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EMBO reports 4, 9, 894–899 (2003)
doi:10.1038/sj.embor.embor919
AOP Published online: 29 August 2003
Loss of eyes in zebrafish caused by mutation of chokh/rx3
Felix Loosli1, Wendy Staub2, Karin C. Finger-Baier2, Elke A. Ober3, Heather Verkade3, Joachim Wittbrodt1 & Herwig Baier2
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1 Developmental Biology Programme, European
Molecular Biology Laboratory, Meyerhofstrasse 1, PO Box
10.2209, 69012 Heidelberg,
Germany
2 Department of Physiology, Programs in Genetics,
Human Genetics, Neuroscience, and Developmental Biology, 513 Parnassus Avenue,
University of California, San Francisco, California
94143 0444, USA
3 Department of Biochemistry, Programs in Genetics,
Human Genetics, Neuroscience, and Developmental Biology, 513 Parnassus Avenue,
University of California, San Francisco, California
94143 0444, USA
To whom correspondence should be addressed
Herwig Baier Tel: +1 415 502 4301; Fax: +1 415 476 4929;
hbaier@itsa.ucsf.edu
Received 30 May 2003; Accepted 15 July 2003; Published online 29 August 2003.
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Abstract
The vertebrate eye forms by specification of the retina anlage and
subsequent morphogenesis of the optic vesicles, from which the neural retina
differentiates. chokh (chk) mutant zebrafish lack eyes from the
earliest stages in development. Marker gene analysis indicates that retinal
fate is specified normally, but optic vesicle evagination and neuronal
differentiation are blocked. We show that the chk gene encodes the
homeodomain-containing transcription factor, Rx3. Loss of Rx3 function in
another teleost, medaka, has also been shown to result in an eyeless phenotype.
The medaka rx3 locus can fully rescue the zebrafish mutant phenotype. We
provide evidence that the regulation of rx3 is evolutionarily conserved,
whereas the downstream cascade contains significant differences in gene
regulation. Thus, these mutations in orthologous genes allow us to study the
evolution of vertebrate eye development at the molecular level.
EMBO reports 4, 9, 894–899 (2003)
doi:10.1038/sj.embor.embor919
AOP Published online: 29 August 2003
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Introduction
The retina and lens of the vertebrate eye originate from an anterior
territory, which comprises the neuroectodermal retina anlage and the abutting
ectodermal lens-competence regions. During neurulation, the cells of the retina
anlage converge at the midline in the prosencephalon, from where they then
evaginate laterally to form the optic vesicles. These, in turn, induce lens
formation in the adjacent competence regions.
Several genes required for the establishment of the retina anlage and
its subsequent transition to optic vesicles have been identified. The
homeodomain-containing transcription factors Six3 and Pax6 are essential for
the determination of retinal fate in the neuroectoderm (Carl
et al., 2002; Gehring, 2002;
Loosli et al., 1999) and also function in
lens formation (Ashery-Padan & Gruss, 2001).
The retinal homeobox transcription factor Rx has a crucial function in the
morphogenesis of the optic primordia to form the optic vesicles (Loosli et al., 2001; Mathers
et al., 1997). Both Six3 and Rx have been shown to control
proliferation in the optic primordium (Andreazzoli et
al., 1999; Carl et al., 2002;
Loosli et al., 2001; Mathers et al., 1997). For proper morphology of the
optic vesicle, the homeobox factor Vsx2/Chx10 is required (Barabino et al., 1997).
Forward genetic screens in fish have been used to isolate an
increasing number of mutations that cause defects in the developing eye
(Malicki et al., 1996; Loosli et al., 2000; for a review, see
Easter & Malicki, 2002). In zebrafish, two
mutants have been described that affect early Wnt
(Wingless/int-related)-signalling-dependent anterior–posterior patterning
of the brain and, as a secondary consequence, result in eyeless or headless
embryos (Kim et al., 2000;
Heisenberg et al., 2001). These mutations
are not specific to eye formation and also lead to severe patterning defects in
other regions of the brain. So far, in the large-scale mutagenesis screens of
zebrafish, surprisingly few mutations were identified that specifically affect
the earliest stage of eye development.
