1 Masai Initiative Research Unit, The Institute of Physical and Chemical
Research (RIKEN), Hirosawa 2-1, Wako-shi, Saitama 351-0198, Japan
2 Department of Anatomy and Developmental Biology, University College London,
Gower Street, London WC1E 6BT, UK
3 Max-Planck-Institute for Immunobiology, Stuebeweg 51, D-79108 Freiburg,
Germany
4 Laboratory for Developmental Gene Regulation, RIKEN Brain Science Institute
and CREST, Japan Science and Technology Corporation (JST), Hirosawa 2-1,
Wako-shi, Saitama 351-0198, Japan
* Author for correspondence (e-mail: imasai{at}postman.riken.go.jp)
Accepted 27 February 2003
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SUMMARY |
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Key words: Axon guidance, Cell adhesion, Coloboma, Danio rerio, Polarity, Zebrafish
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INTRODUCTION |
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The neural retina is initially composed of a single layer of proliferating
cells that extend processes to both apical and basal surfaces of the
neuroepithelium. Nuclei of the proliferative cells translocate along the
apicobasal axis with cell divisions occurring when nuclei are located at the
apical surface. After exiting the cell cycle, post-mitotic cells migrate
basally to differentiate as retinal neurons or glia. Cell fate decisions in
the developing retina are independent of cell lineage and so it seems likely
that multipotent progenitor cells change their competence to generate
different retinal cell-types in response to position and stage-dependent
environmental cues (Livesey and Cepko,
2001; Marquardt and Gruss,
2002
).
Although the mechanisms that regulate retinal lamination are poorly
understood, there has been some recent progress in identifying genes involved.
For example, genetic approaches in zebrafish have shown that retinal
lamination is severely perturbed in fish carrying mutations at the mosaic
eyes (moe), oko meduzy (ome), heart and
soul (has), nagie oko (nok) and glass
onion (glo) loci (Malicki
and Driever, 1999;
Horne-Badovinac et al., 2001
;
Jensen et al., 2001
;
Pujic and Malicki, 2001
;
Peterson et al., 2001
;
Wei and Malicki, 2002
).
Cloning has identified Has as an atypical protein kinase C (aPKC) and Nok as a
member of the membrane associated guanylate kinase (MAGUK) family that is
localized to adherens junctions at the apical ends of the retinal
neuroepithelial cells (Horne-Badovinac et
al., 2001
; Peterson et al.,
2001
; Wei and Malicki,
2002
). In nok mutant retinae, adherens junctions are not
maintained and dividing cells are detached from the apical surface of the
neural retina, leading to severe disorganization of retinal laminae
(Wei and Malicki, 2002
).
Stardust, a fly homolog of Nok, and an interacting protein, Crumbs, are
essential for formation of adherens junctions and epithelial polarity
(Tepass, 2002
), suggesting
that a vertebrate Crumbs/Stardust pathway is involved in retinal lamination.
aPKC is required for maintenance of cell polarity in a number of biological
systems (reviewed by Ohno,
2001
) and in has mutants, junctional complexes in the
retina are disrupted (Horne-Badovinac et
al., 2001
; Peterson et al.,
2001
).
In vitro studies have again implicated proteins that modulate cell adhesion
in the establishment of retinal lamination. For example, function-blocking
N-cadherin (Ncad; Cdh2Zebrafish Information Network) antibodies disrupt
retinal lamination although the local application of high concentrations of
antibodies leaves the issue of specificity open
(Matsunaga et al., 1988a).
Mice lacking Ncad function show defects in development of brain, heart,
somites and pancreas but retinal phenotypes have not been reported
(Radice et al., 1997
;
Esni et a., 2001
;
Luo et al., 2001
). A large
number of studies also implicate Ncad in the regulation of axon growth. For
example, in vitro, RGC axon outgrowth is promoted on Ncad-expressing cells
(Matsunaga et al., 1988b
), and
expression of dominant-negative Ncad in vivo disrupts RGC axon outgrowth
(Riehl et al., 1996
). Genetic
studies in flies (Clandinin and Zipursky,
2002
), worms (Broadbent and
Pettitt, 2002
) and fish (Lele
et al., 2002
) have all confirmed roles for Ncad in establishing
axon trajectories.
A simple model of how Ncad modulates axon extension is that homophilic
binding mediates adhesive interactions between cells, and that differential
adhesion directly modulates axonal growth. However, the mechanisms underlying
regulation of axonal growth by Ncad and other cell adhesion molecules may be
more complex. For example, soluble versions of cell adhesion molecules
containing only the extracellular domains can still stimulate axonal growth
(Doherty et al., 1995),
suggesting that their function may be mediated by activating second messenger
cascades. In particular, several studies have suggested that Ncad-mediated
outgrowth may be regulated through direct interaction of Ncad with Fgf
receptors (Fgfrs) (Williams et al.,
2001
).
In this study, we demonstrate that the zebrafish ncad
(Bitzur et al., 1994;
Lele et al., 2002
;
Liu et al., 2001
) mutant
parachute (pac) shows defects in retinal lamination,
amacrine cell process outgrowth, confinement of cells to appropriate forebrain
compartments, RGC axon guidance, closure of the choroid fissure and maturation
of lens fiber cells. Null mutant pac embryos fail to maintain
adherens junctions between retinal neuroepithelial cells and exhibit abnormal
localization of dividing cells and severe disorganization of retinal laminae.
In a weaker pac allele, proliferation appears to be unaffected, but
RGCs and amacrine cells are nevertheless frequently intermingled and IPL forms
in ectopic locations. In these retinae, amacrine cell processes are
over-elaborated and mis-positioned, suggesting that Ncad modulates the
outgrowth and targeting of these neurites. In addition to these retinal
phenotypes, pac embryos also show defects in closure of the choroid
fissure and in guidance of RGC axons towards their central targets. These
guidance defects are primarily due to a failure to restrict cells to their
appropriate CNS compartments, as many retinal neurons and reticular astrocytes
invade the ventral brain along the trajectory normally taken by the RGC axons.
Disrupting Fgfr function in the retina does not phenocopy pac,
suggesting that most aspects of Ncad function do not require Fgfr
activity.
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MATERIALS AND METHODS |
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Mutagenesis
Mutations were induced in male gametes of RIKEN wild-type fish using
N-ethyl-N-nitrosourea (ENU). Subsequent breeding and screening for recessive
mutations followed standard protocols
(Solnica-Krezel et al., 1994).
To isolate retinal mutants, embryos from F2 pairwise crosses were screened by
labeling with anti-acetylated
-tubulin antibody.
Mapping mutant loci
pac/ embryos were collected from parents
derived from a pacrw95/RIKEN wild type x WIK/WIK
cross and stored in methanol. Genomic DNA was extracted from individual
embryos at 1 day post-fertilization (dpf). PCR analysis with SSLP markers
mapped to each linkage group was performed on DNA from pools of 20 wild-type
and 20 mutant embryos. Map positions were refined by testing additional SSLP
markers on individual embryos.
