1 Carnegie Institution of Washington, Department of Embryology, 3520 San Martin
Drive, Baltimore, MD, 21218, USA
2 IGBMC, CNRS/INSERM/ULP, 1 rue Laurent Fries, BP10142, CU de Strasbourg, 67404
Illkirch cedex, France
Author for correspondence (e-mail:
halpern{at}ciwemb.edu)
Accepted 9 August 2005
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SUMMARY |
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Key words: KCTD gene family, leftover gene, Left-right asymmetry, Habenular nuclei, Fasciculus retroflexus, Interpeduncular nucleus, Diencephalon
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Introduction |
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In lower vertebrates, the epithalamus of the dorsal diencephalon displays
notable asymmetries (Concha and Wilson,
2001; Halpern et al.,
2003
). The epithalamus includes the pineal complex, which in many
fish consists of the pineal organ and an asymmetrically positioned accessory
organ termed the parapineal (Borg et al.,
1983
). Both the pineal and parapineal transcribe genes encoding
melatonin biosynthetic enzymes in a circadian-regulated manner
(Gamse et al., 2003
;
Gothilf et al., 1999
), but the
specific role of the parapineal is unknown. The bilateral habenular nuclei,
which flank the pineal complex, can differ in size and structure
(Harris et al., 1996
;
Concha and Wilson, 2001
). In
some amphibians, the left habenula contains two morphologically distinct
subdomains whereas there is only a single nucleus on the right
(Wehrmaker, 1969
;
Braitenberg and Kemali, 1970
;
Morgan et al., 1973
). The
degree of habenular asymmetry can vary seasonally in frogs, presumably
correlated with the mating period (Kemali
et al., 1990
). In chickens, L-R habenular differences are
sex-dependent and influenced by hormonal levels
(Gurusinghe and Ehrlich, 1985
;
Gurusinghe et al., 1986
).
The habenulae relay impulses from limbic areas of the telencephalon to an
unpaired midbrain nucleus, the interpeduncular nucleus (IPN) via the
fasciculus retroflexus (FR) nerve bundle (of Meynert), in a conduction system
that is conserved evolutionarily
(Sutherland, 1982). The
function of the habenulointerpeduncular connection is poorly understood,
although there is evidence from rodents that it modulates complex behaviors
such as avoidance, reward and feeding
(Sutherland, 1982
).
Progress in determining how epithalamic asymmetry arises and the molecular
pathways involved has come from studies in zebrafish. cyclops
(cyc), which encodes a zebrafish Nodal-related Tgfß signal
(Rebagliati et al., 1998a;
Rebagliati et al., 1988b; Sampath et al.,
1998
), and other genes functioning in this signaling pathway, are
expressed transiently on the left side of the embryonic pineal anlage
(Bisgrove et al., 2000
;
Concha et al., 2000
;
Liang et al., 2000
). Nodal
signals are known to mediate laterality of the heart and visceral organs in
vertebrate embryos and play an important role in gastrulation
(Schier, 2003
). Zebrafish
mutants for one-eyed pinhead (oep), which encodes an
obligatory component of the Nodal receptor complex, can be rescued past the
early requirement for Nodal signaling by injection of oep RNA
(Yan et al., 1999
;
Zhang et al., 1998
). Rescued
embryos (Roep) lack asymmetric gene expression in the embryonic
pineal but develop to adulthood, thus allowing Nodal pathway function in the
brain to be assessed (Concha et al.,
2000
; Liang et al.,
2000
). In Roep fish the directionality of L-R differences
in diencephalic anatomy is randomized
(Concha et al., 2000
;
Liang et al., 2000
;
Gamse et al., 2002
). The
parapineal, located to the left of the pineal anlage in >95% of wild-type
(WT) larvae, develops to the right in approximately half of Roep
larvae and the asymmetric properties of the habenular nuclei, which include
differences in size, neuropil density and gene expression, are L-R reversed.
