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First published online 17 September 2008
doi: 10.1242/dev.024232
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1 Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot
76100, Israel.
2 Medical Research Council Centre for Developmental Neurobiology, King's
College, London SE1 1UL, UK.
3 Department of Neurobiology and Anatomy, University of Utah, Salt Lake City, UT
84132, USA.
* Author for correspondence (e-mail: gil.levkowitz{at}weizmann.ac.il)
Accepted 26 August 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: Cell number, Neuronal specification, Oxytocin, Dopamine, Cell lineage
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). The Wnt signaling pathway plays a major role in the
patterning of the anterior embryonic neuroectoderm, which results in the
subdivision of the developing brain into discrete compartments
(Glinka et al., 1998;
Heisenberg et al., 2001;
Houart et al., 2002;
Lekven et al., 2001;
Mukhopadhyay et al., 2001).
Recent studies in zebrafish have demonstrated that the canonical Wnt signal
performs multiple functions in the anterior neuroectoderm. It acts as a
posteriorizing signal, controlling the fates of the posterior diencephalon and
mesencephalon (Kim et al.,
2002). It interacts with other, non-canonical Wnt signals to
induce eye-field formation (Cavodeassi et
al., 2005). It acts upstream of Lef1 to positively regulate
neurogenesis in the posterior hypothalamus (part of the diencephalon)
(Lee et al., 2006). Finally,
it has been shown that canonical Wnt signaling is important for the
acquisition of hypothalamic versus floor plate identity
(Kapsimali et al., 2004).
Gain-of-function studies in Xenopus and chick further demonstrate
that graded activation of Wnt signaling induces the expression of defined sets
of transcription factors that delineate the various brain regions
(Kiecker and Niehrs, 2001;
Nordstrom et al., 2002).
Despite this wealth of information, the exact contribution of Wnt-mediated
neural plate patterning to neuronal fate determination remains unclear,
particularly regarding the differentiation phenotypes of distinct neuronal
subtypes in the forebrain.
The development of diencephalic dopaminergic (DA) neurons may serve as a
paradigm for how robust patterning signals contribute to diencephalic subtype
specification. Diencephalic DA cells are present in the hypothalamus, ventral
thalamus and caudal diencephalon (Smeets
and Reiner, 1994). In mammals, these neurons are essential for a
variety of vital neural functions, including regulation of sympathetic
preganglionic neurons and hormone secretion, as well as for control of
motivational responses and sexual arousal
(Dominguez and Hull, 2005;
Iversen et al., 2000;
Paredes and Agmo, 2004). In
the mesencephalon, the development of DA neurons is positively regulated by
Wnt signaling, in vitro (Castelo-Branco et
al., 2003; Schulte et al.,
2005) and in vivo (Prakash et
al., 2006). However, the role of Wnt signaling in diencephalic DA
neural development has not been studied. We and others have investigated the
embryonic development of DA neurons in the zebrafish
(Blechman et al., 2007;
Guo et al., 1999;
Holzschuh et al., 2003;
Levkowitz et al., 2003;
Ryu et al., 2007). Unlike in
mouse embryos, the small DA neuron population size in the zebrafish
diencephalon enables accurate quantitative analysis of even small changes in
the number of differentiated neurons.
A crucial question addressed herein is whether forebrain-patterning cues,
such as the canonical Wnt signal, affect the specification and number of
distinct diencephalic neuronal populations. We show that in the zebrafish
embryo, the number of diencephalic DA cells is almost invariant and is subject
to tight control by the Wnt/β-catenin pathway. Our findings show that
Wnt-mediated signaling does not have an overall effect on the differentiation
of neuronal subtypes in the diencephalon. Instead, Wnt signaling limits the
initial pool of DA progenitors at the neural plate stage, during a window of
plasticity that coincides with the onset of primary neurogenesis, earlier than
previously described (Rink and Wullimann,
2002; Smidt and Burbach,
2007). We suggest that the control of neuronal cluster size by
early patterning signals plays a crucial role in delineating the upper and
lower size limits of selected diencephalic neuronal clusters.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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)
containing a heat-shock 70/4 promoter
(Halloran et al., 2000).
