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First published online December 12, 2006
doi: 10.1242/10.1242/dev.02705
1 Programs in Developmental Biology, Neuroscience and Human Genetics, Department
of Biopharmaceutical Sciences, University of California San Francisco, CA
94143-2811, USA.
2 Division of Molecular and Developmental Biology, National Institute of
Genetics, 1111 Yata, Mishima, Shizuoka 411-8540, Japan.
* Author for correspondence (e-mail: su.guo{at}ucsf.edu)
Accepted 18 October 2006
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: fezl, too few, Zebrafish, Brain patterning, Progenitor cells, Forebrain, Diencephalon, ZLI, Prethalamus, Thalamus, Pretectum, Zinc finger
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Rubenstein et al., 1998;
Stern, 2001;
Wilson and Houart, 2004): (1)
the anterior neural tissue acquires a crude initial regional identity by
avoiding exposure to caudalizing factors, such as Wnt and FGF proteins. This
initial regionalization also establishes local organizing centers in the
neural plate, such as the anterior neural border [ANB, also know as the
anterior neural ridge (ANR)] and the midhindbrain boundary (MHB). (2) These
local organizing centers further refine initial regional patterning and lead
to the establishment of subdivisions that later give rise to various
structures in the mature CNS.
Forebrain regionalization is best understood in the context of
telencephalic development (Rallu et al.,
2002; Wilson and Houart,
2004; Wilson and Rubenstein,
2000). It has been shown previously that the establishment of
telencephalic identity requires local suppression of Wnt signaling - an
evolutionarily conserved pathway that regulates diverse processes, including
embryonic patterning, cell fate determination, cancer and synaptogenesis
(Moon et al., 2002;
Patapoutian and Reichardt,
2000). In zebrafish, the proper development of the telencephalon
requires the secreted Wnt antagonist Tlc
(Houart et al., 2002), the
transcriptional repressor Headless and/or Tcf3 (also known as Tcf7l1a -
Zebrafish Information Network) (Kim et
al., 2000) and the Wnt-pathway scaffolding-protein Masterblind
and/or Axin1 (Heisenberg et al.,
2001).
Compared with the telencephalon, much less is known as to how various
subdivisions within the diencephalon are established. Although the embryonic
diencephalon has been proposed to have a segmental organization
(Figdor and Stern, 1993;
Puelles and Rubenstein, 2003),
cell-lineage restriction boundaries are not apparent between some of the
segments (Larsen et al.,
2001). During embryogenesis, a transient boundary-like structure
called the zona limitans intrathalamica (ZLI) is present between the
prethalamus and the thalamus. This region divides the diencephalon into the
anterior (the prethalamus) versus the posterior, or caudal (the thalamus and
pretectum) territories (Kiecker and
Lumsden, 2005). In chick, the ZLI is predicted by the absence of
lunatic fringe and the presence of Wnt8b expression, and, starting
from the mid-somitogenesis stage, is demarcated by the expression of
Shh, which has been shown to play a role in the proper maturation of
the vertebrate diencephalon (Kiecker and
Lumsden, 2004; Scholpp et al.,
2006). High levels of Wnt signaling have also been suggested to
promote posterior diencephalic fates (Braun
et al., 2003; Kiecker and
Niehrs, 2001; Masai et al.,
1997; Nordstrom et al.,
2002). However, the genes and pathways that establish the ZLI and
other diencephalic subdivisions are elusive. Based on mis-expression studies
(Kobayashi et al., 2002), it
has been proposed that crossrepression between the Irx family of homeodomain
proteins - which are expressed in the prospective caudal diencephalon
(including the thalamus and pretectum) and midbrain
(Lecaudey et al., 2005) - and
Six3 homeodomain transcription factors - which are detected early in the
entire anterior forebrain anlage and later mainly in the optic stalk and eye
regions (Seo et al., 1998) -
may contribute to the establishment of the ZLI and other diencephalic
subdivisions. However, six3 (also known as six3a - Zebrafish
Information Network) expression is dynamic and regresses rostrally as
development progresses (Kobayashi et al.,
2002; Seo et al.,
1998), leaving the anterior diencephalic domain free of both irx
gene family and six3 expression.
