Neuron, Vol. 39, 423–438, July 31, 2003, Copyright 2003 by Cell Press
Local Tissue Interactions across the Dorsal
Midline of the Forebrain Establish CNS Laterality
Miguel L. Concha,1,2,8,* Claire Russell,1,8
Jennifer C. Regan,1,9 Marcel Tawk,1,9
Samuel Sidi,3 Darren T. Gilmour,3
Marika Kapsimali,1 Lauro Sumoy,4
Kim Goldstone,5 Enrique Amaya,5
David Kimelman,6 Teresa Nicolson,3
Stefan Gründer,7 Miranda Gomperts,5
Jonathan D.W. Clarke,1 and Stephen W. Wilson1,*
1
Department of Anatomy and Developmental Biology
University College London
Gower Street
London WC1E 6BT
United Kingdom
2
Millennium Nucleus on Integrative Neurosciences
Programa de Morfologı́a
Instituto de Ciencias Biomédicas
Universidad de Chile
P.O. Box 70079
Santiago 7
Chile
3
Max-Planck Institut für Entwicklungsbiologie
Spemannstrasse, 35
D-72076 Tübingen
Germany
4
Laboratori de Microarrays
Centre de Regulació Genòmica
Passeig Maritim 37-49
08003 Barcelona
Spain
5
The Wellcome Trust/Cancer Research UK Institute
Tennis Court Road
Cambridge CB2 1QR
United Kingdom
6
Department of Biochemistry, Box 357350
University of Washington
Seattle, Washington 98195
7
Department of Otolaryngology
Research Group of Sensory Physiology
Elfriede-Aulhorn-Str. 5
72076 Tübingen
Germany
Summary
The mechanisms that establish behavioral, cognitive,
and neuroanatomical asymmetries are poorly understood. In this study, we analyze the events that regulate development of asymmetric nuclei in the dorsal
forebrain. The unilateral parapineal organ has a bilateral origin, and some parapineal precursors migrate
across the midline to form this left-sided nucleus. The
parapineal subsequently innervates the left habenula,
which derives from ventral epithalamic cells adjacent
*Correspondence: mconcha@machi.med.uchile.cl (M.L.C.), s.wilson@
ucl.ac.uk (S.W.W.)
8
These authors contributed equally to this work.
9
These authors contributed equally to this work.
to the parapineal precursors. Ablation of cells in the
left ventral epithalamus can reverse laterality in wildtype embryos and impose the direction of CNS asymmetry in embryos in which laterality is usually randomized. Unilateral modulation of Nodal activity by Lefty1
can also impose the direction of CNS laterality in embryos with bilateral expression of Nodal pathway genes.
From these data, we propose that laterality is determined by a competitive interaction between the left
and right epithalamus and that Nodal signaling biases
the outcome of this competition.
Introduction
Neuroanatomical and functional asymmetries of the
brain are widespread among vertebrates and play important roles in animal behavior (Rogers and Andrew,
2002). CNS asymmetries arise early in development,
suggesting genetic regulation (e.g., Chi et al., 1977; Guglielmotti and Fiorino, 1999; Concha et al., 2000), but
additionally, epigenetic factors can influence asymmetries both in the developing and in the adult brain (e.g.,
Diamond et al., 1983; Melone et al., 1984; Gurusinghe
et al., 1986; Wisniewski, 1998). Although many studies
have demonstrated involvement of Nodal, Bmp, Hedgehog, Fgf, and other signaling pathways in the regulation
of asymmetry of the heart and viscera (reviewed in Burdine and Schier, 2000; Wright, 2001; Hamada et al., 2002),
these analyses have shed little light on the mechanisms
regulating brain laterality. However, recent studies in
zebrafish have shown that a genetic pathway with a
conserved role in the control of asymmetry of the heart
and viscera regulates at least some aspects of brain
asymmetry (Concha et al., 2000; Liang et al., 2000;
Gamse et al., 2002).
The diencephalic epithalamus is a region of the vertebrate brain that exhibits striking neuroanatomical asymmetries. The epithalamus is constituted by the habenular
nuclei and pineal complex. The habenulae form part
of an evolutionarily conserved conduction system that
receives input from the telencephalon via the stria medullaris and relays information to the interpeduncular
nuclei of the ventral midbrain via the fasciculi retroflexus
(reviewed in Sutherland, 1982). Structural asymmetries
between the left and right habenular nuclei are widespread among vertebrates and involve differences in
size, neuronal organization, neurochemistry, and connectivity (reviewed in Concha and Wilson, 2001). The
pineal complex is formed by medial evaginations of the
diencephalic roof plate that include the epiphysis (pineal
organ) and parapineal organ. Both organs contain photoreceptors and projection neurons, express a similar
subset of neurotransmitters, and produce melatonin, a
hormonal regulator of circadian rhythms (reviewed in
Ekström and Meissl, 1997; Falcón, 1999; Concha and
Wilson, 2001). Asymmetries of the pineal complex are
observed in lampreys, teleosts, and lizards in which the
parapineal organ innervates the left habenula (Rüdeberg, 1969; Engbretson et al., 1981; Korf and Wagner,
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424
1981; van Veen, 1982; Yañez et al., 1996, 1999; Concha
et al., 2000). In teleosts, the parapineal organ is also
situated on the left side of the brain (Borg et al., 1983;
Concha et al., 2000), and the pineal stalk develops subtle
morphological asymmetries (Liang et al., 2000).
Epithalamic asymmetries in zebrafish are first evident
as left-sided expression domains of genes encoding the
Nodal ligand Cyclops (Cyc), the negative regulator of
Nodal activity Lefty1 (Lft1), and the downstream effector
of Nodal signaling Pitx2 (Rebagliati et al., 1998; Sampath
et al., 1998; Thisse and Thisse, 1999; Bisgrove et al.,
2000; Concha et al., 2000; Essner et al., 2000; Liang et
al., 2000). At later stages, neuroanatomical asymmetries
consist of a photoreceptive parapineal organ located
on the left side and the left habenula developing more
extensive neuropil (Concha et al., 2000) and more intense expression of several genes (this study and Gamse
et al., 2003) than the right habenula. Zebrafish embryos
with compromised Nodal signaling and/or with midline
defects either lack or show bilateral expression of cyc
and pitx2 in the brain (Bisgrove et al., 2000; Concha et
al., 2000; Liang et al., 2000; Long et al., 2003). Neuroanatomical asymmetries still develop in both situations although laterality of the parapineal organ and habenulae
is randomized (Concha et al., 2000). These results suggest that Nodal signaling is not required for the development of asymmetry per se but is required to determine
the laterality of asymmetry by biasing an otherwise stochastic laterality decision to the left side of the epithalamus (Concha et al., 2000). The cellular and genetic
mechanisms regulating asymmetric development per
se, upon which Nodal signaling influences laterality
within the brain, remain unknown.
In this study, we use fate mapping, tissue ablation,
and electroporation approaches in GFP-transgenic animals to explore how laterality decisions occur within
the field of asymmetric Nodal influence in the zebrafish
forebrain. We show that parapineal cells originate bilaterally in the anterodorsal epithalamus from where they
migrate to become positioned on the left side of the
brain. Development of asymmetry in the habenular neuropil is synchronized in time and space with innervation
by efferent parapineal axons, indicating that the parapineal organ and habenulae interact during the establishment of epithalamic asymmetry. By ablating different
components of the epithalamus, we found that asymmetric habenular neuropil can arise independent of the
parapineal organ. However, removal of ventral epithalamic habenular precursor cells from the left side of the
brain affects direction of migration and connectivity of
the parapineal organ and laterality of the habenulae.
