1 Universität zu Köln, Institut für Genetik, Weyertal 121, 50931
Köln, Germany
2 Universität zu Köln, Institut für Entwicklungsbiologie,
Gyrhofstr. 17, 50923 Köln, Germany
3 Max-Planck Institut für Entwicklungsbiologie, Spemannstrasse 35, 72076
Tübingen, Germany
Author for correspondence (e-mail:
tautz{at}uni-koeln.de)
Accepted 27 May 2003
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SUMMARY |
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Key words: Somitogenesis, bHLH genes, mRNA stability, Enhancer analysis, Morpholino-oligonucleotide mediated knockdown
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INTRODUCTION |
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It has long been speculated that the prepatterning of the somites is
achieved by an oscillator mechanism in the PSM
(Cooke and Zeeman, 1976;
Meinhardt, 1986
) (reviewed by
Dale and Pourquié,
2000
). The first evidence for this oscillator mechanism was
provided by the identification of the c-hairy1 gene (Palmerim et al.,
1997), which is dynamically expressed in the PSM of chicken. Owing to its
cyclic expression, which progresses from the posterior to the anterior PSM,
the cells in the chick embryo undergo several on and off phases of
c-hairy1 transcription before they become a somite. c-hairy1
encodes a bHLH transcription factor, which is a homologue of the
Drosophila pair-rule gene hairy
(Ish-Horowicz et al., 1985
).
More recently, several hairy (h) and Enhancer of split
(E(spl)) related genes have been identified, which also have a dynamic
expression in the vertebrate PSM. This includes the c-hairy2 and
c-Hey2 genes in chick (Jouve et
al., 2000
; Leimeister et al.,
2000
) as well as the Hes1 and the Hes7 genes in
mouse (Jouve et al., 2000
;
Bessho et al., 2001a
;
Bessho et al., 2001b
). In
zebrafish, nine h/E(spl) related genes have been discovered so far
[her1-her6 (von
Weizsäcker, 1994
;
Müller et al., 1996
;
Pasini et al., 2001
),
her7: AF240772; her8a/b: AY007990/AY007991 and her9
(Leve et al., 2001
)] but only
two of them, her1 and her7, show an oscillating expression
in the PSM (Holley et al.,
2000
; Oates and Ho,
2002
). The analysis of a deletion mutant for her1 and
her7 as well as morpholino-oligonucleotide (MO) knockdown studies
suggest that Her1 and Her7 protein function is required for the prepatterning
of the zebrafish PSM (Henry et al.,
2002
; Oates and Ho,
2002
). The loss of Her1/Her7 protein leads to somites that show
alternating weak and strong boundaries
(Henry et al., 2002
). In
addition, a disruption of rostrocaudal polarity within the somites has been
observed (Henry et al., 2002
;
Oates and Ho, 2002
).
The current data suggest that the Delta-Notch signalling pathway
is the major trigger of cyclic gene expression in the vertebrate PSM (for
reviews, see Maroto and Pourquié,
2001; Saga and Takeda,
2001
). In zebrafish, mutants of deltaD (after
eight) and Notch1 (deadly seven), as well as
MO-knockdown of deltaC abolish the cyclic expression of her1
and her7 (Dornseifer et al.,
1997
; van Eeden et al.,
1998
; Takke and Campos-Ortega,
1999
; Holley et al.,
2000
; Holley et al.,
2002
; Oates and Ho,
2002
). In these cases her1 and her7 usually only
show an irregular expression in the anterior PSM and a weak, diffuse
expression in the posterior part of the PSM and the tailbud. Furthermore,
her1 and her7 appear to crossregulate each other, and both
are required for the transcription of deltaC and deltaD
(Holley et al., 2002
;
Oates and Ho, 2002
;
Henry et al., 2002
).
We have focussed here on a better understanding of the differential roles of her1 and her7 in regulating the cyclic gene expression by analysing the effects of MO-knockdown on each other's expression. We have found different roles for Her1 and Her7 in regulating the anterior and posterior parts of cyclic her gene expression. Analysis of the her1 promoter reveals that these anteroposterior differences are the result of separable regulatory elements.
