MRC Centre for Developmental Neurobiology, New Hunt's House, King's College London, Guy's Campus, London SE1 1UL, UK
* Author for correspondence (e-mail: ivor.mason{at}kcl.ac.uk)
Accepted 2 June 2003
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SUMMARY |
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Key words: FGF, Zebrafish, Forebrain, Telencephalon, Diencephalon, Thalamus, Commissure, Zona limitans intrathalamica
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INTRODUCTION |
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Fibroblast growth factors (Fgfs) regulate patterning events in the midbrain
and hindbrain, and have synergistic actions when expressed in overlapping
domains. Notably, Fgf8 and Fgf17, expressed midbrain-hindbrain boundary
(isthmus), regulate the patterning and later aspects of development of the
adjacent territories (Crossley et al.,
1996; Reifers et al.,
1998
; Shamim et al.,
1999
; Martinez et al.,
1999
; Liu et al.,
1999
; Irving and Mason,
1999
; Irving and Mason,
2000
; Xu et al.,
2000
; Reifers et al.,
2000
; Sato et al.,
2001
; Irving et al.,
2002
). There is also a combinatorial role for Fgf8 and Fgf3, from
presumptive rhombomere 4, in patterning the hindbrain
(Walshe et al., 2002
;
Maves et al., 2002
), and in
induction of the adjacent otic placode
(Phillips et al., 2001
;
Maroon et al., 2002
;
Leger and Brand, 2002
).
An emergent theme is one of both unique and combinatorial functions for
Fgfs in brain patterning. Several Fgfs are expressed within the developing
forebrain (Mason et al., 1994;
Mahmood et al., 1995
;
Crossley and Martin, 1995
;
Mahmood et al., 1996
;
McWhirter et al., 1997
;
Reifers et al., 2000
;
Crossley et al., 2001
;
Gimeno et al., 2002
), and Fgf8
has been assigned a role in forebrain development. Mutant mice carrying
hypomorphic fgf8 alleles have smaller forebrains with midline
deletions (Meyers et al.,
1998
). A patterning function is supported by in vitro studies
using both chick and mouse tissues
(Shimamura and Rubenstein,
1997
; Crossley et al.,
2001
), by in vivo studies in the mouse
(Fukuchi-Shimogori and Grove,
2001
), and by analysis of the zebrafish acerebellar
(ace) mutant (Shanmugalingam et
al., 2000
). Although detailed analyses are lacking, fgf3
is also expressed in the forebrain,
(Mahmood et al., 1996
;
Raible and Brand, 2001
;
Walshe et al., 2002
) and its
ectopic expression affects the expression of certain forebrain markers
(Koshida et al., 2002
).
We report a complex and dynamic expression pattern for fgf3 in the zebrafish forebrain, which partially overlaps with that of fgf8. Using morpholino oligonucleotides to inhibit Fgf3 and Fgf8, both individually and together, we identify unique functions for Fgf3 in both telencephalon and several regions of the diencephalon, and in combinatorial actions with Fgf8. In addition, we report further roles for Fgf8 in forebrain development.
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MATERIALS AND METHODS |
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Morpholino oligonucleotide injections
Fgf8, Fgf3 and Fgf control morpholino oligonucleotides (Gene Tools), at a
concentration of 6 µg/µl, were injected into zebrafish embryos as
previously described (Maroon et al.,
2002). Embryos were dechorionated and incubated with the FGFR
inhibitor SU5402, as previously described
(Maroon et al., 2002
), except
that SU5402 stock solutions were prepared at 10 mM and diluted to 0.1 mM for
use.
In situ hybridisation
In situ hybridisation reactions were essentially performed as described
previously (Shamim et al.,
1999; Maroon et al.,
2002
), except that embryos younger than 24 hours post
fertilisation (hpf) were not treated with proteinase K, and the hydrogen
peroxide treatment was omitted.
