1 Division of Developmental Neurobiology, National Institute for Medical
Research, The Ridgeway, Mill Hill, London NW7 7AA, UK
2 Vertebrate Development Laboratory, Imperial Cancer Research Fund, 44 Lincoln's
Inn Fields, London WC2A 3PX, UK
Authors for correspondence (e-mail:
pasini{at}ibdm.univ-mrs.fr
and
dwilkin{at}nimr.mrc.ac.uk)
Accepted 11 December 2003
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SUMMARY |
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Key words: Zebrafish, Somites, Notch, Segmentation clock, her6, her4
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Introduction |
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With the exception of Axin2, all the cyclically expressed genes so
far identified are components of the Delta/Notch pathway. Somitogenesis is
defective in animals in which this pathway is disrupted by either activating
or inactivating mutations (reviewed by
Pourquié, 2000;
Pourquié, 2001
).
Analysis of Notch pathway mutants is complicated by the pleiotropy of Notch
signalling which is required at different steps of the somitogenetic process.
The basic helix-loop-helix (bHLH) protein Mesp2 is required in a
Notch-dependent fashion to establish the identity of anterior and posterior
compartments of the maturing somites in mouse
(Takahashi et al., 2000
). A
role in implementing boundary formation has been attributed to Notch and its
modulator lunatic fringe in the chick
(Sato et al., 2002
). Notch
signalling is also linked to the segmentation clock although the exact nature
of this relationship is not yet understood. Several observations suggest that
Notch signalling is a central component of the oscillatory mechanism: (1) the
cycling expression of mouse Hes1 depends on the Notch ligand
Dll1 (Jouve et al.,
2000
); (2) the activity of the Notch-dependent factors Her1, Her7,
Hes1 and Hes7 is required for their own cyclic expression
(Hirata et al., 2002
;
Holley et al., 2002
;
Oates and Ho, 2002
;
Bessho et al., 2003
); (3) the
oscillatory expression pattern of lunatic fringe, required for somite
segmentation, is under the direct transcriptional control of Notch signalling
and is lost in embryos lacking Dll1 or Hes7
(del Barco Barrantes et al.,
1999
; Bessho et al.,
2001
; Cole et al.,
2002
; Morales et al.,
2002
; Dale et al.,
2003
; Serth et al.,
2003
). Alternatively, it has been proposed that the main function
of Notch signalling is to maintain the synchronisation of cyclic gene
expression in the PSM (Jiang et al.,
2000
). This hypothesis is based on the observation that in
zebrafish that are mutant for Notch pathway components, the dynamic stripes of
deltaC expression are replaced by a single broad stripe in which
cells express deltaC at variable levels, giving rise to a `salt and
pepper' pattern. This phenotype can be explained by assuming that individual
PSM cells still express deltaC in a cyclic fashion, but the
spatiotemporal co-ordination of expression among adjacent cells is lost
(Jiang et al., 2000
).
Among the best characterised effectors of Notch signalling in vertebrates
are the bHLH transcription factors of the Hairy/Enhancer-of-split [E(spl)]
family. These are sequence-specific transcriptional repressors whose activity
is largely dependent upon their interaction with co-repressors of the
Groucho/TLE family (Davis and Turner,
2001). The role of Hairy/E(spl) proteins in somitogenesis is still
unclear and investigations have until now focused on family members expressed
in a cyclic fashion within the PSM (Takke
and Campos-Ortega, 1999
; Jouve
et al., 2000
; Bessho et al.,
2001
; Bessho et al.,
2003
; Henry et al.,
2002
; Oates and Ho,
2002
). We have addressed the role of two Notch-dependent zebrafish
hairy/E(spl)-related genes, her6 and her4, which are
expressed at the transition zone between PSM and somites. We show that these
two genes are necessary for normal paraxial mesoderm segmentation and that the
activities of their protein products are required to maintain synchronisation
of the cyclical expression of both deltaC and her1.
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Materials and methods |
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The construct pCS2+her6-tr was created by PCR-amplifying the her6 cDNA with the primers 5'-AAGGATCCATGGAATTCGAAGATGCCTGCCGATATCATGG and 5'-TTTTCTCGAGCATATGCTAAACGGAGTCTGACGT (restriction sites for NdeI and XhoI and stop codon in bold). This latter primer introduces an in-frame stop codon immediately upstream of the codons encoding the C-terminal WRPW domain of Her6. The resulting fragment was digested with BamHI and XhoI and subcloned into the corresponding sites of the pCS2+ vector.
To generate the construct pCS2+her6-VP16, a plasmid called pCS2+VP16 was first created by subcloning into the BamHI and XbaI sites of pCS2+ the VP16 activation domain excised from pUC18-VP16. A 3'-truncated version of the her6 cDNA was amplified with the primers 5'-AAGGATCCATGGAATTCGAAGATGCCTGCCGATATCATG and 5-GCGGGATCCAACGGAGTCTGACGT (BamHI site in bold), cut with BamHI and subcloned in frame with the VP16 activation domain into the BamHI site of pCS2+VP16.