Furthermore, local modulation of Nodal signaling by
electroporation of lefty1 can impose the direction of CNS
asymmetry in embryos in which laterality is normally
randomized. We propose a model by which local inhibitory interactions between the left and right epithalamus
establish laterality within the forebrain. In this model,
Nodal signaling imposes an initial bias that makes left/
right interactions imbalanced from the outset, thereby
ensuring consistent direction of laterality within the population.
Results
The Epithalamus Is Divided into Dorsal
and Ventral Domains
Asymmetric expression of components of the Nodal signaling pathway is the first manifestation of asymmetry
within the zebrafish forebrain and precedes asymmetric
morphogenesis of the parapineal organ, habenulae
(Concha et al., 2000; Gamse et al., 2003), and pineal
stalk (Liang et al., 2000). Expression of the homeobox
gene floating head (flh) demarcates the territory in which
pineal neurons develop (Masai et al., 1997; Cau and
Wilson, 2003), but the origin of other epithalamic cell
groups is unknown. To enable us to address this issue,
we generated transgenic zebrafish expressing GFP under the control of the flh promoter (see Experimental
Procedures). Comparison of flh:eGFP expression with
that of cyc, lft1, and pitx2 showed that Nodal signaling
overlaps the left, anterior field of the flh expression domain (Concha et al., 2000; Liang et al., 2000) but also
extends further ventral to this domain (Figures 1A–1C
and data not shown). These observations define two
regions of Nodal influence: the dorsal epithalamus, comprising flh-expressing cells, and the ventral epithalamus,
containing cells immediately ventral to the domain of
flh expression.
The Parapineal Organ Has a Bilateral Origin
from the Anterior Dorsal Epithalamus
To establish the origin of epithalamic cell groups, we
followed the fates of cells in the epithalamus of flh:eGFP
fish. At the time of Nodal signaling, flh:eGFP expression
defines a contiguous territory located at the dorsal midline (Figures 1A–1D), but between 28 and 40 hpf this
splits into a large midline domain that corresponds to
the pineal organ and a smaller domain located on the left
side that corresponds to the forming parapineal organ
(Figures 1E–1G). Expression of flh:eGFP in the parapineal organ is then gradually lost (Figures 1H and 1I) and
from around 65 hpf is only detected in the pineal organ
(data not shown). These results suggest that parapineal
precursors express flh when located at the midline but
downregulate expression as they detach from the pineal
complex to become positioned on the left side of the
epithalamus.
At least three mechanisms could explain the absence
of a parapineal nucleus on the right side of the brain:
right-sided cells could die, adopt a different fate, or
could migrate across the midline to contribute to the
left-sided nucleus. To resolve among these possibilities,
we determined the location of parapineal precursors
prior to their appearance on the left side of the pineal
complex. To achieve this, we photoactivated caged fluorescein in different sectors of the flh:eGFP dorsal epithalamic field at about 24 hpf and examined the fate of
cells at 48 hpf. Photoactivation within posterior sectors
of the flh:eGFP domain always labeled cells that remained at the midline within the pineal organ (Table 1
and data not shown). In contrast, left- or right-sided
photoactivation within the anterior 1/3 of the flh:eGFP
expression domain labeled cells that contributed both
to the parapineal organ and to the pineal organ (Figures
Competitive Interactions Establish CNS Laterality
425
Figure 1. The Dorsal and Ventral Epithalamus Give Rise to the Parapineal Organ and
Habenular Nuclei
Dorsal (A and D–O⬘) and frontal (B and C)
views of the dorsal diencephalon with anterior and dorsal toward the top of the panels,
respectively. Epithalamic expression of GFP
in flh:eGFP- (A–I) and flh:eGFP;foxD3:GFP(J–O) transgenic embryos is shown in brown
(A–C) or green (D–O⬘). Stages of development
are indicated in somites (s) or hours postfertilization (h) on the bottom right for each
panel.
(A–C) Relationship between the expression
domains (blue) of pitx2 (A), cyc (B), and lft1
(C), and the domain of flh:eGFP expression
as detected with anti-GFP antibody (brown).
Arrowheads demarcate the limits of pitx2,
cyc, and lft1 expression. Red asterisks and
dashed lines indicate the boundary between
dorsal (d) and ventral (v) epithalamus.
(D–I) Single-depth confocal images showing
early phases of asymmetric morphogenesis
of the parapineal organ in flh:eGFP-transgenic zebrafish. Arrows indicate the position
of the parapineal organ.
(J–L and J⬘–L⬘) Embryos in which caged fluorescein was photoactivated by laser (pseudocolored in red) within the left (J) and right
(K) dorsal epithalamus and labeling was subsequently observed in both pineal (po) and
parapineal (arrowheads) organs (J⬘ and K⬘).
(L) and (L⬘) are schematic representations of
the labeling results.
(M–O and M⬘–O⬘) Embryos in which caged
fluorescein was photoactivated by laser (red)
within the left (M) and right (N) ventral epithalamus and labeling was subsequently observed in the left (M⬘) and right (N⬘) habenula,
respectively. (O) and (O⬘) are schematic representations of the labeling results. Arrowheads indicate the position of the parapineal
organ.
Abbreviations: d, dorsal; LH, left habenula;
po, pineal organ; RH, right habenula; v,
ventral.
1J–1L and 1J⬘–1L⬘ and Table 1). This data demonstrates
that parapineal precursors are located bilaterally in the
dorsal epithalamus within the anterior-most region of
flh expression.
If anteriorly positioned flh-expressing cells are indeed
the source of parapineal precursors, we reasoned that
ablation of these cells should result in an absence of a
parapineal organ. We therefore used laser pulses to
ablate flh:eGFP cells on one or both sides of the anterior
dorsal epithalamus. As flh:eGFP is a poor marker of the
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426
Table 1. Mapping the Location of Parapineal and Habenular Precursors within the Zebrafish Forebrain
Fate at 48 hpf
Position of Labelling at 22–24 hpf
Pineal
Parapineal
Habenulae
n
Dorsal Epithalamus
DLA
DLM
DRA
DRM
87%
100%
86%
100%
91%
–
100%
–
17%
–
5%
–
23
5
21
5
Ventral Epithalamus
VLA
VLM
VRA
VRM
60%
20%
69%
20%
15%
–
15%
–
90%
100%
100%
100%
20
5
13
5
In the schematics, the flh:eGFP-expressing domain is outlined by a solid line in the top panel and by a dashed line in the bottom panel.
Ventral epithalamic cells are positioned underneath (ventral to) the flh:eGFP domain. Abbreviations: D, dorsal; V, ventral; L, left; R, right; A,
anterior; M, middle (between anterior and posterior ends of flh:eGFP expression).
mature parapineal, we performed these experiments in
embryos that also expressed a foxD3:GFP transgene
(Gilmour et al., 2002) that labels mature parapineal neurons (see below) but does not label epithalamic precursor cells. The use of both transgenes allowed pineal
complex cells to be followed throughout their development. A mature but small and sometimes mispositioned
parapineal organ developed on the left side following
ablation of flh:eGFP expressing cells on either the left
side (Figures 2B⬘ and 2B″) or the right side (Figures 2C⬘
and 2C″) of the dorsal anterior epithalamus. In contrast,
bilateral ablation led to complete absence of parapineal
neurons (Figures 2D⬘ and 2D″). These results suggest
that all parapineal precursors are located in the anterior
dorsal epithalamus.