For example, the leftover (lov) gene is typically
transcribed by more cells of the left habenula than the right; however, half
of Roep larvae show the opposite pattern
(Gamse et al., 2003
).
Parapineal L-R position always corresponds with the direction of habenular
laterality. Moreover, following parapineal ablation, the habenulae fail to
develop asymmetrically (Concha et al.,
2003
; Gamse et al.,
2003
).
Even earlier in development, signaling by another Nodal-related factor,
Southpaw (Spaw), influences asymmetric gene expression in the zebrafish
diencephalon. spaw is the earliest known gene to be expressed
unilaterally, with transcripts appearing in the left lateral plate mesoderm
(LPM) by the 10-12 somite stage (Long et
al., 2003). However, expression is not detected in the embryonic
brain. Unlike the zebrafish Nodal-related signals, Cyc and Squint (Sqt), that
mediate tissue specification in the early embryo
(Hatta et al., 1991
;
Feldman et al., 1998
), Spaw
appears to regulate the L-R axis specifically. Embryos deficient for Spaw lack
cyc, pitx2 and lft1 expression in the left diencephalon
(Long et al., 2003
), and
thereby affect morphological asymmetry of the epithalamus. The zebrafish
studies suggest that brain laterality results from a cascade of developmental
events that leads from left-sided Nodal signaling in the pineal anlage to L-R
assignment of parapineal position, which, in turn, directs habenular
asymmetry.
Here, we provide additional evidence for molecular L-R differences in the zebrafish habenular nuclei, which extends to their efferent projections and innervation of the midbrain target, the IPN. Two genes related to lov, right on (ron) and dexter (dex), are also expressed asymmetrically in the habenular nuclei but to a greater extent on the right than the left. Consequently, Lov and Ron proteins are distributed differently in habenular subdomains and in efferent axons within the FR on the left and right sides of the larval brain. Differential dye-labeling of adult habenulae, or visualization of Lov+ and Ron+ immunoreactive axons, demonstrates that L-R habenular efferents project to different regions along the dorsoventral (DV) axis of the IPN. Reversal of habenular laterality by perturbation of Nodal signaling also reverses the L-R origin of projections onto the IPN. Ablation of the parapineal disrupts laterality and the stereotypic DV pattern of IPN connectivity. The results demonstrate that L-R differences of the dorsal forebrain influence the connections and molecular properties of innervating axons at the midbrain target.
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Materials and methods |
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Morpholino injections
Antisense spaw morpholino oligonucleotides (MO) were derived from
the splice acceptor site of the last intron (Spaw-MO2)
(Long et al., 2003). MO stock
solution (10 mg/ml) was diluted (6 ng/nl in dH2O) and
Tg(foxd3:GFP) embryos (1- to 2-cell stage) were pressure injected
with 1 nl to deliver 6 ng of MO. Larvae were presorted according to whether
the GFP+ parapineal was situated to the left or right of the pineal
anlage. Over 90% of MO-injected embryos hatched and, at 4 days, were used for
whole-mount in situ hybridization or antibody labeling with anti-acetylated
tubulin antibody (Concha et al.,
2000
; Gamse et al.,
2003
). Others were raised to adulthood for brain dissection and
hodological analysis.
Dye labeling of the habenulae
After anesthetization with tricaine (170 µg/ml), 2- to 3-month-old
adults (AB) were decapitated with a razor. Brains were dissected out in cold
phosphate-buffered saline (PBS), pinned dorsal side upwards on Sylgard
(Molecular Probes)-coated dishes, and fixed in 4% paraformaldehyde for at
least 24 hours at 4°C. Lipophilic dyes FAST DiI and FAST DiO (Molecular
Probes) were dissolved in dimethylformamide at 50 mg/ml by heating at 50°C
for 5 to 10 minutes. Aliquots of the dye solutions were stored at
80°C, thawed and briefly heated at 42°C before backloading into
glass needles. Prior to dye application, the prominent commissure that extends
between the left and right habenulae was severed with a tungsten needle to
prevent dye passage. Each habenula was impaled and dye applied using a
pressure injector (Aizawa et al.,
2005). Labeled brains were stored in 4% paraformaldehyde at
28°C for 18-21 days because of the distance the lipophilic dyes must
travel along the habenula FR projections to reach the IPN. Dye passage was
monitored under a Leica MZFLIII stereomicroscope. Labeled brains were embedded
in low-melting temperature agarose (4%) in plastic molds for transverse
vibratome (Leica VT1000S) sectioning. Sections (100-150 µm) were mounted on
glass slides in cold Mowiol (Calbiochem) medium (480 mg/ml Mowiol in 25%
glycerol with 0.1 M Tris, pH 8.5) and imaged with a Leica MZFLIII or SP2
confocal microscope.
