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First published online April 10, 2009
doi: 10.1242/10.1242/dev.034793
1 Université de Toulouse, UPS, Centre de Biologie du Développement
(CBD), 118 route de Narbonne, F-31062 Toulouse, France.
2 Department of Cell and Developmental Biology, University College London, Gower
Street, London WC1E 6BT, UK.
* Authors for correspondence (e-mails: s.wilson{at}ucl.ac.uk; blader{at}cict.fr)
Accepted 27 February 2009
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Nodal signalling, Asymmetry, Habenular nuclei, Neurogenesis, Zebrafish
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Levin,
2005). For instance, in humans, aspects of language processing
occur predominantly in the left hemisphere. Although brain lateralisation is
particularly obvious in humans, it is not unique to us, being widespread among
both vertebrates and invertebrates (Cooke,
2004; Vallortigara et al.,
1999).
The molecular mechanisms that underlie the development of brain asymmetry
in vertebrates are beginning to be unravelled, and in this regard the
zebrafish is proving to be a powerful model. Zebrafish exhibit both
neuroanatomical and behavioural asymmetries that are amenable to genetic
analysis (Barth et al., 2005;
Concha, 2004;
Halpern et al., 2003). The
zebrafish dorsal diencephalon (epithalamus) is composed of the pineal complex
and the bilateral habenular nuclei that are part of a highly conserved
conduction system that interconnects sites in the forebrain and ventral
midbrain (Bianco and Wilson,
2009; Facchin et al.,
2009). Both the pineal complex and habenulae display prominent
asymmetries (Concha and Wilson,
2001; Halpern et al.,
2003). The photoreceptive pineal complex consists of the medially
located pineal (epiphysis) and the left-sided parapineal nucleus. During
development, parapineal precursors detach from the anterior pineal anlage and
migrate leftward, coming to lie adjacent to the left habenula
(Concha et al., 2003). This
migration is dependent upon the activity of Fgf8 expressed bilaterally in the
anlagen of the habenulae (Regan et al.,
2009). The left and right habenulae display differences in the
proportion of neuronal subtypes with distinct patterns of gene expression,
axon terminal morphology and connectivity
(Aizawa et al., 2005;
Aizawa et al., 2007;
Bianco et al., 2008;
Concha et al., 2003;
Concha and Wilson, 2001;
Gamse et al., 2005;
Gamse et al., 2003).
The handedness (laterality) of habenular asymmetry is always concordant
with that of the parapineal. Fate mapping and tissue ablation experiments
indicate that the establishment of parapineal and habenulae lateralities is
coordinated through a stepwise mechanism
(Concha et al., 2003;
Gamse et al., 2003). First,
the anlage of the left habenula provides cues that bias the orientation of
Fgf8-dependent parapineal migration towards the left
(Concha et al., 2003;
Regan et al., 2009).
Subsequently, the parapineal promotes the elaboration of left-sided habenular
identity; in the absence of the parapineal, much habenular asymmetry is lost
and both nuclei display predominantly right-sided character
(Bianco et al., 2008;
Concha et al., 2003;
Gamse et al., 2005;
Gamse et al., 2003). However,
some features of habenular neurons, including subtle aspects of axon terminal
morphology, remain asymmetric following parapineal ablation, suggesting that
the parapineal is not an absolute determinant of habenular asymmetry
(Bianco et al., 2008).
The Nodal signalling pathway is required for asymmetric development of the
heart, visceral organs and brain. In zebrafish, a Nodal protein encoded by the
ndr2 (cyclops) gene and its downstream targets are
transiently expressed on the left side of the dorsal diencephalon. However, in
embryos in which Nodal signalling is bilaterally symmetric or absent, the
epithalamus still develops asymmetrically but handedness is randomised: 50%
have normal laterality and 50% are reversed, with the parapineal on the right
and the habenulae showing L/R reversals in gene expression and projection
patterns (Aizawa et al., 2005;
Concha et al., 2000;
Gamse et al., 2005;
Gamse et al., 2003). Although
Nodal signalling is required for specifying the laterality of epithalamic
asymmetry, the manner in which Nodal imposes this bias is not known.