The Habenular Nuclei Originate from Ventral
Epithalamic Cells
flh:eGFP-expressing cells do not significantly contribute
to the habenular nuclei (Table 1), and so, to determine
the location of habenular precursors, we photoactivated
caged fluorescein in small groups of cells directly adjacent to the flh:eGFP domain at about 24 hpf and examined the position of labeling at 48 hpf. Photoactivation
within the epithalamus directly ventral to the flh:eGFPexpression domain always led to staining within the ipsilateral habenula (Figures 1M–1O and 1M⬘–1O⬘ and Table
1). These results demonstrate that, at 24 hpf, precursors
of the left and right habenula are positioned within the
left and right ventral epithalamus, respectively. Altogether, our fate mapping studies indicate that around 24
hpf parapineal and habenular precursors are contiguous
and, on the left side of the brain, both sets of precursors
are likely to be influenced by Nodal activity.
Parapineal Precursors Migrate to the Left Side
of the Brain and Innervate a Region of the Left
Habenula that Develops Asymmetric Neuropil
We have previously shown that the laterality of parapineal and habenular asymmetries is strongly coordinated
(Concha et al., 2000). In order to better understand the
temporal and spatial coordination of parapineal and habenular asymmetries, we performed confocal volumetric
analysis of flh:eGFP- and foxD3:GFP-transgenic fish.
From about 28 hpf, parapineal precursors begin to aggregate and move away from the midline toward the left
side of the epithalamus (Figures 1E–1I). This early phase
of parapineal migration is followed by a second phase
that involves a ventral, posterior, and medial displacement of the parapineal organ, which ends up flanking
the pineal stalk (Figures 3A–3H). Efferent projections
from the parapineal organ are observed from around 50
hpf and develop extensively during the late phase of
parapineal migration to cover a significant fraction of
the left epithalamus (Figures 3E⬘–3H⬘). The abundance
of putative terminals of efferent parapineal axons within
the left epithalamus is most likely due to extensive axonal branching, as the number of parapineal cells is rather
small (13.5 ⫾ 2 cells expressing foxD3:GFP at 96 hpf;
n ⫽ 19).
Habenular asymmetries are characterized by the left
habenula developing a larger area of neuropil than the
right habenula, particularly within the medial and dorsal
aspect of the nucleus (Concha et al., 2000; this study).
The region of enlarged neuropil in the left habenula is
evident from around 70 hpf and becomes more conspicuous contemporaneous with the escalating development of efferent projections from the parapineal organ
(compare Figures 3I–3L and 3E⬘–3H⬘). Indeed, parapineal axons enter the left habenula immediately posterior
to the habenular commissure and distribute extensively
toward medial and dorsal regions of this nucleus to
encompass the area of the left habenula that displays
asymmetric neuropil (Figure 4). Altogether, these results
indicate that the development of neuroanatomical asymmetry in the habenulae is coordinated both in time and
space with the development of efferent parapineal connectivity.
The Laterality of Parapineal Asymmetry Is Disrupted
by Selective Removal of Ventral Cells
from the Left Epithalamus
By definition, the unilateral innervation of the left habenula by parapineal axons contributes to the lateralization
of the habenular nuclei. Indeed, complete removal of
the parapineal organ at 28–30 hpf (when it is already
Competitive Interactions Establish CNS Laterality
427
Figure 2. Bilateral but Not Unilateral Ablation of Anterior Dorsal Epithalamic Cells Abolishes Parapineal Development
Dorsal views of the epithalamus of flh:eGFP;foxD3:GFP-transgenic zebrafish with anterior toward the top of the panels.
(A–D) Schematics indicating the region of the dorsal epithalamus ablated at 22–24 hpf (white dots) in relation to the flh:eGFP expression
domain (green): control (A), anterior left half (B), anterior right half (C), anterior bilateral (D).
(A⬘–D⬘) Confocal volumetric projections from the epithalami of embryos ablated as in (A)–(D). Embryos are labeled with anti-acetylated ␣-tubulin
(red, habenular neuropil) and anti-GFP (green, pineal and parapineal) antibodies. Arrowheads show the position of the parapineal organ. Large
yellow arrows indicate the region of the habenulae displaying enlarged neuropil.
(A″–D″) High-magnification views of (A⬘)–(D⬘) focused on the region where the parapineal organ develops. Arrows indicate efferent parapineal
connectivity.
(A″⬘–D″⬘) lov expression in the left (LH) and right (RH) habenular nuclei. An arrow indicates the region of lov expression that corresponds to
the left dorsomedial neuropil. Expression in this region is reduced but remains asymmetric after parapineal ablation (D″⬘).
Abbreviations: LH and RH, left and right habenula; po, pineal organ; ps, pineal stalk.
located in the left epithalamus) leads to failure to upregulate the marker gene leftover (lov) in the left habenula
(Gamse et al., 2003). We confirmed a requirement of
parapineal cells for left-sided upregulation of lov expression by ablating parapineal precursors at 22–24 hpf (n ⫽
9; Figures 2B″⬘–2D″⬘). However, despite the reduced
levels of expression, we still detected left/right differences in the pattern of lov expression after parapineal
ablation. The dorsomedial aspect of the left habenula
retains an area of staining that is not seen in the right
habenula (Figure 2D″⬘). In addition, embryos lacking parapineal efferents through ablation of flh:eGFP⫹ parapineal precursors in the anterior epithalamus frequently
still exhibit differences in the extent of neuropil (Figure
2D⬘) between left and right habenulae (Figure 2 and
Table 2). Therefore, although the parapineal contributes
to habenular laterality, our results suggest that there
is no absolute requirement for a parapineal organ to
establish differences between left and right habenular
nuclei.
We next asked whether asymmetric migration and/or
connectivity of the parapineal organ depends on habenular development. To test this possibility, we ablated
ventral epithalamic cells that later contribute to the habenulae and assessed the subsequent direction of migration and pattern of connectivity of the parapineal
organ. Ablation of cells within the ventral epithalamus
led to a variable decrease in the extent of neuropil within
the ipsilateral habenula and, in some cases, also affected the development of the habenular commissure
(data not shown). The number of parapineal cells was
also often reduced due to the close proximity between
the parapineal anlage and the ventral epithalamus. As
the severity of these defects was directly correlated
with the quantity of cells ablated (data not shown), we
focused our analyses on ablations localized to a subset
of cells positioned immediately ventral to the flh:eGFP
expression domain. In these cases, the overall morphology of the epithalamus was unaffected and the number
of parapineal cells only mildly reduced (Table 3).
Parapineal asymmetry was disrupted in 68% of embryos following ablation of left ventral epithalamic cells
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Figure 3. Development of Asymmetric Neuropil within the Habenulae Is Spatially and Temporally Coordinated with Parapineal Innervation
Frontal (A–D) and dorsal (E–L) confocal images of the epithalamus with dorsal and anterior toward the top of the panels, respectively. Stages
of development are indicated on the left for each row of images.
(A–H) Confocal volumetric projections showing late phases of parapineal migration in foxD3:GFP-transgenic zebrafish. For clarity, pineal and
parapineal organs have been pseudocolored in red and green, respectively.
(E⬘–H⬘) Development of efferent parapineal connectivity. Images correspond to magnified views of (E)–(H). Green arrows indicate the position
from which axons leave the parapineal organ, and white arrowheads delineate the area of the left epithalamus covered by these axonal
projections.
(I–L) Confocal volumetric projections showing the development of asymmetric neuropil within the habenulae of wild-type zebrafish as revealed
by anti-acetylated ␣-tubulin antibody. Large yellow arrows indicate the region of the left habenula that displays enlarged neuropil.
Abbreviations: hc, habenular commissure; LH, left habenula; pc, posterior commissure; po, pineal organ; RH, right habenula.
(Table 3). These embryos showed either a parapineal
organ located at the dorsal midline (29%, Figures 5C⬘
and 5C″) that projected to the left (4/8), right (3/8), or
both (1/8) habenular nuclei, or, most notably, a parapineal organ with a pattern of migration and efferent connectivity directed toward the right habenula (39%; Figures 5D⬘ and 5D″ and Table 3). Removal of an equivalent
group of ventral cells from the right epithalamus had no
effect on parapineal asymmetry (Table 3 and Figures
5B⬘ and 5B″). These results indicate that left ventral
epithalamic cells are required for normal asymmetric
migration and connectivity of the parapineal organ.