Sequences of primers used to amplify DNA templates for preparation of
digoxigenin (DIG)- or fluorescein (FITC)-labeled probes are available upon
request. The fezl and barhl2 probes have been described
previously (Colombo et al.,
2006; Levkowitz et al.,
2003). The tof m808 mutant, X8
mutant, Tg(1.4dlx5a-dlx6a:gfp) and
Tg(-8.4neurog1:gfp) lines have been described previously
(Blader et al., 2003;
Ghanem et al., 2003;
Lee et al., 2006;
Levkowitz et al., 2003).
Morpholino and mRNA injections
Antisense morpholino oligonucleotides (MOs) (Gene Tools) targeted to the
translation start site of fz8a or wnt8b and to an
RNA-splicing site of lef1 are as previously described
(Houart et al., 2002;
Kim et al., 2002;
Lee et al., 2006).
dkk1 mRNA (20 pg/embryo), fz8a (5 ng/embryo), wnt8b
(3 ng/embryo) and lef1 (2 ng/embryo) MOs were injected at the 1-cell
stage and embryos allowed to develop at 28.5°C.
Quantification of cell number
Cell numbers in confocal images were quantified using ImageJ. Each channel
was analyzed separately and converted to grayscale to maximize contrast. Using
the Point Selection tool, the center of each cell was marked with a blocked
circle (seven pixels in diameter) and its x, y, z position was
automatically logged. Frequent cycling between adjacent z-planes
ensured that no cell was logged more than once. Volocity software
(Improvision, Covington, UK) was used to corroborate these cell counts and to
quantify other large control domains. The software identified as an individual
cell any object with a calculated volume between 100 µm3 and 150
µm3 and signal intensity no lower than 20% of the normalized
distribution of signal intensities in the entire image.
Fate mapping
High-resolution fate mapping of diencephalic progenitor zones was performed
by a two-photon-based photo uncaging procedure using a Zeiss LSM 510
microscope equipped with a 720-nm Mai Tai laser. We used the
neurog1::gfp reporter line [Tg(-8.4neurog1:gfp)] as a live
genetic landmark of the diencephalic territory. neurog1::gfp
transgenic embryos were injected with 1% DMNB-caged fluorescein in 0.2 M KCl
immediately after fertilization, and were then grown in the dark. Embryos were
mounted at the bud stage in 1% low-melting-point agarose in E2 medium. The
caged fluorescein was converted into its fluorescent, active form at discrete
diencephalic domains using the Region of Interest (ROI) function (720 nm, 20
iterations at a scan speed of 25.6 µsec/pixel). The uncaged fluorescein and
DA neurons were visualized at the prim5 stage (24 hpf) using an alkaline
phosphatase-coupled anti-fluorescein antibody followed by Fast Red staining.
The embryos were then stained with anti-tyrosine hydroxylase antibody to
visualize DA neurons.
Cell-proliferation analysis
BrdU labeling (10 mM BrdU in E2 medium) was conducted as described
(Shepard et al., 2004). To
block cell division, embryos were dechorionated and incubated in a combination
of 1 µg/ml aphidicolin and 50 mM 5-hydroxyurea in 4% DMSO. Control embryos
were incubated in 4% DMSO. Cell cycle inhibitors were removed at 24 hpf by
washing three times with embryo medium (E3).
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Hashimoto et al.,
2000a; Houart et al.,
2002; Shinya et al.,
2000). However, the effect of this forebrain mispatterning on the
specification and number of distinct diencephalic neuronal populations has not
been addressed. Members of the canonical Wnt family are necessary for
precursor proliferation, fate decisions and neuronal differentiation of
midbrain DA neurons (reviewed by Prakash
and Wurst, 2006; Smidt and
Burbach, 2007). We therefore examined whether perturbation of
anteroposterior patterning by ectopic expression of the canonical Wnt
antagonist dickkopf 1 (Dkk1), might similarly impair the development of
diencephalic DA neurons.