PCR amplifications were performed with the high fidelity Pfu polymerase
(Promega) and all PCR-generated constructs were sequenced to check for the
absence of mutations. All constructs were in vitro translated with the TNT
Sp6-coupled Reticulocyte Lysate System (Promega) to verify that protein
products of the correct size were expressed. In addition, the transcriptional
activities of the Her6 wild-type and mutant proteins were tested in a
luciferase gene reporter assay carried out in HEK293 cells with the luciferase
vector pHesLuc (Takebayashi et al.,
1994). The luciferase activities were measured using the
Luciferase Reporter Gene Assay Kit (Boehringer Mannheim) according to the
manufacturer's instructions. The pCS2+wther4 construct was a gift from J.-A.
Campos-Ortega.
In vitro mRNA synthesis
All constructs were linearised with NotI and capped mRNAs were
synthesised with the Sp6 RNA polymerase. mRNAs were resuspended in RNAse-free
water (Sigma) at a concentration of 1 mg/ml, aliquoted and stored at
80°C. Immediately prior to injection, synthetic mRNAs were diluted
in RNAse-free water containing 1 µg/µl 2,000,000 Mr
lysinated fluorescein dextran (Molecular Probes). Owing to its very high
molecular weight, this polymer does not leak through the cytoplasmic bridges
or into the yolk and is therefore suitable for lineage tracing in the
zebrafish (Strehlow and Gilbert,
1993).
Morpholinos
The following morpholino-modified antisense oligonucleotides (GeneTools)
were used in this study:
Stock solutions of morpholinos (1 mM) were made in RNAse-free water (Sigma) or in 1xDanieau's solution. Working dilutions were made in 1xDanieau's solution.
The efficiency and specificity of morpholinos were determined on the basis of their ability to block mRNA transcription in the TnT Coupled Reticulocyte Lysate System (Promega).
Embryo production and microinjection
Zebrafish embryos obtained by natural spawning were staged according to
Kimmel et al. (Kimmel et al.,
1995). For injections of mRNA, a volume of about 1-2 nl was
injected with a Picospritzer into the cytoplasm of one cell at the two-cell
stage. Morpholinos were injected into the yolk of one-cell to four-cell stage
embryos. Embryos were allowed to develop to the desired stage, screened for
developmental abnormalities under an Axiophot microscope and photographed
alive or fixed overnight in 4% PFA and processed for whole-mount in situ
hybridisation.
In situ hybridisation
Whole-mount in situ hybridisations were carried out as described by Oxtoby
and Jowett (Oxtoby and Jowett,
1993). Two-colour whole-mount in situ hybridisation with
digoxigenin- and fluorescein-labelled probes were performed according to the
protocol of Hauptmann and Gerster
(Hauptmann and Gerster, 1994
).
When lysinated fluorescein dextran was to added the injected mRNAs as a
lineage tracer, it was detected with an anti-fluorescein antibody coupled to
alkaline phosphatase, using either Fast Red or INT-BCIP (Roche) as chromogenic
substrates.
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Results |
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RNAs coding for wild-type and the two mutant forms of Her6 were injected
into cleavage-stage zebrafish embryos, which were allowed to develop until
10-15 ss and visually screened for mesoderm segmentation abnormalities.
Embryos were considered affected if at least three ipsilateral somites are
irregular in shape and/or size along their AP axis and if at least two
consecutive intersomitic boundaries are absent, incomplete or irregular.
Embryos severely defective in epiboly, gastrulation or convergent extension
were discarded and the concentrations of injected mRNAs were adjusted to
minimise such defects. Defects in paraxial mesoderm segmentation occurred in a
high proportion (53%; 298/559) of embryos injected with wt-her6 mRNA (40-50
pg/embryo) and less frequently (25%; 55/225) in embryos injected with the same
amount of her6tr mRNA. Injection of 40-50 pg her6VP16 mRNA resulted in a high
percentage of embryos with impaired gastrulation but after reducing the
amounts of injected mRNA to 10 pg/embryo specific somitic defects were
observed (37%; 118/321). By contrast, segmentation defects were infrequent
(20%; 33/159) in embryos injected with 40-50 pg of mRNA coding for Her9, a
zebrafish Her protein highly homologous to Her6 but not expressed in the
paraxial mesoderm (Leve et al.,
2001) and rare in embryos injected with lacZ mRNA (7%;
10/147).
In wt-her6- and her6VP16-injected embryos, the paraxial mesoderm was always
segmented, but intersomitic clefts were incomplete or irregular and somites
split or fused (Fig. 3A-C,H-J).
