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First published online 16 April 2008
doi: 10.1242/dev.020693
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1 Instituto de Neurociencias de Alicante (Consejo Superior de Investigaciones
Científicas-Universidad Miguel Hernández, CSIC-UMH). Campus San
Juan, Avd. Ramón y Cajal s/n, Alicante 03550, Spain.
2 Departments of Pathology and Cell Biology, Department of Neuroscience,
Columbia University, College of Physicians and Surgeons, 630 W. 168th Street,
New York, NY 10032, USA.
* Author for correspondence (e-mail: e.herrera{at}umh.es)
Accepted 27 March 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: EphB1, Zic2, Binocular vision, Midline axon divergence, Mouse
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Jeffery, 2001;
Kullander et al., 2003).
The crossing of the midline has frequently been employed as a model to
understand axonal guidance. Studies on how this process occurs have resulted
in the identification of a considerable number of specific axon guidance
ligand-receptor pairs such as netrins and DCC, Slits and Robos, semaphorins
and neuropilins/plexins and ephrins and Ephs, to cite some examples
(Erskine et al., 2000;
Keleman et al., 2002;
Kidd et al., 1998;
Lustig et al., 2001;
Nakagawa et al., 2000;
Oster et al., 2003;
Plump et al., 2002;
Stein et al., 2001;
Williams et al., 2006;
Williams et al., 2003;
Zou et al., 2000). A new level
of analysis of the mechanisms of axonal navigation has revealed that
combinations of transcription factors expressed in neuronal subpopulations
control diverse trajectories to specific targets
(Jessell, 2000;
Shirasaki and Pfaff, 2002).
How the activity of a specific transcription factor dictates axon guidance
choices is still an open question, but in recent years it has become clear
that transcription factors provide a molecular drive by which growth cones can
respond appropriately and differentially to the cues they encounter through
the activation and inhibition of a balance of target axon guidance molecules.
Thus, the transcription factor Even-skipped (Eve), expressed in motoneurons,
dorsalizes ventral axons in part through upregulation of the netrin receptor
Unc-5, which can act with the additional netrin receptor DCC
(Labrador et al., 2005). The
cell adhesion molecule Fasciclin III might be a potential target for Nkx6
(HGTX - FlyBase) in a subset of ventral motoneurons that require this molecule
to exit the central nervous system
(Broihier et al., 2004). In
vertebrate systems, Lim1 (Lhx1) controls motoneuron projections to the limb in
the dorsoventral axis by inducing the expression of EphA4, enabling dorsal
projecting axons to respond to ephrin A expressed by the ventral limb
mesenchyme (Kania and Jessell,
2003), whereas Islet2 seems to be upstream of Slit-induced
fasciculation of sensory neurons (Yeo et
al., 2004). In the murine visual system, Irx4 regulates Slit
proteins to control axonal navigation within the retina
(Jin et al., 2003). The
guidance cue Shh and the cell adhesion molecule L1 have been identified as
targets of the Pou transcription factor Brn3b (Pou4f2), which is essential for
retinal pathfinding at several points along the retinofugal pathway
(Erkman et al., 2000;
Pan et al., 2005).
Misexpression and genetic manipulation of the transcription factors FoxG1,
FoxD1, Vax2 or Tbx5 alter regional specification of visual paths in the
targets by regulation of axon guidance molecules such as Ephs and ephrins
(Herrera et al., 2004;
Koshiba-Takeuchi et al., 2000;
Mui et al., 2002;
Pratt et al., 2004;
Schulte and Cepko, 2000;
Takahashi et al., 2003). These
and other findings describe links between transcription factor expression and
regulation of axon guidance proteins in different scenarios of the developing
nervous system (for a review, see Erskine
and Herrera, 2007). However, to date, there have been no reports
on transcriptional regulation of axon guidance molecules acting on laterality
at the vertebrate midline.
