1 Laboratory of Fish Embryology, Temasek Life Sciences Laboratory, 1 Research
Link, National University of Singapore, Singapore 117604
2 Department of Biological Sciences, 14 Science Drive 4, National University of
Singapore, Singapore 117543
* Present address: Department of Biology, University of Virginia,
Charlottesville, VA, USA
Author for correspondence (e-mail:
karuna{at}tll.org.sg)
Accepted 8 April 2003
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SUMMARY |
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We describe a temperature-sensitive mutation that affects the zebrafish Nodal-related secreted signalling factor, Cyclops, and use it to address the issue of when the floor plate is induced in zebrafish. Zebrafish cyclops regulates the expression of shh in the ventral neural tube. Although null mutations in cyclops result in the lack of the medial floor plate, embryos homozygous for the temperature-sensitive mutation have floor plate cells at the permissive temperature and lack floor plate cells at the restrictive temperature. We use this mutant allele in temperature shift-up and shift-down experiments to answer a central question pertaining to the timing of vertebrate floor plate induction. Abrogation of Cyc/Nodal signalling in the temperature-sensitive mutant embryos at various stages indicates that the floor plate in zebrafish is induced early in development, during gastrulation. In addition, continuous Cyclops signalling is required through gastrulation for a complete ventral neural tube throughout the length of the neuraxis. Finally, by modulation of Nodal signalling levels in mutants and in ectopic overexpression experiments, we show that, similar to the requirements for prechordal plate mesendoderm fates, uninterrupted and high levels of Cyclops signalling are required for induction and specification of a complete ventral neural tube.
Key words: Floor plate, Nodal signalling, cyclops, Zebrafish, Shh, Twhh, Organizer, Temperature-sensitive mutation, Gastrulation, TGFß
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INTRODUCTION |
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Studies in several vertebrates (Placzek
et al., 1990; Lawson and
Pedersen, 1992
; Le Douarin et
al., 1998
) that investigated the development of the floor plate
led to several models for its induction. One model proposes that a signalling
cascade mediated by the Shh protein secreted from the notochord induces the
floor plate in the overlying neural tube
(Placzek et al., 2000
), and
mutations in the mouse Shh gene indeed result in floor plate
deficiencies (Chiang et al.,
1996
). Moreover, grafting experiments in the chick have indicated
that floor-plate markers can be induced in ectopic locations of the neural
tube by signals from the notochord, or the floor plate itself, or by the
expression of SHH protein in ectopic locations of the neural tube
(van Straaten et al., 1985
;
van Straaten et al., 1988
;
Placzek et al., 1990
;
Placzek et al., 1991
;
Yamada et al., 1991
;
Marti et al., 1995
;
Roelink et al., 1995
;
Ericson et al., 1996
).
However, recent experiments in the chick, as well as analyses of zebrafish
mutants suggest that the floor plate may be induced independent of notochord
specification (Halpern et al.,
1997; Le Douarin et al.,
1998
; Le Douarin and Halpern,
2000
; Charrier et al.,
2002
). For example, zygotic mutations in zebrafish
cyclops (cyc), which encodes a Nodal-related secreted
signalling factor (Hatta et al.,
1991
; Rebagliati et al,
1998
; Sampath et al.,
1998
), and one eyed pinhead (oep), which encodes
an essential co-factor for Nodal signalling
(Gritsman et al., 1999
),
result in the lack of a floor plate, in spite of the presence of a
morphologically normal notochord expressing shh
(Strahle et al.,
1997
; Schier et al.,
1997
). On the other hand, mutations in the flh and
ntl genes, which are required for the formation of the notochord,
result in embryos that exhibit a patchy or wider floor plate, respectively
(Halpern et al., 1997
).
Furthermore, medial floor plate cells are not abolished by mutations in the
zebrafish shh gene (Schauerte et
al., 1998
) or its receptor, smoothened
(Chen et al., 2001
;
Varga et al., 2001
), or by
abrogation of Hedgehog signalling using antisense knockdown with
morpholino-modified oligomers (Etheridge
et al., 2001
). Therefore, an alternate model proposes that the
floor plate is induced in the organizer-derived midline precursor cells
(Le Douarin and Halpern, 2000
;
Charrier et al., 2002
). As the
precursor cells give rise to both the notochord and the floor plate, this
model predicts that floor-plate induction takes place early during
gastrulation. A key unresolved issue in the models pertains to the timing of
floor plate induction.