Isolation and sequencing of Ncad mutant cDNAs
Total RNA was extracted from 1 dpf
pacrw95/ embryos using Ultraspec RNA
(Biotecx laboratory). After purification of mRNA with an
oligo-dTTM-MAG mRNA purification kit (TaKaRa Biochemicals),
ncad cDNA was amplified with the RNA LA PCRTM kit (TaKaRa
Biochemicals), using two primers, 5'-GGTTTCTTTATACAGAACGGAATTT-3'
(5'-untranslated region prior to the first Met) and
5'-TCCTCCGGTTCTTACTTGTAGTTCA-3' (3'-untranslated region
after the stop codon). PCR products corresponding to full-length cDNA were
amplified in at least two independent reactions, subcloned into the TA cloning
vector, pCR2.1 (Invitrogen), and sequenced using a ABI PRISMTM 310.
To exclude nucleotide changes derived from polymorphisms, genomic DNA from
male grandparents of the family containing the pacrw95
mutation was also sequenced.
Sectioning
Embryos were fixed, dehydrated to 100% ethanol, embedded in JB4 resin (Agar
Scientific), and sectioned (5 µm) using a Microtome Rotatif, HM335E (MICROM
International GmbH). Sections were stained in 0.1% Toluidine Blue (MERCK). For
cryosectioning, fixed embryos were transferred to 30% sucrose in phosphate
buffer, mounted in OCT compound, and sectioned at 8 µm on a Microtome
cryostat, HM550M-OM (MICROM International GmbH).
Antibody labeling and in situ hybridization
Standard procedures were used (Wilson
et al., 1990) with anti-Pax6 antibody
(Macdonald and Wilson, 1997
)
at 1:400; zn-5 antibody (Oregon Monoclonal Bank) at 1:50; zpr-1 (formerly
FRet43) antibody (Larison and Bremiller,
1990
) (Oregon Monoclonal Bank) at 1:100; anti-acetylated
-tubulin antibody (Sigma) at 1:1000; anti-protein kinase C (PKC)
antibody (sc-209, Santa Cruz Biotechnology) at 1:300; anti-
-tubulin
antibody (Sigma) at 1:200; and anti-phosphorylated histone H3 antibody
(Upstate biotechnology) at 1:500. Sytox Green (Molecular probes) was used at
1:50,000. In situ hybridization to RNA probes was carried out by standard
procedures (Xu et al.,
1994
).
Staining with rhodamine-phalloidin and BODIPY-ceramide
Rhodamine-conjugated phalloidin (Molecular Probes; 107M)
was applied to cryosections of 28 hour post-fertilization (hpf) retinae. After
washing with phosphate buffer, slides were mounted with 70% glycerol and
scanned with a LSM 510 laser-scanning microscope (Carl Zeiss). BODIPY-ceramide
staining of live embryos was carried out as previously described
(Lele et al., 2002).
Cell transplantation
Cell transplantation at late blastula stage was carried out according to
Westerfield (Westerfield,
1995). Approximately 5-20 cells were transplanted to host embryos
from donor embryos labeled at the one to two cell stage with a mix of
rhodamine- and biotin-conjugated dextrans in a 1 to 1 ratio (Molecular Probes;
3
5% w/v in 100 mM KCl). The genotypes of hosts and donors were inferred
by assessing defects in brain morphology at 1 dpf. For analyses of IPL
formation, mosaic embryos were fixed at 3 dpf in 4% paraformaldehyde (PFA) in
PBS. For analyses of RGC axon trajectories, an ath5:GFP transgene was
used to visualize donor retinal cells. As an alternative method to generate
mosaic embryos, retinal cells were directly removed from host optic cups at 24
hpf with glass capillary needles and transplanted into recipient retinae.
Using this protocol, retinal cells did not form columns of neurons in the host
neural retina, but RGCs did differentiate and extend axons into the brain.
Visualization of retinal cells with GFP
Genomic fragments (14 kb) covering the zebrafish ath5
transcription unit (Masai et al.,
2000) were cloned from a genomic library. About 7 kb 5'- and
3'-genomic fragments containing untranslated regions were inserted into
multi-cloning-sites upstream or downstream of the GFP-coding region in the
expression vector pEGFP (Clontech). Injection of one-cell stage embryos with
the linearized construct leads to GFP expression in small numbers of cells in
the developing retinae.
diI and diO injection
Embryos (60 hpf and 4 dpf) were fixed in 4% PFA. diI and diO (C-18,
Molecular Probes; each 2 mg/ml in N, N-dimetylformamide (DMF) or
chloroform) were injected into eyes of fixed embryos using glass capillaries
and scanned by confocal laser microscopy LSM510 (Zeiss).
SU5402 treatment
Previous studies have indicated that median inhibiting concentration
(IC50) values for in vitro kinase inhibition and for inhibition of
neuritogenic effects of Fgf2 are 10 µM and 25 µM SU5402, respectively
(Mohammadi et al., 1997;
Skaper et al., 2000
).
Therefore, for pharmacological inhibition of Fgfr activity, wild-type embryos
were treated with 10 µM-1mM SU5402 (Calbiochem) in the dark. We also
confirmed that this range of SU5402 concentration effectively blocks
expression of sef, a downstream target of Fgf8 signaling
(Tsang et al., 2002
) (data not
shown).
Cell sorting/intermingling assay using zebrafish animal caps
Detailed procedures are described previously
(Mellitzer et al., 1999).
Capped-RNA encoding wild-type and mutant (pacrw95) Ncad
was injected into one cell-stage embryos with Alexa-488 conjugated dextran
(Molecular Probes). Animal caps were dissected and fused with animal caps from
non-injected embryos labeled with Rhodamine-conjugated dextran. The fused cell
masses were cultured in L15 medium at 28.5°C under a coverslip and scanned
by confocal microscopy. Cell sorting/intermingling was quantified by counting
the total number of isolated cells that had crossed the boundary from either
territory into the other.
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RESULTS |
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Injection with Ncad morpholino-antisense oligonucleotides (MO-Ncad) mimics
the brain defects of pac (Lele et
al., 2002). ncad morphants show similar IPL defects to
lyr (data not shown), suggesting that downregulation of Ncad can
mimic the retinal phenotype of lyr. From complementation testing,
mapping, sequencing data, functional analyses of lyr-mutated Ncad
and, finally, MO-Ncad injection, we conclude that lyr is an allele of
pac and call this mutation pacrw95 from here
onwards.
pac mutants show defects in retinal lamination
To elucidate the role that Ncad plays during eye development, we examined
the morphology of pac retinae. In order to identify subtle phenotypic
features that might be masked by the strong adhesion defects of the
pacfr7 null allele, we also examined retinae in
trans-heterozygous pacrw95/fr7 embryos and embryos
homozygous for the weak allele pacrw95. In 3 dpf wild-type
eyes, the three nuclear and two plexiform layers are established
(Fig. 2A) and the IPL forms a
continuous layer of neuropil between the RGC and amacrine cell layers
(Fig. 2E). In both
pacrw95 and pacrw95/fr7 retinae, the
RGC and amacrine cell layers are partially fused, resulting in disruption to
the IPL (Fig. 2B,C). In mutant
eyes, patches of IPL are surrounded by disorganized RGCs, amacrine and bipolar
cells (Fig. 2F,G). Although
different classes of retinal cells are distinguishable by their cell
type-specific morphology in pacrw95 retinae, histological
differences between the retinal neurons are less distinct in
pacrw95/fr7 eyes (Fig.