The optic chiasm, a structure essential for establishing binocular vision,
is a good model for analyzing axon guidance and midline crossing because at
this juncture, retinal fibers arising from each retina diverge to project to
the higher visual targets in the same (ipsilateral) or the opposite
(contralateral) side of the brain. The turn at the midline executed by
ipsilateral axons has been suggested to be mediated by EphB1/ephrin B2
signaling. Ephrin B2 is expressed by glial cells located at the midline,
whereas EphB1 is highly expressed in the ventrotemporal (VT) retina where
ganglion cells giving rise to uncrossed axons are located
(Williams et al., 2003). In
addition to the receptor tyrosine kinase EphB1 and its ligand ephrin B2, the
zinc-finger transcription factor Zic2 has also been implicated as a
determinant of axonal laterality at the chiasmatic midline. Zic2, which is
essential for a wide array of other developmental programs, such as patterning
the neural plate and tube (Aruga et al.,
2002; Brown et al.,
2001), is differentially expressed in the uncrossed but not
crossed retinal pathway. Moreover, reduced levels of Zic2 in vivo lead to a
near absence of the ipsilateral projection
(Herrera et al., 2003). Zic2
expression in the VT retina correlates with the degree of binocularity through
evolution. In mice, which have poor binocular vision, about 3% of retinal
ganglion cell (RGC) axons do not cross the midline and the number of
Zic2-expressing cells during the period of retinal axon outgrowth reflects
this proportion of ipsilateral retinal axons. Animals with a greater extent of
binocular vision than mice, such as the ferret, and, concomitantly, a greater
number of ipsilateral retinal axons, display an equivalent number of
Zic2-positive cells. Species with panoramic vision, such as chick and
zebrafish, have no ipsilateral axons and, accordingly, their RGCs lack Zic2
expression (Herrera et al.,
2003; Seth et al.,
2006). In addition, Islet2, another regulatory gene from
the LIM homeodomain family, has been proposed to affect laterality of the
late-born crossing RGCs in mouse VT retina, by repressing Zic2, EphB1 or both
(Pak et al., 2004), and/or by
putatively regulating expression of NrCAM, which is crucial for the midline
crossing of this late-born population
(Williams et al., 2006).
Since Zic2 and EphB1 expression patterns overlap spatiotemporally during
the formation of the ipsilateral projection, and because in vivo
loss-of-function manipulation for one or the other protein dramatically
reduces the ipsilateral projection
(Herrera et al., 2003;
Williams et al., 2003), we
wondered whether EphB1 might be a target of Zic2 in this process. Here, we
first demonstrate that in mammals, Zic2 expression in RGCs is not only
essential but is sufficient to switch the trajectory of retinal axons from
crossed to uncrossed at the chiasm midline. This control of the ipsilateral
projection by Zic2 is in large part mediated through EphB1, implicating
transcriptional regulation of axon guidance receptors that, in turn, trigger
an axonal response in an intermediate target such as the midline. In addition,
we also report that Zic2 is able to switch axonal laterality at the midline by
an EphB1-independent mechanism.
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MATERIALS AND METHODS |
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SUMMARY
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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) and
Zic2+/kd and Zic2kd/kd mice
(Nagai et al., 2000) kept in a
timed-pregnancy breeding colony at the Instituto de Neurociencias were removed
from anesthetized mothers by caesarean section and staged with E0 defined as
midnight before a plug was found. Animals were treated according to Spanish,
European Union and NIH guidelines for Care and Use of Laboratory Animals.
Animal protocols were approved by the Instituto de Neurociencias Animal Care
and Use Committee.
For electroporation experiments, human ZIC2 and EGFP coding sequences were
cloned in a modified version of the pCAGGS vector
(Borrell et al., 2005;
García-Frigola et al.,
2007) that includes a greater number of restriction sites 3'
to the CAG promoter. The CAG promoter contains a chicken β-actin/rabbit
β-globin hybrid promoter (AG) and the human CMV-IE enhancer. The AG
promoter sequence consists of the chicken β-actin promoter, the first
exon and part of the first intron (that seems to have a strong enhancer-like
activity) linked to a rabbit β-globin fragment, consisting of a 3'
part of the second intron (inclusive of a branch point that is required for
normal splicing reactions) and a 5' part of the third exon
(Niwa et al., 1991). Plasmid
DNA was purified using a conventional Midiprep Kit (Qiagen, Valencia CA) and
resuspended in TE (10 mM Tris pH 8.0, 1 mM EDTA).
