1 Department of Molecular Neurobiology, Graduate School of Life Sciences, Tohoku
University, Seiryo-machi 4-1, Aoba-ku, Sendai 980-8575, Japan
2 Institute of Development, Aging and Cancer, Tohoku University, Seiryo-machi
4-1, Aoba-ku, Sendai 980-8575, Japan
Author for correspondence (e-mail:
nakamura{at}idac.tohoku.ac.jp)
Accepted 28 May 2004
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SUMMARY |
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Key words: Tectum, Cerebellum, Fgf8, Isthmus, Cell signaling, Ras, ERK, Chick
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Introduction |
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There are eight Fgf8 isoforms identified so far
(Crossley and Martin, 1995;
MacArthur et al., 1995b
).
Among these, Fgf8a and Fgf8b are expressed in the chick
isthmus (Sato et al., 2001
).
Fgf8a and Fgf8b have different organizing activities in vivo. Transgenic mice
in which Fgf8a was misexpressed under the control of a Wnt1
enhancer showed overgrowth of the dimesencephalic region
(Lee et al., 1997
). By
contrast, the mesencephalon of Fgf8b-transgenic mice under the
control of a Wnt1 enhancer, showed metencephalic properties
(Liu et al., 1999
), and
Fgf8b-soaked beads can induce the development of cerebellar structures in the
diencephalon and mesencephalon (Martinez
et al., 1999
). Moreover, Fgf8b misexpressed by inovo
electroporation completely transformed the fate of the mesencephalic alar
plate to become cerebellum. Although Fgf8a and Fgf8b exerted
completely different effects, lower doses of Fgf8b exerted similar
effects to those of Fgf8a (Sato
et al., 2001
; Liu et al.,
2003
). This result, together with the result that Fgf8b
has stronger transforming activity than Fgf8a for NIH3T3 cells in
vitro (MacArthur et al.,
1995a
) indicates that the differing effects of Fgf8 may
be attributable to differences in the intensity of the signals.
It is thus of great interest to learn how the Fgf8 signal is transduced in
the metencephalon and mesencephalon for their fate decision. In many systems,
the signal through a receptor tyrosine kinase (RTK) is transduced through the
Rasextracellular-signal-regulated kinase (ERK) pathway to induce cellular
responses, such as proliferation and differentiation (reviewed by
Katz and McCormick, 1997;
Rommel and Hafen, 1998
). Since
the Fgf receptor (Fgfr) is also a tyrosine kinase, we focused our attention on
the Fgf-Ras-ERK signaling pathway. We first examined ERK activation in chick
neural tube using an anti-di-phosphorylated ERK antibody, and found that ERK
was activated strongly in Fgf8 mRNA-expressing regions. We also found
that Fgf8b could activate ERK more strongly than Fgf8a in
the mes/metencephalon. Misexpression of a dominant-negative form of Ras
(RasS17N) was carried out to disrupt the Ras-ERK pathway.
RasS17N changed the fate of the metencephalic alar plate from
cerebellum to tectum. Application of siRNA against Fgf8b by
electroporation resulted in posterior extension of the Otx2
expression domain. We propose that a strong Fgf8 signal activates the Ras-ERK
pathway and ultimately results in cerebellar differentiation.
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Materials and methods |
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In-ovo electroporation
In-ovo electroporation was carried out as described previously
(Funahashi et al., 1999;
Nakamura et al., 2000
;
Nakamura and Funahashi, 2001
).
Briefly, fertilized chicken embryos were incubated in humid conditions at
38°C for 30-36 hours to reach 7-10-somite stages, corresponding to stage
9-10 (Hamburger and Hamilton,
1951
). DNA solution was injected into the lumen of the neural
tube. The electrodes (Unique Medical Imada, Natori, Japan) were placed on the
vitelline membrane at a distance of 4 mm, then a rectangular pulse of 25 V, 50
ms was charged four times by the electroporator (CUY21, Tokiwa Science,
Fukuoka, Japan). To monitor the ectopic expression, the GFP expression vector
(pCA-GAP-GFP) (Niwa et al.,
1991
; Moriyoshi et al.,
1996
) was mixed in the DNA solution (0.35 µg/µl). Since DNA
is negatively charged, only the anode side of the neural tube is transfected.