2C,G) and are absent altogether in pacfr7 eyes
(Fig. 2D,H).
|
To elucidate whether disrupted lamination in pac mutants is due to defective formation of retinal layers or alternatively due to a failure in their maintenance, we examined early steps of IPL formation in living eyes. In 2 dpf wild-type eyes, the first processes of the IPL accumulate at the interface between RGC and amacrine cell layers from the outset (Fig. 2M). By contrast, in pacrw95 and to a greater extent in pacrw95/fr7 eyes, the initial aggregation of neurites occurs at many positions in the amacrine layer (Fig. 2N,O). Over time these initial neuritic foci enlarge until they form the rosettes characteristic of later stages (data not shown). Reflecting later phenotypic severity, initial association of neurites occurs randomly throughout the eyes of pacfr7 embryos (Fig. 2P). Together, these data indicate that Ncad is required for establishment of both retinal cell type morphology and retinal lamination and that inner layers of the retina are most sensitive to reduced levels of Ncad activity.
Neuronal identity is established but patterning is perturbed in
pac mutants
To determine if neuronal specification or patterning is perturbed in
pac embryos, retinae were labeled with retinal neuron-specific
antibodies. In both pacrw95 and
pacrw95/fr7 eyes, the ciliary marginal zone (CMZ) at which
neurons are generated is initially indistinguishable from wild-type
(Fig. 2M-O). Indeed, in all
mutant allele combinations, differentiated RGCs, Müller glia, amacrine,
bipolar and photoreceptor cells are present
(Fig. 3; data not shown).
However, their distribution is progressively more disturbed from mild to
severe alleles. Thus, in pacrw95 retinae, the position of
photoreceptors and bipolar cells is normal and most amacrine cells are
positioned between RGC and bipolar cell layers
(Fig. 3B,F,J). In
pacrw95/fr7 and to a greater extent in
pacfr7 eyes, the different retinal neuron classes are much
more intermingled (Fig.
3C,D,G,H,K,L). Photoreceptors are the neuronal subtype least
affected by ncad mutations but even they are mis-positioned in
pacrw95/fr7 and pacfr7 eyes
(Fig. 3K,L).
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|
Ncad is required to regulate outgrowth of amacrine cell
processes
In pacrw95 eyes, the IPL is severely disrupted despite
the normal establishment of cell polarity, development of the CMZ and
localization of proliferating cells. This suggests that Ncad regulates
additional events during the formation of retinal laminae. The detection of
mispositioned neuropil during formation of the IPL raised the possibility that
outgrowth of amacrine cell processes may be directly perturbed by
ncad mutations. To assess if this is the case, we examined the
morphology of individual retinal neurons in pacrw95
embryos by labeling small numbers of cells with GFP. In wild-type retinae,
neuroepithelial progenitor cells produce columns of neurons in which specific
neuronal classes differentiate at appropriate apicobasal positions
(Fig. 5A,C). Prior to 52 hpf,
there is relatively little outgrowth of amacrine cell processes, and those
that do form are small. In striking contrast, large and irregularly branched
neurites are frequently observed throughout the presumptive INL of
pacrw95 embryos (Fig.
5B) and sorting of different cell-types into laminae is less
distinct (Fig. 5D). Expression
of high levels of Pax6 at 72 hpf confirmed that the neurons with exuberant and
ectopic processes in pacrw95 eyes are indeed amacrine
cells (Fig. 5E,F)
(Macdonald and Wilson, 1997).
Despite the amacrine cell defects, the morphology of cell somata of RGCs and
bipolar cells in pacrw95 eyes resembles wild type at all
stages (Fig. 5G,H). In contrast
to amacrine cell processes, anti-PKC labeled bipolar cell axons are oriented
relatively normally towards the patches of IPL in pacrw95
eyes (Fig. 5I,J). These data
indicate that the irregularly branched neurites belong to amacrine cells, and
suggest that abnormal outgrowth of amacrine cell processes results in
disruption of the IPL in pacrw95.
|
Ncad function is required for guidance of RGC axons
Previous studies have shown that in the absence of Ncad function, RGC axons
exit the eye but may not form a normal optic chiasm
(Lele et al., 2002). To
elucidate RGC axon guidance defects, we determined the trajectories of RGC
axons in pac mutants by combined diI-diO labeling. This analysis
revealed RGC axon defects at many positions along their pathway
(Fig. 6A,B). Within the optic
nerve, RGC axons were sometimes defasciculated and some axons exited the nerve
before reaching the midline to adopt ipsilateral trajectories
(Fig. 6B). Intermingling of
axons from the two eyes at the optic chiasm was often observed
(Fig. 6D). Within the
diencephalon, axons occasionally deviated from the optic tract despite usually
maintaining a course towards the tectum
(Fig. 6B). As RGC axons
approached their target zones, they formed terminal arbors within the tectum.
At the stages examined, both ipsilateral and contralateral axons formed
overlapping terminal arbors within the tecta
(Fig. 6B).
pacrw95/fr7 and pacfr7 embryos showed
similar defects although phenotypic penetrance was lower in
pacrw95/fr7 (Table
1). By contrast, pacrw95 embryos show normal
projections (Table 1),
suggesting that low Ncad activity is sufficient to rescue these trajectory
defects.
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|
In order to address if pathfinding errors by pac mutant RGC axons are alleviated if the diencephalic environment is normal, we transplanted mutant cells into wild-type eyes at 24 hpf. These late transplanted cells did not migrate out of the eye and in cases where they differentiated as RGCs, axons formed tightly fasciculated contralateral projections (n=5/5; Fig. 6J). These data add support to the conclusion that local environmental defects contribute significantly to the axon guidance defects of Ncad deficient RGC axons.
Ncad is required for closure of the choroid fissure
Subsequent to outgrowth of RGC axons, ventral nasal and ventral temporal
retinal tissue fuses to close the choroid fissure
(Fig. 7A,C). This process is
disrupted resulting in severe coloboma in pacfr7 and
pacfr7/rw95 eyes (Fig.
7B; data not shown). In these mutants, retinal neurons stream out
of the back of the eye towards or even into the diencephalon
(Fig. 6F,
Fig. 7D). The choroid fissure
in pacrw95 embryos is macroscopically indistinguishable
from wild type but sectioning reveals some retinal cells detached from the
neural retina through the exit point of the optic nerve (data not shown).
|
Ncad is required for lens fiber formation
In addition to retinal phenotypes lens fiber formation is also perturbed in
pac mutants. In pacfr7, degeneration of lens
fibers occurs, suggesting that Ncad is required for proper differentiation of
the lens (Fig. 8A-C). To
elucidate how lens fiber formation is perturbed in pac mutants, we
analyzed lens cell morphogenesis. Until at least 37 hpf, the lens developed
normally in pacfr7 (31 hpf;
Fig. 8D,E). However, at around
48 hpf, differentiating fiber cells fail to elongate and remain on the basal
side of the lens sphere (Fig.