Here, we describe the identification and analysis of a novel zebrafish
mutant that lacks eyes from the earliest stages. The corresponding gene, named
chokh (chk) after the Bangla word for 'eye', encodes Rx3. In view
of the existing rx mutations in other vertebrate species, the isolation
of a zebrafish rx3 mutation closes an important gap in our understanding
of conserved regulatory networks that underlie optic vesicle morphogenesis and
differentiation. This now provides the opportunity to study the evolution of
vertebrate eye development by cross-species comparison at the molecular
level.
Results
Zebrafish chokh mutants lack eyes
Mutants that are homozygous for the ethylnitrosourea-induced allele
s399 of the chk locus lack eyes at all stages of development
(Fig. 1). This phenotype is similar to medaka
eyeless (el) mutants (Winkler et al.,
2000). Both retinal pigmented epithelium and neuroretina are missing.
A lens forms, but it is markedly reduced in size. The overall morphology of the
head and trunk is not affected by the mutation. In addition, all the main
subdivisions of the brain (that is, the forebrain, midbrain and hindbrain), as
well as the inner ear, seem normal. The chk mutation is recessive and
fully penetrant. Mutants do not show visual responses, but respond to touch and
vibration; they hatch and show apparently normal swimming, but die at
 3–4 weeks.
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Figure 1
Morphological analysis of the chokh phenotype. Wild-type and
mutant embryos are shown at 24 h post-fertilization (h.p.f.), 48 h.p.f. and 6
days post-fertilization (d.p.f.). Eyes are absent at all stages of development
in chokh (chk) mutants. The overall morphology of the head and
trunk is normal in mutants. At 48 h.p.f., a small lens is visible in chk
mutants (arrow). fb, forebrain; hb, hindbrain; mb, midbrain.
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The chokh mutation disrupts rx3
The chk mutation was mapped close to the polymorphic
CA-repeat marker z10432 on chromosome 21 using 41 mutant embryos from a
TL/WIK hybrid cross (see Methods). The zebrafish
rx3 gene had previously been mapped to the same position using the T51
radiation-hybrid panel (Geisler et al.,
1999). The similar phenotypes of chk and medaka el
strongly suggested that rx3 might be a candidate gene. We therefore
sequenced the coding region of rx3 in our mutant and discovered a
nonsense mutation in the homeodomain of the gene (Fig.
2A). This mutation is expected to convert the highly conserved tyrosine
codon at position 479 to a stop codon (Fig. 2B).
Therefore, two-thirds of the homeodomain (including the third helix, which is
indispensable for sequence-specific DNA binding) and the entire
carboxy-terminal portion are deleted, resulting in a null allele (Fig. 2C).
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Figure 2
The chokh gene encodes Rx3. (A) Comparison of the
sequencing trace data from wild-type and chk mutant rx3
complementary DNA. Sequencing reveals a T to A point mutation (arrows).
(B) The predicted translation of the wild-type and mutant rx3
open reading frames. The mutation results in a premature stop codon (indicated
by an asterisk) in the homeobox (red). (C) Comparison of zebrafish (Rx3)
and medaka (OlRx3) proteins. The octapeptide (blue), homeodomain (HD; red) and
the otp–aristaless–rx (OAR) domain (green) are indicated. Sequence
identity at the amino-acid level is indicated for the amino- and
carboxy-terminal regions and the homeodomain. The position of the nonsense
mutation in the homeobox of chk and the frameshift mutation of the
control plasmid (pOlRx3-fs) are indicated (asterisks). The mutation is
predicted to cause a truncation of the protein.
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Expression of pax6 and six3 are unaffected by
loss of rx3
The homeobox transcription factors Six3 and Pax6 are key regulators
of vertebrate retina specification and have been shown to function in a
cross-regulatory interaction (Ashery-Padan & Gruss,
2001; Carl et al., 2002). Similar
to other vertebrate species, in the zebrafish embryo Pax6 and Six3 are
expressed in the eye field starting at late gastrula stages (Kobayashi et al., 1998; Püschel et al., 1992; Seo
et al., 1998). We therefore used the expression of
six3a (six3.1) and pax6a (pax6.1) as markers to
examine the effects of loss of rx3 function on early retina development
in chk mutant embryos.