Differentiation of somitic derivatives appears normal, as shown by
hybridisation with myod. The segmental restriction of gene expression
is impaired, with both markers for the A (lfng, papc) and P
(myod, ephrinb2) somitic compartments expressed in broad, irregular
domains (Fig. 3D-F,K-M). In the
anterior PSM, segmental expression of mespb, which confers anterior
somitic compartment identity (Sawada et
al., 2000), is disrupted but not downregulated in wt-her6-injected
embryos (Fig. 3G) nor expanded
in her6VP16-injected embryos (Fig.
3N). Her6 therefore does not appear to play a role in the
Notch-dependent establishment of A and P compartment identity. Embryos in
which the spatially restricted expression of her6 or the
transcriptional activity of its protein product are altered thus resemble the
Notch pathway mutants beamter (bea), deadly seven
(des), after eight (aei) and mind bomb
(mib), with abnormal AP somite polarity and irregular intersomitic
boundaries (Durbin et al.,
2000
; Jiang et al.,
2000
; Gray et al.,
2001
), rather than the fused somites (fss)
mutant or mespb-injected embryos, in which the A and P somite
compartments, respectively, do not form and intersomitic boundaries are absent
(Durbin et al., 2000
;
Sawada et al., 2000
,
Nikaido et al., 2002
).
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Injection of 40-50 pg/embryo of RNA encoding wild-type Her4 resulted in a higher percentage of embryos with specific paraxial mesoderm segmentation defects (74%; 107/145) than injection of the same amount of wt-her6 (48%; 55/114). However, the morphological somite abnormalities and the expression patterns of the AP somitic polarity markers myod and papc were not different in embryos injected with wt-her4, wt-her6 or her6VP16 (Fig. 3D,F,K,M,O,P). As with injections of wt-her6 or her6VP16 mRNAs, overexpression of Her4 also led to disruptions of the periodical pattern of expression of the deltaC and her1 genes, without affecting their expression levels in the PSM (Fig. 4H-K).
The similarity of the defects observed after disrupting the spatially
restricted expression pattern and activity of Her6 and Her4 suggests that the
two factors may act in the same step of the somitogenetic process. It is
notable that, in embryos injected with her6VP16 or wt-her4, and to lesser
extent in those injected with wt-her6, the domains of expression of
deltaC and her1 often consist of a mixture of cells with
different levels of expression (Fig.
4C-C'',E',G-G'',I',K-K'') reminiscent
of that observed in Notch pathway mutants
(Jiang et al., 2000).
Antisense morpholino oligonucleotides against her6 and her4 disrupt posterior somitogenesis
To test whether Her6 and Her4 are required for somitogenesis, separately or
in combination, we designed antisense morpholino oligonucleotides (MO) to
specifically block translation of their mRNAs. In the absence of antibodies
against the Her6 and Her4 proteins, the efficiency and specificity of the two
MOs were tested in an in vitro translation assay, where 1 µM her4MO was
sufficient to block the translation of her4 mRNA without affecting
that of her6 mRNA, whereas 1 µM her6MO led to a substantial
decrease of Her6 synthesis while leaving the levels of Her4 unchanged
(Fig. 5A). Embryos were then
injected with various amounts of either her6MO or her4MO and stained with an
anti-myosin heavy chain antibody to highlight the morphology of somites and
intersomitic boundaries. Injections of more than 6 ng/embryo of either her6MO
or her4MO result in embryos with arrested epiboly (data not show). We could
not determine whether this phenotype reflects nonspecific toxicity or an early
role of her6 and her4. However, in all the subsequent
experiments, a maximum of 6 ngMO/embryo was injected and embryos showing early
defects were discarded. Both Her6 and Her4 knockdowns result in abnormal CNS
and endodermal development, and embryos injected with 6 ng of her6MO or her4MO
often have a bent notochord. In her6MO-injected embryos the first somites form
normally, but during somitogenesis the newly-formed somites and intersomitic
boundaries become progressively more irregular
(Fig. 5C-C''',D,D').
This phenotype is much less severe in embryos injected with her4MO, in which
only the last 5 to 7 somites are affected
(Fig. 5E,F,F'), and is
only rarely observed in embryos injected with a MO against her9 (data
not shown). As shown in Table
1, the penetrance of the intersomitic boundaries disruption
phenotype depends on three factors: (1) it is dose-dependent; (2) at equal
doses it is higher in her6MO-injected embryos than her4MO-injected embryos;
and (3) within the same batch of her6MO- or her4MO-injected embryos, it
increases with time. When 3 ng each of her6MO and her4MO are injected
together, both the penetrance and the severity of the phenotype increase
cooperatively (Fig. 5G-G''
and Table 1). In all the
injected embryos, the orderly stacking of myosin fibrils was disrupted even in
somites delimited by normal boundaries, possibly reflecting a role for Her6
and Her4 in refining the internal somitic organisation. This phenotype became
aggravated in the more posterior somites, where myofibrils often cross the
defective boundaries (Fig.
5C'',C''',F') and was particularly severe in
her6MO+her4MO-injected embryos, with fibrils spanning the length of several
somites (Fig.