8F,G). These data suggest that N-cadherin is required for
maturation of lens fibers.
|
In embryos treated with SU5402 from 8 to 96 hpf, retinal axons frequently
projected to ipsilateral optic tecta (Table
1; Fig. 9A,B) or in
some cases failed to extend to the midline
(Fig. 9C). Fgf signaling is
required for patterning of midline tissue where the optic axons normally
decussate (Shanmugalingam et al.,
2000) and the domains of slit3 expression that normally
delineate the retinal axon pathway are perturbed by SU5402 treatment
(Fig. 9D), suggesting that
midline defects are likely to contribute to retinal misguidance in SU5402
embryos. When SU5402 was applied at stages after midline patterning had
occurred then retinal axons did project contralaterally. However, they often
failed to enter the tecta (Table
1; Fig. 9E-G),
consistent with previous observations that blocking Fgf signaling slows the
extension of retinal axons and can lead to failure to enter the tecta in frog
(McFarlane et al., 1996
;
Lom et al., 1998
). From these
data, it is likely that environmental cues required for crossing the midline
are affected by abrogation of Fgf signaling as is the ability of retinal axons
to enter their termination zones.
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DISCUSSION |
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Adhesive activity of Ncad is reduced in pac alleles
As the retinal phenotype in pacrw95 is weaker than in
the likely null pacfr7 allele, then Ncad may retain some
activity in pacrw95 embryos. Antibody labeling shows that
Ncad is translated in pacrw95, and the correct
localization of catenin and actin (M.Y. and I.M., unpublished) suggests that
it is able to assemble apical cytoplasmic protein complexes. These data
suggest that the intracellular domain of Ncad is still active in
pacrw95. Nevertheless, pacrw95-Ncad
has reduced cell adhesive activity, consistent with phenotypic severity being
related to the dose of the pacrw95-Ncad allele. As Ncad is
expressed broadly in CNS, this hypomorphic allele will continue to be useful
to elucidate various other roles of Ncad that may be masked in the null
allele.
Biochemical studies have reported that soluble truncated forms of cell
adhesion molecules may function in a dominant-negative manner
(Doherty et al., 1995). In
pacfr7, a stop codon occurs in the extracellular region,
raising the possibility that a soluble, and hence interfering, form of Ncad
may be produced in pacfr7. However, retinal phenotypes are
only observed in embryos homozygous for pacfr7 mutations,
strongly suggesting that pacfr7 eye phenotypes result from
loss-of-function of Ncad. Indeed, Ncad morphants exhibit the same retinal
defects as pacfr7 mutants, supporting previous data
(Lele et al., 2002
) that
pacfr7 is a null mutation.
Ncad is required for localizing apical proteins and cell division in
the neural retina
A feature common to all of the zebrafish mutants affecting retinal
lamination (ome, nok, has, glo and pac) is the disruption to
proteins associated with junctional complexes and apicobasal polarity of
retinal cells (Malicki and Driever,
1999; Horne-Badovinac et al.,
2001
; Pujic and Malicki,
2001
; Peterson et al.,
2001
; Wei and Malicki,
2002
) (this study). In nok, ome and glo retinae,
adherens junctions are not maintained in the retinal neuroepithelium, and it
is thought that this contributes to the abnormal localization of dividing
cells (Malicki and Driever,
1999
; Pujic and Malicki,
2001
; Wei and Malicki,
2002
).
Although the cadherin-catenin complex is a major component of adherens
junctions, it is the maintenance and not the initial establishment of these
junctions that is disrupted in pacfr7 retinae. Perhaps
reflecting the late requirement for Ncad activity, organization of
proliferative neuroepithelial cells at the CMZ of pac mutants is
relatively normal compared with more mature central retina. These observations
raise three possibilities. The first is that cadherins are involved in
establishment of adherens junctions and that other cadherins compensate for
the loss of Ncad function at early stages of retinal maturation. R-cadherin is
one candidate as it heterodimerizes with Ncad in vitro
(Shan et al., 2000) and is
expressed in the eye, although perhaps at stages too late to regulate early
neuroepithelial junctions (Liu et al.,
2001
).
A second possibility is that cadherin function is not essential for the
initial establishment of adherens junctions. Indeed, adherens junctions can
form in the absence of Hmr-1 cadherin, the only classic cadherin in C.
elegans (Costa et al.,
1998) and although DE-cadherin/Shotgun is important for
morphogenesis of epithelial cells in flies, adherens junctions form in zygotic
null shotgun mutants (Uemura et
al., 1996
; Tepass et al.,
1996
). It is possible that other protein complexes compensate for
the absence of cadherin-catenin based cell interactions during adherens
junction formation. For example, a complex containing Nectin and Afadin is
concentrated at adherens junctions and interacts with the actin cytoskeleton
(Mandai et al., 1997
;
Takahashi et al., 1999
) and
both the Par/aPKC and Crumbs/Stardust pathways have key roles in establishment
of the zonula adherens (reviewed by
Tepass, 2002
;
Johnson and Wodarz, 2003
).
Altogether, these data are consistent with our observation that initial
formation of adherens junctions seems to be normal in the null allele of
pac.
A third possibility is that Ncad regulates the renewal of adherens
junctions that may occur following cell divisions. Disruption of adherens
junction integrity begins ventrally and spreads dorsally in
pacfr7 retina with junctional fragments eventually
scattering throughout the retina. This progressive degeneration of adherens
junctions is reminiscent of that seen in the neural tube after Ncad blockage
(Gänzler-Odenthal and Redies,
1998), suggesting similar Ncad-dependent regulation of junctional
complex renewal may occur both in the brain and eye.
Ncad function is required for retinal lamination
Defects in localization of cell divisions and adhesive interactions between
neuroepithelial cells are very likely to contribute to the lamination defects
in pac and other retinal lamination mutants. However, in the
hypomorphic pacrw95 allele, there are no obvious defects
in early neuroepithelial cell organization or in the localization of cell
divisions. Despite this, RGCs and amacrine cells fail to segregate and
consequently differentiate at inappropriate apicobasal positions. This
phenotype suggests that Ncad is also involved in the guidance of RGCs and
amacrine cells to their correct sites of differentiation. The fact that RGC
and amacrine cell organization is disrupted to a greater extent than outer
retinal neurons adds further support to the notion that this phenotype is
distinct from early steps of retinal patterning. In other zebrafish mutants in
which cell division and/or junctional complexes are disrupted, it is generally
the outer retina that is most severely affected, with RGCs showing the least
degree of disorganization (Malicki and
Driever, 1999; Horne-Badovinac
et al., 2001
; Jensen et al.,
2001
; Pujic and Malicki,
2001
; Peterson et al.,
2001
; Wei and Malicki,
2002
).