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), a secreted antagonist of Wnt signaling
(Glinka et al., 1998).
fezl has a paralog named fez, and both genes exhibit
remarkable evolutionary conservation from flies to men
(Matsuo-Takasaki et al.,
2000). Little is known about the role of the Drosophila
Fezl protein. In mammals, Fezl and Fez have been shown to
regulate cortical neuronal differentiation
(Chen et al., 2005a;
Chen et al., 2005b;
Hirata et al., 2004;
Molyneaux et al., 2005) and
olfactory-bulb development (Hirata et al.,
2006b), respectively, but their early roles in brain
regionalization are unclear, probably as a result of their extensively
overlapping expression patterns. In zebrafish, a hypomorphic allele of
fezl, too few (tof), reveals its role in monoaminergic
neuron development (Guo et al.,
1999; Jeong et al.,
2006; Levkowitz et al.,
2003). Moreover, the expression of zebrafish fezl (but
not fez) is detected in distinct domains of forebrain primordia in
early zebrafish embryos (Hashimoto et al.,
2000) (Fig. 1, and
data not shown). These observations intrigued us to investigate whether
fezl has a role in early forebrain regionalization. Here, we report that fezl is expressed exclusively in the presumptive telencephalon, diencephalon and hypothalamus shortly after gastrulation. Reduced activity of fezl results in a deficit of the prethalamus and a corresponding anterior expansion of the ZLI. Gal4-UAS-mediated fezl overexpression in late gastrula is capable of expanding the prethalamus, optic stalk, telencephalon and hypothalamus at the expense of the eyes, ZLI and posterior fore- and/or mid-brain regions. The enlargement of these forebrain subdivisions is preceded by an early downregulation of wnt expression in the prospective diencephalon. Finally, fezl overexpression is able to restore anterior forebrain and downregulate wnt1 expression in Headless- and/or Tcf3-deficient embryos. Our findings reveal a crucial role of Fezl in establishing regional subdivisions within the diencephalon, and also uncover the capability of Fezl in repressing Wnt proteins and in promoting the development of the telencephalon and hypothalamus.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). The resulting plasmid DNA was injected, together with
transposase RNA, into one-cell-stage embryos. Zebrafish embryos were raised at
28.5°C and staged according to Kimmel et al.
(Kimmel et al., 1995).
UAS-fezl-carrying transgenic founders were identified by PCR on
pooled progeny and propagated by crossing with wild-type fish.
UAS-fezl transgenic lines were subsequently crossed with
hsp-gal4 containing transgenic fish to yield double-transgenic
embryos.
Morpholino designs and analyses of morphants
The sequences for fezl splicing-blocking morpholinos (MOs) and the
effectiveness of MOs to block fezl-RNA splicing was as previously
described (Jeong et al.,
2006). Embryos were injected with 3-4 nl of 0.1 mM fezl
sMO at the one-to four-cell stages. The hdl/tcf3 MO was synthesized
according to published information (Dorsky
et al., 2003), and 3-4 nl of 0.3 mM was used per embryo.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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75% epiboly exclusively
in anterior neuroectoderm (Fig.
1A); by the tailbud stage, it was confined to the prospective
telencephalon, hypothalamus and diencephalon
(Fig. 1B). Its expression in
these regions was maintained throughout the segmentation stages
(Fig. 1C,D). The expression of
fezl overlapped with the telencephalic marker foxg1
(Fig. 1E and see Fig. S1A in
the supplementary material) and anterior forebrain marker six3
(Fig. 1F and data not shown,
and see Fig. S1B in the supplementary material) but not with the eye field
marker rx3 (see Fig. S1C,D in the supplementary material). During
early somitogenesis, whereas six3 and fezl expression
overlapped in the prospective telencephalon, only fezl expression was
detected in the prospective diencephalon
(Fig. 1F and see Fig. S1E,F in
the supplementary material). By late somitogenesis stages, fezl was
strongly expressed in the dorsal telencephalon, prethalamus and hypothalamus
(Fig. 1D), whereas
six3 expression was largely confined to the optic stalk and eye
region (see Fig. 1G,H in the supplementary material). Compared with
posteriorly expressed genes, the fezl domain at the tailbud stage was
separated from that of wnt1 and wnt8b by an approximately
two- to three-cell diameter space; at least part of this area may represent
the presumptive ZLI (Fig.
