbrain and cerebellum require different levels
of FGF signaling for their development. This conclusion is consistent with the
results of a recent analysis showing that when En1/En2 function,
which is apparently downstream of FGF signaling from the IsO, is progressively
compromised, specific functional domains of the tectum and vermis are lost in
a dose-dependent manner (Sgaier et al.,
2007). In addition, we present evidence that FGF signaling
influences the balance between vermis and roof-plate development in anterior
r1 via an effect on BMP signaling.
The effects of Spry2 gain-of-function in mes/r1 differ in mouse and chicken embryos
The phenotypes we observed in our mutant mice differ from those reported in
chicken embryos, in which ectopic expression of Spry2 caused cells in
r1 to express Otx2, a marker for the mes, and to develop into
midbrain tissue (Suzuki-Hirano et al.,
2005). We did observe that Otx2 expression was abnormal
in mes/r1-S2GOF;F8 embryos, in that the boundary between
Otx2-positive and Otx2- negative cells was not as sharp as
in control embryos and there were a few Otx2-positive cells scattered
in r1 (see Fig. S2 in the supplementary material). However, we found no
evidence that r1 cells took on a midbrain fate. Instead, misexpressing
Spry2 in mes/r1 resulted in defects in vermis development. Although
there is evidence that the forced expression of Otx2 in r1 from E8.75
can transform anterior r1 into midbrain tissue in the mouse embryo
(Broccoli et al., 1999), there
is as yet no direct evidence that mouse r1 can give rise to midbrain tissue
when FGF signaling is reduced. The disparity between our findings and those of
Suzuki-Hirano et al. (Suzuki-Hirano et
al., 2005) might be due to differences in the methods employed to
obtain ectopic sprouty gene expression, i.e. sustained expression of an
inherited transgene in the mouse versus transient expression of a transgene
introduced by electroporation in the chicken embryo. Alternatively, it is
possible that the different results reflect differences in the mechanisms that
control the level of FGF signaling in mouse versus chicken brain
development.
Loss of the posterior tectum caused by reducing FGF signaling can be explained by death of anterior cells and mis-specification of posterior cells as anterior tectum
A specific loss of the IC, with normal development of the SC, is a feature
common to mutants in which FGF signaling is moderately reduced in mes/r1,
including Fgf17-/-;Fgf8+/- embryos
(Xu et al., 2000),
Fgf8neo/neo embryos
(Chi et al., 2003) and embryos
in which Fgfr1 has been inactivated in mes/r1
(Trokovic et al., 2003), but
the mechanism by which this loss occurs is not known. Death of IC progenitors
is one possible explanation, as we previously showed that inactivation of
Fgf8 in mes/r1 by the 10-somite stage results in apoptosis throughout
the mes (Chi et al., 2003).
However, cell death was not detected at E9.5 in the mesencephalon when
Fgfr1 was inactivated (Trokovic
et al., 2003).
Here, we show that a moderate reduction in FGF signaling does cause abnormal cell death prior to E9.25, but that the dying cells are detected only in the anterior mes. To explain this localization, we propose that there is a minimum level of FGF signaling below which cells in the mes die. In normal embryos, there is sufficient FGF signaling to sustain survival, even of cells that are furthest from the source of FGFs in the IsO (Fig. 6A). However, when FGF signaling is moderately reduced, as for example in mes/r1-S2GOF mutants, only cells close to the IsO attain the level of FGF signaling required for survival. At early stages, when the mes is relatively small, all the cells are sufficiently close to the IsO. But as the mes increases in size and the anterior-most cells in such mutants are displaced progressively further from the IsO, they reach a point at which they are too far from the IsO to attain the level of FGF signaling required for survival, and therefore they die.
The observation that cell death is restricted to the anterior mes suggests
the following explanation for the loss of the IC in mutants with reduced FGF
signaling in the mes: AP cell fates have not yet been determined in the mes at
the stage when the cells are dying, and the remaining posterior cells are
subsequently specified as SC because of the low level of FGF signaling.