5G',G''). Counting the number of normal somites in
injected embryos at 48 hour stage shows that the onset of boundary disruption
occurs on average between somites 20 and 22 in embryos injected with her6MO,
between somites 23 and 25 in embryos injected with her4MO and around somite 11
in embryos coinjected with her6+her4MO
(Table 1).
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A reduction of Her6 function disrupts notch1a/des expression in the anterior PSM
To determine if a decrease in Her6 activity affects the expression of other
Notch pathway components in the somites and anteriormost PSM, embryos injected
with 6 ng her6MO were probed for notch1a/des
(Bierkamp and Campos-Ortega,
1993), notch1b, notch5, notch6
(Westin and Lardelli, 1997
) or
lfng (Prince et al.,
2001
) expression at the 10-15 ss. We analysed the embryos at a
stage preceding the onset of morphologically recognisable her6MO-induced
phenotype in order to identify early alterations of expression patterns which
presage, and could be the cause of, somitogenesis defects, rather than late
disruptions which could be secondary to abnormal somite morphology. As shown
in Fig. 6, of all the genes
analysed only notch1a/des shows a clear disruption of its expression
pattern. The PSM of control embryos can be subdivided into three territories
with varying levels of notch1a/des expression: strong in the tailbud
and posterior PSM, intermediate in the intermediate PSM and low in the
anterior PSM, at the level of somite 0
(Fig. 6A,C). In her6MO-injected
embryos, this progressive downregulation is impaired: notch1a/des is
expressed at equally strong levels throughout the PSM and its expression
domain is not delimited by a sharp boundary between somites 0 and 1
(Fig. 6B,D). The expression
patterns of notch1b, notch5 and notch6 are not affected
(Fig. 6G-L). The pattern of
lfng expression is not altered, but its levels are increased in
her6MO-injected embryos (Fig.
6E,F). Thus, the activity of Her6 is required to progressively
downregulate notch1a/des within the intermediate and anterior PSM and
to refine the boundary of its expression domain at the transition between PSM
and somites.
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Discussion |
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Differences in the PSM expression pattern of Notch pathway components
between zebrafish on one hand and mouse and chicken on the other have already
been noticed. Zebrafish lfng, in contrast to its mouse and chicken
homologues, does not cycle in the PSM
(Prince et al., 2001), whereas
Delta genes cycle in zebrafish but not in mouse or chicken
(Jiang et al., 2000
). One
possible explanation for these discrepancies is that different vertebrate
classes exploit different cycling components of Notch pathway to fulfil the
same functions during somitogenesis. Alternatively, it is possible that
Hes1 and chick hairy2 exert different functions in the PSM
and in the segmented somites and that in zebrafish such functions have been
shared among distinct hairy/E(Spl)-related genes, of which some
her1 and her7 cycle within the PSM
(Holley et al., 2002
;
Oates and Ho, 2002
), while
others, such as her6, are expressed in a static fashion within the
anterior PSM and the somites.
Our data show that the pattern of expression of her6 is dependent
on the integrity of the Notch signalling pathway and on its spatially
restricted activation: a block of the Notch signal by dominant-negative Su(H)
results in a loss of her6 expression in somites and the anterior PSM,
while ubiquitous and sustained activation of Notch signalling by
constitutively active Su(H) leads to ectopic expression of her6
throughout the PSM. Four zebrafish Notch genes with spatially restricted
expression patterns have been identified to date
(Bierkamp and Campos-Ortega,
1993; Westin and Lardelli,
1997
). Ubiquitous expression of constitutively active Notch1a,
NIC, which leads to increased and ectopic expression of her1 and
her4 (Takke et al.,
1999
; Takke and Campos-Ortega,
1999
), fails to induce an ectopic expression of her6 in
the posterior PSM (A. P., Y.-J. J. and D. G. W., unpublished). However, the
expression pattern of her6 in the anterior PSM and the segmented
somites is remarkably similar to that of notch5
(Westin and Lardelli, 1997
).
Thus, it is possible that Her6 is a specific effector of the Notch5-mediated
signal.
The function of Her6 and its relation to the segmentation clock
Her6 is unlikely to be involved in the determination of somitic compartment identity
The pattern of her6 expression and its dependence upon Notch
signalling suggest that this gene could be an output of the segmentation
clock, required to determine the identity of the posterior somitic
compartment, thus providing a link between the mechanism setting the tempo for
the generation of the somites and their internal patterning. We addressed this
hypothesis by injecting zebrafish embryos with RNAs coding for wild-type or an
antimorph form of the Her6 protein. Paraxial mesoderm forms normally in all
the injected embryos but its segmentation is imperfect. Internal somitic AP
patterning required for the formation and positioning of intersomitic clefts
is disrupted in embryos in which the function of Her6 or its expression
profile are altered. However, in contrast to what is expected if Her6 were
required for determining the P compartment identity by repressing A
compartment identity markers, both A and P compartment markers are expressed
throughout the segmented paraxial mesoderm. Similarly, mespb, which
codes for a bHLH protein determining the identity of A compartment
(Sawada et al., 2000), is not
downregulated in wt-her6-injected embryos nor up-regulated in
her6VP16-injected ones. Thus, we can conclude that her6 does not play
a major role in the determination of the P somitic compartment.