1G,H). By early somitogenesis, the fezl-expressing domain
was separated by a gap (the presumptive ZLI) from wnt1-expressing
cells in the roof plate and MHB (Fig.
1I,K), and from irx3a-expressing cells in the posterior
diencephalon (Fig. 1L);
moreover, fez1 expression abutted wnt8b expression in the
prospective ZLI (Fig. 1J).
These analyses indicate that fezl expression is initiated early and
exclusively in the developing forebrain, and is subsequently maintained in
discrete forebrain subdivisions throughout the somitogenesis stages. These
observations led us to hypothesize that fezl might have a role in
forebrain regionalization.
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): both MOs
gave similar results, whereas the control MOs had no effect. fezl
MO-injected embryos (hereafter referred to as morphants) were examined at
28 hours post fertilization (hpf), when brain subdivisions are distinct
by morphology and gene expression; they were also examined at earlier stages
(neural-plate and mid-somitogenesis) in order to better define the timing of
fezl action.
In 28 hpf fezl morphants, we found that dlx2a
(Akimenko et al., 1994)
expression in the prethalamus was significantly reduced in a dose-dependent
fashion (Fig. 2B, 97%,
n=65; and data not shown), while remaining unaffected in the
pharyngeal arch progenitors (insets of Fig.
2B). In addition to dlx2a, the expression of
lhx5 (Peng and Westerfield,
2006; Scholpp et al.,
2006; Toyama et al.,
1995) in the prethalamus region was significantly reduced, but its
expression in the posterior tubercular area remained largely normal
(Fig. 2D, 90%, n=58).
Despite the strong expression of fezl in the telencephalon and
hypothalamus (Fig. 1B,D),
expression of the telencephalic foxg1
(Fig. 2F, 98%, n=60)
and the hypothalamic nk2.1a (also known as titf1a -
Zebrafish Information Network) (Fig.
2H, 98%, n=81) appeared largely normal (perhaps slightly
reduced); the expression of six3b also appeared largely normal
(Fig. 2J, 88%,
n=33).
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A corresponding anterior expansion of ZLI accompanies the loss of prethalamus in the fezl morphants
Since proliferation and cell death were not significantly affected in the
fezl morphants (data not shown), we next asked whether the
prethalamic defects might be a result of altered regionalization within the
diencephalon. We examined the expression of shha (also known as
shh - Zebrafish Information Network, and hereafter referred to as
shh) and foxa2, two markers of the ZLI, which is located
immediately posterior to the prethalamus. In control embryos, a gap occupied
by the prethalamus existed between shh-expressing cells and the
telencephalic boundary (Fig.
3A,C,E), but in 28 hpf fezl morphants, this gap was
filled in by expanded shh expression
(Fig. 3B,D, 99%, n=83;
Fig. 3F, 98%, n=46).
The expression of foxa2 was also expanded to abut the telencephalon
(Fig. 3H, 95%, n=86),
but foxa2 expansion was noticeably most pronounced in the dorsal
region, suggesting perhaps an easier transformation of the dorsal area as
compared with the more ventral territory of the prethalamus. In addition to
shh and foxa2, the expression of dbx1a - which is
detected in the anterior diencephalon adjacent to or overlapping with the ZLI,
and also in the posterior diencephalon (the thalamus)
(Scholpp et al., 2006) - was
significantly expanded in the anterior diencephalon
(Fig. 3J, arrow, 90%,
n=40), and moderately increased in the thalamic region
(Fig. 3J, arrowhead, 90%,
n=40). These analyses suggest that fezl activity is required
to repress the fate of ZLI and possibly also the posterior diencephalon.
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). In
16-somite stage fezl morphants, dbx1a expression
adjacent to the ZLI appeared slightly increased
(Fig. 3L, 85%, n=42);
moreover, an increased and anteriorly expanded shh expression in the
prospective ZLI was readily discernible, which was accompanied by the loss of
the gap between the hypothalamic- and ZLI-expression, whereas its expression
in the anterior hypothalamus was moderately decreased
(Fig. 3N, 88%, n=50).
irx3a expression, which is in the prospective caudal diencephalon and
midbrain and in close proximity to shh-expressing prospective ZLI
cells (Lecaudey et al., 2005),
was moderately expanded anteriorly; enhanced irx3a expression was
observed in a ventral location that might be overlapping with the
shh-expressing prospective ZLI cells; in addition, a small cluster of
irx3a+ cells was detected in the anterior forebrain,
possibly in the prospective telencephalon or in the dorsal prethalamus
(Fig. 3P, 99%, n=40).
