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First published online 24 July 2008
doi: 10.1242/dev.021618
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RIKEN Brain Science Institute, 2-1 Hirosawa, Wako-Shi, Saitama 351-0198, Japan.
* Author for correspondence (tshimogori{at}brain.riken.jp)
Accepted 26 June 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Diencephalon, Thalamus, Nuclei, Homeobox genes, Shh, Fgf8, Mouse
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INTRODUCTION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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). The central nervous system (CNS) is a complex tissue
including multiple types of neurons, which participate in elaborate neuronal
circuits. Signaling molecules secreted from distinct signaling centers, which
establish positional information and regulate regional growth, can allocate
neuronal cell identities, as well as regional identities. For example,
regional signaling centers have been shown to establish cortical area maps,
midbrain nucleogenesis, dorsal ventral patterning in the spinal cord, and the
compartmentalization of the diencephalon
(Fukuchi-Shimogori and Grove,
2001; Agarwala et al.,
2001; Chiang et al.,
1996; Kiecker and Lumsden,
2004).
The thalamic region comprises three functionally distinct zones, the
prethalamus, the thalamus proper and the pretectum, along the
anterior-posterior (A/P) axis of the diencephalon
(Figdor and Stern, 1993;
Puelles and Rubenstein, 2003;
Larsen et al., 2001). The
thalamus has been considered as a relay center in which specific thalamic
nuclei receive and project particular sets of fibers to targeted cortical
fields. Sensory inputs are, in turn, transmitted to the somatosensory cortex
through a relay station in the ventroposterior (VP) nucleus complex, known as
the barreloid of the thalamus (Erzurumlu
and Kind, 2001). By contrast, the prethalamus, which includes the
reticular nucleus, zona incerta and ventral lateral geniculate nucleus (vLGN),
does not send axons to cortex.
The progenitor region of diencephalon has been subdivided into transverse
domains defined by morphological and molecular criteria, including p1 (the
presumptive pretectum), p2 (presumptive thalamus) and p3 (presumptive
prethalamus). Recent studies have established the identity of molecules that
may control the patterning of the diencephalon in mice
(Nakagawa and O'Leary, 2001;
Nakagawa and O'Leary, 2003;
Fode et al., 2000;
Miyashita-Lin et al., 1999;
Puelles et al., 2006;
Suda et al., 2001), monkey
(Jones and Rubenstein, 2004)
and chick (Kobayashi et al.,
2002; Lim and Golden,
2002). The zona limitans intrathalamica (ZLI), a neuroepithelial
domain in the alar plate of the diencephalon, has been suggested to separate
p3 from p2 (Larsen et al.,
2001), and may function as a secondary organizer
(Vieira et al., 2005). The
expression of the signaling molecule sonic hedgehog (Shh) in ZLI is a key
source of signals that pattern the thalamus in mice
(Ishibashi and McMahon, 2002),
chick (Kiecker and Lumsden,
2004; Lim and Golden,
2007; Vieira et al.,
2005; Hashimoto-Torii et al.,
2003; Zeltser,
2005) and zebrafish (Scholpp
et al., 2006). Additionally, Wnt expression in thalamus is also
required for normal development, especially for the establishment of regional
thalamic identities (Braun et al.,
2003; Zhou et al.,
2004). However, the molecular and cellular mechanisms that pattern
functional nuclei within each region in the diencephalon are still largely
unknown.
Fibroblast growth factor 8 (Fgf8) is expressed in the dorsal part of the
diencephalon, as already described in previous studies and also in the present
study (Fig. 1C), though its
true function in the diencephalon is largely unknown
(Crossley et al., 2001).