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MATERIALS AND METHODS |
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Sequence comparisons and phylogeny
Amino acid sequences were aligned using the Pileup program of the GCG
software package (Devereux et al.,
1984; Senger et al.,
1998
). Similarity trees were generated using PAUP, calculations
are based on Pileup alignments. Trees were displayed using Treeview
(Page, 1996
). The accession
numbers of the compared genes are: c-hairy1: AF032966,
c-hairy2 (Jouve et al.,
2000
), Drosophila hairy: X15905, her1:
X97329, her4: X97332, her6: X97333, her7: AF240772,
her9: AF301264, mouse Hes1: NM008235, mouse Hes7:
AB049065, X-hairy1: U36194, X-hairy2A: AF383159, human
HES1: Q14469, human HES7: NM032580.
Whole-mount in situ hybridisation and histological methods
Fish were bred at 28.5°C in a 14 hour light/10 hour dark cycle. Embryos
were collected by natural spawning and staged according to Kimmel et al.
(Kimmel et al., 1995). For
automated in situ hybridisations we followed the protocol of Leve et al.
(Leve et al., 2001
) using a
programmable liquid handling system described by Plickert et al. (Plickert et
al., 1997). The hybridisation temperature for the her1 intron probe
had to be reduced to 50°C, because of its high AT content. Digoxigenin- or
fluorescein-labelled RNA probes were prepared using RNA labelling kits
(Roche). Staining was performed with BM purple (Roche) for single in situ
hybridisations or, for double fluorescence in situ hybridisations, Vector®
Red Alkaline Phosphatase Substrate Kit I (Vector Laboratories) and the
ELF®-97 mRNA In Situ Hybridisation Kit (Molecular Probes) were used
according to the method of Jowett and Yan
(Jowett and Yan, 1996
).
Whole-mount embryos were observed under a stereomicroscope (Leica) and
digitally photographed (Axiocam, Zeiss). Flat-mounted embryos were observed
with an Axioplan2 microscope (Zeiss). For observation of the Vector® Red
staining or the ELF®-97 precipitate a rhodamine filter set or a DAPI
filter set was used, respectively.
Reporter gene constructs and transgenic lines
A 10.9 kb NcoI fragment from the upstream region of the
her-1 gene was in-frame subcloned into the start methionine of the
coding sequence of pEGFP (Clontech). After digestion with PstI and
EcoRI, the promoter-reporter construct containing 8.6 kb upstream
sequence and the EGFP reporter was inserted into pHSREM1 (acc. no. ATCC37642,
kindly provided by D. Knipple) to yield construct I. Transgenic lines were
produced by injection of a PstI-linearized fragment of this
construct, or of PCR-amplified promoter deletions of it, into single cell
embryos. For PCR-amplification the Expand High Fidelity PCR System (Roche) was
used. For generation of constructs II-VI the same downstream primer (M13for:
5'-GTA AAA CGA CGG CCA GT-3') was used in combination with
upstream primer II (5'-TAA ACT TTC CCC AGT CAG-3'), upstream
primer III (5'-AAA GCC ACA TCA AAG CCC-3'), upstream primer IV
(5'-TTA GCC ATG AAC GAT GCC-3'), upstream primer V (5'-AGC
AAC TCC ATA AAA TCC-3'), upstream primer VI (5'-CTA TGA GAC AAC
GAT GAG-3'), respectively. Between five and 15 transgenic lines were
obtained in each case, but not all showed a sufficiently strong expression.
Four, two and one line were eventually analysed for constructs I-III. There
were only quantitative (expression level) but no qualitative differences
between the lines. The DNA fragments were gel purified prior to injection
using a gel extraction kit (Bio-Rad, Gibco BRL). The DNA concentration of the
injected solutions was between 80-100 ng/µl in water containing 0.2% phenol
red and 0.1 M KCl. Injections were carried out using FemtoJet® and a
Micromanipulator (Eppendorf). To test for possible transgenic animals, DNA of
100 embryos was extracted as described previously
(Meng et al., 1999). Positive
PCR controls were the Wnt5a sense primer 5'-CAG TTC TCA CGT CTG CTA CTT
GCA-3' and the Wnt5a antisense primer 5'-ACT TCC GGC GTG TTG GAG
AAT TC-3'. For the transgene test, two GFP primers were used (GFPfor3:
5'-CGG CAA CTA CAA GAC CCG CG-3' and GFPrev3: 5'-GTC CTC GAT
GTT GTG GCG GA-3'). The following PCR profile was carried out: 95°C
for 2 minutes, then 35 cycles 95°C for 15 seconds, 55°C for 15 seconds
and 72°C for 30 seconds followed by an elongation step of 72°C for 5
minutes after the 35 cycles.