The other side is used as a control. In some cases, the cathode (Unique
Medical Imada) was inserted into the lumen of the neural tube and a
rectangular pulse of 15 V, 25 ms was given three times to transfect
efficiently in the ventral side of the neural tube.
Bead implantation
AG1-X2 ion-exchange resin beads (BioRad) were washed with DMSO three times,
then incubated for 20 minutes with 10 mM SU5402 (Calbiochem) in DMSO. An
SU5402-soaked bead was implanted in the isthmus. At 1 or 2 hours after
implantation, embryos were fixed with 4% paraformaldehyde/PBS.
siRNA to specifically silence Fgf8a and Fgf8b
Recently, it was shown that siRNA could specifically disrupt target mRNA by
introducing siRNA expression vector
(Katahira and Nakamura, 2003).
Since the difference between Fgf8a and Fgf8b is only the
presence of 33 bases in Fgf8b, target sequence specific for
Fgf8a and Fgf8b siRNA is limited, and was determined as
shown in Fig. 7A. The 19-mer
sense and antisense siRNA sequences were linked with nine nucleotide spacer
(TTCAAGAGA) as a loop, and six T and A bases were added as the terminal signal
to the 3' end of the forward oligonucleotides, and 5' end of the
reverse oligonucleotides, respectively. EcoRI and ApaI
restriction site was added to the 5' and 3' end of the reverse
oligonucleotides, respectively. The forward and reverse oligonucleotides were
annealed, and were inserted into the pSilencer 1.0-U6 (Ambion).
|
Immunohistochemistry
For whole-mount immunohistochemistry, the following monoclonal antibodies
were used as primary antibodies: anti-En2 antibody, 4D9 (American Type Culture
Collection), anti-di-phosphorylated ERK (Sigma), anti-neurofilament antibody,
3A10 (Developmental Studies Hybridoma Bank) and anti-HA antibody (Boehringer).
For detection of En2 and activated ERK, horseradish peroxidase
(HRP)-conjugated anti-mouse IgG (Jackson) and biotinylated anti-mouse IgG were
used as secondary antibodies, respectively. For detection of neurofilament,
Cy3-conjugated anti-mouse IgG (Jackson) was used as a secondary antibody. For
detection of HA-tag, HRP-conjugated anti-rat IgG was used. Immunoreactivity
for activated ERK was detected using the ABC-Elite system (Vector
Laboratories). DAB (3,3'-diaminobenzidine) was adopted as the chromogen
for HRP.
Histology
Embryos were fixed in 4% paraformaldehyde and embedded in Historesin
(Leica). Serial sections at 5 µm were stained with hematoxylin and
eosin.
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Results |
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Misexpression of a dominant-negative form of Ras causes differentiation of the tectum instead of the cerebellum
Since ERK was activated by Fgf8b, we assumed that the Ras-ERK signaling
pathway plays a pivotal role in mes/metencephalic development. In order to
check this assumption, we misexpressed a dominant-negative form of Ras
(RasS17N), which was shown to disrupt the Ras-ERK pathway
(Feig and Cooper, 1988).
Indeed, the activation level of ERK was decreased after misexpression of
RasS17N by in-ovo electroporation in 7-10-somite stage embryos
(n=8/11) (Fig. 3A).
Misexpression of RasS17N exerted drastic effects on the development
of the presumptive metencephalon (Fig.
3B-D). At E10.5 (HH36-37), a large swelling with a smooth surface
was observed in the metencephalic region on the experimental side
(n=7/7) (Fig. 3B,D).
On the control side, the swelling displayed typical fissures of the cerebellum
(Fig. 3B,C), and had an
external granular layer (egl), which is also characteristic of the cerebellum
at E10.5 (Fig. 3E,F). On the
experimental side of the metencephalic region, the swelling lacked the
external granular layer (Fig.