Although, Fgf8-coated bead placement in caudal diencephalon induces an ectopic
midbrain/hindbrain boundary (Crossley et
al., 1996), the function of endogenous Fgf8 in diencephalon has
not been explored. Fgf8 is also expressed in other parts of the CNS in various
animals for appropriate control of development, suggesting important roles for
it in the developing diencephalon (Chi et
al., 2003; Garel et al.,
2003; Shanmugalingam et al.,
2000; Shimamura and
Rubenstein, 1997; Storm et
al., 2006; Walshe and Mason,
2003; Ye et al.,
1998). We explored this possibility by employing mouse in utero
electroporation to manipulate Fgf8 in the developing diencephalon in a
temporally and spatially restricted manner. We found that Fgf8 activity
controls the patterning of thalamic nuclei and also that of some of the
prethalamic nuclei on the A/P axis. However, analysis of electroporated brains
in the early embryonic stage demonstrated that regional shifts due to FGF
activity occur only in the p2 progenitor region. Our detailed gene analysis in
the early embryonic stage suggests the identity of the progenitor regions of
nuclei shifted by FGF activity. These findings aid in determination of the
molecular and cellular mechanisms of nucleogenesis of the diencephalon.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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In situ hybridization
For section in situ hybridization, brains were removed, fixed overnight in
30% sucrose/4% paraformaldehyde, and sectioned in a semi-horizontal plane
(Fig. 2A) on a Leica sledge
microtome at 40 µm. Each section was mounted on slides and hybridized to
visualize expression of different gene. For whole-mount in situ hybridization,
embryos are collected and fixed overnight. Each pattern of gene expression was
confirmed in at least four to six replications/age. Single- or two-color
non-radioactive in situ hybridization was performed using a method described
previously (Grove et al.,
1998), including use of the chromagens nitroblue tetrazolium
(Nacalai, Japan; 350 mg/ml) and tetranitroblue tetrazolium (Research Organics;
350 mg/ml).
Fluorescent in situ hybridization
Fluorescent in situ hybridization was performed with slight modification of
in situ hybridization (using a protocol kindly provided by Drs Parnaik and
Ragsdale). Riboprobes incorporating digoxigenin- (DIG), fluorescein- (FL) or
dinitrophenyl (DNP)-labeled nucleotides were hybridized overnight and detected
with each antibody conjugated to HRP. Signals were detected using the TSA plus
system (Perkin Elmer). Images were acquired with a DP30 (Olympus) camera and
processed using Lumiavision software.
Electroporation
Electroporation was performed as described previously, with some
modifications for electroporation of the diencephalon
(Fukuchi-Shimogori and Grove,
2001). DNA solution was injected into the 3rd ventricle followed
by negative electrode insertion in the lumen of the 3rd ventricle. The
positive electrode was placed outside, near the head of the embryo. A series
of three square-wave current pulses (7V, 100 ms) was delivered, resulting in
gene transfection into one side of the diencephalic wall.
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RESULTS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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) as well as expression towards ZLI
(Fig. 1C, orange arrow)
(defined as F11 in chick) (Crossley et
al., 2001). However, Shh in ZLI exhibited similar patterns of
expression in chick and mouse brain, whereas Fgf8 expression in diencephalon
differed between them. In both F11 and F12 in chick brains, extensive Fgf8
expression was observed (Crossley et al.,
2001), although in mouse the pattern of Fgf8 expression in both
diencephalic domains was less extended (see Fig. S1A in the supplementary
material). We further performed two-color in situ hybridization for direct
comparison of the domains of Fgf8 and Shh mRNA expression.
This revealed that the Fgf8 expression toward ZLI was exclusive and
slightly anterior to Shh expression
(Fig. 1E, arrow). However,
Spry2, an Fgf8 target, spread on both sides of ZLI and exhibited a
wider pattern of expression than Fgf8
(Fig. 1F, arrows). The spatial
relationship of the patterns of expression of Shh and Fgf8
is clearly revealed in sectioned brains
(Fig. 1G-I). Fgf17 and
Fgf18, members of the Fgf8 subfamily with similar receptor affinities
and functions in other systems, are also expressed in the diencephalon in
similar manner (see Fig. S1B,C in the supplementary material)
(Maruoka et al., 1998), which
suggests that multiple ligands might contribute to FGF activity in the
developing diencephalon. Furthermore, a high level of expression of Fgf
receptors was observed in diencephalon at E10.5 (see Fig. S1D-F in the
supplementary material). Among three receptors (Fgfr1, Fgfr2 and Fgfr3), Fgfr1
exhibited expression only in p3, whereas both Fgfr2 and Fgfr3 exhibited a
gradient of expression in p2 and p3 concentrically from the Fgf8-expressing
domain in diencephalon. This also suggests the importance of Fgf activity in
developing diencephalon.