Morpholino injections
Antisense morpholino-modified oligonucleotides (GeneTools) were designed
against the first 25 nucleotides of the 5'-UTR and against the start of
the ORF of both the her1 cDNA (X97329) and the her7 cDNA
(AF240772). Sequence for her1-anti5' morpholino: 5'-AGT
ATT GTA TTC CCG CTG ATC TGT C-3, sequence for the her1-antiATG
morpholino: 5'-CAT GGC TGA AAA TCG GAA GAA GAC G-3', sequence for
her7-anti5' morpholino: 5'-ATG CAG GTG GAG GTC TTT CAT
CGA G-3', sequence for the her7-antiATG morpholino: 5'-CAT TGC ACG
TGT ACT CCA ATA GTT G-3'. 0.5 mM of the her7mos and
1 mM of the her1mos were injected into single-cell stage
embryos. The injection solution additionally contained 0.1 M KCl and 0.2%
phenol red. Control injections were done with the morpholino-modified
oligonucleotide recommended by GeneTools, or with buffer. The death rate
caused by injection of the different morpholinos was usually between 5 and
11%.
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RESULTS |
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Embryos injected with the her7mo also show disruption of her1 and her7 cycling, but with different effects on each and differences when compared to her1 morphants. her1 is expressed in a punctate pattern in the PSM of her7 morphants and a more pronounced domain is observed in the tailbud (Fig. 4O,P,Q). her7 also shows a pronounced tailbud expression in her7 morphants, and at best a residual weak signal without punctate pattern in the intermediate PSM (Fig. 4R,S). Thus, it appears that the mRNA stabilisation effect is much weaker than that for her1. However, the perturbation of her1 and her7 wave generation in posterior PSM of the her7 morphants, which is not seen in her1 morphants, indicates a unique role for the Her7 protein in this process.
her1 regulation by distinct promoter elements
The above results indicate that at least her1 regulation is
governed by two distinct phases. This is supported by reporter gene constructs
in transgenic lines. her1 upstream fragments of various size were
fused to GFP as reporter and the DNA was injected into early embryos. Stably
transformed lines were established from these and analysed for their
regulatory effects by in situ hybridisation to the GFP mRNA. Lines containing
an 8.6 kb upstream fragment (construct I, see
Fig. 1) confer the full normal
cyclic pattern (Fig. 5). Double
hybridisation with the her1 probe shows that it is essentially
indistinguishable from the endogenous expression
(Fig. 5G,H) indicating that all
important elements for in phase cycling are present. However, the transgenic
embryos of all 8.6 kb lines show an additional strong expression in the
notochord, which is not seen for the her1 gene itself, suggesting
that construct I is missing a notochord-specific repressor. Furthermore, the
expression in the most posterior domain is more persistent than for the
her1 RNA. It is unclear whether this is due to slight differences in
the RNA stability, or to differences in the action of the enhancer included in
our construct.
|
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Regulation of reporter gene constructs
To analyse whether the reporter gene constructs depend on her1 and
her7 regulation in the same way as the endogenous genes, we have used
MO knockdown of her1 and her7 in the background of the
respective transgenic lines. For lines containing the 8.6 kb construct we find
that the stripes in the anterior PSM are indeed disrupted by
her1mo and her7mo injection, while the
expression in the posterior PSM persists
(Fig. 7B,C). A similar picture
is seen in lines with the 3.3 kb construct. The stripe formation is clearly
disrupted and a broad domain persists instead
(Fig. 7E,F). These results
confirm that the essential elements of the stripe regulation must be included
in our constructs. In particular, the binding site(s) for Her7, which regulate
the stripes of her1 expression, must be included in construct II
(compare Fig. 7D with F).
|
Evidence for cyclic her1 expression
Although the analysis of carefully staged embryos has provided a clear
indication that her1 expression is dynamic in the PSM
(Holley et al., 2000), this
can be demonstrated more directly in the transgenic lines by comparing the GFP
mRNA expression with the GFP protein expression. Because the GFP protein is
much more stable than the GFP mRNA, it persists in all cells in which the mRNA
was at least transiently expressed. Accordingly, if the her1
expression moves across the PSM in the same way as it was demonstrated for
hairy-like genes in chicken
(Palmeirim et al., 1997
), then
we would expect all somites to express GFP protein. The somites and the PSM
show GFP fluorescence in the 8.6 kb line, with a fading of the signal towards
the oldest somites (Fig. 8A,B).