In this study, we have explored the involvement of Nodal signalling in the
establishment of very early epithalamic asymmetry by focussing on the earliest
stages of habenular neurogenesis. We describe a novel marker of habenular
progenitors/neurons, C-X-C chemokine receptor 4b (cxcr4b),
and show that the expression of cxcr4b appears earlier in the left
habenula than in the right. This is consistent with previous results showing
asymmetries in the generation of neuronal subtypes between left and right
habenulae (Aizawa et al.,
2007). The temporal L/R difference in neurogenesis revealed by
cxcr4b expression occurs prior to the leftward migration of the
parapineal and is still detected if the parapineal is ablated. By contrast,
removing the L/R bias in Nodal signalling renders habenular neurogenesis
symmetric. Our results provide a clear example of a role for Nodal signalling
in promoting an asymmetry per se, rather than in directing laterality.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). The
transgenic lines Tg(huC:gfp), Tg(ngn1:gfp),
Tg(flh:egfp) and Tg(foxD3:gfp) have been described
previously (Blader et al.,
2003; Concha et al.,
2003; Gilmour et al.,
2002; Park et al.,
2000). Embryos were fixed overnight at 4°C in 4%
paraformaldehyde, after which they were dehydrated through an ethanol series
and stored at -20°C.
In situ hybridisation (ISH) and antibody labelling
Antisense probes for lov
(Gamse et al., 2003),
brn3a (Aizawa et al.,
2005), cxcr4b (David
et al., 2002), ngn1
(Blader et al., 1997),
lft1 (Thisse and Thisse,
1999), ndr2 and pitx2
(Essner et al., 2000) were
generated using standard procedures. Embryos were stained using BCIP and NBT
(Roche) or Fast Red (Sigma) as substrate. In certain cases, embryos were
further stained with rabbit anti-GFP antibody (1/500, Torrey Pines Biolabs) or
anti-HuC/D antibody (16A11, Molecular Probes) in blocking buffer (1xPBS,
0.5% Triton X-100, 10% goat serum, 1% DMSO) and visualised with an Alexa Fluor
488-conjugated secondary antibody (1/500, Molecular Probes). For nuclear
staining, embryos were incubated in 1xPBS, 0.5% Triton X-100, 1% bovine
serum albumin containing ToPro (1/1000, Molecular Probes).
BrdU labelling
Embryos at 48 hpf were incubated in 15% DMSO and 10 mM 5-bromo
2'-deoxyuridine (BrdU) for 90 minutes at 4°C, rinsed and development
allowed to continue at 28°C until 60 hpf, at which time embryos were
fixed. BrdU incorporation was detected as previously described
(Shepard et al., 2004) using
anti-BrdU antibody (1/100, G3G4 Hybridoma Bank) and Alexa Fluor 548-conjugated
secondary antibody (1/500, Molecular Probes).
Parapineal ablation
Laser ablation of parapineal precursors was performed at 24-28 hpf in
Tg(flh:egfp); Tg(foxD3:gfp) double transgenic embryos as
described previously (Concha et al.,
2003). Larvae were subsequently fixed at 36-38 hpf and
immunostained with anti-HuC/D antibody in order to count habenular
neurons.
Morpholino injections and temperature shifts
Morpholino oligonucleotides (MOs) were dissolved in water at 1 mM
(ntl MO) (Nasevicius and Ekker,
2000) or 3 mM (spaw MO)
(Long et al., 2003). The
resulting stock solution was diluted to working concentrations (0.12 mM and
1.2 mM, respectively) in water and Phenol Red before injection (0.5-2 nl) into
eggs at the 1 cell stage. For temperature shifts, wild-type embryos were
placed at 22°C 2 hours after laying. Embryos were subsequently fixed and
processed for ISH or antibody labelling.
SB431542 treatment
For inhibition of the Nodal signalling pathway, Tg(huC:gfp) or
wild-type embryos were treated with SB431542, a selective inhibitor of the
Tgfβ type I receptor (Ho et al.,
2006; Inman et al.,
2002). SB431542 (Tocris Bioscience) was dissolved at 50 mM in DMSO
and aliquots stored at -20°C. Embryos were dechorionated at 10 hpf or 16
hpf and incubated in 100 µM SB431542 or in DMSO alone at 28°C until
fixation. Treated embryos were fixed at 21 hpf or 23 hpf and processed for ISH
to reveal Nodal pathway gene expression or at 34-38 hpf to determine the L/R
ratio of HuC:GFP+ or HuC/D+ cells.