Little is known of the signals that position different neuronal classes
after they are born in the retina. From genetic analyses of nok and
moe zebrafish mutants, it has been speculated that guidance cues
exist on neuroepithelial cells (Wei and
Malicki, 2002) and that positioning signals pass between pigmented
epithelium and neural retina (Jensen et
al., 2001
). Furthermore, the absence of Müller glia is
closely associated with formation of photoreceptor rosettes, suggesting that
Müller glia may also be a source of guidance signals
(Wang et al., 2002
). However,
in all cases, the identity of the actual guidance cues is unknown. We suggest
that Ncad contributes to the ability of cells to sort into laminae containing
discrete neuronal classes, perhaps by promoting cell class specific homotypic
interactions.
There is extensive literature showing that cadherins contribute to cell
type specific sorting (reviewed by Redies,
2000) and recent studies have provided compelling evidence that
cadherins mediate sorting of pools of different classes of motoneurons in the
lateral motor column (Price et al.,
2002
). Indeed, our cell sorting/intermingling assays suggest
levels of Ncad influence cell affinities. This suggests that even though
ncad is widely expressed (Liu et
al., 2001
), subtle cell-type specific differences in the activity
of Ncad protein may contribute to sorting of different neuronal classes.
Ncad modulates outgrowth of amacrine cell dendrites
A surprising phenotype in the hypomorphic pacrw95
allele is the exuberant and undirected extension of amacrine cell processes
within the INL. With the exception of interplexiform cells, the only neurites
elaborated from all classes of amacrine cells are horizontally directed
processes within one or more laminae of the IPL. The mechanisms controlling
the initial directed outgrowth of amacrine processes are unknown but are
unlikely to depend upon adjacent RGCs as an IPL forms in the zebrafish
lakritz mutant which lacks RGCs
(Kay et al., 2001). In
pac mutants, amacrine cell morphology is more severely disrupted than
other neuronal cell types, suggesting that Ncad activity has specific roles in
directing the outgrowth of amacrine cell processes. As pac amacrine
cells can adopt more normal morphologies in a wild-type environment, then it
is likely that Ncad is a local environmental signal that is read by the growth
cones of the amacrine processes. This is consistent with the observation that
Ncad is expressed at high levels in the IPL
(Liu et al., 2001
) and that
neurites expressing Ncad preferentially elongate along substrates that express
the highest levels of Ncad (Redies,
2000
).
Perhaps analogous to the amacrine cell phenotype, in flies, mosaic analyses
have revealed roles for Ncad in the selection by retinal axons of appropriate
target laminae within the optic lobe (reviewed by
Clandinin and Zipursky, 2002).
However, in this case, over-elaboration of processes is not observed and the
morphology of terminals is relatively normal
(Lee et al., 2001
). Similarly,
application of function blocking Ncad antibodies to avian retinal axons and
their tectal targets disrupts lamina selection by RGC growth cones
(Inoue and Sanes, 1997
).
Recent studies using cultured vertebrate hippocampal neurons have also
implicated cadherin function in modulating dendritic morphology
(Togashi et al., 2002
) but
even in situations where all cadherin activity is likely to be blocked,
overall organization and length of dendritic processes are not significantly
affected. The alterations in amacrine cell processes in fish pac
mutants are considerably more severe than these other situations.
Ncad is required for epithelial fusion of the choroid fissure
Although there are few, if any, studies on the cellular basis of choroid
fissure closure, it almost certainly involves a series of events similar to
those that characterize other epithelial fusions
(Martin and Wood, 2002). The
initial interactions between leading edge epithelial cells may be mediated by
a variety of different proteins that include, at least in vertebrates, Eph
receptors and ephrins (reviewed by
Holmberg and Frisén,
2002
). Subsequent to these events, adherens junctions stabilize
the connections between the apposing epithelia
(Simske and Hardin, 2001
).
Within the ventral eye, the abrogation of Ncad function may compromise the
stabilization of any interactions between ventronasal and ventrotemporal
retinal cells leading to a failure of epithelial fusion.
It is likely that the epithelial cells that mediate choroid fissure closure
are Pax2+ prospective astrocytes that line the choroid fissure
(Sanyanusin et al., 1995;
Torres et al., 1996
;
Macdonald et al., 1997
). The
exodus of Pax2.1+ cells from the optic nerve into the brain of
pac mutants suggests that the normal adhesive interactions that
maintain the cohesion of the prospective astrocytic network of the nascent
optic nerve are compromised. Indeed, the reduced convergence of ventronasal
and ventrotemporal retinal cells is likely to compromise further the ability
of such cells to undergo fusion. Together, these observations strongly suggest
that the mis-localization of Pax2.1+ prospective astrocytes coupled
with compromised adhesive interactions/junction formation between these cells
is the cause of the coloboma in pac mutants.
Ncad maintains boundaries between retina, optic nerve and ventral
brain
Perhaps the most dramatic of all the eye phenotypes after abrogation of
Ncad activity is the invasion of the brain by retinal neurons and prospective
optic nerve astrocytes. This phenotype implies that retinal neurons no longer
respect the tissue compartment boundaries between retina, optic stalk/ nerve
and ventral brain. Even in chimaeric embryos in which mutant cells are in a
predominantly wild-type environment, pac mutant retinal neurons
invade the ventral brain.
It is widely believed that classical cadherins contribute to the separation
of cell compartments during embryonic development
(Redies, 2000). For example,
within the telencephalon, R-cadherin and cadherin 6 are thought to contribute
to the sorting of cells between pallial and sub-pallial territories
(Inoue et al., 2001
). Ncad
may, therefore, contribute to the maintenance of boundaries between neural
retina, optic stalk and ventral brain. However, how it would achieve this is
uncertain as no differences in Ncad expression have been reported between
these different regions. A common feature of the retina/optic nerve and optic
nerve/ventral brain interfaces is the presence of Pax2.1+ cells
defining the boundaries. We have previously shown that these
Pax2.1+ cells provide barriers to axonal navigation
(Macdonald et al., 1997
). Our
current studies indicate that Pax2.1+ cells fail to tightly
associate with each other in pac mutants, suggesting that the optic
stalk tissue may play a more comprehensive role in restricting both axonal
extension and neuronal migration.
One further possibility is that retinal neurons are drawn out of the eye by exiting RGC axons in pac mutants. Indeed, there appears to be little invasion of the brain by retinal cells prior to outgrowth of the optic nerve (data not shown). If neuronal contacts within the retina are weakened, then perhaps there is less anchoring of the neurons within the neural retina and a greater propensity to be drawn out of the eye.
Ncad activity is required non-autonomously for guidance of RGC
axons
pac mutants exhibit severe retinotectal pathfinding defects with
axons adopting ectopic trajectories both ipsilaterally and within the optic
tract. Axons from the two eyes are frequently intermingled at the optic
chiasm, no doubt contributing to the ipsilateral axon guidance defects.
Although many other studies have suggested that Ncad may be required for
navigation of RGC axons (Matsunaga et al.,
1988b; Riehl et al.,
1996
), we found that pac mutant RGC axons could establish
a tightly fasciculated contralateral projection in a wild-type background.