The Laterality of Habenular Asymmetry Is Disrupted
by Selective Removal of Ventral Cells
from the Left Epithalamus
To test the possibility that the left ventral epithalamus
not only influences parapineal laterality but more glob-
ally affects epithalamic asymmetry, we determined
whether habenular laterality was also affected by ablation of ventral epithalamic cells. For this, we examined
the extent of habenular neuropil and the expression pattern of hest1 (habenularEST1), a novel genetic marker
of habenular asymmetry (see Experimental Procedures).
hest1 is normally expressed bilaterally in the habenulae
but shows more intense expression in the dorsomedial
aspect of the left nucleus (Figure 5A″⬘).
In 48% of cases, removal of left ventral epithalamic
cells led to the right dorsomedial habenular nucleus
developing a region of enlarged neuropil resembling that
normally found in the left habenular nucleus (Figures
5C⬘ and 5D⬘ and Table 4). In these embryos, hest1 expression was consistently reduced compared to wildtype but, in many cases, remaining expression was
stronger in the right than in the left habenula (45%;
Figures 5C″⬘ and 5D″⬘ and Table 4). When the right ha-
Competitive Interactions Establish CNS Laterality
429
Figure 4. The Parapineal Organ Projects to a
Region of the Left Habenula that Develops
Asymmetric Neuropil
Dorsal views of the epithalamus in a
foxD3:GFP-transgenic zebrafish at 96 hpf
with anterior toward the top of the panels.
The habenular neuropil has been labeled with
anti-acetylated ␣-tubulin antibody (red) and
the pineal complex with anti-GFP antibody.
For clarity, pineal and parapineal organs have
been pseudocolored in blue and green, respectively.
(A) Confocal volumetric projection. The habenular commisure (hc) normally runs between the left (LH) and right (RH) habenular
nuclei but has broken during the labeling procedure.
(B–E) Single optical sections of the region encircled in (A). The depth of each section is
indicated in the bottom left corner. The arrow
indicates the position from which axons leave
the parapineal organ, and white arrowheads
delineate the area of the left habenula covered by these axonal projections.
Abbreviations: hc, habenular commissure;
LH, left habenula; pineal, pineal organ; pp,
parapineal organ; RH, right habenula.
benula was transformed into a nucleus with left characteristics, this was usually accompanied by migration and
connectivity of the parapineal organ toward the right
epithalamus (9/11 embryos; Figures 5D⬘ and 5D″). In
these experiments, we did not assess whether the left
habenula showed left- or right-sided character due to
the reduction of neuropil after ablation. In contrast to
the laterality reversals induced by left-sided removal of
habenular precursors, ablation of right ventral epithalamic cells had no effect on the laterality of habenular
neuropil and only minor effects on hest1 expression
(Figures 5B⬘ and 5B″⬘ and Table 4).
Altogether, these results demonstrate that laterality
is not fixed at the time of ablation and show that local
tissue interactions between the left and right epithalamus are able to determine forebrain laterality.
Ventral Epithalamic Ablations Impose Handedness
upon Embryos that Normally
Exhibit Heterotaxia
In wild-type embryos, ablation of left-sided ventral epithalamic cells sometimes leads to reversal of epithalamic laterality. We next wished to address whether such
ablations could impose laterality in embryos where the
outcome of the laterality is not already biased to the
left by unilateral Nodal activity (about 95% of wild-type
Table 2. Effect of Parapineal Ablation on the Development of Habenular Asymmetry
Asymmetry of the Habenulae
Severity of Parapineal Ablation
None (control)
Mild
Intermediate
Severe
Complete
13.5 ⫾ 2 cells
9–12 cells
5–8 cells
1–4 cells
0 cells
L⫹/R
L/R
L⫹/R⫹
L/R⫹
n
85%
87%
90%
80%
81%
5%
–
–
10%
13%
5%
–
–
–
6%
5%
13%
10%
10%
–
19
8
10
10
16
The severity of parapineal ablation was assessed by counting the number of remaining parapineal cells expressing GFP at 96 hpf. Asymmetry
of the habenulae was determined by examination of the content of neuropil using anti-acetylated ␣-tubulin antibody at 96 hpf (Concha et al.,
2000). Laterality of the habenula: normal (left ⬎ right, L⫹/R), reversed (left ⬍ right; L/R⫹), left isomerism (right habenula of left characteristics;
L⫹/R⫹), right isomerism (left habenula of right characteristics; L/R).
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Table 3. Ventral Epithalamic Cell Ablation Affects the Laterality of Parapineal Migration
Laterality of Parapineal Migration
Laterality of Ablation
Wild-type
ntl-MO
LZoep⫺/⫺
none (control)
right
left
none (control)
right
none (control)
right
Left
Midline
Right
n
95%
91% (9 ⫾ 3 cells)
32% (7 ⫾ 2 cells)
45%
82% (7 ⫾ 2 cells)
52.5%
74% (8.4 ⫾ 3 cells)
–
–
29% (7 ⫾ 3 cells)
–
–
–
13% (11 ⫾ 1 cells)
5%
9% (12 ⫾ 0 cells)
39% (6 ⫾ 3 cells)
55%
18% (4 ⫾ 0 cells)
47.5%
13% (11 ⫾ 1 cells)
38
11
28
22
11
40
23
Laser ablation of ventral epithalamic cells was performed in wild-type flh:eGFP;foxD3:GFP, ntl morphant (ntl-MO) flh:eGFP;foxD3:GFP, and
LZoep⫺/⫺; foxD3:GFP embryos at 22–24 hpf. The laterality of parapineal migration was assessed at 96 hpf by live confocal imaging or antiGFP immunostaining. Due to the close proximity between the ventral epithalamus and the parapineal anlage, the ablation procedure often
involved some loss of parapineal cells. To take this limitation into account, we give in brackets the average number of foxD3:GFP⫹ parapineal
cells remaining after ablation. The average number of parapineal cells in control embryos is 13.5 ⫾ 2.
embryos show left-sided parapineal and an enlarged
left habenula; Concha et al., 2000). To address this issue,
we ablated ventral epithalamic cells in situations where
Nodal activity within the brain is either bilaterally symmetric (embryos lacking No-tail [Ntl] activity) (Bisgrove
et al., 2000; Concha et al., 2000; Liang et al., 2000) or
absent (embryos lacking Late Zygotic activity of Oneeyed-pinhead, LZoep⫺/⫺) (Concha et al., 2000; Liang et
al., 2000). In both classes of embryos, parapineal and
habenular laterality is normally randomized (Concha et
al., 2000; Gamse et al., 2002, 2003), suggesting that
there is no initial left/right bias in Nodal signaling or in
the subsequent allocation of left/right laterality.
In virtually all cases, ablation of right-sided ventral
epithalamic cells in ntl morphants led to left-sided migration and connectivity of the parapineal (Figures 5E⬘ and
5E″ and Table 3), left-sided enlargement of habenular
neuropil (Figure 5E⬘, Table 4), and high levels of habenular hest1 expression on the left (Figure 5E″⬘, Table 4). A
shift in laterality was also observed after ablations in
LZoep⫺/⫺ embryos (Figures 5F⬘–5F″⬘, Tables 3 and 4),
suggesting that Nodal activity is not required for the
ablation-mediated imposition of laterality. These results
indicate that in situations where it is likely that left and
right sides of the dorsal forebrain are initially equivalent,
ablation of ventral epithalamic cells leads to parapineal
migration and expanded habenular development on the
opposite side of the brain. As we discuss below, this
suggests that a competitive mechanism between left
and right epithalamus determines laterality in the dorsal
forebrain.