Identification of leftover-related genes
Individual clones from an adult zebrafish kidney cDNA library containing
inserts between EcoRI and XhoI sites of pBK-CMV (Stratagene)
were assayed for expression during embryonic stages by whole-mount in situ
hybridization. A partial ron cDNA was isolated and the transcript
5' end identified by RNA-ligase RT-PCR (RLM-RACE Kit, Ambion) using
total RNA from 4-day larvae. Scanning of the zebrafish genome database
(Zv3;http://www.sanger.ac.uk/Projects/D_rerio/)
for sequences similar to lov and ron, yielded
dexter (dex) and homologues of KCTD12b, 16a and
16b. Primers specific for dex, kctd12b, 16a and 16b
sequences were used to amplify the open reading frame by RT-PCR (Retroscript
Kit, Ambion), products were subcloned into pCRII (Invitrogen), and confirmed
by sequencing. kctd16a, kctd12b and dex map to the same arm
of linkage group 14.
RNA in situ hybridization
Protocols were followed as in Gamse et al.
(Gamse et al., 2002), using
reagents from Roche Molecular Biochemicals. To synthesize antisense
digoxigenin or fluorescein RNA probes, insulin
(Stafford and Prince, 2002
),
pBS-otx5 and pBK-CMV-lov were linearized with EcoRI and transcribed
with T7 RNA polymerase, pBK-CMV-ron with BamHI and T7 RNA polymerase,
pCRII-KCTD16a and pCRII-KCTD12b with SpeI and T7 RNA polymerase, and
pCRII-KCTD16b and pCRII-dex with XhoI and SP6 RNA polymerase.
Hybridized probes were detected using alkaline phosphatase-conjugated
antibodies and visualized by 4-nitro blue tetrazolium (NBT) and
5-bromo-4-chloro-3-indolyl-phosphate (BCIP) staining for single labeling, or
NBT/BCIP followed by iodonitrotetrazolium (INT) and BCIP staining for double
labeling. Larvae were embedded in 4% low-melt agarose (Cambrex Rockland, ME)
for vibratome sectioning (100-150 µm), or in London Resin Gold (Ted Pella,
Redding, CA) and sectioned using a Reichert ultramicrotome (8-10 µm)
followed by Basic Fuchsin (Sigma) counterstaining.
Lov and Ron antibodies
Partial lov and ron open reading frames were subcloned
into pDEST17 (Invitrogen), which adds a 6-histidine tag to the amino (N)
terminus. Tagged protein was not recovered at high levels from bacterial cells
unless the T1 domain was also deleted. T1-deleted constructs were expressed in
BL21(DE3)pLysS bacteria (Stratagene), the protein purified on nickel-NTA
columns (Qiagen) and injected into rabbits (Spring Valley Laboratories) to
produce polyclonal antibodies against Lov or Ron. Resultant sera were purified
using HiTrap affinity columns (Amersham). Specificity was confirmed by western
blotting using standard methods (Gallagher
et al., 1989) and by immunofluorescence in embryos injected with
lov or ron morpholinos.
