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First published online January 25, 2006
doi: 10.1242/10.1242/dev.02244
1 Biology Department, University of Massachusetts, Amherst, MA 01003-9297,
USA.
2 ETH Zurich and Brain Research Institute, University Zurich, Switzerland.
3 Department of Developmental Biology, University Freiburg, Freiburg,
Germany.
* Corresponding author (e-mail: Karlstrom{at}bio.umass.edu)
Accepted 19 December 2005
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SUMMARY |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: AC, Chiasm, Commissure, Ephrin, Gfap, Glial bridge, Morpholino, Netrin, POC, Semaphorin, Slit
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INTRODUCTION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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), whereas the AC forms as lateral telencephalic neurons
extend axons anteriorly across the midline of the ventral/anterior
telencephalon (reviewed by Chitnis and
Kuwada, 1990; Wilson et al.,
1990). Soon after the formation of these forebrain commissures,
retinal ganglion cell (RGC) axons from the eye cross the midline of the
diencephalon near the POC to form the optic nerve, optic chiasm and optic
tract (Burrill and Easter,
1995). In monocular organisms such as zebrafish, all retinal axons
cross the ventral midline, whereas in binocular organisms some retinal axons
turn ipsilaterally at the midline and innervate ipsilateral targets in the
brain (reviewed by Williams et al.,
2004). In humans, congenital defects in chiasm formation lead to
visual defects, including nystagmus and loss of depth perception
(Apkarian and Bour, 2001).
The cellular and molecular cues that guide midline-crossing axons in the
forebrain are poorly understood. Glial cells may provide the cellular
substrate for midline crossing axons and help establish the position of
commissures, as glial structures have been found associated with many brain
commissures, including the corpus callosum
(Shu et al., 2003), chiasm
(Marcus et al., 1995), AC and
POC (Barresi et al., 2005).
This role in guiding commissural axons may be conserved through evolution, as
midline glial cells are known to help guide axons toward the midline in the
Drosophila CNS (Chotard and
Salecker, 2004; Hidalgo and
Booth, 2000).
Compared with these cellular cues, more is known about the molecular
guidance cues that influence commissure formation. In the vertebrate spinal
cord, a combination of attractive and repulsive cues regulates commissural
axon crossing (Dickson, 2002).
Netrin and Sonic hedgehog (Shh) act as attractants for spinal commissural
axons (Salinas, 2003), whereas
Slit molecules prevent non-commissural axons from crossing the midline
(Brose et al., 1999). In the
vertebrate forebrain, netrin expression in the telencephalon is
consistent with a possible role in AC formation; however, the lack of
netrin expression in the diencephalon suggests it does not play a
role in POC or chiasm formation
(Lauderdale et al., 1997).
Slit genes are expressed in bands across the midline of both the telencephalon
and diencephalon, and these proteins act to channel Robo-expressing retinal
axons during chiasm formation (reviewed by
Rasband et al., 2003;
Richards, 2002). This
repellent function also helps to position glial cells and axons in the POC
region (Barresi et al., 2005).
The transcription factor Zic2 is expressed in a subset of retinal axons that
grow ipsilaterally in binocular organisms, and Zic2 may regulate EphB1
receptors that receive the repulsive EphrinB2 cues from the midline, thus
preventing these axons from crossing the midline
(Herrera et al., 2003).
The precise expression of axon guidance cues in the eye and forebrain is
dependent upon complex cellular differentiation events that lead to an
exquisitely patterned neural tube. A large number of transcription factors
interact to help pattern the forebrain
(Dodd et al., 1988;
Herrera et al., 2004;
Shimamura et al., 1997). These
include several members of the Lim-Homeodomain (LHD) transcription factor
family that are involved in the related processes of neural patterning, cell
fate determination and axon pathfinding (reviewed by
Sockanathan, 2003). Among
these, Lhx2 is required for mouse forebrain patterning and eye formation, with
Lhx2 knock-out (KO) mice having a highly reduced telencephalon and no
eyes (Porter et al., 1997).
Although the telencephalic and eye phenotypes in Lhx2 KO are well
documented, little information is available about the role of Lhx2 in
forebrain axon guidance or in the formation of the diencephalon, a region
where it is also strongly expressed.