Cell death and division
Dividing cells were detected using an anti-phosphorylated histone H3 (ser
128) antiseurm (Calbiochem), and apoptotic cells were detected using the
DeadEndTM colourimetric detection kit (Promega) as described
(Maroon et al., 2002). Numbers
of dividing cells, within an area measuring 200 µm by 300 µm
encompassing the presumptive forebrain from four embryos at tailbud stage
injected with either control morpholinos, Fgf8 morpholinos (Fgf8mo), Fgf3
morpholinos (Fgf3mo), or both Fgf8mo and Fgf3mo, were determined and subjected
to a Student's t-test for statistical analysis.
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RESULTS |
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Descriptions of fgf3 expression in zebrafish embryos have been
reported previously (Phillips et al.,
2001; Raible and Brand,
2001
; Shinya et al.,
2001
; Maroon et al.,
2002
; Walshe et al.,
2002
; Maves et al.,
2002
; Leger and Brand,
2002
); however, detailed analyses of the developing forebrain were
not included in those studies. fgf3 transcripts were first detected
in anterior neuroectoderm at 80% epiboly
(Fig. 1G), and at 90% epiboly
fgf3 expression was detected in cells of the presumptive forebrain
and underlying prechordal hypoblast (Fig.
1H) (Phillips et al.,
2001
). At the beginning of somitogenesis, fgf3 was
expressed in cells of the anterior neural boundary (row 1 cells;
Fig. 1H,I). At early somite
stages, transcripts became confined to the dorsal telencephalon and the
polster located anterior to the forebrain
(Fig. 1J). At late somite
stages, very low level fgf3 expression remained in the telencephalon,
while a new expression domain appeared in the ventral hypothalamus
(Fig. 1K). fgf3
transcripts remained within the ventral hypothalamus until at least 30 hpf and
were additionally present in the optic stalks at this stage
(Fig. 1L).
Morpholino oligonucleotides effectively inhibit Fgf3 and Fgf8
functions but do not result in increased cell death or division in the
forebrain
To analyse the involvement of Fgf3 and Fgf8 in forebrain development, 1- to
4-cell stage zebrafish embryos were injected with morpholino oligonucleotides
to inhibit their translation. We have previously shown that these morpholino
oligonucleotides compromise Fgf8 and Fgf3 function, phenocopying the
ace (fgf8) mutant and rendering Fgf3 protein undetectable,
respectively (Maroon et al.,
2002; Walshe et al.,
2002
).
Fgf morpholino injection did not appear to affect cell division in the forebrain at either the tailbud or 10-somite stage (10s; Fig. 2A-H); this was confirmed by Student's t-test analyses. In addition, no changes in cell death were detected in forebrains at 10s (Fig. 2I-P) or at 24 hpf (data not shown).
|
|
dlx2 is expressed in the zebrafish telencephalon and ventral
thalamus from 13 hpf (Akimenko et al.,
1994), and is required for the development of specific
telencephalic neurons in mice (Qiu et al.,
1995
; Anderson et al.,
1997
). Injection of Fgf3mo resulted in variable reduction of
dlx2 expression in the telencephalon at 24 hpf (n=6/9), but
complete loss of expression by 28 hpf (n=22/23), whereas
Fgf8mo-injection had no effect on telencephalic dlx2 expression at
either 24 hpf (n=11/11) or 28 hpf (n=21/21;
Fig. 3I-L,Q-T). Double in situ
hybridisation results demonstrated that expanded tbr1 expression in
the subpallial telencephalon following Fgf8mo injection was largely anterior
and lateral to the more medially-located dlx2 domain in that region
(Fig. 3J).
Fgf3 and Fgf8 regulate the transcription of each other in the hindbrain
(Walshe et al., 2002), and we
found that in the absence of Fgf8, fgf3 transcripts were strongly
upregulated in the telencephalon at 28 hpf, whereas the absence of Fgf3
protein did not produce such an effect. Upregulated fgf3 transcripts
were not detected in the telencephalons of embryos lacking both Fgf8 and Fgf3,
although normal expression was detected in the hypothalamus (n=23/23;
Fig. 3I-L). Taken together, our
data suggest that these two factors perform both combinatorial and unique
functions in telencephalic patterning.