Immunofluorescence
Four-day larvae fixed overnight in 4% paraformaldehyde were stored in
methanol at 20°C for up to 2 weeks. Following rehydration through a
methanol/PBS series, samples were permeabilized with proteinase K (10
µg/ml; Roche) for 30 minutes, refixed, blocked in PBS/0.1%TritonX-100/10%
sheep serum (PBSTS), and incubated overnight in primary antibody diluted 1:500
in PBSTS. Primary antibody was detected using goat-anti-rabbit:Cy3 (Jackson
Immunoresearch) or goat-anti-rabbit:Alexa Fluor 488 (Molecular Probes). For
double labeling with Lov and Ron antibodies, the first primary was detected
with goat-anti-rabbit Fab fragment:Rhodamine (Jackson Immunoresearch), then
blocked with unlabeled goat-anti-rabbit antibody. The protocol for the second
primary was the same as single labeling. Larvae were mounted in glycerol and
images collected on a Leica SP2 confocal microscope. For vibratome sectioning,
brains of adult fish derived from spaw MO-injected embryos or WT
controls were dissected, fixed overnight at 4°C and embedded as above.
Sections were arranged on precleaned Superfrost slides (VWR), dried briefly
and double labeled with Lov and Ron antibody.
Laser-mediated cell ablation
Parapineal ablation was performed using
Tg(flh:GFP)c161, Tg(flh:GFP)c162 or
Tg(flh:GFP)c161/+;Tg(foxd3:GFP)/+ 28- to
32-hour-old embryos, mounted dorsal side up in 1.2% agar on glass slides. The
parapineal was visualized by GFP fluorescence and approximately 15-20 cells
ablated by 5-10 pulses/cell from a 440 nm laser beam (Photonic Instruments)
focused through a 40x water immersion objective mounted on a Zeiss
Axiophot microscope. An equivalent number of cells contralateral to the
parapineal were ablated in controls.
Accession numbers
GenBank accession numbers: KCTD16a, AY763407; KCTD12b,
AY763408; KCTD16b, AY763409; dex, AY763410; ron,
AY763411.
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Results |
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Upon subsequent screening, another gene, right on (ron), was found to show asymmetric expression in the habenulae. Remarkably, ron shares a high degree of sequence homology with lov, particularly in two discrete domains of the predicted protein coding sequence (Fig. 1A). Scanning of available zebrafish genomic sequence and degenerate PCR yielded four additional homologous genes (Fig. 1A). Only one, dexter (dex), is expressed in the habenulae (see below). In 4-day larvae, ron is also expressed in the gall bladder and posterior border of the optic tectum, and dex in paired groups of cells in the ventral diencephalon (data not shown).
Related genes from mammals were grouped as the potassium channel
tetramerization domain containing (KCTD) gene family
(Marchler-Bauer et al., 2003).
Structurally, KCTD proteins contain a N-terminal sequence homologous to the T1
tetramerization domain of the Shaker class of voltage-gated potassium channels
(Papazian, 1999
). However,
they have no other features of channel proteins, such as transmembrane
domains. Phylogenetic comparison with human and mouse genes reveals that the
zebrafish proteins (Fig. 1B,
boxed) fall into four subclasses of the larger KCTD family: Kctd16a/b;
Kctd12b; Lov/Ron/Kctd12/Pfetin and Dex/Kctd8. The zebrafish kctd12b,
16a and 16b genes are strongly expressed in the retina.
Additionally, kctd16a is expressed in the dorsal forebrain and optic
tectum and kctd16b in olfactory epithelium (data not shown).
In humans, KCTD12/PFET expression was detected in the fetal
cochlea (Resendes et al.,
2004), a structure absent in the zebrafish
(Whitfield et al., 2002
). We
found that Kcdt12/Pfet is also expressed in the medial habenulae of
the fetal mouse brain and Kcdt8 transcripts specifically localize to
the medial habenulae of the adult rat brain (Fig. S1 in supplementary
material).