In zebrafish, DA groups develop in a ventral diencephalic area termed the
posterior tuberculum (PT) (Holzschuh et
al., 2001; Rink and Wullimann,
2002). Within this domain, distinct DA clusters can be readily
identified in the 1- to 5-day-old zebrafish embryo
(Fig. 1A)
(Rink and Wullimann, 2002). We
analyzed the effects of Dkk1 on the two major diencephalic DA clusters, group
2 and groups 3-6, using a transgenic line expressing GFP under the control of
the dlx6a promoter (Ghanem et
al., 2003). These transgenic embryos (denoted dlx6a::gfp)
express GFP in the telencephalon and in two discrete domains of the
diencephalon (Fig. 1C,E,I). An
anterior expression domain, located just posterior to the
telencephalon-diencephalon boundary, demarcates the proliferating zone that
gives rise to the group 2 DA cluster, which appears 22-24 hours
post-fertilization (hpf) (data not shown), and an expression domain
perpendicular to this boundary demarcates a proliferating zone contiguous with
group 3-6 DA neurons, which appear at 30-48 hpf
(Fig. 1C,E,I).
Surprisingly, when mRNA encoding the Dkk1 protein was injected into zebrafish embryos, we were still able to detect all known neuronal clusters of diencephalic DA cells (Fig. 1B,D,F). We then took advantage of the small and virtually invariant cell population size in the zebrafish diencephalon to examine whether DA cell number is altered following attenuation of canonical Wnt signaling. Quantitative analysis revealed that dkk1-injected embryos had a two-fold increase in tyrosine hydroxylase (TH)-positive group 2 DA neurons and a moderate increase in the number of group 3-6 DA cells (Figs 1B,D,F and see Fig. S1 in the supplementary material). Similarly, a two-fold increase in dopamine transporter (dat; slc6a3)-positive neurons was observed, starting from the earliest stage in which differentiated group 2 DA neurons are detected (22 hpf) and persisting through 5 days of development (Fig. 1G and data not shown). In contrast to DA neurons, the number of Dlx6a+ cells in the basal diencephalon was unaltered by Wnt attenuation, suggesting that not all ventral diencephalic progenitors are affected by Wnt perturbation (Fig. 1E,F,H). Thus, attenuation of the canonical Wnt signal by Dkk1 specifically alters the number of diencephalic DA neurons.
Wnt attenuation specifically elevates DA cell number without affecting the progeny of adjacent progenitor populations
Dkk1 might have an overall effect on the specification of diencephalic
fates, including DA neurons. We therefore examined whether Dkk1 affects
terminal differentiation as well as cell number of other diencephalic cell
types that develop in juxtaposition to DA neurons. We observed that
oxytocinergic neurons [termed isotocinergic (IT) in fish] develop just
anterior to DA group 2, whereas hypocretin (Hcrt)-secreting neurons are formed
in a slightly ventral position (Fig.
2A,C,G). Somatostatin (SS)-producing cells appear in several
clusters located in the ventral (preoptic area) and dorsal (thalamus)
diencephalon (Fig. 2E,G)
(Blechman et al., 2007).
Simultaneous examination of DA neurons, together with each of the other cell
types, revealed that ectopic expression of Dkk1 had no effect on the cellular
organization and number of either IT, Hcrt or SS producing neurons
(Fig. 2B,D,F,H). These results
suggest that although inhibition of canonical Wnt signaling affects forebrain
patterning, it does not affect the fate and number of all diencephalic cell
populations.
|
). As Staudt and Houart showed that some gene expression
domains in the neural plate seem to be retained by the same cells up to the
prim5 stage (24 hpf), we sought genetic landmarks expressed in the
diencephalic anlage at bud stage (end of gastrulation), as well as in the PT
of embryos at 24 hpf. Thus, triple staining of the pro-neural marker
neurogenin 1 (neurog1), the diencephalic marker
BarH-like 2 (barhl2) and TH in 24 hpf embryos showed that
the neurog1+ group 2 DA cluster appeared adjacent to the
barhl2+ PT domain (see Fig. S2 in the supplementary
material).