In situ hybridization and immunohistochemistry
The in situ hybridization protocol was modified from reported methods
(Schaeren-Wiemers and Gerfin-Moser,
1993) to perform fluorescent instead of the colorimetric reaction
[using the TSA system (Perkin Elmer), following the manufacturer's
instructions].
Immunohistochemistry using anti-Zic2 antibodies (gift of S. Brown,
University of Vermont) was performed as described
(Herrera et al., 2003).
In utero electroporation and axon quantification
Timed-pregnant mouse females were anesthetized with sodium pentobarbital
(0.625 mg per 10 g body weight) and Rytodrine (0.1 ml of a 14 mg/ml solution)
intraperitoneally, the abdomen cut open and the uterine horns exposed. A DNA
solution (1 µg/µl CAG-EGFP plasmid alone, or in combination with 1
µg/µl CAG-Zic2 plasmid and 0.03% Fast Green in PBS) was injected into
one eye of each embryo using a pulled glass micropipette. The head of each
embryo was placed between tweezer-type electrodes (CUY650-P5 Nepa GENE, Chiba,
Japan) and five square electric pulses (50 milliseconds) were passed at
1-second intervals using an electroporator (CUY21E, Nepa GENE). Voltage
conditions depended on the developmental stage of the embryo (38V and 48 V
were applied to electroporate E13 and E14 embryos, respectively). The
abdominal cavity was suture-closed and embryos allowed to develop normally.
Three days after electroporation, pregnant mothers were sacrificed and embryos
removed and fixed in 4% paraformaldehyde overnight at 4°C. Brains were
dissected and the optic chiasm viewed en face in whole-mount with a
fluorescence dissecting scope.
Quantification of the degree of co-localization of markers after
co-electroporation of two plasmids into retinal cells was performed as
follows. Retinal sections from E16 embryos (n=4) that were
co-electroporated at E13 with CAG-Zic2 and CAG-EGFP plasmids were analyzed. A
total of 25 sections and 914 cells were included in the study. Each given
percentage was calculated as the average percentage ±s.e.m.
Quantification of the crossed and uncrossed projection was performed as
described (Herrera et al.,
2003) in whole-mounts of the optic chiasm area.
Quantitative RT-PCR
Retinas of E13.5 wild-type embryos were electroporated in utero with
CAG-GFP alone or together with CAG-Zic2 plasmids. Embryos were allowed to
develop for 2 days and then their retinas were removed. Each electroporated
retina was observed under a fluorescence microscope and the electroporated
GFP-positive area was dissected out and kept for RNA extraction. An average of
six retinal fragments was used for each experiment. Total RNA was extracted
using the RNeasy Mini Kit (Qiagen), DNaseI digested and retrotranscribed using
the Reverse Transcription System (Promega) the following manufacturer's
recommendations. Quantitative (q) PCR was performed using an ABI PRISM 7000
sequence detection system with the SYBR Green method. Primers were designed
using Primer Express (software v2.0, Applied Biosystems): mouse EphB1 forward,
5'-CGCTCTATCCCAGACTTCACG-3'; mouse EphB1 reverse,
5'-GTGAAGCCTGCGGTGAGG-3'; mouse Gapdh forward,
5'-CTTCACCACCATGGAGAAGGC-3'; mouse Gapdh reverse,
5'-CATGGACTGTGGTCATGAGCC-3'. Transcript levels were calculated
using the comparative Ct method normalized to Gapdh. The final
results were expressed relative to calibrator (control embryos electroporated
with CAG-GFP) using the 2-(
Ct) ±s.e.m.
formula.