Left-right differences in habenular gene expression patterns
Zebrafish ron and dex are expressed asymmetrically in the
habenular nuclei; however, in contrast to lov, they are transcribed
in more cells of the right habenula than the left. Expression, which is
asymmetric from the outset, is first detected at 2 days
(Fig. 1C,G), and increases
significantly by 4 days (Fig.
1D,H). Parasagittal sections reveal that ron and
dex are expressed in the dorsal region of the right but not the left
habenula (compare Fig. 1E and I with F and
J). Expression of dex extends more laterally in both
habenulae than expression of ron
(Fig. 1D,H).
Double label in situ hybridization allows further refinement of habenular gene expression subdomains (Fig. 1K-N, and not shown). Only cells in the dorsal region of the left habenula express lov, while those in the ventral region express ron and dex. In contrast, cells in both dorsal and ventral regions of the right habenula express lov, ron and dex. All three genes are transcribed in medial and posterior regions of the right habenula, but only ron and dex expression is detected in anterior and lateral regions.
Molecular L-R specialization of habenulointerpeduncular connections
To determine whether asymmetric gene expression leads to L-R differences in
protein distribution, and to evaluate subcellular localization, we generated
polyclonal antibodies against Lov and Ron. Rabbit sera reacted specifically
against each protein on western blots and in Lov or Ron MO-depleted embryos
(Fig. S2 in supplementary material).
Confocal microscopy of Lov and Ron immunolabeled larvae demonstrated that protein levels differ between the left and right habenulae in a pattern closely reflecting mRNA distribution (Fig. 2A-C). Although transcripts were restricted to the cell bodies of habenular neurons, protein was also detected within ventrocaudally projecting axons that course through the FR, and in synaptic terminals at the midbrain target, the interpeduncular nucleus. Lov+ growth cones reach the IPN by 2 days (Fig. 2D). By 4 days, habenulointerpeduncular connections are well formed: the Lov+ axonal bundle is larger within the left FR than the right, and Lov+ (Fig. 2E,F) and Ron+ (Fig. 2G) habenular efferents project extensively along the IPN. Innervation of the target was distinguished more clearly in lateral views of the larval brain. Lov+ axons project to the dorsal and ventral IPN (Fig. 2I), while most, if not all, Ron+ axons project to the ventral IPN (Fig. 2J). Double labeling confirmed that Lov+ axons traverse the dorsal and ventral regions of the midbrain target and Ron+ axons are confined ventrally (Fig. 2K).
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In agreement with transcriptional patterns, the asymmetric distribution of Lov and Ron proteins in the habenulae and FR was similar to WT in half of Roep larvae (n=39, Fig. 3G,I). The other half (n=31) showed a L-R reversal in protein levels (Fig. 3H,J) that extended to habenular efferents within the FR.
We examined whether altered habenular laterality affected innervation along the DV axis of the IPN. Lov+ efferents project to the dorsal and ventral IPN, while Ron+ fibers innervate the ventral IPN in Roep larvae that have a left-positioned parapineal (Fig. 3K) and those that are L-R reversed, with the parapineal on the right (Fig. 3L). In such larvae, however, input to the IPN shows a mirror image reversal (Fig. 3H). Thus, when the L-R origin of habenular efferents is opposite that of WT, the DV pattern of immunoreactive connections to the IPN is still preserved.
Southpaw regulates directional asymmetry of habenulointerpeduncular projections
Targeted depletion of Spaw in zebrafish embryos was previously shown to
alter laterality of the heart and pancreas and prevent expression of Nodal
pathway genes in the left LPM and diencephalon
(Long et al., 2003). Because
disruption of diencephalic gene expression causes L-R randomization of
epithalamic asymmetry (Liang et al.,
2000
; Gamse et al.,
2003
; Concha et al.,
2000
; Concha et al.,
2003
), lack of Spaw should also affect directional asymmetry of
the pineal complex and habenular nuclei.
spaw MO was injected into embryos bearing the foxD3:GFP
transgene (Gilmour et al.,
2002), which is highly expressed in the pineal complex at 2-3 days
(Concha et al., 2003
) and
allows unambiguous assignment of the L-R position of the parapineal relative
to the midline pineal, throughout larval stages (Fig. S3A,B in supplementary
material). The direction of habenular asymmetry was determined at 4 days by
assessing L-R differences in neuropil density (Fig. S3C,D in supplementary
material) or lov expression (Fig. S3E,F in supplementary material).