We have isolated a temperature-sensitive mutation in the zebrafish cyc locus. In contrast to null mutations in cyc, embryos homozygous for the cycsg1 mutation manifest variable cyc phenotypes at 22°C. Mutant embryos exhibit variably fused eyes, ventral curvature and patchy to complete floor plate, with motoneurones that may or may not be at their normal positions. At 28.5°C, cycsg1 mutant embryos are indistinguishable from cyc-null mutant embryos, with fused eyes, lack of medial floor plate cells and ventral curvature. Using this allele in temperature shift-up and shift-down experiments, we show that Cyc function is essential at gastrulation to induce the floor plate in zebrafish. By modulating Nodal signalling levels in mutants, and by overexpressing cyc in wild-type embryos, we show that high levels of Cyc signalling are required for induction of the floor plate. Furthermore, we show that continuous and high levels of Cyc signalling during gastrulation are essential for formation of a complete ventral neural tube. These results show that the floor plate inducing activity of Cyc is essential during gastrulation, and that it is required at multiple steps of the floor plate induction pathway for the development of a complete ventral neural tube.
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MATERIALS AND METHODS |
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cyc allele screen, mapping and sequencing
Adult zebrafish males of the AB strain were mutagenized with the chemical
ethyl nitrosourea as described (Riley and
Grunwald, 1995), and mosaic F1 progeny were screened for
non-complementation with cyctf219/+ fish. Putative mutant
fish were subsequently tested with other known cyc alleles as well as
other mutants affecting the Nodal signalling pathway. Identified heterozygous
fish were out-crossed to AB or to WIK fish. Embryos from identified
heterozygous fish in the next generation were split into two groups at the
one-cell stage, allowed to develop at 28.5°C or at 22°C until prim-5
stage, and analysed for cyc phenotypes of fused eyes, ventral
curvature and the floor plate. Mapping was carried out using PCR on a AB/WIK
mutant panel (n=80 haploid and 1200 diploid embryos) using primers
flanking a CA repeat in the 3'-untranslated region of cyc
(Sampath et al., 1998
). For
identifying the mutation, genomic DNA was isolated from single
cycsg1 mutant embryos at 24 hours post-fertilization (hpf)
and used as templates for sequencing. In addition, DNA and RNA were extracted
from individual cycsg1 homozygous embryos at shield stages
using TRIZOL reagent (Gibco, BRL), and single embryos were genotyped using the
cyc CA repeat marker. RT-PCR was carried out using pooled RNA from
identified cycsg1mutant embryos and the nucleotide
sequence was determined.
Genotyping
The genotype of sqtcz35 mutant embryos and
cycm294 mutant embryos was determined as described
(Feldman et al., 1998;
Sampath et al., 1998
). For
determining the genotype of cycsg1 homozygous mutant
embryos, genomic DNA was isolated from single embryos (live or after analysis
of in situ hybridization patterns), and PCR was performed with the primers
5'-AACAGGAGCTACCGAGCAGGC-3' and
5'-ACTGGCCCCGTCCTGCTGCT-3'. The PCR products were digested with
the restriction enzyme PvuII (New England Biolabs), and analysed by
agarose gel electrophoresis.
Temperature shift experiments
Embryos obtained from matings of cycsg1/+ fish were
split into two groups at the one-cell stage, and raised at 22°C and
28.5°C, respectively. At regular intervals from 50% epiboly to 10-somite
stages, embryos at 22°C were shifted to 28.5°C. Conversely, embryos
raised at 28.5°C were shifted to 22°C at the same intervals. For
temperature pulse experiments, embryos were incubated at either 22°C or
28.5°C with a brief shift-up or shift-down period during mid-gastrulation.
Embryos were fixed at 100% epiboly or prim-5 stages for in situ hybridization
with various markers.