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RESULTS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
However, Zic2 is also expressed in the chiasmatic region
(Brown et al., 2003;
Herrera et al., 2004) as well
as in cycling cells at the ciliary margin zone
(Herrera et al., 2003), and
our experiments with Zic2 hypomorphic mutants could not distinguish whether
the reduced ipsilateral projection was attributable to the decreased
expression of Zic2 in RGCs, in resident cells at the optic chiasm region or in
cycling cells in the ciliary margin zone.
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;
Saito and Nakatsuji, 2001) and
postnatal retina (Matsuda and Cepko,
2004), we have adapted it for gene delivery in embryonic retina
(García-Frigola et al.,
2007). Plasmids bearing EGFP alone or together with plasmids
containing the Zic2 coding sequence under the regulation of the CAG promoter
(Niwa et al., 1991) (see
Materials and methods) were monocularly delivered to embryonic retinas
(Fig. 1 and see Materials and
methods). From previous work, we know that as little as 1 day after
electroporation, EGFP expression can be detected in targeted cells
(García-Frigola et al.,
2007). Two or 3 days after electroporation, pregnant mothers were
sacrificed and embryos analyzed. In embryos co-electroporated with CAG-EGFP
and CAG-Zic2 plasmids, Zic2 was expressed in around 85-90% of the
EGFP-expressing cells (Fig.
1C), indicating that EGFP can be considered as a surrogate
indicator of neurons that express ectopic Zic2 when both plasmids are injected
together. In flattened whole-mount retinas, targeted cells can be visualized
in the center of the retina and axons of targeted cells can be followed by
EGFP fluorescence up to and through the optic chiasm
(Fig. 1).
In the mouse retina, the wave of differentiation and subsequent
axonogenesis occur in a central-to-peripheral manner. Thus, axons from the
central retina reach the midline sooner than axons from peripheral retinal
regions. Because many axons from the central retina reach the midline at E13.5
(Colello and Guillery, 1990;
Godement et al., 1990;
Marcus et al., 1995;
Sretavan, 1990;
Sretavan, 1993), gene delivery
was performed at this age. Pregnant mothers were sacrificed 3 or 4 days later
and embryos analyzed to check the site of electroporation into the retina.
Embryos electroporated only in the central retina were chosen. In E16.5 or
E17.5 embryos electroporated at E13.5 with EGFP, nearly all the green axons
projected contralaterally (Table
1). By contrast, in embryos electroporated with Zic2 and EGFP, a
large proportion of axons changed their behavior at the midline to project
ipsilaterally (Table 1,
Fig. 2).
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The quantification of these results (Table 1) confirms that: (1) There is a significant increase in axons that project ipsilaterally after ectopic expression of Zic2 in central retina at E13.5, as compared with ectopic expression of EGFP in the same area; (2) this increase is much lower when Zic2 ectopic induction is performed 1 day later at E14.5. Together, these results demonstrate that in vivo Zic2 expression is sufficient to switch the trajectory of axons that have not yet reached the midline, but cannot alter the trajectory of axons that have already passed the midline region.
EphB1 as a candidate effector of Zic2
In the mouse, RGCs whose axons do not cross the midline are located
exclusively in the VT region of the developing retina, whereas the cell bodies
giving rise to crossing RGC axons are found across the entire retina. We have
previously reported that the zinc-finger transcription factor Zic2, and the
receptor tyrosine kinase EphB1, are expressed in the VT portion of embryonic
retina and that each protein is essential for the formation of the ipsilateral
projection (Herrera et al.,
2003; Williams et al.,
2003). Zic2 expression in VT retina is initiated at E14.5 and it
is maintained until E17.5, the period when uncrossed RGCs traverse the midline
(Herrera et al., 2003). EphB1
is dynamically expressed in the embryonic retina, commencing at E13.5 in the
dorsocentral retina. One day later, dorsocentral expression of EphB1
disappears, but then EphB1 is highly expressed in the VT region from E14.5
until approximately E17.5. Subsequently, EphB1 expression spreads throughout
the entire retina but not in RGCs
(Williams et al., 2003)
(Fig. 3). Thus, based on
temporal and spatial expression, EphB1 is a good candidate for a downstream
target of Zic2. Based on the above findings, we examined more closely the
spatiotemporal coincidence of Zic2 and EphB1 expression by utilizing
immunohistochemistry against Zic2 combined with in situ hybridization for
EphB1. At E15.5, Zic2 alone is expressed by a group of RGCs at the
edge of the neural retina, whereas both Zic2 and EphB1 are expressed in RGCs
located more centrally (Fig.