Local Modulation of Nodal Signaling Directs
Epithalamic Laterality
We have previously shown that mutations affecting
Nodal signaling randomize CNS laterality (Concha et al.,
2000). However, it is unclear where, when, and how Nodal
signaling is acting to modulate CNS laterality. Although
Nodal pathway components are asymmetrically expressed in the epithalamus (previous studies), and parapineal and habenular precursors are both likely to be
influenced by Nodal signaling (this study, Figures 1A–
1C), there is no direct evidence that epithalamic Nodal
activity can directly influence laterality decisions. To test
this possibility, we examined the consequences on laterality of unilaterally abrogating Nodal signaling locally
within the epithalamus of embryos that show bilateral
Nodal expression and which normally exhibit randomization of CNS asymmetries. To do this, we electroporated DNA encoding a secreted antagonist of Nodal signaling (Lefty1) together with a reporter (⬀-tubulin GFP)
in the right epithalamus of ntl morphant embryos. Lefty1
is a very potent secreted Nodal-signaling antagonist
(Solnica-Krezel, 2003), making it a good reagent to locally modulate Nodal activity. After electroporation, high
levels of exogenous lefty1 mRNA expression could be
detected in small numbers of cells on the right side of
the epithalamus (Figure 6B), colocalizing with the site
of ⬀-tubulin GFP⫹ clones (Figure 6B, inset). As Lefty1
can function at a distance from its site of expression
(Chen and Schier, 2002), then it is likely that exogenous
Lefty1 activity spreads beyond the immediate vicinity
of the electroporated cells.
In all cases where cells expressing ⬀-tubulin GFP were
visualized on the right side of the pineal complex (n ⫽
10), the parapineal organ migrated to the contralateral
(left) side and showed connectivity directed to the contralateral (left) habenula (Figure 6D). Conversely, coelectroporation of ⬀-tubulin GFP⫹ DsRed DNAs in ntl morphant control embryos had no effect on laterality and
parapineal migration remained randomized (L ⫽ 52%,
R ⫽ 48%; n ⫽ 29) (Figure 6C). We therefore conclude
that local asymmetric modulation of epithalamic Nodal
signaling is sufficient to impose laterality on embryos
that would otherwise develop randomized CNS asymmetries.
Discussion
In this study, we have used fate mapping, tissue ablation, and electroporation approaches in GFP-transgenic
animals to elucidate the mechanisms by which laterality
decisions are made in the vertebrate forebrain. We show
that local tissue interactions regulate the laterality of
asymmetry of the diencephalic habenular nuclei and
the photoreceptive parapineal organ. Below, we discuss
how both ipsilateral and contralateral interactions influence morphogenesis of these nuclei and propose a model
in which signals passing across the dorsal midline of
the diencephalon ensure epithalamic asymmetry.
Competitive Interactions Establish CNS Laterality
431
Figure 5. The Laterality of Parapineal and Habenular Asymmetries Is Affected by Removal of Cells from the Left Ventral Epithalamus
Dorsal views (A–F, A⬘–F⬘, and A″–F″) and transverse sections (A″⬘–F″⬘) of the epithalamus with anterior and dorsal toward the top of the panels,
respectively. Embryos are flh:eGFP;foxD3:GFP- (A–D), ntl morphant flh:eGFP;foxD3:GFP- (E), and LZoep⫺/⫺ ;foxD3:GFP- (F) transgenic zebrafish.
(A–F) Schematics indicating the region of the ventral epithalamus ablated at 22–24 hpf (white dots) in relation to the flh:eGFP expression
domain (green): control nonablated (A), ablated on the anterior right side (B) and anterior left side (C and D) in wild-type, and anterior right
side in ntl morphant (E) and LZoep⫺/⫺ embryos (F).
(A⬘–F⬘) Confocal volumetric projections of the epithalami in embryos ablated as in (A)–(F). Embryos are labeled with anti-acetylated ␣-tubulin
(red, habenular neuropil) and anti-GFP (green, pineal complex) antibodies. Arrowheads show the position of the parapineal organ. Large yellow
arrows indicate the region of the habenulae displaying an enlarged neuropil.
(A″–F″) High-magnification views of (A⬘)–(F⬘) focused on the region where the parapineal organ develops. Arrows indicate efferent parapineal
axons.
(A″⬘–F″⬘) hest1 expression in the epithalamus. There is extensive expression of hest1 in regions of the brain ventral to right (RH) and left (LH)
habenular nuclei. Large arrows indicate the side of the habenulae displaying more intense hest1 labeling.
Abbreviations: po, pineal organ; ps, pineal stalk; LH, left habenula; RH, right habenula.
Coordinated Development of Habenular
and Parapineal Asymmetries
Our studies have shown a remarkable degree of coordination in development of the parapineal and habenular
nuclei (Figure 7A). Precursors of both nuclei are contiguous and on the left side of the brain both are likely to
be directly influenced by Nodal activity. At the stage
when left-sided activation of Nodal signaling occurs, the
epithalamus is symmetric, but this soon changes as
parapineal cells begin to aggregate on the left side of
the pineal complex. This is followed by migration to the
left side of the brain and subsequently by the develop-
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432
Table 4. Ventral Epithalamic Cell Ablation Affects Laterality of Habenular Asymmetry
Laterality of Habenular Asymmetry
Laterality of Ablation
Wild-type
ntl-MO
LZoep⫺/⫺
none (control)
right
right
left
left
none (control)
right
right
none (control)
right
right
Probe
L⫹/R
L/R
L⫹/R⫹
L/R⫹
n
␣-tub/hest1
␣-tub
hest1
␣-tub
hest1
␣-tub/hest1
␣-tub
hest1
␣-tub/hest1
␣-tub
hest1
91%
91%
92%
24%
10%
44%
80%
100%
50%
73%
60%
1%
9%
8%
28%
45%
4%
10%
–
–
8%
–
–
–
–
17%
–
4%
–
–
–
4%
–
8%
–
–
31%
45%
48%
10%
–
50%
15%
40%
66
11
12
29
20
25
11
7
76
26
15
Laser ablation of ventral epithalamic cells was performed in wild-type flh:eGFP;foxD3:GFP, ntl morphant (ntl-MO) flh:eGFP;foxD3:GFP, and
LZoep⫺/⫺-foxD3:GFP embryos at 22–24 hpf. The laterality of habenular asymmetry was assessed at 96 hpf by examination of the content of
neuropil using anti-acetylated ␣-tubulin antibody (␣-tub; Concha et al., 2000) or by hest1 mRNA in situ hybridization. Laterality of the habenulae:
normal (left ⬎ right, L⫹/R), reversed (left ⬍ right; L/R⫹), left isomerism (L⫹/R⫹), right isomerism (L/R).
ment of efferent connectivity with the left habenula. The
latter is temporally synchronized and topographically
coordinated with the development of asymmetric neuropil in the left habenula.
In virtually all situations, the laterality of habenular
and parapineal asymmetries is coordinated (Concha et
al., 2000; this study; Gamse et al., 2003). There are at
least three mechanisms that could explain this: the parapineal determines the laterality of the habenular nuclei,
the habenular nuclei determine the laterality of the parapineal, or a common mechanism influences laterality of
both structures. We think that the first two, and probably
all three, mechanisms contribute to the final morphogenesis of the epithalamic nuclei. The evidence that a common mechanism influences both nuclei is circumstantial––precursors of both nuclei are contiguous at the
time at which laterality is allocated, and the only bona
fide signaling pathway known to influence laterality
(Nodal) is activated in both sets of precursors.