Cell counting and statistical analysis
To detect habenular neurons, we used immunostaining with either an anti-GFP
antibody in Tg(huC:gfp) embryos or an anti-HuC/D antibody and
counterstained cell nuclei with ToPro to facilitate counting. Left and right
habenular neurons were counted using ImageJ software (NIH) from confocal
stacks acquired every 1 µm. For each embryo, we plotted the numbers of left
versus right HuC:GFP+ or HuC/D+ neurons and linear
regressions were performed using Prism 4 software (GraphPad Software). To test
whether individual datasets were asymmetric, we performed a Wilcoxon
signed-rank test
(http://faculty.vassar.edu/lowry/wilcoxon.html).
To determine the asymmetry index (AI) for individual embryos, the number of
left neurons (L) minus the number of right neurons (R) was divided by the sum
of both left and right neurons, i.e. (L-R)/(L+R). To compare AI values between
two datasets, we performed a Kolmogorov-Smirnov (KS) statistical test
(www.physics.csbsju.edu/stats/KS-test.n.plot_form.html),
except for the spaw MO experiment for which we used a Student's
t-test (two-tailed) owing to the small sample size.
Imaging
For differential interference contrast pictures, embryos were mounted in
glycerol and analysed on a Zeiss Axiophot microscope using a Nikon digital
camera. For all fluorescent labelling, embryos were mounted in glycerol and
habenular nuclei were imaged from a dorsal view using a Leica SP2
laser-scanning confocal microscope with a x63 oil-immersion objective.
Transverse views were generated using Leica software from z-stacks
acquired at 0.5 µm intervals.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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36 hours post-fertilisation (hpf), becomes
broader at 48 and 72 hpf, and is largely symmetrical
(Fig. 1A,D,G; data not shown)
(Aizawa et al., 2005;
Gamse et al., 2003;
Roussigne and Blader, 2006).
The second category comprises genes such as leftover (lov),
right on (ron) and dexter (dex)
(kctd12.1, kctd12.2 and kctd8 - ZFIN) that are expressed
from 36 hpf or later in a strongly asymmetric manner
(Fig. 1B,E,H; data not shown)
(Gamse et al., 2005;
Gamse et al., 2003). Many
habenular neurons are born before 36 hpf
(Aizawa et al., 2007), so in
order to find earlier markers for these neurons we searched the expression
pattern database on ZFIN
(http://zfin.org)
for genes expressed in the habenulae. This directed our focus to
cxcr4b. The spatiotemporal dynamics of cxcr4b expression do
not fit into either of the two categories mentioned above because expression
is already robust at 36 hpf, peaks at 48 hpf and then decreases, becoming
restricted to a small medial domain at 3 days of development
(Fig. 1C,F,I). These results
suggest that cxcr4b expression defines a different cell population
within the habenulae than those identified by the markers described to
date.
To better appreciate the expression domains of the various markers within
the developing habenulae, we mapped their expression relative to that of the
transgene Tg(huC:gfp). huC is a marker of post-mitotic
neurons and the transgene recapitulates the expression of the endogenous gene
(Park et al., 2000). At 48
hpf, transcripts of brn3a and lov were found to completely
overlap with the domain of GFP transgene expression, confirming that these
genes are expressed in differentiated habenular neurons
(Fig. 1J-J'',K-K'').
By contrast, cxcr4b was expressed in cells located more ventrally,
closer to the ventricular surface of the neuroepithelium, than those
expressing the transgene (Fig.
1L-L'', Fig.
2C).
The location and dynamic nature of cxcr4b expression, broad early
and decaying with time, suggests that cxcr4b could be a transient
early marker of nascent habenular neurons or neuronal progenitors. Neuronal
progenitors are characterised by the expression of proneural genes that encode
members of the basic helix-loop-helix (bHLH) family of transcriptional
regulators (Bertrand et al.,
2002; Chapouton and
Bally-Cuif, 2004). The proneural genes neurogenin 1
(ngn1; neurog1) and achaete-scute complex-like 1b
(ascl1b) are expressed in the habenulae
(Mueller and Wullimann, 2003),
and we compared their expression with that of the huC:gfp transgene.
As with cxcr4b, the ngn1 and ascl1b transcripts
were located ventral to GFP+ neurons, suggesting that these
proneural genes are coexpressed with cxcr4b
(Fig. 2A-A'',D; data not
shown). In support of this conclusion, expression of cxcr4b partially
overlaps with the expression of GFP driven by ngn1 regulatory
sequences (Fig. 2B-B'')
(Blader et al., 2003;
Mueller and Wullimann, 2003).