Since the GFP protein derived from the posterior U-shaped domain could cause
this effect alone in the 8.6 kb line, we monitored GFP fluorescence in the 3.3
kb line, which lacks the posterior PSM expression. This line also shows
continuous fluorescence, apart of the signal in the posterior part of the PSM
(Fig. 8C,D). We can therefore
conclude that the stripes in the intermediate and anterior PSM also move
across all cells, confirming that the stripes seen in the RNA pattern are
indeed solely due to a very short half-life of the mRNA.
|
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DISCUSSION |
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Different roles of her1 and her7
The MO knockdown results suggest that her1 and her7 act
in a common pathway, as they both affect the other's expression, as well as
deltaC and deltaD expression. The effect on the latter two
genes is almost indistinguishable between her1 and her7.
Knockdown of these bHLH proteins disrupts deltaC and deltaD
expression (Fig. 4). Since
overexpression of her1 leads to a decreased transcript level of these
delta genes (Takke and
Campos-Ortega, 1999), the results are consistent with the proposed
role of a Her-linked Delta-Notch feedback loop
(Holley et al., 2002
;
Oates and Ho, 2002
).
However, different mutual effects are observed for her1 and
her7, on each other, as well as on their own expression. Lack of Her1
protein results in a specific loss of the stripes in the intermediate and
anterior PSM, while the dynamic expression in the tailbud and posterior PSM of
both bHLH genes appears nearly unaffected. In contrast to previous suggestions
(Holley et al., 2002;
Oates and Ho, 2002
),
her1 thus acts formally as an activator rather than a repressor, not
only on its own but also on her7 transcription. Since the Her1
protein possesses all features of bHLH repressors (see alignment in
supplemental data at
http://dev.biologists.org/supplemental/)
and experimental evidence also supports this, another yet unidentified
component must be postulated to act in intermediate and anterior PSM. Two
working models seem most likely: Her1 might repress transcription of another
repressor resulting in own-stripe activation, or, a modulator protein might
switch the function of Her1.
The role of her7 is more complex. Loss of Her7 protein leads to disruption of her1 and her7 cycling, indicating that Her7 is needed for the wave generation of both genes in the posterior PSM. Only residual tailbud expression of her7 is visible in her7 morphants and in intermediate and anterior PSM her7 is very weakly expressed suggesting that stabilisation of this transcript plays a less important role than in the case of her1 in her1 morphants. her1 also loses its dynamic expression upon Her7 knockdown, but is expressed throughout the PSM. This would lead to the conclusion that Her7 plays different roles in regulating the two bHLH genes in intermediate and anterior PSM, while Her1 regulates both genes in a similar manner. Formally, Her7 acts as an interstripe repressor on her1, but as an activator on itself. Again, Her7 displays all features known for bHLH repressors (see alignment in supplemental data at http://dev.biologists.org/supplemental/) suggesting that Her7-mediated activation is indirect and might involve an unknown component. Whether this component is the same as postulated for Her1-mediated activation remains to be investigated.
Separable promoter elements of her1
The results discussed above suggest that her7 is specifically
required to initiate the dynamic expression wave, while her1 is
required to carry it further on. This points towards a functional separation
of the regulation in the posterior PSM from that in the intermediate and
anterior PSM. The analysis of the promoter elements of her1 confirms
this.