This suggests that the majority of the severe guidance defects can be
attributed to pathway defects within the pac optic nerve, chiasm and
tract. Indeed the ectopic distribution of Pax2.1+ cells and retinal
neurons along the RGC axon pathway are very likely to disrupt growth cone
navigation. Our studies do not discount the possibility that Ncad is
cell-autonomously required within RGCs for more subtle aspects of guidance,
such as selective fasciculation (Treubert-Zimmerman et al., 2002) or target
selection (Lee et al.,
2001
).
Ncad is required for lens fiber differentiation
Lens fiber differentiation consists of a series of tightly coordinated
events that include the spatially regulated proliferation of superficial lens
epidermal cells, elongation and migration of lens fiber cells, and fusion of
maturing fiber cells at the medial lens suture
(Fig. 8A). Although it has been
suggested that cell adhesion molecules including Ncad are involved in
morphogenesis of the lens (Bassnett et al.,
1999; Leong et al.,
2000
), early steps in lens vesicle formation proceed normally in
pacfr7. It is the elongation and differentiation of lens
fiber cells that requires Ncad function. The late appearance of lens defects
is consistent with the fact that, as within the retina, adherens junctions
initially form in the lens vesicle of pac embryos (M.Y. and I.M.,
unpublished). As adherens junctions form at the apical ends of lens fiber
cells (Lo et al., 2000
),
absence of Ncad may reduce the ability of the elongating lens fiber cells to
absorb mechanical stresses with the consequence that they lose apical
attachments and accumulate basally within the lens.
Ncad function is largely independent of Fgfr function during eye
development
Although it was originally thought that cadherins regulate adhesion solely
through homophilic interactions, it is likely that the situation is more
complex. For example, there is now ample evidence of heterophilic interactions
between different cadherins and, in flies, a receptor-type protein tyrosine
phosphatase called DLAR is intimately associated with Ncad activity (reviewed
by Redies, 2000;
Clandinin and Zipursky,
2002
).
An interaction between Ncad and Fgfrs was initially suggested as they share
HAV binding motifs (Williams et al.,
1994), previously thought only necessary to mediate homophilic
interactions. The HAV motif in the EC1 domain of Ncad is involved in
homophilic interactions (Shapiro et al.,
1995
; Overduin et al.,
1995
), whereas the one in EC4 is implicated in the interaction
with Fgfrs (Williams et al.,
2001
). As the pacrw95 mutation is within the
EC4 domain, it is likely to disrupt any interactions of this domain. Given
these findings, we attempted to address whether Ncad function requires Fgfr
function by comparing Ncad mutant phenotypes to Fgfr blockade phenotypes.
Within the eye, none of the pac mutant phenotypes was phenocopied by Fgfr inhibition, suggesting that Ncad and Fgfrs function independently during retinal development. Furthermore, although there are superficial similarities between the RGC axon phenotypes in pac mutants and SU5402-treated embryos, in both cases the major defects are likely to be due to problems within the environment through which the RGC axons extend. It remains possible that more subtle aspects of Ncad function during RGC axon extension do require Fgfr function and indeed for motor axons, we observe similar Ncad and Fgfr phenotypes (Z.L. and S.W.W., unpublished). However, it is reasonable to conclude that Ncad functions are largely independent of Fgfr activity with respect to most/all of the roles we have revealed for Ncad during eye development.
In conclusion, through the use of severe and weak pac mutant alleles we have revealed a variety of crucial functions for Ncad during the formation of the vertebrate eye. Indeed, given the complexity of the pac mutant phenotypes, it is very likely that there will be further roles for Ncad in this region of the CNS. Our work identifies Ncad activity as being absolutely central to the establishment of a functional visual system.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
Recent studies have shown that glass onion is allelic with
parachute (Malicki et al.,
2003).
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REFERENCES |
---|
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---|
Bassnett, S., Missey, H. and Vucemilo, I.
(1999). Molecular architecture of the lens fiber cell basal
membrane complex. J. Cell Sci.
112,2155
-2165.
Bitzur, S., Kam, Z. and Geiger, B. (1994). Structure and distribution of N-cadherin in developing zebrafish embryos: morphogenetic effects of ectopic over-expression. Dev. Dyn. 201,121 -136.[Medline]
Brand, M., Heisenberg, C.-P., Jiang, Y. J., Beuchle, D., Lun,
K., Furutani-Seiki, M., Granato, M., Haffter, P., Hammerschmidt, M., Kane, D.
A. et al. (1996). Mutations in zebrafish genes affecting the
formation of the boundary between midbrain and hindbrain.
Development 123,179
-190.
Broadbent, I. D. and Pettitt, J. (2002). The C. elegans hmr-1 gene can encode a neuronal classic cadherin involved in the regulation of axon fasciculation. Curr. Biol. 12, 59-63.[CrossRef][Medline]
Clandinin, T. R. and Zipursky, S. L. (2002). Making connections in the fly visual system. Neuron 35,827 -841.[Medline]
Costa, M., Raich, W., Agbunag, C., Leung, B., Hardin, J. and
Priess, J. R. (1998). A putative catenin-cadherin system
mediates morphogenesis of the Caenorhabditis elegans embryo.
J. Cell Biol. 141,297
-308.
Doherty, P., Williams, E. and Walsh, F. S. (1995). A soluble chimeric form of the L1 glycoprotein stimulates neurite outgrowth. Neuron 14, 57-66.[Medline]
Esni, F., Johansson, B. R., Radice, G. L. and Semb, H. (2001). Dorsal pancreas agenesis in N-cadherin-deficient mice. Dev. Biol. 238,202 -212.[CrossRef][Medline]
Gänzler-Odenthal, S. I. I. and Redies, C.
(1998). Blocking N-cadherin function disrupts the epithelial
structure of differentiating neural tissue in the embryonic chick brain.
J. Neurosci. 18,5415
-5425.
Holmberg, J. and Frisén, J. (2002). Epherins are not only unattractive. Trends Neurosci. 25,239 -243.[CrossRef][Medline]
Horne-Badovinac, S., Lin, D., Waldron, S., Schwarz, M., Mbamalu,
G., Pawson, T., Jan, Y.-N., Stainier, D. Y. R. and Abdelilah-Seyfried, S.
(2001). Positional cloning of heart and soul reveals
multiple roles for PKC in zebrafish organogenesis. Curr.
Biol. 11,1492
-1502.[CrossRef][Medline]
Hutson, L. D. and Chien, C.-B. (2002). Pathfinding and error correction by retinal axons: the role of astray/robo2. Neuron 33,205 -217.[Medline]
Inoue, A. and Sanes, J. R. (1997).
Lamina-specific connectivity in the brain: regulation by N-cadherin,
neurotrophins, and glycocojugates. Science
276,1428
-1431.
Inoue, T., Tanaka, T., Takeichi, M., Chisaka, O., Nakamura, S.
and Osumi, N. (2001). Role of cadherins in maintaining the
compartment boundary between the cortex and striatum during development.
Development 128,561
-569.
Jensen, A. M., Walker, C. and Westerfield, M.