As cells located ventral to the huC:GFP+ neurons
incorporate BrdU (Fig. 2E), we
suggest that cxcr4b-expressing cells are either neural progenitors or
newly born neurons that have only recently finished their last S phase.
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), we observed that at 48 hpf there are more
huC:GFP+ neurons in the left compared with the right
habenula (data not shown). To address whether this asymmetry arises from
accelerated neurogenesis in the left habenula, we determined the earliest time
at which this L/R difference could be detected. An asymmetry in the pool of
Tg(huC:gfp)+ neurons is present as early as 36 hpf
(Fig. 3A)
(Aizawa et al., 2007). At 32
hpf, GFP+ habenular neurons were often only detected on the left
(Fig. 3B), and prior to 32 hpf
no GFP+ neurons were detected in either the left or right habenula
(Fig. 3C). We also observed
asymmetry in the expression of cxcr4b at 36 and 32 hpf
(Fig. 3D,E). However, compared
with huC:GFP, the asymmetric expression of cxcr4b extended
to earlier stages of habenular development: whereas no
Tg(huC:gfp)-expressing cells were detected prior to 32 hpf,
cxcr4b-expressing cells were consistently found in the left habenula
at 28 hpf (Fig. 3C versus
3F). Thus, cxcr4b mRNA
reveals an asymmetry in habenular neurogenesis at least 4 hours earlier than
Tg(huC:gfp) or the endogenous HuC protein
(Fig. 3; data not shown).
The parapineal does not determine early habenular asymmetry
The parapineal plays an instructive role in the elaboration of asymmetries
between the left and right habenula
(Halpern et al., 2003). To
address whether the parapineal is involved in the generation of the asymmetry
in early habenular neurogenesis, we first assessed the position of the
migrating parapineal relative to the onset of lateralised cxcr4b
expression in the left habenula. Parapineal cells display a rosette
organisation from the onset of their migration that is readily visible in
fixed embryos after staining nuclei (Fig.
3D',E',F'). In 28 hpf embryos, in which
left-sided expression of cxcr4b is already detected, the parapineal
is either at the midline or just beginning to orient to the left
(Fig. 3F versus
3F')
(Concha et al., 2003). These
results indicate that left-sided neurogenesis begins before, or is concomitant
with, left-sided parapineal migration, raising the possibility that this
asymmetry might not be dependent upon the parapineal.
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). Subsequently, early habenular neurogenesis was
analysed by counting the number of HuC/D+ (Elav3/4+)
neurons in the left and right habenulae and nuclear staining was performed to
confirm that the parapineal had been successfully ablated
(Fig. 4A-B').
In all control embryos, a greater number of HuC/D-expressing neurons was
detected in the left as compared with the right habenula (P=0.002,
Wilcoxon signed-rank sum test) (Fig.
4C). An overall left bias was retained after ablation of
parapineal progenitor cells (P=0.005, Wilcoxon signed-rank sum test)
(Fig. 4C). This leftwards
asymmetry, in both control and ablated embryos, can also be quantitatively
expressed as an asymmetry index (AI), defined as the difference between the
number of neurons on the left versus the right side, normalised to the total
number of neurons [AI=(L-R)/(L+R)] (Fig.
4D). Our data do not demonstrate a significant difference in the
degree of asymmetry between control and ablated embryos [median AI for
parapineal ablated=0.18, n=14; median AI for controls=0.26,
n=13; P=0.131, Kolmogorov-Smirnov (KS) test]. Thus, in
contrast to its role in later habenular gene expression and axonal targeting
asymmetries (Bianco et al.,
2008; Concha et al.,
2003; Gamse et al.,
2005; Gamse et al.,
2003), our results suggest that the parapineal is dispensable for
the establishment of early asymmetry in the timing of neurogenesis between the
left and right habenulae.
Perturbations in Nodal signalling correlate with disruptions in asymmetric habenular neurogenesis
Asymmetric epithalamic Nodal signalling determines the laterality of
epithalamic asymmetries but is not thought to be required for the elaboration
of asymmetry per se (Concha et al.,
2000). Accordingly, genetic or other manipulations that remove the
asymmetric bias in Nodal signalling (such that signalling is either bilateral
or absent) randomise epithalamic laterality, whereas L/R reversal in biased
Nodal signalling reverses laterality
(Aizawa et al., 2005;
Carl et al., 2007). To address
whether Nodal signalling is involved in determining either the asymmetry of
early neurogenesis or its laterality, we assessed neurogenesis in situations
in which epithalamic Nodal signalling is disrupted.