This anterior forebrain expression appeared to be transient and might
represent an incomplete fate switch because it was not observed in 28 hpf
fezl morphants (data not shown), and because other thalamic markers,
such as dbx1a, were not detected in the corresponding location (data
not shown). These results suggest that fezl plays a role in
repressing the early ZLI and posterior diencephalic fate, beginning around the
mid-somitogenesis stage.
Mis-expression of fezl results in expansion of the prethalamus and elimination of the ZLI
To further elucidate the role of fezl in forebrain
regionalization, we investigated the consequence of overexpressing
fezl. As the delivery of fezl mRNA into one- to
four-cell-stage embryos led to severe embryonic deformity (data not shown)
(Levkowitz et al., 2003;
Yang et al., 2001), we
employed the heat inducible Gal4-UAS system
(Scheer and Camnos-Ortega,
1999) to achieve a temporal control of fezl
overexpression. We established transgenic lines carrying the UAS-fezl
transgene and crossed them with another line carrying the hsp-gal4
transgene. The embryos derived from this cross were subjected to heat shock
for 15, 30 or 45 minutes at 75% epiboly, analyzed for gene expression at
28 hpf, and subsequently genotyped to determine the presence or absence
of the transgenes. Heat shock at later stages (e.g. 10-somite and 24 hpf) had
little or no effect on brain regionalization (data not shown). Because the
earliest time when the endogenous fezl expression was observed is at
75% epiboly, our analyses were focused on the embryos that were heat
shocked at this stage.
Ubiquitous fezl expression was detected 2 hours post
heat-shock through to 6 hours (Fig. S3 in the supplementary material). In the
heat-shocked double-transgenic embryos, prethalamic and hypothalamic
dlx2a expression was expanded in proportion to the duration of heat
shock, whereas pharyngeal arch dlx2a expression was relatively normal
(Fig. 4A-D, n=62). To
determine whether the expansion of dlx2a-expressing domains might be
at the expense of more posterior brain tissues, we examined the expression of
shh. Remarkably, shh-expressing ZLI was shifted posteriorly,
reduced or eliminated in the double-transgenic embryos in proportion to the
duration of heat shock, whereas the floor plate expression of shh was
unaffected (Fig. 4E-H,
n=34). These analyses indicate that fezl overexpression is
capable of expanding the prethalamus at the expense of the ZLI.
Mis-expression of fezl expands the telencephalon and hypothalamus at the expense of other fore- and mid-brain regions
To further characterize the fezl gain-of-function (GOF) phenotype,
we assessed the extent to which fezl overexpression could alter brain
regionalization by examining additional region-specific genes: the expression
of foxg1 (Fig. 5B,
n=24) in the telencephalon and nk2.1a in the hypothalamus
(Fig. 5D, n=42), as
well as pax2a in the optic stalk
(Fig. 5F, n=10), were
all expanded; by contrast, the expression of pax2a
(Fig. 5F, n=10) and
engrailed 2 (also known as engrailed 2a - Zebrafish
Information Network) (Fig. 5J,
n=22) in the MHB, and irx3a in the caudal diencephalon and
midbrain (Fig. 5H,
n=10) were lost, and the eyes were significantly reduced
(Fig. 5F, n=10).
However, krox-20 (also known as egr2a - Zebrafish Information
Network) expression in the hindbrain rhombomeres r3 and r5 remained
(Fig. 5J, n=22).
Therefore, it appears that ectopic expression of fezl at late
gastrulation can expand the telencephalon, hypothalamus and prethalamus at the
expense of the eyes, ZLI, caudal diencephalon, midbrain and MHB.
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28 hpf.
fezl overexpression is sufficient to restore anterior forebrain gene expression in Headless- and/or Tcf3-deficient embryos
headless and/or tcf3 (hdl/tcf3), which encode a
transcriptional repressor of Wnt target genes (headless being the
mutant form of tcf3), has been shown previously to be crucial for the
development of the forebrain: its inactivation leads to a headless phenotype
and a dramatic expansion of wnt1 expression into anterior forebrain
(Kim et al., 2000). Injection
of six3 mRNA, a known repressor of wnt1 transcription, into
Hdl/Tcf3-deficient embryos has been shown to rescue the eyes, as well as the
head, based on gross morphology, but the extent of head rescue by
six3 has not been examined at the molecular level
(Lagutin et al., 2003).