Consistent with this hypothesis, it has been shown that fate changes can occur
in explants of E9.5 mouse midbrains (Liu
et al., 1999). Presumably, if cells in the anterior mes had died
at a stage after the fate of all mes cells had been determined, a normal SC
would not have formed. This model further suggests that specification of IC
progenitors requires a higher level of FGF signaling than specification of SC
progenitors. This idea is supported by data showing that increasing FGF
signaling, by inserting beads loaded with FGF protein in the anterior mes of
chicken embryos, induces anterior cells to take on a posterior fate
(Martinez et al., 1999;
Shamim et al., 1999).
Loss of the vermis caused by reducing FGF signaling can be explained by an increase in BMP signaling and expansion of the roofplate
A loss of the entire vermis is observed when FGF signaling is reduced to a
level lower than in Fgf17-/-;Fgf8+/-
and mes/r1-S2GOF mutants. Our data indicate that abnormal cell death is not
responsible for the loss of the vermis in such mutant embryos. Instead, the
vermis may be absent because a specific minimum level of FGF signaling is
required for specification and/or expansion of vermis progenitors
(Liu et al., 2003), and that
level is not attained in such mutants. Another possibility is based on our
observation that the roof plate in r1 is abnormally expanded in
mes/r1-S2GOF;F8 embryos. Although this abnormality might be secondary to a
failure of vermis development, we favor the hypothesis that the converse is
true, and that the observed expansion of the roof plate is the primary cause
of loss of the vermis (Fig.
6B).
|
; Chizhikov and Millen,
2004c; Chizhikov and Millen,
2005), that overexpression of Lmx1a in mouse r1 is
associated with increased BMP signaling and increased roof plate formation
(Chizhikov et al., 2006), and
that BMP signaling can be inhibited by FGF signaling in numerous developmental
settings, including the forebrain (Storm
et al., 2003) and midbrain
(Alexandre et al., 2006). We
propose that in r1, roof plate formation is likewise controlled by BMP
signaling from the ectoderm, which in turn is inhibited by FGF signaling from
the IsO. This inhibitory interaction could explain why the roof plate is
normally so narrow in anterior r1, adjacent to the IsO where FGF ligands are
produced, but is much wider in more posterior r1. Accordingly, when FGF
signaling is sufficiently reduced, as in mes/r1-S2GOF;F8 embryos, BMP
signaling increases, and the roof plate in anterior r1 becomes abnormally wide
(Fig. 6C). In support of this
idea, we found that in mes/r1-S2GOF;F8 embryos, BMP signaling is increased, as
evidenced by an expansion of the Msx1 expression domain. Moreover, we
observed that a dramatic reduction in expression of the BMP antagonist
Grem1 precedes abnormal expansion of the roof plate by 12 hours,
suggesting a molecular mechanism by which FGF signaling could exert a negative
effect on BMP signaling. However, GREM1 alone is unlikely to be the sole
factor responsible for mediating the proposed inhibitory effect of FGF
signaling on BMP signaling and roof plate expansion in r1, because we found
that roof plate development appears normal in Grem1-null embryos (not
shown).
An important question is how might the expansion of the roof plate observed
in mes/r1-S2GOF;F8 mutants compromise vermis development? One possibility is
based on the fact that the roof plate itself expresses BMPs
(Alder et al., 1999;
Lee and Jessell, 1999;
Alexandre et al., 2006), and an
increase in the size of the cell population producing these potent signaling
molecules might in turn cause the nearby vermis progenitors to slow their
proliferation or differentiate prematurely, leading ultimately to absence of
the vermis (Alder et al., 1999;
Krizhanovsky and Ben-Arie,
2006; Machold et al.,
2007). Alternatively, as misexpression of the BMP effector MSX1
results in the conversion of spinal cord neuroepithelum into roof plate
(Liu et al., 2004), it is
possible that the increase in Msx1 expression in mes/r1-S2GOF;F8
mutants functions to stimulate roof plate development at the expense of the
vermis by converting vermis progenitors to a roof plate fate. Further studies
will be needed to distinguish between these possibilities.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/5/889/DC1
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
|
REFERENCES |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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