Our study provides compelling evidence that habenular precursors are asymmetric within a few hours of
unilateral Nodal signaling and that this asymmetry influences parapineal migration. It is likely that the habenular
nuclei continue to influence parapineal neurons at later
stages, for instance, by providing guidance cues to attract parapineal axons. Conversely, unilateral parapineal
innervation, by definition, affects later asymmetry of the
habenular nuclei. Parapineal cells also influence lateralized gene expression within the habenular nuclei
(Gamse et al., 2003). Indeed, the extent of habenular
asymmetry in reptiles is strictly correlated with the presence or absence of a parapineal organ/parietal eye
Figure 6. Electroporation of lefty1 in the Epithalamus Directs Laterality of Parapineal Migration
Dorsal views of the dorsal diencephalon in
ntl morphant foxD3:GFP-transgenic zebrafish,
with anterior toward the top of the panels. Embryos were electroporated with ␣-tubulin
GFP ⫹ pCS2DsRed DNAs in the left epithalamus (A and C) or ␣-tubulin GFP ⫹ lefty1 DNAs
in the right epithalamus (B and D).
(A and B) Whole-mount in situ hybridization
for lefty1 mRNA at 40 hpf. Exogenous lefty1
mRNA is detected after lefty1 DNA electroporation (arrowhead in [B]), colocalizing with
␣-tubulin GFP⫹ cells (brown immunostaining, arrow in inset).
(C and D) Epithalamic expression of
foxD3:GFP transgene (pineal and parapineal
organs), electroporated DsRed (arrow in [C]),
and electroporated ␣-tubulin GFP (arrow in
[D]) in living embryos at 48 hpf (C) and 96
hpf (D). For clarity, the pineal organ has been
pseudocolored in blue, the parapineal organ
in green, and electroporated cells in either
red (DsRed in [C]) or green (␣-tubulin GFP in
[D]).
Abbreviations: DsRed, pCS2DsRed; po, pineal organ; pp, parapineal organ; tub-GFP,
␣-tubulin GFP.
Competitive Interactions Establish CNS Laterality
433
Figure 7. A Model of Tissue and Molecular Interactions Underlying Forebrain Laterality
(A) Schematics illustrating the development of epithalamic asymmetry. The earliest known manifestation of asymmetry is the activation of
Nodal signaling on the left side of the brain (cyc, crosshatching), encompassing both parapineal (yellow) and habenular (pink) precursors.
Shortly after this, parapineal cells aggregate on the left side of the pineal complex. Although no markers of asymmetry of the prospective
habenulae at this stage have been identified, these nuclei are already likely to be lateralized. Subsequent parapineal migration to the left is
dependent upon habenular precursor cells on the left side of the brain. During later development, left-sided parapineal and habenular cells
continue to interact with parapineal axons innervating the left dorsomedial (LDM) neuropil of the habenulae (this study) and influencing gene
expression in this nucleus (Gamse et al., 2003).
(B) Schematics illustrating a mutual inhibition model of CNS lateralization. The top row shows events occurring in the epithalamus at early
stages and the bottom panels show the outcomes of these events. Central to this model is that the left and right sides of the epithalamus
compete, with the outcome of the competition determining the direction of parapineal migration and the enlargement of the ipsilateral habenular
nucleus.
(Bi) In the absence of Nodal signaling in the epithalamus (LZoep⫺/⫺; Concha et al., 2000; Liang et al., 2000), both left and right epithalamus
compete on equal terms, and laterality is determined on a stochastic basis.
(Bii) In wild-type embryos, lateralized Nodal signaling gives the left side an advantage, and the outcome of the competition is always biased
to the left.
(Biii) In embryos with bilateral Nodal signaling in the brain (ntl-MO), both sides of the epithalamus receive the advantage provided by Nodal
activity, and once again, the competition is equal and outcome is stochastic.
(Biv) Ablation of left-sided ventral epithalamic precursors in wild-type embryos negates the advantage provided by Nodal signaling so that,
in some cases, the right side of the epithalamus wins the competition, and laterality is to the right.
(Bv) In embryos with ablation of right-sided ventral epithalamic precursors, the left side still wins the competition, and laterality is the same
as in unablated embryos.
(Bvi) Ablation of right-sided ventral epithalamic precursors in embryos lacking Nodal signaling (LZoep⫺/⫺) imposes an advantage to the left
epithalamus, and laterality is subsequently biased to the left.
(Bvii) In embryos with bilateral activation of Nodal signaling (ntl morphants, ntl-MO), either ablation of right-sided ventral epithalamic precursors
or inhibition of Nodal signaling by exogenous Lefty1 on the right restores the advantage to the left epithalamus, and laterality is subsequently
biased to the left.
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434
(Huber and Crosby, 1926; Cruce, 1974; Engbretson et
al., 1981; ten Donkelaar, 1998). Whether the parapineal
has any earlier role in the initial determination of habenular laterality is less clear. For instance, habenular asymmetries still develop in many vertebrate groups that apparently lack a parapineal organ (reviewed in Concha
and Wilson, 2001). Furthermore, in our ablation studies,
some embryos developed asymmetric habenular neuropil and asymmetric lov expression despite the apparent absence of parapineal neurons. It seems likely that
reciprocal interactions between parapineal and habenular cells serve to reinforce and ensure coordination of
initial molecular asymmetries, thereby guaranteeing appropriate functional development of asymmetric neuronal circuits.
Communication Occurs between Left and Right
Sides of the Epithalmus
Several lines of evidence suggest that cells on the left
and right sides of the diencephalon communicate
across the dorsal midline of the CNS. The fact that epithalamic isomerism is rare in embryos carrying mutations affecting Nodal signaling or following disruption
to axial midline tissue (Concha et al., 2000; Liang et al.,
2000; Gamse et al., 2002) suggests that if one side of
the CNS acquires a certain handedness (left or right),
then the other is inhibited from adopting the same. Intuitively, it makes sense if this inhibitory interaction occurs
locally between left and right sides of the dorsal diencephalon. However, all mutations examined to date
globally affect laterality, and so we cannot be absolutely
certain where, when, or how laterality in the CNS is
determined in these mutant embryos.
Our fate mapping studies provide the most compelling
evidence that communication occurs between left and
right epithalamus. Parapineal migration is dependent
upon left-sided ventral epithalamic cells, and so, signals
that promote left-sided migration are able to directly or
indirectly attract right-sided cells to the left. Given that
right-sided parapineal precursors (which do not express
the Nodal ligand Cyc) can still cross the midline in the
absence of left-sided (Cyc-expressing) parapineal precursors, it seems likely that tropic signals can act across
the midline. However, as midline tissue itself may be
damaged in these experiments, we should not discount
the possibility that left-sided parapineal precursors may
promote the movement of right-sided precursors to the
left. Altogether, current evidence favors the notion that
signals can traverse the dorsal midline during establishment of epithalamic laterality. As we discuss below,
midline-crossing signals may influence initial establishment of left/right identity as well as subsequent morphogenetic events.
Nodal Signaling in the Dorsal Diencephalon
Directs Laterality
The Nodal signaling pathway has conserved roles in
regulating asymmetry and laterality throughout vertebrates (Hamada et al., 2002; Boorman and Shimeld,
2002). However, this signaling pathway has many roles
during early development and indeed is even used reiteratively during the establishment of asymmetry (e.g.,
Brennan et al., 2002; Yamamoto et al., 2003). This has
made it challenging to determine where, when, and how
Nodal signals influence laterality of various structures.
Previous work has shown that mutations affecting Nodal
signaling disrupt epithalamic laterality (Concha et al.,
2000; Liang et al., 2000; Gamse et al., 2002) but have
not determined whether local Nodal pathway activity in
the diencephalon is responsible for regulating laterality.