Our results suggest that there are at least two distinct elements
controlling the PSM expression of her1. One mediates a specific
activation in the most posterior region of the PSM and the second mediates the
expression in the intermediate and anterior PSM. Genetic analysis of various
mutants (van Eeden et al.,
1998; Holley et al.,
2000
), as well as additional experiments
(Holley et al., 2002
) suggest
a threephase model for the activation and action of her1. The first
phase is activation through deltaD and deltaC in the most
posterior part of the PSM. The second is the generation of the dynamical
stripe pattern and the third is the stabilisation of the stripes during the
early stages of somite boundary formation
(Holley et al., 2002
). Our
transgenic lines provide support for at least the first two of these phases. A
possible enhancer that is required for the activation of the cycles could be
located in the region between -8.6 to -3.3 kb. This would explain the absence
of the most posterior expression of GFP mRNA in transgenic embryos containing
the 3.3 kb construct. We note, however, that this enhancer will still have to
be better defined, because the respective construct leads in addition to an
ectopic expression in the notochord. The fact that the 3.3 kb construct
specifically drives the dynamic expression of the stripes supports the notion
that the second phase of expression is driven by a separate enhancer, which
includes activating and repressing subelements. The presence of specific
activator elements is suggested by the GFP mRNA expression pattern in the line
containing the 2.8 kb construct, which shows only a broad domain, but no
distinct stripes in the respective region (see
Fig. 1). A distinct enhancer
for the most anterior stripe, and thus evidence for the third phase of
expression, was not detected in our experiments.
mRNA stability
Our promoter studies confirm the notion that the dynamic expression of the
her1 gene is caused by differential transcriptional regulation,
rather than differential mRNA stability, a result that is in line with
comparable experiments on lunatic fringe regulation in the mouse
(Cole et al., 2002;
Morales et al., 2002
).
However, it is clear that the her1 mRNA must be very unstable, since
it would otherwise accumulate in the PSM, like the stable GFP reporter
protein. It is thus likely that there is a specific element in the mRNA that
causes this instability. A specific 3'-UTR degradation signal has been
identified for the gene xhairy2a in Xenopus
(Davis et al., 2001
). A
sequence of 25 bases in the 3'-UTR of this gene seems to be necessary
and sufficient for the rapid turnover. Similarities with this 25 base block
are found in some other hairy-related mRNAs, like c-hairy1
and human Hes4. However, we do not find this motif in the 3'-
or 5'-UTRs of her1 and her7. Interestingly, two copies
of the motif occur in the 3'-UTR of her9, a gene that is
closely related to the c-hairy1 gene, but that is not expressed in
the PSM of zebrafish (Leve et al.,
2001
).
The mRNA of our GFP reporter gene appears to be equally unstable as the
endogenous her1 mRNA. This reporter gene contains only the
5'-UTR of her1, suggesting that the destabilisation signal must
be located there. Also the fact that the her1 mRNA is stabilised by
the her1mo that binds in the 5' region of the mRNA
would suggest that a destabilisation signal is linked to the 5'-UTR. MOs
against her7 5' mRNA regions stabilise coinjected her7
mRNA as well as a GFP hybrid mRNA that contained only the her7
5'-UTR (Oates and Ho,
2002) indicating that the destabilisation signal for her7
could also be linked to the 5'-UTR. In contrast, the stabilisation
effect of MOs on the endogenous her7 mRNA is apparently only weak, at
least when compared to her1 (see
Fig. 4).
Models of cyclic gene regulation
Holley et al. (Holley et al.,
2002) and Oates and Ho (Oates
and Ho, 2002
) have proposed models that integrate the effects of
Delta-Notch signalling in the regulation of her1 and her7 in
the PSM. A core component of these models is that the Her proteins repress
their own transcription as well as the transcription of the delta
genes, leading to oscillatory gene expression. Our data suggest that
her1 and her7 do not act as repressors, but formally as
activators. However, since both proteins belong to a family of bHLH proteins,
which are only known as repressors, we have to postulate additional components
(see above).
Lewis (Lewis, 2003) has
proposed a model in which autoinhibition of her1 and her7
coupled with transcriptional delay could serve as the basis of an
intracellular oscillation, which would be brought into phase by Delta-Notch
signalling (Jiang et al.,
2000
). This model is also not fully in line with our findings,
because it requires a direct repression effect of both genes on themselves and
a fully equivalent role of both genes. However, the model seems sufficiently
flexible to allow for indirect repression and for the possibility of slightly
different roles of her1 and her7.
It is clear that any model that takes only Delta-Notch signalling
components into account cannot be complete. Holley et al.