Another possibility is that Her6 represents an output of the clock directly
involved in establishing and/or maintaining the intersomitic boundaries. An
inductive interaction dependent on the spatial restriction of dynamic
lunatic fringe expression and Notch pathway activation and
responsible for the morphological implementation of intersomitic boundaries
has been recently described in chicken embryos
(Sato et al., 2002). Notch
targets of the Hairy/E(Spl) family are likely to be among the effectors of
such an activity. If a comparable phenomenon exists in zebrafish, Her6 could
be a candidate for regulating the expression of boundary-forming cell surface
molecules. However, in this report we focus our attention on a different
aspect of Her6 function, its effect on the activity and synchronisation of the
segmentation clock.
Her6 is required (together with Her4) to maintain the coordinate cyclic expression of deltaC and her1
Altering the restricted expression pattern of Her6 or its transcriptional
repressor function disrupts the dynamic expression of deltaC and
her1 in the PSM. To explore the possibility that Her6 acts as an
effector of Notch signalling which feeds back on the clock to control the
cyclic expression of genes in the PSM, we knocked down its activity by
injection of morpholino oligonucleotides. A reduction of Her6 function led to
disruption of the posterior paraxial mesoderm segmentation in a dose-dependent
manner, the abnormalities arising earlier during somitogenesis in embryos
injected with higher doses of her6MO. In her6MO-injected embryos, the onset of
morphological defects correlates with the first signs of a progressive
breakdown of coordinated cycling expression of deltaC and
her1. The appearance of both the morphological and the molecular
defects is cooperatively accelerated when the functions of her6 and
of a second her gene expressed in the anterior PSM, her4,
are simultaneously reduced by coinjection of morpholinos against her6
and her4.
The exact relationship between Notch signalling and the somite segmentation
clock is still unclear. In particular, it is debated whether Notch signalling
is a central component of the clock, which is necessary for the establishment
and/or maintenance of its oscillatory behaviour, or whether it acts only as a
coupling system, which is required to synchronise the oscillation among
individual, autonomously cycling PSM cells
(Jiang et al., 2000;
Holley et al., 2002
;
Aulehla et al., 2003
).
Alternatively, Notch signalling could exert a dual action, being responsible
for both the initiation and maintenance of the clock oscillations and for
keeping their synchrony (Oates and Ho,
2002
). In this case, it will be interesting to understand whether
the two functions can be separated and attributed to distinct components of
the Notch cascade or whether they represent two aspects of the same molecular
interaction(s). In addition, `Notch signalling' has often been used as a
general byword for the sum of signalling events triggered by the different
Notch receptors expressed in the PSM and only few attempts have been made at
identifying the respective contribution of the different Notch genes and their
different effectors during somitogenesis
(Henry et al., 2002
;
Oates and Ho, 2002
). In
zebrafish, at least four notch genes (notch1a, notch1b,
notch5 and notch6) and four her genes (her1, her4,
her6 and her7) are expressed in the PSM with spatially
restricted expression patterns (Bierkamp
and Campos-Ortega, 1993
;
Westin and Lardelli, 1997
;
Takke et al., 1999
;
Holley et al., 2000
;
Pasini et al., 2001
;
Oates and Ho, 2002
),
suggesting that distinct Notch receptors could regulate the transcription of
specific Her nuclear effectors to mediate distinct aspects of a general Notch
signalling function. her6 and her4 are only expressed in the
tailbud and as two pairs of static stripes in the anterior PSM
(Takke et al., 1999
;
Pasini et al., 2001
), and are
therefore unlikely to represent central elements of the clock oscillatory
mechanism. Accordingly, in both Her6- and Her6+Her4-depleted embryos, the
coordinated periodic pattern of deltaC and her1 stripes is
correctly initiated. However, this pattern fails to be maintained throughout
somitogenesis and is gradually replaced by an irregular mixture of cells
expressing deltaC and her1 at variable levels. This
phenotype resembles the one described in the Notch pathway mutants bea,
deltaD/aei, notch1a/des and mib or in Her7-depleted embryos and
interpreted as the result of a progressive loss of synchronisation among PSM
cells cycling between a deltaC-positive and a
deltaC-negative status (Jiang et
al., 2000
; Oates and Ho,
2002
). Therefore, Her6 and Her4 represent components of the
Notch-dependent machinery which feed back on the clock but are only required
to maintain the synchronisation among cycling PSM cells. In Her6-, Her4- or
Her6+Her4-depleted embryos, the onset of morphological abnormalities and
deltaC pattern disruption is delayed compared with the
notch1a/des and deltaD/aei mutants. This is consistent with
the hypothesis that her6 and her4 only account for part of
the Notch-dependent response, and that in the absence of their protein
products the synchronisation of gene expression oscillation in the PSM is
maintained over a longer period of time than when a more central component is
withdrawn. However, our finding that the onset of morphological abnormalities
and of deltaC pattern disruption is shifted caudally in embryos
injected with decreasing amounts of her6MO or her4MO shows that a partial
depletion of Her6 or Her4 allows the coordination of deltaC dynamic
expression to be maintained over an even longer time and thus highlights the
importance of Her6 and Her4 protein dosage. Therefore, an alternative
explanation of the relatively weak phenotype elicited by 6 ng/embryo
injections of her6MO and her4MO is that these lead to a downregulation rather
than a complete suppression of her6 and her4 translation and
the residual amounts of proteins synthesised are sufficient to maintain clock
synchronisation over a certain number of cycles. If this was the case,
injecting MO doses higher that 6 ng/embryo would shift anteriorly the onset of
somitogenesis defects. This hypothesis could not be tested, since we found
that high her6MO or her4MO doses lead to an early arrest of epiboly.