As summarized in Table 1, Tg(foxD3:GFP) controls did not show reversals in habenular laterality
(n=300). However, almost half of larvae derived from spaw
MO-injected embryos had L-R reversals in parapineal position and habenular
asymmetry (45%, n=418). A smaller number (3%) exhibited bilaterally
symmetric lov expression in the habenulae. These findings confirm
that signaling by Spaw regulates directional asymmetry of the developing
epithalamus.
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Despite alterations in the L-R axis and presumptive heterotaxia in a significant fraction of the population, the majority of Spaw-depleted embryos hatched, developed swim bladders (>90%) and survived to adulthood (>60%). Injection of spaw MO into foxD3:GFP embryos was therefore a useful method for generating larvae with L-R randomized diencephalic asymmetry that could be screened on the basis of a left-sided or right-sided parapineal, separated, and raised to adulthood. This allowed habenular projections to be traced in the brains of adult fish that had a pre-determined parapineal position.
|
In adult fish derived from spaw MO-injected embryos, the brains from those that had formed the parapineal on the left side (n=4) (Fig. 5D-F) showed an IPN innervation pattern similar to WT. Brains with the parapineal on the right (n=4) exhibited a reversed pattern, in which efferents from the left habenula (green) projected solely to the ventral region of the IPN and those from the right habenula projected along the entire DV axis (Fig. 5G-I). Similar to Roep larvae, the overall DV pattern of efferents was preserved in adult brains even when the habenula of origin was L-R reversed.
The adult projection pattern onto the IPN was invariant in WT fish and closely paralleled by the distribution of Lov and Ron protein in habenular axons. Lov+ immunoreactive habenular efferents coursed throughout the DV extent of the adult IPN, while Ron+ axons were confined to the ventral region (Fig. 6A-C, n=3). This DV pattern of immunolabeled habenular projections was unaltered in adults derived from spaw MO-injected embryos (n=6), irrespective of the direction of epithalamic laterality (compare Fig. 6D-F and G-I).
Habenular asymmetry directs IPN connectivity
Prior studies demonstrated that habenular asymmetry is dependent on the
parapineal (Concha et al.,
2003; Gamse et al.,
2003
). Following parapineal ablation, the left habenula fails to
adopt its characteristic properties, such as a larger size, expanded dense
neuropil and increased lov expression relative to the right habenula.
We examined whether the parapineal also influences properties associated with
the right habenula by assaying ron and dex expression in
larvae lacking the parapineal.
|
In parapineal-ablated larvae, Lov protein levels were reduced and Ron+ domains expanded in the left habenula, relative to control-ablated larvae (compare Fig. 7G,H and I,J). Fewer Lov+ axons projected within the left FR, whose immunolabeling now resembled that of the right FR. In contrast to L-R reversal of parapineal position, ablation of the parapineal disrupted the DV distribution of habenular efferents onto the IPN. The two Lov+ domains normally observed at the dorsal IPN of control-ablated larvae (asterisk and arrowhead, Fig. 7K) were reduced to one small anterior domain (asterisk in Fig. 7L) and an increase in immunofluorescence was detected at the ventral IPN (Fig. 7L). These results are consistent with left habenular efferents adopting an IPN projection pattern more characteristic of those derived from the right habenula.
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Discussion |
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In the present study, we have shown that the zebrafish lov-related
gene family includes three members, lov, ron and dex that
are expressed differently by the left and right habenulae. By comparing their
patterns, six asymmetric subdomains could be assigned to the left and right
habenulae (designated i-vi in Fig.
8A). One distinctive feature is that the left habenula exhibits DV
compartmentalization in gene expression not observed for the right.