Here, we show that the zebrafish axon guidance mutant belladonna
(bel) encodes Lhx2. In bel mutants, both POC and RGC axons
fail to cross the midline of the forebrain and no optic chiasm forms.
bel(lhx2) mutant embryos have subtle eye defects, but have no other
morphological defects and can grow to adulthood
(Karlstrom et al., 1996).
bel(lhx2) mutants also have a reversed optokinetic response, similar
to defects in human achiasmats (Rick et
al., 2000). We show that bel(lhx2) mutants have subtle
forebrain patterning defects that are restricted to regions of the forebrain
where the AC, POC and optic chiasm form. Our detailed analysis of forebrain
defects in bel(lhx2) mutants indicates that disorganization of
midline glia and the misexpression of a subset of known axon guidance
molecules accompany retinal and commissural axon guidance defects. These
results demonstrate a role for bel(lhx2) in forebrain axon
guidance and in the patterning of the diencephalon and eye, and help to
characterize the guidance substrate for commissural and retinal axons in the
forebrain.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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), whereas belb700 was isolated in a
genetic screen for forebrain patterning mutations (Z.M.V., unpublished). For
genetic mapping, beltv42 heterozygotes in the Tü
background were crossed into polymorphic TL and WIK strains
(Rauch et al., 1997). Mutant
embryos were identified by axon guidance defects or visible eye defects at 5
dpf. PCR was performed on embryonic DNA using simple sequence repeat (SSR)
markers (Knapik et al., 1998).
Polymorphisms were visualized on 2.5% agarose gels or by single-strand
conformation polymorphism (SSCP) analysis
(Karlstrom et al., 1999).
In situ hybridization and immunohistochemistry
Embryos were maintained at 28.5°C and 0.003% 1-phenyl-2-thiourea (PTU)
was added to block melanin biosynthesis
(Westerfield, 1993). Embryos
were staged (Kimmel et al.,
1995) and ages designated as hours post fertilization (hpf) or
days post fertilization (dpf). In situ labeling was performed as previously
described (Karlstrom et al.,
1999). Probes used were: dlx2
(Akimenko et al., 1994),
erm (Munchberg et al.,
1999), fgf8
(Furthauer et al., 1997),
netrin1 (Lauderdale et al.,
1997), nk2.1b (Rohr
et al., 2001), nk2.2
(Barth and Wilson, 1995),
pax2.1 (Krauss et al.,
1991), sema3d
(Halloran et al., 1998),
slit2 (Yeo et al.,
2001), vax2
(Take-uchi et al., 2003) and
zic2.1 (Grinblat and Sive,
2001). Antisense lhx2 probes were generated against the
full-length cDNA cloned into the pCR4TOPO vector (see below).
Immunohistochemistry was performed as described for whole-mount embryos
(Karlstrom et al., 1999) and
frozen sections (Devoto et al.,
1996). Antibodies used were: ZN-5 (1:25) to label RGCs (University
of Oregon Monoclonal Antibody Facility)
(Laessing et al., 1994),
anti-acetylated tubulin (1:1000, Sigma) to label axons
(Wilson et al., 1990),
anti-GFAP (1:400) to label glial cells
(Nona et al., 1989), and
anti-phosphohistone H3 (pH3) (1:100, Sigma) to label mitotic cells
(Nechiporuk and Keating,
2002). Individual pH3-labeled cells were counted in the
telencephalon (dorsal to the optic recess and first ventricle) and preoptic
area of the diencephalon. Cell numbers were compared between wild type and
bel mutants using Student's paired t-test. Sections (7
µm) of 5 dpf larvae and adult eyes were embedded in epon/araldite
(Mollenhauer, 1964) and
counterstained with Toluidine Blue.
Positional cloning of the bel locus
The zebrafish CHORI-211 BAC library (RZPD, Germany) was screened by PCR,
using closely linked zMarkers, according to manufacturer's instructions. BAC
ends were sequenced directly or obtained from the Sanger zebrafish genome
database
(http://www.sanger.ac.uk/Projects/D_rerio/).
PCR primers were designed using these sequenced ends and the library was
re-screened until a BAC was identified that spanned the bel genetic
interval (zC142I8). Genescan analysis
(http://genes.mit.edu/GENSCAN.html)
of the zC142I8 sequence revealed a single coding sequence encoding Lhx2.
The lhx2 coding region was amplified from first-strand cDNA (Clonetech RT-PCR kit) using 5'UTR and 3'UTR PCR primers (lhx2.5Fw, 5'-GGGTTGCAGATCTGACGG-3'; lhx2.21Rv, 5'-GCAGTGGGTAAAATGATGG-3'). The gel-purified 1191 bp PCR product was cloned into the pCR4TOPO cloning vector (Invitrogen) and sequenced (GenBank Accession number 725255). The predicted Lhx2 protein sequence was compared with Lhx2 sequences from other species using ClustalW analysis (Biology Workbench, http://workbench.sdsc.edu/). To sequence the lhx2 gene in the two bel alleles, primers flanking each of the five lhx2 exons were used to amplify genomic DNA from beltv42 siblings, beltv42 mutants, belb700 siblings and belb700 mutants.