At 10.5 h post-fertilization (h.p.f.), just before optic vesicle
evagination, six3a expression in the anterior neural plate comprises the
retina anlage. Whole-mount in situ hybridization showed that
six3a expression is unaltered in chk mutant embryos (data not
shown). At 11.5 h.p.f., the optic vesicles start to evaginate in wild-type
embryos. This morphogenetic process does not occur in chk mutant embryos. At
this stage, pax6a is expressed in the prosencephalon, which is adjacent
to the head ectoderm, and more posteriorly in the hindbrain and spinal cord. In
chk mutant embryos, pax6a is expressed normally in all of these domains
(data not shown). Hence, the normal early expression of six3a and
pax6a indicate that forebrain patterning, and particularly retinal
specification, occur normally in the absence of rx3 function.
At 19 h.p.f., pax6a expression is prominent in the
diencephalon, the evaginated optic vesicles and the abutting lens placodes
(Fig. 3A). In chk mutant embryos, the diencephalic
expression domain extends further anteriorly, which suggests that retinal
progenitor cells remain in the forebrain instead of evaginating. The expression
in the lens placodes is unaltered in mutant embryos, which indicates that
rx3 is not required for lens induction (Fig. 3B).
Consistent with this, small lenses are visible at 48 h.p.f. (see also
Fig. 1). More posterior pax6a expression in the
hindbrain and spinal cord is unaffected at this stage, too, which is a further
indication that central nervous system (CNS) regionalization is not affected by
loss of rx3.
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Figure 3
Whole-mount in situ hybridization analysis of marker genes in
wild-type embryos and chokh mutants. (A–J) Dorsal views.
Lateral views are shown in (K) and (L), and in the insets in
(A) and (B). Anterior is to the left in all panels. (A,B)
Wild-type (WT) (A) and mutant (B) pax6a expression in the
forebrain and optic vesicles at 19 h.p.f. Note the anteriorly extended
pax6a expression in the mutant. Arrows indicate pax6a expression
in the lens placodes. pax6a expression along the
anterior–posterior axis is identical in wild-type and mutant embryos
(insets in (A) and (B), respectively). (C,D) rx3
expression in the ventral forebrain and optic vesicles of wild-type (C)
and mutant (D) embryos at 14 h.p.f. Note the ectopic rx3
expression in the forebrain of mutants (D) (arrow). (E,F)
rx1 is expressed in the optic vesicles of wild-type embryos (E)
and in the forebrain of mutant embryos (F) at 14 h.p.f. Expression in
the forebrain of the mutant (F) coincides with the position of the
evaginated optic vesicles in wild-type embryos (E). (G,H)
rx2 is expressed in wild-type optic vesicles (G) but not in
mutants (H) at 14 h.p.f. (I–L) vsx2 expression in
the optic cup and ventral midbrain and hindbrain (arrows) of wild-type
(I,K) and mutant (J,L) embryos at 26 h.p.f. Midbrain and
hindbrain expression is unaffected by the mutation.
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Rx3 is required for normal expression of Rx1 and
Rx2
The enlarged pax6a expression domain in the forebrain of
mutant embryos suggested that retinal progenitor cells, once specified, remain
in the forebrain. To test this further, we used other marker genes that are
expressed in retinal progenitor cells. rx3 is initially expressed in the
anteriormost neural plate, in a region which gives rise to the ventral
forebrain and the optic primordia (Chuang et al.,
1999). At 14 h.p.f., its expression is detectable in the presumptive
anterior hypothalamus and the proximal optic vesicles (Fig.
3C). In chk mutant embryos, the rx3 expression domain in
the ventral forebrain extends posteriorly into the region from which the optic
vesicles should evaginate, consistent with the abnormal location of retinal
progenitor cells (Fig. 3D). Furthermore, the finding that
rx3 is expressed normally both in the presumptive hypothalamus and in
retinal progenitor cells shows that rx3 does not function in an
autoregulatory feedback loop, similar to the situation in medaka (Loosli et al., 2001).