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). We have employed micro in utero electroporation at E10.5
unilaterally (Fukuchi-Shimogori and Grove,
2001) to overexpress Fgf8 in a spatially and temporally restricted
manner. One-third of the surviving embryos with strong transgene expression
throughout p2 exhibit morphology consistent with ectopic induction of midbrain
(see Fig. S2A in the supplementary material, white arrow). This conversion is
also suggested by the ectopic induction of En2 expression in
diencephalon (see Fig. S2C,D in the supplementary material). Another one-third
of surviving embryos in each litter exhibited strong expression of transgene
only in the region close to ZLI without morphological conversion of the
diencephalon (see Fig. S2B in the supplementary material). We also confirmed
with BrdU labeling or TUNEL assay that in these brains there is no ectopic
cell growth or cell death at E13.5 (see Fig. S2E-H in the supplementary
material). To test whether Fgf8 activity is restricted to around ZLI, we
examined the expression of Spry2, the readout of Fgf8 activity, and
observed increased expression close to ZLI alone (see Fig. S2I in the
supplementary material, arrow). To inhibit FGF activity, we electroporated a
DNA construct carrying a truncated Fgfr3 receptor (sFgfr3) described
previously (Fukuchi-Shimogori and Grove,
2001; Ye et al.,
1998). Electroporation was performed at E9.5, before Fgf8 is
expressed (Fig. 1A), for
complete suppression of Fgf8 activity. We observed decreased Spry2 in
diencephalon, suggesting successful inhibition of FGF activity by
electroporation of sFgfr3 construct at E9.5 (see Fig. S2J in the supplementary
material, arrow). We have also tested the effect of Fgf8 at E9.5 and detected
similar effect at E10.5; however, targeting only close to the ZLI is difficult
due to the size (data not shown). Furthermore, FGF inhibition at E10.5, after
endogenous Fgf8 expression starts, yielded suppression of Spry2,
though its effect was less than that produced by electroporation at E9.5 (data
not shown). We therefore performed the two types of electroporation at
different embryonic ages in this study: Fgf8 overexpression at E10.5 and Fgf8
inhibition at E9.5. Sites of electroporation are shown in the
Fig. 2E,F; insets are
hybridized with GFP probe, which has been co-electroporated with target
plasmid.
Fgf8 activity alters neither Shh nor Wnt activity in diencephalon
Previous studies demonstrated that Shh activity from ZLI and Wnt activity
from p2 are important in establishing regional identity in the developing
diencephalon (Kiecker and Lumsden,
2004; Zhou et al.,
2004; Braun et al.,
2003). However, we demonstrated that expression of Wnt3a,
Shh and its activity are not altered by Fgf8 overexpression (see Fig.
S3A-H in the supplementary material). Wnt8b, which is expressed in
ZLI in chick (Garcia-Lopez et al.,
2004), was not detected in mouse brain at all (data not shown).
These findings suggest that Fgf8 activity alters neither Shh nor
Wnt expression, nor their activity. To understand the role of Fgf8 in
developing diencephalon, we next tested gene expression after manipulating FGF
activity in diencephalon at E12.5, when nucleogenesis takes place
(Fig. 2). To visualize more
clearly the A/P divisions (Figdor and
Stern, 1993; Puelles and
Rubenstein, 2003; Bulfone et
al., 1993), we decided to section brains perpendicular to ZLI and
selected a section plane around the midway point of ZLI along the D/V axis
(Fig. 2A, arrow). This enables
visualization of all three major regions of the diencephalon (p1, p2 and p3)
in a single section (Fig.
2B).