(Holley et al., 2002) have
shown that fused somites (fss) and beamter
(bea) are involved in gene regulation in the anterior PSM.
Furthermore it is known that an FGF gradient is required for coordinating the
segmentation process and the differentiation of cells in the anterior PSM
(Dubrulle et al., 2001
;
Sawada et al., 2001
). Finally,
results from the mouse indicate that the wnt-signalling pathway could be an
essential component that is linked to the Delta-Notch pathway genes
(Aulehla et al., 2003
).
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ACKNOWLEDGMENTS |
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Footnotes |
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* These authors contributed equally to this work
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REFERENCES |
---|
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---|
Aoyama, H. and Asamoto, K. (1988). Determination of somite cells: independence of cell differentiation and morphogenesis. Development 104, 15-28.[Abstract]
Aulehla, A., Wehrle, C., Brand-Saberi, B., Kemler, R., Gossler, A., Kanzler, B. and Herrmann, B. G. (2003). Wnt3a plays a major role in the segmentation clock controlling somitogenesis. Dev. Cell 3,395 -406.
Bessho, Y., Miyoshi, G., Sakata, R. and Kageyama, R.
(2001a). Hes7: a bHLH-type repressor gene regulated by
Notch and expressed in the presomitic mesoderm. Genes
Cells 6,175
-185.
Bessho, Y., Sakata, R., Komatsu, S., Shiota, K., Yamada, S. and
Kageyama, R. (2001b). Dynamic expression and essential
functions of Hes7 in somite formation. Genes
Dev. 15,2642
-2647.
Cole, S. E., Levorse, J. M., Tilghman, S. M. and Vogt, T. F. (2002). Clock regulatory elements control cyclic expression of Lunatic fringe during somitogenesis. Dev. Cell 3,75 -84.[Medline]
Cooke, J. and Zeeman, E. C. (1976). A clock and wavefront model for control of the number of repeated structures during animal morphogenesis. J. Theor. Biol. 58,455 -476.[Medline]
Dale, J. K. and Pourquié, O. (2000). A clock-work somite. BioEssays 22, 72-83.[CrossRef][Medline]
Dale, J. K., Maroto, M., Dequeant, M.-L., Malapert, P., McGrew, M. and Pourquié, O. (2003) Periodic Notch inhibition by Lunatic Fringe underlies the chick segmentation clock. Nature 421,275 -278.[CrossRef][Medline]
Davis, R. L., Turner, D. L., Evans, L. M. and Kirschner, M. W. (2001). Molecular targets of vertebrate segmentation: two mechanisms control segmental expression of Xenopus hairy2 during somite formation. Dev. Cell 1, 553-565.[Medline]
Dawson, S. R., Turner, D. L., Weintraub, H. and Parkhurst, S. M. (1995). Specificity for the hairy/enhancer of split basic helix-loop-helix (bHLH) proteins maps outside the bHLH domain and suggests two separable modes of transcriptional repression. Mol. Cell. Biol. 15,6923 -6931.[Abstract]
Devereux, J., Haeberli, P. and Smithies, O. (1984). A comprehensive set of sequence analysis programs for the VAX. Nucl. Acids Res. 12,387 -395.[Abstract]
Dornseifer, P., Takke, C. and Campos-Ortega, J. A. (1997). Overexpression of a zebrafish homologue of the Drosophila neurogenic gene Delta perturbs differentiation of primary neurons and somite development. Mech. Dev. 63,159 -171.[CrossRef][Medline]
Dubrulle, J., McGrew, M. J. and Pourquié, O. (2001). FGF signalling controls somite boundary position and regulates segmentation clock control of spatiotemporal Hox gene activation. Cell 106,219 -232.[Medline]
Fisher, A. L., Ohsako, S. and Caudy, M. (1996). The WRPW motif of the hairy-related basic helix-loop-helix repressor proteins acts as a 4-aminoacid transcription repression and protein-protein interaction domain. Mol. Cell. Biol. 16,2670 -2677.[Abstract]
Gajewski, M. and Voolstra, C. (2002). Comparative analysis of somitogenesis related genes of the hairy/Enhancer of split class in Fugu and zebrafish. BMC Genomics 3, 21.[CrossRef][Medline]
Henry, C. A., Urban, M. K., Dill, K. K., Merlie, J. P., Page, M.