Generation of constructs
The cycsg1 mutation was introduced into
pCS2cyc+ (Sampath et
al., 1998) by PCR-based mutagenesis. The Flag epitope-tagged
pCS2cyc+FLAG and pCS2cycsg1FLAG
constructs were generated by PCR-based methods and their nucleotide sequence
was confirmed. In both constructs, the Flag epitope was fused in frame after
the cleavage site, between Val 385 and Arg 386 in Cyc.
Embryo injections and animal cap assays
The plasmids pCS2cyc+,
pCS2cyc+FLAG, pCS2cycm294,
pCS2cycsg1, and pCS2cycsg1FLAG were
linearized with NotI, and sense strand capped mRNA was synthesized
with SP6 RNA polymerase using the mMESSAGE mMACHINE system (Ambion). In vitro
synthesised RNA was injected into one- to four-cell stage wild-type embryos.
Animal caps were dissected at late blastula stages and cultured as described
(Sagerstrom et al., 1996;
Dheen et al., 1999
) until
sibling stage 80% epiboly at 22°C or 28.5°C. Animal cap explants and
embryos at various stages were fixed for antibody staining or in situ
hybridization with various markers.
Cell culture
Cos-7 cells were cultured at 37°C in DMEM (Gibco-BRL) containing 10%
foetal bovine serum (Sigma), 10 U/ml penicillin and 10 mg/ml streptomycin
sulphate (Sigma). The plamids pCS2cyc+FLAG and
pCS2cycsg1FLAG were transfected into Cos-7 cells using the
Superfect transfection reagent (Qiagen). Cells were fixed after 24 hours in 4%
paraformaldehyde and processed for detection of the Flag epitope.
In situ hybridization
Whole-mount in situ hybridization was performed as described
(Sampath et al., 1998) on
embryos fixed at 100% epiboly (10 hpf at 28.5°C or 20 hpf at 22°C),
six-somite (12 hpf at 28.5°C or 24 hpf at 22°C) or prim-5 stages (24
hpf at 28.5°C or 48 hpf at 22°C). The following plasmids were
linearized and antisense probes were synthesized by in vitro transcription:
pBSshh (EcoRI, T7) (Krauss et
al., 1993
), pBStwhh (PstI, T7)
(Ekker et al., 1995
),
pBSislet2 (EcoRI, T7) (Appel et
al., 1995
), pBShgg1 (XbaI, T7)
(Thisse et al., 1994
), pBSgsc
(EcoRI, T7) (Stachel et al.,
1993
), pBSflh (EcoRI, T7)
(Talbot et al., 1995
). Single-
or double-colour in situ hybridization was performed as described
(Sampath et al., 1998
). For
digoxigenin-labelled probes, BM purple substrate (Roche) was used; for
fluorescein-labelled probes, fast red (Roche) or 4-iodonitrotetrazolium violet
(Molecular Probes) were used. For cryosections, whole-mount bicolour in situ
hybridized embryos were embedded in 1.5% agarose:30% sucrose blocks. The
blocks were frozen and sections were obtained on a Leica CM 1900 cryomicrotome
at 16 µm intervals.
Immunostaining
Embryos and animal caps were fixed in 4% paraformaldehyle at 4°C
overnight. Embryos were incubated with a monoclonal antibody raised against
the zn-5 epitope (Trevarrow et al.,
1990), and colour was developed using the ABC kit (Pierce) with
the substrate diaminobenzidine (Sigma). Animal caps and Cos-7 cells were
incubated with an anti-Flag polyclonal antibody (Sigma), and detected with an
anti-rabbit secondary antibody conjugated with Alexa 568 (Molecular Probes).
Optical sections were obtained at 0.5 µm intervals on a Zeiss Axiovert 200M
microscope, and images were processed using the Zeiss LSM image browser
software.
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RESULTS |
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Analysis of markers of mesendoderm, ventral neural tube and
motoneurones in cycsg1 mutant embryos
Expression of cyc transcripts in gastrula stage
cycsg1 mutant embryos at 22°C is similar to that seen
in wild type embryos (Fig.
3A,B) or reduced (Fig.