Indeed, recent data suggests that Nodal signaling within
lateral plate mesoderm can influence laterality in the
epithalamus (Long et al., 2003).
Our experiments unilaterally modulating Nodal activity
in the brain provide strong evidence that diencephalic
Nodal activity does have a critical role in mediating CNS
laterality. In our experiments, we used Lefty1 to abrogate
Nodal signaling in the right epithalamus of ntl morphants
that normally show bilateral activation of Nodal pathway
target genes (e.g., Bisgrove et al., 2000; Concha et al.,
2000; Liang et al., 2000). In these experiments, laterality
was directed to the side of the brain opposite to that of
exogenous lefty1 expression. The consistency of this
experimental result may be due to the fact that Nodal
activity is probably equal on left and right sides of the
epithalamus of ntl morphants, and so, introducing even
a small bias in Nodal activity is sufficient to impose a
difference between left and right sides. Furthermore,
Lefty1 is a potent secreted antagonist of Nodal activity
(Solnica-Krezel, 2003), and so, stable expression of exogenous lefty1 within very few cells is likely to significantly influence the ability of Nodals to signal in the
vicinity of the Lefty-expressing cells.
A Mutual Inhibition Model of Laterality
Determination in the CNS
We propose a model that can account for the laterality
phenotypes in wild-type and mutant embryos, with or
without ablation of ventral epithalamic precursor cells.
Central to this model is the notion that competitive interactions between left and right epithalamus ensure lateralization by preventing symmetric development (isomerism) of the habenulae and parapineal nucleus. This
interaction can be conceptualized as both left and right
sides of the epithalamus producing signals that inhibit
the ability of the contralateral side to differentiate with
left-sided character.
We suggest that in the absence of Nodal signaling
within the brain, both sides of the epithalamus are equivalent and there is a stochastic outcome to the competition (Figure 7Bi). Fifty percent of the time the left epithalamus “wins” and parapineal migration and expanded
habenular development occur on the left, and fifty percent of the time there is the opposite outcome. In wildtype embryos, unilateral Nodal signaling gives the left
side of the epithalamus an advantage in the competition
so that laterality is consistently biased to the left (Figure
7Bii). Conversely, in embryos where Nodal signaling is
bilateral, both sides of the epithalamus receive the boost
provided by Nodal activity, and the competition is again
balanced, resulting in stochastic allocation of laterality
(Figure 7Biii). Thus, although embryos lacking Nodal signaling may be considered initially “double right-sided”
(with respect to Nodal activity) and embryos with bilateral Nodal signaling “double left-sided,” in both cases,
this initial unstable symmetry resolves to asymmetric
differentiation of epithalamic nuclei.
Competitive Interactions Establish CNS Laterality
435
In this paper, we show that ablation of ventral epithalamic cells can profoundly influence the outcome of the
interactions between left and right epithalamus. We suggest that ablation of these cells on the left in wild-type
embryos negates the advantage provided by ipsilateral
Nodal signaling. This makes the outcome of the competition less certain, and in some cases, parapineal and
habenular laterality is switched to the right (Figure 7Biv).
Ablation of equivalent cells on the right side of wildtype embryos has no consequence on laterality, as the
left side is already at an advantage (Figure 7Bv). Conversely, ablation of right-sided ventral epithalamic cells
in embryos lacking Nodal signaling gives an advantage
to the left, and laterality is subsequently directed to this
side (Figure 7Bvi). Similarly, in embryos with bilateral
Nodal signaling, either ablation of right-sided ventral
epithalamic cells or right-sided inhibition of Nodal signaling by exogenous Lefty1 influences the outcome of
laterality decisions. In these embryos, the initial equivalence between left and right sides is disturbed with the
consequence that epithalamic asymmetry is biased to
the left side (Figure 7Bvii).
One outcome of consistently activating Nodal signaling on the left side of the brain is that within wild-type
populations most fish will show the same direction of
CNS laterality. Conversely, fish populations in which
Nodal activity is not asymmetric will have equal proportions of “left-sided” and “right-sided” fish. This raises
the intriguing possibility that Nodal signaling in the brain
is more important for coordination of laterality between
different members of the species than for the determination of CNS laterality per se. Although data is limited,
coordinated CNS laterality between individuals appears
to be more common in social species than in animals
with solitary life styles (Vallortigara et al., 1999). It will
be interesting to assess if lateralized Nodal signaling
varies between species in which laterality is coordinated
between individuals and those with randomly allocated
CNS laterality.
Symmetry May Be Intrinsically Unstable
in Many Tissues/Organs
Symmetric development (isomerism) of some organs,
including the heart and gut and possibly also the brain,
is not compatible with normal function. Mechanisms
favoring asymmetry over symmetry may therefore have
been fixed during vertebrate evolution. It is likely that
redundant mechanisms ensure asymmetric development since perturbation of different pathways regulating
asymmetry more often results in randomization of asymmetry than in isomerisms (e.g., Nonaka et al., 1998; Ryan
et al., 1998; Meyers and Martin, 1999; Yan et al., 1999;
Concha et al., 2000; Levin et al., 2002). It is intriguing
that in embryos in which left/right signaling is disrupted,
organs such as the lungs, for which asymmetries appear
to have no obvious functional consequence, often display isomerism (Lin et al., 1999; Gaio et al., 1999; Rankin
et al., 2000), whereas organs dependent on asymmetry
for correct function, such as the heart and gut, exhibit
heterotaxia. Symmetry may thus have evolved as an
intrinsically unstable condition, and therefore an unlikely
outcome, especially for structures for which asymmetry
is inherently linked to function. As most such structures
develop from cells on both sides of the midline, it may
be that small, stochastic differences between left and
right sides tip the balance in favor of left-handed or
right-handed subsequent development. In each case,
there would need to be a mechanism of communication
across the midline to ensure coordinated development
of left- and right-sided precursors.
Mechanisms Underlying Mutual Inhibition
Our model for assignment of laterality in the brain has
several key features that need to be taken into account
when considering the signals that might be involved.
First, there is an initial bilaterally symmetric but unstable
condition. This initial symmetry is broken either by a
stochastic mechanism or by the influence of other factors (such as unilateral Nodal signaling). The induced
asymmetry is subsequently reinforced and amplified to
ensure generation of two different cell populations. Amplification and stabilization of small initial asymmetries
has been described for other signaling events, such as
lateral inhibition-mediated selection of cell fates (Lewis,
1998) and axon contact-dependent induction of asymmetric cell fates in worms (Troemel et al., 1999; Hobert
et al., 2002). Such an outcome can also be theoretically
achieved by reaction/diffusion mechanisms (e.g., Hamada et al., 2002).
If we consider a reaction/diffusion mechanism is responsible for establishing laterality in the brain, then we
need to envisage a signal (or signals) capable of inducing
itself and also inducing the expression of an antagonist
that can spread more effectively than the signal itself.
In this way, a stochastic increase in the signal on one
side of the brain would lead to enhanced production of
the antagonist that diffuses and suppresses contralateral expression. The Nodal pathway seems to have the
criteria required for establishing a reaction/diffusion
mode of activity (Chen and Schier, 2002; Solnica-Krezel,
2003). However, even if this is the case, it is unlikely
that Nodal signaling constitutes the only mechanism
for communication across the midline, as laterality is
established perfectly well in the absence of expression
of several known components of the Nodal pathway in
the brain (Concha et al., 2000). One possibility is that
Nodal signaling unilaterally modulates some other signaling pathway that operates between the two sides of
the brain. As both Wnts and Fgfs show localized bilateral
expression in the epithalamus, then both are candidates
for mediating the interactions we have defined in this
study.