F., Kimmel, C. B. and Amacher, S. L. (2002). Zebrafish her1
and her7 function together to refine alternating somite boundaries.
Development 129,3693
-3704.
Holley, S. A., Geisler, R. and Nüsslein-Volhard, C.
(2000). Control of her1 expression during zebrafish
somitogenesis by a Delta-dependent oscillator and an independent wave
front activity. Genes Dev.
14,1678
-1690.
Holley, S. A., Jülich, D., Rauch, G.-J., Geisler, R. and
Nüsslein-Volhard, C. (2002). her1 and the
Notch pathway function within the oscillator mechanism that regulates
zebrafish somitogenesis. Development
129,1175
-1183.
Ish-Horowicz, D., Howard, K. R., Pinchin, S. M. and Ingham, P. W. (1985) Molecular and genetic analysis of the hairy locus in Drosophila. Cold Spring Harb. Symp. Quant. Biol. 50,135 -144.[Medline]
Jen, W. C., Wettstein, D., Turner, D., Chitnis, A. and Kintner,
C. (1997). The Notch ligand, X-Delta-2,
mediates segmentation of the paraxial mesoderm in Xenopus embryos.
Development 124,1169
-1178.
Jiang, Y. J., Aerne, B. L., Smithers, L., Haddan, C., Ish-Horowicz D. and Lewis, J. (2000). Notch signalling and the synchronization of the somite segmentation clock. Nature 408,475 -479.[CrossRef][Medline]
Jouve, C., Palmeirim, I., Henrique, D., Beckers, J., Gossler,
A., Ish-Horowicz, D. and Pourquié, O. (2000).
Notch signalling is required for cyclic expression of the
hairy-like gene HES1 in the presomitic mesoderm.
Development 127,1421
-1429.
Jowett, T. and Yan, Y. L. (1996). Double fluorescence in situ hybridisation to zebrafish embryos. Trends Genet. 12,387 -389.[Medline]
Kelly, P. D., Chu, F., Woods, I. G., Ngo-Hazelett, P., Cardozo,
T., Huang, H., Kimm, F., Liao, L., Yan, Y.-L., Zhou, Y., Johnson, S. L.,
Abagyan, R., Schier, A. F., Postlethwait, J. H. and Talbot, W. S.
(2000). Genetic linkage mapping of zebrafish genes and ESTs.
Genome Res. 10,558
-567.
Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B. and Schilling, T. H. (1995). Stages of embryonic development of the zebrafish. Dev. Dyn. 203,253 -310.[Medline]
Leimeister, C., Dale, K., Fischer, A., Klamt, B., Hrabe de Angelis, M., Radtke, F., McGrew, M. J., Pourquié, O. and Gessler, M. (2000). Oscillating expression of c-Hey2 in the presomitic mesoderm suggests that the segmentation clock may use combinatorial signaling through multiple interacting bHLH factors. Dev. Biol. 227,91 -103.[CrossRef][Medline]
Leve, C., Gajewski, M., Rohr, K. B. and Tautz, D. (2001). Homologues of c-hairy1 (her9) and lunatic fringe in zebrafish are expressed in the developing central nervous system, but not in the presomitic mesoderm. Dev. Genes Evol. 211,493 -500.[CrossRef][Medline]
Lewis, J. (2003). Autoinhibition with transcriptional delay: a simple mechanism for the zebrafish somitogenesis oscillator. Curr. Biol. (in press).
Maroto, M. and Pourquié, O. (2001). A molecular clock involved in somite segmentation. Curr. Top. Dev. Biol. 51,221 -248.[Medline]
Meinhardt, H. (1986). Models of segmentation. In Somites in developing embryos, NATO ASI Series 118, (ed. R. Bellairs, D. A. Ede and J. W. Lash), pp.179 -189. New York: Plenum.
Meng, A., Jessen, J. R. and Lin, S. (1999). Transgenesis. in: In The Zebrafish Genetics and Genomics, Methods in Cell Biology 60 (ed. H. W. Detrich III., M. Westerfield and L. I. Zon), pp. 133-148.
Morales, A. V., Yasuda, Y. and Ish-Horowicz, D. (2002). Periodic Lunatic fringe expression is controlled during segmentation by a cyclic transcriptional enhancer responsive to Notch signaling. Dev. Cell 3,63 -74.[Medline]
Müller, M., Weizsäcker, E. and Campos-Ortega, J.