Abrogation of no tail (ntl) function results in either
bilateral or absent epithalamic Nodal pathway gene expression and randomised
epithalamic laterality (Concha et al.,
2000) (Table 1). In
ntl morpholino (MO)-injected embryos, pools of left- and right-sided
habenular huC+ neurons were significantly more symmetric than in
control embryos (Fig. 5A,B)
(median AI for ntl morphants=0.00, n=46; median AI for
controls=0.38, n=17; P<0.001, KS test). For example,
60% (n=27/46) of injected embryos had a small AI of between 0.15
and -0.15 versus 14% in control embryos (n=3/21)
(Fig. 5B).
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) and again
to randomised epithalamic laterality
(Gamse et al., 2005). In
spaw morphants, we observed a significant reduction in the asymmetry
of early neurogenesis between the left and right habenulae
(Fig. 5C,D) (median AI for
spaw morphants=0.00, n=17; median AI for controls=0.21,
n=9; P=0.006, Student's t-test; see Materials and
methods). Thus, although neuroanatomical asymmetry is eventually established
in ntl and spaw morphants, the loss of unilateral Nodal
signalling correlates with a loss of the initial asymmetry in
neurogenesis.
Embryos raised at low temperature (22°C instead of 28°C) also show
randomised parapineal/habenular laterality, but in contrast to ntl
and spaw morphants, epithalamic Nodal signalling is randomised
(Table 1)
(Liang et al., 2000). The
distribution of AI values for cold-treated embryos was statistically different
from controls (P<0.001, KS test). Interestingly, similar
proportions of cold-treated embryos displayed leftwards asymmetry (35%),
rightwards asymmetry (35%), and symmetric pools of neurons (30%) (where we
classified -0.15<AI<0.15 as symmetric, AI>0.15 as left-sided and
AI<-0.15 as right-sided; n=20)
(Fig. 5E,F). Thus, whereas
ntl and spaw morphants display symmetric Nodal signalling
and symmetric neurogenesis, the majority of temperature-shifted embryos
display randomised unilateral Nodal signalling and asymmetric neurogenesis
with randomised laterality (Fig.
5, Table 1).
Altogether, these results strongly suggest that although Nodal signalling is not required for the eventual elaboration of neuroanatomical asymmetries, it is required for the initial asymmetry in habenular neurogenesis.
Nodal signalling is required for asymmetric habenular neurogenesis
To test the hypothesis that lateralised Nodal signalling leads to
asymmetric neurogenesis, we analysed embryos in which Nodal signalling was
compromised pharmacologically using the small-molecule inhibitor SB431542
(Inman et al., 2002).
Zebrafish embryos grown from early stages in SB431542 display a phenotype
identical to that of embryos genetically deficient for Nodal signalling
(Ho et al., 2006). Embryos
treated with SB431542 from 10 hpf, after the early requirement for Nodal
signalling in gastrulation, developed normally but lefty1
(lft1) expression was no longer detected in the epithalamus
(Table 1), consistent with the
drug blocking Nodal signalling in the LPM and brain. In embryos treated with
SB431542 from 10 hpf until 34-38 hpf, pools of early-born
huC:GFP+ habenular neurons were symmetric (Wilcoxon
sign-rank sum test for SB431542, P=0.749, n=17; for DMSO
controls, P=0.001, n=16)
(Fig. 6).
Supporting the conclusions from the analysis of spaw morphants, this indicates that Nodal signalling is required for asymmetric neurogenesis but does resolve whether it is Nodal signalling from the LPM or within the epithalamus that is required. To address this point, we treated embryos with SB431542 from 16 hpf, just prior to the initiation of expression of ndr2 in the dorsal diencephalon, but after spaw has been expressed asymmetrically in the LPM. As for early treatment, addition of SB431542 at later stages also abolished the epithalamic expression of Nodal targets. Although habenular neurogenesis continued to display mild asymmetry in SB431542-treated embryos (Wilcoxon sign-rank test for SB431542, P=0.024, n=22; for DMSO controls, P=0.001, n=16) (Table 1, Fig. 6K,L), the specific inhibition of epithalamic Nodal signalling caused a significant reduction in the degree of asymmetric neurogenesis (SB431542 median AI=0.07 and DMSO median AI=0.23, P=0.001, KS test).