3E,G). Instead, the swelling had a structure similar to that of
the tectum. The proper tectum at E10.5 has ten layers in addition to the
neuroepithelium (Fig. 3H). In
the structure on the experimental side of the metencephalic region
(Fig. 3G), we could discern
nine of the layers characteristic of the tectum. The outermost layer x, which
is composed of optic fibers, could not be discerned. Layer x was not
differentiated, possibly because all the optic fibers had already projected to
the proper tectum and hence could not reach the ectopic tectum. These results
indicate that Ras signaling is needed for differentiation of the cerebellum,
and that disruption of Ras signaling converts the fate of the metencephalic
alar plate to differentiate to the tectum. The posterior part of the swelling
at the experimental side consisted of cerebellar structure.
|
Alteration of gene expression by disruption of Ras signaling
We examined the effects of the misexpression of RasS17N on
molecular markers for the mesencephalon and the metencephalon. In normal
embryos, the homeobox genes, Otx2 and Gbx2, are expressed in
the mesencephalon and the metencephalon, respectively
(Simeone et al., 1992;
Bally-Cuif et al., 1995
;
Millet et al., 1996
;
Bouillet et al., 1995
;
Niss and Leutz, 1998
;
Shamim and Mason, 1998
;
Hidalgo-Sanchez et al., 1999
).
It has been suggested that repressive interaction between Otx2 and
Gbx2 determines the MHB, and that Fgf8 mRNA is induced at
the interface of Otx2 and Gbx2 expression overlapping with
Gbx2 expression (Millet et al.,
1999
; Broccoli et al.,
1999
; Katahira et al.,
2000
; Li and Joyner,
2001
; Ye et al.,
2001
). At 24 hours after electroporation of RasS17N
(E2.5, HH17), induction of Otx2 and repression of Gbx2 in
the metencephalon were observed (Otx2; n=9/9, Gbx2;
n=7/8) (Fig. 4A-F).
Expression of Fgf8 was also repressed by RasS17N, but was
induced in the caudal part of its expression belt so that the Fgf8 expression
belt became wider (n=3/3) (Fig.
4G-I).
|
Next, we examined the effect on Wnt1 expression. At E2.5, Wnt1 was expressed in the dorsal midline of the mesencephalon and in the caudal mesencephalon. Since the dorsal midline of the metencephalon does not express Wnt1, it is a good marker to discriminate between metencephalon and mesencephalon (Fig. 5A, control side). After disruption of Ras signaling, Wnt1 was induced in the dorsal metencephalon on the experimental side (n=3/4) (Fig. 5A,B). These effects of RasS17N on marker gene expression also support the notion that disruption of Ras signaling changes the fate of the metencephalon to that of the mesencephalon.
|
The Ras signaling pathway functions downstream of FgF8b, but not Fgf8a
Morphological and gene expression analyses indicate that the Ras signaling
pathway plays an important role in mes/metencephalic fate determination. To
ascertain if the Ras signaling pathway functions at the downstream of the
FgF8b signal, we carried out co-transfection of Fgf8b with
RasS17N. If Ras functions at the downstream of the Fgf8b
signal, cotransfection of RasS17N with Fgf8b may
cancel the effects of Fgf8b misexpression. Conversely, if Ras does
not transduce the Fgf8b signal, co-transfection may exert additive effects.
After co-transfection, some large swellings were observed on the experimental
side of the mes/metencephalon of E10.5 embryos (n=4/4)
(Fig. 6A,B). Histologically,
these swellings showed a tectal structure (compare
Fig. 6C,D with
6E,F), in agreement with our
prediction. The anterior part of the presumptive metencephalon differentiated
into the tectum (Fig. 6A,E). In
the posterior part of the presumptive metencephalon, target genes of ERK may
have been already turned prior to expression of the introduced gene product,
explaining why cerebellar differentiation may have occurred in this
region.
|
Differential silencing of Fgf8a and Fgf8b by siRNA method
We previously showed that Fgf8b could change the fate of the mesencephalon
to the metencephalon (Sato et al.,
2001), and have shown in the present study that disruption of the
Ras-ERK signaling pathway resulted in the fate change of the metencephalon to
the mesencephalon. These results suggest that the Fgf8b signal activates the
Ras-ERK signal pathway to organize the metencephalic differentiation. To
confirm this notion, we tried differential disruption of Fgf8a and
Fgf8b by the siRNA method. Since the vector-based-siRNA method has
been realized recently (Katahira and
Nakamura, 2003
), we introduced the siRNA expression vectors to the
metencephalon and mesencephalon by electroporation
(Fig. 7A).