For genotyping bel carriers, fin clip or embryo DNA was amplified using allele specific primers. beltv42 genotyping primers (tv42.GT.Fw, 5'-GCTGCAACATAAGAGAG-3'; and tv42.GT.BsmAI.Rv, 5'-CTCAGACTCCAGGTTCAGTTTACAGTC-3') amplify a 248 bp fragment with the mutant sequence containing a restriction site for BsmAI. belb700 genotyping primers (lhx2.37Fw, 5'-CAATCACACGGATGTAGC-3'; and lhx2.18Rv, 5'-CAGTTAACCAGCAGCAAC-3') flank the 22 bp deletion. DNA fragments were resolved on 3.5% Metaphor (Cambrex) or 4% agarose (Sigma) gels.
Antisense oligonucleotide injections and cell transplantation
Phosphorothioated antisense oligonucleotides (S-oligos)
(Stenkamp and Frey, 2003) were
generated against three different regions in zebrafish lhx2 coding
sequence. S-oligo sequences were: CCTgtaggacgcgcttGGTg, GCAtgtgccattgcttGTCc,
and CGCctccaggcagaccGTGg (Sigma-Genosys). Capital letters signify bases joined
by thioester bonds. An lhx2 splice blocking morpholino (MO)
(GeneTools) (5'-CTTTTCTCCTACCGTCTCTGTTTCC-3'; 8-15 ng), an
unrelated mismatch control MO (10 ng), or a cocktail of the three S-oligos
(1-1.5 pg each) was injected into one- to two-cell embryos. Embryos were
incubated at 28.5°C until the desired time points, fixed in 4%
paraformaldehyde, and processed for in situ labeling.
For cell transplantation, wild-type or bel embryos were injected with rhodamine-dextran (2.5%) at the one- to two-cell stage and used as donors. Ten to 20 cells were transplanted from the donor animal pole at the dome stage into the same region of unlabeled wild-type or mutant hosts. Donor and host embryos were maintained as pairs in 24-well dishes in Danieau solution [1x Danieau: 58 mM NaCl, 0.7 mM KCl, 0.4 mM MgSO4, 0.6 mM Ca(NO3)2] containing penicillin and streptomycin (1% each). At the Prim-5 stage (24 hpf), donors and hosts were analyzed for dlx2 expression by in situ hybridization. Genotypes were determined by dlx2 expression or by PCR-based genotyping of tail tissue, as described above.
FGF signaling inhibition
To block Fgf signaling, embryos were treated with 20 µM SU5402
(Calbiochem) diluted in embryos raising medium for described time intervals at
28.5°C. Control embryos were treated with DMSO (SU5402 carrier). Embryos
were placed in 12-well plates (30 embryos per well) with 0.5 ml of medium.
After treatments, embryos were fixed and processed for in situ hybridization.
As a control for SU5402 efficacy, we examined expression of the Fgf-regulated
gene erm (Raible and Brand,
2001) in similarly treated embryos.
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RESULTS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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) (Fig. 1A,B).
To understand the nature and onset of bel axon guidance defects, we
examined retinal axon growth at earlier time-points. As retinal axons grew
toward the midline (32-36 hpf) two distinct phenotypes were seen in
bel mutants; retinal axons either projected ipsilaterally immediately
after leaving the eye [16/39 optic nerves (ONs) examined], or they projected
toward the midline (23/39 ONs; Fig.
1D,D inset). When examined at 38-48 hpf of development, RGC axons
failed to cross the midline in 88% of bel mutants (n=276
embryos; Fig. 1F) and almost no
midline growth was seen. Thus, in bel mutants RGC axons often
approached the midline at early stages of development, but subsequently axons
failed to cross the midline and instead projected ipsilaterally.
We next examined the formation of the forebrain commissures in bel
mutants (Chitnis and Kuwada,
1990; Wilson et al.,
1990). In 28 hpf wild-type embryos, axons from the nucleus of the
tract of the post-optic commissure (ntPOC) and axons of the nucleus of the
tract of the AC (ntAC) have crossed the midline to form the POC and the AC,
respectively (Fig. 1G)
(Bak and Fraser, 2003). In
bel mutants, both ntPOC and ntAC axons were present but failed to
extend axons across the midline and no forebrain commissures were formed
(Fig. 1H). All other major axon
pathways appeared to be normal in bel mutants. These include the
posterior commissure, hindbrain commissures, Mauthner cells, spinal cord
commissures and peripheral axons (data not shown). Thus mutation of
bel affects midline axon crossing only in the forebrain.