Based on these gene expression data, we hypothesized that DA progenitors
originate in or around this area. We then performed fate-mapping analyses
using the neurog1::gfp transgenic line
(Blader et al., 2003), which
served as a live landmark for high-resolution visualization of the
neurog1+ cells in the diencephalic anlage during
neurulation (Fig. 3A). To carry
out high-resolution fate mapping of diencephalic progenitors, we developed a
new photo-activation (i.e. uncaging) strategy using two-photon microscopy,
which enables activation of fluorescein-conjugated tracer dye at highly
localized focal points. We precisely activated the fluorescein tracer in a
small group of cells at the basal diencephalic anlage of the
neurog1::gfp transgenic embryos at the 1- to 3-somite stage
(Fig. 3A and see Fig. S3 in the
supplementary material). We then allowed the embryos to develop until 24 hpf,
at which time terminally differentiated DA neurons are readily detectable
(Fig. 3B,C). Using this method,
we located three discrete diencephalic progenitor pools; one of these, denoted
domain II, reproducibly labeled Neurog1+ TH+ DA neurons
in both wild-type (WT) and reduced Wnt conditions
(Fig. 3A-D, n=6, and
see Fig. S3 in the supplementary material). Furthermore, uncaging other
diencephalic domains around domains I and II in Dkk1-overexpressing embryos
did not label DA cells, indicating that DA progenitors remain in the same
neural plate position under reduced Wnt conditions (data not shown).
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Cellular responses underlying Wnt-mediated restriction of DA cell number
The identity and number of progenitor cells may be influenced by local
instructive cues affecting the progenitor zone and by transcription factors
determining the timing of the last cell division of the progenitors
(Guillemot, 2007). We next
wished to elucidate the type(s) of cellular behavior that leads to the changes
in DA cell number in response to Wnt attenuation.
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;
Guo et al., 1999;
Jeong et al., 2006;
Lee et al., 2006;
Rink and Guo, 2004). The
supernumerary DA cell phenotype reported herein might occur because DA
progenitors undergo an extra cell division. However, the exact developmental
stage at which zebrafish DA neurons exit the cell cycle (termed birthdate) is
not known. We therefore performed birthdating analysis on WT and
dkk1-injected embryos. Embryos were labeled with
5-bromo-2'-deoxyuridine (BrdU) at different developmental time points,
and DA neurons were immunostained subsequent to their terminal differentiation
(Fig. 5 and see Table S1 in the
supplementary material). We then analyzed the proportion of proliferating DA
progenitors by double immunofluorescence staining of progenitors with
anti-BrdU (nuclear staining) and of differentiated DA cells with anti-TH
(cytoplasmic staining) antibodies. This analysis revealed that the first DA
cells became postmitotic as early as 10 hpf (bud/1-somite stage). By 16 hours
(14- to 15-somite stage) and thereafter, the DA progenitors that gave rise to
the TH+ neurons detectable at 48 hpf did not incorporate BrdU
(Fig. 5A,C,E,G). In accordance
with the developmental stage at which terminally differentiated DA neurons
appear in the diencephalon, we found that the group 2 DA cluster exits the
cell cycle earlier than the group 3-6 cluster
(Fig. 5G). Surprisingly,
birthdating analysis of DA progenitors in dkk1-injected embryos
showed that these neurons exhibited identical cell cycle withdrawal kinetics
to their WT siblings (Fig.
5B,D,F,G). Furthermore, WT and dkk1-injected embryos had
the same proportion of cycling DA progenitors at each of the sampled
developmental stages (Fig. 5G).
Thus, DA progenitors become postmitotic between 10 and 16 hpf, and the
expansion of the DA cell population following Wnt inhibition is not due to
delayed cell cycle exit of DA progenitors.
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20% of DA progenitors are in a state of proliferation
(Fig. 5G and
Fig. 6H). Consistent with our
birthdating analyses, blocking cell division at 6 hours had the greatest
effect on DA cell number, whereas application of the cell cycle inhibitors at
14 hours had a minimal effect on DA cell development
(Fig. 6A,C,E,G). A comparison
between WT and dkk1-injected embryos showed that the number of DA
neurons was doubled in dkk1-injected embryos treated with cell
division blockers at 6, 10 or 14 hours of development
(Fig. 6B,D,F,G). Notably, Dkk1
gain-of-function was able to recover the reduction in the number of group 3-6
DA clusters in inhibitor-treated embryos. However, the number of cells in the
restored DA group never exceeded the number in untreated WT embryos,
suggesting that Wnt does not restrict the final population size of group 3-6
clusters (Fig. 6 and see Fig.
S5 in the supplementary material). That Dkk1 induced the same proportional
increase (two-fold) in DA neurons in the presence or absence of cell cycle
blockers, regardless of the developmental stage at which the inhibitors were
administrated, suggests that Dkk1 does not affect the rate of DA progenitor
proliferation. The observed Dkk1-induced doubling of DA cell number in embryos in which cell division was blocked at 6 hours of development, together with our finding that Wnt inhibition does not delay proper exit of DA progenitors from the cell cycle, suggest that the increase in the number of DA neurons might be due to an effect on early DA progenitors.