3B). The wave of RGC generation progresses from central to
peripheral retina and the youngest cells are located more peripherally.
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Zic2 is required for EphB1 expression in VT retina
Since Zic2 expression precedes EphB1 induction in VT RGCs in the developing
retina, we wondered whether this transcription factor is required for EphB1
expression.
As Zic2 expression is barely detectable in the VT retina of
Zic2-knockdown mice (Zic2kd/kd)
(Herrera et al., 2003), we
used these mice to perform in situ hybridization for EphB1. At E16.5,
RGCs uniquely expressing Zic2 are not found in wild-type embryos and the
most-peripheral cells co-expressed Zic2 and EphB1. By contrast,
Zic2kd/kd RGCs in VT retina did not exhibit EphB1
expression. In mice heterozygous for the Zic2 mutation
(Zic+/kd) in which very low levels of Zic2 expression are
detected in VT retina, EphB1 expression was accordingly reduced
(Fig. 4A). These data show that
Zic2 expression in the developing neural retina is essential for the
expression of EphB1 in VT RGCs.
In E16.5 Zic2kd/kd embryos, however, EphB1 was detected in other brain areas in a pattern very similar to that in wild-type embryos. Both Zic2+/+ and Zic2kd/kd show high expression levels of EphB1 in entorhinal cortex, hippocampus and lateral globus pallidus (Fig. 4B), suggesting that Zic2 selectively regulates EphB1 expression in RGCs.
Ectopic expression of Zic2 in non-VT retina upregulates EphB1 levels
To test whether EphB1 is induced after ectopic expression of Zic2 in vivo,
we compared EphB1 mRNA levels after ectopic electroporation of Zic2
in the center of embryonic retinas. Two days after electroporation of
CAG-Zic2/CAG-EGFP or CAG-EGFP plasmids in E13.5 embryo retinas, the
EGFP-positive central portions of the retinas were dissected. Quantitative
RT-PCR (qRT-PCR) was performed to detect EphB1 mRNA levels in these
samples, as well as in non-electroporated central regions of retina. Segments
from the center of the retina electroporated with Zic2/EGFP showed a
statistically significant increase of 1.4±0.116-fold in the levels of
EphB1 mRNA as compared with non-electroporated retinal segments or
those electroporated with EGFP alone. These results demonstrate that in vivo,
Zic2 is sufficient to enhance EphB1 expression
(Fig. 5A).
Zic2 is sufficient to induce axon turning at the midline in the absence of EphB1
Since our in vivo assays indicated that Zic2 can regulate EphB1 expression,
we decided to test whether the ipsilateral phenotype induced by Zic2 in
wild-type embryos depends on EphB1 expression. If Zic2 controls ipsilaterality
through EphB1 expression, ectopic expression of Zic2 in the absence of the
EphB1 gene should not result in an increased ipsilateral projection
in vivo. To test this hypothesis, we repeated the in utero electroporation
experiments expressing Zic2 ectopically in RGCs of E13.5 mutant mouse embryos
lacking EphB1. In EphB1-/- embryos electroporated with
CAG-EGFP plasmids in the central retina at E13.5 and sacrificed 3 days later,
almost no ipsilateral axons were seen at the chiasm level, similar to what was
observed for wild-type embryos electroporated with EGFP
(Fig. 5B). When
co-electroporation of Zic2/EGFP was performed in EphB1-null embryonic
retinas, a statistically significant increase in the proportion of ipsilateral
axons was observed, as compared with electroporation of EGFP alone.
Nevertheless, the amount of axons projecting ipsilaterally after Zic2/EGFP
introduction was significantly smaller in EphB1-null embryos than
when wild-type retinas were electroporated with Zic2/EGFP
(Fig. 5B). The quantification
of these experiments is shown in Table
1.