Patterning of the diencephalon requires Fgf3 and Fgf8 function
We examined the effects of Fgf3 and Fgf8 inhibition on regional
diencephalic development using markers of the hypothalamus [shh,
nk2.1b (titf1b - Zebrafish Information Network), fgf3],
ventral thalamus [nk2.1b, shh, twhh, dlx2, pax6.1 (pax6a -
Zebrafish Information Network)], dorsal thalamus (pax6.1), zona
limitans intrathalamica (shh) and optic stalks (pax2.1;
pax2a - Zebrafish Information Network).
Hypothalamus
shh is expressed in the hypothalamus, as well as in other regions
of the brain at 30 hpf (Fig.
4A) (Krauss et al.,
1993; Mathieu et al.,
2002
). Expression was substantially reduced in embryos injected
with both Fgf8 and Fgf3 morpholinos (n=14/20), but not in embryos
injected with either morpholino alone (Fig.
4A-D). By contrast, hypothalamic fgf3 expression was
unaffected in embryos injected with the Fgf8mo and/or Fgf3mo
(Fig. 3Q-T). nk2.1b,
required for ventral forebrain development in mice
(Kimura et al., 1996
), is
expressed in the zebrafish hypothalamus, as well as in the anterior ventral
thalamus and subpallial telencephalon at 30 hpf
(Fig. 4E). Previous studies
showed reduced expression in the forebrains of zebrafish embryos deficient in
shh signalling (Rohr et al.,
2001
). Unexpectedly, embryos co-injected with Fgf8mo and Fgf3mo
had normal nk2.1b expression in the hypothalamic (and telencephalic)
regions (Fig. 4H), despite
shh being greatly reduced when both Fgf8 and Fgf3 were inhibited
(Fig. 4D). These results
suggest that residual Shh in embryos injected with both morpholinos may have
been sufficient to regulate nk2.1b, or that expression of the latter
was dependent upon earlier Shh signalling.
|
To examine patterning of the posterior ventral thalamus, dlx2 and
pax6.1 were analysed. As in telencephalon, injection of Fgf3mo
resulted in reduced expression of dlx2 in the ventral thalamus at 24
hpf (n=6/9), and complete loss of expression by 28 hpf
(n=23/23), whereas Fgf8mo injection had no effect at either 24 hpf
(n=11/11) or 28 hpf (n=21/21;
Fig. 3I-L,Q-T). pax6.1
expression marks posterior ventral thalamus, dorsal thalamus and pretectum at
28 hpf (Fig. 4M)
(Püschel et al., 1992;
Nornes et al., 1998
;
Hauptmann et al., 2002
).
pax6.1 transcripts were detected in the diencephalons of all embryos
at 28 hpf; however, the extent and pattern of expression was altered in
embryos lacking either Fgf3, or both Fgf3 and Fgf8. In these embryos, ventral
thalamic and dorsal thalamic domains of expression were reduced, and there was
no clear separation between them, which is suggestive of defects in the ZLI
(Fig. 4M-P). Overall, we found
patterning defects in both the anterior and posterior ventral thalamus in
embryos lacking Fgf3, with more severe defects in the anterior ventral
thalamus in embryos lacking both Fgf8 and Fgf3.
Zona limitans intrathalamica
The ZLI expresses shh, may pattern the adjacent ventral and dorsal
thalamus, and, in chick, has been identified as a lineage-restricted
compartment (Zeltser et al.,
2001). Embryos injected with either Fgf3mo (n=14/16), or
both Fgf3mo and Fgf8mo (n=20/20), had substantially reduced
expression of shh in the ZLI region at 30 hpf. In particular,
expression was undetectable in the dorsal ZLI
(Fig.
4A-D,A'-D').
Optic stalks
The optic stalks are transitory structures through which retinal axons
extend to the diencephalon. pax2.1 is expressed in the optic stalks
and is required for their development
(Krauss et al., 1991;
Macdonald et al., 1997
). At 18
hpf, two well-separated lateral domains of pax2.1 expression
corresponding to the optic stalks were present in embryos lacking either Fgf8
(n=13/13) or Fgf3 (n=15/15). By contrast, when both Fgfs
were depleted these expression domains were fused at the midline
(n=10/14), providing further evidence for a patterning defect in the
ventral thalamic midline and a potential problem with separation of the eye
field (Fig. 4Q-T), although
there was no evidence of cyclopia.