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A single nucleotide polymorphism (SNP) in the first intron of the predicted lhx2 sequence co-segregated with the bel locus (0 recombination events in 2500 meioses). We therefore sequenced the lhx2 gene in two bel alleles and found genetic lesions leading to severe protein truncations in both cases. In beltv42, a point mutation (C to A) in the second exon introduces a premature stop codon that would truncate the Lhx2 protein in the first LIM domain (Fig. 2E). In belb700, a 22 bp deletion in the third exon results in a frame-shift that leads to a stop codon after the second LIM domain (Fig. 2E).
To verify that lhx2 is the gene mutated in bel embryos, we used antisense thioester oligos (S-oligos) and a splice blocking morpholino (MOs) to reduce Lhx2 function in wild-type embryos. Injection of the MO or of a cocktail of three S-oligos (SO) led to reduced dlx2 (see Fig. 5A inset, Table 1) and sema3d (see Fig. 6D inset) expression in the ventral forebrain that was very similar to that seen in homozygous bel mutants. These results further support the idea that bel disrupts lhx2 and suggest that these two bel alleles result in a loss of Lhx2 function. These antisense injections also led to subtle and variable axon defects, including POC defasciculation and RGC guidance errors at the midline (data not shown). Ipsilateral projections were not seen, suggesting antisense injections may not reduce Lhx2 function as completely as do the bel mutations.
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), starting prior to the onset of lhx2
expression (6 hpf). This SU5402 treatment nearly eliminated lhx2
expression in the forebrain at 10 hpf, with expression remaining in only a few
ventral cells (Fig. 4A,B).
Treating embryos with SU5402 starting at 10 hpf led to a major reduction of
lhx2 expression at 24 hpf, showing a continued role for Fgf in the
expression of lhx2 (Fig.
4C,D). In contrast to these results, elimination of Hh signaling,
either in Shh pathway mutants or using the alkaloid Hh signal blocker CyA, had
little affect on lhx2 expression (data not shown). These results show
that Fgf signaling is required for both the onset and maintenance of
lhx2 gene expression.
zebrafish lhx2 function is required cell autonomously for forebrain patterning
The identification of bel as a LHD forebrain transcription factor
led us to look for forebrain patterning defects that might underlie the
observed axon guidance defects, focusing on the regions where commissural and
retinal axons cross the midline. The distal-less related transcription factor
dlx2 is expressed in the ventral telencephalon adjacent to the AC,
and in the preoptic area of the diencephalon adjacent to the POC and chiasm
prior to commissure formation (Fig.
5A) (Akimenko et al.,
1994; Ellies et al.,
1997). In bel mutants, dlx2 expression is absent
from the diencephalon in the preoptic area
(Fig. 5B). The homeodomain
transcription factor nk2.1b is also expressed in the anterior
telencephalon and diencephalon (Fig.
5C) (Rohr et al.,
2001). In bel mutants, nk2.1b expression is
subtly disrupted in the preoptic area, with a small region adjacent to the
optic recess ectopically expressing nk2.1b and a small region in
lateral diencephalon adjacent to the tract of the POC lacking nk2.1b
expression (Fig. 5D). In the
telencephalon, expression of dlx2 is slightly reduced in the AC
region (Fig. 5B), whereas
expression of nk2.1b appears unaffected
(Fig. 5D), indicating that
lhx2 is also required for ventral telencephalon formation. These
forebrain patterning defects are apparent starting at 24 hpf, as POC axons are
crossing the midline, suggesting that bel(lhx2) is required for
patterning the neural growth substrate that provides guidance cues for retinal
and commissural axons.
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; Culverwell and
Karlstrom, 2002; Karlstrom et
al., 1999; Karlstrom et al.,
2003; Shanmugalingam et al.,
2000; Tyurina et al.,
2005; Walshe and Mason,
2003). Furthermore, it was previously suggested that bel
might mediate Fgf and Hh signaling in the regulation of vax
expression in the preoptic area (Take-uchi
et al., 2003). To determine whether the forebrain defects seen in
bel may be due to defects in Hh or Fgf signaling, we examined the
expression of Hh and Fgf signaling molecules, or their downstream targets, in
bel mutants. fgf8 expression is lost in the preoptic area of
bel mutants (Fig. 5).