These results demonstrate that Zic2 can change retinal axon laterality in vivo primarily through upregulation of EphB1, and also indicate that Zic2 is able to change axonal laterality through an undetermined pathway that is independent of EphB1 signaling.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). Transcriptional regulation of axon guidance receptor
expression could provide a means for modulating the interpretation of the
myriad of guidance cues faced by navigating axons. Here we report the
induction of EphB1 by Zic2 as the first described link between an axon
guidance molecule and a transcription factor controlling laterality at the
vertebrate midline.
|
).
However, because Zic2 is also expressed in the optic chiasm region, in the
retinal ciliary margin zone and in the early optic cup
(Herrera et al., 2004;
Nagai et al., 1997), it was
unclear whether postmitotic expression of Zic2 in VT retina is responsible for
determining axonal behavior at the midline. Our in vivo electroporation
experiments at E13.5 show that misexpression of Zic2 in the central retina
leads to the development of an ectopic ipsilateral projection, but when Zic2
is introduced 1 day later the number of axons that switch behavior at the
midline from a crossed to uncrossed trajectory was very modest. These findings
demonstrate that expression of Zic2 in RGCs is sufficient, in a
cell-autonomous and temporally restricted manner, to determine an ipsilateral
projection at the chiasmatic midline. We also observed that when wild-type
embryos are electroporated with Zic2/EGFP, a percentage of targeted cells
still project contralaterally. It is likely that these remaining contralateral
axons represent axons from the few targeted RGCs that express EGFP but do not
co-express Zic2, together with fibers that had already passed through the
chiasm region when the ectopic Zic2 protein began to induce its target genes.
Electroporation experiments performed at earlier stages might more
definitively demonstrate that Zic2 can change the behavior of the
contralateral axons before their growth cones reach the midline.
Unfortunately, the efficiency of the electroporation technique is very low at
stages earlier than E13.5. In addition, because the reported ligand for EphB1,
ephrin B2, is not expressed at the midline until E13.5-14.5, the outcome of
such experiments would be difficult to interpret. Despite this, our
observations present compelling evidence that Zic2 is sufficient to control
laterality at the midline and that its regulation is highly coordinated with
the navigation of retinal axons through the optic chiasm midline.
Recent studies have suggested that the VT region of retina is the domain
uniquely competent to project ipsilaterally and that RGCs in this region are
genetically distinct from those in the remainder of the retina
(Herrera et al., 2003;
Herrera et al., 2004;
Pak et al., 2004;
Pratt, 2004;
Williams et al., 2006;
Williams et al., 2003). Our
results confirm that Zic2, a protein uniquely expressed in the VT quadrant, is
in fact responsible for the repulsion of retinal axons at the midline. Since
Zic2 is essential for the ipsilateral projection to form and ectopic
expression of Zic2 in the central retina is sufficient to change axonal
behavior at the chiasm, it is likely that all of the upstream dissimilarities
between the VT and the remainder of the retina converge at Zic2 expression and
that once this molecule is expressed, other differences are not relevant for
determining laterality of axonal projection at the midline.
Transcriptional regulation of expression of a guidance receptor that functions at the mammalian midline
Zic2 is expressed in brain regions that do not express EphB1 and,
conversely, EphB1 is expressed in areas of the developing nervous system
negative for Zic2 expression. In fact, EphB1 expression is not affected in the
cortex or in the globus pallidus in the absence of Zic2. Thus, in addition to
Zic2, other transcription factors might participate in the control of EphB1
expression. Moreover, our electroporation experiments on the
EphB1-knockout background show that a reduced proportion of
Zic2-positive RGCs are still able to direct their axons ipsilaterally,
supporting the view that Zic2 might regulate other proteins in addition to
EphB1 during retinal axonal decisions at the midline. It is possible that Zic2
regulates EphB1 in VT retina, but when Zic2 is expressed in non-VT regions
other molecules replace EphB1 to promote ipsilaterality. Our findings,
together with previous observations that EphB1-null mice still have a
remaining ipsilateral projection (Williams
et al., 2003), whereas almost no ipsilateral axons are detected in
the Zic2 hypomorphic mutants
(Herrera et al., 2003) (our
unpublished observations), strongly suggest that EphB1 is not the only target
for Zic2 in the VT retina.