We used the retina-specific expression of the homeobox genes
rx1 and rx2 to examine retina development in chk mutants
in more detail. From 14 h.p.f. onwards, rx1 and rx2 are
specifically expressed in the retinal progenitor cells of the optic vesicle
(Fig. 3E,G). At that stage, rx1 is expressed,
although at reduced levels, in the ventral forebrain of mutant embryos at the
position from which the optic vesicles normally evaginate, whereas at 26 h.p.f.
rx1 expression is absent (Fig. 3F; and data not
shown). rx2 expression, however, is completely abolished at all stages
in mutant embryos (Fig. 3H). Thus, the retinal
progenitor-specific expression of rx1 in the forebrain of chk
mutant embryos provides further independent evidence for abnormally located
retinal progenitor cells due to failure of optic vesicle evagination.
Retinal progenitors do not differentiate in chk
mutants
We examined whether retinal progenitor cells in the ventral
forebrain of chk mutant embryos undergo differentiation. At 26 h.p.f.,
the homeobox gene vsx2 is expressed in mitotically active retinal
progenitor cells before terminal differentiation. In the midbrain and
hindbrain, vsx2 is expressed in a ventrally located, paired row of cells
(Barabino et al., 1997). Retinal expression
of vsx2 is not detectable at 26 h.p.f. in chk mutant embryos,
whereas the expression in the double row of the midbrain and hindbrain is not
affected (Fig. 3J,L). Thus, retinal progenitor cells do
not reach this early stage of development in mutant embryos.
The basic helix–loop–helix gene ath5 (lak,
atoh7) is the earliest known marker for differentiating retinal ganglion
cells and is essential for their development during the first differentiation
wave in the retina (Kay et al., 2001;
Masai et al., 2000). In wild-type embryos
at 36 h.p.f., ath5 is expressed in the proximal retina in presumptive
ganglion cells (Fig. 4D). In mutant embryos, ath5
expression is not detectable, indicating that the first step of retinal
differentiation is already disrupted in chk mutant embryos (Fig. 4E).
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Figure 4
Rescue of chokh mutants by the medaka rx3 locus.
(A–C) Lateral views, with the anterior to the left. (D) and
(F) show transverse sections at the level of the eye. (E) is a
frontal view. (A,B) Medaka rx3 (Olrx3) expression at 12 h
post-fertilization in wild-type (WT) embryos injected with the rescue plasmid
(pOlRx3; A) and control plasmid (pOlRx3-fs; B). Note the specific
expression in the ventral forebrain and optic vesicles of injected embryos.
(C) The same Olrx3 probe does not cross-hybridize with uninjected
control embryos. (D–F) ath5 is expressed in ganglion cells
of wild-type retinae (D) at 36 h.p.f., but not in the mutant (E)
(whole-mount, to show the absence of staining). The dark spots in (E)
are due to pigmentation. Injection of pOlRx3 restores morphology and wild-type
ath5 expression (F, compare with D).
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The chk mutant is rescued by medaka
rx3
We analysed whether injection at the one-cell stage of a plasmid
containing medaka rx3 and its regulatory regions (Loosli et al., 2001) is able to rescue the
chk phenotype. As a control, we injected a frameshift derivative of the
plasmid (Loosli et al., 2001). Because a
conserved BspEI restriction site was used to introduce the frameshift
mutation, the same portion of the medaka rx3 protein was deleted in the
control plasmid and by the chk mutation (Fig. 2C).
Wild-type embryos injected with the control plasmid were analysed for medaka
rx3 (Olrx3) expression at 12 h.p.f. by whole-mount in situ
hybridization. For both plasmids, specific and strong Olrx3 expression
was detectable in the ventral forebrain and evaginating optic vesicles at this
stage (Fig. 4A–C). Thus, both plasmids contain the
essential regulatory elements for stage- and region-specific rx3
expression in zebrafish embryos.