In summary, diencephalic patterning defects were observed in the ventral thalamus in the absence of Fgf3, and were more severe in the absence of both Fgf3 and Fgf8. Additional Fgf3-dependent defects were found in the ZLI, where shh was reduced and adjacent thalamic pax6.1 domains merged.
Axon tract formation in the forebrain is disrupted in embryos lacking
Fgf3 and Fgf8
At 16 hpf neurons appear as bilateral clusters within the forebrain
(Wilson et al., 1990;
Ross et al., 1992
). A pair of
ventrorostral clusters, positioned ventral to the optic stalks within the
diencephalon, extend axons towards the midline at 18 hpf to form the
post-optic commissure (POC). Soon after, a pair of dorsorostral clusters
within the telencephalon extend axons to form the anterior commissure (AC;
Fig. 5A-C). Because morpholino
injections affected forebrain midline gene expression patterns, we examined
the formation of these commissures. The AC and POC were visualised using an
acetylated ß-tubulin antibody at 34 hpf, following Fgf morpholino
injection (Fig. 5D-F). The AC
did not form properly in embryos injected with Fgf8mo (n=8/19), and
axons with abnormal trajectories were observed in the space between the two
commissures, which is normally devoid of axons, in agreement with previous
results (Shanmugalingam et al.,
2000
). The POC developed normally in the majority of these embryos
(n=18/19; Fig. 5G-I).
Commissure formation was more severely affected in embryos injected with
Fgf3mo: both the AC (n=14/16) and POC (n=12/16) failed to
form, and axons projected abnormally. In less severely affected embryos, the
AC and POC were situated abnormally close together at the midline
(Fig. 5J-L). Both commissures
failed to form and axons projected abnormally after co-injection of Fgf3mo and
Fgf8mo (n=9/12), and in the most severe cases no axons were observed
in the midline (Fig. 5M-O).
|
zash1a (asha - Zebrafish Information Network), a member of the achaete-scute family of transcription factors, is expressed in the zebrafish ventral forebrain from 9 hpf, and by 18 hpf it is expressed in dorsorostral and ventrorostral clusters, and in presumptive epiphysis. zash1a is proposed to have a proneural function, and expression precedes neuronal differentiation (Allende and Weinberg et al., 1994). We examined zash1a expression at 18 hpf when the first forebrain neurons differentiate. A minority of embryos injected with Fgf8mo had reduced expression in the dorsorostral and ventrorostral clusters, with increased expression of zash1a in presumptive epiphysis (n=5/13). Injection of Fgf3mo (n=13/13), or Fgf3mo and Fgf8mo (n=15/15), resulted in a more dramatic phenotype; dorsorostral and ventrorostral cluster expression was severely reduced or absent, whereas epiphysial expression was expanded in these embryos (Fig. 6A-D). These results indicated a protential problem with neuronal specification in dorsorostral and ventrorostral clusters in embryos lacking functional Fgfs, in particular in embryos lacking Fgf3.
|
Forebrain patterning requires Fgf signalling at multiple
developmental stages
Morpholinos can interfere with gene function from the time of their
injection. Therefore, to provide an indication of the temporal requirement for
Fgf signals, and also an independent assay of Fgf function, embryos were
treated at different stages with the FGFR inhibitor SU5402
(Mohammadi et al., 1997) and
subsequently analysed for changes in gene expression corresponding to those
observed with the Fgf morpholinos. Embryos were treated from 50% epiboly until
80% epiboly [corresponding with fgf expression in the shield
(Walshe et al., 2002
), and
prior to expression in the presumptive forebrain], from 80% epiboly until
tailbud (when only fgf3 is expressed in the presumptive forebrain),
continuously from 50% epiboly until tailbud, from tailbud until 8s (when
fgf3 and fgf8 expression overlaps in the telencephalon), or
from 13s until 18s (when distinct fgf3 and fgf8 expression begins in
the diencephalon). Sister embryos were taken from each batch and analysed for
erm expression immediately following treatment. erm is a
transcription factor downstream of MAPK that is dependent upon Fgf signalling
for its transcription in the zebrafish embryo
(Raible and Brand, 2001
;
Roehl and Nüsslein-Volhard,
2001
). In all cases, erm expression was eliminated from
the forebrain (and other regions) of the embryo following SU5402 treatment,
which indicated the effective inhibition of Fgf receptor (Fgfr) signalling
(Fig. 7A-H; data not
shown).