Massive degradation of Fgf8 mRNA could not be detected after Fgf8b-siRNA application, but downregulation of Fgf8 mRNA to some extent could be detected (n=5/7) (Fig. 7B-E). Degradation of Fgf8 mRNA by Fgf8a-siRNA could not be detected (n=8/8) (Fig. 7F-I). Efficient degradation of Fgf8 mRNA could be observed after application of a mixture of Fgf8a- and Fgf8b-siRNA (n=3/5) (Fig. 7J-M). Since the Fgf8 probe hybridizes to both Fgf8a and Fgf8b mRNA, disruption of each Fgf8 mRNA after application of each siRNA may be more than we could observe.
Next we checked the effects of differential disruption of Fgf8a and Fgf8b by siRNA on ERK activation. The activation level of ERK was decreased after electroporation of FGF8bsiRNA (n=7/10) (Fig. 7N) and of both Fgf8a- and Fgf8bsiRNA (n=7/11) (Fig. 7P). Fgf8a-siRNA alone did not affect the activation level of ERK (n=11/14) (Fig. 7O). RasS17N more effectively repressed ERK activation than Fgf8b-siRNA (compare Fig. 3A with Fig. 7N). The difference may be due to the fact that Fgf8 exerts its effects non-cell autonomously but that RasS17N exerts its effects cell autonomously. If Fgf8 mRNA is degraded by siRNA in some cells, the Fgf8 signal from the adjacent intact cells may take its place. However, RasS17N shuts off the downstream Ras signal of the transfected cell.
The effects of siRNA on Otx2 expression were examined, since
Otx2 misexpression in the metencephalon changes its fate to the
mesencephalon (Katahira et al.,
2000). Transfection of Fgf8b-siRNA resulted in induction of
Otx2 expression in the isthmic region (n=4/14)
(Fig. 7Q-S); that is, the
Otx2 expression domain extended caudally, although the effect is very
subtle because of the above-mentioned reason. Transfection of Fgf8a-siRNA did
not affect Otx2 expression (n=9/9)
(Fig. 7T-V). The effect of
Fgf8b-siRNA on Otx2 expression also suggests that disruption of
Fgf8 mRNA may have occurred more than we could detect.
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Discussion |
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Eight isoforms of Fgf8 have been identified to date
(Crossley and Martin, 1995;
MacArthur et al., 1995b
), with
Fgf8a and Fgf8b being expressed in the isthmus
(Sato et al., 2001
). Fgf8a and
Fgf8b possess different organizing activities for brain development.
Fgf8b-soaked beads implanted in the presumptive diencephalon induce cerebellar
structures closest to the bead with tectum around the mini cerebellum
(Martinez et al., 1999
).
Transgenic mice in which Fgf8b was misexpressed under the control of
a Wnt1 enhancer changed the property of the presumptive diencephalon
and mesencephalon to that of the metencephalon
(Liu et al., 1999
). Moreover,
misexpression of Fgf8b by electroporation completely changed the fate
of the mesencephalic alar plate so that it differentiated into cerebellum
(Sato et al., 2001
;
Liu et al., 2003
).
Misexpression of Fgf8a caused expansion of the midbrain
(Lee et al., 1997
;
Sato et al., 2001
). Although
Fgf8a and Fgf8b show different organizing activities, lower
doses of Fgf8b exert similar effects to those of Fgf8a; the
tectum was induced around the mini cerebellum in Fgf8b-bead implantation
experiments, and electroporation with lower doses of Fgf8b exerted
similar effects to those seen with Fgf8a
(Sato et al., 2001
;
Liu et al., 2003
). These
results suggest that the difference in organizing activity between Fgf8a and
Fgf8b is attributable to the difference in the intensity of the signal. This
notion was further confirmed by the results of this study. Misexpression of
Fgf8b at 1 µg/µl resulted in activation of ERK at
Fgf8b misexpressing sites throughout the diencephalon and
metencephalon, while misexpression of Fgf8b at 0.01 µg/µl or
Fgf8a at 1 µg/µl resulted in activation of ERK in only a
portion of the diencephalon.