According to a recent report (Gajewski
et al., 2003), a single her6 gene and three
her4-related genes are present in the zebrafish genome. No data are
at present available regarding the expression profile of the two previously
unknown her4 pseudoalleles but, should they be expressed in the PSM,
it is possible that they escape targeting by her4MO and provide protein
products capable of partially rescuing clock synchronisation, thus providing
an alternative explanation for the weak effect of her4MO.
The mechanism by which Her6 and Her4 exert their function is likely to
involve the transcriptional repression of clock components. Although no bona
fide target of Her6 and/or Her4 transcription repressor activity is yet known,
our data show that the progressive downregulation of notch1a/des
within the anteriormost PSM and the establishment of the boundary of its
expression domain at the transition between PSM and somites are impaired by a
decrease in the function of Her6. notch1a/des regulates the
expression of the cycling gene her1
(Takke and Campos-Ortega,
1999). The Her1 protein negatively regulates the transcription of
her1 itself and of the Notch ligand deltaC.
(Holley et al., 2002
;
Oates and Ho, 2002
).
Regardless of whether such an autoregulatory loop constitutes the core
oscillator responsible for clock activity
(Oates and Ho, 2002
) or is
exclusively required as a synchronising mechanism
(Jiang et al., 2000
), its
localised disruption within the anterior PSM of Her6-depleted embryos can
account for the somitogenesis defects observed.
Our data show that, starting from about 14-somite stage, a `salt and
pepper' pattern of deltaC expression is present not only in the
anterior PSM, but also in the tailbud of her6+her4MO-injected embryos. As
expression of both her6 and her4 is essentially restricted
to the anterior PSM, it may seem surprising that a loss of function of their
protein products results in a loss of clock synchronisation throughout the
PSM. However, oscillator synchronicity is by definition a non cell-autonomous
phenomenon, which requires a fast and accurate intercellular coupling element.
Notch signalling-based synchronising activities are likely to rely on
intercellular feedback loops analogous to those proposed to explain the
generation of oscillations (Oates and Ho,
2002; Dale et al.,
2003
). In such models, local disruptions can be corrected by
community effects as long as they affect a number of cells below a given
threshold, beyond which they override the whole system. It is therefore
conceivable that a localised failure of the ability of some cells to cycle in
synchrony with their neighbours is amplified and gradually spread to the
entire PSM by the same Delta/Notch intercellular signalling that is normally
responsible for maintaining synchrony. An alternative explanation is that the
early and transient expression of her6 and her4 in the
involuting marginal zone and in the tailbud
(Fig. 1A-E) (Takke et al., 1999
) provides
enough transcription factor products sufficient and necessary for maintaining
the clock synchronisation in the posterior PSM throughout several cycles of
deltaC expression. In this case, Her6 and Her4 would be independently
required within the anterior and the posterior PSM and morpholino-mediated
block of their transient early synthesis would be enough to affect later
synchrony of deltaC expression in the posterior PSM.
Co-operation between Her6 and Her4
Morpholino-mediated depletion experiments indicate that Her6 and Her4 are
partially redundant in maintaining the coordination of deltaC dynamic
expression. This synergy could underlie a direct cooperation between the Her6
and Her4 transcription factors. Indeed, hairy/E(spl)-related proteins are
known to homo- and/or heterodimerise (reviewed by
Davis and Turner, 2001) and it
has been suggested that this phenomenon is the basis of a combinatorial
network among cyclically expressed Hairy/E(spl)-related factors in the chicken
PSM (Leimeister et al., 2000
).