We reasoned that, if Fezl were a crucial player in establishing forebrain
subdivisions, overexpression of fezl should restore these
subdivisions to Hdl/Tcf3-deficient embryos. Because the maternal zygotic
hdl/tcf3 mutant had a rather variable severity of head loss (our
unpublished observations), we delivered the hdl/tcf3 MO, which was
previously shown to be effective and specific in knocking down
hdl/tcf3 activity (Dorsky et al.,
2003; Dorsky et al.,
2002), to embryos derived from the
hsp-gal4/+xuas-fezl cross. These hdl/tcf3
morphants were subjected to heat shock at 75% epiboly, analyzed for
forebrain gene expression at 28 hpf, and subsequently genotyped for the
presence or absence of the transgenes. At the 3-somite stage, fezl
expression was dramatically reduced and wnt1 expression was
significantly increased in the hdl/tcf3 morphants
(Fig. 7A,B, n=43).
fezl overexpression was sufficient to repress wnt1
expression in the hdl/tcf3 morphant
(Fig. 7C, 81%, n=36).
Similarly, whereas the lack of hdl/tcf3 activity led to severe
deficits in the expression of foxg1
(Fig. 7D, 62%, n=52),
dlx2a (Fig. 7G, 67%,
n=42) and nk2.1a (Fig.
7J, 45%, n=29) in anterior brain regions, fezl
overexpression was not only able to restore, but was also able to expand, the
telencephalic foxg1 expression
(Fig. 7F, 80%, n=35),
prethalamic dlx2a expression (Fig.
7I, 91%, n=34) and hypothalamic nk2.1a
expression (Fig. 7L, 84%,
n=13) in the hdl/tcf3 morphants. These analyses demonstrate
that Fezl overexpression is sufficient to repress wnt1 and to promote
telencephalic hypothalamic and prethalamic identity even in the absence of the
wnt signaling repressor Hdl/Tcf3.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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In addition to affecting the diencephalon, Fezl GOF is also able to expand
the telencephalon and hypothalamus at the expense of eyes and other fore-
and/or mid-brain regions. However, the telencephalon and hypothalamus remain
largely normal in fezl morphants. Although we cannot fully rule out
the possibility that the GOF phenotypes observed for the telencephalon and
hypothalamus are `artifacts' due to overexpression of fez1, several
lines of evidence suggest that it is not the case. First, such GOF phenotypes
were not observed in embryos that were heat-shocked at later stages,
suggesting that these phenotypes are not simply due to an overproduction of
Fezl. Second, whereas anterior-posterior patterning is disturbed in
fezl GOF embryos, other axial development (e.g. dorsoventral
patterning) appears largely normal. Finally, endogenous fezl
expression was detected strongly in both the telencephalon and hypothalamus
during the early development of these embryos. Therefore, one plausible
explanation for the strong GOF but weak LOF phenotypes in the telencephalon
and hypothalamus is that other forebrain-expressed factors may compensate for
the loss of Fezl in these brain regions. One such factor might be Six3, which
is essential for murine forebrain development
(Lagutin et al., 2003) and
medaka fish eye formation (Carl et al.,
2002; Del Bene et al.,
2004), and has also been shown to regulate forebrain development
in zebrafish (Ando et al.,
2005; Kobayashi et al.,
1998). Another compensating factor may be lhx5, which was
recently shown to promote forebrain development via the transcriptional
activation of secreted Wnt antagonists
(Peng and Westerfield, 2006).