Only tissues posterior to ZLI respond to Fgf8 activity
At E12.5, the major divisions of the diencephalon can be easily detected by
the restriction of expression of some marker genes
(Puelles and Rubenstein,
2003). We tested expression of these marker genes after Fgf8
overexpression or inhibition and demonstrated that the expression of the p3
markers Dlx1 and Dlx2 is altered by neither Fgf8
overexpression nor inhibition (Fig.
2C,D; data not shown). Nakagawa and his colleagues characterized
distinctive progenitor domains in developing thalamus, which are marked by
Mash1 and Ngn2 (Vue et
al., 2007). We therefore examined the pattern of expression of
Mash1 and Ngn2 in Fgf8-overexpressed and Fgf8-inhibited
brains at E12.5 by fluorescent in situ hybridization. Surprisingly, restricted
Fgf8 electroporation (Fig. 2E,
inset) expanded the small Mash1+ population posterior to ZLI
[(Fig. 2E, red bracket) defined
as pTH-R in Vue et al. (Vue et al.,
2007)] and resulted in concomitant reduction of Ngn2 + VZ
(Fig. 2E, green bracket). In
complementary experiments, sFgfr3-electroporated brains
(Fig. 2F, inset) displayed
shrinkage of Mash1 + VZ (Fig.
2F, red arrow and bracket) and expansion of Ngn2 + VZ
(Fig. 2F, green brackets). To
examine how this change in gene expression in progenitor regions reflects the
patterning of postmitotic cells, we further tested genes that are expressed
flanking ZLI and also in postmitotic regions, such as Nkx2.2
(Kiecker and Lumsden, 2004;
Kitamura et al., 1997) and
Sox14 (Hashimoto-Torii et al.,
2003). In Fgf8-overexpressed brains, Nkx2.2+ and
Sox14+ populations in the postmitotic region are both expanded
(Fig. 2G,I, arrow). In
FGF-inhibited brains, Nkx2.2 and Sox14 had smaller domains
of expression (Fig. 2H,J,
arrow). However, control GFP electroporated brains exhibited no defects in
Nkx2.2 expression (see Fig. S4A,B in the supplementary material).
Given the unique phenotypes of the populations that flank ZLI only posteriorly
and are controlled by FGF signaling, we have termed this region the `Rim
(Nkx2.2+ and Sox14+)' and have determined its identity in
further experiments. Furthermore, Fgf8 overexpression reduced the size not
only of Ngn2+VZ in p2 but also the postmitotic region of p2, labeled
by markers such as Lhx9 (see Fig. S4C in the supplementary material).
These findings indicate that Fgf8 activity controls only the pattern of the p2
region, which includes Ngn2+ VZ, the post-mitotic region in p2 and
the Rim. Furthermore, diencephalic tissue exhibited responses to Shh
electroporation that were different from responses to Fgf8
electroporation (see Fig. S4E,F in the supplementary material). We also
electroporated Shh, and found that Shh expression does not alter Fgf8
expression (data not shown), suggesting that Fgf8 has specific patterning
effects in developing diencephalon restricted to p2.
The Rim is multi-complex
To determine the molecular identity of the Rim, we further investigated its
gene expression signature. While searching for genes expressed in the
diencephalon, we found that the homeodomain transcription factor Six3
(Fig. 3A, arrow), the
vertebrate ortholog of Aristaless (Arx)
(Cobos et al., 2005)
(Fig. 3B, arrow) and
Gad67 exhibit distinct patterns of expression in the Rim
(Fig. 3C, arrow). Direct
comparison of expression of Gad67, Six3, Arx and the Rim marker
Nkx2.2 was performed by two-color in situ hybridization and
fluorescent in situ hybridization, and revealed that Gad67 and
Six3 expression occurs in the medial region of the Rim
(Fig. 3D,E, arrow and inset),
while the Arx+ population is distinctive to the Rim
(Fig. 3F, arrow and inset). We
also demonstrated expression of other marker genes around ZLI, including
Tal2 (T-cell acute leukemia 2)
(Bucher et al., 2000),
Pitx2 (Kitamura et al.,
1997) and Sim1
(Epstein et al., 2000)
(Fig. 3G,I; data not shown).