Ras, a member of a group of small GTP-binding proteins, is activated
downstream of various RTKs and activates Raf, Mek and ERK in turn (reviewed by
Katz and McCormick, 1997;
Rommel and Hafen, 1998
). Since
ERK was activated in the isthmus and sites of Fgf8b misexpression, we
assumed that activation of the Ras-ERK pathway is necessary for metencephalic
fate determination. Fate change of the alar plate is easily identifiable
because of the distinct structures of the tectum and cerebellum. As expected,
disruption of the Ras-ERK pathway by a dominant-negative form of Ras
(RasS17N) in the alar plate of the metencephalon caused its fate
change to the tectum. To follow the development of the basal plate, we paid
attention to the oculomotor and trochlear nerves. In
RasS17N-transfected embryos, nerve fibers running a similar course
to that of the trochlear nerve arose from the caudal end of the ectopic
swelling(s) in the metencephalic region. Additional nerve trunks similar to
the oculomotor nerve originated from the ventral metencephalon in some cases.
Moreover, at 24 hours after electroporation of RasS17N, induction
of Otx2 and repression of Gbx2 in the metencephalon
occurred. Thus, we concluded that disruption of Ras signaling by a
dominant-negative form of Ras converted the fate of the presumptive
metencephalon to that of the mesencephalon. As in the case of Otx2
misexpression (Katahira et al.,
2000
), Otx2 induction was patchy at first, but a large
tectum differentiated in the most effective case. Repression of Gbx2
and Fgf8 was also patchy at first (24 hours after electroporation),
but a wide region in which Gbx2 and Fgf8 were not expressed
appeared just posterior to the proper mesencephalon (42 hours after
electroporation). Fgf8 expression line(s) were established posterior
to the Fgf8-free region. The results indicate that regulation of
Otx2, Gbx2 and Fgf8 expression may have taken place. Thus
new Fgf8 line(s) may have served as a new organizer, and most of the
presumptive r1 region may have changed its property to that of the
mesencephalon. In some cases, fate change to mesencephalon may have occurred
patchily because a number of trochlear nerves differentiated in the
metencephalic region (see Fig.
3M,N).
Further evidence to support the hypothesis that the Ras-ERK pathway is activated by Fgf8b to result in metencephalic differentiation comes from co-transfection studies with RasS17N and Fgf8b. If the Ras-ERK pathway does indeed transduce the Fgf8 signal, then co-transfection may cancel the Fgf8 signal. However, if the Ras-ERK pathway does not transduce the Fgf8 signal, co-transfection may exert additive effects. Accordingly, co-transfection of RasS17N and Fgf8b canceled the effects of Fgf8b misexpression, while cotransfection of Fgf8a and RasS17N caused differentiation of the ectopic tectum in the diencephalon and in the metencephalon, displaying the additive effects of Fgf8a and RasS17N misexpression. Distinct disruption of Fgf8a and Fgf8b also supports the notion that Fgf8b activates Ras-ERK signaling pathway to organize cerebellar differentiation. Disruption of Fgf8b by its specific siRNA resulted in a decrease in the activation level of Erk, and in caudal extension of the Otx2 expression domain. siRNA for Fgf8a did not affect the activity of ERK. In conclusion, the results indicate that Fgf8b functions as the organizer for the metencephalon by activating the Ras-ERK pathway.