Control of DA population size begins during gastrulation
Our fate mapping and analysis of DA progenitor proliferation suggested that
Wnt signaling modulates the size of the early DA progenitor pool in the neural
plate. To further define the window during which the Wnt/β-catenin
pathway acts to control DA cell number, we used a heat-shock protein 70
(hsp70) promoter to induce the expression of Dkk1 at discrete
developmental stages. By means of this expression system, we detected ectopic
transcription of dkk1 mRNA as early as 15-30 minutes after shifting
the temperature from 28°C to 37°C (data not shown). We also observed
that a temperature pulse of 37°C for 30-60 minutes produced the same DA
cell number phenotype as that caused by injection of dkk1 mRNA
(Fig. 7A,B). Temporal induction
of dkk1 at various points in development revealed that 6-7 hpf is the
latest developmental stage at which Hsp70-driven dkk1 can elicit a
change in DA cell number (Fig.
7C). We conclude that the critical period of competence during
which canonical Wnt signaling is able to modulate DA progenitor number occurs
between late gastrulation and early somitogenesis, indicating that
specification of DA cell number is modulated concomitantly with the
establishment of the earliest proneural field.
|
). We sought candidate Wnt ligand-receptor complexes that
might constitute the target for Dkk1-mediated control of DA cell number. We
focused on the Wnt8b ligand and its receptor frizzled 8a (Fz8a; Fzd8a), as
both proteins are expressed in the prospective diencephalon both before and
during DA differentiation, and are known to be involved in modulating regional
fates in the anterior neural plate (Houart
et al., 2002; Kelly et al.,
1995; Kim et al.,
2002). Targeted knockdown of wnt8b using antisense
morpholino oligonucleotides (MOs) led to an elevation in DA cell number,
phenocopying dkk1-injected embryos
(Fig. 8B,F). Downregulation of
Wnt8b had no effect on the barhl2+ cell population, which
defines a discrete progenitor population juxtaposed to the DA precursor domain
(Fig. 8G,H and see Fig. S2 in
the supplementary material). MO-based knockdown of fz8a, which is
known to functionally interact with Wnt8b
(Cavodeassi et al., 2005;
Kim et al., 2002), led to a
similar increase in DA cell number (Fig.
8C,F). Double knockdown of wnt8b and fz8a did
not further enhance the single-morphant phenotype
(Fig. 8F), suggesting that the
Wnt8b ligand, through the Fz8a receptor, induces a regulatory cascade that
limits the number of DA progenitors.
|
). The Lef1 transcription factor is a crucial determinant of
neurogenesis and of neural progenitor fates in the posterior-ventral
hypothalamus (Galceran et al.,
2000; Lee et al.,
2006; van Genderen et al.,
1994). Furthermore, both Wnt8b and Lef1 regulate
β-catenin-dependent transcription of neural progenitor genes in the basal
diencephalon (Lee et al.,
2006). To study the possible involvement of Lef1 in the control of
diencephalic DA cell number, we quantified DA cells in the
lef1-deficient mutant, denoted X8
(Lee et al., 2006), and in the
lef1 morphant. This analysis revealed that lef1-deficient
embryos had an elevated number of DA neurons
(Fig. 8D-F). Similar to
wnt8b, inhibition of lef1 gene activity had no effect on the
adjacent barhl2 expression domain (see Fig. S6 in the supplementary
material). Taken together, these results show that the Wnt8b/Lef1 signaling pathway restricts DA cell number in the PT with no effect on the adjacent barhl2 expression domain.
fezl (too few) is epistatic to Wnt signaling
We have previously characterized the zebrafish mutant too few
(tof m808), in which there is a marked reduction in cell
number of the majority of diencephalic DA clusters owing to a recessive
mutation in the gene encoding the Fezl (also known as Fezf2)
zinc-finger-containing protein (Levkowitz
et al., 2003). Having demonstrated that, like fezl,
components of the canonical Wnt signaling cascade are necessary in order to
maintain the correct number of DA neurons, we next examined whether
fezl and Wnt(s) share a common genetic pathway.