Tal2 is expressed in the VZ of the Rim
(Fig. 3G, arrow and inset),
with complete overlap with Mash1+ Rim VZ shown on triple fluorescent
in situ hybridization (Fig. 3H,
arrow). Pitx2 and Sim1 were expressed lateral to
Shh (Fig. 3I, arrow;
data not shown). To recognize the distinct characteristics of the Pitx2- and
Sim1-positive population, we termed it the `Stream' in this study. To
determine the direct spatial relationships between the Six3+ medial
Rim, Pitx2+ Stream region and ZLI, we performed triple fluorescent in
situ hybridization and found that these three populations exhibited no overlap
in expression (Fig. 3J).
Fgf8 electroporation expands the Rim
As indicated above, we have shown that Fgf8 activity controls the size of
the Rim (Fig. 2E-J). To test
whether FGF activity controls only the Rim or also controls other structures,
we examined specific markers around the Rim in brains electroporated with Fgf8
or sFgfr3 (Fig. 4). To focus on
a restricted area, only the experimental side of the diencephalon is shown in
high magnification around ZLI (with VZ on the right side). Fgf8-overexpressing
brains exhibited expansion of the Rim VZ marked by Tal2 and reduction
in Fgf8-inhibited brains (Fig.
4A-C, arrows). The medial Rim, marked by Six3
(Fig. 4D-F, green arrow) and
Gad67 (Fig. 4G-I; pink
arrow), was also found to be controlled by FGF activity. However, the
population posterior to ZLI but distinctive to the Rim marked by the
Arx or Pitx2+ Stream was resistant to FGF activity
(Fig. 4D-F, orange arrows;
Fig. 4G-I, blue arrows). Taken
together, these findings suggest that FGF activity in the diencephalon
controls the patterning of the p2 progenitor region, which includes the Rim
VZ, Ngn2+ VZ and medial Rim, but not the Arx+ and
Pitx2+ stream regions.
Posterior vLGN and EML respond to FGF activity
To determine the effects of the shift in the progenitor region by FGF
activity in the postmitotic region, we examined brains at E15.5, the earliest
age at which individual thalamic nuclei can be defined by gene expression
patterns (Fig. 5)
(Nakagawa and O'Leary, 2001).
Unfortunately, we could no longer detect expression of Tal2, a
specific marker of the Rim VZ at E15.5 with in situ hybridization (data not
shown). However, examination of the Tal2-lacZ mouse, which contains
the lacZ gene in the Tal2 locus, demonstrated X-gal staining
at E16.5 in the posterior part of vLGN and the external medullary lamina
(EML), a structure that divides the thalamus and prethalamus in the adult
(Bucher et al., 2000). This was
also suggested by analysis of Mash1-EGFP transgenic mouse brain by Nakagawa
and colleagues (Vue et al.,
2007). However, expression of the Rim marker Nkx2.2 and
the medial Rim marker Gad67 was detectable at E15.5, and both were
expressed in posterior vLGN (Fig.
5A,B,E,F) and the EML (Fig.
5B, blue arrow). These findings suggest that the Rim might give
rise to the posterior vLGN and the EML. However, Gad67 is expressed
not only in the medial Rim but also in the p3 region at E12.5
(Fig. 3C), and no Six3
expression is detected in the posterior vLGN or EML, which make it difficult
to test our hypothesis. Owing to the lack of mouse lines appropriate for true
cell lineage analyses of the Rim, we further examined Fgf8-manipulated brains
with these markers at E15.5 (Fig.
5C,D,G,H). In Fgf8-overexpressing brains, expansion of the
posterior vLGN and a posterior shift of the EML were observed
(Fig. 5C,G, arrows), while
inhibition of FGF reduced the size of posterior vLGN
(Fig. 5D,H, arrows).
Furthermore, marker genes such as Pitx2 and Arx, which did
not respond to Fgf8 activity at E12.5, also exhibited no differences in
expression at E15.5 compared with electroporated brains and control brains
(Fig. 5B-D,F-H, yellow and
green arrows, brown staining). These findings strongly suggest that the Rim
tissue that responds to FGF activity generates the posterior part of vLGN and
EML.