(2001). mosaic eyes: a zebrafish gene required in
pigmented epithelium for apical localization of retinal cell division and
lamination. Development
128,95
-105.
Jiang, Y.-J., Brand, M., Heisenberg, C.-P., Beuchle, D., Frutani-Seiki, M., Kelsh, R. N., Warga, R. M., Granato, M., Haffter, P., Hammerschmidt, M. et al. (1996). Mutations affecting neurogenesis and brain morphology in the zebrafish, Danio rerio. Development 123,205 -216.
Johnson, K. and Wodarz, A. (2003). A genetic hierarchy controlling cell polarity. Nat. Cell Biol. 5, 12-14.[CrossRef][Medline]
Kay, J. N., Finger-Baier, K. C., Roeser, T., Staub, W. and Baier, H. (2001). Retinal ganglion cell genesis requires lakritz, a zebrafish atonal homolog. Neuron 30,725 -736.[CrossRef][Medline]
Larison, K. D. and Bremiller, R. (1990). Early onset of phenotype and cell patterning in the embryonic zebrafish retina. Development 109,567 -576.[Abstract]
Lee, C.-H., Herman, T., Clandinin, T. R., Lee, R. and Zipursky, S. L. (2001). N-cadherin regulates target specificity in the Drosophila visual system. Neuron 30,437 -450.[CrossRef][Medline]
Lele, Z., Folchert, A., Concha, M., Rauch, G.-J., Geisler, R., Rosa, F., Wilson, S. W., Hammerschmidt, M. and Bally-Cuif, L. (2002). parachute/n-cadherin is required for morphogenesis and maintained integrity of the zebrafish neural tube. Development 129,3281 -3294[Medline]
Leong, L., Menko, A. S. and Grunwald, G. B. (2000). Differential expression of N- and B-cadherin during lens development. Invest. Opth. Vis. Sci. 41,3503 -3510.
Liu, Q., Babb, S. G., Novince, Z. M., Doedens, A. L., Marrs, J. and Raymond, P. A. (2001). Differential expression of cadherin-2 and cadherin-4 in the developing and adult zebrafish visual system. Vis. Neurosci. 18,923 -933.[Medline]
Livesey, F. J. and Cepko, C. L. (2001). Vertebrate neural cell-fate determination: lessons from the retina. Nat. Rev. Neurosci. 2,109 -118.[CrossRef][Medline]
Lo, W.-K., Shaw, A. P., Paulsen, D. F. and Mills, A. (2000). Spatiotemporal distribution of zonulae adherens and associated actin bundles in both epithelium and fiber cells during chick lens development. Exp. Eye Res. 71, 45-55.[CrossRef][Medline]
Lom, B., Höpker, V., McFarlane, S., Bixby, J. L. and Holt, C. E. (1998). Fibroblast growth factor receptor signaling in Xenopus retinal axon extension. J. Neurobiol. 37,633 -641.[CrossRef][Medline]
Luo, Y., Ferreira-Cornwell, M. C., Baldwin, H. S., Kostetskii,
I., Lenox, J. M., Lieberman, M. and Radice, G. L. (2001).
Rescuing the N-cadherin knockout by cardiac-specific expression of N- or
E-cadherin. Development
128,459
-469.
Macdonald, R., Scholes, J., Strähle, U., Brennan, C.,
Holder, N., Brand, M. and Wilson, S. W. (1997). The Pax
protein Noi is required for commissural axon pathway formation in the rostral
forebrain. Development
124,2397
-2408.
Macdonald, R. and Wilson, S. W. (1997). Distribution of Pax6 protein during eye development suggests discrete roles in proliferative and differentiated visual cells. Dev. Genes Evol. 206,363 -369.[CrossRef]
Malicki, J. and Driever, W. (1999). oko
meduzy mutations affect neuronal patterning in the zebrafish retina and
reveal cell-cell interactions of the retinal neuroepithelial sheet.
Development 126,1235
-1246.
Malicki, J., Jo, H. and Pujic, Z. (2003). The zebrafish N-cadherin, encoded by the glass onion locus, plays an essential role in retinal patterning. Dev. Biol. (in press).
Mandai, K., Nakanishi, H., Satoh, A., Obaishi, H., Wada, M.,
Nishioka, H., Itoh, M., Mizoguchi, A., Aoki, T., Fujimoto, T. et al.
(1997). Afadin: a novel actin filament-binding protein with
one PDZ domain localized at cadherin-based cell-to-cell adherens junction.
J. Cell Biol. 139,517
-528.
Marquardt, T. and Gruss, P. (2002). Generating neuronal diversity in the retina: one for nearly all. Trends Neurosci. 25,32 -38.[CrossRef][Medline]
Martin, P. and Wood, W. (2002). Epithelial fusions in the embryo. Curr. Opin. Cell Biol. 14,569 -574.[CrossRef][Medline]
Masai, I., Stemple, D. L., Okamoto, H. and Wilson, S. W. (2000). Midline signals regulate retinal neurogenesis in zebrafish. Neuron 27,251 -263.[Medline]
Matsunaga, M., Hatta, K. and Takeichi, M. (1988a). Role of N-cadherin cell adhesion molecules in the histogenesis of neural retina. Neuron 1, 289-295.[Medline]
Matsunaga, M., Hatta, K., Nagafuchi, A. and Takeichi, M. (1988b). Guidance of optic nerve fibres by N-cadherin adhesion molecules. Nature 334,62 -64.[CrossRef][Medline]
McFarlane, S., Cornel, E., Amaya, E. and Holt, C. E. (1996). Inhibition of FGF receptor activity in retinal ganglion cell axons causes errors in target recognition. Neuron 17,245 -254.[Medline]
Mellitzer, G., Xu, Q. and Wilkinson, D. G. (1999). Eph receptors and ephrins restrict cell intermingling and communication Nature 400, 77-81.[CrossRef][Medline]
Mohammadi, M., McMahon, G., Sun, L., Tang, C., Hirth, P., Yeh,
B. K., Hubbard, S. R. and Schlessinger, J. (1997). Structure
of the tyrosine kinase domain of fibroblast growth factor receptor in complex
with inhibitors. Science
276,955
-960.