Thus, it is possible that fezl, six3 and lhx5 together may
ensure the proper establishment of multiple anterior neural subdivisions that
include the telencephalon, eyes, prethalamus and hypothalamus.
|
) a
cell non-autonomous function of fezl has been previously proposed for
their development. However, recent evidence favors the hypothesis that
fezl is expressed in the dopaminergic progenitor cells to cell
autonomously control their development
(Jeong et al., 2006). Several
lines of evidence suggest that fezl may also act cell autonomously in
the specification of the prethalamus. First, fezl is expressed in the
prethalamus. Second, fezl LOF leads to a deficit of prethalamus,
whereas fezl GOF leads to an expansion of prethalamus. Third,
fezl has previously been shown to cell autonomously induce the
expression of dlx2a, a prethalamic marker
(Yang et al., 2001). Whereas
fezl is likely to act cell autonomously in the specification of the
prethalamus, its requirement for repressing ZLI may be cell non-autonomous
because fezl appears not detected in this region.
What are the mechanisms by which fezl regulates diencephalic
regionalization? One possible avenue is through repressing the target genes of
the wnt and/or ß-catenin signaling pathway, which has been shown to
repress the anterior, and promote the posterior, neural fate. Our LOF analysis
shows an expansion of irx3a, a gene induced by wnt signaling
(Braun et al., 2003). Our GOF
analysis demonstrates the capability of fezl to repress wnt1
and pax2a, which is followed by the loss of two important organizers
- the ZLI and MHB - which may be the cause of the GOF phenotypes. Moreover,
the rescue of the headless/tcf3-deficient embryos by fezl
overexpression further substantiates the ability of Fezl to repress
wnt signaling. The largely unaffected wnt expression in the
fezl morphants (see Fig. S2E-J in the supplementary material) may be
due to genetic compensation, possibly provided by six3 or
lhx5. Alternatively, the interaction between fezl- and
wnt-signaling may not be at the level of direct transcriptional regulation of
the wnt family of genes. Taken together and consistent with a suggested
requirement of inhibiting wnt activity for the development of the
prethalamus (Braun et al.,
2003; Kiecker and Niehrs,
2001; Lagutin et al.,
2003), our results provide strong evidence that such repression of
wnt activity can be achieved by fezl. Finally, it is worth
pointing out that our data are consistent with the possibility that
fezl may also play a role in directly promoting the prethalamic fate
independent from repressing wnt activity.
A clear understanding of the biochemical mechanisms underlying
fezl function requires the future identification of its direct target
genes. Moreover, it is also of great interest to know mechanistically how
fezl expression is restricted to distinct anterior forebrain
subdivisions. Consistent with the Wnt gradient hypothesis in forebrain
regionalization (Wilson and Houart,
2004), fezl is inducible by Dkk1
(Hashimoto et al., 2000),
again, making the Wnt pathway an attractive candidate in regulating
fezl expression. Future studies will provide crucial insights into
possible cross regulations between fezl and wnt
signaling.
Our study uncovers an essential role of fezl in diencephalic
patterning, prior to its function in neuronal subtype differentiation
(Chen et al., 2005a;
Chen et al., 2005b;
Guo et al., 1999;
Hirata et al., 2004;
Jeong et al., 2006;
Levkowitz et al., 2003;
Molyneaux et al., 2005). Given
its restricted expression in the vertebrate forebrain and a demonstrated role
in regulating reward-associated behaviors
(Lau et al., 2006), it would
be interesting to test in the future whether the deregulation of Fezl may be
involved in human neurological disorders that have a developmental origin.
While our manuscript was under review, it was reported that the mouse Fez
and Fezl proteins together also play a crucial role in the establishment of
the diencephalon divisions (Hirata et al.,
2006a), suggesting an evolutionarily conserved role of the Fez-
and Fezlgene families. Interestingly, a notable difference exists between
zebrafish and mice: whereas our LOF and GOF analyses indicate a clear role of
zebrafish fezl in promoting the prethalamus and in repressing the
ZLI, fez-fezl- double-mutant mouse embryos had
a loss of both prethalamus and ZLI, but an expansion of caudal diencephalon
(the thalamus and pretectum). However, similar to our GOF data in zebrafish,
overexpression of mouse fezl or fez abolished
shh-expressing ZLI. This difference in LOF phenotypes could be
species-dependent. Alternatively, the zebrafish fezl morphant perhaps
represents a weaker LOF of fezl (also with fez being
intact), whereas the fez-fezl- double-mutant
mice are null conditions. Therefore, it is attractive to hypothesize that the
levels of fezl and/or fez activity may determine distinct
subdivisions along the rostrocaudal axis. Future experiments are necessary to
test this hypothesis.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/1/02705/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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