In the rescue experiments, we genotyped the injected embryos by PCR,
followed by digestion with BspEI, which cuts the wild-type DNA, but not
the chk mutant DNA. In a representative experiment, on injecting the
pOlRx3 plasmid, we obtained 15 eyeless (non-rescued) mutants among 68 injected
embryos at 48 h.p.f. We genotyped the 53 embryos that had eyes and found that 7
of them had homozygous mutant genotypes. This corresponds to a rescue rate of
32% (7 out of 22 injected mutants), which is similar to the result obtained
with the same plasmid in medaka el mutants (37%; Loosli et al., 2001). Injection of the control
plasmid, pOlRx3-fs (which contains a frameshift mutation in rx3), did
not rescue any of the mutants. For this plasmid, all 34 genotyped embryos (with
eyes) were either heterozygous or were homozygous wild-type.
Interestingly, most rescued mutants (six out of seven in the
experiment described above) seemed to be morphologically normal. The size and
morphology of their eyes were indistinguishable from wild-type embryos. This
result was reproducible and was obtained by double-blind scoring of rescued,
control-injected and uninjected embryos.
We analysed ath5 expression in chk mutant embryos that
were injected with the pOlRx3 plasmid and found that ath5 expression is
restored in the optic cups of rescued embryos at 36 h.p.f. (Fig.
4F). Thus, molecular analysis confirms the result of our morphological
inspections, showing that morphogenesis and differentiation of retinal
progenitor cells in chk mutants is fully restored by the medaka
rx3 locus.
Discussion
We have identified a novel point mutation in zebrafish that results
specifically in a lack of eyes. In summary, map position, sequence analysis and
rescue by complementation show that the chk gene encodes the zebrafish
orthologue of the homeodomain transcription factor Rx3. The nonsense mutation
that we identified is predicted to lead to a complete loss of function, as it
deletes most of the DNA-binding homeodomain and the entire C-terminal portion.
We showed that retinal progenitor cells fail to evaginate as optic vesicles,
and that subsequent differentiation of the optic primordia is blocked. Other
regions of the brain are not affected. Thus, in contrast to other zebrafish
genes that affect anterior brain development, chk/rx3 is specific
for the developing optic primordia.
Our analysis of marker gene expression shows that rx3
requirement is limited to the portion of the neural plate that will give rise
to the optic primordia. Retinal specification and the formation of the lens
placode are normal, consistent with neural-plate-specific expression of
rx3.
Recently, a loss-of-function mutation of rx3 has been described
in the teleost medaka that also results in the complete absence of retinae
(Loosli et al., 2001). In cross-species
rescue experiments, we show that injection of the medaka rx3 locus
results in stage- and region-specific expression of this gene in the zebrafish
embryo that resembles the endogenous pattern. This indicates that, in addition
to the open reading frame of rx3, the respective regulatory elements are
also highly conserved. This is further supported by the high frequency of
complete rescues that were obtained. Therefore, the regulatory region of
rx3 can be expected to share conserved motifs, an issue that will be
addressed in the future.
In zebrafish, all three rx paralogues are expressed in the
evaginating optic vesicles (Chuang et al.,
1999), whereas in medaka only rx3 is expressed during optic
vesicle evagination. Medaka rx1 and rx2 are first expressed in
the fully evaginated optic vesicle just before the transition to optic cups
(Loosli et al., 2001; F.L. and J.W.,
unpublished data). Thus, there is considerably more temporal overlap of
rx gene expression during the critical period in zebrafish than in
medaka. However, the phenotypic consequences of loss of rx3 are equally
dramatic in both species. This suggests that the weak and transient rx1
expression in chk mutants is not sufficient for optic vesicle
evagination because either the expression level is too low or, alternatively,
rx1 and rx3 have non-redundant functions. Future experiments will
address this issue.
The closely related genes rx1 and rx2 are specifically
expressed in retinal progenitor cells of both species. Interestingly,
rx1 and rx2 are differentially affected by the respective
rx3 mutations. Whereas in the medaka el mutation both rx1
and rx2 are still expressed, albeit at reduced levels (Loosli et al., 2001; F.L. and J.W., unpublished
data), only rx1 is initially expressed in the zebrafish chk
mutant, whereas rx2 expression is completely abolished. Hence, only in
zebrafish are rx1 and rx2 completely dependent on rx3
function. This finding shows a different transcriptional control of orthologues
in these two vertebrates, which predicts an interesting divergence of the
respective regulatory elements.