3D,E). At 28.5°C, similar to ENU-induced null mutations in the
cyc locus (Rebagliati et al.,
1998; Sampath et al.,
1998
), cyc transcripts in cycsg1
mutants are reduced by mid-gastrula stages
(Fig. 3C), and are not detected
by the end of gastrulation (Fig.
3F). Analysis of expression of goosecoid (gsc),
a marker of the prechordal plate mesendoderm
(Thisse et al., 1994
), which
is reduced in cyc null mutants, reveals variably reduced
(Fig. 3I,J) to normal
(Fig. 3G) prechordal plate
mesendoderm in cycsg1 mutants at 22°C, compared with
those at 28.5°C (Fig.
3H).
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Interestingly, we found that embryos that were shifted down to the permissive temperature at 50% or 60% epiboly showed a complete floor plate and ventral neural tube, throughout the entire length of the neuraxis (Fig. 6E), whereas shift-down at later stages resulted in rescue of patches of floor-plate cells (Fig. 6F). The patches were distributed throughout the length of the neuraxis, regardless of the stage at which the embryos were shifted. In addition, embryos from 28-22 shift-down experiments at mid-gastrula stages typically showed longer stretches of cells expressing floor plate markers than embryos from 22-28 shifts (Fig. 6C,F). These results suggest that Cyclops function may be required first for induction of the floor plate from its precursors early during gastrulation, and, subsequently, for the complete development of the floor plate along the entire length of the neuraxis.
Continual Cyclops signalling is required during gastrulation for
formation of a complete floor plate
To confirm the above observations that the floor-plate inducing activity of
Cyclops is essential during mid-gastrulation, we addressed whether a transient
pulse at the permissive temperature during mid-gastrulation was sufficient for
induction of the floor plate, or, conversely, if a brief incubation at the
restrictive temperature could abrogate floor-plate fates in
cycsg1 mutant embryos. When embryos were incubated at
28.5°C throughout gastrulation and segmentation, with a brief shift-down
period at 22°C during mid-gastrulation, 27/29 mutant embryos (93%) that
were incubated at 22°C between 70 and 80% epiboly showed rescue of medial
floor plate cells, determined by the presence of shh- or
twhh-expressing cells (Fig.
7A,D,H). However, the extent of rescue as determined by
shh expression at prim-5 stage was usually patches of three or four
cells, throughout the length of the neuraxis
(Fig. 7H). If the pulse of
22°C was given between 60 and 90% epiboly, all mutant embryos
(n=17) showed floor-plate cells
(Fig. 7A,B,F). Furthermore, the
embryos showed longer stretches of cells expressing shh
(Fig. 7F), with a complete
floor plate in the trunk in all mutant embryos. Conversely, only 9/30 (30%) of
the mutant embryos that were raised at 22°C and incubated at 28.5°C
between 70 and 80% epiboly showed floor plate cells
(Fig. 7A,E,I). The number of
cells expressing twhh or shh were also fewer, with large
gaps between patches of floor plate cells
(Fig. 7E,I). If the pulse of
28.5°C was given between 60 and 90% epiboly, all mutant embryos
(n=24) lacked floor plate cells
(Fig. 7C,G). Thus, although a
transient pulse of Cyclops signalling between 70-80% epiboly is sufficient to
initiate floor plate fates, continuous Cyclops signalling is required between
60 and 90% epiboly for a complete floor plate.
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DISCUSSION |
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Interestingly, the molecular lesion in cycsg1 is a
transversion which results in a premature stop codon in the pro domain. The
receptor-binding functional moiety of TGFß family proteins is thought to
lie within the C terminus mature domain
(Kingsley, 1994), and this
region should be lacking in the Cycsg1 mutant protein. However,
cycsg1 is functional at 22°C. Accordingly, we find
that Flag-epitope tagged Cycsg1 mutant protein is detected in
zebrafish animal cap explants at 22°C, but not at 28.5°C. In addition,
in Cos-7 cells, wild-type Cyc+ protein shows subcellular
localization in a pattern reminiscent of the Golgi complex, whereas the mutant
protein is not detected. Because an alternate start site (met 336) is present
within the pro region after the stop codon, it is possible that the N
terminus-truncated Cyc protein generated from this site is stable and
functional in zebrafish embryos at 22°C but not at 28.5°C.