Experimental Procedures
Zebrafish Lines
Embryos and fry were obtained by natural spawning from wild-type,
MZoep (Gritsman et al., 1999), foxD3:GFP (Gilmour et al., 2002),
flh:eGFPu1 (see below), and flh:eGFPu1 ⫻ foxD3:GFP fish. ntl morphants (Feldman and Stemple, 2001) and LZoep⫺/⫺ fish (Concha et
al., 2000) were obtained as previously described.
The flh gene was characterized from two zebrafish flh genomic
clones, 1A and 9A, kindly provided by Will Talbot (Stanford University). These clones were isolated by screening a genomic library
made in DASH-II (Stratagene) constructed by Martin Petkovich
(Queen’s University, Kingston, Ontario, Canada) with the zebrafish
flh cDNA (Talbot et al., 1995). Clones were restriction mapped and
subcloned into pBluescript2SK⫹ (Stratagene). DNA sequence was
Neuron
436
derived by primer walking using BigDye terminators (Applied Biosystems) from a 5.5 kb EcoRI subclone of 9A encompassing 1.6 kb of
sequence upstream of the putative TATA box, three exons containing the entire coding region, and 1.9 kb of sequence downstream
of the stop codon. At least three reads per base with at least one
read per strand were performed. The genomic sequence of the
zebrafish flh gene can be obtained from GenBank (accession number AY214391).
In order to generate flh:eGFP-transgenic fish, a membrane tethered eGFP reporter (gap43eGFP, Clontech) was cloned into the
5⬘UTR of the 5.5 kb EcoRI flh subclone between the TATA box and
the initiation methionine. Fertilized eggs were injected with linearized 5.5-flh:eGFP plasmid DNA. This construct was sufficient to
recapitulate endogenous epithalamic flh expression in both Xenopus (M.G., L.S., K.G., E.A., and D.K., unpublished data) and zebrafish.
Injected embryos were raised, and pairwise crosses identified a fish
that passed the transgene onto its progeny. This fish was used to
establish a transgenic line.
Isolation of hest1
A clone with asymmetric expression in the habenular nuclei was
obtained through screening a variety of genes and ESTs for expression in sensory organs. The gene fragment has sequence homology
with asic (acid-sensing ion channel) receptor encoding genes, and,
until we are more certain of its orthology with related genes in other
species, we have named it habenular EST1 (hest1). Sequence of
the fragment is available from EMBL (accession number AJ558033).
Phenotypic Analysis
In situ hybridizations were performed using standard procedures.
For antibody labeling, mouse anti-acetylated ␣-tubulin (Sigma),
mouse anti-fluorescein (Boheringer), and rabbit anti-GFP (Chemokine) antibodies were used at 1:1000 dilutions. Light microscopy
images were acquired using a Polaroid DMC camera attached to a
Nikon upright microscope and analyzed using Photoshop 5.5
(Adobe) and Canvas 7 (Deneba) softwares. For quantitation of asymmetries determined by ␣-tubulin and hest1 labeling, only fry that
could be unequivocally scored are counted. Confocal analysis was
performed on a Leica TCS SP Confocal Microscope using 25⫻ oil
and 20⫻/40⫻ water immersion objectives. Three-dimensional series
of images were typically acquired at 1–2 ␮m intervals and the selected depths then projected by a combination of maximum intensity
and opacity rendering methods using Volocity (Improvison)
software.
Fate Mapping
flh:eGFP- or flh:eGFP;foxD3:GFP-transgenic embryos were injected
at the 1 cell stage with DMNB-caged Fluorescein Dextran (D-7146,
Molecular Probes), kept in the dark at 28⬚C until 22–24 hpf, anesthetized with 0.003% tricaine, and mounted on a custom-made chamber for experimentation. The region of photoactivation was determined relative to the flh:eGFP-expression domain using a 40⫻
Achroplan water immersion objective on an upright Axioplan Zeiss
microscope. For photoactivation of caged fluorescein, a UV-nitrogen laser microbeam (VSL-337, Laser Sciences) tuned to 365 ␩m
by means of the BPBD-365 dye (Exciton Inc.) was focused on the
selected region and laser pulses delivered at a frequency of 5–10
Hz for 10–20 s (Rohr and Concha, 2000). Embryos were kept in the
dark until 48 hpf and the position of the labeling within the epithalamus examined by anti-fluorescein immunostaining.
Laser Ablation
foxD3:GFP- or flh:eGFP;foxD3:GFP-transgenic embryos were prepared as for fate mapping. For laser ablation, a UV-nitrogen laser
microbeam (VSL-337, Laser Sciences) tuned to 440 ␩m by means
of the Coumarin-440 dye (Exciton Inc.) was focused on the selected
region and laser pulses delivered at a frequency of 10 Hz for 50–120
s. Ablation was performed in seven to ten consecutive neighboring
regions by controlling the displacement of a motorized XY stage
(Ludl) using custom-made routines written in Openlab (Improvision).
Ablation was confirmed directly under DIC optics and embryos kept
at 28⬚C until further examination.
DNA Electroporation
The electroporation technique was adapted from Haas et al. (2001)
to enable the efficient transfer of DNA to a small group of cells in
embryonic zebrafish CNS. ntl morphant foxD3:GFP embryos were
mounted in 1.5% low melting point agarose (Sigma). Using a surgical
blade (John Weiss), a small chamber of agarose was cut out to
expose an area of the dorsal diencephalon. Micropipettes with a
tip diameter of 1–2 ␮m were pulled on a P-87 Micropipette puller
(Sutter Instrument Company, CA) and filled with a solution containing purified plasmid DNA (Qiagen Maxiprep purification Kit) resuspended in dH2O. Electroporation was performed on the right
side of the epithalamus. Control embryos were electroporated with
0.5 ␮g/␮l ␣-tubulin GFP and 0.8 ␮g/␮l pCS2DsRed (final DNA concentration, 1.3 ␮g/␮l) and experimental embryos with 0.5 ␮g/␮l
␣-tubulin GFP and 0.8 ␮g/␮l lefty1 (final DNA concentration, 1.3 ␮g/
␮l). Coelectroporation of two plasmids from the same pipette results
in expression of both proteins in the same cells (⬎95% of cases; M.T.
and J.D.W.C., unpublished data). For exogenous DNA to generate
protein during the time of endogenous lefty1 expression (20–22 hpf),
we performed electroporation at 10–14 somites (electroporated GFP
constructs express the protein at a level that can be visualized
after 10 hr, and this persists for many days [data not shown]). The
following stimulation parameters were used: 1 s long trains of 1 ms
square pulses at 200 Hz and a voltage of 45 V. Trains were delivered
five times with 1 s interval between trains. Pulses were generated
with a Grass SD9 stimulator (Grass-Telefactor, West Warwick, RI).
After electroporation, embryos were cut out from the agarose and
returned to embryo medium to recover.
Acknowledgments
We thank Alex Schier for valuable suggestions for experiments;
members of our groups for discussion of the project; Carole Wilson
and her team for care of the fish; and Marnie Halpern for communication of results prior to publication. This work was supported by
grants from the Wellcome Trust, Biotechnology and Biological Sciences Research Council, and European Community (to S.W.W.);
support from the Wellcome Trust, Fondecyt (1020902), and Fundación Andes (C-13760) (to M.L.C.); from the Wellcome Trust (to M.G.
and E.A.); and from the Attempto Research Group program (to S.G.).
M.T. and J.D.W.C. are supported by the Medical Research Council.
S.W.W. is a Wellcome Trust Senior Research Fellow, and M.L.C.
is a Wellcome Trust International Research Development Award
Fellow.
Received: December 23, 2002
Revised: May 5, 2003
Accepted: June 20, 2003
Published: July 30, 2003
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