By contrast, preoptic expression of the Hh target genes ptc1 and
nk2.2 was unaffected in bel mutants (data not shown), which
suggests that loss of Lhx2 function does not affect Hh signaling in the POA.
Thus bel(lhx2) might affect forebrain patterning and axon guidance,
at least partially, via Fgf8 signaling.
We next examined optic stalk expression of vax2, zic2.1 and
pax2.1. As previously documented, we showed that bel mutants
lack vax2 expression only in preoptic area and medial optic stalk
(data not shown) (Take-uchi et al.,
2003). pax2.1 is expressed prior to vax2
expression in the optic stalk and eye
(Krauss et al., 1991). Similar
to vax2, pax2.1 was missing in bel mutants in the medial
regions, but was unaffected in more lateral regions and the ventral retina
(Fig. 5G,H). Another
transcription factor involved in establishing retinal projections in mice is
Zic2, acting both in the retina (Herrera
et al., 2003) and ventral diencephalon
(Williams et al., 2004). In
zebrafish, zic2.1 is expressed in the optic stalk but not in RGCs.
Similar to pax2.1, bel mutants lack expression of zic2.1 in
the optic stalk (Fig.
5I,J).
Given the multiple roles for Lhx genes in cell differentiation and axon guidance, we next wondered whether lhx2 affected cell differentiation cell autonomously in the brain. When transplanted into a wild-type forebrain, bel mutant cells were unable to correctly express dlx2 (Fig. 5S,U). Conversely, wild-type cells were able to express dlx2 appropriately when transplanted into a bel mutant forebrain (Fig. 5R,T). These results indicate that lhx2 function is cell-autonomously required for cell differentiation in the forebrain.
Reduced cell proliferation in bel(lhx2) mutants
Loss of lhx2 function in mouse leads to a highly reduced cortex
and reduced cell proliferation in the telencephalon
(Porter et al., 1997). We
therefore examined bel mutants to determine whether zebrafish
lhx2 similarly affects cell proliferation in the forebrain. Labeling
mitotic cells with the anti-phosphohistone3 antibody revealed that
bel mutants have regionally reduced cell proliferation in the ventral
forebrain (Fig. 5K,L). In the
diencephalon, bel mutants had less than half the number of
proliferating cells when compared with the wild-type siblings. However, in the
telencephalon, there was no statistical difference in the number of
proliferating cells (Fig. 5M).
No differences were seen in cell death in the forebrain (data not shown). Thus
defects in ventral forebrain patterning, including loss of dlx2, vax2
and pax2.1, may, at least partially result, from the failure of
precursor cells to proliferate in the preoptic area of the forebrain.
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). At
21hpf, prior to axonogenesis in the forebrain, these midline glial cells are
present but highly disorganized in bel mutants, spreading dorsally to
fill the preoptic area (Fig.
6A,B). The disorganization of GFAP expressing cells was also
observed at later ages, and AC and POC axons appear to grow in association
with the mis-placed glial cells (Fig.
6A,B insets).
To further link bel forebrain defects to the observed axon
guidance defects, we analyzed the expression pattern of several axon guidance
molecules that are expressed in the ventral forebrain near the AC, POC and/or
chiasm. Semaphorin 3d (sema3d) is expressed at the midline
of the diencephalon immediately ventral to the POC and chiasm
(Fig. 4C)
(Halloran et al., 1998). In
bel mutants, sema3d expression is absent in the
diencephalon, whereas expression is unaffected in the midbrain
(Fig. 6D). netrin1a is
normally absent in the diencephalon where the POC and optic chiasm form
(Fig. 6E) (Lauderdale et al., 1997). In
bel mutants, netrin1a expression was unaffected in the
telencephalon but was expanded across the optic recess into the preoptic area
of the diencephalon (Fig. 6F).
Slit2 is normally expressed in domains that surround the optic nerve,
tract and POC (Erskine et al.,
2000; Nguyen-Ba-Charvet and
Chedotal, 2002; Plump et al.,
2002; Rasband et al.,
2003; Richards,
2002). In bel mutants, slit2 expression is
expanded in the region where RGC axons cross the ventral midline
(Fig. 6G,H). Although Ephrin
and Eph receptors are known to influence ipsilateral retinal axon growth
(Nakagawa et al., 2000;
Williams et al., 2003), their
role in guiding axons toward or across the midline is not known. In
bel mutants, EphB2 expression was reduced in parts of the
diencephalon, but was largely unaffected in the chiasm region.