Ohno, S. (2001). Intercellular junctions and cellular polarity: the PAR-aPKC complex, a conserved core cassette playing fundamental roles in cell polarity. Curr. Opin. Cell Biol. 13,641 -648.[CrossRef][Medline]
Overduin, M., Harvey, T. S., Bagby, S., Tong, K. I., Yau, P., Takeichi, M. and Ikura, M. (1995). Solution structure of the epithelial cadherin domain responsible for selective cell adhesion. Science 267,386 -389.[Medline]
Peterson, R. T., Mably, J. D., Chen, J.-N. and Fishman, M. C. (2001). Convergence of distinct pathways to heart patterning revealed by the small molecule concentramide and the mutation heart-and-soul. Curr. Biol. 11,1481 -1491.[CrossRef][Medline]
Plump, A. S., Erskine, L., Sabatier, C., Brose, K., Epstein, C. J., Goodman, C. S., Mason, C. A. and Tessier-Lavigne, M. (2002). Slit1 and Slit2 cooperate to prevent premature midline crossing of retinal axons in the mouse visual system. Neuron 33,219 -232.[Medline]
Price, S. R., de Marco Garcia, N. V., Ranscht, B. and Jessell, T. M. (2002). Regulation of motor neuron pool sorting by differential expression of type II cadherins. Cell 109,205 -216.[Medline]
Pujic, Z. and Malicki, J. (2001). Mutation of the zebrafish glass onion locus causes early cell-nonautonomous loss of neuroepithelial integrity followed by severe neuronal patterning defects in the retina. Dev. Biol. 234,454 -469.[CrossRef][Medline]
Radice, G. L., Rayburn, H., Matsunami, H., Kundsen, K. A., Takeichi, M. and Hynes, R. O. (1997). Developmental defects in mouse embryos lacking N-cadherin. Dev. Biol. 181, 64-78.[CrossRef][Medline]
Redies, C. (2000). Cadherins in the central nervous system. Prog. Neurobiol. 61,611 -648.[CrossRef][Medline]
Riehl, R., Johnson, K., Bradley, R., Grunwald, G. B., Cornel, E., Lilienbaum, A. and Holt, C. E. (1996). Cadherin function is required for axon outgrowth in retinal ganglion cell in vivo. Neuron 17,979 -990.[Medline]
Sanyanusin, P., Schimmenti, L. A., McNoe, L. A., Ward, T. A., Pierpont, M. E. M., Sullivan, M. J., Dobyns, W. B. and Eccles, M. R. (1995). Mutation of the PAX2 gene in a family with optic nerve colobomas, renal anomalies and vesicoureteral reflux. Nat. Genet. 9,358 -363.[Medline]
Shan, W. S., Tanaka, H., Phillips, G. R., Arndt, K., Yoshida,
M., Colman, D. R. and Shapiro, L. (2000). Functional
cis-heterodimers of N- and R-cadherins. J. Cell Biol.
148,579
-590.
Shanmugalingam, S., Houart, C., Picker, A., Reifers, F.,
Macdonald, R., Barth, A., Griffin, K., Brand, M. and Wilson, S. W.
(2000). Ace/Fgf8 is required for forebrain commissure formation
and patterning of the telencephalon. Development
127,2549
-2561.
Shapiro, L., Fannon, A. M., Kwong, P. D., Thompson, A., Lehmann, M. S., Grübel, G., Legrand, J.-F., Als-Nielsen, J., Colman, D. R. and Hendrickson, W. A. (1995). Structural basis of cell-cell adhesion by cadherins. Nature 374,327 -337.[CrossRef][Medline]
Simske, J. S. and Hardin, J. (2001). Getting into shape: epidermal morphogenesis in Caenorhabditis elegans embryos. BioEssays 22,12 -23.[CrossRef]
Skaper, S. D., Kee, W. J., Facci, L., Macdonald, G., Doherty, P. and Walsh, F. (2000). The FGFR1 inhibitor PD 173074 selectively and potently antagonizes FGF-2 neurotrophic and neurotropic effects. J. Neurochem. 75,1520 -1527.[CrossRef][Medline]
Solnica-Krezel, L., Schier, A. F. and Driever, W.
(1994). Efficient recovery of ENU-induced mutations from the
zebrafish germline. Genetics
136,1401
-1420.
Takahashi, K., Nakanishi, H., Miyahara, M., Mandai, K., Satoh,
K., Satoh, A., Nishioka, H., Aoki, J., Nomoto, A., Mizoguchi, A. et al.
(1999). Nectin/PRR: an immunoglobulin-like cell adhesion molecule
recruited to cadherin-based adherens junctions through interaction with
Afadin, a PDZ domain-containing protein. J. Cell Biol.
145,539
-549.
Tepass, U., Gruszynski-DeFeo, E., Haag, T. A., Omatyar, L., Török, T. and Hartenstein, V. (1996). shotgun encodes Drosophila E-cadherin and is preferentially required during cell rearrangement in the neuroectoderm and other morphogenetically active epithelia. Gene Dev. 10,672 -685.[Abstract]
Tepass, U. (2002). Adherens junctions: new insight into assembly, modulation and function. BioEssays 24,690 -695.[CrossRef][Medline]
Togashi, H., Abe, K., Mizoguchi, A., Takaoka, K., Chisaka, O. and Takeichi, M. (2002). Cadherin regulates dendritic spine morphogenesis. Neuron 35, 77-89.[Medline]
Torres, M., Gómez-Pardo, E. and Gruss, P.
(1996). Pax2 contributes to inner ear patterning and
optic nerve trajectory. Development
122,3381
-3391.
Treubert-Zimmermann, U., Heyers, D. and Redies, C.
(2002). Targeting axons to specific fiber tracts in vivo
by altering cadherin expression. J. Neurosci.
22,7617
-7626.
Tsang, M., Friesel, R., Kudoh, T. and Dawid, I. B. (2002). Identification of Sef, a novel modulator of FGF signalling. Nat. Cell Biol. 4, 165-169.[CrossRef][Medline]
Uemura, T., Oda, H., Kraut, R., Hayashi, S., Kataoka, Y. and Takeichi, M. (1996). Zygotic Drosophila E-cadherin expression is required for processes of dynamic epithelial cell rearrangement in the Drosophila embryo. Gene Dev. 10,659 -671.[Abstract]
Wang, Y. P., Dakubo, G., Howley, P., Campsall, K. D., Mazarolle, C. J., Shiga, S. A., Lewis, P. M., McMahon, A. P. and Wallace, V. A. (2002). Development of normal retinal organization depends on Sonic hedgehog signaling from ganglion cells. Nat. Neurosci. 5,831 -832.[Medline]
Wei, Y. and Allis, C. D. (1998). Pictures in cell biology. Trends Cell Biol. 8, 226.
Wei, X. and Malicki, J. (2002). nagie oko, encoding a MAGUK-family protein, is essential for cellular patterning of the retina. Nat. Genet. 31,150 -157.[CrossRef][Medline]
Westerfield, M. (1995). The Zebrafish Book. Salem, OR: University of Oregon Press.
Williams, E. J., Furness, J., Walsh, F. S. and Doherty, P. (1994). Activation of the FGF receptor underlies neurite outgrowth stimulated by L1, N-CAM, and N-cadherin. Neuron 13,583 -594.[Medline]
Williams, E. J., Williams, G., Howell, F. V., Skaper, S. D.,
Walsh, F. S. and Doherty, P. (2001). Identification of an
N-cadherin motif that can interact with the fibroblast growth factor receptor
and is required for axonal growth. J. Biol. Chem.
276,43879
-43886.
Wilson, S. W., Ross, L. S., Parrett, T. and Easter, S. S., Jr (1990). The development of a simple scaffold of axon tracts in the brain of the embryonic zebrafish, Brachydanio rerio.Development 108,121 -145.[Abstract]
Xu, Q., Holder, N., Patient, R. and Wilson, S. W.
(1994). Spatially regulated expression of three receptor tyrosine
kinase genes during gastrulation in zebrafish.
Development 120,287
-299.