Since Fgf8 mutant mice or zebrafish show disruption of the
mes/metencephalon (Meyers et al.,
1998; Reifers et al.,
1998
; Chi et al.,
2003
), it was thought that Fgf8 might also be necessary
for the development of the mesencephalon. However, our results show that
disruption of the Ras signaling pathway did not affect the fate of the
presumptive mesencephalon, and actually changed the fate of the presumptive
metencephalon to that of the mesencephalon. This suggests that Ras signaling
does not play a role in fate determination of the mesencephalon. To accord our
assumption, animal cap assay indicated that the PLC
signaling pathway
through Fgf receptor IV (FgfR4) is responsible for the fate decision of the
mesencephalon (Umbhauer et al.,
2000
). However, it was suggested that FgfR1 is the receptor for
the Fgf8 signal in the isthmus region (Liu
et al., 2003
; Trokovic et al.,
2003
). So far, it is not reported that FgfR4 is expressed in the
isthmic region as suggested (Walshe and
Mason, 2000
). Further study is needed to determine what signaling
pathway is responsible for the mesencephalic determination.
In the mesencephalon, the Fgf8-Ras-ERK signaling pathway may be involved in
rostrocaudal polarity formation. For the rostrocaudal polarity formation, it
is suggested that En confers caudal property to the tectal anlage so that the
rostrocaudal polarity of the tectum is determined according to a gradient of
En (Itasaki and Nakamura,
1996; Friedman and O'Leary,
1996
). ERK is activated in a gradient in the mesencephalon, as
revealed by whole-mount immunohistochemistry. The gradient corresponds to that
of En2 expression at the 14-somite stage, when the rostrocaudal axis is still
plastic. Pax2/5, En1/2 and Fgf8 act in a positive feedback
loop (reviewed by Nakamura,
2001
). Disruption of Ras signaling caused repression of
Pax2/5 and En1/2 expression. Thus, the Ras-ERK pathway,
which is activated by Fgf8, may play a crucial role in formation of the
rostrocaudal polarity of the tectum.
In normal embryos around the 8-somite stage, ERK was activated in the
region where Fgf8 mRNA was expressed. In the metencephalon, ERK
became inactivated by the 14-somite stage, while it remained activated in the
mesencephalon. This indicates that the gene expression cascade favoring
cerebellar differentiation has proceeded by the 10-somite stage, meaning that
the fate of the metencephalon is determined by this time. This notion is
supported by ectopic transplantation studies that show that while the
rostrocaudal axis of the mesencephalon is not fixed at the 10-somite stage,
the fate of the mesencephalon and metencephalon is already determined
(Nakamura et al., 1986;
Nakamura et al., 1988
;
Ichijo et al., 1990
;
Matsuno et al., 1990
).
Focusing on mechanisms of mes/metencephalic development
(Fig. 8), expression of
Fgf8 mRNA is induced at the interface of Otx2 and
Gbx2 expression, overlapping with Gbx2 expression; that is,
at the presumptive metencephalon (Millet
et al., 1999; Broccoli et al.,
1999
; Hidalgo-Sanchez et al.,
1999
; Irving and Mason,
2000
; Katahira et al.,
2000
; Ye et al.,
2001
; Garda et al.,
2001
; Li and Joyner,
2001
; Martinez-Barbera et al.,
2001
; Li et al.,
2002
). Consequently, the presumptive metencephalic region may
receive a strong Fgf8 signal, in turn activating the Ras-ERK pathway, which
may result in turning on the gene cascade favoring development of the
cerebellum. The cascade might be turned on before the 10-somite stage, because
ERK becomes strongly activated at around the 8-somite stage, its activity
gradually weakening thereafter. Our results correspond well to the classical
transplantation experiments that show that the fate of the mesencephalon and
metencephalon is determined before the 10-somite stage. In the mesencephalon,
the gene expression cascade toward cerebellar differentiation may not be
turned on because Otx2 is expressed there. Fgf17 and Fgf18 that are induced by
Fgf8 together with Fgf8a may regulate proliferation of the mesencephalon and
metencephalon (Xu et al.,
2000
; Liu et al.,
2003
). In the mesencephalon, ERK activity remains in a gradient
distribution after the 10-somite stage, and may contribute to the
determination of the rostrocaudal axis of the tectum.
|
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ACKNOWLEDGMENTS |
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Footnotes |
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