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Nodal signalling asymmetrically biases neurogenesis
Our results indicate that the Nodal pathway is required for the L/R
asymmetry in the generation of early habenular neurons. However, the
production of neurons continues in the absence of Nodal signalling, indicating
that the signalling pathway asymmetrically biases a generic programme of
habenular neurogenesis and is not required for neurogenesis per se. How might
Nodal signalling drive asymmetry in habenular progenitors and/or early
post-mitotic habenular neurons? One possibility is that Nodal signals might
mediate L/R differences in proliferation, as has been found in the visceral
endoderm (Yamamoto et al.,
2004). However, no significant L/R differences in the mitotic
index have been detected in the dorsal diencephalon
(Aizawa et al., 2007).
Alternatively, early Nodal signalling could influence the size of the
neurogenic territory that gives rise to left habenular neurons by modulating
the expression or activity of proneural factors or components of the Notch
signalling pathway. Although Notch signalling has been examined in the
epithalamus at stages after the onset of early asymmetric neurogenesis
(Aizawa et al., 2007), it
remains unclear whether the pathway is asymmetrically activated earlier.
Further investigation of the basic programme of early neurogenesis in this
system should help address these questions. Finally, the generation of
chimaeric embryos in which habenular cells lack the ability to respond to
Nodal signals will allow us to determine whether Nodal acts directly or
indirectly upon habenular precursors.
The expression of cxcr4b, which encodes a chemokine receptor, in
habenular neuronal progenitors/newly born neurons raises the intriguing
question of the role of chemokine signalling in the development of the
epithalamus. Cxcr4b and its ligand, Cxcl12 (Sdf1), are implicated in various
aspects of brain development including migration, axon guidance, proliferation
and neuronal survival (Klein and Rubin,
2004; Lazarini et al.,
2003). The zebrafish cxcr4b mutant odysseus
(ody) (Knaut et al.,
2003) displays no obvious phenotype with respect to markers of L/R
epithalamic asymmetry such as lov (our unpublished observations);
whether ody mutants display subtle changes in the number,
organisation or connectivity of different subtypes of habenular neurons
remains to be investigated.
The Nodal pathway asymmetrically biases both habenular neurogenesis and parapineal migration
If asymmetric Nodal signalling is removed, the parapineal migrates with
equal frequency to the left and right sides of the brain
(Concha and Wilson, 2001).
Therefore, either Nodal signalling directly influences migrating parapineal
cells or the pathway indirectly influences migration by introducing an
asymmetry elsewhere in the epithalamus. Supporting an indirect role for the
Nodal pathway, parapineal laterality is compromised by partial ablation of the
prospective left habenula shortly after unilateral Nodal signalling
(Concha et al., 2003). Thus,
an early L/R asymmetry exists in the prospective habenulae and this asymmetry
appears to bias the orientation of parapineal migration towards the left.
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) show an early asymmetry in the timing of
neurogenesis between left and right habenulae prior to parapineal migration.
Furthermore, the stage at which asymmetric neurogenesis is first detected
(from 24 hpf) correlates well with that at which ablation experiments reveal
an asymmetric activity that biases the orientation of parapineal migration
(Concha et al., 2003). Here,
we show that Nodal signalling is required for this early asymmetric
neurogenesis. The role of Nodal in biasing parapineal laterality might,
therefore, be an indirect consequence of its effect on habenular
neurogenesis.
How might an early excess of precursors/neurons in the left habenula
influence the orientation of migration of the parapineal? One possibility is
that the precocious progenitors in the left habenula express parapineal
guidance cues. Alternatively, the guidance cue might be expressed by all
habenular precursors and it is the early asymmetry in the numbers of such
cells that provides a left-biased difference in the overall level of the cue.
We have recently shown that fgf8 is required for parapineal migration
and that L/R differences in the levels of Fgf8 can bias the direction of
migration (Regan et al.,
2009). fgf8 is expressed bilaterally, but there appear to
be subtle asymmetries between left and right habenulae. It will therefore be
important to determine whether Nodal signalling biases the levels of Fgf8
activity, for instance through promoting a L/R asymmetry in the number of
fgf8-expressing habenular precursors. An alternative possibility is
that asymmetric neurogenesis and directed parapineal migration are independent
consequences of a Nodal-dependent asymmetry in Fgf8 activity.