Alternatively, a translational read-though mechanism similar to that described
in mammalian cells (Laski et al.,
1982
; Hryniewicz and Vonder
Haar, 1983
; Phillips-Jones et
al., 1995
) may function at 22°C, the permissive temperature
for cycsg1, allowing Cyc function at this temperature. It
is also possible that the pro domain of Cyc has some activities that have not
been previously identified. Analysis of Cycsg1 can therefore
provide valuable insights into the functions of various domains of Cyc/Nodal
proteins.
Cyclops is required at multiple steps of floor-plate
specification
By abrogation of Cyclops signalling in cycsg1
temperature-sensitive mutant embryos at various stages of early development,
we have determined that the crucial window for Cyc function in inducing the
zebrafish floor plate is during gastrulation. Disruption of Cyc function
during gastrulation by temperature shift experiments and modulation of the
level of Cyclops signalling results in patchy or no medial floor plate marker
gene expression. Interestingly, the cycsg1 mutant embryos
with patchy floor plate exhibit groups of floor-plate cells that are
distributed throughout the length of the neuraxis. The presence of patches of
floor plate cells throughout the length of the neuraxis suggests that the
entire ventral neural tube arises from a group of precursors that are
distributed throughout the length of the embryo, and that their
differentiation into floor plate cells requires continuous Cyclops signalling
during gastrulation. Previous observations by Hatta et al.
(Hatta et al., 1991) where
transplanted wild-type cells adopted floor-plate fates in cyc mutant
hosts, and were able to recruit adjacent mutant host cells into floor plate
fates, indicated that once specified, mutant cells had the ability to
differentiate into floor plate cells. Our data indicates that sustained and
high levels of Cyclops signalling are essential for the complete specification
of floor-plate cells. Thus, in addition to being required for induction of
cells of the floor plate and ventral neural tube, Cyclops signalling is also
required for the development of a complete ventral neural tube throughout the
entire length of the embryo.
Prechordal plate mesendoderm and the floor plate inducing activity of
Cyc
Signalling in the anterior organizer cells, which give rise to the
prechordal plate mesendoderm, has been implicated in induction of the floor
plate (Sampath et al., 1998;
Amacher et al., 2002
). We find
that deficiencies of the prechordal plate did not affect induction of the
floor plate by Cyclops in sqtcz35/sqtcz35,
cycsg1/cycsg1;sqtcz35/sqtcz35,
or cycm294/cycsg1;
sqtcz35/sqtcz35 mutant embryos. It is possible that
the precursors of the prechordal plate cells or the remaining prechordal plate
cells in these mutants are able to mediate floor plate induction via Cyclops
signalling. Similar to the requirements for specification of prechordal plate
mesendoderm (Gritsman et al.,
2000
), we find that uninterrupted and high levels of Cyclops
signalling during gastrulation are crucial for induction and complete
development of the floor plate in zebrafish. Sustained and high levels of
Cyc/Nodal signalling during gastrulation can specify both floor plate and
prechordal plate mesoderm fates. Therefore, we cannot rule out the possibility
that it is the high level of Cyclops signalling, rather than signalling in the
prechordal plate mesendoderm, which is responsible for floor plate
induction.
Using the temperature-sensitive cycsg1 allele, we have
conclusively provided evidence that the medial floor plate is induced during
gastrulation in zebrafish. It will be important to identify the cells in which
Cyc/Nodal signalling is required for inducing the floor plate, the molecules
that function downstream of Cyclops signalling to mediate this process in
fish, and its similarities and differences with floor plate induction in
amniotes. Given that several aspects of Nodal signalling are highly conserved
(Schier and Shen, 2000), and
given that axial/FoxA2/HNF3ß is a common downstream effector of the
floor-plate induction pathways in zebrafish as well as mice
(Rastegar et al., 2002
),
similar mechanisms and timing of floor-plate induction are also likely in
other vertebrates.
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ACKNOWLEDGMENTS |
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