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). The Fgf8 construct was co-electroporated at E10.5
with Cre (encoding Cre recombinase) into embryos from the R26R reporter mouse
line (Soriano, 1999).
Expression of β-galactosidase, visualized with X-gal histochemistry, was
restricted to the thalamus/prethalamus border
(Fig. 6A, left side of brain).
Fgf8 overexpression shifted the barreloid closer to the pretectum (posterior
shift) and compressed its shape as revealed by cytochrome oxidase (CO) assay
and Nissl staining (Fig. 6B,C,
brackets). By contrast, blockade of FGF signaling by sFgfr3 at E9.5 reduced
the size of barreloid slightly, and shifted its position closer to the
prethalamus (anterior shift) (Fig.
6E,F, brackets). The site of electroporation with sFgfr3 construct
is detected by YFP fluorescence (Fig.
6D). A higher-magnification view of CO assay of VP shows the shift
in position of the barreloid by the distance from the MMT (mammillothalamic
tract) and its structural change (Fig.
6G-I, double-headed arrow and arrowheads). These effects on
patterning of Fgf8 were also examined by the expression of genes that mark
specific areas of the thalamus at E18.5
(Fig. 7). Shrinkage of the
thalamic region is demonstrated by Steel expression
(Fig. 7A,D), whereas expansion
of the GABAergic prethalamic region is shown by Gad67 expression
(Fig. 7B,E). Finally,
Cdh6, which marks VP at E18.5, was also shifted posteriorly and
compressed (Fig. 7C,F). In this
study, we have shown that local Fgf8 expression in the diencephalon controls
gene expression only in p2 (Fig.
2). However, the direct involvement of Fgf8 activity in the
patterning of thalamic nuclei has not yet been demonstrated. To determine its
involvement, we examined expression of the Rim marker genes and FGF activity
in the early embryonic stage. At E10.5, when expression of Fgf8 begins in
diencephalon (Fig. 1C), the
progenitor region of p2 was already divided in two, and the Rim markers
Nkx2.2 and Mash1 were expressed (see Fig. S5 in the
supplementary material). However, Fgf8 activity did not overlap with the Rim
markers along the D/V axis, suggesting that the mechanism of patterning of the
Rim by FGF activity is not direct and requires downstream effectors for
completion. These findings suggest that FGF activity in diencephalon controls
thalamic nuclear patterning along the A/P axis, which is generated in the p2
progenitor region (Fig. 8).
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Puelles and Rubenstein, 2003).
The only true lineage restriction in the diencephalon occurs at ZLI, which
divides p2 and p3, and also exhibits expression of Shh. It has been
suggested in previous studies that expression of signaling molecules such as
Shh (Kiecker and Lumsden,
2004) and Wnt proteins (Braun
et al., 2003) is important for determination of regional identity
in the developing diencephalon. In our study, we focused on another signaling
molecule, Fgf8, which is also locally expressed in diencephalon
(Crossley et al., 2001). Fgf8
is expected to affect tissue at a distance, as it is a secreted molecule.
However, expression site-dependent, tissue-specific responses were noted in
our in utero Fgf8 electroporation experiments (see Fig. S2A-D in the
supplementary material). When Fgf8 was specifically electroporated close to
ZLI, no growth defect was observed in the diencephalon. However, when strong
expression was observed in the wide p2 region, diencephalic size and
morphology were dramatically altered (see Fig. S2A-D in the supplementary
material). In this study, we focused on the parts of brain that receive strong
Fgf8 expression close to ZLI, and demonstrated that Fgf8 controls the specific
population posterior to the ZLI marked by Nkx2.2
(Shimamura et al., 1995) and
Sox14 (Kobayashi et al.,
2002), which has been defined as a Rim in this study. It has been
previously reported that Sox14 and Nkx2.2 respond to
Shh in dose-dependent manner
(Kiecker and Lumsden, 2004;
Kobayashi et al., 2002).