|
nk2.1b expression in the telencephalon was also examined in treated embryos. Whereas telencephalic nk2.1b expression was not dependent upon Fgf3 or Fgf8 function (see Fig. 4E-H), transcripts were absent in embryos treated with SU5402 between 80% and tailbud (n=21/21), or tailbud and 8s (n=30/30), and reduced following treatment between 13s and 18s (n=14/14; Fig. 7M-P). These results suggest that Fgfr signalling is required continuously between 50% epiboly and 18s for correct telencephalic patterning, and that inhibition results in either loss or alteration of gene expression domains. They also indicate that other activators of Fgfrs, in addition to Fgf8 and Fgf3, regulate telencephalic patterning.
Expression of nk2.1b in the ventral thalamus and hypothalamus was also analysed in embryos treated with SU5402. A proportion of embryos treated between 80% epiboly and tailbud failed to express nk2.1b in the ventral thalamus and hypothalamus (n=7/21), whereas remaining embryos retained some hypothalamic expression. Embryos treated between tailbud and 8s (n=27/30), and between 13s and 18s (n=14/14), had reduced expression in the ventral thalamus and normal expression in the hypothalamus (Fig. 7M-P). These results supported those obtained using Fgf morpholinos, which indicated the requirement for Fgf3 and Fgf8 signalling in the ventral thalamus. In addition, the results suggest that Fgfr signalling, possibly involving other Fgfs, is required for hypothalamic development during gastrulation stages, but is not required at later stages.
To examine the requirement for Fgf signalling specifically within dorsorostral and ventrorostral clusters, SU5402-treated embryos were analysed for expression of isl1 at 30 hpf. Transcripts were absent or greatly reduced following treatment between 80% epiboly and tailbud (n=16/16), and between tailbud and 8s (n=22/22). Notably, expression in the epiphysis was expanded in embryos treated at these stages. isl1 expression was virtually normal in the dorsorostral and ventrorosral clusters of embryos treated between 13s and 18s (n=26/26; Fig. 7Q-T). These data suggest that neurons within the dorsorostral and ventrorostral clusters depend upon Fgfr signalling prior to 8s for specification. Moreover, they are consistent with the data obtained using Fgf3 and Fgf8 morpholinos, as both techniques resulted in reduced isl1 expression in the dorsorostral and ventrorostral clusters, and in expanded epiphysial expression.
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DISCUSSION |
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In the zebrafish telencephalon, Fgf8 appears to be primarily involved in
establishing anteroposterior (AP) polarity, with loss of some subpallial
(anterior) markers and expansion of pallial (posterior) markers when
inhibited. Thus, sema3D and net1 (ntn1 - Zebrafish
Information Network) (Shanmugalingam et
al., 2000) are downregulated in ace mutants, whereas
emx1, tbr1 and fgf3 expand anteriorly (this study). Overall,
these data are consistent with functions ascribed to Fgf8 in mouse embryos. In
addition, our results with SU5402 show that subpallial expression of
nk2.1b is also dependent upon Fgf signalling. This may be mediated in
part by Fgf8, as others report slightly downregulated expression in the
ace mutant and/or morpholino-injected embryos
(Shanmugalingam et al., 2000
;
Shinya et al., 2001
); however,
it was unaffected in our study.