(Fig. 6I,J). Similarly,
EphB3, ephrinA4, sdf1a (cxcl12a - Zebrafish Information
Network), and sdf1b (cxcl12b - Zebrafish Information
Network) expression appeared normal at the midline (data not shown).
In summary, sema3d expression is reduced in the chiasm region in bel mutants. By contrast, netrin1a and slit2 expression is expanded across the commissure region where axons fail to cross the midline in bel mutants. Several other guidance molecules were not mis-expressed in bel mutants, indicating that bel(lhx2) affects only some but not all axon guidance molecules in the forebrain.
bel(lhx2) is required for eye development
belladonna mutants have a `dilated pupil' phenotype at 5 dpf
(Karlstrom et al., 1996) and
thus they were named after the plant whose extract causes pupil dilation in
humans (Duncan and Collison,
2003; Feinsod,
2000). To understand the eye defects in bel mutants, we
sectioned 5 dpf wild-type and mutant eyes. This analysis showed that the
pigmented epithelium (PE) fails to contact the lens in mutants, leaving a gap
between PE and the lens (Fig.
7A,B). bel mutant eyes are also shorter in the
dorsoventral axis and wider in the mediolateral axis than are wild-type eyes;
the ratio of eye width (mediolateral axis) to eye height (dorsoventral axis)
is 15% greater in bel mutants than in wild types. Finally, in nearly
all bel eyes we observed an acellular aggregate near a normal looking
lens that was labeled with the lens protein-specific Zl-1 antibody
(Fig. 7A,B insets).
Although all cell layers appear to be present in bel mutant eyes,
labeling with an in situ probe to apoE
(Babin et al., 1997) revealed
that amacrine cells are mostly absent, with only a few cells remaining in the
ventral retina (Fig. 7C,D).
Labeling of Müller glial cells showed that these cells are disorganized
(data not shown). These subtle eye morphogenesis defects become more severe in
those bel mutants that survived into adulthood. bel mutant
eyes are much smaller than in wild type, with highly disorganized retinal
layers that appear to fold over on themselves, most likely due to the failure
of the fluid filled posterior compartment to form
(Fig. 7E,F). In some adults,
vascularized retinal tissue protrudes from the eye adjacent to the lens
(Fig. 7F, left inset).
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Nohno et al., 1997). Our
analyses show that lhx2 is disrupted in bel mutants and that
two bel alleles encode severely truncated Lhx2 proteins that lack the
highly conserved homeodomain. Both alleles appear to be loss-of-function
mutants based on two criteria: first, neither allele appears to have any
dominant activity; and second, bel forebrain patterning defects are
similar to those seen following MO- or S-oligo-induced loss of Lhx2 function.
Analysis of the bel phenotype reveals that zebrafish Lhx2 function is
required for proper patterning of the anterior forebrain and eye, and for the
proper expression of several axon guidance molecules in the preoptic area of
the diencephalon. Coincident with these localized gene expression defects,
retinal axons fail to cross the midline and instead project ipsilaterally, and
forebrain commissural neurons fail to cross the midline.
Lhx2 and neural patterning
Our analysis shows that, similar to in mouse, Lhx2 is required for both
proper gene expression and for cell proliferation in the forebrain in
zebrafish (Fig. 5).
fgf8, one of the genes downregulated in the forebrain of bel
mutants, is known to regulate cell proliferation
(Xu et al., 1993). Thus,
reduced fgf8 signaling may account for the reduced proliferation
observed in bel mutants (Fig.
5K,L). However, because the loss of the optic stalk marker
pax2.1 occurs prior to the loss of fgf8 expression in this
region (data not shown), it appears that cell differentiation defects may
precede these cell proliferation defects. Furthermore, although it is possible
that regional reductions in cell proliferation could account for the observed
losses in forebrain gene expression (Fig.
5), we show that the ventral telencephalic markers nk2.1b
and netrin1 are expanded into the preoptic area of the diencephalon.
Thus, lhx2 may directly regulate cell specification and regional
identity. This is consistent with the model for Lhx2 function in the mouse
cortex, in which Lhx2 is initially thought to function in the specification of
cortical cells, as distinct from the cortical hem, and later is required for
the proliferation of cortical precursors
(Bulchand et al., 2001).
Whether lhx2 and other forebrain patterning genes regulate neural
patterning primarily by affecting precursor cell differentiation or by
selectively regulating precursor proliferation, or both, is an important issue
that remains to be determined.