Subdivision of the right habenula is largely along the anteroposterior axis.
DV regionalization of the left habenula has been noted in other lower
vertebrates. A lateral subnucleus occupies most of the dorsal left habenula in
the stingray brain, but is restricted to a smaller medial posterior region on
the right (Iwahori et al.,
1991a; Iwahori et al.,
1991b
). There is also a dorsoventral difference in the
distribution of the calcium-binding protein calretinin A in the left habenula
of Rana esculenta (Guglielmotti
et al., 2004
). The significance of asymmetry and the relationship
of gene expression subdomains with afferent input, habenular ultrastructure,
and connectivity are important issues to pursue. For example, it is known that
the parapineal selectively innervates the left habenula in several fish
species (see Concha and Wilson,
2001
) and may connect with neurons within a specific
subdomain.
Determining the purpose of molecular specialization of the zebrafish
habenulae will require a greater understanding of the functions of Lov-related
proteins. The conserved N-terminal T1 domain, a protein-protein interaction
motif that promotes oligomerization
(Collins et al., 2001), could
provide useful clues. In the Shaker family of voltage-gated potassium
channels, it is required for tetramerization of alpha subunits into a
functional channel and for axonal localization
(Gu et al., 2003
;
Li et al., 1992
). Preliminary
data from yeast two-hybrid assays suggest that zebrafish Lov and Ron proteins
form homophilic and heterophilic dimers, dependent on the presence of the T1
domain (J.T.G., unpublished observations). Since the T1 domain of Lov-related
KCTD proteins is conserved with that of voltage-gated K+ channel
subunits [e.g. approximately 50% amino acid identity between zebrafish Lov and
Drosophila Shaker (Gamse et al.,
2003
)], these proteins might interact and thereby modulate channel
assembly or activity. Such a role was recently proposed for KCNRG
(K+ channel regulator encoding gene), which encodes a protein
structurally similar to Lov-related proteins
(Ivanov et al., 2003
). Another
potential function is in modulating neuronal responses to secreted signals
from other cells. KCTD11/REN represses signaling by the secreted protein
Hedgehog by preventing the downstream transcription factor GLI from entering
the nucleus (Di Marcotullio et al.,
2004
). Two-hybrid library screening, immunoprecipitation and MO
depletion should shed more light on the cellular functions of zebrafish
Lov-related proteins.
|
The discovery of molecular asymmetry in the zebrafish habenulae
(Gamse et al., 2003) suggested
that L-R differences could extend to habenular efferents and influence target
recognition. By selective labeling with lipophilic dyes, we corroborated the
recent findings of Aizawa et al. (Aizawa et
al., 2005
) that the left and right habenular axons project along
different DV extents of the IPN. Those authors concluded that there is a
"topographic projection of left-sided habenular axons to the dorsal
region of the IPN and of the right-sided habenular axons to the ventral
IPN." While our data support the projection of axons from the L and R
medial habenulae to topographically different domains along the DV axis of the
zebrafish IPN, we find that there is substantial overlap in L and R
projections within the ventral region. Thus, the assertion that left and right
information is laterotopically represented onto discrete dorsal and ventral
regions of the target nucleus (Aizawa et
al., 2005
) is an oversimplification of the pattern of connectivity
documented in our experiments.
Furthermore, the finding that left habenular neurons project along the entire DV extent of the IPN and right habenular neurons project predominantly to the ventral IPN is strongly supported by their molecular specificity. Anti-Lov and anti-Ron sera label different DV domains of the IPN, domains that closely resemble the pattern of left and right habenular projections, respectively. Lov+ efferents are found throughout the DV extent of the IPN, while Ron+ efferents are confined to the ventral region.