In most respects, inhibition of Fgf8 with morpholinos in this study
reproduced effects on the forebrain that had been previously reported by
others (Shanmugalingam et al.,
2000). Notable exceptions were the failure of morpholinos to
downregulate the expression of transcripts for twhh and
nk2.1b. Although other explanations are possible, this may indicate
incomplete inhibition of Fgf8 by the morpholino and raises the possibility of
dose-dependent requirements for this ligand in forebrain development.
Unfortunately, this could not be investigated further as higher concentrations
of the morpholino proved to be lethal at gastrulation stages (I.M.,
unpublished).
We also find roles for Fgf signalling in posterior telencephalic development, with Fgf3 required for pallial tbr1 and dlx2 expression, while Fgf3 and Fgf8 are required together for eom expression.
We and others have examined roles of Fgf signalling in telencephalic
development using dominant-negative Fgfrs (dnFgfrs), Fgfr inhibitors or
dominant-negative Ras isoforms, an effector of Fgfr signalling (this study)
(Shinya et al., 2001).
Inhibition of Ras activity results in a loss of telencephalic dlx2
and nk2.1b, and anterior expansion of emx1, eom and
tbr1 (Shinya et al.,
2001
). This is not entirely consistent with the inhibition of Fgf3
and Fgf8, either singly or together: in the absence of Fgf8, tbr1 and
emx1 expand anteriorly, whereas eom is downregulated by both
Fgf morpholinos. Although Ras may function downstream of other receptors,
anterior expansion of tbr1 was also seen following ectopic expression
of dnFgfr1iiic or dnFgfr4 (also a iiic isoform)
(Shinya et al., 2001
), which
is consistent with a loss of Fgf8 function, but not a loss of Fgf3, or of both
Fgf8 and Fgf3. This might reflect the specificity of Fgf3 for iiib Fgfr
isoforms (Kiefer et al., 1996
;
Ornitz et al., 1996
); because
dnFgfrs are thought to function as ligand-dependent inhibitors, it is possible
that the iiic Fgfr isoforms would not have inhibited the Fgf3 signalling
required for telencephalic tbr1 expression. Thus, the complexity of
Fgf3 and Fgf8 functions in forebrain development may reflect not only to their
dynamic expression patterns, but also Fgfr ligand specificity and expression
dynamics. All Fgfrs are expressed in the developing zebrafish forebrain but
data is unavailable for individual Fgfr isoforms, although we have reported
iiib and iiic isoforms during avian forebrain development
(Thisse et al., 1995
;
Walshe and Mason, 2000
;
Sleptsova-Friedrich et al.,
2001
; Tonou-Fujimori et al.,
2002
).
Surprisingly, we also found that loss of Fgf8 upregulated telencephalic
fgf3, whereas loss of Fgf3 did not affect expression, and loss of
both Fgf3 and Fgf8 together resulted in an absence of ectopic fgf3.
These data suggest a complex interplay of Fgf activities in regulating
fgf expression. This appears to be a common theme in the developing
brain as Fgf3 and Fgf8 regulate the transcription of each other in the
hindbrain (Walshe et al.,
2002), and Fgf8 regulates its own transcription in the forebrain
(Shanmugalingam et al.,
2000
).
We found that Fgf3 and Fgf8 are required for the patterning of multiple
diencephalic derivatives. In ventral thalamus, the most striking defects
occurred in embryos lacking both Fgf8 and Fgf3. These embryos lacked
transcripts for nk2.1b, twhh and dlx2, and shh
expression was reduced, with midline expression of pax2.1 expanded.
Although shh expression was greatly reduced, the presence of
nk2.1b transcripts [Shh-dependent in the mouse
(Shimamura and Rubenstein,
1997; Ericson et al.,
1995
)] and pax2.1 [Shh-dependent in zebrafish
(Macdonald et al., 1995
)],
indicated either that forebrain Shh function was not fully compromised or that
Shh activity was required at an earlier developmental stage. Our results also
confirm a previous study, which identified a role for Fgf8 in patterning the
ventral thalamic midline (Shanmugalingam
et al., 2000
), and extend those data to provide evidence that Fgf3
is also required to pattern that tissue.