|
;
Herrera et al., 2004;
Jin et al., 2003;
Kania and Jessell, 2003;
Labrador et al., 2005;
Pan et al., 2005;
Pratt, 2004;
Schulte and Cepko, 2000;
Takahashi et al., 2003;
Yeo et al., 2004). In many of
these cases, whether the transcription factor plays a role in direct
regulation of axon guidance cues or in general patterning in early neural
differentiation is not clear. At the invertebrate midline, Longitudinals
lacking (Lola) appears to be an example of a class of transcription factor
that controls axon targeting by coordinating the regulation of multiple (Slit,
as well as its axonal receptor Robo) guidance genes, ensuring that they are
co-expressed at the correct time, place and relative level
(Crowner et al., 2002). Lola
homologs have not been described in vertebrates, but we have found a
transcription factor that precisely regulates one particular axon guidance
decision by the expression of at least one axon guidance receptor, EphB1, to
specifically control laterality at the vertebrate midline.
Previous studies have suggested that the LIM homeodomain transcription
factor Islet2 might be part of the program that controls laterality at the
midline. In the embryonic retina, crossed projections originate from RGCs
located over the entire retina except for the VT region, up to E17.5-18.5.
Ganglion cells born later in the VT segment project contralaterally and
intermingle with already differentiated ipsilaterally projecting RGCs in the
VT retina (Guillery, 1995).
Islet2 is expressed in RGCs that project contralaterally, including the
late-born crossed RGCs in the VT crescent, but not in those projecting
ipsilaterally. The role of Islet2 in the rest of the retina is not clear yet,
but it appears to affect the laterality of the later-born crossing RGCs
arising from the VT segment. Mice deficient for Islet2 show an increase in the
number of ipsilateral axons originating in the VT retinal segment, which is
accompanied by increased levels of Zic2 and EphB1 expression in that segment
of the retina (Pak et al.,
2004). Thus, Islet2 might repress an ipsilateral axon pathfinding
program exclusively in the VT retina, which involves repression of Zic2 or
EphB1 expression, or both. Although we cannot rule out the possibility that
Islet2 represses EphB1, our results suggest that determination of
ipsilaterality is, at least partially, a consequence of direct activation of
EphB1 by Zic2, the latter potentially repressed by Islet2, rather than Islet2
repressing EphB1.
Transcription events can affect axon guidance decisions at distant choice points
Several transcriptional mechanisms have been suggested for regulating
axonal pathways at intermediate targets. At the nuclear level, transcriptional
regulation at early stages of differentiation might provide the complete
repertoire of mRNAs that are required by a growth cone to navigate its entire
pathway. According to this notion, transcriptional regulation could specify
the pathway, whereas post-transcriptional regulation at the level of the axon
or growth cone (local translation, protein modification, etc.) would control
growth and directionality along the path, priming the axon with the cellular
components necessary for `on-site' post-transcriptional responses
(Butler and Tear, 2006;
Dickson and Gilestro, 2006;
Polleux et al., 2007).
Alternatively, specific receptors might be directly activated by different
transcription factors (controlled by a master gene specifying the entire
pathway) expressed in a hierarchical manner as the axon grows. Whether or not
transcription factors directly activate guidance receptors in a sequential
manner at different points along the journey of an axon is not clear.
Nevertheless, because the midline is not the first choice-point encountered by
the RGC axons, as they must first orient and navigate towards the optic disc
to then exit the retina, our work clearly supports the idea that initial
transcriptional events can affect guidance decisions at distant choice points
without disturbing previous axon decisions.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Aruga, J., Inoue, T., Hoshino, J. and Mikoshiba, K.
(2002). Zic2 controls cerebellar development in cooperation with
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