The parapineal is not the sole determinant of habenular asymmetry
The habenulae contain medial and lateral subnuclei that are L/R asymmetric
with respect to the expression of differentiation markers and targeting of
projections within the interpeduncular nucleus (IPN); the left habenula
contains predominantly neurons of lateral subnucleus identity that project to
the dorsal IPN, whereas the right habenula is mostly composed of neurons of
medial subnucleus identity that project to the ventral IPN
(Aizawa et al., 2005;
Bianco et al., 2008) [see Gamse
et al. for slightly different conclusions
(Gamse et al., 2005)]. The
elaboration of habenular asymmetries requires instructive input from the
parapineal, as ablation of the parapineal anlage results in both habenulae
displaying an overtly right-sided phenotype with respect to gene expression
and connectivity (Bianco et al.,
2008; Concha et al.,
2003; Gamse et al.,
2005; Gamse et al.,
2003). However, some subtle differences remain between the left
and right habenulae of parapineal-ablated embryos with respect to molecular
markers, neuropil organisation (Concha et
al., 2003; Gamse et al.,
2005) and early neurogenesis (this study). Moreover, although left
habenula neurons project to the ventral IPN in parapineal-ablated embryos,
their axons retain distinct lateralised morphologies
(Bianco et al., 2008). Thus,
although the parapineal is important for the lateralisation of habenular
circuitry and marker expression, it is not the sole determinant of L/R
differences between the habenulae.
How the parapineal drives habenular lateralisation is unclear, but it has
been proposed that the birthdate of habenular neurons influences their
lateral/medial subnuclear character (Aizawa
et al., 2007). Early neurogenesis occurs predominantly in the left
habenula and gives rise to lov+ neurons that will populate
the lateral subnucleus, whereas later neurogenesis is L/R symmetric and
produces neurons of both lateral and medial subnuclear character. Thus, one
hypothesis is that the parapineal directs habenular lateralisation by
regulating the timing of neurogenesis. Our study argues against such a simple
mechanism as we show that the L/R asymmetry in early-born neurons is not
dependent on the parapineal. Indeed, we show that this early asymmetry in
neurogenesis is dependent on Nodal signalling. However, if this
Nodal-dependent asymmetry were the sole mechanism generating the difference in
size of the left and right lateral subnuclei, one would expect that blocking
Nodal signalling would result in symmetric lateral subnuclei, which is not the
case (Concha et al., 2003;
Gamse et al., 2003). Thus, the
Nodal-dependent L/R asymmetry of early-born habenular neurons is not the
primary determinant of later left and right habenular asymmetries. It could,
nonetheless, still be responsible for the subtle differences that remain
between the left and right habenula in parapineal-ablated embryos
(Bianco et al., 2008;
Concha et al., 2003).
|
). However, a left bias in BrdU-pulse-labelled habenular
neurons is detected until 32-36 hpf, suggesting that an asymmetry in the pool
of HuC+ neurons lasts for at least 4 hours after the stage at which
we analysed neuronal number; this asymmetry could be parapineal dependent.
Thus, the parapineal might be responsible for maintaining an asymmetry in
habenular neurogenesis that is initiated by Nodal signalling. Although this
possibility is attractive, it is unlikely, however, that the primary role for
the parapineal in promoting habenular asymmetry is simply to regulate the
timing of neurogenesis. Although early and late waves of neurogenesis, as
described in Aizawa's study (Aizawa et al.,
2007), appear to produce different ratios of neurons destined for
the lateral and medial subnuclei, these two waves overlap extensively,
resulting in both lov+ and ron+
neurons being generated contemporaneously for many hours. It is hard to
reconcile this with a model in which timing of neurogenesis alone (driven by
Nodal early and by the parapineal later) regulates the subnuclear identity of
neurons. It is probable, therefore, that the generation of different ratios of
specific neurons involves additional mechanisms, such as a direct influence of
the parapineal in the specification of neuronal identity. From our data and previous studies, we propose a model (Fig. 7) in which asymmetric Nodal signalling acts upon prospective habenular cells to promote left-sided neurogenesis. Early, Nodal-dependent asymmetric neurogenesis may bias parapineal migration towards the left and also be responsible for the subtle parapineal-independent differences between the left and right habenulae. Once migration is initiated, the parapineal may maintain the left bias in habenular neurogenesis and/or directly influence the specification of lateral versus medial habenular neural subtypes.
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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