It was previously shown that the expression of fezl in the
prospective telencephalon and diencephalon is enhanced following
overexpression of Dkk1, indicating that fezl expression is repressed
by the canonical Wnt/β-catenin pathway
(Hashimoto et al., 2000b). We
therefore examined whether fezl gene activity is required for
Wnt-mediated restriction of DA cell number by inhibiting Wnt signaling in a
fezl-deficient genetic background. Because tof
m808 is a weak loss-of-function allele
(Levkowitz et al., 2003),
these mutant embryos display a reduction in the group 2 DA cluster;
nevertheless, DA neurons are still specified in the tof
m808 mutant (Fig.
9A,D,G,I). We examined whether attenuation of Wnt affected the
remaining group 2 DA neurons in tof m808 mutants.
Injection of either dkk1 mRNA or wnt8b MO into tof
m808 embryos did not affect the number of dat+
DA neurons, demonstrating that the effect of Wnt attenuation on DA cells is
dependent upon intact fezl gene activity and that Fezl is a key
mediator of DA cell regulation downstream of the Wnt pathway
(Fig. 9A-K). Thus, a canonical
Wnt signal(s) acts upstream of fezl, suggesting that attaining the
correct number of DA neurons depends on a balanced activity of Wnt8b and
Fezl.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Relationships between early neural plate patterning, cell identity and cell number
Previous studies have shown that early induction of diencephalic DA fates
is governed by patterning cues, such as nodal, Bmp7, Shh and Fgf8
(Holzschuh et al., 2003;
Mathieu et al., 2002;
Ohyama et al., 2005;
Ye et al., 1998). The present
study demonstrates, for the first time, that the determination of precise
numbers of diencephalic DA progenitor cells during primary neurogenesis is
specifically controlled by canonical Wnt signaling. Notably, induction of
diencephalic DA neuronal identity by Shh and Bmp7 in the chick embryo requires
the forebrain-specific transcriptional repressor Six3
(Ohyama et al., 2005), a
protein that in mouse directly binds to the wnt1 promoter to repress
its expression (Lagutin et al.,
2003).
One could conceivably attribute the alterations in DA cell number reported
herein to the robust action of Wnt on neural patterning. Our study, however,
shows that the differential response of distinct neuronal populations to the
Wnt signal is not a simple interpretation of their relative anteroposterior
position. We propose that diencephalic DA cell number in the PT area is
specifically determined by the Wnt8b-Fz8a signaling complex, concomitant with
Wnt-mediated regional patterning events. Several lines of evidence support
this model. Firstly, we show that attenuation of Wnt signaling leads to a
selective expansion of DA cell clusters that are generated in the basal plate
of the diencephalon (i.e. the PT; Figs
1,
2,
3 and
4). Secondly, gain-of-function
of several Wnt antagonists at an early developmental stage does not alter the
population size or fate of neighboring diencephalic cell types such as IT, SS
and Hcrt producing cells (Fig.
2 and data not shown), and of broad diencephalic domains
expressing dlx6a or neurog1 (Figs
1,
4). This specificity might be
due to differing responses of the neuronal progenitors to the signal, or to a
difference in the time of induction of the progenitor pools, which might also
determine the nature of the inductive signals to which the progenitors are
exposed. Thirdly, Wnt8b/Lef1 activity restricts DA cell number in the PT
(Fig. 8), and these embryos
have reduced neurogenesis in the adjacent sox3+ ventral
hypothalamic domain (Lee et al.,
2006) and normal barhl2 expression in the neighboring
ventral hypothalamus (Fig. 8
and see Fig. S6 in the supplementary material). Finally, inhibiting canonical
Wnt signaling in a tof m808 genetic background led to an
enlarged telencephalon without affecting DA cell number
(Fig. 9 and data not shown).
Hence, the tof m808 hypomorph enables us to uncouple the
role of Wnt in anteroposterior axis patterning from its role in the control of
DA cell number. In a manner similar to Wnt, Fezl might play a role in both
neural specification and patterning events (see below).
|
; Baker et al.,
1983; Vadasz et al.,
1982), the exact mechanisms and cellular events modulating the
number of mesencephalic or diencephalic DA progenitors are currently unknown.
Zebrafish DA neurons develop extremely rapidly relative to those of other
vertebrate species; most diencephalic DA progenitors are in a postmitotic
state by 12-14 hpf, and by 4-5 days all DA clusters that appear in the adult
brain are already detectable (Fig.