Directional asymmetry of the epithalamus influences target recognition
Our results support a role for Nodal signaling in setting the direction of
asymmetry in the zebrafish epithalamus that extends to epithalamic
projections. In Roep larvae, the ability to respond to Nodal
signaling is partially restored but is insufficient to direct brain
laterality. Therefore, at a population level, the direction of brain asymmetry
is L-R randomized. However, even in individuals that show a mirror image
reversal of habenular asymmetry and in the L-R pattern of immunoreactive
Lov+ and Ron+ habenular efferents, DV connections onto
the IPN appear unaffected (Fig.
8C). Parallel studies on adult zebrafish that lacked the Nodal
signal Southpaw as embryos also indicate that reversal of habenular asymmetry
changes the L-R origin of inputs to the IPN, but not DV innervation of the
IPN. Therefore, development of the midbrain target and its putative DV
guidance cues appear intact, suggesting that the IPN is not directly modified
by Nodal signals.
Habenular asymmetry determines the dorsoventral pattern of IPN connectivity
In contrast to genetically altered larvae, where global or early-acting
effects of Nodal activity on the developing neural tube cannot be completely
ruled out, selective ablation of the parapineal provides a rigorous test of
the correlation between habenular laterality and IPN connectivity.
After parapineal ablation, the left habenula showed increased expression of
ron and dex and reduced expression of lov, a
molecular profile more characteristic of the right habenula. Thus, the
parapineal normally functions not only to promote the acquisition of
left-specific gene expression (Gamse et
al., 2003), but to repress right-specific gene expression in the
adjacent left habenula.
Loss of the parapineal also affects habenular connections. Lov+ axons innervating the dorsal IPN were reduced while Ron immunoreactivity increased in the ventral IPN, consistent with left habenular neurons adopting the projection pattern of right habenular neurons (Fig. 8D). However, this transformation may not be complete because some Lov+ projections persisted in the anterior region of the dorsal IPN. Dye labeling of larval or adult brains derived from parapineal-ablated embryos should resolve the L-R origin of the Lov+ axonal endings that remain at the dorsal IPN.
Parapineal ablation causes a local perturbation of diencephalic asymmetry
that is not expected to affect the properties of the midbrain target directly,
such as the expression of attractive or repulsive guidance cues. Therefore, we
conclude that laterality of the habenular nuclei influences IPN connectivity
by altering the molecular properties of habenular axons and presumably their
choice of which target subdomains to innervate. How efferents from the right
habenula are targeted to only the ventral IPN domain avoiding the dorsal one,
while left habenula efferents innervate both, is a problem that will require
more information about the types and distribution of axon guidance molecules
at the IPN and neighboring brain regions. Members of the neuropilin
gene family and the netrin 1 receptor Dcc (Deleted in colorectal cancer) are
strongly expressed in the habenular nuclei of mouse embryos
(Funato et al., 2000;
Shu et al., 2000
). Repulsive
Semaphorin 3F (Sema3F) and attractive Netrin 1 signals have been implicated in
directing the ventroposterior outgrowth of habenular efferents along the
diencephalic neuromere boundary (Funato et
al., 2000
). Moreover, mutations of neuropilin 2 or
Sema3F lead to defasciculation of the FR
(Giger et al., 2000
;
Sahay et al., 2003
). Although
the studies in mice provide information about FR navigation toward the
midbrain, little is known about the nature of guidance cues habenular axons
receive at the IPN. With the aid of GFP transgenes that highlight habenular
projections (Parinov et al.,
2004
), forward genetic screens in zebrafish may identify such
cues.
The demonstration that directional asymmetry in one region of the brain
guides connectivity in a distant region has interesting implications for
deciphering the origin of laterality in the mammalian cortex. Further
exploration into the generation of L-R connectivity differences could also
prove relevant for understanding human neurological disorders that have been
associated with abnormal neuroanatomical asymmetry
(Bruder, 2003;
Green et al., 2003
).
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
Supplementary material available online at http://dev.biologists.org/cgi/content/full/132/21/4869/DC1
* Present address: Vanderbilt University, Department of Biological Sciences,
VU Station B, Box 35-1634, Nashville TN 37235-1634, USA
These authors contributed equally to this work
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