However, our findings revealed that Nkx2.2 and Sox14 respond
to FGF activity without alteration of expression of Shh or its
activity (see Fig. S3A-F in the supplementary material). Furthermore, our Shh
electroporation experiments also revealed that p2 tissue responds to Shh
activity but in a manner different from the response to Fgf8 (see Fig. S4E,F
in the supplementary material). These findings indicate that FGF activity
controls the patterning of the p2 progenitor region. Finally, the extra-toes
(J) [Xt(J)] mouse mutant, which carries a deletion in the Shh
inhibitor Gli family member Gli3, also exhibits no expansion of the Rim (A.K.
and T.S., unpublished). This finding suggests that the effects of patterning
by Fgf8 are independent of other signaling molecules such as Shh and Wnt
proteins.
The bHLH transcription factors Mash1 and Ngn2 have
distinct roles in specification of neurons in spinal cord and telencephalon
(Parras et al., 2002;
Fode et al., 2000).
Interestingly, Mash1 and Ngn2 expression is also observed in
the diencephalon at E12.5 in an alternating manner
(Fig. 2E,F)
(Vue et al., 2007). We have
shown that Mash1 expression in p2 overlaps with the Rim VZ marker
(Fig. 3H), suggesting that the
Rim contains GABAergic neurons. This is also supported by the work of
Guillemot and colleagues, who showed that replacing Ngn2 with
Mash1 leads to GABAergic differentiation of the thalamus
(Fode et al., 2000).
Interestingly, lack of Otx2 causes the release of repression of
Mash1 expression in p2, which in turn causes ectopic expression of
GABAergic markers in the thalamus (Puelles
et al., 2006). This finding indicates that the bHLH transcription
factors play important roles in determining the neuronal characteristics of
the diencephalon as well. Furthermore, Martin and colleagues showed that Fgf8
represses Otx2 expression in midbrain
(Martinez et al., 1999). These
finding suggests that Mash1 upregulation in p2 by overexpression of
Fgf8 is caused by repression of Otx2. To test this, we checked the
expression of Otx2 after Fgf8 overexpression at E12.5, and
demonstrated that it represses Otx2 expression (see Fig. S4D in the
supplementary material). This finding suggests that Mash1+ progenitor
cells for the Rim provide a GABAergic cell population posterior to ZLI, which
is controlled by FGF activity via Otx2. To understand the role of the
Rim in p2 domain in thalamic patterning, we examined the pattern of expression
of the Rim marker genes at E15.5 after augmenting and reducing Fgf8 signal.
When the diencephalon received excess FGF activity, posterior vLGN and the EML
were expanded and shifted posteriorly (Fig.
5C,G). By contrast, these structures were specifically reduced in
size in FGF-inhibited brains (Fig.
5D,H). These findings suggest that some of the prethalamic nuclei
arise from the p2 domain, which is derived from the Rim. It is reported that
vLGN contains several nuclei in the ground squirrel and tree shrew
(Agarwala et al., 1992), but
not in mouse. Further examination is required to determine which specific vLGN
populations respond to FGF activity. Furthermore, a similar pattern of
expression of Rim marker genes was reported in the lamprey Lampetra
fluviatilis (Osorio et al.,
2005) and in Xenopus brain
(Bachy et al., 2001). These
observations suggest that mechanisms of patterning are conserved across
species in diencephalic development.
In this study, we have shown that local Fgf8 expression in the diencephalon
controls gene expression on the A/P axis in p2 region
(Fig. 2). This is also similar
to the tissue responses of midbrain and hindbrain to Fgf8 activity in the
isthmus (Liu et al., 1999).
However, the Fgf8 protein and its activity appear to spread in both the p2 and
p3 regions, and the mechanism of p2-specific responses to Fgf8 activity is
still unclear. The ability of the tissue to respond differently to the same
signaling molecule is suggested by tissue-dependent competency
(Reim and Brand, 2002).
Identifying the correct patterning mechanism in developing diencephalon
(without loss of appropriate regional identity) will require intensive
study.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/17/2873/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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