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;
Bach, 2000;
Gill, 2003;
Lumsden, 1995;
Sockanathan, 2003). Similarly,
overlapping expression of a large number of LHD and Lim domain only (LMO)
genes in the forebrain helps to define prosomeric boundaries
(Rubenstein and Beachy, 1998),
and combinatorial Lhx function may control the specification of postmitotic
thalamic neurons, as well as thalamocortical axon projections
(Nakagawa and O'Leary, 2001).
Overlapping expression may also result in overlapping functionality. In fact,
although zebrafish lhx2 is expressed in the midbrain, hindbrain,
epiphysis and fin buds, we did not observe any early developmental defects in
these structures. This is similar to the situation in mice, where it has been
speculated that the highly related LHD gene lhx9 may compensate for
the loss of lhx2 function in tissues that express both genes
(Bachy et al., 2001;
Retaux et al., 1999).
Consistent with this idea, our search of the zebrafish genome sequence
database uncovered a zebrafish expressed sequence tag (EST) on chromosome 22
that is highly similar to lhx9
(Fig. 2) and that may
compensate for the loss of lhx2 function in regions of overlapping
expression.
|
;
Nohno et al., 1997), but no
gene expression or axon guidance defects have yet been reported in
lhx2 mutant mice (Bulchand et
al., 2003; Vyas et al.,
2003). Although development of the anterior diencephalon and optic
stalk have not yet been explicitly described in mouse lhx2 mutants,
published reports show disrupted dlx2 expression in more posterior
regions of the diencephalon (Vyas et al.,
2003), suggesting lhx2 function is likely to be conserved
in this region.
Some aspects of zebrafish Hh
(Culverwell and Karlstrom,
2002) and Fgf (Shanmugalingam
et al., 2000) pathway mutant phenotypes resemble those seen in
bel mutants. Furthermore, previous studies suggested that
bel might act downstream of Hh and in parallel to Fgf signaling
(Take-uchi et al., 2003). We
thus examined whether lhx2 is regulated by, or regulates, Fgf and Hh
signaling in the diencephalon. lhx2 expression is largely unaffected
in yot and dtr mutations that block Hh signaling
(Karlstrom et al., 2003), and
bel mutants have normal expression of the Hh regulated genes
nk2.2 and ptc1 (data not shown). This indicates that
lhx2 is not regulated by Hh and strongly suggests that defects seen
in bel mutants are not due to defects in Hh signaling. By contrast,
we show that Fgf signaling is required for both the induction and maintenance
of lhx2 expression.
Lhx2 and telencephalon development
Lhx2 function is best studied in the mammalian cortex, where loss of
lhx2 function leads to a general reduction in the dorsal
telencephalon (neocortex or pallium), but not more medial and ventral
telencephalic structures (paleocortex or subpallium)
(Vyas et al., 2003). Loss of
cortical tissue in mouse lhx2 mutants was originally attributed to a
general reduction in telencephalic cell proliferation
(Porter et al., 1997). More
recent work showed that the cortical hem is expanded at the expense of cortex
in lhx2 mutant mice, suggesting that Lhx2 may also pattern the cortex
through selectively influencing precursor cell proliferation
(Bulchand et al., 2003). It
was also suggested that Lhx2 influences cell fate decisions at later times
(Porter et al., 1997). In
zebrafish, loss of Lhx2 function subtly disrupts ventral telencephalon
patterning but does not grossly affect the telencephalon. However, because
teleosts lack a cortex, this difference is likely to reflect the structural
differences between species.
|
), although
this phenotype has not been described in detail. A more detailed analysis of
ventral forebrain development in lhx2 mutant mice may thus reveal a
conserved role for Lhx2 in neural patterning in this region.
Lhx2 and eye development
Lhx2 is one of the six known eye field transcription factors (EFTFs) that
establish the presumptive eye field and can induce ectopic eyes in
Xenopus (Zuber et al.,
2003). Mice lacking lhx2 completely lack eyes at birth
(Porter et al., 1997).
However, this phenotype does not represent a complete loss of eye development,
as pax6 expressing optic vesicles are formed in these embryos. Eye
development arrests before the optic cup forms, at which time the defective
eye tissue is reabsorbed (Porter et al.,
1997). By contrast, bel mutants have relatively
well-developed, functional eyes (Karlstrom
et al., 1996; Rick et al.,
2000) (Fig. 7).
Although this seemingly large phenotypic difference may represent a divergence
in Lhx2 function between species, it is more likely explained by the fact that
zebrafish embryos do not reabsorb defective tissue, as is the case for mouse
embryos.