A similar network comprising Her6, Her4 and other cyclically or non cyclically
expressed bHLH and hairy/E(spl)-related molecules could function within the
anterior PSM and be responsible for coordinating the synchronisation of the
dynamic waves of expression of cycling genes. Total loss or partial reduction
in the function of one or more of the network components will result in a more
or less rapid disruption of the cycling coordination among PSM cells, and this
will in turn lead to intersomitic defects arising at different points along
the rostrocaudal axis.
Conclusions
The zebrafish transcription factor Her6 is an output of the Notch
signalling pathway that feeds back by regulating the expression of
notch1a in the anterior PSM. Together with another Notch-dependent
hairy/E(Spl) factor, Her4, Her6 is required for maintaining the
synchronisation of cyclic gene expression among adjacent cells within the PSM.
Future studies of their transcriptional regulation as well as identification
of their molecular partners and transcriptional targets will clarify the
relationship between Notch signalling and the segmentation clock.
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ACKNOWLEDGMENTS |
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Footnotes |
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Present address: IMCB, 30 Medical Drive, Singapore 117609, Republic of
Singapore
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REFERENCES |
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---|
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 4, 395-406.[Medline]
Bessho, Y., Sakata, R., Komatsu, S., Shiota, K., Yamada, S.
and Kageyama, R. (2001). Dynamic expression and
essential functions of Hes7 in somite segmentation. Genes
Dev. 15,2642
-2647.
Bessho, Y., Hirata, H., Masamizu, Y. and Kageyama, R.
(2003). Periodic repression by the bHLH factor Hes7 is an
essential mechanism for the somite segmentation clock. Genes.
Dev. 17,1451
-1456.
Bierkamp C. and Campos-Ortega J-A. (1993). A zebrafish homologue of the Drosophila neurogenic gene Notch and its pattern of transcription during early embryogenesis. Mech. Dev. 43,87 -100.[CrossRef][Medline]
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]
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. and Turner, D. L. (2001). Vertebrate Hairy and Enhancer of split related proteins: transcriptional repressors regulating cellular differentiation and embryonic patterning. Oncogene 20,8342 -8357.[CrossRef][Medline]
Davis. R. L., Turner. D. L., Evans. L. M. and Kirschner. M. (2001). Molecular targets of vertebrate segmentation: two mechanisms control segmental expression of Xenopus hairy2 during somite formation. Dev. Cell 1,553 -565.[Medline]
del Barco Barrantes, I., Elia, A. J., Wünsch, K., Hrabe de Angelis. M., Mak, T. W., Rossant, J., Conlon, R. A., Gossler, A. and de la Pompa, J. L. (1999). Interaction between Notch signalling and Lunatic fringe during somite boundary formation in the mouse. Curr. Biol. 9,470 -480.[CrossRef][Medline]
Durbin, L., Sordino, P., Barrios, A., Gering, M., Thisse, C.,
Thisse, B., Brennan, C., Green, A., Wilson, S. and Holder, N.
(2000). Anteroposterior patterning is required within segments
for the somite boundary formation in developing zebrafish.
Development 127,1703
-1713.
Fisher, A. L., Ohsako, S. and Caudy, M. (1996). The WRPW motif of the Hairy-related basic helix-loop-helix proteins acts as a 4-amino-acids transcription repression and protein-protein interaction domain. Mol. Cell. Biol. 16,2670 -2677.[Abstract]
Forsberg, H., Crozet, F. and Brown, N. A. (1998). Waves of mouse Lunatic fringe expression, in four-hour cycles at two-hour intervals, precede somite boundary formation. Curr. Biol. 8,1027 -1030.[Medline]
Gajewski, M., Sieger, D., Alt, B., Leve, C., Hans, S., Wolff,
C., Rohr, K. B. and Tautz, D. (2003). Anterior and posterior
waves of cyclic her1 gene expression are differentially regulated in
the presomitic mesoderm of zebrafish. Development
130,4269
-4278.
Gray, M., Moens, C. B., Amacher S. L., Eisen, J. S. and Beattie C. E. (2001). Zebrafish deadly seven functions in neurogenesis. Dev. Biol. 237,306 -323.[CrossRef][Medline]
Hauptmann, G. and Gerster, T. (1994). Two-color whole-mount in situ hybridization to vertebrate and Drosphila embryos. Trends Genet. 10,266 -267.[CrossRef][Medline]
Henry, C. A, Crawford, B. D., Yan, Y. L., Postlethwait, J., Cooper, M. S. and Hille, M. B. (2001). Roles for zebrafish focal adhesion kinase in notochord and somite morphogenesis. Dev. Biol. 240,474 -487.[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). Two linked
hairy/Enhancer of split-related zebrafish genes, her1 and
her7, function together to refine alternating somite boundaries.
Development 129,3693
-3704.
Hirata, H., Yoshiura, S., Ohtsuka, T., Bessho, Y., Harada, T.,
Yoshikawa, K. and Kageyama, R. (2002). Oscillatory
expression of the bHLH factor Hes1 regulated by a negative feedback loop.