In contrast to rx1 and rx2, retinal vsx2 is lost
in both el and chk mutants, suggesting that residual rx1
and rx2 expression cannot rescue vsx2 expression in the medaka
embryo. This indicates that, probably indirectly, vsx2 depends mainly on
rx3 function.
Although they are highly conserved in both structure and expression
pattern, several genes of the downstream cascade are differentially affected by
the medaka and zebrafish rx3 mutations. The availability of rx3
mutations in both species offers a unique paradigm to study the divergence of
gene function by changes in gene regulation. The ongoing whole-genome
sequencing projects of medaka and zebrafish will provide the tools that are
needed to compare the regulatory elements of these genes and address the
evolution of regulatory interactions at the molecular level.
Methods
Fish stocks and mapping.
The chks399 mutation was originally discovered
in D. Stainier's laboratory in an unrelated screen for ethylnitrosourea-induced
mutations that disrupt organ development (H.A. Field, E.O., H.V. and D.
Stainier, unpublished data) and is now maintained in a TL background. The
mutant name chokh (chk) was submitted to the Zebrafish
Information Network database (http://www.zfin.org). For mapping, an
s399 TL carrier was crossed to a WIK wild-type fish, and heterozygous
F1 progeny were identified in the next generation. Bulk-segregant
analysis was performed on 41 homozygous mutants and 48 siblings from one cross
to determine the linkage group, using a panel of 192 simple-sequence repeat
markers (CA repeats), which were chosen for their high rate of polymorphisms
between TL and WIK strains (K.F.-B. and H.B., unpublished data). Single-embryo
analysis was used to confirm bulk linkage.
Cloning of zebrafish rx3.
Total RNA was isolated from 2–3-day wild-type and chk
mutant embryos using Trizol (Gibco BRL). The BD Biosciences SMART RACE
complementary DNA amplification kit was used to generate 5' cDNA. The
coding region of rx3 was amplified by nested PCR (outer primers: NUP
primer and 5'-AGTGGCCAGCTGCATTGT-3'; 35 cycles of 94 °C for 30
s, 52 °C for 30 s, 72 °C for 1.5 min; inner primers:
5'-CTTTTCTGCCGGGACAGTCT-3' and
5'-AAAACGGATCCAGACCACTG-3', 35 cycles of 94 °C for 30 s, 55
°C for 30 s, 72 °C for 1.5 min). The PCR product was gel extracted and
sequenced using internal primers of the nested PCR reaction.
DNA injections and genotyping of rescued embryos.
One-cell embryos from an incross of two heterozygous chk
carriers were injected with 200 ng  l-1 of a plasmid
that contains medaka rx3 and regulatory regions (pOlRx3) or a control
plasmid that contains a frameshift mutation in rx3 (pOlRx3-fs). pOlRx3
carries the entire rx3 locus. This plasmid fully rescues the medaka
el mutant, whereas the pOlRx3-fs plasmid has no rescue activity
(Loosli et al., 2001). Embryos were scored
for the presence or absence of eyes at 48 h.p.f., and DNA was isolated from
individual embryos. A 426-bp fragment of rx3 was amplified by PCR with
the following primers: 5'-GGATGATGAAAACCCGAAGA-3' and
5'-TGGGCTGAATAAACAAACG-3' (35 cycles of 94 °C for 30 s, 52
°C for 30 s, 72 °C for 1 min). PCR products were digested with
BspE1 overnight and run on a 2% agarose gel.
In situ hybridizations and vibratome
sections.
Whole-mount in situ hybridizations using digoxygenin-labelled
antisense RNA probes and vibratome sections were carried out as described in
Loosli et al. (2001).
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Acknowledgements
We thank D. Stainier and H.A. Field for generously providing the
chokh mutant, and P. Page-McCaw for assistance with primer design and
sequence analysis. We thank S. Wilson, M. Jamrich and C. Neumann for probes, M.
Carl and C. Neumann for critical comments on the manuscript, and A. Krone and
E. Grzebisz for excellent technical assistance. This work was supported by
grants from the National Insitutes of Health (EY13855, to H.B.), the Packard
foundation (to H.B), the European Union (to J.W.) and the Human Frontier
Science Program Organization (to J.W.).
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