Our analysis of bel mutants has thus allowed us to identify new
roles for lhx2 in vertebrate eye development. Although most cell
layers form normally in bel(lhx2) mutants, amacrine cell numbers are
extremely reduced (Fig. 7D).
The most obvious defect in bel mutant adults is the small size of the
eye and the disorganization of the retinal layers
(Fig. 7). In teleosts, the eye
grows throughout life, with new cells being added at the ciliary marginal zone
(CMZ). We did not observe major cell proliferation defects in the eye using
the phospo-histone antibody (data not shown), and at 5 dpf bel mutant
eyes are normal in size (Fig.
7), suggesting that the small size of bel eyes at later
ages is not due to proliferation defects but is due to the failure of the
posterior compartment to form. The posterior compartment is filled with
vitreous humor, which is produced by cells of the CMZ (reviewed by
Bishop et al., 2002). CMZ
cells appear to differentiate appropriately based on 
-collagen
expression in bel mutants (data not shown); however, this region of
the eye is clearly disrupted, as evidenced by the gap between the PE and the
lens, and by the presence of ectopic lens proteins
(Fig. 7B). Thus, failure of the
posterior chamber to form in bel mutants may result from defects in
CMZ cell function, and/or from defects in the formation of the barrier to
contain the vitreous humor in the eye.
Lhx2 and forebrain axon guidance
Despite the fact that numerous guidance systems are affected by mutations
in bel, the observed axon guidance defects are remarkably specific.
Axon defects are limited to the midline, with retinal axon pathways forming
normally on the ipsilateral side of the brain. This indicates that dorsal
guidance systems are unaffected by defects in midline axon crossing and is in
contrast to other mutations affecting single axon guidance systems in the
forebrain. In mutants affecting only Robo/Slit-mediated repulsion, defects in
axon crossing are accompanied by axon wandering in the rostrocaudal axis and
the formation of ectopic chiasm (Plump et
al., 2002; Richards,
2002). Loss of Netrin results in lack of the hippocampal
commissure, the corpus callosum and the anterior commissure in the forebrain
(Mitchell et al., 1996).
Similarly, mice with disrupted ephrin function show a wide range of defects,
including guidance errors, incorrect mapping in the tectum or lateral
geniculate nucleus, and defasciculation
(O'Leary and McLaughlin,
2005).
Despite extensive work on LHD proteins and forebrain patterning, little is
known about how LHD-mediated forebrain specification affects neural
connectivity. LHD transcription factors have been shown to regulate the
expression of the axon guidance molecule receptor EphA in motoneurons, thus
directly affecting the response to the guidance molecule EphrinA
(Kania and Jessell, 2003).
Although it is possible that cell fate changes or changes in guidance
receptors in commissural and retinal neurons could account for the lack of
midline crossing in bel(lhx2) mutants, RGCs appear to be specified
correctly in bel mutants and can form functional projections on the
incorrect tectal lobe (Rick et al.,
2000) (Fig. 1).
Combined with the observed gene expression defects in the preoptic area, this
strongly suggests that forebrain patterning defects underlie the observed axon
defects in bel(lhx2) mutants.
The midline defects seen in bel mutants are in fact surprisingly
similar to those seen in the hedgehog pathway mutant you-too
(yot) (Barresi et al.,
2005). Both mutants have similar defects in midline-spanning glial
bridges that provide the cellular substrate for commissural and retinal axons
(Barresi et al., 2005). In
addition, slit guidance molecule expression is expanded across the
commissure regions in both yot and bel mutants. Slit2 and
Slit3 have been shown to directly influence the position of the forebrain
commissures (Barresi et al.,
2005) and the optic chiasm
(Rasband et al., 2003) by a
surround/repulsion mechanism. Expanded slit expression appears to be
the major cause of axon defects in yot, as reducing Slit function in
yot mutants largely rescues commissure formation
(Barresi et al., 2005). Because
Slit repulsion also helps to position the glial bridges
(Barresi et al., 2005), the
expansion of slit genes across the midline in bel mutants
could affect midline crossing by disrupting the cellular substrate for axon
growth and/or by directly repelling midline crossing axons. Finally,
misexpression of the axon guidance genes sema3d and netrin1
in bel mutants may also contribute to the observed axon guidance
defects. Further analysis of the causes of axon guidance defects in
bel thus promises to shed light on the relative importance of
multiple guidance systems in the vertebrate forebrain.
|
ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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