Shh expression is a characteristic of the ZLI, which develops as a
compartment between dorsal and ventral thalami
(Larsen et al., 2001). Because
of its tightly regulated compartmentation, and expression of genes such as
shh and the Wnt genes (Garda et
al., 2002
), the ZLI is postulated to be a signalling centre within
the diencephalon. Our results show that Fgf3 function is required for the
expression of shh in dorsal ZLI, and for the separation of adjacent
ventral and dorsal thalamic pax6.1 expression domains. Taken
together, these results suggest that ventral and dorsal ZLI formation may be
differentially regulated, and that Fgf3 function is required for dorsal ZLI
formation. However, it seems unlikely that Fgf3 directly regulates dorsal ZLI
formation as it is only detected in the ventral diencephalon at relevant
stages, instead factors crucial for ZLI formation may depend upon earlier Fgf3
function for their expression or function.
Both fgf8 and fgf3 come to be expressed in the hypothalamus during somitogenesis. However, shh expression was reduced, but not abrogated, following injection with both Fgf8mo and Fgf3mo, and all other markers were unaffected, which suggested that fgf8 and fgf3 were not essential for most aspects of hypothalamic development. This was supported by the Fgfr inhibition studies.
The midline tissue of the ventral thalamus and subpallial telencephalon
provides an important conduit for axons. pax2.1 transcripts were
upregulated in the ventral thalamic midline between the optic stalks in the
absence of Fgf8 and Fgf3, a phenotype also observed in embryos after elevation
of Shh, resulting in midline tissue with optic stalk morphology
(Macdonald et al., 1995).
Optic stalk pax2.1 expression begins at 6-7s
(MacDonald et al., 1997
), when
fgf3 and fgf8 are expressed in adjacent telencephalic
tissue. Thus, Fgf3 and Fgf8 may serve to antagonise Shh, and to repress
inappropriate pax2.1 expression in midline tissue.
We found that, in the absence of Fgf8, formation of the anterior commissure
was severely compromised, whereas formation of the post-optic commissure was
less affected. This is consistent with a role for Fgf8 in patterning midline
tissue of the subpallial telencephalon through which the anterior commissure
forms. Others also identified a requirement for Fgf8 in the formation of the
anterior commissure, and proposed that this was probably because of defects in
the midline tissue rather than in the axons themselves
(Shanmugalingam et al., 2000).
In support of this, our analyses showed that lack of Fgf8 had little effect on
neuronal gene expression in either ventral or dorsal rostral clusters. Loss of
Fgf3 affected both commissures, consistent with a role for Fgf3 in patterning
both subpallial telencephalon and ventral thalamus. However, a lack of Fgf3
also resulted in reduction or loss of proneural gene expression and
differentiation markers in dorsorostral and ventrorostral clusters, but not in
the loss of the clusters themselves, indicating that a problem with neuronal
specification may have contributed to the commissural defects. Embryos
deficient in both Fgf8 and Fgf3 exhibited more extreme commissural phenotypes
but showed similar effects on neuronal markers as Fgf3mo alone.
Whereas axons extended within the midline territory following inhibition of Fgf3, inhibition of both Fgf3 and Fgf8 frequently resulted in a complete absence of axons from the midline territory.
Dynamic spatial and temporal expression of fgf3 and fgf8,
coupled with both unique and combinatorial actions in forebrain development,
suggested that Fgf signalling is required during multiple stages of forebrain
morphogenesis. This was investigated by pharmacological inhibition of Fgfr
activity during different periods of development. Our results confirmed a
requirement for Fgfr activity in forebrain from at least the beginning of
gastrulation until 18s. This contrasts with studies that indicate that Fgf
patterning activities in hindbrain, isthmus and otic induction only require
signalling during a brief, 2 hour period from late epiboly
(Walshe et al., 2002;
Maroon et al., 2002
). It
should be noted that SU5402 also produced some additional effects on gene
expression and embryo morphology not observed when Fgf3 and Fgf8 were
specifically inhibited. These are indicative of additional functions of Fgf
signalling in forebrain development, most likely mediated by other Fgf
ligands.
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ACKNOWLEDGMENTS |
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REFERENCES |
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