A. (1996). Expression domains of a zebrafish homolog of the
Drosophila pair-rule gene hairy correspond to the primordia
of alternating somites. Development
122,2071
-2078.
Oates, A. C. and Ho, R. K. (2002). Hairy/E(spl)-related (Her) genes are central components of the segmentation oscillator and display redundancy with the Delta/Notch signaling pathway in the formation of anterior segmental boundaries in the zebrafish. Development 129,2929 -2946.[Medline]
Oka, C., Nakano, T., Wakeham, A., de la Pompa, J. L., Mori, C.,
Sakai, T., Okazaki, S., Kawaichi, M., Shiota, K., Mak, T. W. and Honjo, T.
(1995). Disruption of the mouse RBP-J kappa gene results in early
embryonic death. Development
121,3291
-3301.
Page, R. D. M. (1996). TREEVIEW: An application to display phylogenetic trees on personal computers. Comp. Appl. Biosci. 12,357 -358.[Medline]
Palmeirim, I., Henrique, D., Ish-Horowicz, D. and Pourquié, O. (1997). Avian hairy gene expression identifies a molecular clock linked to vertebrate segmentation and somitogenesis. Cell 91,639 -648.[Medline]
Pasini, A., Henrique, D. and Wilkinson, D. G. (2001). The zebrafish Hairy/Enhancer-of-split-related gene her6 is segmentally expressed during the early development of hindbrain and somites. Mech. Dev. 100,317 -321.[CrossRef][Medline]
Plickert, G., Gajewski, M., Gehrke, G., Gausepohl, H., Schlossherr, J. and Ibrahim, H. (1999). Automated in situ detection (AISD) of biomolecules. Dev. Genes Evol. 207,362 -367.[CrossRef]
Prince, V. E., Holley, S. A., Bally-Cuif, L., Prabhakaran, B., Oates, A. C., Ho, R. K. and Vogt, T. F. (2001). Zebrafish lunatic fringe demarcates segmental boundaries. Mech. Dev. 105,175 -180.[CrossRef][Medline]
Saga, Y. and Takeda, H. (2001). The making of the somite: molecular events in vertebrate segmentation. Nature Reviews 2,835 -845.
Sawada, A., Shinya, M., Jiang, Y.-J., Kawakami, A., Kuriowa, A.
and Takeda, H. (2001). Fgf/MAPK signalling is a crucial
positional cue in somite boundary formation.
Development 128,4873
-4880.
Senger, M., Flores, T., Glatting, K., Ernst, P., Hotz-Wagenblatt, A. and Suhai, S. (1998). W2H: WWW interface to the GCG sequence analysis package. Bioinformatics 14,452 -457.[Abstract]
Sieger, D., Tautz, D. and Gajewski, M. (2003). The role of Suppressor of Hairless in Notch mediated signalling during zebrafish somitogenesis. Mech. Dev. (in press).
Sprague, J., Doerry, E., Douglas, S. and Westerfield, M.
(2001). The zebrafish information network (ZFIN): a source for
genetic, genomic and developmental research. Nucleic Acids
Res. 29,87
-90.
Stern, C. D. and Keynes, R. J. (1987). Interactions between somite cells: the formation and maintenance of segment boundaries in the chick embryo. Development 99,261 -272.[Abstract]
Takke, C. and Campos-Ortega, J. A. (1999).
her1, a zebrafish pair-rule like gene, acts downstream of notch
signalling to control somite development. Development
126,3005
-3014.
Tam, P. P. and Trainor, P. A. (1994). Specification and segmentation of the paraxial mesoderm. Anat. Embryol. 189,275 -305.[Medline]
van Eeden, F. J. M., Holley, S. A., Haffter, P. and Nüsslein-Volhard, C. (1998). Zebrafish segmentation and pair-rule patterning. Dev. Genet. 23, 65-76.[CrossRef][Medline]
von Weizsäcker, E. (1994). Molekulargenetische Untersuchungen an sechs Zebrafisch-Genen mit Homologie zur Enhancer of split Gen-Familie von Drosophila. PhD thesis, Universität zu Köln.