5). These characteristics of the zebrafish brain are compatible
with the findings of our study, which is the first demonstration that DA cell
number is already tightly regulated at the initial stages of anterior neural
plate specification.
|
; Johnson,
2003). For the most part, these processes occur during embryonic
neurogenesis or in the neonatal brain, after the neural plate has subdivided
into prospective rostrocaudal and dorsoventral brain territories. Regulation
of neuronal population size has been mainly studied in the mammalian retina
and in the cortex, where it is largely determined by the proportion of
neuronal precursors that re-enter the cell cycle
(Ohnuma and Harris, 2003;
Rakic, 1995). Activation of
Wnt/β-catenin signaling increases the size of the cortex through the
propagation of cortical neurogenesis
(Chenn and Walsh, 2002;
Zechner et al., 2003).
However, the increased zebrafish DA population shown herein is not due to a
delay in cell cycle exit, an increased proportion of dividing DA progenitors,
or a higher rate of proliferation (Figs
5,
6). Analysis of DA cell number
following temporal inhibition of Wnt signaling and cell proliferation
indicates that the initial pool of DA progenitors is restricted by the Wnt
pathway. These findings suggest that the early embryonic patterning machinery
(i.e. Wnt/β-catenin) also controls the number of neural progenitors that
will later assume a DA fate. Thus, the number of DA progenitors is
predetermined during primary neurogenesis by patterning signals that control
the size of specific neurogenic fields.
The role of the zinc-finger protein Fezl in forebrain and DA neuron development
The forebrain-specific embryonic zinc-finger transcription factor Fezl
plays an import role in neural patterning and specification
(Chen, B. et al., 2005;
Chen, J. G. et al., 2005;
Hirata et al., 2006;
Hirata et al., 2004;
Jeong et al., 2007;
Molyneaux et al., 2005). Fezl
is one of the earliest markers delineating the prospective forebrain
(Hashimoto et al., 2000b;
Yang et al., 2001). A
hypomorphic allele of fezl (tof m808) results in
a marked decrease in some DA clusters, whereas other diencephalic DA subtypes
remain unaffected (Blechman et al.,
2007; Levkowitz et al.,
2003; Rink and Guo,
2004). Moreover, Wnt8b negatively regulates the expression of Fezl
in the neural plate (Hashimoto et al.,
2000b). We now extend these findings by showing that Wnt8b acts
upstream of Fezl in controlling DA cell number
(Fig. 9). The question remains
as to whether the canonical Wnt signal regulates Fezl directly or indirectly?
Indirect inhibition would suggest that an unknown Wnt/β-catenin target
gene represses fezl transcription. A direct repression of
fezl by Wnt would imply atypical transcriptional repression activity
of the β-catenin-Lef1 complex, as was recently shown in the case of
pituitary organogenesis (Olson et al.,
2006).
Despite evidence for extensive neurogenesis in the adult zebrafish
forebrain, the missing DA neurons in tof m808 mutants are
not regenerated in adult animals (Adolf et
al., 2006; Grandel et al.,
2006; Rink and Guo,
2004). Thus, the activity of Fezl during early neural
specification (gastrulation) is crucial in regulating DA cell number. The
period of time during which activation of Dkk1 affects DA cell number
correlates with the developmental stage at which Fezl affects diencephalic
patterning (Fig. 7)
(Jeong et al., 2007).
Furthermore, it has been shown that the Fezl protein acts upstream of Neurog1,
which is the first proneural gene product known to appear in the zebrafish
forebrain; moreover, Neurog1 is necessary for diencephalic DA neuron
development (Jeong et al.,
2006). Similar to Wnt inhibitors, the tof m808
mutant specifically affects TH+ Neurog1+ DA
neurons in the PT (Blechman et al.,
2007). Hence, the genetic interaction between Wnt and Fezl
provides a link between early patterning of the forebrain and specific
regulation of DA cell number.
In conclusion, our analyses of the role of Wnt activity in diencephalic neuronal subtype decisions reveal a new function for early canonical Wnt signaling in DA neuron development. We present an example of how early neural patterning signals affect the size of a defined neuronal population, concomitantly with the initial regionalization of the neural plate.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/20/3401/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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