Science 298,840
-843.
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.
Jiang, Y.-J., Aerne, B. L., Smithers, L., Haddon, C., Ish-Horowicz, D. and Lewis J. (2000). Notch signalling and the synchronization of the somite segmentation clock. Nature 408,475 -479.[CrossRef][Medline]
Jen, W.-C., Gawantka, V., Pollet, N., Niehrs, C. and Kintner,
C. (1999). Periodic repression of Notch pathway genes governs
the segmentation of Xenopus embryos. Genes
Dev. 13,1486
-1499.
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.
Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B. and Schilling, T. F. (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, M., 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 signalling 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]
McGrew, M. J., Dale, J. K., Fraboulet, S. and Pourquié, O. (1998). The lunatic fringe gene is a target of the molecular clock linked to somite segmentation in avian embryos. Curr. Biol. 8,979 -982.[Medline]
Maroto, M. and Pourquié, O. (2001). A molecular clock involved in somite segmentation. Curr. Top. Dev. Biol. 51,221 -248.[Medline]
Morales, A., Yasuda, Y. and Ish-Horowicz, D. (2002). Periodic Lunatic fringe expression is controlled by a cyclic transcriptional enhancer responsive to Notch signalling. Dev. Cell 3,63 -74.[Medline]
Nikaido, M., Kawakami, A., Sawada, A., Furutani-Seiki, M., Takeda, H. and Araki, K. (2002). Tbx24, encoding a T-box protein, is mutated in the zebrafish somite-segmentation mutant fused somites. Nat. Genet. 31,195 -199.[CrossRef][Medline]
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 signalling pathway in the formation of anterior segmental boundaries in the zebrafish. Development 129,2929 -2946.[Medline]
Oxtoby, E. and Jowett, T. (1993). Cloning of the zebrafish krox-20 gene (krx-20) and its expression during hindbrain development. Nucleic Acids Res. 21,1087 -1095.[Abstract]
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]
Pourquié, O. (2000). Notch around the clock. Curr. Opin. Genet. Dev. 9, 559-565.[CrossRef]
Pourquié, O. (2001). Vertebrate somitogenesis. Annu. Rev. Cell Dev. Biol. 17,311 -350.[CrossRef][Medline]
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]
Sato, Y., Yasuda, K. and Takahashi, Y. (2002).
Morphological boundary forms by a novel inductive event mediated by Lunatic
fringe and Notch during somitic segmentation.
Development 129,3633
-3644.
Sawada, A., Fritz, A., Jiang, Y.-J., Yamamoto, A., Yamasu, K.,
Kuroiwa, A., Saga, Y. and Takeda, H. (2000). Zebrafish
Mesp family genes, mesp-a and mesp-b, are
segmentally expressed in the presomitic mesoderm and Mesp-b confers the
anterior identity to the developing somites.
Development 127,1691
-1702.
Serth, K., Schuster-Gossler, K., Cordes, R. and Gossler, A.
(2003). Transcriptional oscillation of Lunatic fringe is
eessential for somitogenesis. Genes Dev.
17,912
-925.
Strehlow, D. and Gilbert, W. (1993). A fate map for the first cleavages of the zebrafish. Nature 361,451 -453.[CrossRef]
Takahashi, Y., Koizumi, K., Takagi, A., Kitajima, S., Inoue, T., Koseki, H. and Saga, Y. (2000). Mesp2 initiates somite segmentation through the Notch signalling pathway. Nat. Genet. 25,390 -396.[CrossRef][Medline]
Takebayashi, K., Sasai, Y., Sakai Y., Watanabe T., Nakanishi S.
and Kageyama, R. (1994). Structure, chromosomal locus
and promoter analysis of the gene encoding the mouse helix-loop-helix factor
HES-1. J. Biol. Chem.
269,5150
-5156.
Takke, C., Dornseifer, P., v. Weizsäcker, E. and
Campos-Ortega, J. A. (1999). her4, a zebrafish
homologue of the Drosophila neurogenic gene E(spl), is a
target of NOTCH signalling. Development
126,1811
-1821.
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.
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]
Weinberg E. S., Allende M. L., Kelly C. S., Abdelhamid A.,
Murakami T., Andermann P., Geoffrey Doerre O., Grunwald D. and
Riggleman B. (1996). Developmental regulation of zebrafish
myoD in wild-type, no tail and spadetail embryos.
Development 122,271
-280.
Westin, J. and Lardelli, M. (1997). Three novel Notch genes in zebrafish: implications for vertebrate Notch gene evolution and function. Dev. Genes Evol. 207, 51-63.[CrossRef]
Yamamoto, A., Amacher, S. L., Kim, S-H., Geissert, D., Kimmel,
C. B. and De Robertis, E. M. (1998). Zebrafish
paraxial protocadherin is a downstream target of spadetail
involved in morphogenesis of gastrula mesoderm.
Development 125,3389
-3397.